Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells from Type2 Diabetes Patients: Rescue by Alpha-Ketoglutarate and TET-TDG Functional Reactivation Circ. Res. (IF 13.965) Pub Date : 2017-11-20 Francesco Spallotta, Chiara Cencioni, Sandra Atlante, Davide Garella, Mattia Cocco, Mattia Mori, Raffaella Mastrocola, Carsten Künne, Stefan Günther, Simona Nanni, Valerio Azzimato, Sven Zukunft, Angela Kornberger, Duran Sueruen, Frank Schnutgen, Harald von Melchner, Antonella Di Stilo, Manuela Aragno, Maarten Braspenning, Wim Van Criekinge, Miles J De Blasio, Rebecca H Ritchie, Germana Zaccagnini, Fabio Martelli, Antonella Farsetti, Ingrid Fleming, Thomas Braun, Andres Beiras-Fernandez, Bruno Botta, Massimo Collino, Massimo Bertinaria, Andreas M Zeiher, Carlo Gaetano
Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically-relevant primary cell population. Diabetes compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes.Objective: To investigate the role of α-ketoglutarate (αKG) in the epi-metabolic control of DNA demethylation in CMSCs.Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing and gene specific GC methylation detection revealed an accumulation of 5mC, 5hmC and 5fC in the genomic DNA of human CMSCs isolated from diabetic (D) donors (D-CMSCs). Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high fat diet (HFD), injected with streptozotocin (STZ) or both in combination (STZ-HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in D-CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised thymine DNA glycosylase (TDG) and ten eleven translocation protein 1 (TET1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5fC. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5fC accumulation thus partially mimicking the diabetic epigenetic landscape in cells of non-diabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG-dehydrogenase, increased the αKG level in D-CMSCs and in the heart of HFD and STZ mice eliciting, in HFD, DNA demethylation, glucose uptake and insulin response.Conclusions: Restoring the epi-metabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.
Quaking Inhibits Doxorubicin-Mediated Cardiotoxicity Through Regulation of Cardiac Circular RNA Expression Circ. Res. (IF 13.965) Pub Date : 2017-11-13 Shashi K Gupta, Ankita Garg, Christian Bär, Shambhabi Chatterjee, Ariana Foinquinos, Hendrik Milting, Katrin Streckfuss-Bömeke, Jan Fiedler, Thomas Thum
Rationale: RNA-binding proteins (RBPs) have been described to be expressed and regulated in various organs including the heart. Little is known about the role of RBPs in heart failure induced by the chemotherapy drug doxorubicin and their interaction with circular RNAs.Objective: We aimed to identify key RBPs involved in doxorubicin-mediated heart failure and to elucidate their function.Methods and Results: Global transcriptome profiling from murine myocardium exposed to doxorubicin identified five differentially expressed RBPs. Expression of the RNA-binding protein Quaking (QKI) in response to doxorubicin was strongly downregulated in rodent cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes in vitro and in vivo in mice. Knockdown of Qki in primary cardiomyocytes increased apoptosis and atrophy after treatment with doxorubicin, while lentiviral-mediated overexpression of Qki5 inhibited the doxorubicin-induced apoptosis in cardiomyocytes. In vivo, AAV9-mediated cardiac overexpression of Qki5 prevented cardiac apoptosis and cardiac atrophy induced by doxorubicin and improved cardiac function. Mechanistically, by lentiviral-based overexpression and CRISPR/Cas9-mediated silencing of Qki5 we identified regulated expression of specific circular RNAs derived from Ttn, Fhod3, and Strn3. Moreover, inhibition of Tt derived circular RNA increased the susceptibility of cardiomyocytes to doxorubicin.Conclusions: We here show that overexpression of Qki5 strongly attenuates the toxic effect of doxorubicin via regulating a set of circular RNAs. Qki5 is thus an interesting target molecule to combat doxorubicin-induced cardiotoxicity.
In this Issue Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Ruth Williams
### Khalafalla et al rejuvenate cardiac progenitor cells via nucleotide receptor signaling.The use of autologous cardiac progenitor cells (CPCs) for repairing failing hearts has shown promising results in animal models, but trials in patients with heart failure have provided modest, if any, clinical benefits. One reason for the limited efficacy of CPCs may be that CPCs from patients carry genetic predispositions for heart failure, or have reduced regenerative capacity because of patient’s age or other characteristics. Boosting the potency of autologous CPCs may, therefore, improve their clinical performance. Regeneration in a variety of tissues has been shown to be promoted by the nucleotide receptor P2Y2R, which is activated by extracellular UTP. Now, Khalafalla and colleagues have discovered that P2Y2R is more …
Meet the First Authors Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Lippincott Williams & Wilkins
Dr Farid G. Khalafalla is currently a postdoctoral fellow in Dr Mark Sussman’s laboratory at San Diego State University, conducting research into unraveling purinergic signaling in murine cardiomyocytes and human cardiac progenitor cells derived from heart failure (HF) patients. Farid earned his BS in Pharmaceutical Sciences from the School of Pharmacy, Cairo University, Egypt, and he continued his research career in the United States by pursuing a PhD in Biochemistry under the supervision of Dr Gary Weisman at the University of Missouri-Columbia. His graduate research focused on the role of P2Y2 nucleotide receptor (P2Y2R) in salivary gland regeneration. Farid’s lifelong passion for scientific research was inspired by his father, Dr Galal Khalafalla, a professor of Microbiology at Cairo University. Farid’s goal is to establish an independent research career with emphasis on studying cellular responses to extracellular nucleotide stress signals in the myocardium and developing novel translational …
To Seek the Holy Grail of Cardiac Progenitor Cells Circ. Res. (IF 13.965) Pub Date : 2017-11-10 A.J. Marian
Discoveries in cardiovascular stem cell biology, myocyte regeneration, and cell transplantation have enormous clinical potentials, yet the field is embroiled in amusing controversies.1–3 The existence of cardiac myocyte progenitor cells (CPCs) in the adult heart, let alone the identity of the genuine CPCs, has been debated and so has the relevance of the primary candidate, namely the resident cardiac progenitor cells expressing the KIT antigen (KITpos cells), to cardiac myocyte generation.1,2,4 Similarly, the choice of the cells, delivery approach, and efficacy of cell transplantation in improving cardiovascular outcomes have remained unsettled as has been the elusive nature of the paracrine factors that are presumably responsible for the improvement in cardiac function post-cell transplantation. Consequently, given these ambiguities, basic and clinical investigators are in the quest to seek the holy grail of CPCs; cells that possess genuine capacities to survive, thrive, differentiate to working myocytes, properly couple with the neighboring resident myocytes; electrically and mechanically, enhance cardiac function, and improve the clinical outcomes, without causing serious adverse events. Similarly, there is an intense focus on defining the responsible mechanisms and identifying and characterizing the elusive paracrine factors.Article, see p 1224 The Hippo pathway, first discovered in Drosophila and aptly named after hippopotamus because of its key role in determining organ size, is a contact-dependent pathway that regulates cell growth, survival, proliferation, and differentiation.5,6 The pathway comprises a series of upstream molecules that are linked to cell junctions and are sensitive to cell–cell contact. The upstream molecules are also responsive to mechanical properties of cell environment and cell cytoskeleton, particularly actin filament organization. On sensing the stimuli, the upstream Hippo molecules activate a …
Cortical Bone Stem Cells Administered at Reperfusion Attenuate Remote Zone Myocyte Remodeling Circ. Res. (IF 13.965) Pub Date : 2017-11-10 John M. Canty, Brian R. Weil
Heart failure arises from intrinsic diseases of cardiac muscle or idiopathic dilated cardiomyopathy as well as consequences of coronary artery disease after acute myocardial infarction and chronic ischemia. In patients with ST-segment–elevation myocardial infarction (MI) from a coronary thrombus, immediate reperfusion is the most effective therapy to prevent the loss of myocytes. The routine use of β-blockers, angiotensin inhibition therapy, and spironolactone blocks neurohormonal activation and prevents subsequent left ventricular (LV) remodeling. Stem cell therapy has emerged as an adjunctive approach to further attenuate postinfarction remodeling by stimulating myocyte proliferation and mitigating myocyte loss arising from within and outside of the infarct region. Despite favorable preclinical and early clinical studies in humans using bone marrow mononuclear cells, meta analyses of completed trials have failed to demonstrate a significant impact on clinical outcomes and LV function.1 Because substantial myocyte loss occurs during the first few days after infarction, the lack of efficacy may reflect delays in administering therapy that arise from synthesizing autologous cell products after the event. This and the increasing recognition of the role that paracrine effects play led to interest in allogeneic cells, such as cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs). These are relatively immune privileged and can be administered at the time of percutaneous coronary intervention and reperfusion. Cortical bone stem cells (CBSCs) are a particularly proliferative mesenchymal cell subtype2 that, in vitro, have characteristics similar to murine cKit+/Sca1+ cells and can to some extent transdifferentiate into cardiac myocytes.3 Enriching the cKit+ CBSC population could potentially provide an allogeneic cell therapy platform similar to cardiac stem cells (CSCs) without the need for a myocardial biopsy. Completed studies in nonreperfused murine infarct models have demonstrated that they attenuate postinfarction remodeling.3Article, see p 1263 In this issue of …
Posology for Regenerative Therapy Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Andre Terzic, Atta Behfar
> Sola dosis facit venenum (The dosage makes it either a poison or a remedy)> > —Paracelsus (1493–1541)The science of dosage or posology (from Greek posos, how much, and logos, study) is a branch of pharmacology and therapeutics concerned with treatment dosage and dosage regimen. Establishing optimum dosage underpins every clinical development plan for novel therapeutic candidates. Failure to select the adequate drug dose is a leading culprit for regulatory delays or denial of initial applications for new drugs and, more generally, inadequate dose selection contributes to the high attrition rate of pivotal clinical trials.1Article, see p 1279 Regulatory agencies are committed to facilitate the development and ultimate licensure of safe and effective regenerative therapies.2 To this end, dedicated programs applicable to stem cell therapies have been designed to expedite the advancement and approval of new products (eg, breakthrough therapy designation, accelerated approval).3 However, with limited grasp on the disposition (pharmacokinetics) and action (pharmacodynamics) of stem cells, posology applied to the development of cardiovascular regenerative therapies is still a nascent area of investigation.Without the establishment of standardized dose regimens, clinical trials continue to evaluate wide dose ranges.4 A case in point are clinical studies that have shown rather paradoxical results about the relationship between the stem cell dose and clinical benefit in the setting of heart disease.5 Accordingly, scientific, regulatory and medical communities remain challenged with critical gaps …
A Special Report on the NHLBI Initiative to Study Cellular and Molecular Mechanisms of Arterial Stiffness and Its Association With Hypertension Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Young S. Oh, Dan E. Berkowitz, Richard A. Cohen, C. Alberto Figueroa, David G. Harrison, Jay D. Humphrey, Douglas F. Larson, Jane A. Leopold, Robert P. Mecham, Nelson Ruiz-Opazo, Lakshmi Santhanam, Francesca Seta, John Y.J. Shyy, Zhongjie Sun, Philip S. Tsao, Jessica E. Wagenseil, Zorina S. Galis
Large arteries (especially the aorta) lose elasticity and thicken with aging and as a consequence of other conditions, thus leading to central arterial stiffening and associated adverse effects on blood flow and pressure. Arterial stiffness can be defined and measured in different ways, at a local level or systemically. Increases in either the intrinsic (material) stiffness or net structural (combined geometric and material) arterial stiffness, or both, can increase the velocity at which the pressure pulse travels along the arterial tree and central pulse pressure, which can negatively impact downstream resistance vessels and organs (ie, heart, brain, and kidney). Clarifying temporal and causal relationships between arterial stiffening and hypertension was identified by NHLBI as an important gap of knowledge, with a potential for clinical translation. NIH (National Institutes of Health)-funded studies, more than half of them supported by the NHLBI (Online Figure), have investigated various aspects of arterial stiffening in humans and in experimental models. To enable a more focused research effort on this topic, NHLBI launched a Request for Applications (RFA) HL-10 -027, entitled Cellular and Molecular Mechanisms of Arterial Stiffening and Its Relationship to Development of Hypertension (R01). This initiative supported 11 R01 awards during 2010 to 2015 (Online Table II; cumulative ≈ $20 million dollars in total costs), which represented a significant component of the overall NHLBI investment in this field. Here, we report a summary of important scientific findings that resulted from this NHLBI-initiated research effort, constituting the basis of > 200 original research and review articles (Online Table II), some highlighted here, many conference presentations, and several patents .In humans, increased arterial stiffness, or loss of elastic compliance of large arteries, has been linked to an increased risk of myocardial infarction, heart failure, stroke, and kidney disease, among other conditions. Pulse wave velocity (PWV), …
Onur Kanisicak Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Pam Goldberg-Smith
Hoping to leave a legacy of scientists who work smart as much as they work hard, Onur Kanisicak is well on his way to changing the world. Onur earned his BS in Biomedical Sciences at Istanbul University in his home country of Turkey. He then achieved both his MS in Genetics and PhD in Genetics and Genomics at the University of Connecticut. Onur is currently a postdoctoral research fellow for the Molkentin Laboratory at the Children’s Hospital Medical Center in Cincinnati, OH. He is proud of being a collaborative scientist and the recognitions of his research, such as his published work, and the Postdoctoral fellowship award from the American Heart Association.Onur Kanisicak If you ask my mom, she would say I was always curious about science. She has plenty of stories where I disassembled my toys to figure out how they work; when I wanted to do the same with insects, she patiently showed me how and where to keep them. My family lived in the city, but we would often visit parks and the country where I could roam freely for hours to explore what nature had to offer. As early as I can remember, I wanted to be a medical doctor—that is the normal path in Turkey if you were interested in biology. Eventually, my interest grew more toward understanding how the human body works rather than just seeing patients. For college, I was able to get into a unique program at the Istanbul University Cerrahpasa School of Medicine in the Department of Biomedical Sciences. This program aimed to generate scientists within the human medicine field. While taking classes, I volunteered in various laboratories to be exposed to research as much as possible, which inspired me to establish my own research group one day. Unfortunately, the scope …
Synergistic Research Between the Center of Arrhythmia Research and the Michigan Biology of Cardiovascular Aging at the University of Michigan Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Daniel R. Goldstein, José Jalife
The Center for Arrhythmia Research (CAR) and the Michigan Biology of Cardiovascular Aging (MBoCA) program at the University of Michigan (UM) are performing synergistic research to examine how aging impacts arrhythmias, in particular atrial fibrillation (AF). This collaborative research endeavor has been driven by a very clear increasing clinical demand posed by the aging of our society. Specifically, by the year 2050, the number of older people over 65 years of age will exceed the number of younger people for the first time in history. Given this important trend, the growing healthcare needs of the aging population exacerbated by cardiovascular diseases will pose an ever-increasing burden on our healthcare resources. Cardiovascular diseases in the aging population have well surpassed other age-associated diseases such as susceptibility to infection, chronic lung disease, and cancer as a cause of morbidity and mortality.1 Investigation into the field of aging and cardiovascular diseases has tremendous potential to impact the health and quality of life of older people as no therapies exist that explicitly target aging-specific processes that enhance cardiovascular diseases such as AF.Advanced age is the most critical factor for the development of AF; 10% of patients in their eighth decade have AF and 50% of patients with AF are 80 years of age or older. AF is largely a geriatric condition, and older patients with AF have associated mortality and morbidity not only because of hemodynamic effects and thromboembolism but also because of side effects of therapy including an increased propensity for bleeding with anticoagulation and falls from heart rate–controlling medications.2 Not only does AF in older people pose a large healthcare morbidity and mortality burden, but AF with aging is costly. Thus, research is urgently needed to understand how aging predisposes to AF.The UM CAR (http://www.med.umich.edu/arrhythmia_research/) is a multidisciplinary …
P2Y2 Nucleotide Receptor Prompts Human Cardiac Progenitor Cell Activation by Modulating Hippo SignalingNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Farid G. Khalafalla, Steven Greene, Hashim Khan, Kelli Ilves, Megan M. Monsanto, Roberto Alvarez, Monica Chavarria, Jonathan Nguyen, Benjamin Norman, Walter P. Dembitsky, Mark A. Sussman
Rationale: Autologous stem cell therapy using human c-Kit+ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF.Objective: To improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y2 nucleotide receptor (P2Y2R) activated by extracellular ATP and UTP molecules released following injury/stress.Methods and Results: c-Kit+ hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y2R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)—the downstream effector of Hippo signaling pathway.Conclusions: Proliferation and migration of functionally impaired hCPCs are enhanced by P2Y2R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling—a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y2R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF.
Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic CellsNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Ming-Tao Zhao, Ning-Yi Shao, Shijun Hu, Ning Ma, Rajini Srinivasan, Fereshteh Jahanbani, Jaecheol Lee, Sophia L. Zhang, Michael P. Snyder, Joseph C. Wu
Rationale: Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type–specific gene expression in the human heart.Objective: We aimed to decipher the cell type–specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression.Methods and Results: We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31+CD144+), cardiac progenitor cells (Sca-1+), fibroblasts (DDR2+), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type–specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type–specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression.Conclusions: Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases.
Lactate Promotes Synthetic Phenotype in Vascular Smooth Muscle CellsNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Libang Yang, Ling Gao, Thomas Nickel, Jing Yang, Jingyi Zhou, Adam Gilbertsen, Zhaohui Geng, Caitlin Johnson, Bernice Young, Craig Henke, Glenn R. Gourley, Jianyi Zhang
Rationale: The phenotypes of vascular smooth muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood.Objective: To determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs.Methods and Results: Experiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L−) medium. Compared with the L− medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters and were generally attenuated both by the blockade of monocarboxylic acid transporter activity and by transfection with iRNA for NDRG (N-myc downstream regulated gene). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+-cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate-dehydrogenase levels, vSMC proliferation, and monocarboxylic acid transporter and NDRG expression were greater in the ischemic zone than in nonischemic tissues.Conclusions: These results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.
Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial InfarctionNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Thomas E. Sharp, Giana J. Schena, Alexander R. Hobby, Timothy Starosta, Remus M. Berretta, Markus Wallner, Giulia Borghetti, Polina Gross, Daohai Yu, Jaslyn Johnson, Eric Feldsott, Danielle M. Trappanese, Amir Toib, Joseph E. Rabinowitz, Jon C. George, Hajime Kubo, Sadia Mohsin, Steven R. Houser
Rationale: Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early-stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients.Objective: To determine whether post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model.Methods and Results: Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia–reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n=9; 2×107 cells total) or placebo (vehicle; n=9) through NOGA-guided transendocardial injections. 5–ethynyl–2′deoxyuridine (EdU)—a thymidine analog—containing minipumps were inserted at the time of MI induction. At 72 hours (n=8), initial injury and cell retention were assessed. At 3 months post-MI, cardiac structure and function were evaluated by serial echocardiography and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72 hours post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes, and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular volumes and ejection fraction were significantly more preserved in CBSC-treated hearts, and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC- versus vehicle- treated animals.Conclusions: CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves left ventricular functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction.
Dose Comparison Study of Allogeneic Mesenchymal Stem Cells in Patients With Ischemic Cardiomyopathy (The TRIDENT Study)Novelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Victoria Florea, Angela C. Rieger, Darcy L. DiFede, Jill El-Khorazaty, Makoto Natsumeda, Monisha N. Banerjee, Bryon A. Tompkins, Aisha Khan, Ivonne H. Schulman, Ana Marie Landin, Muzammil Mushtaq, Samuel Golpanian, Maureen H. Lowery, John J. Byrnes, Robert C. Hendel, Mauricio G. Cohen, Krystalenia Valasaki, Marietsy V. Pujol, Eduard Ghersin, Roberto Miki, Cindy Delgado, Fouad Abuzeid, Mayra Vidro-Casiano, Russell G. Saltzman, Daniel DaFonseca, Lina V. Caceres, Kevin N. Ramdas, Adam Mendizabal, Alan W. Heldman, Raul D. Mitrani, Joshua M. Hare
Rationale: Cell dose and concentration play crucial roles in phenotypic responses to cell-based therapy for heart failure.Objective: To compare the safety and efficacy of 2 doses of allogeneic bone marrow–derived human mesenchymal stem cells identically delivered in patients with ischemic cardiomyopathy.Methods and Results: Thirty patients with ischemic cardiomyopathy received in a blinded manner either 20 million (n=15) or 100 million (n=15) allogeneic human mesenchymal stem cells via transendocardial injection (0.5 cc per injection × 10 injections per patient). Patients were followed for 12 months for safety and efficacy end points. There were no treatment-emergent serious adverse events at 30 days or treatment-related serious adverse events at 12 months. The Major Adverse Cardiac Event rate was 20.0% (95% confidence interval [CI], 6.9% to 50.0%) in 20 million and 13.3% (95% CI, 3.5% to 43.6%) in 100 million (P=0.58). Worsening heart failure rehospitalization was 20.0% (95% CI, 6.9% to 50.0%) in 20 million and 7.1% (95% CI, 1.0% to 40.9%) in 100 million (P=0.27). Whereas scar size reduced to a similar degree in both groups: 20 million by −6.4 g (interquartile range, −13.5 to −3.4 g; P=0.001) and 100 million by −6.1 g (interquartile range, −8.1 to −4.6 g; P=0.0002), the ejection fraction improved only with 100 million by 3.7 U (interquartile range, 1.1 to 6.1; P=0.04). New York Heart Association class improved at 12 months in 35.7% (95% CI, 12.7% to 64.9%) in 20 million and 42.9% (95% CI, 17.7% to 71.1%) in 100 million. Importantly, proBNP (pro-brain natriuretic peptide) increased at 12 months in 20 million by 0.32 log pg/mL (95% CI, 0.02 to 0.62; P=0.039), but not in 100 million (−0.07 log pg/mL; 95% CI, −0.36 to 0.23; P=0.65; between group P=0.07).Conclusions: Although both cell doses reduced scar size, only the 100 million dose increased ejection fraction. This study highlights the crucial role of cell dose in the responses to cell therapy. Determining optimal dose and delivery is essential to advance the field, decipher mechanism(s) of action and enhance planning of pivotal Phase III trials.Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02013674.
Correction to: Association of Serum Retinoic Acid With Risk of Mortality in Patients With Coronary Artery Disease Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Lippincott Williams & Wilkins
In the article by Liu et al, “Association of Serum Retinoic Acid With Risk of Mortality in Patients With Coronary Artery Disease,” which published in the August 5, 2016 issue of the journal ( Circ Res . 2016;119:557–563. DOI: 10.1161/CIRCRESAHA.116.308781), corrections were needed.The authors report the following error in the published paper: in the Methods section, description …
Correction to: Association of Plasma 7-Ketocholesterol With Cardiovascular Outcomes and Total Mortality in Patients With Coronary Artery Disease Circ. Res. (IF 13.965) Pub Date : 2017-11-10 Lippincott Williams & Wilkins
In the article by Song et al, “Association of Plasma 7-Ketocholesterol With Cardiovascular Outcomes and Total Mortality in Patients With Coronary Artery Disease,” which published in the May 12, 2017 issue of the journal ( Circ Res . 2017;120:1622–1631. DOI: 10.1161/CIRCRESAHA.117.311049), corrections were needed.The authors report the following error in the published paper: in the …
Induced Pluripotent Stem Cell (iPSC)-Derived Extracellular Vesicles Are Safer and More Effective for Cardiac Repair than iPSCs Circ. Res. (IF 13.965) Pub Date : 2017-11-08 Marta Adamiak, Guangming Cheng, Sylwia Bobis-Wozowicz, Lin Zhao, Sylwia Kedracka-Krok, Anweshan Samanta, Elzbieta Karnas, Yu-Ting Xuan, Bozena Skupien-Rabian, Xing Chen, Urszula Jankowska, Magdy Girgis, Malgorzata Sekula, Arash Davani, Slawomir Lasota, Robert J Vincent, Michal Sarna, Kathy L Newell, Ou-Li Wang, Nathaniel Dudley, Zbigniew Madeja, Buddhadeb Dawn, Ewa K Zuba-Surma
Rationale: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs.Objective:To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro; and compare the safety and efficacy of iPSC-EVs and iPSCs for cardiac repair in vivo.Methods and Results: Murine iPSCs were generated and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time qRT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 h after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved LV function at 35 d after MI, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in LV mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, while reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, while iPSC-EV injection was safe.Conclusions: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to LV function, vascularization, and amelioration of apoptosis and hypertrophy. Due to their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.
Mitochondrial Oxidative Stress Reduces the Immunopotency of Mesenchymal Stromal Cells in Adults with Coronary Artery Disease Circ. Res. (IF 13.965) Pub Date : 2017-11-07 Ozge K Kizilay Mancini, Maximilien Lora, Alexanne Cuillerier, Dominique Shum-Tim, Reggie C Hamdy, Yan Burelle, Marc J Servant, Ursula Stochaj, Ines Colmegna
Rationale: Mesenchymal stromal cells (MSCs) are promising therapeutic strategies for coronary artery disease (CAD), however, donor-related variability in cell quality is a main cause of discrepancies in preclinical studies. In vitro, MSCs from individuals with CAD have reduced ability to suppress activated T-cells. The mechanisms underlying the altered immunomodulatory capacity of MSCs in the context of atherosclerosis (ATH) remain elusive.Objective: The aim of this study was to assess the role of mitochondrial dysfunction in the impaired immunomodulatory properties of MSCs from ATH patients.Methods and Results: Adipose tissue-derived MSCs were isolated from ATH (n=38) and Non ATH (n=42) donors. MSCs:CD4+ T-cell suppression, was assessed in allogeneic co-culture systems. Compared to Non ATH-, ATH-MSCs displayed higher levels of both intracellular (p=0.006) and mitochondrial (p=0.03) reactive oxygen species (ROS) reflecting altered mitochondrial function. The increased mitochondrial ROS levels of ATH-MSCs promoted a phenotypic switch characterized by enhanced glycolysis and an altered cytokine secretion (interleukin-6 (IL-6) p<0.0001, IL-8/CXCL8 p=0.04, and monocyte chemoattractant protein-1 (MCP-1/CCL2) p=0.01). Furthermore, treatment of ATH-MSCs with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced the levels of IL-6, IL-8/CXCL8, and MCP-1/CCL2 in the MSC secretome and improved MSCs immunosuppressive capacity (p=0.03).Conclusions: An impaired mitochondrial function of ATH-MSCs underlies their altered secretome, and reduced immunopotency. Interventions aimed at restoring the mitochondrial function of ATH-MSCs improve their in vitro immunosuppressive ability, and may translate into enhanced therapeutic efficiency.
Associations of Endogenous Estradiol and Testosterone Levels with Plaque Composition and Risk of Stroke in Subjects with Carotid Atherosclerosis Circ. Res. (IF 13.965) Pub Date : 2017-11-02 Marija Glisic, Blerim Mujaj, Oscar L Rueda-Ochoa, Eralda Asllanaj, Joop S Laven, Maryam Kavousi, M. Kamran Ikram, Meike W Vernooij, M Arfan Ikram, Oscar H Franco, Daniel Bos, Taulant Muka
Rationale: Sex steroids may play a role in plaque composition and in stroke incidence.Objective: To study the associations of endogenous estradiol and testosterone with carotid plaque composition in elderly men and postmenopausal women with carotid atherosclerosis, as well as with risk of stroke in this population.Methods and Results: Data of 1023 postmenopausal women and 1124 men (≥45 years) with carotid atherosclerosis, from prospective population-based Rotterdam Study, were available. At baseline, total estradiol (TE) and total testosterone (TT) were measured. Carotid atherosclerosis was assessed by ultrasound, whereas plaque composition (presence of calcification, lipid core and intraplaque hemorrhage) was assessed by MRI. TE and TT were not associated with calcified carotid plaques in either sex. TE was associated with presence of lipid core in both sexes (in women odds ratio (OR), 95% CI: 1.48 [1.02, 2.15], in men OR, 95% CI: 1.23 (1.03, 1.46]), whereas no association was found between TT and lipid core in either sex. Higher TE (OR, 95% CI: 1.58 [1.03, 2.40]) and lower TT (OR, 95% CI: 0.82 [0.68, 0.98]) were associated with intraplaque hemorrhage in women but not in men. In women, TE was associated with increased risk of stroke (hazard ratio (HR), 95% CI: 1.98 [1.01, 3.88], whereas no association was found in men. TT was not associated with risk of stroke in either sex.Conclusions: TE was associated with presence of vulnerable carotid plaque as well as increased risk of stroke in women, whereas no consistent associations were found for TT in either sex.
Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induces Post-Translational Modifications of AKAP121, DRP1 and OPA1 That Promote Mitochondrial Fission Circ. Res. (IF 13.965) Pub Date : 2017-11-01 Kensuke Tsushima, Heiko Bugger, Adam R Wende, Jamie Soto, Gregory A Jenson, Austin R Tor, Rose McGlauflin, Helena C kenny, Yuan Zhang, Rhonda Souvenir, Xiao X Hu, Crystal L Black, Renata O Pereira, Vitor A Lira, Kenneth Spitzer, Terry L Sharp, Kooresh I Shoghi, Genevieve C Sparagna, Eva A Rog-Zielinska, Peter Kohl, Oleh Khalimonchuk, Jean E Schaffer, E. Dale Abel
Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood.Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo.Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes (NRVCs). Palmitate exposure to NRVCs initially activates mitochondrial respiration, coupled with increased mitochondrial membrane potential and adenosine triphosphate (ATP) synthesis. However, long-term exposure to palmitate (> 8h) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of A-kinase anchor protein (AKAP121) leading to reduced phosphorylation of DRP1 at Ser637 and altered proteolytic processing of OPA1. Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro.Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy.
Unspliced XBP1 Confers VSMC Homeostasis and Prevents Aortic Aneurysm Formation via FoxO4 Interaction Circ. Res. (IF 13.965) Pub Date : 2017-10-31 Guizhen Zhao, Yi Fu, Zeyu Cai, Fang Yu, Ze Gong, Rongbo Dai, Yanhua Hu, Lingfang Zeng, Qingbo Xu, Wei Kong
Rationale: Although not fully understood, the phenotypic transition of vascular smooth muscle cells exhibits at the early onset of the pathology of aortic aneurysms. Exploring the key regulators that are responsible for maintaining the contractile phenotype of VSMCs may confer vascular homeostasis and prevent aneurysmal disease. X-box binding protein 1, which exists in a transcriptionally inactive unspliced form (XBP1u) and a spliced active form (XBP1s), is a key component in response to endoplasmic reticular (ER) stress. Compared to XBP1s, little is known about the role of XBP1u in vascular homeostasis and disease.Objective: We aim to investigate the role of XBP1u in VSMC phenotypic switching and the pathogenesis of aortic aneurysms.Methods and Results: XBP1u, but not XBP1s, was markedly repressed in the aorta during the early onset of aortic aneurysm in both angiotensin II-infused ApoE-/- and CaPO4-induced C57BL/6J murine models, in parallel with a decrease in SMC contractile apparatus proteins. In vivo studies revealed that XBP1 deficiency in SMCs caused VSMC dedifferentiation, enhanced vascular inflammation and proteolytic activity, and significantly aggravated both thoracic and abdominal aortic aneurysms in mice. XBP1 deficiency, but not an inhibition of XBP1 splicing, induced VSMC switching from the contractile phenotype to a proinflammatory and proteolytic phenotype. Mechanically, in the cytoplasm, XBP1u directly associated with the N-terminus of FoxO4, a recognized repressor of VSMC differentiation via the interaction and inhibition of myocardin. Blocking the XBP1u-FoxO4 interaction facilitated nuclear translocation of FoxO4, repressed SMC marker genes expression, promoted proinflammatory and proteolytic phenotypic transitioning in vitro and stimulated aortic aneurysm formation in vivo.Conclusions: Our study revealed the pivotal role of the XBP1u-FoxO4-myocardin axis in maintaining the VSMC contractile phenotype and providing protection from aortic aneurysm formation.
Thyroid Function and the Risk of Atherosclerotic Cardiovascular Morbidity and Mortality: The Rotterdam Study Circ. Res. (IF 13.965) Pub Date : 2017-10-31 Arjola Bano, Layal Chaker, Francesco U Mattace-Raso, Aad van der Lugt, M Arfan Ikram, Oscar H Franco, Robin P Peeters, Maryam Kavousi
Rationale: Thyroid hormones have been linked with various proatherogenic and antiatherogenic processes. However, the relationship of thyroid function with manifestations of atherosclerosis remains unclear.Objective: To investigate the association of thyroid function with atherosclerosis throughout its spectrum; i.e. subclinical atherosclerosis, incident atherosclerotic cardiovascular (ASCV) events and ASCV mortality.Methods and Results: This population-based study was embedded within the Rotterdam Study. The risk of atherosclerosis was evaluated by measuring: 1. Presence of subclinical atherosclerosis, assessed by coronary artery calcification (CAC) score >100 AU; 2. ASCV events, defined as fatal and nonfatal myocardial infarction, other coronary heart disease (CHD) mortality or stroke; 3. ASCV mortality, defined as death due to CHD, cerebrovascular or other atherosclerotic diseases. Associations of thyroid-stimulating hormone (TSH) and free thyroxine (FT4) with the outcomes were assessed through logistic regression and Cox proportional-hazard models, adjusted for potential confounders including cardiovascular risk factors. A total of 9420 community-dwelling participants (mean age±SD, 64.8±9.7 years) were included. During a median follow-up of 8.8 years (interquartile range:4.5-11.8 years), 934 incident ASCV events and 612 ASCV deaths occurred. FT4 levels were positively associated with high CAC score (odds ratio [OR]; 95% confidence interval [CI]: 2.28; 1.30-4.02) and incident ASCV events (hazard ratio [HR]; CI: 1.87; 1.34-2.59). The risk of ASCV mortality increased in a linear manner with higher FT4 levels (HR; CI: 2.41; 1.68-3.47 per 1 ng/dl) and lower TSH levels (HR; CI: 0.92; 0.84-1.00 per 1 logTSH). Results remained similar or became stronger among euthyroid participants.Conclusions: FT4 levels in middle-aged and elderly subjects were positively associated with atherosclerosis throughout the whole disease spectrum, independently of cardiovascular risk factors.
In This Issue Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Ruth Williams
### Li et al uncover an unexpected effect of protein kinase C deletion on atherosclerosis. Excess lipids in the blood can activate monocytes and enhance their uptake into artery walls, thus promoting the development of atherosclerotic plaques. This lipid-induced activation of monocytes involves the signal transduction factor protein kinase C (PKC); but PKC has a number of isoforms, so exactly which enzyme is involved in the process, and how, remains unclear. To find out, Li and colleagues examined rats with hyperlipidemia. They showed that PKC levels were indeed increased in the animals’ monocytes, and that most of this was accounted for by an increase in PKCδ. To determine the physiological significance of this increase, the team generated transgenic atherosclerosis-prone mice with macrophages lacking PKCδ. To their surprise, …
Meet the First Authors Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Lippincott Williams & Wilkins
Kuei-Ting Chang is a PhD candidate in the Molecular Medicine at National Yang-Ming University and Academia Sinica, Taipei, Taiwan, under the mentorship of Dr Guey-Shen Wang. She earned her MS in Nutrition at Chung Shan Medical University. She is currently engaged in research investigating the cardiac pathogenesis of myotonic dystrophy (DM). While Kuei-Ting was studying the causal mechanism for heart failure, one of the major DM heart features, she wondered if the pathogenic mechanism could be common to other cardiovascular diseases. In addition, she thought it could be her own niche if she worked on “understanding the disruption of gene regulation at the post-transcriptional level in cardiovascular disease.” Her favorite motto is, “doubt is the key to knowledge.” In her free time, she enjoys rock music and movies. Dr Qian Li earned his PhD at Peking University of China under the supervision of Dr Ruiping Xiao in 2006. He continued his training with Dr Paul Huang at the Cardiovascular Research Center of Massachusetts …
PVDOMICS Drive the Pulmonary Hypertension Field Into the Precision Medicine Era Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Evangelos D. Michelakis
Pulmonary hypertension (PHT) is a heterogeneous complex vascular disease that has intrigued vascular biologists and clinicians for decades. The need for collaborative efforts and research networks became apparent early on. An NIH-driven effort in 1981 resulted in the first registry of idiopathic pulmonary arterial hypertension (PAH), one of the most mysterious and deadly subtypes of PHT. Its findings defined the magnitude of the clinical problem and triggered an explosion of research efforts that have been steadily increasing since then. A few years earlier (1973), a WHO-sponsored international collaborative effort, first attempted to define the clinical phenotypes of PHT. Since this first WHO classification, several have followed producing a regularly updated classification system, based on standard clinical data. The hypothesis was that many PHT subtypes within each of the 5 WHO classes share a common pathogenesis (and thus may benefit from the same therapy), partly because they have similar basic lung pathology, particularly those in the WHO Class I PHT that included idiopathic PAH. The WHO classifications could not clearly define the phenotype of PHT subtypes, and the diagnosis of idiopathic PAH remained that of exclusion (ie, PHT that does not fit the diagnostic criteria of the other WHO classes). Article, see p 1136 The WHO classifications were very important in the community’s initial efforts to understand the clinical complexity of PHT, which was being diagnosed earlier and more accurately because of advances in imaging and the emergence of specialized PHT clinics. The weakness of the WHO classification-driven approach became apparent as preclinical research revealed a tremendous diversity of molecular mechanisms in animal models and human tissues (despite similarities in lung pathology) and clinical experience exposed many gray zones among PHT subtypes. Most of the clinical trials to date have been based on the WHO classification paradigm and produced drugs that …
Monocyte Recruitment Versus Macrophage Proliferation in Atherosclerosis Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Jenny E. Kanter
Atherosclerosis, the underlying pathology in most cardiovascular disease, is a chronic inflammatory disease,1 characterized by accumulation of macrophages in the subendothelial space. Macrophages are protagonists of the disease, both early in the disease as well as during later stages of atherosclerotic lesion progression, with a continuous turnover of the cells.2 Blood monocytes enter the artery wall and differentiate into macrophages, contributing to atherosclerotic lesion growth,3 but local proliferation and survival of existing artery wall macrophages also contribute to lesional macrophage plaque burden (Figure).2 Figure. Macrophages play an important role in all phases of atherosclerosis. Monocyte recruitment is critical during early atherosclerotic disease (and perhaps during certain other situations; left ), and thus at this stage of the disease, blood monocyte levels are an important determining factor. Local macrophage proliferation dominates macrophage accumulation in later stages of atherosclerosis ( right ). Macrophage protein kinase C (PKC) δ is induced by modified lipids that can be found in the plaque. Myeloid cell PKCδ primarily impairs macrophage survival and proliferation, via regulation of phosphorylation of Akt and Foxo3a and ultimately the apoptotic regulator Bim. Deletion knockout (KO) of PKCδ results in increased proliferation and survival, both in the artery wall and in the spleen resulting in more atherosclerosis and enlargement of the spleen. SMC indicates smooth muscle cells. Article, see p …
Considerations for Cardiac CRISPR Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Kelli J. Carroll, Eric N. Olson
The recently identified 2-component clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the ease with which genome editing can be performed, greatly simplifying the process of generating knockout animal models.1,2 However, in spite of the great advances made, fewer studies have focused on the efficacy of the system for editing postnatal or adult tissues as a means to model adult onset diseases or to provide potential therapeutic intervention. In particular, the use of the system to perform genome editing in a tissue-specific manner remains to be thoroughly evaluated, especially for tissues that are less amenable to viral delivery of the CRISPR/Cas9 components, a common strategy for delivery in adult tissues.3 Because the heart is responsible for a large disease burden, it represents a particularly attractive tissue to target for therapeutic purposes.4 Although several studies have shown that cardiomyocytes can be edited in the postnatal murine heart using viral delivery of CRISPR/Cas9 system components,5–7 the efficiency of this phenomenon has been less well characterized. Article, see p 1168 A study in this issue by Johansen et al8 from Eva van Rooij’s group performed an in-depth analysis of CRISPR/Cas9-mediated gene editing in postnatal mouse myocytes. Cardiac-restricted expression of Cas9 was inducing using mice containing a floxed Cas9 cassette with a GFP (green fluorescent protein) reporter knocked into the Rosa26 locus together with an αMHC-Cre (alpha-myosin heavy chain-Cre) transgene. Consistent with other models that have expressed Cas9 in either a broad or tissue restricted manner,5,6,9 no changes in baseline cardiac function were observed after Cas9 overexpression, suggesting that Cas9 …
AMP-Activated Protein Kinase γ2 to the Rescue in Ischemic Heart Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Ye Ding, Ming-Hui Zou
Activation of the highly conserved 5′-adenosine monophosphate–activated protein kinase (AMPK) is a fundamental component of the response to stresses that threatens cellular viability and therefore modulates many aspects of cell metabolism.1 This serine/threonine protein kinase is activated after stresses that deplete cellular ATP levels such as low glucose, hypoxia, ischemia, and heat shock. AMPK activation positively regulates signaling pathways that replenish cellular ATP including fatty acid oxidation and autophagy, whereas it negatively regulates ATP-consuming biosynthetic processes including gluconeogenesis, and lipid and protein synthesis. A growing body of evidence, however, show that AMPK also responds to glucose deprivation and oxidants stress without altering energy nucleotide levels in a wide variety of cells. For example, physiologically or pathologically relevant concentrations of free radicals and oxidants such as superoxide anion-derived hydrogen peroxide and peroxynitrites can activate AMPK without altering AMP/ATP (redox sensor).2,3 In turn, oxidant-activated AMPK feedback suppresses superoxide anion production by inhibiting NAD(P)H (nicotinamide adenine dinucleotide phosphate) oxidase and mitochondria-derived oxidants (redox modulator). As such, AMPK is considered to be a sensor and master regulator of both energy and redox homeostasis.2,3 Article, see p 1182 AMPK is a heterotrimeric protein complex consisting of 3 subunits, α, β, and γ, each encoded by distinct genes. These isoforms play specific roles in AMPK stability and activity, and all 3 are necessary for full activity.1–3 The α subunit (2 isoforms) contains the catalytic site; β (2 isoforms) and γ subunits (3 isoforms) are regulatory subunits. If ATP generation is impaired or cellular demand is increased, a higher cellular AMP: ATP ratio causes AMP to displace ATP from the exchangeable sites. AMP or ADP (adenosine-5'-diphosphate) then binds the γ subunit of AMPK and activates the kinase by 3 main mechanisms: allosteric activation of the α catalytic …
Cardiac Cell Therapy Evolving From Complex to Straightforward Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Keith L. March, Carl J. Pepine
Numerous trials have evaluated mesenchymal stem/stromal cells (MSCs) derived from bone marrow (BM-MSCs) for a wide range of indications, including diseases of the cardiovascular system, as recently reviewed.1,2 Earlier this year, a seminal study evaluated the safety and feasibility of intravenous BM-MSC administration in nonischemic cardiomyopathy,3 and gathered initial evidence supporting the ability of such an approach to ameliorate inflammation and improve functional status. As described in that study and accompanying editorial,4 the notion of a heart failure therapy based on an allogeneic cell product delivered by intravenous infusion is extremely attractive, given the practicality and affordability of such delivery on a standard hospital unit, or even in an outpatient setting. Article, see p 1192 In parallel with the advance of clinical studies involving BM-MSCs, basic and translational efforts have advanced our understanding of mesenchymal stem/stromal cells from the early identification of these cells as bone marrow–resident cells functioning particularly in support of marrow hematopoiesis.5 It is now well-recognized that MSCs are distributed on the vascular network in all vascularized tissues, thus playing a broad tissue-supportive role throughout the body.6–10 This recognition has in turn prompted a quest for tissue sources of both autologous and allogeneic MSCs that might have advantages with respect to either practical or biological properties that support their use as therapeutic agents. Several such sources have been identified that involve tissues that can readily be obtained with limited or no morbidity, as medical waste. These include adipose tissue obtained from minimally invasive lipoaspiration, placental tissue, and umbilical cord tissue.2 The umbilical cord and its components are recognized as a ready source of MSCs that are present immediately surrounding the cord vessels, as well as distributed throughout the Wharton’s jelly in which those vessels are suspended.11 …
CANTOS Trial Validates the Inflammatory Pathogenesis of Atherosclerosis Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Christian Weber, Philipp von Hundelshausen
> Now this is not the end. It is not even the beginning of the end. But it is, perhaps, the end of the beginning . > > —Winston Churchill The CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcome Study) provides intriguing support for the inflammatory hypothesis of atherosclerosis and cancer in man, demonstrating diverse clinical benefits of inhibiting IL (interleukin)-1β for cardiovascular events and lung cancer. Limited effects on cardiovascular mortality and safety concerns raised by a higher incidence of fatal infection warrant further studies to identify patient subgroups which profit most from anti-inflammatory therapy and a careful pursuit of alternative targets. Abundant evidence from experimental and clinical studies has lend strong support to the hypothesis that inflammation contributes to the pathogenesis of atherosclerotic disease in conjunction with or beyond elevated lipid levels; however, ultimate proof by selective anti-inflammatory treatment in a clinical trial remained elusive for decades. This has now changed with the results of the CANTOS, a double-blind trial involving high-risk patients with prior myocardial infarction (MI) and a residual inflammatory response (defined by hsCRP [high-sensitivity C-reactive protein] levels >2 mg/L despite intensive statin therapy), who were randomized to canakinumab (50, 150, or 300 mg every 3 months) or placebo.1 Canakinumab, a human monoclonal antibody targeting IL-1β as the master cytokine of innate immunity, dose dependently reduced hsCRP and IL-6 levels by ≤43% from baseline. At 150 mg (but not the other doses), canakinumab significantly lowered the risk for the primary (MI, stroke, cardiovascular death) and secondary end points (hazard ratio [HR], 0.85 and 0.83, respectively). Adversely, canakinumab was associated with leukopenia and a higher incidence of fatal infection. Whereas no significant difference in all-cause or cardiovascular mortality was observed, canakinumab strikingly reduced cancer mortality (exploratory results indicating HR 0.23, for lung cancer), supporting a role of inflammation in cancer progression. …
Researcher, Heal Thyself? Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Mary Burchett Sheppard
“You see doctor, when I found out you had Marfan syndrome and I saw that you were pregnant, I felt so jealous I could not stand it. Then I realized, if you did it, maybe I could, too.” My mind raced with ways to respond. I did not answer as quickly as usual. She took my pause as an opportunity to continue… “You know, I love my foster babies. I feel so blessed. It’s just hard to wake up every morning and remember that they can be taken away. I have three now…they are siblings. Their mother is addicted to drugs. But I think she is pregnant again, so I might be able to have four children.” My patient looked into my eyes intently. I continued to listen. “You give me so much hope. I thought I would die if I tried to have a baby, so my husband got a vasectomy. But if it is possible for me to carry a baby, he will try to have it reversed. Please doctor, tell me the truth. Is it safe for me to have a baby?” I usually do not disclose my diagnosis to my patients. I have always felt that my role as a physician is to ease THEIR suffering, not mine. I learned very early in my training that patient visits should …
Elizabeth Murphy Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Jaclyn Jansen
For even the most accomplished researcher, the path to discovery is more often marked with failure than success. Rather than be deterred, Elizabeth Murphy, Senior Investigator at the National Institutes of Health Heart, Lung, and Blood Institute, sees each challenge as an opportunity to uncover new and unexpected insights into human biology. It is this sense of perseverance and optimism that has made her a successful scientist and a leader in cardiac research. Elizabeth Murphy Murphy (who is better known as Tish among her peers) has dedicated her research to understanding the molecular changes that occur during and after a heart attack. Her early work focused on the ion changes that lead to cell death during ischemia and reperfusion. She found that an increase in sodium leads to a subsequent increase in intracellular calcium,1–3 which triggers cell death pathways in the mitochondria.4 Her work has helped to define many of the basic pathways that are responsible for cardiac cell death. Recently, Murphy turned her attention to the mechanisms that protect cells from ischemia and reperfusion. Her lab focused on the effects of preconditioning: brief intermittent periods of ischemia and reperfusion that protect the heart from subsequent, more sustained attacks. Murphy and her colleagues identified several post-translational modifications and signaling pathways are activated during preconditioning.5–7 Even with this success, her work took an unexpected turn. At the time, Murphy was like most researchers, using male mice exclusively to study ischemia reperfusion. For one set of experiments, a collaborator offered her female mice as well. When Heather Cross, a postdoctoral researcher in Murphy’s lab, compared the males and females, she was surprised to find that males were much more sensitive to ischemia–reperfusion injury than female mice.8 Murphy and Cross were quick to follow-up on …
Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Junichi Sadoshima
Rutgers New Jersey Medical School (NJMS; Figure A) is located in Newark, NJ. Rutgers NJMS is 1 of the 2 major medical schools in the state of NJ, and it belongs to Rutgers Biomedical and Health Sciences. The Department of Cell Biology and Molecular Medicine is 1 of 4 basic science departments in NJMS. Here, the author provides an overview of the department and discusses its strategies for conducting high quality research and improving research productivity. Figure. Department of Cell Biology and Molecular Medicine at the Rutgers New Jersey Medical School. A , The Medical Science Building where the department is located. B , Faculty members of the Department of Cell Biology and Molecular Medicine. (Four members are not pictured). C , Diagram of research projects and the current National Institutes of Health (NIH)–funded investigators. The research projects on cardiac development are currently funded by non-NIH funding. Our research projects are supported by core facilities both within (indicated by blue) and outside (indicated by green) the department. AAV indicates adeno-associated virus; and iPS, inducible pluripotent stem cell. The goal in this department is to perform high quality research into human diseases, including, but not limited to, cardiovascular disease. Although many investigators in this department focus on cardiovascular disease, others study stem cells, aging, cancer, and development, allowing for research using interdisciplinary approaches. Currently, our department has 21 faculty members (Figure B), 7 postdoctoral fellows, and 7 PhD students. In 2016, 53% of the departmental budget came from external grants, 32% from the state of NJ, and the rest from overheads and tuitions. Many of our investigators are supported by grants from the National Institutes of Health (NIH) and other sources, including the American Heart Association, the Muscular Dystrophy Association, and the Leducq Fondation. Our department ranked 21st of 78 cell biology/anatomy …
Identity Crisis for Regenerative Cardiac cKit+ Cells Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Onur Kanisicak, Ronald J. Vagnozzi, Jeffery D. Molkentin
The concept that cardiac-derived cKit+ cells can regenerate the injured adult heart by transdifferentiating into functioning cardiomyocytes was proposed 14 years ago although it remains controversial because of negative data from multiple independent laboratories. Irreproducibility of cardiac cKit+ cell studies was attributed to the differences in cell isolation, selection, and expansion before in vivo application. This Viewpoint will discuss recent results that again change the formula for how cardiac cKit+ cells must be isolated and processed to be cardiomyogenic, as well as discuss the uncertain in vivo relevance of cKit+ cells as putative cardiomyocyte-producing stem cells. The regenerative and myogenic potential of cKit+ cardiac-resident stem cells (CSCs) remains a dominant and contentious topic. In vitro expanded cKit+ CSCs were originally described 14 years ago as a source for robust new myocyte generation when injected directly into the adult rodent heart after myocardial infarction,1 and although some laboratories have confirmed the basic principle, the magnitude of the effect remains unclear.2 More recently, Ellison et al3 showed that a clonal cKit+ cell line derived from a population of cardiac CD45− cKit+ cells was capable of widely regenerating isoproterenol-injured adult rodent hearts with abundant new cardiomyocytes, after simple tail vein infusion. However, many independent laboratories reported that adult cardiac-derived cKit+ cells did not generate appreciable new cardiomyocytes when directly injected back into the injured rat or mouse heart.4–7 In addition to controversies surrounding the regenerative potential of injected cKit+ cell formulations, until recently the field has not attempted to directly address if endogenous cardiac cKit+ cells are true cardiomyocyte producing stem cells of meaningful significance.8 …
Novel Antithrombotic Drugs on the Horizon Circ. Res. (IF 13.965) Pub Date : 2017-10-27 James D. McFadyen, Karlheinz Peter
Diseases associated with arterial thrombosis or venous thrombosis are leading causes of mortality and morbidity globally. Since the 1930s, anti thrombotic therapy has been the cornerstone of medical therapy for thrombotic diseases. However, the success of anti thrombotic therapy has come at the cost of one of the most dreaded iatrogenic complications— bleeding. Recently, new evidence has emerged on potentially important differences between thrombosis and hemostasis, thereby raising the possibility of developing new anti thrombotic drugs that do not cause bleeding. All currently used antithrombotic drugs in the clinic are associated with an inherent risk of bleeding. For example, the risk of serious major bleeding with a non–vitamin K antagonist oral anticoagulant is ≈2% to 3%, with the risk of intracranial hemorrhage of ≈0.3 to 0.5% per annum.1 Likewise nonmajor bleeding with aspirin approximates 2%, and the rates of major and life-threatening bleeding rises significantly in those over 75 years of age to >2% per year.2 Thus, a significant proportion of patients at high risk of thrombotic disease are vulnerable to bleeding complications and often miss out on potentially beneficial antithrombotic therapy. Importantly, aside from the mortality and morbidity directly linked to the index bleeding event, there is a large body of evidence demonstrating that hemorrhagic complications are associated with adverse clinical outcomes.3 Despite the introduction of new anticoagulants, such as the non–vitamin K antagonist oral anticoagulants and newer antiplatelet drugs, we have reached a tipping point with regard to the achievable balance between antithrombotic potency and bleeding risk with current antithrombotic approaches. Therefore, there remains an unmet clinical need for new antithrombotic approaches that maintain efficacy while preserving hemostasis. Recently, major progress has been achieved in our understanding of the factors that may differentially regulate pathological thrombosis from hemostasis based on the introduction of intravital microscopy, …
PVDOMICS Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Anna R. Hemnes, Gerald J. Beck, John H. Newman, Aiden Abidov, Micheala A. Aldred, John Barnard, Erika Berman Rosenzweig, Barry A. Borlaug, Wendy K. Chung, Suzy A.A. Comhair, Serpil C. Erzurum, Robert P. Frantz, Michael P. Gray, Gabriele Grunig, Paul M. Hassoun, Nicholas S. Hill, Evelyn M. Horn, Bo Hu, Jason K. Lempel, Bradley A. Maron, Stephen C. Mathai, Mitchell A. Olman, Franz P. Rischard, David M. Systrom, W.H. Wilson Tang, Aaron B. Waxman, Lei Xiao, Jason X.-J. Yuan, Jane A. Leopold
The National Institutes of Health (NIH)/National Heart, Lung and Blood institute (NHLBI) launched an initiative, PVDOMICS (Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics) that aims to augment the current pulmonary hypertension (PH) classification based on shared biological features. PVDOMICS will enroll 1500 participants with PH and disease and healthy comparators. Enrollees will undergo deep clinical phenotyping, and blood will be acquired for comprehensive omic analyses that will focus on discovery of molecular-based subtypes of pulmonary vascular disease (PVD) through application of high dimensional model-based clustering methods. In addition to an updated, molecular classification of PVD, the phenomic data generated will be a rich resource to the broad community of heart and lung disease investigators. Editorial, see p 1106 PH is a hemodynamic condition that causes increased blood pressure in the pulmonary arteries and the right heart leading to adverse clinical outcomes. The current World Symposium on Pulmonary Hypertension (WSPH) classification of PH is based on a combination of patient characteristics, clinical features, and cardiopulmonary hemodynamics, and these features are used to inform treatment options.1 Aside from heritable pulmonary arterial hypertension, this classification is not tied to molecular or cellular pathobiologic mechanism to explain the pathogenesis of PH. The NIH has a vested interest in understanding the causes and natural history of PH, as well as the discovery of effective treatment options. Since the first large NIH registry of patients with pulmonary arterial hypertension >30 years ago,2 significant advances in scientific knowledge and translational medicine have occurred, highlighting a need for updating the current clinical classification system. The NHLBI has sponsored several workshops focusing on PVD research strategic planning over the past decade. PVD encompasses PH and PVD without PH, for example, pulmonary vasculitis and pathological pulmonary vascular remodeling without hemodynamic criteria for PH. Experts identified the need …
CELF1 Mediates Connexin 43 mRNA Degradation in Dilated CardiomyopathyNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Kuei-Ting Chang, Ching-Feng Cheng, Pei-Chih King, Shin-Yi Liu, Guey-Shin Wang
Rationale: Downregulation of Cx43 (connexin 43), the major cardiac gap junction protein, is often associated with arrhythmia, dilated cardiomyopathy (DCM), and heart failure. However, the cause of the reduced expression remains elusive. Reinduction of a nuclear RNA-binding protein CELF1 (CUGBP Elav-like family member 1) in the adult heart has been implicated in the cardiac pathogenesis of myotonic dystrophy type 1. However, how elevated CELF1 level leads to cardiac dysfunction, such as conduction defect, DCM, and heart failure, remains unclear. Objective: We investigated the mechanism of CELF1-mediated Cx43 mRNA degradation and determined whether elevated CELF1 expression is also a shared feature of the DCM heart. Methods and Results: RNA immunoprecipitation revealed the involvement of CELF1-regulated genes, including Cx43, in controlling contractility and conduction. CELF1 mediated Cx43 mRNA degradation by binding the UG-rich element in the 3′ untranslated region of Cx43. Mutation of the nuclear localization signal in CELF1 abolished the ability to downregulate Cx43 mRNA, so nuclear localization was required for its function. We further identified a 3′ to 5′ exoribonuclease, RRP6 (ribosomal RNA processing protein 6), as a CELF1-interacting protein. The interaction of CELF1 and RRP6 was RNA-independent and nucleus specific. With knockdown of endogenous RRP6, CELF1 failed to downregulate Cx43 mRNA, which suggests that RRP6 was required for CELF1-mediated Cx43 mRNA degradation. In addition, increased CELF1 level accompanied upregulated RRP6, and reduced Cx43 level was detected in mouse models with DCM, including myotonic dystrophy type 1 and CELF1 overexpression models and a myocardial infarction model. Importantly, depletion of CELF1 in the infarcted heart preserved Cx43 mRNA level and ameliorated the cardiac phenotypes of the infarcted heart. Conclusions: Our results suggest a mechanism for increased CELF1 expression downregulating Cx43 mRNA level and a pathogenic role for elevated CELF1 level in the DCM heart.
Regulation of Macrophage Apoptosis and Atherosclerosis by Lipid-Induced PKCδ Isoform ActivationNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Qian Li, Kyoungmin Park, Yu Xia, Motonobu Matsumoto, Weier Qi, Jialin Fu, Hisashi Yokomizo, Mogher Khamaisi, Xuanchun Wang, Christian Rask-Madsen, George L. King
Rationale: Activation of monocytes/macrophages by hyperlipidemia associated with diabetes mellitus and obesity contributes to the development of atherosclerosis. PKCδ (protein kinase C δ) expression and activity in monocytes were increased by hyperlipidemia and diabetes mellitus with unknown consequences to atherosclerosis. Objective: To investigate the effect of PKCδ activation in macrophages on the severity of atherosclerosis. Methods and Results: PKCδ expression and activity were increased in Zucker diabetic rats. Mice with selective deletion of PKCδ in macrophages were generated by breeding PKCδ flox/flox mice with LyzM-Cre and ApoE−/− mice (MPKCδKO/ApoE−/− mice) and studied in atherogenic (AD) and high-fat diet (HFD). Mice fed AD and HFD exhibited hyperlipidemia, but only HFD-fed mice had insulin resistance and mild diabetes mellitus. Surprisingly, MPKCδKO/ApoE−/− mice exhibited accelerated aortic atherosclerotic lesions by 2-fold versus ApoE−/− mice on AD or HFD. Splenomegaly was observed in MPKCδKO/ApoE−/− mice on AD and HFD but not on regular chow. Both the AD or HFD increased macrophage number in aortic plaques and spleen by 1.7- and 2-fold, respectively, in MPKCδKO/ApoE−/− versus ApoE−/− mice because of decreased apoptosis (62%) and increased proliferation (1.9-fold), and not because of uptake, with parallel increased expressions of inflammatory cytokines. Mechanisms for the increased macrophages in MPKCδKO/ApoE−/− were associated with elevated phosphorylation levels of prosurvival cell-signaling proteins, Akt and FoxO3a, with reduction of proapoptotic protein Bim associated with PKCδ induced inhibition of P85/PI3K. Conclusions: Accelerated development of atherosclerosis induced by insulin resistance and hyperlipidemia may be partially limited by PKCδ isoform activation in the monocytes, which decreased its number and inflammatory responses in the arterial wall.
Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene DisruptionNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Anne Katrine Johansen, Bas Molenaar, Danielle Versteeg, Ana Rita Leitoguinho, Charlotte Demkes, Bastiaan Spanjaard, Hesther de Ruiter, Farhad Akbari Moqadam, Lieneke Kooijman, Lorena Zentilin, Mauro Giacca, Eva van Rooij
Rationale: CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)–based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized. Objective: To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice. Methods and Results: We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6, Sav1, and Tbx20, using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1, which increased the editing efficiency. Conclusions: Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo.
Activation of γ2-AMPK Suppresses Ribosome Biogenesis and Protects Against Myocardial Ischemia/Reperfusion InjuryNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Yang Cao, Naveen Bojjireddy, Maengjo Kim, Tao Li, Peiyong Zhai, Narayani Nagarajan, Junichi Sadoshima, Richard D. Palmiter, Rong Tian
Rationale: AMPK (AMP-activated protein kinase) is a heterotrimeric protein that plays an important role in energy homeostasis and cardioprotection. Two isoforms of each subunit are expressed in the heart, but the isoform-specific function of AMPK remains unclear. Objective: We sought to determine the role of γ2-AMPK in cardiac stress response using bioengineered cell lines and mouse models containing either isoform of the γ-subunit in the heart. Methods and Results: We found that γ2 but not γ1 or γ3 subunit translocated into nucleus on AMPK activation. Nuclear accumulation of AMPK complexes containing γ2-subunit phosphorylated and inactivated RNA Pol I (polymerase I)–associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes for rDNA. The subsequent downregulation of pre-rRNA level led to attenuated endoplasmic reticulum (ER) stress and cell death. Deleting γ2-AMPK led to increases in pre-rRNA level, ER stress markers, and cell death during glucose deprivation, which could be rescued by inhibition of rRNA processing or ER stress. To study the function of γ2-AMPK in the heart, we generated a mouse model with cardiac-specific deletion of γ2-AMPK (cardiac knockout [cKO]). Although the total AMPK activity was unaltered in cKO hearts because of upregulation of γ1-AMPK, the lack of γ2-AMPK sensitizes the heart to myocardial ischemia/reperfusion injury. The cKO failed to suppress pre-rRNA level during ischemia/reperfusion and showed a greater infarct size. Conversely, cardiac-specific overexpression of γ2-AMPK decreased ribosome biosynthesis and ER stress during ischemia/reperfusion insult, and the infarct size was reduced. Conclusions: The γ2-AMPK translocates into the nucleus to suppress pre-rRNA transcription and ribosome biosynthesis during stress, thus ameliorating ER stress and cell death. Increased γ2-AMPK activity is required to protect against ischemia/reperfusion injury. Our study reveals an isoform-specific function of γ2-AMPK in modulating ribosome biosynthesis, cell survival, and cardioprotection.
Safety and Efficacy of the Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells in Patients With Heart FailureNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-27 Jorge Bartolucci, Fernando J. Verdugo, Paz L. González, Ricardo E. Larrea, Ema Abarzua, Carlos Goset, Pamela Rojo, Ivan Palma, Ruben Lamich, Pablo A. Pedreros, Gloria Valdivia, Valentina M. Lopez, Carolina Nazzal, Francisca Alcayaga-Miranda, Jimena Cuenca, Matthew J. Brobeck, Amit N. Patel, Fernando E. Figueroa, Maroun Khoury
Rationale: Umbilical cord–derived mesenchymal stem cells (UC-MSC) are easily accessible and expanded in vitro, possess distinct properties, and improve myocardial remodeling and function in experimental models of cardiovascular disease. Although bone marrow–derived mesenchymal stem cells have been previously assessed for their therapeutic potential in individuals with heart failure and reduced ejection fraction, no clinical trial has evaluated intravenous infusion of UC-MSCs in these patients. Objective: Evaluate the safety and efficacy of the intravenous infusion of UC-MSC in patients with chronic stable heart failure and reduced ejection fraction. Methods and Results: Patients with heart failure and reduced ejection fraction under optimal medical treatment were randomized to intravenous infusion of allogenic UC-MSCs (Cellistem, Cells for Cells S.A., Santiago, Chile; 1×106 cells/kg) or placebo (n=15 per group). UC-MSCs in vitro, compared with bone marrow–derived mesenchymal stem cells, displayed a 55-fold increase in the expression of hepatocyte growth factor, known to be involved in myogenesis, cell migration, and immunoregulation. UC-MSC–treated patients presented no adverse events related to the cell infusion, and none of the patients tested at 0, 15, and 90 days presented alloantibodies to the UC-MSCs (n=7). Only the UC-MSC–treated group exhibited significant improvements in left ventricular ejection fraction at 3, 6, and 12 months of follow-up assessed both through transthoracic echocardiography (P=0.0167 versus baseline) and cardiac MRI (P=0.025 versus baseline). Echocardiographic left ventricular ejection fraction change from baseline to month 12 differed significantly between groups (+7.07±6.22% versus +1.85±5.60%; P=0.028). In addition, at all follow-up time points, UC-MSC–treated patients displayed improvements of New York Heart Association functional class (P=0.0167 versus baseline) and Minnesota Living with Heart Failure Questionnaire (P<0.05 versus baseline). At study completion, groups did not differ in mortality, heart failure admissions, arrhythmias, or incident malignancy. Conclusions: Intravenous infusion of UC-MSC was safe in this group of patients with stable heart failure and reduced ejection fraction under optimal medical treatment. Improvements in left ventricular function, functional status, and quality of life were observed in patients treated with UC-MSCs. Clinical Trial Registration: URL: https://www.clinicaltrials.gov/ct2/show/NCT01739777. Unique identifier: NCT01739777
Genetic Depletion or Hyperresponsiveness of Natural Killer Cells Do Not Affect Atherosclerosis Development Circ. Res. (IF 13.965) Pub Date : 2017-10-18 Wared Nour Eldine, Jeremie Joffre, Kazem Zibara, Andreas Giraud, Bruno Esposito, Lynda Zeboudj, José Vilar, Megumi Terada, Patrick Bruneval, Eric Vivier, Hafid Ait-Oufella, Ziad Mallat, Sophie Ugolini, Alain Tedgui
Rationale: Chronic inflammation is central in the development of atherosclerosis. Both innate and adaptive immunity are involved. Although several studies have evaluated the functions of NK cells in experimental animal models of atherosclerosis, it is not yet clear whether NK cells behave as protective or pro-atherogenic effectors. One of the main caveats of previous studies was the lack of specificity in targeting loss- or gain-of-function of NK cells.Objective: We used two selective genetic approaches to investigate the role of NK cells in atherosclerosis: 1) Ncr1iCre/+R26lsl-DTA/+ mice in which NK cells were depleted, 2) Noé mice in which NK cells are hyperresponsive.Methods and Results: No difference in atherosclerotic lesion size was found in Ldlr-/- mice transplanted with bone marrow cells from Ncr1iCreR26Rlsl-DTA, Noé or WT mice. Also, no difference was observed in plaque composition in terms of collagen content, macrophage infiltration or the immune profile, although Noé chimera had more IFN-γ-producing NK cells, compared with WT mice. Then, we investigated the NK cell selectivity of anti-asialo GM1 anti-serum, which was previously used to conclude to the pro-atherogenicity of NK cells. Anti-asialo GM1 treatment decreased atherosclerosis in both Ldlr-/- mice transplanted with Ncr1iCreR26Rlsl-DTA or WT BM, indicating that its anti-atherogenic effects are unrelated to NK cell depletion, but to CD8+ T and NKT cells. Finally, to determine whether NK cells could contribute to the disease in conditions of pathological NK cell overactivation, we treated irradiated Ldlr-/- mice reconstituted with either WT or Ncr1iCreR26Rlsl-DTA BM with the viral mimic poly(I:C) and found a significant reduction of plaque size in NK-cell deficient chimeric mice.Conclusions: Our findings, using state-of-the-art mouse models, demonstrate that NK cells have no direct effect on the natural development of hypercholesterolemia-induced atherosclerosis, but may play a role when an additional systemic NK cell overactivation occurs.
Attenuation of Myeloid Specific TGFβ Signaling Induces Inflammatory Cerebrovascular Disease and Stroke Circ. Res. (IF 13.965) Pub Date : 2017-10-19 M C Hollander, Lawrence L Latour, Dan Yang, Hiroki Ishii, Zhiguang Xiao, Yongfen Min, Abhik Ray-Choudhury, Jeeva Munasinghe, Anand S Merchant, P C Lin, John Hallenbeck, Manfred Boehm, Li Yang
Rationale: Cryptogenic strokes, those of unknown cause, have been estimated as high as 30-40% of strokes. Inflammation has been suggested as a critical etiological factor. However, there is lack of experimental evidence.Objective: In this study, we investigated inflammation associated stroke etiology using a mouse model that developed spontaneous stroke due to myeloid deficiency of TGFβ signaling.Methods and Results: We report that mice with deletion of Tgfbr2 in myeloid cells (Tgfbr2Myeko) developed cerebrovascular inflammation in the absence of significant pathology in other tissues, culminating in stroke and severe neurological deficits with 100% penetrance. The stroke phenotype can be transferred to syngeneic wild type mice via Tgfbr2Myeko bone marrow transplant, and can be rescued in Tgfbr2Myeko mice with wild-type bone marrow. The underlying mechanisms involved an increased type 1 inflammation, and cerebral endotheliopathy, characterized by elevated NFΚB activation and TNF production by myeloid cells. A high fat diet accelerated stroke incidence. Anti-TNF treatment, as well as metformin and methotrexate, which are associated with decreased stroke risk in population studies, delayed stroke occurrence.Conclusions: Our studies show that TGFβ signaling in myeloid cells is required for maintenance of vascular health, and provide insight into inflammation-mediated cerebrovascular disease and stroke.
In This Issue Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Ruth Williams
### The kinase LKB1 slows foam cell formation and atherosclerosis progression in mice, report Liu et al. Atherosclerosis begins with the gradual accumulation of fatty deposits on blood vessel walls. Monocytes are recruited to the lesions where they convert to macrophages to phagocytose lipids in the lesions. Continued lipid uptake by the macrophages transforms them into foam cells, which is a critical step in plaque development. However, mechanisms underlying this transformation remain unclear. Liu and colleagues now show that the kinase LKB1, previously identified as having cardioprotective and anti-inflammatory effects, is a negative regulator of foam cell development. They found that, in mice prone to atherosclerosis, the levels of LKB1 in plaque macrophages decreases with disease progression. This decrease prevented LKB1-directed phosphorylation of scavenger receptor A (SRA)—a …
Meet the First Authors Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Lippincott Williams & Wilkins
Dr Zhaoyu Liu earned her PhD from the University of Oklahoma Health Science Center, mentored by Dr Ming-hui Zou. Prior to earning her PhD, she earned her BS and MS in Biochemistry and Molecular Biology from Jinan University, Guangzhou, China. Dr Liu is a recipient of an American Heart Association Postdoc fellowship, and is pursuing postdoctoral training at Georgia State University with Dr Ming-hui Zou, where she is studying the role of macrophages and other immune cells in the development and progression of atherosclerosis. She became interested in cardiovascular diseases after witnessing her aunt’s battle with the disease. She believes that success is only achieved through sustained interest and insistence. She is a sports fan and likes to play various sports, both for fun and also to build and maintain a healthy body for science. Dr Alicia D’Souza is a cardiac physiologist and a Research Fellow at the University of Manchester. She joined Professor Mark Boyett’s group after completing a PhD in Physiology …
Liver Kinase B1 Links Macrophage Metabolism Sensing and Atherosclerosis Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Vishal Kothari, Karin E. Bornfeldt
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of cholesterol-laden macrophages at susceptible sites in the artery wall. Altered cellular metabolism plays an important role in the conversion of a macrophage into a lipid-laden foam cell and also in the ability of the macrophage to mount an inflammatory response. In the atherosclerotic lesion environment, the macrophage is exposed to a plethora of extracellular molecules that govern its phenotype, including cytokines, modified lipids, nutrients, and extracellular matrix components. Intracellular metabolic sensors allow the macrophage to adjust to the lesional environment and to alter its cellular functions accordingly. The molecular mechanisms underlying pathophysiological changes in the metabolism sensing machinery are not fully understood. In the current issue of Circulation Research ,1 Liu et al enhance our understanding of foam cell formation by reporting that loss of expression of the metabolic sensor LKB1 (liver kinase B1) in macrophages results in increased uptake of modified lipoproteins, increased foam cell formation, and subsequently, increased atherosclerosis in 2 different mouse models. This study provides new links between macrophage metabolic sensing and atherosclerosis. Article, see p 1047 LKB1, also known as STK11 (serine/threonine kinase 11), is a kinase originally identified as a tumor suppressor in patients with Peutz–Jeghers syndrome—an autosomal dominant genetic disorder associated with increased risk of developing cancer in the gastrointestinal tract and other organs.2 Later studies revealed that LKB1 acts in part as an upstream activator of AMPK (AMP-activated protein kinase)—an intracellular energy sensor activated by low nutrient status in cells as a mechanism to preserve energy.3 However, LKB1 also has AMPK-independent targets in cells. Recent studies have shown that the LKB1/AMPK pathway is activated by glucose deprivation and that reduced levels of the glycolytic intermediate fructose-1,6-bisphosphate …
Novel Pathways for Regulation of Sinoatrial Node Plasticity and Heart Rate Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Peter J. Mohler, Thomas J. Hund
Heart rate is an amazingly adaptive process with redundant pathways for both acute and chronic regulation in response to a wide range of environmental stimuli. Dramatic changes to basal heart rate are common not only in cardiovascular disease but also under physiological conditions (eg, exercise) as a way to match cardiac output with demand. Driving these changes in heart rate is alterations in activity of the sinoatrial node (SAN), a heterogeneous collection of specialized myocytes located in the right atrium. Defects in SAN function are increasingly common in an aging population (effects ≈1 in 600 patients over the age of 65 years) and manifest as prominent issues with heart rate control.1 Given the growing prevalence of SAN dysfunction and the limitations of available therapies, there is a great need to better understand the various ways the heart has evolved to control intrinsic rate. Article, see p 1058 Normal SAN function depends on a delicate balance between the activity of a relatively small number of automatic cells capable of spontaneously generating an action potential (AP) and the demands of surrounding atrial cells that require external stimulus for activation. This balance (and therefore heart rate) is readily tuned by perturbations at the cell and tissue level. SAN cell automaticity itself depends on a distinct ion channel expression profile that differs substantially from the ion channel signature present in atrial or ventricular cells.2 In contrast to ventricular and atrial cells, the SAN myocyte expresses virtually no inward rectifier K …
Unfriendly Fire From Neutrophils Promiscuously Potentiates Cardiovascular Inflammation Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Aaron P. Kithcart, Peter Libby
Polymorphonuclear leukocytes (PMNs) comprise the vast majority of white blood cells and serve as the first responders during acute inflammatory responses. These cells constitute an essential component of the innate immune response, classically conceived of as antimicrobial stewards that deliver their toxic payload at the site of injury. These cells contain granules, hence their membership in the granulocyte family of leukocytes. The granules contain enzymes such as myeloperoxidase, which generates the strong oxidant hypochlorous acid, and proteinases, which degrade foreign peptides, among other functions. PMNs also possess machinery that potently produces other reactive oxygen species (ROS) such as superoxide anion. Combined with phagocytosis, the mediators elaborated by PMNs provide a bulwark of defenses to combat foreign invaders and also to aid mop up operations in injured tissues. Article, see p 1081 Yet, friendly fire from PMNs can aggravate or cause disease when these cells undergo excessive or inappropriate activation. Cardiovascular and allied conditions provide examples of the adverse consequences of collateral damage because of the unleashing of PMNs’ potent actions (Figure). The dreaded mechanical complications of myocardial infarction such as rupture of the intraventricular septum, papillary muscles, or of the ventricle itself may result, in part, from excessive extracellular matrix dissolution and extensive cell damage wrought by PMN mediators.1 Coronary microvascular damage caused by PMN can contribute to no reflow situations. PMNs …
Cardiovascular Risk Prediction Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Nina P. Paynter
Accurate assessment of cardiovascular risk in the clinical setting is important for appropriate management and counseling of patients and for identification of biological pathways. Classic models, including the Framingham risk score,1 have combined strong correlates of risk, such as age, with modifiable factors, such as blood pressure and lipids, into a single model. That structure has been retained by the most recent pooled cohort equations from the American College of Cardiology/American Heart Association guidelines.2 The article by Ambale-Venkatesh et al3 in the current issue uses a different framework for prediction and risk factor identification and results in an array of identified risk factors across multiple outcomes. Article, see p 1092 Ambale-Venkatesh et al build on the strengths of MESA (Multi-Ethnic Study of Atherosclerosis), which collected extensive baseline and outcome data on the participants and included an ethnically diverse cohort by design. The MESA cohort has been a source of key insights in the use of markers, such as coronary calcium to predict risk,4 and the findings in MESA have been widely replicated in other cohorts. In this article, the authors took advantage of as much of the information as possible, examining 735 variables across a range of different domains, including …
Elena Matsa Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Pam Goldberg-Smith
Elena Matsa attended the University of Nottingham for her BSc (Hons) degree in Biochemistry and Genetics. She then earned her MSc at Imperial College, London, in Human Molecular Genetics before returning to the University of Nottingham for her PhD in Stem Cell Biology. After a 2-year postdoctoral fellowship in Nottingham, she moved onto Stanford University as a postdoctoral research fellow. Elena currently works as an Instructor for the Joseph Wu lab and teaches courses on Bioengineering, and Progenitor Cell Biology at Stanford, where she encourages her students to keep the fire in their belly burning. Elena Matsa Yes, most of my time is spent in the lab, and I am currently working on two main projects, both dealing with precision medicine.1 One project2,3 tries to figure out new treatments for people who are born with heart disease and have a mutation that causes the disease. This project is especially exciting since it’s what I’ve been working on since my postdoctoral fellowship in the UK under my mentor Chris Denning. Right now, at Stanford, we have progressed to using nanomaterials to deliver the new treatment to the heart. The other project4 relates to how our genetic makeup affects our response to medications. Some people are fine, but others may experience side effects when taking a drug, which can lead to heart complications. Fourteen percent of people who take antidiabetic medications have heart …
Current State of Basic and Translational Cardiovascular Research in Spain Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Enrique Lara-Pezzi, Vicente Andrés, Francisco Fernández-Avilés, Valentín Fuster, Borja Ibáñez
Although the risk of developing cardiovascular disease (CVD) is reduced by the Mediterranean diet,1 CVD prevalence in Spain is rising. CVD is a major cause of mortality and morbidity in this country, and there is a growing awareness in the scientific community of the need for advances in knowledge, diagnosis, and therapy. Spain has a strong tradition of basic research in molecular biology, immunology, development, neuroscience, and oncology, but historically there have been few research groups dedicated to basic cardiovascular research, and this has been reflected in limited funding. Over the last 20 years, a national strategy to address this situation has seen the creation of a cardiovascular center of excellence and research networks focused on CVD. This significant boost to Spanish cardiovascular research has generated closer collaboration between the clinical and basic research communities, with already tangible beneficial results for patients. Here, we outline current trends in cardiovascular research in Spain, highlighting achievements, opportunities, and challenges. ### Grants and Funding Despite increasing CVD incidence and prevalence, public research funding in Spain has decreased over the last decade. This cut in funds follows a general trend in Spain in the wake of the global financial crisis. In 2016, the Ministry of Economy and Competitiveness, which is the main public funding body in Spain, spent around 380 million euros on different project grants, mainly in the so-called Excellence, Societal Challenges, and Health Research Projects programs. The Excellence call funded >700 grants (42% success rate), averaging around 100 000 euros per project. The Societal Challenges awarded 1500 grants (49% success rate) with 145 000 euros each on average. The Health Research Projects scheme funded >600 projects with an average of 95 000 euros each (36% success rate). These projects are 3 to 4 years long and were awarded to 1, or sometimes 2, principal investigators. Over the last …
General Overview of the 14th International Symposium on Stem Cell Therapy and Cardiovascular Innovations Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Ricardo Sanz-Ruiz, Andreu M. Climent, Maria Eugenia Fernández-Santos, Adolfo Villa Arranz, Enrique Gutiérrez Ibañes, Maria Eugenia Vázquez-Álvarez, Francisco Fernández-Avilés
Regenerative therapies with cardiac and vascular applications represent one of the most promising options in the fight against cardiovascular failure. Since 2004, the International Symposium on Cell Therapy and Cardiovascular Innovations (TECAM, www.cardiovascularcelltherapy.com) has been held in Spain on a yearly basis to gather the most important research groups of clinicians and scientists worldwide and to discuss the progress and hurdles of Cardiovascular Regenerative Medicine (CRM) in an open forum. The latest edition of the TECAM conference took place in Madrid on June the 15th and the 16th. Recent breakthroughs and future steps were debated during the meeting, with a special focus on the most recent achievements of the Transnational AllianCe for regenerative Therapies In Cardiovascular Syndromes (TACTICS, www.tacticsalliance.org) group and are summarized in this article. The opening session started with a keynote conference delivered by Dr Michael Laflamme, who reflected on the difficulties for obtaining real cardiac regeneration with old types of stem cells. He therefore focused his talk on pluripotent stem cells, the one cell type that has demonstrated robust differentiation into cardiomyocytes. After presenting the important functional benefits of human embryonic stem cell (ESC)–derived cardiomyocytes in murine models and recognizing safety concerns (ie, ventricular arrhythmias) in primates and swine models of myocardial scar, he concluded that large-scale remuscularization is possible using this type of human-derived cells. In the second talk, Dr Juan Carlos Izpisúa presented the latest approaches to manipulate the genome in cells that do not divide. After describing how CRISPR/Cas9 can be used to induce double-strand breaks and nonhomologous end joining-based repair on the DNA, he explained the Homology Independent Targeted Integration technology that has been designed by his group. This novel tool for gene editing has been used to correct mutations in nondividing cells such as neurons and cardiomyocytes, with impressive …
Paracrine-Mediated Systemic Anti-Inflammatory Activity of Intravenously Administered Mesenchymal Stem Cells Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Stephen E. Epstein, Dror Luger, Michael J. Lipinski
Stem cell therapy as a treatment option for acute myocardial infarction or heart failure caused by ischemic or nonischemic cardiomyopathy has focused on direct cardiac delivery of stem cells to facilitate cardiac engraftment. Once engrafted, it was believed that these cells would either transdifferentiate into new functioning myocytes or stimulate the expansion of resident myocardial stem cells. However, a sea change in thinking about cell therapy in cardiovascular disease has occurred as mounting evidence indicates that (1) inflammation is a major mechanism contributing to the progressive myocardial dysfunction seen in patients post-acute myocardial infarction and in patients with cardiomyopathy, and (2) systemic paracrine-mediated anti-inflammatory effects of stem cells can drive beneficial cardiac effects in these diseases. These concepts lead to a potentially transformative strategy that intravenous delivery of stem cells, through systemic anti-inflammatory mechanisms, improves myocardial function and thereby obviates the need for invasive methods of stem cell delivery. Until recently, the prevailing view of the mechanism responsible for any potential benefit of stem cells in patients with acute myocardial infarction (AMI) or with heart failure (HF) caused by ischemic or nonischemic cardiomyopathy (ICM/NICM) was that benefit derived from local effects—once engrafted in damaged myocardium, the stem cells either transdifferentiate into functional myocardium, stimulate resident myocardial stem cells to expand, and repopulate the heart with functioning myocytes or secrete substances leading to myocardial healing. This mechanistic perspective implied that the greater the number of engrafted cells in the myocardium, the greater the cardiac benefit. Because few intravenously administered stem cells engraft in injured myocardium, invasive strategies providing direct delivery of stem cells to the heart were uniformly adopted. This necessarily involved either catheter-based delivery (intracoronary or transendocardial injection) or surgical delivery (direct intramyocardial injection). A transformative concept relating to stem cell treatment of patients with AMI or ICM/NICM has recently …
Macrophage Liver Kinase B1 Inhibits Foam Cell Formation and AtherosclerosisNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Zhaoyu Liu, Huaiping Zhu, Xiaoyan Dai, Cheng Wang, Ye Ding, Ping Song, Ming-Hui Zou
Rationale: LKB1 (liver kinase B1) is a serine/threonine kinase and tumor suppressor, which regulates the homeostasis of hematopoietic cells and immune responses. Macrophages transform into foam cells upon taking-in lipids. No role for LKB1 in foam cell formation has previously been reported. Objective: We sought to establish the role of LKB1 in atherosclerotic foam cell formation. Methods and Results: LKB1 expression was examined in human carotid atherosclerotic plaques and in western diet–fed atherosclerosis-prone Ldlr−/− and ApoE−/− mice. LKB1 expression was markedly reduced in human plaques when compared with nonatherosclerotic vessels. Consistently, time-dependent reduction of LKB1 levels occurred in atherosclerotic lesions in western diet–fed Ldlr−/− and ApoE−/− mice. Exposure of macrophages to oxidized low-density lipoprotein downregulated LKB1 in vitro. Furthermore, LKB1 deficiency in macrophages significantly increased the expression of SRA (scavenger receptor A), modified low-density lipoprotein uptake and foam cell formation, all of which were abolished by blocking SRA. Further, we found LKB1 phosphorylates SRA resulting in its lysosome degradation. To further investigate the role of macrophage LKB1 in vivo, ApoE−/−LKB1fl/flLysMcre and ApoE−/−LKB1fl/fl mice were fed with western diet for 16 weeks. Compared with ApoE−/−LKB1fl/fl wild-type control, ApoE−/−LKB1fl/flLysMcre mice developed more atherosclerotic lesions in whole aorta and aortic root area, with markedly increased SRA expression in aortic root lesions. Conclusions: We conclude that macrophage LKB1 reduction caused by oxidized low-density lipoprotein promotes foam cell formation and the progression of atherosclerosis.
Targeting miR-423-5p Reverses Exercise Training–Induced HCN4 Channel Remodeling and Sinus BradycardiaNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Alicia D’Souza, Charles M. Pearman, Yanwen Wang, Shu Nakao, Sunil Jit R.J. Logantha, Charlotte Cox, Hayley Bennett, Yu Zhang, Anne Berit Johnsen, Nora Linscheid, Pi Camilla Poulsen, Jonathan Elliott, Jessica Coulson, Jamie McPhee, Abigail Robertson, Paula A. da Costa Martins, Ashraf Kitmitto, Ulrik Wisløff, Elizabeth J. Cartwright, Oliver Monfredi, Alicia Lundby, Halina Dobrzynski, Delvac Oceandy, Gwilym M. Morris, Mark R. Boyett
Rationale: Downregulation of the pacemaking ion channel, HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4), and the corresponding ionic current, If, underlies exercise training–induced sinus bradycardia in rodents. If this occurs in humans, it could explain the increased incidence of bradyarrhythmias in veteran athletes, and it will be important to understand the underlying processes. Objective: To test the role of HCN4 in the training-induced bradycardia in human athletes and investigate the role of microRNAs (miRs) in the repression of HCN4. Methods and Results: As in rodents, the intrinsic heart rate was significantly lower in human athletes than in nonathletes, and in all subjects, the rate-lowering effect of the HCN selective blocker, ivabradine, was significantly correlated with the intrinsic heart rate, consistent with HCN repression in athletes. Next-generation sequencing and quantitative real-time reverse transcription polymerase chain reaction showed remodeling of miRs in the sinus node of swim-trained mice. Computational predictions highlighted a prominent role for miR-423-5p. Interaction between miR-423-5p and HCN4 was confirmed by a dose-dependent reduction in HCN4 3′-untranslated region luciferase reporter activity on cotransfection with precursor miR-423-5p (abolished by mutation of predicted recognition elements). Knockdown of miR-423-5p with anti-miR-423-5p reversed training-induced bradycardia via rescue of HCN4 and If. Further experiments showed that in the sinus node of swim-trained mice, upregulation of miR-423-5p (intronic miR) and its host gene, NSRP1, is driven by an upregulation of the transcription factor Nkx2.5. Conclusions: HCN remodeling likely occurs in human athletes, as well as in rodent models. miR-423-5p contributes to training-induced bradycardia by targeting HCN4. This work presents the first evidence of miR control of HCN4 and heart rate. miR-423-5p could be a therapeutic target for pathological sinus node dysfunction in veteran athletes.
GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated DiscsNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Wassim A. Basheer, Shaohua Xiao, Irina Epifantseva, Ying Fu, Andre G. Kleber, TingTing Hong, Robin M. Shaw
Rationale: Delivery of Cx43 (connexin 43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubule highways, vesicles of Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has also been identified that actin provides rest stops for Cx43 forward trafficking and that Cx43 has a 20 kDa internally translated small C terminus isoform, GJA1-20k (Gap Junction Protein Alpha 1- 20 kDa), which is required for full-length Cx43 trafficking, but by an unknown mechanism. Objective: We explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs. Methods and Results: Using an in vivo Adeno-associated virus serotype 9–mediated gene transfer system, we confirmed in whole animal that GJA1-20k markedly increases endogenous myocardial Cx43 gap junction plaque size at the intercalated discs. In micropatterned cell pairing systems, we found that exogenous GJA1-20k expression stabilizes filamentous actin without affecting actin protein expression and that GJA1-20k complexes with both actin and tubulin. We also found that filamentous actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A disrupts the targeting of microtubules to cell–cell junctions. GJA1-20k protects actin filament from latrunculin A disruption, preserving microtubule trajectory to the cell–cell border. For therapeutic implications, we found that prior in vivo Adeno-associated virus serotype 9–mediated gene delivery of GJA1-20k to the heart protects Cx43 localization to the intercalated discs against acute ischemic injury. Conclusions: The internally translated GJA1-20k isoform stabilizes actin filaments, which guides growth trajectories of the Cx43 microtubule trafficking machinery, increasing delivery of Cx43 hemichannels to cardiac intercalated discs. Exogenous GJA1-20k helps to maintain cell–cell coupling in instances of anticipated myocardial ischemia.
Neutrophil Activation of Endothelial Cell-Expressed TRPM2 Mediates Transendothelial Neutrophil Migration and Vascular InjuryNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Manish Mittal, Saroj Nepal, Yoshikazu Tsukasaki, Claudie M. Hecquet, Dheeraj Soni, Jalees Rehman, Chinnaswamy Tiruppathi, Asrar B. Malik
Rationale: TRPM2 (transient receptor potential melastatin-2) expressed in endothelial cells (ECs) is a cation channel mediating Ca2+ entry in response to intracellular generation of adenosine diphosphoribose—the TRPM2 ligand. Objective: Because polymorphonuclear neutrophils (PMN) interaction with ECs generates reactive oxygen species, we addressed the possible role of TRPM2 expressed in ECs in the mechanism of transendothelial migration of PMNs. Methods and Results: We observed defective PMN transmigration in response to lipopolysaccharide challenge in adult mice in which the EC expressed TRPM2 is conditionally deleted (Trpm2iΔEC). PMN interaction with ECs induced the entry of Ca2+ in ECs via the EC-expressed TRPM2. Prevention of generation of adenosine diphosphoribose in ECs significantly reduced Ca2+ entry in response to PMN activation of TRPM2 in ECs. PMNs isolated from gp91phox−/− mice significantly reduced Ca2+ entry in ECs via TRPM2 as compared with wild-type PMNs and failed to induce PMN transmigration. Overexpression of the adenosine diphosphoribose insensitive TRPM2 mutant channel (C1008→A) in ECs suppressed the Ca2+ entry response. Further, the forced expression of TRPM2 mutant channel (C1008→A) or silencing of poly ADP-ribose polymerase in ECs of mice prevented PMN transmigration. Conclusions: Thus, endotoxin-induced transmigration of PMNs was secondary to TRPM2-activated Ca2+ signaling and VE-cadherin phosphorylation resulting in the disassembly of adherens junctions and opening of the paracellular pathways. These results suggest blocking TRPM2 activation in ECs is a potentially important means of therapeutically modifying PMN-mediated vascular inflammation.
Cardiovascular Event Prediction by Machine LearningNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Bharath Ambale-Venkatesh, Xiaoying Yang, Colin O. Wu, Kiang Liu, W. Gregory Hundley, Robyn McClelland, Antoinette S. Gomes, Aaron R. Folsom, Steven Shea, Eliseo Guallar, David A. Bluemke, João A.C. Lima
Rationale: Machine learning may be useful to characterize cardiovascular risk, predict outcomes, and identify biomarkers in population studies. Objective: To test the ability of random survival forests, a machine learning technique, to predict 6 cardiovascular outcomes in comparison to standard cardiovascular risk scores. Methods and Results: We included participants from the MESA (Multi-Ethnic Study of Atherosclerosis). Baseline measurements were used to predict cardiovascular outcomes over 12 years of follow-up. MESA was designed to study progression of subclinical disease to cardiovascular events where participants were initially free of cardiovascular disease. All 6814 participants from MESA, aged 45 to 84 years, from 4 ethnicities, and 6 centers across the United States were included. Seven-hundred thirty-five variables from imaging and noninvasive tests, questionnaires, and biomarker panels were obtained. We used the random survival forests technique to identify the top-20 predictors of each outcome. Imaging, electrocardiography, and serum biomarkers featured heavily on the top-20 lists as opposed to traditional cardiovascular risk factors. Age was the most important predictor for all-cause mortality. Fasting glucose levels and carotid ultrasonography measures were important predictors of stroke. Coronary Artery Calcium score was the most important predictor of coronary heart disease and all atherosclerotic cardiovascular disease combined outcomes. Left ventricular structure and function and cardiac troponin-T were among the top predictors for incident heart failure. Creatinine, age, and ankle-brachial index were among the top predictors of atrial fibrillation. TNF-α (tissue necrosis factor-α) and IL (interleukin)-2 soluble receptors and NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) levels were important across all outcomes. The random survival forests technique performed better than established risk scores with increased prediction accuracy (decreased Brier score by 10%–25%). Conclusions: Machine learning in conjunction with deep phenotyping improves prediction accuracy in cardiovascular event prediction in an initially asymptomatic population. These methods may lead to greater insights on subclinical disease markers without apriori assumptions of causality. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00005487.
Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells Circ. Res. (IF 13.965) Pub Date : 2017-10-13 Mingtao Zhao, Ning-Yi Shao, Shijun Hu, Ning Ma, Rajini Srinivasan, Fereshteh Jahanbani, Jaecheol Lee, Sophia L Zhang, Michael P Snyder, Joseph C Wu
Rationale: Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is a mystery how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart.Objective: We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression.Methods and Results: We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (n=84) in human endothelial cells (ECs: CD31+CD144+), cardiac progenitor cells (CPCs: Sca1+), fibroblasts (FBs: DDR2+), and their respective induced pluripotent stem cells (iPSCs). We uncovered two classes of regulatory DNA elements: Class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas Class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both Class I and Class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, Class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human iPSCs and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells, and identified a few enhancers that could possibly regulate the cardiac-specific gene expression.Conclusions: Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases.
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