PDK4 Inhibits Cardiac Pyruvate Oxidation in Late Pregnancy Circ. Res. (IF 13.965) Pub Date : 2017-09-19 Laura Liu, Glenn C Rowe, Steven Yang, Jian Li, Federico Damilano, Mun Chun Chan, Wenyun Lu, Cholsoon Jang, Shogo Wada, Michael Morley, Michael Hesse, Bernd K Fleischmann, Joshua Rabinowitz, Saumya Das, Anthony Rosenzweig, Zoltan Arany
Rationale: Pregnancy profoundly alters maternal physiology. The heart hypertrophies during pregnancy, but its metabolic adaptations are not well understood.Objective: To determine the mechanisms underlying cardiac substrate use during pregnancy.Methods and Results: We use here 13C-glucose, 13C-lactate, and 13C-fatty acid tracing analyses to show that hearts in late pregnant mice increase fatty acid uptake and oxidation into the tricarboxylic acid (TCA) cycle, while reducing glucose and lactate oxidation. Mitochondrial quantity, morphology, and function do not appear altered. Insulin signaling appears intact, and the abundance and localization of the major fatty acid and glucose transporters, CD36 and GLUT4, are also unchanged. Rather, we find that the pregnancy hormone progesterone induces pyruvate dehydrogenase kinase (PDK)-4 in cardiomyocytes, and that elevated PDK4 levels in late pregnancy lead to inhibition of pyruvate dehydrogenase (PDH) and pyruvate flux into the TCA cycle. Blocking PDK4 reverses the metabolic changes seen in hearts in late pregnancy.Conclusions: Taken together, these data indicate that the hormonal environment of late pregnancy promotes metabolic remodeling in the heart at the level of PDH, rather than at the level of insulin signaling.
GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs Circ. Res. (IF 13.965) Pub Date : 2017-09-18 Wassim A Basheer, Shaohua Xiao, Irina Epifantseva, Ying Fu, Andre G Kleber, TingTing Hong, Robin M Shaw
Rationale: Delivery of connexin 43 (Cx43) 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 20kDa internally translated small C-terminus isoform (GJA1-20k) 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 AAV9-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 (F-actin) without affecting actin protein expression, and that GJA1-20k complexes with both actin and tubulin. We also found that F-actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A (LatA) disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from LatA disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo AAV9-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.
P2Y2 Nucleotide Receptor Prompts Human Cardiac Progenitor Cell Activation by Modulating Hippo Signaling Circ. Res. (IF 13.965) Pub Date : 2017-09-18 Farid G Khalafalla, Steven R Greene, Hashim Khan, Kelli Ilves, Megan M Monsanto, Roberto Alvarez, Monica Chavarria, Jonathan H 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 HF patients with genetic predispositions and/or comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impairs overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with pro-regenerative molecules ex vivo is crucial for improving the therapeutic outcome in HF patients.Objective: To improve hCPC proliferation and migration responses that are critical for regeneration by targeting pro-regenerative 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 HF patients undergoing left ventricular assist device (LVAD) 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 to fast-growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent upon activation of yes-associated protein (YAP), 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 HF patients.
Dose Comparison Study of Allogeneic Mesenchymal Stem Cells in Patients with Ischemic Cardiomyopathy (The TRIDENT Study) Circ. Res. (IF 13.965) Pub Date : 2017-09-18 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 M Landin, Muzammil Mushtaq, Maureen H Lowery, John Byrnes, Robert C Hendel, Mauricio G Cohen, Krystalenia Valasaki, Marietsy V Pujol, Eduard Ghersin, Roberto Miki, Cindy Delgado, Fouad A Abuzeid, Mayra Vidro-Casiano, Russell 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 two doses of allogeneic bone marrow-derived human mesenchymal stem cells (hMSC) identically delivered in patients with ischemic cardiomyopathy (ICM).Methods and Results: Thirty patients with ICM received in a blinded manner either 20 million (20M, n=15) or 100 million (100M, n=15) allogeneic hMSCs via transendocardial injection (10 0.5 cc injections/patient). Patients were followed for 12-months for safety and efficacy endpoints. There were no treatment-emergent serious adverse events (SAE) at 30 days or treatment related SAEs at 12 months. The Major Adverse Cardiac Event rate was 20.0% (95% CI, 6.9%, 50.0%) in 20M and 13.3% (95% CI, 3.5%, 43.6%) in 100M (p=0.58). Worsening heart failure re-hospitalization was 20.0% (95% CI, 6.9%, 50.0%) in 20M and 7.1% (95% CI, 1.0%, 40.9%) in 100M (p=0.27). Whereas scar size reduced to a similar degree in both groups: 20M by -6.4g (IQR, -13.5g, -3.4g, p=0.001) and 100M by -6.1g (IQR, -8.1g, -4.6g, p=0.0002), the ejection fraction (EF) improved only with 100M by 3.7 units (IQR, 1.1, 6.1, p=0.04). NYHA class improved at 12 months in 35.7% (95% CI, 12.7%, 64.9%) in 20M and 42.9% (95% CI, 17.7%, 71.1%) in 100M. Importantly, pro-BNP increased at 12 months in 20M by 0.32 log pg/mL (95% CI, 0.02, 0.62, p=0.039), but not in 100M (-0.07 log pg/mL; 95% CI, -0.36, 0.23, p=0.65; between group p=0.07).Conclusions: Although both cell doses reduced scar size, only the 100M dose increased EF. 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: NCTO2013674 [https://clinicaltrials.gov/ct2/show/NCT02013674]
Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial Infarction Circ. Res. (IF 13.965) Pub Date : 2017-09-14 Thomas E Sharp, Giana J Schena, Alexander R Hoachlandr-Hobby, Timothy Starosta, Remus M Berretta, Markus Wallner, Giulia Borghetti, Polina Gross, Daohai Yu, Jaslyn Johnson, Eric A 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 if 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) (2x107 cells total) or placebo (vehicle; VEH, 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 72hrs (n=8) initial injury and cell retention were assessed. At 3 Months post-MI, cardiac structure and function was evaluated by serial echocardiography, and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72hrs 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 (LV) 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- vs. VEH- treated animals.Conclusions: CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves LV functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction (HFrEF). es that can lead to heart failure with reduced ejection fraction (HFrEF).
Cardiomyopathies Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Eugene Braunwald
The nonischemic cardiomyopathies are a diverse group of cardiac disorders that frequently cause heart failure and death and are now recognized with increasing frequency. There has been substantial progress in the clinical recognition and understanding of the natural history of these conditions. Well-established and new techniques of cardiac imaging are also helpful in this regard. Basic scientists are elucidating the pathogenesis and pathobiology of individual cardiomyopathies. In this compendium, some of the most important advances in this field are reviewed. Scientific opportunities to enhance further collaborative research to accelerate progress are identified.
Classification, Epidemiology, and Global Burden of Cardiomyopathies Circ. Res. (IF 13.965) Pub Date : 2017-09-15 William J. McKenna, Barry J. Maron, Gaetano Thiene
In the past 25 years, major advances were achieved in the nosography of cardiomyopathies, influencing the definition and taxonomy of this important chapter of cardiovascular disease. Nearly, 50% of patients dying suddenly in childhood or adolescence or undergoing cardiac transplantation are affected by cardiomyopathies. Novel cardiomyopathies have been discovered (arrhythmogenic, restrictive, and noncompacted) and added to update the World Health Organization classification. Myocarditis has also been named inflammatory cardiomyopathy. Extraordinary progress accomplished in molecular genetics of inherited cardiomyopathies allowed establishment of dilated cardiomyopathy as mostly cytoskeleton, force transmission disease; hypertrophic–restrictive cardiomyopathies as sarcomeric, force generation disease; and arrhythmogenic cardiomyopathy as desmosome, cell junction disease. Channelopathies (short and long QT, Brugada, and catecholaminergic polymorphic ventricular tachycardia syndromes) should also be considered cardiomyopathies because of electric myocyte dysfunction. Cardiomyopathies are easily diagnosed but treated only with palliative pharmacological or invasive therapy. Curative therapy, thanks to insights into the molecular pathogenesis, has to target the fundamental mechanisms involved in the onset and progression of these conditions.
Dilated Cardiomyopathy Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Elizabeth M. McNally, Luisa Mestroni
Nonischemic dilated cardiomyopathy (DCM) often has a genetic pathogenesis. Because of the large number of genes and alleles attributed to DCM, comprehensive genetic testing encompasses ever-increasing gene panels. Genetic diagnosis can help predict prognosis, especially with regard to arrhythmia risk for certain subtypes. Moreover, cascade genetic testing in family members can identify those who are at risk or with early stage disease, offering the opportunity for early intervention. This review will address diagnosis and management of DCM, including the role of genetic evaluation. We will also overview distinct genetic pathways linked to DCM and their pathogenetic mechanisms. Historically, cardiac morphology has been used to classify cardiomyopathy subtypes. Determining genetic variants is emerging as an additional adjunct to help further refine subtypes of DCM, especially where arrhythmia risk is increased, and ultimately contribute to clinical management.
Hypertrophic Cardiomyopathy Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Ali J. Marian, Eugene Braunwald
Hypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by left ventricular hypertrophy unexplained by secondary causes and a nondilated left ventricle with preserved or increased ejection fraction. It is commonly asymmetrical with the most severe hypertrophy involving the basal interventricular septum. Left ventricular outflow tract obstruction is present at rest in about one third of the patients and can be provoked in another third. The histological features of HCM include myocyte hypertrophy and disarray, as well as interstitial fibrosis. The hypertrophy is also frequently associated with left ventricular diastolic dysfunction. In the majority of patients, HCM has a relatively benign course. However, HCM is also an important cause of sudden cardiac death, particularly in adolescents and young adults. Nonsustained ventricular tachycardia, syncope, a family history of sudden cardiac death, and severe cardiac hypertrophy are major risk factors for sudden cardiac death. This complication can usually be averted by implantation of a cardioverter-defibrillator in appropriate high-risk patients. Atrial fibrillation is also a common complication and is not well tolerated. Mutations in over a dozen genes encoding sarcomere-associated proteins cause HCM. MYH7 and MYBPC3, encoding β-myosin heavy chain and myosin-binding protein C, respectively, are the 2 most common genes involved, together accounting for ≈50% of the HCM families. In ≈40% of HCM patients, the causal genes remain to be identified. Mutations in genes responsible for storage diseases also cause a phenotype resembling HCM (genocopy or phenocopy). The routine applications of genetic testing and preclinical identification of family members represents an important advance. The genetic discoveries have enhanced understanding of the molecular pathogenesis of HCM and have stimulated efforts designed to identify new therapeutic agents.
Hypertrophic Obstructive Cardiomyopathy Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Rick A. Nishimura, Hubert Seggewiss, Hartzell V. Schaff
Hypertrophic cardiomyopathy is a genetic disorder characterized by marked hypertrophy of the myocardium. It is frequently accompanied by dynamic left ventricular outflow tract obstruction and symptoms of dyspnea, angina, and syncope. The initial therapy for symptomatic patients with obstruction is medical therapy with β-blockers and calcium antagonists. However, there remain a subset of patients who have continued severe symptoms, which are unresponsive to medical therapy. These patients can be treated with septal reduction therapy, either surgical septal myectomy or alcohol septal ablation. When performed by experienced operators working in high-volume centers, septal myectomy is highly effective with a >90% relief of obstruction and improvement in symptoms. The perioperative mortality rate for isolated septal myectomy in most centers is <1%. Alcohol septal ablation is a less invasive treatment. In many patients, the hemodynamic and clinical results are comparable to that of septal myectomy. However, the results of alcohol septal ablation are dependent on the septal perforator artery supplying the area of the contact between the hypertrophied septum and the anterior leaflet of the mitral valve. There are some patients, particularly younger patients with severe hypertrophy, who do not uniformly experience complete relief of obstruction and symptoms. Both techniques of septal reduction therapy are highly operator dependent. The final decision as to which approach should be selected in any given patient is dependent up patient preference and the availability and experience of the operator and institution at which the patient is being treated.
Arrhythmogenic Cardiomyopathy Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Domenico Corrado, Cristina Basso, Daniel P. Judge
Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder, predisposing to sudden cardiac death, particularly in young patients and athletes. Pathological features include loss of myocytes and fibrofatty replacement of right ventricular myocardium; biventricular involvement is often observed. It is a cell-to-cell junction cardiomyopathy, typically caused by genetically determined abnormalities of cardiac desmosomes, which leads to detachment of myocytes and alteration of intracellular signal transduction. The diagnosis of arrhythmogenic cardiomyopathy does not rely on a single gold standard test but is achieved using a scoring system, which encompasses familial and genetic factors, ECG abnormalities, arrhythmias, and structural/functional ventricular alterations. The main goal of treatment is the prevention of sudden cardiac death. Implantable cardioverter defibrillator is the only proven lifesaving therapy; however, it is associated with significant morbidity because of device-related complications and inappropriate implantable cardioverter defibrillator interventions. Selection of patients who are the best candidates for implantable cardioverter defibrillator implantation is one of the most challenging issues in the clinical management.
Inflammatory Cardiomyopathic Syndromes Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Barry H. Trachtenberg, Joshua M. Hare
Inflammatory activation occurs in nearly all forms of myocardial injury. In contrast, inflammatory cardiomyopathies refer to a diverse group of disorders in which inflammation of the heart (or myocarditis) is the proximate cause of myocardial dysfunction, causing injury that can range from a fully recoverable syndrome to one that leads to chronic remodeling and dilated cardiomyopathy. The most common cause of inflammatory cardiomyopathies in developed countries is lymphocytic myocarditis most commonly caused by a viral pathogenesis. In Latin America, cardiomyopathy caused by Chagas disease is endemic. The true incidence of myocarditis is unknown to the limited utilization and the poor sensitivity of endomyocardial biopsies (especially for patchy diseases such as lymphocytic myocarditis and sarcoidosis) using the gold-standard Dallas criteria. Emerging immunohistochemistry criteria and molecular diagnostic techniques are being developed that will improve diagnostic yield, provide additional clues into the pathophysiology, and offer an application of precision medicine to these important syndromes. Immunosuppression is recommended for patients with cardiac sarcoidosis, giant cell myocarditis, and myocarditis associated with connective tissue disorders and may be beneficial in chronic viral myocarditis once virus is cleared. Further trials of immunosuppression, antiviral, and immunomodulating therapies are needed. Together, with new molecular-based diagnostics and therapies tailored to specific pathogeneses, the outcome of patients with these disorders may improve.
Restrictive Cardiomyopathy Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Eli Muchtar, Lori A. Blauwet, Morie A. Gertz
Restrictive cardiomyopathy (RCM) is characterized by nondilated left or right ventricle with diastolic dysfunction. The restrictive cardiomyopathies are a heterogenous group of myocardial diseases that vary according to pathogenesis, clinical presentation, diagnostic evaluation and criteria, treatment, and prognosis. In this review, an overview of RCMs will be presented followed by a detailed discussion on 3 major causes of RCM, for which tailored interventions are available: cardiac amyloidosis, cardiac sarcoidosis, and cardiac hemochromatosis. Each of these 3 RCMs is challenging to diagnose, and recognition of each disease entity is frequently delayed. Clinical clues to promote recognition of cardiac amyloidosis, cardiac sarcoidosis, and cardiac hemochromatosis and imaging techniques used to facilitate diagnosis are discussed. Disease-specific therapies are reviewed. Early recognition remains a key barrier to improving survival in all RCMs.
Cardiomyopathies Due to Left Ventricular Noncompaction, Mitochondrial and Storage Diseases, and Inborn Errors of Metabolism Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Jeffrey A. Towbin, John Lynn Jefferies
The normal function of the human myocardium requires the proper generation and utilization of energy and relies on a series of complex metabolic processes to achieve this normal function. When metabolic processes fail to work properly or effectively, heart muscle dysfunction can occur with or without accompanying functional abnormalities of other organ systems, particularly skeletal muscle. These metabolic derangements can result in structural, functional, and infiltrative deficiencies of the heart muscle. Mitochondrial and enzyme defects predominate as disease-related etiologies. In this review, left ventricular noncompaction cardiomyopathy, which is often caused by mutations in sarcomere and cytoskeletal proteins and is also associated with metabolic abnormalities, is discussed. In addition, cardiomyopathies resulting from mitochondrial dysfunction, metabolic abnormalities, storage diseases, and inborn errors of metabolism are described.
Pediatric Cardiomyopathies Circ. Res. (IF 13.965) Pub Date : 2017-09-15 Teresa M. Lee, Daphne T. Hsu, Paul Kantor, Jeffrey A. Towbin, Stephanie M. Ware, Steven D. Colan, Wendy K. Chung, John L. Jefferies, Joseph W. Rossano, Chesney D. Castleberry, Linda J. Addonizio, Ashwin K. Lal, Jacqueline M. Lamour, Erin M. Miller, Philip T. Thrush, Jason D. Czachor, Hiedy Razoky, Ashley Hill, Steven E. Lipshultz
Pediatric cardiomyopathies are rare diseases with an annual incidence of 1.1 to 1.5 per 100 000. Dilated and hypertrophic cardiomyopathies are the most common; restrictive, noncompaction, and mixed cardiomyopathies occur infrequently; and arrhythmogenic right ventricular cardiomyopathy is rare. Pediatric cardiomyopathies can result from coronary artery abnormalities, tachyarrhythmias, exposure to infection or toxins, or secondary to other underlying disorders. Increasingly, the importance of genetic mutations in the pathogenesis of isolated or syndromic pediatric cardiomyopathies is becoming apparent. Pediatric cardiomyopathies often occur in the absence of comorbidities, such as atherosclerosis, hypertension, renal dysfunction, and diabetes mellitus; as a result, they offer insights into the primary pathogenesis of myocardial dysfunction. Large international registries have characterized the epidemiology, cause, and outcomes of pediatric cardiomyopathies. Although adult and pediatric cardiomyopathies have similar morphological and clinical manifestations, their outcomes differ significantly. Within 2 years of presentation, normalization of function occurs in 20% of children with dilated cardiomyopathy, and 40% die or undergo transplantation. Infants with hypertrophic cardiomyopathy have a 2-year mortality of 30%, whereas death is rare in older children. Sudden death is rare. Molecular evidence indicates that gene expression differs between adult and pediatric cardiomyopathies, suggesting that treatment response may differ as well. Clinical trials to support evidence-based treatments and the development of disease-specific therapies for pediatric cardiomyopathies are in their infancy. This compendium summarizes current knowledge of the genetic and molecular origins, clinical course, and outcomes of the most common phenotypic presentations of pediatric cardiomyopathies and highlights key areas where additional research is required. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT02549664 and NCT01912534.
Modern Imaging Techniques in Cardiomyopathies Circ. Res. (IF 13.965) Pub Date : 2017-09-15 M. Fuad Jan, A. Jamil Tajik
Modern advanced imaging techniques have allowed increasingly more rigorous assessment of the cardiac structure and function of several types of cardiomyopathies. In contemporary cardiology practice, echocardiography and cardiac magnetic resonance imaging are widely used to provide a basic framework in the evaluation and management of cardiomyopathies. Echocardiography is the quintessential imaging technique owing to its unique ability to provide real-time images of the beating heart with good temporal resolution, combined with its noninvasive nature, cost-effectiveness, availability, and portability. Cardiac magnetic resonance imaging provides data that are both complementary and uniquely distinct, thus allowing for insights into the disease process that until recently were not possible. The new catchphrase in the evaluation of cardiomyopathies is multimodality imaging, which is purported to be the efficient integration of various methods of cardiovascular imaging to improve the ability to diagnose, guide therapy, or predict outcomes. It usually involves an integrated approach to the use of echocardiography and cardiac magnetic resonance imaging for the assessment of cardiomyopathies, and, on occasion, single-photon emission computed tomography and such specialized techniques as pyrophosphate scanning.
CELF1 Mediates Connexin 43 MicroRNA Degradation in Dilated Cardiomyopathy Circ. Res. (IF 13.965) Pub Date : 2017-09-05 Kuei-Ting Chang, Ching-Feng Cheng, Pei-Chih King, Shin-Yi Liu, Guey-Shin Wang
Rationale: Downregulation of connexin 43 (Cx43), 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. Re-induction of a nuclear RNA-binding protein CUGBP, Elav-like family member 1 (CELF1) in the adult heart has been implicated in the cardiac pathogenesis of myotonic dystrophy type 1 (DM1). 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, ribosomal RNA processing protein 6 (RRP6), 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 DM1 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.
In This Issue Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Ruth Williams
### McLendon et al identify factors influencing protein aggregation within cardiomyocytes. Accumulations of misfolded proteins within cells can impair cellular functions. Cellular aggregates of misfolded proteins are cleared mainly by the autophagic and proteasomal pathways, and this clearance is particularly important in postmitotic cells, such as those in the brain and heart. Indeed, protein aggregates are a hallmark of certain neurodegenerative disorders as well as heart conditions including desmin-related myopathy (DRM)—a disease characterized by heart and skeletal muscle degeneration. Using a mouse cardiomyocyte model of DRM and a library of silencing RNAs, McLendon and colleagues have now carried out an unbiased genome-wide screen for novel genes that either prevent protein aggregates or promote their clearance. The team identified a …
Meet the First Authors Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Lippincott Williams & Wilkins
Dr Patrick McLendon earned his BS in Biochemistry and his PhD in Biological Chemistry from Virginia Tech. His postdoctoral work, with Dr Jeffrey Robbins at Cincinnati Children’s Hospital Medical Center, focused on mechanisms of cardiac protein degradation. He currently works for UES, Inc, developing high-content assays for predicting personalized toxicological risks. Patrick’s primary research interests are using high-throughput cellular imaging to develop agnostic/unbiased approaches to address biological questions: these approaches are necessary to drive forward new biological discoveries and discover new mechanisms. In his spare time, Patrick enjoys hiking, traveling, and catching wild yeasts for fermenting beer. Dr Atsushi Tachibana is currently a PhD student in Radiological Sciences at the Tokyo Metropolitan University (TMU), Japan. He received his BS in Healthcare Sciences and Radiological Technologist degree from the Komazawa University, Japan, and an MS in Radiological Sciences from TMU. In 2014, he joined Dr Phillip C. Yang’s lab in the Department of Cardiovascular Medicine at Stanford School of Medicine as a Research Associate to work on …
Untangle a Broken Heart via Janus Kinase 1 Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Chen Gao, Yibin Wang
Protein homeostasis and quality control, as balanced by synthesis and degradation, is critical to cellular health and normal cardiac function. Similar to amyloid-related neurodegenerative diseases, the familial forms of amyloid cardiomyopathy are characterized by protein aggregate accumulation in cardiomyocytes. These protein aggregates trigger proteotoxicity in cardiomyocytes, leading to cardiac remodeling and dysfunction.1 Beyond these familial forms of cardiomyopathies, protein aggregation is also a common and perhaps functionally significant pathological feature in the diseased hearts induced by a variety of pathological stressors, including pressure overload and ischemia.2,3 Therefore, understanding the pathogenic mechanisms of cardiac proteotoxicity induced by protein aggregation in cardiomyocytes could lead to significant progress to develop potential therapeutic interventions, not only for the rare cases of amyloid cardiomyopathies but also for the common forms of heart failure in general. Article, see p 604 In this issue, McLendon et al4 from Robbins’s laboratory performed an unbiased genome-wide screening to identify genes affecting protein aggregation in primary cardiomyocytes. Taking advantage of a previously established αB-Crystallin mutant protein (CryABR120G) which possesses a strong propensity to form desmin-related protein aggregates in cardiomyocytes,5,6 they developed a high-throughput screening platform to measure protein aggregation quantitatively based on fluorescent signal intensity from the exogenously expressed GFP (green fluorescent protein) CryAB …
Smooth Muscle Differentiation Control Comes Full Circle Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Mary C.M. Weiser-Evans
Atherosclerosis is a chronic inflammatory disease that progresses to complex, unstable arterial lesions.1 Restenosis is an acute inflammatory vascular disease and a major limitation of percutaneous angioplasty procedures.2 Both are characterized by dedifferentiation of vascular smooth muscle cells (SMCs) resulting in neointimal hyperplasia and vessel occlusion. Differentiated SMCs are highly specialized cells whose primary role is to maintain vessel homeostasis, vessel tone, blood pressure, and blood flow distribution.3 This function is driven through expression of SMC-specific contractile and contractile-related proteins, including SMMHC (smooth muscle myosin heavy chain/ Myh11 ), α-SMA (α-smooth muscle actin/ Acta2 ), SM22α ( Tagln1 ), and calponin ( Cnn1 ), among others. Unlike terminally differentiated cardiac and skeletal muscle, SMCs retain a significant degree of phenotypic plasticity, exhibiting the ability to undergo extensive changes in phenotype in response to specific stimuli (ie, dedifferentiated SMC). SMC dedifferentiation is associated with a transition to a highly proliferative, inflammatory phenotype characterized by downregulation of SMC-specific genes and increased production of multiple inflammatory and matrix-associated mediators. Thus, SMCs are major contributors to vascular disease progression, and defining molecular mechanisms regulating SMC phenotypic transitions is critical to define novel therapeutics for the treatment of vascular disease. Article, see p 628 Regulation of SMC differentiation is complex, involving multiple signaling pathways and transcriptional regulators. Most SMC-specific genes are under transcriptional control by the transcription factor, serum response factor (SRF), and its cardiac and SMC-specific cofactor, myocardin, the SRF–myocardin axis.3–5 SRF binds the serum response element or CArG box, in which one or more are present within promoter and intronic regions of SMC-specific genes.3,4 In contrast, myocardin does not directly bind DNA, but transactivates SMC-specific genes through its interaction with SRF.5 Although the SRF–myocardin axis is central to transcriptional regulation of SMC genes, additional factors and mechanisms have been …
Dynamic Regulation of the Subunit Composition of BK Channels in Smooth Muscle Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Gregory M. Dick, Johnathan D. Tune
Ion channels in vascular smooth muscle cells control membrane potential, which is a major determinant of the intracellular Ca2+ concentration and thus vascular tone. Membrane potential is modulated by a complex collection of endogenous vasodilator and vasoconstrictor pathways that ultimately dictate regional vascular resistance, organ blood flow, and systemic arterial pressure. Membrane potential in vascular smooth muscle is dominated by multiple types of K+ channels including those regulated by Ca2+, voltage, and ATP.1 It is not surprising then that K+ channels in vascular smooth muscle have been studied extensively as targets in normal vasoregulatory pathways and as end-effectors gone awry in diseases. Most investigations to date have focused on traditional intracellular signaling systems (eg, cyclic nucleotides and their associated kinases2) and chronic changes in expression patterns (eg, downregulation of subunits3). Article, see p 650 In the present issue of Circulation Research , Zhai et al4 examine the novel hypothesis that endothelin-1 elicits vasoconstriction, in part, by rapidly altering the subunit composition of vascular smooth muscle K+ channels, thereby reducing their functional activity. The K+ channel under investigation is the large conductance Ca2+-activated K+ channel, known colloquially as BK (K+ channels of big conductance).1 The subunit composition of BK …
The Endless Summer Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Kevin J. Woollard, Andrew J. Murphy
People with metabolic syndrome and obesity are at an increased risk of mortality from cardiovascular disease. Over the past decade, there has been intensive research into new ways to stimulate weight loss to lower this risk. One of the key tissues is the metabolically active brown adipose tissue (BAT). Given that this is a thermogenic tissue, it can be stimulated by cold and is suggested by some as a new frontier in weight loss and improvements in other aspects of human health. But what does this mean for the health of our arteries? In the current issue of Circulation Research , Williams et al,1 reveal that ambient cool temperature may not correlate to benefit in our blood vessels. By comparing atherosclerotic lesion development over a series of temperatures and in 2 atherosclerotic prone models, they reveal stark differences in plaque size, such that larger lesions are found in animals under cold conditions. Their studies reveal a novel temperature-sensitive modulation of monocyte release from the bone marrow that ultimately impacts atherogenesis. Importantly, Williams et al1 have also uncoupled the metabolic protective role of the heat-generating UCP1 (uncoupling protein 1) from atherosclerosis. Article, see p 662 Cooler temperatures have long been associated with an increased mortality because of cardiovascular disease; however, the causality has been indirect, rationalized by changes in traditional risk factors (eg, lipids and blood pressure).2 In line with this, Dong et al3 originally found that cold housing (4°C) compared with thermoneutrality (the temperature at which energy expenditure is not needed to maintain body temperature) stimulated BAT and the browning of white adipose tissue. Cold temperature through UCP1 caused lipolysis and resulted in elevated levels of atherogenic lipoproteins. However, the role of ambient temperature and cholesterol seems controversial, with the current study reporting no changes.1 …
Pearl Quijada Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Pam Goldberg-Smith
As one who was not raised in a scientific background, Pearl Quijada is living proof that the science community benefits when taking a chance on those who exhibit great passion for the field. Pearl is a postdoctoral research fellow in the lab of Eric Small at the University of Rochester Medical Center—Cardiovascular Research Institute. She earned her BS in Biology at the University of California, Riverside. Pearl then achieved her MS in Cell and Molecular Biology at San Diego State University, before attending the Joint Doctoral Program at both San Diego State University and the University of California, San Diego for her PhD. Most recently, she has won the 2016 BCVS Abstract Travel Award.1 I moved across the country to attend the University of Rochester, and I joined the lab of Dr Eric Small. He had a strong background in the role of epicardium during development, and he was also interested in studying its role in cardiac injury, similar to what I had studied back in California. We are investigating how epicardium—the layer around the heart that nurtures progenitor cells—plays a role during cardiac repair.2 Novel molecular markers may help us to develop new models and study heart regeneration more effectively. I like that I am taking a step back to understand more about the basic biology of progenitor cells in the heart. Some people think these cells are insignificant, that they are already well known in certain tissues, but I am applying new technology to validate that they can be beneficial in cardiac repair. My teaching assistants at University of California, Riverside were always so excited in their discussions about helping people, doing field work, and collecting specimens. They seemed stoked, and I thought “I want to do that.” Right after college, I thought about getting a …
The Myofilament Field Revisited in the Age of Cellular and Molecular Biology Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Sakthivel Sadayappan
In the not too distant past, myofilament research attracted considerable attention after the discovery that mutations in myofilament genes cause cardiomyopathies. However, basic research has not been completely translated into therapies. This viewpoint discusses the need to develop innovative and integrative technologies and generate translatable models to uncover the complex biology of myofilament proteins to advance the field. The sarcomere, composed of myofilaments, is the fundamental contractile unit of striated skeletal and cardiac muscle.1 Myofilaments, occupying 70% of heart tissue, are composed of thick and thin filament proteins. Post-translational modifications of myofilament proteins regulate the rate and force of contraction.2 Mutations in myofilament proteins cause contractile dysfunction leading to cardiomyopathies.3 Therefore, studying the structure and function of myofilament proteins is essential to understand basic muscle physiology. Since the late 1800s, scientists have studied myofilament biology, and interest in these proteins dramatically increased after the discovery of a point mutation in myosin that caused hypertrophic cardiomyopathy.3 From the late 1990s to the early 2000s, scientists, flush with research funding, made several significant discoveries. Unfortunately, continued reductions in funding and changing interests have conspired to reduce the number of active muscle researchers and the enthusiasm to work in the muscle biology field. Yet, the need for muscle research remains high. The arrangement and interaction of thick and thin filament proteins in the myofilament are not completely defined.4 Increasing prevalence of heart failure demands new solutions. In response, the combination of advanced technology, improved scientific rigor, and interdisciplinary collaboration, including a renewed link to clinicians, is needed to reinvigorate myofilament research. The early pioneers of this field laid the fundamental groundwork for our understanding of myofilament properties by testing conditions that affect muscle behavior. These findings were almost always based on the development of new techniques to study …
An Unbiased High-Throughput Screen to Identify Novel Effectors That Impact on Cardiomyocyte Aggregate LevelsNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Patrick M. McLendon, Gregory Davis, James Gulick, Sonia R. Singh, Na Xu, Nathan Salomonis, Jeffery D. Molkentin, Jeffrey Robbins
Rationale: Postmitotic cells, such as cardiomyocytes, seem to be particularly susceptible to proteotoxic stimuli, and large, proteinaceous deposits are characteristic of the desmin-related cardiomyopathies and crystallin cardiomyopathic diseases. Increased activity of protein clearance pathways in the cardiomyocyte, such as proteasomal degradation and autophagy, has proven to be beneficial in maintaining cellular and cardiac function in the face of multiple proteotoxic insults, holding open the possibility of targeting these processes for the development of effective therapeutics. Objective: Here, we undertake an unbiased, total genome screen for RNA transcripts and their protein products that affect aggregate accumulations in the cardiomyocytes. Methods and Results: Primary mouse cardiomyocytes that accumulate aggregates as a result of a mutant CryAB (αB-crystallin) causative for human desmin-related cardiomyopathy were used for a total genome-wide screen to identify gene products that affected aggregate formation. We infected cardiomyocytes using a short hairpin RNA lentivirus library in which the mouse genome was represented. The screen identified multiple candidates in many cell signaling pathways that were able to mediate significant decreases in aggregate levels. Conclusions: Subsequent validation of one of these candidates, Jak1 (Janus kinase 1), a tyrosine kinase of the nonreceptor type, confirmed the usefulness of this approach in identifying previously unsuspected players in proteotoxic processes.
Paracrine Effects of the Pluripotent Stem Cell-Derived Cardiac Myocytes Salvage the Injured MyocardiumNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Atsushi Tachibana, Michelle R. Santoso, Morteza Mahmoudi, Praveen Shukla, Lei Wang, Mihoko Bennett, Andrew B. Goldstone, Mouer Wang, Masahiro Fukushi, Antje D. Ebert, Y. Joseph Woo, Eric Rulifson, Phillip C. Yang
Rationale: Cardiac myocytes derived from pluripotent stem cells have demonstrated the potential to mitigate damage of the infarcted myocardium and improve left ventricular ejection fraction. However, the mechanism underlying the functional benefit is unclear. Objective: To evaluate whether the transplantation of cardiac-lineage differentiated derivatives enhance myocardial viability and restore left ventricular ejection fraction more effectively than undifferentiated pluripotent stem cells after a myocardial injury. Herein, we utilize novel multimodality evaluation of human embryonic stem cells (hESCs), hESC-derived cardiac myocytes (hCMs), human induced pluripotent stem cells (iPSCs), and iPSC-derived cardiac myocytes (iCMs) in a murine myocardial injury model. Methods and Results: Permanent ligation of the left anterior descending coronary artery was induced in immunosuppressed mice. Intramyocardial injection was performed with (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14), and (5) PBS control (n=10). Left ventricular ejection fraction and myocardial viability, measured by cardiac magnetic resonance imaging and manganese-enhanced magnetic resonance imaging, respectively, was significantly improved in hCM- and iCM-treated mice compared with pluripotent stem cell- or control-treated mice. Bioluminescence imaging revealed limited cell engraftment in all treated groups, suggesting that the cell secretions may underlie the repair mechanism. To determine the paracrine effects of the transplanted cells, cytokines from supernatants from all groups were assessed in vitro. Gene expression and immunohistochemistry analyses of the murine myocardium demonstrated significant upregulation of the promigratory, proangiogenic, and antiapoptotic targets in groups treated with cardiac lineage cells compared with pluripotent stem cell and control groups. Conclusions: This study demonstrates that the cardiac phenotype of hCMs and iCMs salvages the injured myocardium effectively than undifferentiated stem cells through their differential paracrine effects.
EphrinB2 Regulates Cardiac Fibrosis Through Modulating the Interaction of Stat3 and TGF-β/Smad3 SignalingNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Sheng-an Su, Du Yang, Yue Wu, Yao Xie, Wei Zhu, Zhejun Cai, Jian Shen, Zurong Fu, Yaping Wang, Liangliang Jia, Yidong Wang, Jian-an Wang, Meixiang Xiang
Rationale: Cardiac fibrosis is a common feature in left ventricular remodeling that leads to heart failure, regardless of the cause. EphrinB2 (erythropoietin-producing hepatoma interactor B2), a pivotal bidirectional signaling molecule ubiquitously expressed in mammals, is crucial in angiogenesis during development and disease progression. Recently, EphrinB2 was reported to protect kidneys from injury-induced fibrogenesis. However, its role in cardiac fibrosis remains to be clarified. Objective: We sought to determine the role of EphrinB2 in cardiac fibrosis and the underlying mechanisms during the pathological remodeling process. Methods and Results: EphrinB2 was highly expressed in the myocardium of patients with advanced heart failure, as well as in mouse models of myocardial infarction and cardiac hypertrophy induced by angiotensin II infusion, which was accompanied by myofibroblast activation and collagen fiber deposition. In contrast, intramyocardial injection of lentiviruses carrying EphrinB2-shRNA ameliorated cardiac fibrosis and improved cardiac function in mouse model of myocardial infarction. Furthermore, in vitro studies in cultured cardiac fibroblasts demonstrated that EphrinB2 promoted the differentiation of cardiac fibroblasts into myofibroblasts in normoxic and hypoxic conditions. Mechanistically, the profibrotic effect of EphrinB2 on cardiac fibroblast was determined via activating the Stat3 (signal transducer and activator of transcription 3) and TGF-β (transforming growth factor-β)/Smad3 (mothers against decapentaplegic homolog 3) signaling. We further determined that EphrinB2 modulated the interaction between Stat3 and Smad3 and identified that the MAD homology 2 domain of Smad3 and the coil–coil domain and DNA-binding domain of Stat3 mediated the interaction. Conclusions: This study uncovered a previously unrecognized profibrotic role of EphrinB2 in cardiac fibrosis, which is achieved through the interaction of Stat3 with TGF-β/Smad3 signaling, implying a promising therapeutic target in fibrotic diseases and heart failure.
A Novel Regulatory Mechanism of Smooth Muscle α-Actin Expression by NRG-1/circACTA2/miR-548f-5p AxisNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Yan Sun, Zhan Yang, Bin Zheng, Xin-hua Zhang, Man-li Zhang, Xue-shan Zhao, Hong-ye Zhao, Toru Suzuki, Jin-kun Wen
Rationale: Neuregulin-1 (NRG-1) includes an extracellular epidermal growth factor–like domain and an intracellular domain (NRG-1-ICD). In response to transforming growth factor-β1, its cleavage by proteolytic enzymes releases a bioactive fragment, which suppresses the vascular smooth muscle cell (VSMC) proliferation by activating ErbB (erythroblastic leukemia viral oncogene homolog) receptor. However, NRG-1-ICD function in VSMCs remains unknown. Objective: Here, we characterize the function of NRG-1-ICD and underlying mechanisms in VSMCs. Methods and Results: Immunofluorescence staining, Western blotting, and quantitative real-time polymerase chain reaction showed that NRG-1 was expressed in rat, mouse, and human VSMCs and was upregulated and cleaved in response to transforming growth factor-β1. In the cytoplasm of HASMCs (human aortic smooth muscle cells), the NRG-1-ICD participated in filamentous actin formation by interacting with α-SMA (smooth muscle α-actin). In the nucleus, the Nrg-1-ICD induced circular ACTA2 (alpha-actin-2; circACTA2) formation by recruitment of the zinc-finger transcription factor IKZF1 (IKAROS family zinc finger 1) to the first intron of α-SMA gene. We further confirmed that circACTA2, acting as a sponge binding microRNA (miR)-548f-5p, interacted with miR-548f-5p targeting 3′ untranslated region of α-SMA mRNA, which in turn relieves miR-548f-5p repression of the α-SMA expression and thus upregulates α-SMA expression, thereby facilitating stress fiber formation and cell contraction in HASMCs. Accordingly, in vivo studies demonstrated that the localization of the interaction of circACTA2 with miR-548f-5p is significantly decreased in human intimal hyperplastic arteries compared with normal arteries, implicating that dysregulation of circACTA2 and miR-548f-5p expression is involved in intimal hyperplasia. Conclusions: These results suggest that circACTA2 mediates NRG-1-ICD regulation of α-SMA expression in HASMCs via the NRG-1-ICD/circACTA2/miR-548f-5p axis. Our data provide a molecular basis for fine-tuning α-SMA expression and VSMC contraction by transcription factor, circular RNA, and microRNA.
SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH SignalingNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Xiaolong Zhu, Sha Ding, Cong Qiu, Yanna Shi, Lin Song, Yueyue Wang, Yuewen Wang, Jinying Li, Yiran Wang, Yi Sun, Lingfeng Qin, Jun Chen, Michael Simons, Wang Min, Luyang Yu
Rationale: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell–cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. Objective: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. Methods and Results: Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)–deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. Conclusions: These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.
TPL2 (Therapeutic Targeting Tumor Progression Locus-2)/ATF4 (Activating Transcription Factor-4)/SDF1α (Chemokine Stromal Cell-Derived Factor-α) Axis Suppresses Diabetic RetinopathyNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 De-Wei Lai, Keng-Hung Lin, Wayne Huey-Herng Sheu, Maw-Rong Lee, Chung-Yu Chen, Wen-Jane Lee, Yi-Wen Hung, Chin-Chang Shen, Tsung-Ju Chung, Shing-Hwa Liu, Meei-Ling Sheu
Rationale: Diabetic retinopathy is characterized by vasopermeability, vascular leakage, inflammation, blood–retinal barrier breakdown, capillary degeneration, and neovascularization. However, the mechanisms underlying the association between diabetes mellitus and progression retinopathy remain unclear. Objective: TPL2 (tumor progression locus 2), a serine-threonine protein kinase, exerts a pathological effect on vascular angiogenesis. This study investigated the role of Nε-(carboxymethyl)lysine, a major advanced glycation end products, and the involved TPL2-related molecular signals in diabetic retinopathy using models of in vitro and in vivo and human samples. Methods and Results: Serum Nε-(carboxymethyl)lysine levels and TPL2 kinase activity were significantly increased in clinical patients and experimental animals with diabetic retinopathy. Intravitreal administration of pharmacological blocker or neutralizing antibody inhibited TPL2 and effectively suppressed the pathological characteristics of retinopathy in streptozotocin-induced diabetic animal models. Intravitreal VEGF (vascular endothelial growth factor) neutralization also suppressed the diabetic retinopathy in diabetic animal models. Mechanistic studies in primary human umbilical vein endothelial cells and primary retinal microvascular endothelial cells from streptozotocin-diabetic rats, db/db mice, and samples from patients with diabetic retinopathy revealed a positive parallel correlation between Nε-(carboxymethyl)lysine and the TPL2/chemokine SDF1α (stromal cell–derived factor-α) axis that is dependent on endoplasmic reticulum stress-related molecules, especially ATF4 (activating transcription factor-4). Conclusions: This study demonstrates that inhibiting the Nε-(carboxymethyl)lysine-induced TPL2/ATF4/SDF1α axis can effectively prevent diabetes mellitus–mediated retinal microvascular dysfunction. This signaling axis may include the therapeutic potential for other diseases involving pathological neovascularization or macular edema.
Endothelin-1 Stimulates Vasoconstriction Through Rab11A Serine 177 PhosphorylationNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Xue Zhai, M. Dennis Leo, Jonathan H. Jaggar
Rationale: Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BKα and auxiliary β1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BKα is primarily plasma membrane localized, whereas the cellular location of β1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. Objective: Test the hypothesis that ET-1 inhibits BK channels by altering BKα and β1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. Methods and Results: ET-1, through activation of PKC (protein kinase C), reduced surface β1 abundance and the proximity of β1 to surface BKα in myocytes. In contrast, ET-1 did not alter surface BKα, total β1, or total BKα proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1–induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface β1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of β1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. Conclusions: ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of β1 subunits. The decrease in surface β1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.
Thermoneutrality but Not UCP1 Deficiency Suppresses Monocyte Mobilization Into BloodNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Jesse W. Williams, Andrew Elvington, Stoyan Ivanov, Skyler Kessler, Hannah Luehmann, Osamu Baba, Brian T. Saunders, Ki-Wook Kim, Michael W. Johnson, Clarissa S. Craft, Jae-Hoon Choi, Mary G. Sorci-Thomas, Bernd H. Zinselmeyer, Jonathan R. Brestoff, Yongjian Liu, Gwendalyn J. Randolph
Rationale: Ambient temperature is a risk factor for cardiovascular disease. Cold weather increases cardiovascular events, but paradoxically, cold exposure is metabolically protective because of UCP1 (uncoupling protein 1)-dependent thermogenesis. Objective: We sought to determine the differential effects of ambient environmental temperature challenge and UCP1 activation in relation to cardiovascular disease progression. Methods and Results: Using mouse models of atherosclerosis housed at 3 different ambient temperatures, we observed that cold temperature enhanced, whereas thermoneutral housing temperature inhibited atherosclerotic plaque growth, as did deficiency in UCP1. However, whereas UCP1 deficiency promoted poor glucose tolerance, thermoneutral housing enhanced glucose tolerance, and this effect held even in the context of UCP1 deficiency. In conditions of thermoneutrality, but not UCP1 deficiency, circulating monocyte counts were reduced, likely accounting for fewer monocytes entering plaques. Reductions in circulating blood monocytes were also found in a large human cohort in correlation with environmental temperature. By contrast, reduced plaque growth in mice lacking UCP1 was linked to lower cholesterol. Through application of a positron emission tomographic tracer to track CCR2+ cell localization and intravital 2-photon imaging of bone marrow, we associated thermoneutrality with an increased monocyte retention in bone marrow. Pharmacological activation of β3-adrenergic receptors applied to mice housed at thermoneutrality induced UCP1 in beige fat pads but failed to promote monocyte egress from the marrow. Conclusions: Warm ambient temperature is, like UCP1 deficiency, atheroprotective, but the mechanisms of action differ. Thermoneutrality associates with reduced monocyte egress from the bone marrow in a UCP1-dependent manner in mice and likewise may also suppress blood monocyte counts in man.
Reducing the Global Burden of Cardiovascular Disease, Part 1 Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Philip Joseph, Darryl Leong, Martin McKee, Sonia S. Anand, Jon-David Schwalm, Koon Teo, Andrew Mente, Salim Yusuf
Current global health policy goals include a 25% reduction in premature mortality from noncommunicable diseases by 2025. In this 2-part review, we provide an overview of the current epidemiological data on cardiovascular diseases (CVD), its risk factors, and describe strategies aimed at reducing its burden. In part 1, we examine the global epidemiology of cardiac conditions that have the greatest impact on CVD mortality; the predominant risk factors; and the impact of upstream, societal health determinants (eg, environmental factors, health policy, and health systems) on CVD. Although age-standardized mortality from CVD has decreased in many regions of the world, the absolute number of deaths continues to increase, with the majority now occurring in middle- and low-income countries. It is evident that multiple factors are causally related to CVD, including traditional individual level risk factors (mainly tobacco use, lipids, and elevated blood pressure) and societal level health determinants (eg, health systems, health policies, and barriers to CVD prevention and care). Both individual and societal risk factors vary considerably between different regions of the world and economic settings. However, reliable data to estimate CVD burden are lacking in many regions of the world, which hampers the establishment of nationwide prevention and management strategies. A 25% reduction in premature CVD mortality globally is feasible but will require better implementation of evidence-based policies (particularly tobacco control) and integrated health systems strategies that improve CVD prevention and management. In addition, there is a need for better health information to monitor progress and guide health policy decisions.
Reducing the Global Burden of Cardiovascular Disease, Part 2 Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Darryl P. Leong, Philip G. Joseph, Martin McKee, Sonia S. Anand, Koon K. Teo, Jon-David Schwalm, Salim Yusuf
In this second part of a 2-part series on the global burden of cardiovascular disease, we review the proven, effective approaches to the prevention and treatment of cardiovascular disease. We specifically review the management of acute cardiovascular diseases, including acute coronary syndromes and stroke; the care of cardiovascular disease in the ambulatory setting, including medical strategies for vascular disease, atrial fibrillation, and heart failure; surgical strategies for arterial revascularization, rheumatic and other valvular heart disease, and symptomatic bradyarrhythmia; and approaches to the prevention of cardiovascular disease, including lifestyle factors, blood pressure control, cholesterol-lowering, antithrombotic therapy, and fixed-dose combination therapy. We also discuss cardiovascular disease prevention in diabetes mellitus; digital health interventions; the importance of socioeconomic status and universal health coverage. We review building capacity for conduction cardiovascular intervention through strengthening healthcare systems, priority setting, and the role of cost effectiveness.
Recommendation on Design, Execution, and Reporting of Animal Atherosclerosis Studies: A Scientific Statement From the American Heart Association Circ. Res. (IF 13.965) Pub Date : 2017-09-01 Alan Daugherty, Alan R. Tall, Mat J.A.P. Daemen, Erling Falk, Edward A. Fisher, Guillermo García-Cardeña, Aldons J. Lusis, A. Phillip Owens, Michael E. Rosenfeld, Renu Virmani
Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.
Interleukin 17A Exacerbates Atherosclerosis by Promoting Fatty Acid-Binding Protein 4–Mediated ER Stress in Macrophages Circ. Res. (IF 13.965) Pub Date : 2012-01-01 Qi Gao, Yang Jiang, Shen Dai, Bo Wang, Fei Gao, Chun Guo, Faliang Zhu, Qun Wang, Xiaoyan Wang, Jining Wang, Yongyu Shi, Yun Zhang, Wanjun Chen, Lining Zhang
Rationale: Apoptosis and fatty acid-binding protein-4 (FABP4) induced-endoplasmic reticulum (ER) stress in macrophage is an important pathological process in several vascular occlusive diseases, including atherosclerosis, both of which are accelerated by lipids or inflammatory cytokines. Objective: To determine whether interleukin 17A (IL-17A) accelerates atherosclerosis through activating FABP4-mediated ER stress in macrophage. Methods and Results: We show here that IL-17A induced ER stress in both murine and human-derived macrophages in vitro, and in the atherosclerotic lesions of ApoE-/- mice. Treating ApoE-/- mice with a chemical chaperone alleviated IL-17A–mediated ER stress and macrophage apoptosis, which was accompanied by recovered atherogenesis. Mechanistically, IL-17A up-regulated the expression of FABP4 (aP2), a cytosolic lipid chaperone that is able to promote lipid-induced macrophage ER stress, through NF-κB and ERK/p38 mitogen-activated protein kinase (MAPK) pathways in macrophages. The inhibition of aP2 expression with a specific chemical inhibitor significantly blocked IL-17A-accelerated ER stress and apoptosis in plaque, and partially rescued IL17A-induced atherogenesis. Conclusions: The data collectively establish a previously unrecognized link between IL-17A and ER stress through cytosolic lipid chaperone aP2 in macrophages and provide a new insight for understanding the role of IL-17A in atherosclerosis.
In This Issue Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Ruth Williams
### Veerman et al determine the likely effect of a Brugada syndrome risk allele. Brugada syndrome is characterized by abnormal electrical signals in the heart and a heightened risk of sudden death. A genome-wide association study (GWAS) has identified a Brugada-linked single nucleotide polymorphism (SNP) at the genetic locus 6q22.31. This SNP lies close to the gene for transcription factor HEY2; however, whether HEY2 contributes to Brugada syndrome is unclear. Veerman and colleagues examined gene expression data from 190 human left ventricle samples and found that, of 8 genes in the 6q22.31 region, only HEY2 showed significant association—increased expression—with the Brugada risk allele. Further, genome-wide coexpression analyses showed that, of 15 617 genes examined, the expression of KCNIP2 , which encodes an ion …
Meet the First Authors Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Lippincott Williams & Wilkins
Dr Jennifer Thompson is a newly appointed Assistant Professor with the Libin Cardiovascular Institute in the Cumming School of Medicine at the University of Calgary. She earned her BS in Kinesiology and PhD in Physiology from Western University, Ontario. Her PhD project focused on the influence of intrauterine growth restriction on cardiovascular development and later cardiovascular function. Her postdoctoral work, supported by the NIH Pathway to Independence Award (K99/R00) and the American Heart Association, was undertaken at Augusta University. Dr Thompson’s current research aims to understand how the maternal metabolic state acts through the intrauterine environment to shape disease risk in succeeding generations, an emerging concept with implications for the treatment and prevention of heart disease. Dr Thompson has 2 children and enjoys spending time with her family when she is not in the lab. Dr Renjing Liu earned her BS in Biochemistry from the University of Sidney and her PhD at the same institution in 2010. She completed her postdoctoral training at the Yale Stem Cell Center with …
New Initiatives to Improve the Rigor and Reproducibility of Articles Published in Circulation Research Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Roberto Bolli
The rampant irreproducibility of scientific articles is arguably the most serious problem that bedevils biomedical research.1–12 It is a veritable plague that undermines the credibility of published studies, hinders clinical translation of basic work, and impedes the progress of medicine. Disquietingly, the problem seems to be getting worse rather than better.10 Although the causes of irreproducibility are multifarious, inadequate rigor is probably the most important2,11; thus, it will be impossible to augment reproducibility without augmenting methodological rigor. It is not the purpose of this editorial to revisit the nature, origins, mechanisms, and consequences of irreproducibility, all of which have been discussed innumerable times in recent years, both in the literature and in ad hoc workshops.1–12 There has been enough discussion; now it is time for action. Much of this action must come from editors. Publication of methodologically flawed or suboptimal research can be limited by promulgating, and diligently applying, higher standards during editorial evaluation of submitted work. The editors of Circulation Research believe that the journal has a responsibility to implement initiatives that promote more rigorous and, therefore, more reproducible scientific work. It is for this reason that Circulation Research has published several reviews, editorials, Viewpoints, and News & Views articles focused on this issue.1,2,7–12 It is for this reason that the journal has joined other leading journals to promote reproducibility of biomedical research4 by endorsing the National Institutes of Health principles and guidelines for reporting preclinical research,6 and it is for this very reason that we have recently chosen to publish a study showing that only a minority of preclinical animal experiments reported in leading cardiovascular journals (including Circulation Research ) adheres to basic criteria necessary to assure rigor: for …
Allele-Specific Gene Silencing Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Stanley Nattel
Cardiac arrhythmias are a major healthcare problem in the developed world. The American Heart Association 2017 statistical report indicates that atrial fibrillation, the most common cardiac rhythm disorder, has an ≈25% lifetime incidence and annual costs of ≈26 billion dollars, whereas sudden cardiac death (usually caused by malignant arrhythmias) affects fewer individuals (just under 200 000 Americans/year) but has more disastrous consequences.1 There have been a variety of improvements in arrhythmia therapy over the past decades, particularly in the realm of nonpharmacological approaches, but many challenges remain.2 Article, see p 525 One area with great promise is the induced modification of cardiac gene expression to produce the targeted downregulation or overexpression of specific gene products (gene therapy).3 The first reported experimental application of gene therapy was for the control of ventricular response rate in atrial fibrillation.4 Since then, a wide range of developments has occurred in the design of gene therapy, the development of gene delivery systems (including cardiac-selective delivery vectors, promoter-selection and product design, and production methods), and therapeutic-targeting strategy.3 Inherited arrhythmia syndromes caused by ion-channel dysfunction lend themselves naturally to gene therapy because most result from gain or loss of function of a single gene product that can be targeted specifically. In 2014, Denegri et al5 reported a fascinating proof-of-principle study demonstrating that a single injection of a cardiotropic adeno-associated virus (serotype-9, AAV9) carrying wild-type calsequestrin-2 is able to produce long-term suppression (over the full animal life span) of the arrhythmic phenotype in mice with a loss-of-function mutation causing catecholaminergic polymorphic ventricular tachycardia (CPVT). The mutation that was targeted produces a recessive form of CPVT, in which affected individuals produce no functional protein and the only curative treatment possible is to restore the full functional wild-type gene product in the heart. The wild-type …
When High Throughput Meets Mechanistic Studies Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Bettina Heidecker
The greatest insights in science are obtained through integrative approaches that combine the best aspects emerging and traditional technologies. The study by Veerman et al1 that appears in this issue of Circulation Research thoughtfully integrates big data high throughput technology, small data molecular approaches, and electrophysiological methods to draw conclusions important both to the understanding of Brugada syndrome and normal ventricular function. Article, see p 537 More than 2 decades ago, Josep and Pedro Brugada first described a syndrome of ST-segment elevation, right bundle branch block, a high incidence of polymorphic ventricular tachycardia and ventricular fibrillation, and increased risk of sudden cardiac death.2 Research dating back to 1991 has described that the induction of phase 2 reentry by sodium channel block which was thought to be the cause of life-threatening arrhythmias in this syndrome,3 and which later became known as Brugada syndrome.4,5 In 1998, Chen et al6 showed linkage of the Brugada syndrome with a mutation in the SCN5A gene encoding the α subunit of the cardiac sodium channel protein. Because of the dynamic and temporary nature of the Brugada ECG pattern, sodium channel blockers (eg, ajmaline, flecainide, or procainamide) have been administered to unmask type 1 ECG patterns.7 Since the discovery of the SCN5A mutation, a total of 12 Brugada syndrome susceptibility genes have been identified (Brugada susceptibility [BrS] 1–12). Type 1 BrS or BrS1, caused by the loss-of-function mutation in the SCN5A-encoded subunit of the sodium channel, represents the most common genetic substrate for BrS accounting for ≤30% of the disorder.8,9 Furthermore, mutations of the L-type calcium channel α1, β2, and α2δ subunits encoded by CACNA1, CACNB2B, and CACNA2B are estimated to cause 10% to 15% of Brugada syndrome.10,11 Functional analyses have shown that …
Besides Imprinting Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Shizuka Uchida
It is now firmly recognized that although the majority of mammalian genome is transcribed to RNA, protein-coding transcripts occupy only a minor part of mammalian transcriptome. Instead, increasing evidence suggests that the majority of mammalian RNAs is comprised noncoding RNAs (ncRNAs). ncRNAs are a diverse class of RNAs with many subclasses involved in a wide variety of physiological functions, including those in the heart. Although the functions of small RNAs, including microRNAs, have been elucidated in the past 2 decades, those of longer ncRNAs are increasing being investigated in various fields of studies, including cardiovascular medicine. Among longer ncRNAs, those longer than 200 nucleotides are classified as long noncoding RNAs (lncRNAs). Numerous screening studies have identified several thousand lncRNAs expressed in the heart using microarrays and next-generation sequencing (eg, RNA sequencing), yet only handful have been characterized.1 This lack of characterized lncRNAs is partially because of the low sequence conservation of lncRNAs, making it difficult to translate screening of lncRNAs in humans (eg, patients with cardiovascular disease versus healthy donors) to model organisms (eg, mice, rat, and zebrafish) to study the target lncRNAs in vivo because homologs of lncRNAs cannot easily be identified. However, some well-conserved lncRNAs do exist among humans and other organisms, especially those that are involved in genomic imprinting, which is an epigenetic phenomenon whereby genes are expressed in a parent-of-origin dependent manner.2 Although their function in imprinting is well characterized, the functions outside of imprinting are still under investigation, which should broaden our understanding of …
Training for Success Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Robert N. Correll
As scientists, the decision about where to postdoc is among the most important that we will make in our professional development. Postdoctoral training, after all, is where young scientists are set loose to utilize those skills obtained during their graduate studies and create a trajectory of inquiry that will guide the early stages of their independent careers. So how does a senior graduate student or a newly-minted PhD go about picking the best postdoctoral environment? For that matter, how does one decide whether or not a postdoc is even right for them? Fortunately, the graduate school experience itself goes a long way toward informing this decision. Not only is it an important formative period that develops bench skills and teaches students how to formulate hypotheses and develop effective experimental strategies, but graduate education should also provide the young scientist with a better understanding of their own interests and what additional scientific training they require to meet their career goals. When it was time for me to find a lab in which to do my postdoctoral training, I was a senior graduate student in biochemistry at the University of Kentucky in the lab of Doug Andres. Over a period of 6 years in his laboratory, Dr Andres provided a tremendous …
Michael Potente Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Ruth Williams
Michael Potente rose quickly through the ranks of Frankfurt’s Goethe University—first pursuing a medical degree, then combining post-doctoral research with his clinical career—before being snapped up by the Max Planck Institute for Heart and Lung Research in Bad Nauheim at the age of 35 years. Potente’s rapid career ascent is no surprise once you get talking to him. He comes across as decisive, a man on a mission. He suggested to Circulation Research that this attitude may have been shaped by his clinical cardiology experience. In medicine, he explained, there is no time for lengthy decisions. It is a question of getting things done. At the Max Planck Institute, Potente heads the Angiogenesis and Metabolism Laboratory, where his team studies the development and growth of blood vessels and how the vasculature changes during aging and disease. In particular, Potente asks how the underlying metabolic mechanisms within endothelial cells control vessel growth, maintenance, and function.1–5 He describes the subject as both fascinating and beautiful. Even in a fast-paced laboratory, there is time for aesthetics, he says. Michael Potente ### Tell Me About Your Childhood I was born in Aachen, West Germany, but only lived there until I was 4 years old, when my father, who is a professor of engineering, got a new position at the University of Paderborn. That is where I spent most of my formative years. As a teenager, I had some wild times. I was heavily into skateboarding and hip-hop culture, I was into music, and—you may not believe this—I even had dreadlocks. So I looked different than I do today. ### How Did This Hip-Hop Dude Turn Into a Doctor? In high school, I was thinking about what to do, and medicine just seemed attractive to me. I always liked science, but the engineering side of it, which my father had introduced me to, was not really for me. …
Cardiovascular Research in Germany Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Gerd Heusch, Thomas Eschenhagen, Stefanie Dimmeler
### Introduction: Development of Cardiovascular Research German academic cardiovascular medicine has been traditionally rigidly separated into clinical cardiology, where the top clinicians had some early career basic science exposure but practiced exclusively clinical medicine, and classic basic science departments, that is, physiology, pharmacology, and pathology, where the chair had a cardiovascular focus. The institutional funding by the universities, which are, with few exceptions, state institutions in Germany, was sufficient to provide budget/resources for personnel, equipment and consumables to conduct basic research, and was in the range of 1 to 2 Mio € per year for such institutions. This situation has changed progressively but profoundly during the past 3 decades: almost all university clinics of cardiology now practice not only clinical cardiology but perform also basic science studies. They find it, however, increasingly difficult to recruit young physicians to a clinician scientist path of career. Basic cardiovascular science is no longer restricted to the classic university departments of physiology, pharmacology, pathology, but many chairs for experimental cardiovascular medicine under a variety of names/designations have been founded (>10 chairs in the past decade). Here, it has become extremely difficult to recruit young physicians into such basic science departments, and their personnel has now mostly a natural sciences rather than a medical background. Institutional funding for a basic cardiovascular research department has been restricted and now rarely exceeds 300 000 € to 600 000 € per year. Thus, there is an increasing pressure for competitive external funding. Along with these changes, the rigid hierarchical system of German academic medicine with a single professor and chair has become more flexible and more similar to the Anglo-American system where many professors with a different specialization coexist in the same department. Such more flexible system has in part evolved because many clinicians and researchers have …
Large Animal Model Efficacy Testing Is Needed Prior to Launch of a Stem Cell Clinical Trial Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Stephen E. Epstein, Dror Luger, Michael J. Lipinski
Stem cell therapeutics, as other therapeutic strategies, must surmount multiple requirements before reaching the phase of clinical trials. These requirements can be onerous in terms of time and funding and could, thereby, lead to abandonment of many promising therapeutics. One of these requirements is the perceived necessity of conducting large animal efficacy studies—especially pig studies—prior to initiating a clinical trial. The value of such a strategy, despite acceptance by experts, has never been demonstrated scientifically, raising the question of whether bias rather than evidence hampers the development of new therapeutic strategies. Of the questions facing investigators involved in developing new treatment strategies, one is common across disease disciplines and therapeutic approaches: What animal models should be used to determine whether the likelihood a therapeutic will be clinically successful is high enough to justify spending years of effort and tens, or even hundreds, of millions of dollars in clinical trials? A particularly vexing extension of this question is whether it is necessary to test efficacy in a large animal model prior to clinical trial launch. The purpose of this Viewpoint is to challenge the prevailing view, epitomized by the following quote: “Translational studies in large animal models (usually pigs) are rare because they are expensive, complex, time-consuming, technically demanding, slow, and usually not suitable for mechanistic investigations; nevertheless, because they are conducted in settings closer to the human situation than those found in rodent models, these studies are essential to justify the risks and costs of clinical trials.”1 This quote reflects current unofficial dogma that once efficacy of an intervention is established in mouse or other rodent models, efficacy needs confirmation in a large animal model—the most popular one being the pig.1–3 This is not a trivial conclusion because adequately powered pig studies are expensive from both …
Application of PCSK9 Inhibitors in Practice Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Tina M. Kaufman, P. Barton Duell, Jonathan Q. Purnell, Cezary Wójcik, Sergio Fazio, Michael D. Shapiro
Although the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9) and development of therapeutic antagonists represent a major triumph of modern clinical medicine, efforts to implement PCSK9 inhibitors (PCSK9i) in patient care have been sobering. This practical guide examines the barriers and opportunities for the successful application of pharmacological inhibition of PCSK9 in clinical practice through introduction of a new model of care delivery—the PCSK9i clinic. Historically, the foundation of primary and secondary prevention of atherosclerotic cardiovascular disease (ASCVD) has consisted of therapeutic lifestyle changes in combination with pharmacological therapy focused on lipid modulation, specifically low-density lipoprotein cholesterol (LDL-C) lowering.1 In 2015, the Food and Drug Administration (FDA) approved a new class of cholesterol-lowering medications, PCSK9i, to great anticipation. The seminal discovery in 2003 by Abifadel et al2 linked gain-of-function mutations in the PCSK9 gene with autosomal dominant hypercholesterolemia. This finding uncovered PCSK9 as a key player in cholesterol homeostasis, a circulating protein with the strongest influence on plasma LDL-C concentration.3 PCSK9 directly interacts with the low-density lipoprotein receptor and enhances its degradation by targeting it for destruction by the lysosome and halting its efficient recycling. Because PCSK9 causes degradation of the low-density lipoprotein receptor, inhibiting its action prolongs the lifespan of the low-density lipoprotein receptor and leads to profound reductions in plasma LDL-C levels. The ultimate culmination of this work was the regulatory approval of 2 monoclonal antibody inhibitors of PCSK9 (alirocumab and evolocumab). More recently, the randomized, placebo-controlled trial, FOURIER (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk), demonstrated improved ASCVD outcomes when evolocumab was added to background treatment with a statin. The combination of statin plus evolocumab resulted in a significant absolute and relative risk reduction in both the primary composite end point (cardiovascular death, myocardial infarction, stroke, and hospitalization …
Genetic Deletion of NADPH Oxidase 1 Rescues Microvascular Function in Mice With Metabolic DiseaseNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Jennifer A. Thompson, Sebastian Larion, James D. Mintz, Eric J. Belin de Chantemèle, David J. Fulton, David W. Stepp
Rationale: Early vascular changes in metabolic disease that precipitate the development of cardiovascular complications are largely driven by reactive oxygen species accumulation, yet the extent to which excess reactive oxygen species derive from specific NADPH oxidase isoforms remains ill defined. Objective: Identify the role of Nox1 in the development of microvascular dysfunction in metabolic disease. Methods and Results: Four genotypes were generated by breeding Nox1 knockout mice with db/db mice: lean (HdbWnox1), lean Nox1 knockout (HdbKnox1), obese (KdbWnox1), and obese KK (KdbKnox1). The degree of adiposity, insulin resistance, and dyslipidemia in KW mice was not influenced by Nox1 deletion as determined by nuclear magnetic resonance spectroscopy, glucose tolerance tests, and plasma analyses. Endothelium-dependent responses to acetylcholine in pressurized mesenteric arteries were reduced in KW versus HW (P<0.01), whereas deletion of Nox1 in KW mice normalized dilation. Vasodilator responses after inhibition of NO synthase blunted acetylcholine responses in KK and lean controls, but had no impact in KW, attributing recovered dilatory capacity in KK to normalization of NO. Acetylcholine responses were improved (P<0.05) with Tempol, and histochemistry revealed oxidative stress in KW animals, whereas Tempol had no impact and reactive oxygen species staining was negligible in KK. Blunted dilatory responses to an NO donor and loss of myogenic tone in KW animals were also rescued with Nox1 deletion. Conclusions: Nox1 deletion reduces oxidant load and restores microvascular health in db/db mice without influencing the degree of metabolic dysfunction. Therefore, targeted Nox1 inhibition may be effective in the prevention of vascular complications.
ARHGAP18 Protects Against Thoracic Aortic Aneurysm Formation by Mitigating the Synthetic and Proinflammatory Smooth Muscle Cell PhenotypeNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Renjing Liu, Lisa Lo, Angelina J. Lay, Yang Zhao, Ka Ka Ting, Elizabeth N. Robertson, Andrew G. Sherrah, Sorour Jarrah, Haibo Li, Zhaoxiong Zhou, Brett D. Hambly, David R. Richmond, Richmond W. Jeremy, Paul G. Bannon, Mathew A. Vadas, Jennifer R. Gamble
Rationale: Thoracic aortic aneurysm (TAA) is a potentially lethal condition, which can affect individuals of all ages. TAA may be complicated by the sudden onset of life-threatening dissection or rupture. The underlying mechanisms leading to TAA formation, particularly in the nonsyndromal idiopathic group of patients, are not well understood. Thus, identification of new genes and targets that are involved in TAA pathogenesis are required to help prevent and reverse the disease phenotype. Objective: Here we explore the role of ARHGAP18, a novel Rho GAP expressed by smooth muscle cells (SMCs), in the pathogenesis of TAA. Methods and Results: Using human and mouse aortic samples, we report that ARHGAP18 levels were significantly reduced in the SMC layer of aortic aneurysms. Arhgap18 global knockout (Arhgap18−/−) mice exhibited a highly synthetic, proteolytic, and proinflammatory smooth muscle phenotype under basal conditions and when challenged with angiotensin II, developed TAA with increased frequency and severity compared with littermate controls. Chromatin immunoprecipitation studies revealed this phenotype is partly associated with strong enrichment of H3K4me3 and depletion of H3K27me3 at the MMP2 and TNF-α promoters in Arhgap18-deficient SMC. We further show that TAA formation in the Arhgap18−/− mice is associated with loss of Akt activation. The abnormal SMC phenotype observed in the Arhgap18−/− mice can be partially rescued by pharmacological treatment with the mTORC1 inhibitor rapamycin, which reduces the synthetic and proinflammatory phenotype of Arhgap18-deficient SMC. Conclusion: We have identified ARHGAP18 as a novel protective gene against TAA formation and define an additional target for the future development of treatments to limit TAA pathogenesis.
Allele-Specific Silencing of Mutant mRNA Rescues Ultrastructural and Arrhythmic Phenotype in Mice Carriers of the R4496C Mutation in the Ryanodine Receptor Gene (RYR2)Novelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Rossana Bongianino, Marco Denegri, Andrea Mazzanti, Francesco Lodola, Alessandra Vollero, Simona Boncompagni, Silvia Fasciano, Giulia Rizzo, Damiano Mangione, Serena Barbaro, Alessia Di Fonso, Carlo Napolitano, Alberto Auricchio, Feliciano Protasi, Silvia G. Priori
Rationale: Mutations in the cardiac Ryanodine Receptor gene (RYR2) cause dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals exposed to emotions or physical exercise. Objective: We investigated the efficacy of allele-specific silencing by RNA interference to prevent CPVT phenotypic manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2. Methods and Results: We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2-R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 (Ryanodine Receptor type 2 protein) function after in vivo delivery by intraperitoneal injection in neonatal and adult RyR2R4496C/+ (mice heterozygous for the R4496C mutation in the RyR2) heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10, the ratio between wild-type and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2-R4496C mRNA, whereas protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively (1) reduced isoproterenol-induced delayed afterdepolarizations and triggered activity in infected cells, (2) reduced adrenergically mediated ventricular tachycardia in treated mice, (3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and transverse tubules, and (4) attenuated mitochondrial abnormalities. Conclusions: The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice, suggesting that the reduction of mutant RyR2 may be a novel therapeutic approach for CPVT.
The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical HeterogeneityNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Christiaan C. Veerman, Svitlana Podliesna, Rafik Tadros, Elisabeth M. Lodder, Isabella Mengarelli, Berend de Jonge, Leander Beekman, Julien Barc, Ronald Wilders, Arthur A.M. Wilde, Bastiaan J. Boukens, Ruben Coronel, Arie O. Verkerk, Carol Ann Remme, Connie R. Bezzina
Rationale: Genome-wide association studies previously identified an association of rs9388451 at chromosome 6q22.3 (near HEY2) with Brugada syndrome. The causal gene and underlying mechanism remain unresolved. Objective: We used an integrative approach entailing transcriptomic studies in human hearts and electrophysiological studies in Hey2+/− (Hey2 heterozygous knockout) mice to dissect the underpinnings of the 6q22.31 association with Brugada syndrome. Methods and Results: We queried expression quantitative trait locus data acquired in 190 human left ventricular samples from the genotype-tissue expression consortium for cis-expression quantitative trait locus effects of rs9388451, which revealed an association between Brugada syndrome risk allele dosage and HEY2 expression (β=+0.159; P=0.0036). In the same transcriptomic data, we conducted genome-wide coexpression analysis for HEY2, which uncovered KCNIP2, encoding the β-subunit of the channel underlying the transient outward current (Ito), as the transcript most robustly correlating with HEY2 expression (β=+1.47; P=2×10−34). Transcript abundance of Hey2 and the Ito subunits Kcnip2 and Kcnd2, assessed by quantitative reverse transcription–polymerase chain reaction, was higher in subepicardium versus subendocardium in both left and right ventricles, with lower levels in Hey2+/− mice compared with wild type. Surface ECG measurements showed less prominent J waves in Hey2+/− mice compared with wild-type. In wild-type mice, patch-clamp electrophysiological studies on cardiomyocytes from right ventricle demonstrated a shorter action potential duration and a lower Vmax in subepicardium compared with subendocardium cardiomyocytes, which was paralleled by a higher Ito and a lower sodium current (INa) density in subepicardium versus subendocardium. These transmural differences were diminished in Hey2+/− mice because of changes in subepicardial cardiomyocytes. Conclusions: This study uncovers a role of HEY2 in the normal transmural electrophysiological gradient in the ventricle and provides compelling evidence that genetic variation at 6q22.31 (rs9388451) is associated with Brugada syndrome through a HEY2-dependent alteration of ion channel expression across the cardiac ventricular wall.
Transient Notch Activation Induces Long-Term Gene Expression Changes Leading to Sick Sinus Syndrome in MiceNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Yun Qiao, Catherine Lipovsky, Stephanie Hicks, Somya Bhatnagar, Gang Li, Aditi Khandekar, Robert Guzy, Kel Vin Woo, Colin G. Nichols, Igor R. Efimov, Stacey Rentschler
Rationale: Notch signaling programs cardiac conduction during development, and in the adult ventricle, injury-induced Notch reactivation initiates global transcriptional and epigenetic changes. Objective: To determine whether Notch reactivation may stably alter atrial ion channel gene expression and arrhythmia inducibility. Methods and Results: To model an injury response and determine the effects of Notch signaling on atrial electrophysiology, we transiently activate Notch signaling within adult myocardium using a doxycycline-inducible genetic system (inducible Notch intracellular domain [iNICD]). Significant heart rate slowing and frequent sinus pauses are observed in iNICD mice when compared with controls. iNICD mice have structurally normal atria and preserved sinus node architecture, but expression of key transcriptional regulators of sinus node and atrial conduction, including Nkx2-5 (NK2 homeobox 5), Tbx3, and Tbx5 are dysregulated. To determine whether the induced electrical changes are stable, we transiently activated Notch followed by a prolonged washout period and observed that, in addition to decreased heart rate, atrial conduction velocity is persistently slower than control. Consistent with conduction slowing, genes encoding molecular determinants of atrial conduction velocity, including Scn5a (Nav1.5) and Gja5 (connexin 40), are persistently downregulated long after a transient Notch pulse. Consistent with the reduction in Scn5a transcript, Notch induces global changes in the atrial action potential, including a reduced dVm/dtmax. In addition, programmed electrical stimulation near the murine pulmonary vein demonstrates increased susceptibility to atrial arrhythmias in mice where Notch has been transiently activated. Taken together, these results suggest that transient Notch activation persistently alters ion channel gene expression and atrial electrophysiology and predisposes to an arrhythmogenic substrate. Conclusions: Our data provide evidence that Notch signaling regulates transcription factor and ion channel gene expression within adult atrial myocardium. Notch reactivation induces electrical changes, resulting in sinus bradycardia, sinus pauses, and a susceptibility to atrial arrhythmias, which contribute to a phenotype resembling sick sinus syndrome.
Sirt3 Impairment and SOD2 Hyperacetylation in Vascular Oxidative Stress and HypertensionNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Anna E. Dikalova, Hana A. Itani, Rafal R. Nazarewicz, William G. McMaster, Charles R. Flynn, Roman Uzhachenko, Joshua P. Fessel, Jorge L. Gamboa, David G. Harrison, Sergey I. Dikalov
Rationale: Clinical studies have shown that Sirt3 (Sirtuin 3) expression declines by 40% by 65 years of age paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 because of increased NADH (nicotinamide adenine dinucleotide, reduced form) and acetyl-CoA levels. Sirt3 impairment reduces the activity of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2) because of hyperacetylation. Objective: In this study, we examined whether the loss of Sirt3 activity increases vascular oxidative stress because of SOD2 hyperacetylation and promotes endothelial dysfunction and hypertension. Methods and Results: Hypertension was markedly increased in Sirt3-knockout (Sirt3−/−) and SOD2-depleted (SOD2+/−) mice in response to low dose of angiotensin II (0.3 mg/kg per day) compared with wild-type C57Bl/6J mice. Sirt3 depletion increased SOD2 acetylation, elevated mitochondrial O2· –, and diminished endothelial nitric oxide. Angiotensin II-induced hypertension was associated with Sirt3 S-glutathionylation, acetylation of vascular SOD2, and reduced SOD2 activity. Scavenging of mitochondrial H2O2 in mCAT mice expressing mitochondria-targeted catalase prevented Sirt3 and SOD2 impairment and attenuated hypertension. Treatment of mice after onset of hypertension with a mitochondria-targeted H2O2 scavenger, mitochondria-targeted hydrogen peroxide scavenger ebselen, reduced Sirt3 S-glutathionylation, diminished SOD2 acetylation, and reduced blood pressure in wild-type but not in Sirt3−/− mice, whereas an SOD2 mimetic, (2-[2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino]-2-oxoethyl) triphenylphosphonium (mitoTEMPO), reduced blood pressure and improved vasorelaxation both in Sirt3−/− and wild-type mice. SOD2 acetylation had an inverse correlation with SOD2 activity and a direct correlation with the severity of hypertension. Analysis of human subjects with essential hypertension showed 2.6-fold increase in SOD2 acetylation and 1.4-fold decrease in Sirt3 levels, whereas SOD2 expression was not affected. Conclusions: Our data suggest that diminished Sirt3 expression and redox inactivation of Sirt3 lead to SOD2 inactivation and contributes to the pathogenesis of hypertension.
Inhibition of the Cardiac Fibroblast–Enriched lncRNA Meg3 Prevents Cardiac Fibrosis and Diastolic DysfunctionNovelty and Significance Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Maria-Teresa Piccoli, Shashi Kumar Gupta, Janika Viereck, Ariana Foinquinos, Sabine Samolovac, Freya Luise Kramer, Ankita Garg, Janet Remke, Karina Zimmer, Sandor Batkai, Thomas Thum
Rationale: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing. Objective: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction. Methods and Results: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-β I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-β I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance. Conclusions: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling.
Letter by El-Battrawy et al Regarding Article, “The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical Heterogeneity” Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Ibrahim El-Battrawy, Siegfried Lang, Martin Borggrefe, Xia-Bo Zhou, Ibrahim Akin
The article by Veerman et al1 published in Circulation Research describing the role of HEY2 in modulation of ion channel expression in Brugada syndrome elicited great interest among members of our research working group. The authors found that the transmural differences of action potential characteristics, Ito (transient outward pottasium current), and INa (voltage-dependent sodium current) were diminished in Hey2+/− mice. Certainly, it …
Response by Veerman et al to Letter Regarding Article, “The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical Heterogeneity” Circ. Res. (IF 13.965) Pub Date : 2017-08-18 Christiaan C. Veerman, Ronald Wilders, Arthur A. Wilde, Ruben Coronel, Carol Ann Remme, Arie O. Verkerk, Connie R. Bezzina
We welcome the opportunity to respond to the question raised by El-Battrawy et al in their letter concerning our study entitled “The Brugada Syndrome Susceptibility Gene HEY2 Modulates Cardiac Transmural Ion Channel Patterning and Electrical Heterogeneity.”1 In their letter, El-Battrawy et al state that the transmural differences observed in Hey2+/− mice as compared with wild-type could be explained by the fact that the sodium current (INa) was measured at room temperature, whereas the transient outward potassium current (Ito) and action potential were evaluated at 36°C. The authors comment that sodium channel expression and sodium current density are modulated by temperature in Brugada Syndrome …
Regulation of Macrophage Apoptosis and Atherosclerosis by Lipid Induced PKCδ Isoform Activation Circ. Res. (IF 13.965) Pub Date : 2017-08-30 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 and obesity contributes to the development of atherosclerosis. PKCδ expression and activity in monocytes were increased by hyperlipidemia and diabetes 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 very high fat diet (HFD). Mice fed AD and HFD exhibited hyperlipidemia, but only HFD fed mice had insulin resistance and mild diabetes. Surprisingly, MPKCδKO/ApoE-/- mice exhibited accelerated aortic atherosclerotic lesions by 2-fold vs. 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 numbers in aortic plaques and spleen by 1.7 and 2-fold, respectively, in MPKCδKO/ApoE-/- vs. ApoE--/- mice due to decreased apoptosis (62%) and increased proliferation (1.9 fold), and not due to uptake, with parallel increased expressions of inflammatory cytokines. Mechanisms for the increased macrophages in MPKCδKO/ApoE-/- were associated with elevated phosphorylation levels of pro-survival cell signaling proteins, Akt and FoxO3a, with reduction of pro-apoptotic 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 Disruption Circ. Res. (IF 13.965) Pub Date : 2017-08-29 Anne K Johansen, Bas Molenaar, Danielle Versteeg, Ana R Leitoguinho, Charlotte J Demkes, Bastiaan Spanjaard, Hesther de Ruiter, Farhad A Akbari Moqadam, Lieneke Kooijman, Lorena Zentilin, Mauro Giacca, Eva van Rooij
Rationale: CRISPR/Cas9-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 (sgRNAs) targeting three genes critical for cardiac physiology, Myh6, Sav1 and Tbx20, using a cardiotropic adeno-associated viral vector (AAV9). Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of sgRNA 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 sgRNA approach to effectively delete en 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 AAV9 to deliver a single sgRNA 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 in order to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo.
MerTK Cleavage on Resident Cardiac Macrophages Compromises Repair after Myocardial Ischemia Reperfusion Injury Circ. Res. (IF 13.965) Pub Date : 2017-08-29 Matthew DeBerge, Xin-Yi Yeap, Shirley Dehn, Shuang Zhang, Lubov S Grigoryeva, Sol Misener, Daniele Procissi, Xin Zhou, Daniel C Lee, William A Muller, Xunrong Luo, Carla Rothlin, Ira Tabas, Edward B Thorp
Rationale: Clinical benefits of reperfusion after myocardial infarction (MI) are offset by maladaptive innate immune cell function and therapeutic interventions are lacking.Objective: We sought to test the significance of phagocytic clearance by resident and recruited phagocytes after myocardial ischemia reperfusion (I/R).Methods and Results: In humans, we discovered that clinical reperfusion after MI led to significant elevation of the soluble form of MerTK (i.e. solMER), a critical biomarker of compromised phagocytosis by innate macrophages. In reperfused mice, macrophage Mertk-deficiency led to decreased cardiac wound debridement, increased infarct size, and depressed cardiac function, newly implicating MerTK in cardiac repair after myocardial I/R. More notably, Mertk(CR) mice, which are resistant to cleavage, showed significantly reduced infarct sizes and improved systolic function. In contrast to other cardiac phagocyte subsets, resident cardiac MHCIILOCCR2- macrophages expressed higher levels of MerTK and when exposed to apoptotic cells, secreted pro-reparative cytokines, including TGF-β. Mertk-deficiency compromised the accumulation of MHCIILO phagocytes, and this was rescued in Mertk(CR) mice. Interestingly, blockade of CCR2-dependent monocyte infiltration into the heart reduced solMER levels post I/R.Conclusions: Our data implicate monocyte-induced MerTK-cleavage on pro-reparative MHCIILO cardiac macrophages as a novel contributor and therapeutic target of reperfusion injury.
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