Biochemical and Biophysical Research Communications
The effects of membrane potential and extracellular matrix composition on vascular differentiation of cardiac progenitor cells
Introduction
Substantial progress has been made in recent years in elucidating the role of microenvironmental signals from the extracellular matrix (ECM) in cardiac development, disease, and regeneration [[1], [2], [3]]. With progress in biomaterial design, strides have been made toward therapeutic cardiovascular tissue engineering [4]. Even with these advances, there is an incomplete understanding of how the matrix interacts with other mechanisms, such as bioelectric signaling [[5], [6], [7], [8]]. This is despite the knowledge that resting membrane potential (RMP) plays a fundamental role in developmental processes such as myogenesis and left-right heart patterning [9,10] and that manipulation of bioelectric signals may be a method with which to promote functional regeneration [5,11]. Thus, a complete understanding of how the microenvironment interacts with bioelectric signals is crucial in developing a practical understanding of development and regeneration [7,8].
Despite disagreement on their exact identity [12], cardiac progenitor cells (CPCs) have previously been utilized to study the role of microenvironmental signals in differentiation [[13], [14], [15], [16], [17]] due to their ability to differentiate into all three major cardiovascular lineages. Recent lineage tracing studies suggest CPCs are vascular progenitors with in vivo benefits attributed to neovascularization and paracrine signaling [[18], [19], [20]]. In line with this perspective, our lab previously explored the effect of ECM developmental age and stiffness on CPCs [21]. We found that CPCs expressing the receptor tyrosine kinase c-kit, a commonly employed CPC marker [12], had significantly altered vascular gene expression when exposed to matrices derived from different developmental stages, but only minor changes in cardiac gene expression.
Investigation into the role of bioelectric signaling in CPCs has been more limited, but initial efforts have found that hyperpolarization of CPCs enhances cardiac gene expression via calcineurin signaling [22] and depolarization can enhance CPC mobilization [23]. Similarly, a variety of studies examining skeletal muscle progenitors have suggested that hyperpolarization precedes myogenic gene expression and differentiation [9,24]. While these studies establish a role for bioelectric signaling in progenitor differentiation, the effects of matrix-bioelectric cross talk have not yet been studied in CPCs.
This study examines the interplay of cellular microenvironment and resting membrane potential in the context of CPC differentiation. Pediatric c-kit+ CPCs were differentiated with or without ion-channel targeting agents on substrates coated with cardiac ECM from multiple developmental ages. We found that differentiation in depolarizing conditions enhanced smooth muscle cell gene expression but left endothelial or cardiac expression unaltered. Hyperpolarization resulted in the opposite trend, reducing smooth muscle differentiation. Smooth muscle protein expression underwent similar changes to gene expression but was further affected by exposure to ECM. Ultimately, our work suggests that the developmental function of matrix-bioelectric cross talk can be applied to modify the differentiation potential of progenitor cells in vitro.
Section snippets
CPC isolation and maintenance culture
The isolation and use of human CPCs were approved by the Institutional Review Boards at Boston Children’s Hospital and Tufts University. Isolation of CPCs was performed employing protocols previously utilized by our lab [17,21]. De-identified, discarded samples from right atrial appendages of pediatric cardiac patients at Boston Children’s Hospital were serially digested in collagenase type 2 (Worthington Biochemical Corporation) in phosphate buffered saline (PBS) with 20 mM glucose. The
Modulation of K+ alters CPC resting membrane potential
We modulated the K+ flux across the membrane due to its role in maintaining cellular resting membrane potential via treatment with either 20 μM diazoxide or 20 mM potassium gluconate. Acute response was established via whole cell patch clamping on TCP (Fig. 1). The RMP of untreated CPCs was found to be −26.7 ± 5.9 mV. Change in RMP was calculated per cell from pre- and post-treatment RMP and compared to vehicle (DMSO) and osmolarity (NMDG) controls, respectively. CPCs were significantly
Discussion
While changes in both ECM and RMP have been shown to direct portions of cardiac development [2,10] as well as CPC phenotype [[14], [15], [16], [17],[21], [22], [23]], additional work is needed to illuminate their interaction [7,8]. We sought to investigate the combined effect of changes in cardiac ECM composition and RMP in CPC differentiation. We leveraged our previous experience employing decellularized cardiac ECM derived from both fetal and adult hearts, which have been shown to possess
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Science Foundation (NSF1351241 and NSF1603524 to LDB), the Department of Defense (W81XWH-16-1-0304 to LDB), the National Institutes of Health (P41EB027062 to DLK), and the Paul G. Allen Family Foundation (2171 to DLK). We thank Dr. Sitaram Emani at Boston Children’s Hospital in acquiring the tissue samples for CPC isolation.
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