Recent studies have identified a novel ?fibroblast-like? cell in several organs that express the receptor tyrosine kinase, PDGFRa (platelet-derived growth factor receptor-a). Using a mouse strain (Pdgfratm11(EGFP)Sor/J) in which PDGFR?+ cells are constitutively labeled by expression of a transgene encoding a histone 2B-eGFP fusion protein driven by the endogenous, cell-specific promoter for Pdgfra, we are able to isolate, purify, and study the function and phenotype of fibroblast-like cells from a variety of organs, including the heart. We have demonstrated that these cells are excitable and respond to agonists with rapid activation of large ionic currents. We have further confirmed that the eGFP reporter is exclusive to PDGFRa+ cells by immunodetection using antibodies against PDGFR?, thus definitively establishing these fibroblast-like cells as PDGFR?+ cells. In preliminary studies for this proposal, we found that PDGFR?+ cells are widely distributed in cardiac muscles, especially in sinoatrial nodal (SAN) and atrial tissues. A significant population of PDGFRa+ cells expresses the classical fibroblast marker, vimentin, and the same population is labeled by PDGFRa antibodies in the primate heart, indicating that this population corresponds to the cardiac fibroblast-like cells (CFCs) of the heart. Using the eGFP reporter, we isolated and purified cardiac PDGFRa+ cells by FACS from SAN and atrial tissues. Our subsequent characterization of PDGFRa+ cells in the SAN revealed that these cells are excitable and are capable of generating spontaneous pacemaker activity through activation of a nonselective cation channel (NSCC). Notably, we found that these cells form gap junctions with neighboring myocytes. Further investigation of the automaticity of PDGFRa+ cells and more extensive characterization of the phenotypes of these cells will be pursued in two Specific Aims: 1) Determine the genetic transcript signature of cardiac PDGFR?+ cells that determine this cellular phenotype. 2) Determine the role(s) of NSCCs in generating spontaneous inward currents in PDGFR?+ CFCs and in modulating cardiac AP waveform. Novel insights provided by successful completion of this research proposal will facilitate a better understanding of the automaticity and phenotype of PDGFRa+ CFCs in SAN and atrial tissues and may support the development of novel therapeutic targets for pacemaker deficiencies.
Spontaneous cardiac rhythmicity is indispensable for life. Abnormal pacing and propagation of cardiac action potentials leads to serious health problems. The existence of large populations of non-myocyte phenotypes that make up the majority of cells in cardiac tissues raises questions about the physiological roles these cells play in heart function. In this proposal, we provide multifaceted experimental approaches to evaluate the functional role(s) of PDGFR?+ cardiac fibroblast cells (CFCs) in heart rhythmicity. Data obtained from this proposal will provide valuable information on the contribution of these cells to cardiac rhythmicity under normal conditions and provide a foundation for investigating changes that occur in these cellular networks in cardiac dysrhythmias. !
