Abstract 574: The Secretomes from Cardiac Stem Cells and Neonatal Myocytes: Proteomics-based Identification of Novel Paracrine Factors
Soluble proteins secreted from stem cells serve as interactive signals to the local environment, influencing survival, differentiation and engraftment. To characterize the secreted proteome (“secretome”), proteins found in the media conditioned by adult cardiac stem cells (CSCs) or neonatal rat ventricular myocytes (NRVMs) were obtained under optimized conditions that minimize cell lysis, allowing distinction between secreted proteins and those artificially liberated with cell death.
Methods: CSCs were grown from rat septal or left ventricular explants and NRVMs were isolated under conditions with minimal cell death (18±2% [CSCs, n=3] and 10±1% [NRVMs, n=3] by Annexin V labeling). Conditioned media (1% serum) was collected after 48 hours, filtered, concentrated, resuspended in 20% (v/v) acetontile:1%TFA and separated by reversed phase HPLC. Collected fractions were digested with trypsin and analyzed by ESI/MS/MS for protein identification and quantification.
Results: 90–110 proteins were identified exclusively in media from CSCs or NRVMs, with the majority (>85%) comprising known membrane, extracellular or secreted proteins. Of these, >2 fold more proteins were detected in CSCs than NRVMs (60 vs. 27, respectively). Functional protein classes were conserved between the cell types, although proteins and/or their isoforms could differ. Of interest, 10 different collagen isoforms were observed with 5 being cell-specific. Cell-specificity was also reflected with collagen regulation as TIMP2 and MMP2 were observed in NRVMs while TIMP1 was uniquely present in CSCs. The signaling molecules, insulin-like growth factor binding protein 6 and 3, were present in CSCs, but only isoform 7 in NRVMs. Interestingly, the cardiac specific natriuretic hormone ANP was only detected in NRVMs and not CSCs, while the soluble interleukin-1 receptor family member ST2 was exclusive to CSCs. ST2 is known to increase in myocytes with mechanical stress and heart failure where it has been linked to neurohormonal activation.
Conclusion: This first report of CSC and NRVM-specific secretomes displays unique functionality including differential secretion of two cardiovascular hormones.