Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes

Abstract

In the heart, the proteomes secreted by both cardiac stem cells (CSCs) and cardiac myocytes could act synergistically, but the identification and functionality of the proteins comprising the individual secretomes have not yet been described. In this study, we have identified proteins present in the media obtained from cultured rat CSCs and from cultured neonatal rat ventricular myocytes (NRVMs) and compared them with proteins identified in the media alone. Briefly, 83 unique proteins were identified after analysis by RPLC and MS. In total 49 and 23% were NRVM-specific or CSC-specific proteins, respectively, and 63% of total 83 proteins were integral plasma membrane and/or known secreted proteins. Fifteen proteins met our criteria for paracrine/autocrine factors: (i) robust protein identification, (ii) cell specific and (iii) known to be secreted. Most of these proteins have not been previously linked to stem cells. NRVM-specific proteins atrial natriuretic factor (ANP) and connective tissue growth factor, and CSC-specific protein interleukin-1 receptor-like 1 (ST2) were found to affect rat CSC proliferation. These findings suggest that relative concentration of each protein may be crucial for cellular intertalk and for the final outcome of cardiac cell therapy.

Figure 1

Strategyof protein analysis in conditioned media of CSCs and NRVMs.

Figure 2

Representative chromatograms obtained by RPLC analysis. A - Chromatogram of CDC conditioned media analyzed in 8 subsequent runs (100 μg/run) with 9 final fractions used for MS protein identification after trypsin digestion. Eight chromatograms are overlayed to demostrate the reproducibility. Fraction containing albumin was excluded. B - Comparison of chromatograms obtained during analysis of control media and conditioned media of NRVMs and CSCs (CDCs and CSps), respectively, in time window from 14 to 22 minutes.

Figure 3

A - Distribution of 83 unique proteins present in the conditioned media from CSCs and/or NRVMs. B - Cellular locations of 83 unique proteins as found in protein databases.

Figure 4

Proteomic and functional data for 3 identified secreted proteins (CTGF, ST2 and ANP). Growth curves at different protein concentrations of CTGF, ST2 and ANP are shown in graphs. Western blots for ANP: panel A-protein amounts in NRVM media and lysate loaded into gel were 75 μg and 180 μg, respectively; panel B-protein amounts in CSp, CDC and NRVM media were 130 μg, 50 μg and 75 μg, respectively. Additional experimental conditions for proliferation assays and Westen blots are given in detail in Online Supporting Information (Expanded Materials and Methods).