Proteome Analysis of Secreted Proteins during Embryonal P19CLONE6 Cell Differentiation into Cardiac Myocytes Using 2-DE/MALDI-TOF

Secreted proteins by cardiomyocytes, including paracrine factors and extracellular matrix proteins provide a means of communication in the heart between myocytes and non-myocytes. Heart disease encompasses a broad spectrum of pathological conditions and abnormal changes to the proteome can reflect these various disease states. In this project we set out to identify differentially secreted proteins that mediate cardiac myocyte differentiation using a cell model. The secretome of in vitro cardiaclike cells during differentiation of the multipotent embryonal carcinoma mouse cell line P19 clone 6, was subjected to 2-DE analysis. P19cl6 cells were differentiated in culture into beating cardiac myocytes by treatment with 1% dimethyl sulfoxide (DMSO). On day 10 –12 of in vitro differentiation, RT-PCR analysis and immunohistochemical staining gave evidence that these cells were of cardiac origin as they expressed cardiac-specific markers: troponin T, beta myosin heavy chain and F-actin fibres were highly expressed in the treated cells. Serum-free supernatants from undifferentiated and differentiated cells were harvested, concentrated and desalted on Vivaspin columns at 5kDA MWCO. Secreted proteins were submitted to 2-DE coupled with MALDI-TOF peptide mass fingerprinting. Preliminary 2-DE results showed 17 proteins that were differentially expressed with five proteins only present in the control (not differentiated) cultures and 12 proteins only present in the differentiated beating cardiac myocyte cultures. Protein identification by peptide mass fingerprinting is currently under way. In conclusion, this study provides a tool for the characterization of the major proteins released by cardiac cells upon differentiation. 15.2 Proteomic Analysis of Membrane Microdomains Derived from Both Failing and Non-failing Human Hearts M. Brioschi, R. Wait, P. Fratto, G. Polvani, L. Mussoni, E. Tremoli, and C. Banfi University of Milan and Monzino Cardiologic Center IRCCS, Milan, Italy; Imperial College London, London, United Kingdom; Niguarda Hospital, Milan, Italy; Monzino Cardiologic Center IRCCS, Milan, Italy; University of Milan, Milan, Italy