Abstract
Although ceramide accumulation in the heart is considered a major factor in promoting apoptosis and cardiac disorders, including heart failure, lipotoxicity and ischemia–reperfusion injury, little is known about ceramide’s role in mediating changes in contractility. In the present study, we measured the functional consequences of acute exposure of isolated field-stimulated adult rat cardiomyocytes to C6-ceramide. Exogenous ceramide treatment depressed the peak amplitude and the maximal velocity of shortening without altering intracellular calcium levels or kinetics. The inactive ceramide analog C6-dihydroceramide had no effect on myocyte shortening or [Ca2+]i transients. Experiments testing a potential role for C6-ceramide-mediated effects on activation of protein kinase C (PKC) demonstrated evidence for signaling through the calcium-independent isoform, PKCε. We employed 2-dimensional electrophoresis and anti-phospho-peptide antibodies to test whether treatment of the cardiomyocytes with C6-ceramide altered myocyte shortening via PKC-dependent phosphorylation of myofilament proteins. Compared to controls, myocytes treated with ceramide exhibited increased phosphorylation of myosin binding protein-C (cMyBP-C), specifically at Ser273 and Ser302, and troponin I (cTnI) at sites apart from Ser23/24, which could be attenuated with PKC inhibition. We conclude that the altered myofilament response to calcium resulting from multiple sites of PKC-dependent phosphorylation contributes to contractile dysfunction that is associated with cardiac diseases in which elevations in ceramides are present.
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Acknowledgments
The authors would like to acknowledge Dr. Suresh Govidan and Dr. Xiang Ji for their technical assistance with the cMyBP-C immunoblots. This research was supported by an American Heart Association, Midwest Affiliate pre-doctoral fellowship to JNS, NIH research grants R01 HL-64035 (RJS and BMW), R01 HL-105826 and K02 HL-114749 (SS), RO1 HL-081680 (DFW), and PO1 HL-62426 (Project 1 and Core C to RJS and CMW).
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Simon, J.N., Chowdhury, S.A.K., Warren, C.M. et al. Ceramide-mediated depression in cardiomyocyte contractility through PKC activation and modulation of myofilament protein phosphorylation. Basic Res Cardiol 109, 445 (2014). https://doi.org/10.1007/s00395-014-0445-6
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DOI: https://doi.org/10.1007/s00395-014-0445-6