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1 Internal Medicine, University of Virginia, Charlottesville, Virginia, United States
2 Cellular and Molecular Physiology, Pennsylvania State University, Hershey, United States; Weis Center for Research, Geisinger Medical Center, Danville, United States
3 Weis Center for Research, Geisinger Medical Center, Danville, United States
4 Cellular and Molecular Physiology, Pennsylvania State University, Hershey, United States; Medicine, Pennsylvania State University, Hershey, United States; Weis Center for Research, Geisinger Medical Center, Danville, United States
* To whom correspondence should be addressed. E-mail: jyc1{at}psu.edu.
Phospholemman (PLM), a 72-amino acid sarcolemmal protein, has been shown to regulate contractility and Ca2+ homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice back-bred to a pure congenic C57BL/6 background. Cell length, cell width and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared to wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na+/Ca2+ exchanger, sarcoplasmic reticulum (SR) Ca2+-ATPase,
1-subunit of Na+-K+-ATPase and calsequestrin levels in PLM-null myocytes. At 5mM extracellular [Ca2+] concentration ([Ca2+]o), contraction and cytosolic [Ca2+] ([Ca2+]i) transient amplitudes and SR [Ca2+] contents in PLM-null myocytes were significantly (p<0.0004) higher than wild-type myocytes. At 0.6 mM [Ca2+]o, however, contraction and [Ca2+]i transient amplitudes and SR [Ca2+] contents were significantly lower in PLM-null than wild-type myocytes. At 1.8 mM [Ca2+]o, the differences in contraction and [Ca2+]i transient amplitudes were no longer apparent. This pattern of contractile and [Ca2+]i transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na+/Ca2+ exchanger. Indeed, we have previosuly reported that Na+/Ca2+ exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotrophy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (p<0.04) in PLM-null myocytes. Whole cell Ca2+ current densities were similar between wild-type and PLM-null myocytes, as were the fast and slow inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca2+ fluxes, likely by modulating Na+/Ca2+ exchange activity.
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