Novel Functions of Protein Kinase D in Cardiac Excitation-Contraction Coupling
Abstract
While the function of protein kinase D (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 and CREB implicating this kinase in the development of dysfunction seen in heart failure. Here we significantly extend our understanding of PKD signaling in heart using a molecular genetic approach to examine PKD through adenoviral vector expression of wild type (wt), constitutively active (ca) or dominant negative (dn) PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated-reticular pattern suggesting the importance of PKD in Ca<super>2+</super> signaling in heart. Thus, an initial hypothesis was that PKD plays unappreciated roles in regulation of excitation-contraction coupling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca<super>2+</super> sensitivity with an unexpected electrical stimulus-dependence to this response. This desensitization was accompanied by stimulus-dependent increases in phosphorylation of cTnI and of regulatory site, Ser916, on PKD. The functional importance of this phospho-Ser916 event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. dnPKD expression resulted in sensitization of the myofilaments to Ca<super>2+</super> and blocked stimulus-dependent increases in cTnI phosphorylation. Furthermore, steady-state Ca<super>2+</super> transients were markedly increased in dnPKD cells and are explained, in part, by a marked increase in sarcoplasmic reticulum (SR) Ca<super>2+</super> load. In addition, changes in the cardiac Ca<super>2+</super> current (I<sub>Ca</sub>) and behavior of the phosphatase inhibitor, calyculin A (CalyA), support a role for PKD as a dynamic regulatory kinase of the L-type Ca<super>2+</super> channel (LTCC). Together, these data suggest a complex collection of novel functions carried out by PKD to dynamically regulate several components of the excitation-contraction coupling cascade in cardiomyocytes to allow for precise fine-tuning of cardiac cell function. Given that PKD activity is elevated in failing hearts, it will be important to determine the role of increased signaling at these newly appreciated cellular locales in the development of heart disease.Description
University of Maryland in Baltimore. Molecular Medicine. Ph.D. 2010Keyword
cardiac musclephosphorylation enzymes
protein kinase D
Calcium Signaling
Contractile Proteins
Excitation Contraction Coupling
Myocardium