Abstract
Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative 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(2+) signaling 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(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.
Highlights
The precise phosphorylation sites for these changes in Ca2ϩ signaling are not yet defined, these results reveal a broader scope of functions for this kinase in the regulation of excitation-contraction coupling in cardiac myocytes
Given that PKD is up-regulated in failing human heart and its inhibition is proposed as a promising therapeutic strategy for heart disease (2, 4, 19), it is critical to identify the hierarchy of functional targets for PKD throughout the cardiac myocyte
Through a combination of molecular genetics, functional assays, and complementary in situ molecular studies, we define a novel role for PKD in stimulus-dependent dynamic regulation of myofilament Ca2ϩ sensitivity in cultured ventricular cardiac myocytes
Summary
Construction of Recombinant Adenoviruses—Production of recombinant adenovirus (Ad5)-containing wild type PKD (wtPKD) cDNA, constitutively active PKD (caPKD) cDNA, or kinase-deficient dominant negative PKD (dnPKD) cDNA and the corresponding fluorescent fusion proteins with the redshifted GFP mutant, Plum, was carried out according to methods described previously (24). Each of these recombinant adenoviral constructs contained an N-terminal hemagglutinin (HA) epitope tag to monitor heterologous PKD expression in cultured adult rat ventricular myocytes (ARVMs). Unpaired Student’s t test and one-way analysis of variance with Newman-Keuls test were applied when appropriate to determine statistical significance of the differences. p Ͻ 0.05 was considered statistically significant, except where noted
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