Novel Functions of Protein Kinase D in Cardiac Excitation-Contraction Coupling

While the dynamic function of protein kinase D (PKD) has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 and CREB in the heart and has been implicated in the maintenance of cellular dysfunction that develops in heart failure. Here we significantly extend our understanding of PKD signaling in heart by investigating the cytosolic targeting of PKD in adult rat ventricular myocytes (ARVMs) using a molecular genetic approach to drive adenovirus-dependent expression of wild type (wt), constitutively active (ca) or dominant negative (dn) PKD in cultured ARVMs. Confocal imaging reveals a significant distribution of PKD in a non-nuclear, striated-reticular pattern in steady-state ARVMs with changes in PKD spatial distribution as PKD activity changes. Consistent with an established role of PKD in targeting cardiac troponin I, caPKD expression led to a marked decrease in contractile myofilament Ca2+ sensitivity. Steady-state Ca2+ transients were markedly increased in dnPKD cells and are explained in part by a marked increase in sarcoplasmic reticulum (SR) Ca2+ load. In addition, changes in the cardiac Ca2+ current (ICa) and behavior of the phosphatase inhibitor calyculin A (CalyA) support a role for PKD as a dynamic regulatory kinase of the L-type Ca2+ channel (LTCC). Whole-cell voltage clamp studies illustrate a marked increase in ICa throughout the entire voltage range in caPKD cells. Dynamic analyses of ICa reveal that, unlike control cells, the Ca2+ current in caPKD cells was maximally activated and did not further increase after phosphatase inhibition, while there was a loss of the CalyA stimulatory response in dnPKD cells. Taken together with our new findings, work to date suggests a complex collection of functions carried out by PKD that can best be explained by a new model that requires specific spatially-resolved subcellular targeting.