Cardiac Protein Kinase D1 ablation alters the myocytes ß-adrenergic response.

ß-adrenergic (ß-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute ß-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte ß-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal ß-adrenergic responses in Ca2+ handling.

Endurance training restores spatially distinct cardiac mitochondrial function and myocardial contractility in ovariectomized rats.

We previously demonstrated that the loss of female hormones induces cardiac and mitochondrial dysfunction in the female heart. Here, we show the impact of endurance training for twelve weeks, a nonpharmacological therapy against cardiovascular disease caused by ovariectomy and its contribution to cardiac contractility, mitochondrial quality control, bioenergetics and oxidative damage. We found that ovariectomy induced cardiac hypertrophy and dysfunction by decreasing SERCA2 and increasing phospholamban protein expression. Endurance training restored myocardial contractility, SERCA2 levels, increased calcium transient in ovariectomized rats but did not change phospholamban protein expression or cardiac hypertrophy. Additionally, ovariectomy decreased the amount of intermyofibrillar mitochondria and induced mitochondrial fragmentation that were accompanied by decreased levels of mitofusin 1, PGC-1α, NRF-1, total AMPK-α and mitochondrial Tfam. Endurance training prevented all these features except for mitofusin 1. Ovariectomy reduced O2 consumption, elevated O2.- release and increased Ca2+-induced mitochondrial permeability transition pore opening in both mitochondrial subpopulations. Ovariectomy also increased NOX-4 protein expression in the heart, reduced mitochondrial Mn-SOD, catalase protein expression and increased protein carbonylation in both mitochondrial subpopulations, which were prevented by endurance training. Taken together, our findings show that endurance training prevented cardiac contractile dysfunction and mitochondrial quality control in ovariectomized rats.

Action potential duration determines sarcoplasmic reticulum Ca2+ reloading in mammalian ventricular myocytes.

After sarcoplasmic reticulum (SR) Ca2+ depletion in intact ventricular myocytes, electrical activity promotes SR Ca2+ reloading and recovery of twitch amplitude. In ferret, recovery of twitch and caffeine-induced contracture required fewer twitches than in rabbit or rat. In rat, there was no difference in action potential duration at 90% repolarization (APD90) at steady state (SS) versus at the first post-depletion (PD) twitch. The SS APD90 was similar in ferret and rabbit (but longer than in rat). However, compared to SS, the PD APD90 was lengthened in ferret, but shortened in rabbit. When rabbit myocytes were subjected to AP-clamp patterns during SR Ca2+ reloading (ferret- or rabbit-type APs), reloading was much faster using the ferret AP templates. We conclude that the faster SR Ca2+ refilling in ferret is due to the increased Ca2+ influx during the longer PD AP. The PD versus SS APD90 difference was suppressed by thapsigargin in ferret (indicating Ca2+ dependence). In rabbit, the PD AP shortening depended on the preceding diastolic interval (rather than Ca2+), because rest produced the same AP shortening, and SS APD90 increased as a function of frequency (in contrast to ferret). Transient outward current (Ito) was larger and recovered from inactivation much faster in ferret than in rabbit. Moreover, slow Ito recovery (tau approximately 3 s) in rabbit was a much larger fraction of Ito. Our data and a computational model (including two Ito components) suggest that in rabbit the slowly recovering Ito is responsible for short post-rest and PD APs, for the unusual frequency dependence of APD90, and ultimately for the slower post-depletion SR Ca2+ reloading.