SR Ca-ATPase Research Articles

Intracellular calcium levels are unchanged in the diabetic heart.

The quality control indices of myocyte isolation (viability, yield, survival time, cell response, etc.) suggest that the adult rat myocyte model is stable and useful in [Ca2+]i measurements and functional studies at the cellular level. Moreover, diabetic cardiomyocytes are a valuable model for studying cellular functions of the diabetic heart as they retain most of the features of cardiac dysfunction of intact rat. Data from our studies indicate that the basal [Ca2+]i in both quiescent and electrically-stimulated cells is not changed. Thus, resting levels of [Ca2+]i and basal [Ca2+]i transients may not reflect the abnormalities observed in diabetes until the system is challenged by certain stimuli. [Ca2+]i responses to isoproterenol are depressed in both resting and stimulated diabetic cells. This suggests an alteration in the beta-adrenergic pathway, possibly related to the beta-adrenoceptor deficiency reported in the diabetic heart. SR Ca-ATPase is also involved in the isoproterenol-induced [Ca2+]i changes. Moreover, the decreased maximum response to 8-bromo-cAMP provides evidence of a post-receptor alteration in the pathway. Diabetic myocytes are more sensitive to ouabain, whereas the maximum response to ouabain was depressed. This may be the result of depressed Na-K ATPase and increased [Na+]i. In diabetic myocytes, rapid cooling contractures and caffeine contractures are depressed, whereas caffeine-induced Ca2+ transients are decreased. Ryanodine binding suggests a decreased number of high-affinity binding sites in the SR of diabetic myocytes. Additionally, there are indications that SR releasable calcium is reduced and that the major functions of SR, notably uptake, release and storage, may be depressed in diabetic myocytes. Finally, L-type Ca(2+)-channels are quantitatively and qualitatively altered in diabetes. Insulin treatment normalizes most of the diabetes-induced changes in cardiomyocytes, suggesting that metabolic alterations due to insulin deficiency play an important role in diabetic cardiomyopathy. Results from several studies show that in diabetes the function of major organelles which handle [Ca2+]i in myocytes is depressed, which in turn causes the alteration of [Ca2+]i mobilization in myocytes. Different second messenger systems involved in E-C coupling may also be altered due to the metabolic impairments. The rapid increase in our understanding of the pathophysiology of calcium homeostasis in cardiomyocytes will be forthcoming as the powerful new tools of molecular and structural biology are used to investigate the regulation of the Ca2+ transport system.

Ca transport during contraction and relaxation in mammalian ventricular muscle.

During relaxation of cardiac muscle four Ca transport systems can compete to remove Ca from the myoplasm. These are 1) the SR Ca-ATPase, 2) the sarcolemmal Na/Ca exchange, 3) the sarcolemmal Ca-ATPase, and 4) the mitochondrial Ca uniporter. Isolated ventricular myocytes loaded with the intracellular fluorescent Ca indicator indo-1 were used to study [Ca]i decline during relaxation. By selective inhibition of the various Ca transporters above the dynamic interaction of these systems during relaxation was evaluated. Quantitatively the SR Ca-ATPase and Na/Ca exchange are clearly the most important (accounting for > 95% of Ca removal). However, the balance of Ca fluxes between these systems vary in a species dependent manner. For example, the SR is much more strongly dominant in rat ventricular myocytes, where ~ 92% of Ca removal is via SR Ca-ATPase and only 7% via Na/Ca exchange during a twitch. In other species (rabbit, ferret, cat, and guinea-pig) the balance is more in the range of 70–75% SR Ca-ATPase and 25–30% Na/Ca exchange. Ferret ventricular myocytes also exhibit a unusually strong sarcolemmal Ca-ATPase. During the normal steady state cardiac contraction-relaxation cycle the same amount of Ca must leave the cell as enters over a cardiac cycle. This implies that 25–30% of the Ca required to activate contraction must enter the cell at each cardiac cycle. Experiments using voltage clamp to measure both Ca current and Na/Ca exchange current demonstrate that this amount of Ca may be supplied by the L-type Ca current.

Ionic dependency of membrane potential and autorhythmicity in the atrium of the whelk Busycon canaliculatum

1. 1. Calcium-free media usually caused a cessation of all electrical and mechanical activity of the Busycon atrium. Where any electrical activity survived, the action potential consisted of a pre-and plateau-like potential devoid of the usual terminal spike. 2. 2. High Ca salines induced tonic force, membrane depolarization and reduction in generation of spontaneous action potentials. The Ca ionophore A23187 enhanced contractions and the SR CaATP-ase inhibitor cyclopiazonic acid induced slight depolarization, tonic contractures and increased action potential firing. 3. 3. The inorganic Ca antagonist Co 2+ was without effect on the preparations, although the lanthanide Gd 3+ inhibited contractions and spontaneous action potentials as well as inducing membrane potential depolarization. 4. 4. The organic Ca entry-blocker nifedipine enhanced both spontaneous action potential amplitude and the phasic contractions they generated. 5. 5. High K salines considerably depolarized atrial preparations with accompanying large tonic contractures and suppression of action potentials. The K channel-blocker 4AP enhanced action potential amplitude with slight increase in contractions, and TEA depolarized the atrium, and enhanced action potentials and rhythmic contractions. 6. 6. Sodium-free salines strongly hyperpolarized atrial preparations and abolished spontaneous action potentials and, on washout, the membrane potential became temporarily unstable. In 2 preparations, low chloride and chloride-free media induced significant membrane potential hyperpolarization. 7. 7. It is concluded that, in the atrium, the resting membrane potential is largely determined by the transmembrane K gradient, but with significant conductances to Na and C1 though probably not Ca. The action potential spike appears to be a Ca-dependent event and the plateau-like phase may be a Na-dependent event.

Na-Ca exchange and Ca fluxes during contraction and relaxation in mammalian ventricular muscle.

There are four cellular Ca transport systems which compete to remove Ca from the myoplasm in mammalian ventricular myocytes. These are 1) the SR Ca-ATPase, 2) the sarcolemmal Na-Ca exchange, 3) the sarcolemmal Ca-ATPase and 4) the mitochondrial Ca uniporter. Using multiple experimental approaches we have evaluated the dynamic interaction of these systems during the normal cardiac contraction-relaxation cycle. The SR Ca-ATPase and Na-Ca exchange are clearly the most important, quantitatively; however, the relative roles vary in a species-dependent manner. In particular, the SR is much more strongly dominant in rat ventricular myocytes, where approximately 92% of Ca removal is via SR Ca-ATPase and only 7% via Na-Ca exchange during a twitch. In other species (rabbit, ferret, cat, and guinea pig) the balance is more in the range of 70% SR CA-ATPase and 25-30% Na-Ca exchange. Ferret ventricular myocytes also exhibit an unusually strong sarcolemmal Ca-ATPase. During the steady state the same amount of Ca must leave the cell as enters over a cardiac cycle. This implies that 25-30% of the Ca required to activate contraction must enter the cell, and experiments demonstrate that this amount of Ca may be supplied by the L-type Ca current.

Accumulation of nitrotyrosine on the SERCA2a isoform of SR Ca-ATPase of rat skeletal muscle during aging: a peroxynitrite-mediated process?

The SR Ca-ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca-ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca-ATPase even in the presence of excess SERCA1a. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of O 2. These data suggest the in vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue imolies an age-associated increase in susceptibility to oxidation by this species.