Sarcoplasmic Reticulum Calcium Leak Research Articles

High-Fat Diet–Induced Mitochondrial Biogenesis Is Regulated by Mitochondrial-Derived Reactive Oxygen Species Activation of CaMKII

Calcium/calmodulin-dependent protein kinase (CaMK) activation induces mitochondrial biogenesis in response to increasing cytosolic calcium concentrations. Calcium leak from the ryanodine receptor (RyR) is regulated by reactive oxygen species (ROS), which is increased with high-fat feeding. We examined whether ROS-induced CaMKII-mediated signaling induced skeletal muscle mitochondrial biogenesis in selected models of lipid oversupply. In obese Zucker rats and high-fat-fed rodents, in which muscle mitochondrial content was upregulated, CaMKII phosphorylation was increased independent of changes in calcium uptake because sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) protein expression or activity was not altered, implicating altered sarcoplasmic reticulum (SR) calcium leak in the activation of CaMKII. In support of this, we found that high-fat feeding increased mitochondrial ROS emission and S-nitrosylation of the RyR, whereas hydrogen peroxide induced SR calcium leak from the RyR and activation of CaMKII. Moreover, administration of a mitochondrial-specific antioxidant, SkQ, prevented high-fat diet-induced phosphorylation of CaMKII and the induction of mitochondrial biogenesis. Altogether, these data suggest that increased mitochondrial ROS emission is required for the induction of SR calcium leak, activation of CaMKII, and induction of mitochondrial biogenesis in response to excess lipid availability.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance*

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (Δ[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.

Sarcoplasmic Reticulum Calcium Leak in Normal and Dystrophic Skeletal Muscle

Under resting conditions, external calcium is known to enter skeletal muscle cells while calcium stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the resting sarcolemmal calcium entry and in the SR calcium leak are still debated but several lines of evidence suggest that an up-regulation of these calcium fluxes occurs in Duchenne Muscular Dystrophy (DMD). We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the murine model of DMD. Using the cytosolic calcium dye Fura2, we first demonstrated that the rate of calcium increase induced by CPA (cyclopiazonic acid) inhibition of SR Ca2+-ATPases at resting potential is significantly higher in mdx fibers suggesting an elevated SR passive calcium leak. However, in these experiments, sarcolemmal calcium influx may contribute to the CPA-induced calcium increase and another series of experiments indicated that CPA-induced SR calcium leak was deeply modified in the absence of external calcium. Fibers were then loaded with the low affinity calcium dye Fluo5N-AM and dialyzed with 50 mM EGTA to measure intraluminal SR calcium changes. Depolarization pulses evoked voltage-dependent Fluo5N fluorescence decreases followed by a recovery phase which was inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR calcium changes. Voltage-dependence and magnitude of depolarization-induced SR calcium depletion were found to be unchanged in mdx fibers but the rate of the recovery phase that followed depletion was found to be faster, suggesting a higher SR calcium reuptake capacity in mdx fibers. Finally, CPA-induced SR calcium leak at −80 mV was found to be significantly higher in mdx fibers. The elevated SR passive calcium leak may participate to the muscle degenerative process in mdx muscle.

Abstract P087: Hydralazine and Nitroglycerin Combination Improves Excitation-Contraction Coupling in Failing Cardiomyocytes

Although hydralazine and organic nitrates have proven clinical benefits for heart failure, the underlying mechanism of action of this combination remains obscure. Spontaneously Hypertensive Heart Failure (SHHF) rats are a representative heart failure model which shares common phenotypic features with human failing hearts. We have recently shown that SHHF cardiomyocytes exhibit depressed contractility and calcium transient amplitude in response to increasing rate and increased sarcoplasmic reticulum (SR) calcium leak, typical characteristics observed in heart failure. The purpose of this study was to test the hypothesis that hydralazine, alone or in combination with nitroglycerin, restores calcium cycling and contractile performance in cardiomyocytes from SHHF rats. We measured sarcomere length (SL) shortening and calcium transient amplitude (Δ[Ca 2+ ] i ) in isolated cardiac myocytes from male SHHF rats (22–24 months old, n=4) and their normotensive controls Wistar-Kyoto rats (18–22 months old, n=4) during pacing (0.5–4 Hz). Both SL shortening and Δ[Ca 2+ ] i responses to increasing pacing in SHHF cardiomyocytes were augmented by 10 μM hydralazine. The same concentration of nitroglycerin alone did not significantly affect these parameters. In response to the combination of these two drugs, there was a strong trend toward improved contractility and normal Δ[Ca 2+ ] i . We also assessed SR Ca 2+ leak by a brief incubation with tetracaine following by a caffeine challenge, a regimen designed to test SR Ca 2+ load. At matched SR Ca 2+ content, Ca 2+ leak was not significantly changed by nitroglycerin or hydralazine alone. However, in combination they restored the leak in SHHF myocytes to the normal levels seen in WKY myocytes. Hydralazine improves cardiac excitation-contraction coupling that is impaired in this model, whereas nitroglycerin does not exert a significant effect on this response. However, when administered together, nitroglycerin appears to modulate the enhancing effect induced by hydralazine and helps to restore normal SR calcium leakage. These data offer new insights into the mechanisms underlying the actions of hydralazine and organic nitrates on heart failure.

SERCA2a Gene Transfer Decreases Sarcoplasmic Reticulum Calcium Leak and Reduces Ventricular Arrhythmias in a Model of Chronic Heart Failure

Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca(2+)) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca(2+) homeostasis and spontaneous SR Ca(2+) leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca(2+) leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca(2+) load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca(2+) leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. SERCA2a gene therapy stabilizes SR Ca(2+) load, reduces ryanodine receptor phosphorylation and decreases SR Ca(2+) leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

How Does RyR2-Mediated SR Calcium Leak Fail to Cause Sparks?

Recent experiments have shown that Ca leak from the sarcoplasmic reticulum (SR) can occur via spontaneous Ca sparks, non spark ryanodine receptor (RyR) mediated leak, and RyR independent pathways (Zima et al. BJ, 94:104a, 2008). Opening of a single RyR is expected to increase local [Ca] in the cleft space rapidly (<< 1 ms) to levels that are expected to activate neighboring RyR in that cleft via Ca induced Ca release. Our question is how and why RyR-mediated SR Ca leak becomes mainly non-spark mediated at moderate to low SR Ca loads. To answer this question, we analyze Ca flux through RyRs using a physiologically detailed mathematical model of junctional SR Ca release in which RyR gating is regulated by intra SR and cleft [Ca] ([Ca]SR & [Ca]Cleft). In this model, there are one hundred RyR channels in one Ca release unit. Each RyR opens stochastically depending on [Ca]Cleft and [Ca]SR . We find that several factors contribute to the failure of one RyR channel opening to opens adjacent RyRs as [Ca]SR declines: 1) the lower [Ca]SR reduces driving force and thus limits local [Ca]Cleft (both absolute level and rate of rise), 2) low [Ca]SR can inhibit RyR open time (τO) which further reduces local [Ca]Cleft attained, 3) the low τO and fast [Ca]Cleft dissipation after closure shorten the opportunity for neighboring RyR activation, 4) at low [Ca]SR the RyR2 exhibits reduced [Ca]Cleft sensitivity. We conclude that all of these factors conspire to reduce the probability of Ca sparks as [Ca]SR declines, despite continued RyR-mediated SR Ca leak.

Impaired S-Nitrosylation of the Ryanodine Receptor Caused by Xanthine Oxidase Activity Contributes to Calcium Leak in Heart Failure

S-Nitrosylation is a ubiquitous post-translational modification that regulates diverse biologic processes. In skeletal muscle, hypernitrosylation of the ryanodine receptor (RyR) causes sarcoplasmic reticulum (SR) calcium leak, but whether abnormalities of cardiac RyR nitrosylation contribute to dysfunction of cardiac excitation-contraction coupling remains controversial. In this study, we tested the hypothesis that cardiac RyR2 is hyponitrosylated in heart failure, because of nitroso-redox imbalance. We evaluated excitation-contraction coupling and nitroso-redox balance in spontaneously hypertensive heart failure rats with dilated cardiomyopathy and age-matched Wistar-Kyoto rats. Spontaneously hypertensive heart failure myocytes were characterized by depressed contractility, increased diastolic Ca(2+) leak, hyponitrosylation of RyR2, and enhanced xanthine oxidase derived superoxide. Global S-nitrosylation was decreased in failing hearts compared with nonfailing. Xanthine oxidase inhibition restored global and RyR2 nitrosylation and reversed the diastolic SR Ca(2+) leak, improving Ca(2+) handling and contractility. Together these findings demonstrate that nitroso-redox imbalance causes RyR2 oxidation, hyponitrosylation, and SR Ca(2+) leak, a hallmark of cardiac dysfunction. The reversal of this phenotype by inhibition of xanthine oxidase has important pathophysiologic and therapeutic implications.

Limitations of FKBP12.6-directed treatment strategies for maladaptive cardiac remodeling and heart failure

Sarcoplasmic reticulum (SR) calcium (Ca) leak can be reduced by enhancing FKBP12.6 binding to SR Ca release channels (RyR2) and expression of a “sticky” FKBP12.6D37S mutant may correct reduced binding stoichiometry in RyR2 from failing hearts. Both calcium/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and protein kinase A (PKA) are activated in heart failure and promote SR Ca leak at RyR2. It is possible that FKBP12.6 dissociation from RyR2 may promote remodeling and that interventions to reassociate FKBP12.6 with RyR2 reflect a future therapeutic strategy. We created transgenic (TG) mice expressing FKBP12.6D37S and tested their capacity to improve intracellular Ca handling and pathological remodeling in vivo. FKBP12.6D37S TG mice were cross-bred with CaMKIIδc TG mice, which are known to exhibit pronounced RyR2 dysfunction and heart failure. We observed a significant improvement of post-rest Ca transients and a higher SR Ca content in FKBP12.6D37S TG mice. In double-TG mice, a marked reduction of SR Ca spark frequency indicated reduced SR Ca leak but neither SR Ca transient amplitude, SR Ca content nor morphological or functional parameters improved in vivo. Likewise, FKBP12.6D37S TG mice subjected to increased afterload after aortic banding exhibited higher SR Ca load but did not exhibit any improvement in hypertrophic growth or functional decline. Enhancement of FKBP12.6-RyR2 binding markedly reduced RyR2 Ca leak in CaMKIIδc-induced heart failure and in pressure overload. Our data suggest that activation of CaMKIIδc and pressure overload confer significant resistance towards approaches aiming at FKBP12.6-RyR2 reconstitution in heart failure and maladaptive remodeling, although RyR2 Ca leak can be reduced.

Nitric Oxide and the Ryanodine Receptor Ca-Release Channel

To the Editor: I have read with interest the recent review on the role of S -nitrosylation in cardiovascular signaling, in which Lima et al1 provide a very useful digest on the current status of research in this important but burgeoning field of cardiovascular physiology. On the other hand, I believe that one of the many functions of a scientific review, especially when summarizing the status of a field with a certain degree of ambiguity, is to promote further progress. This is, perhaps, best achieved if all the available data relevant to the field are summarized and discussed, even those that seem to be contradictory to the intended conclusion of the review. Although the review in question cites almost 200 works, it …

Does Protein Kinase A–Mediated Phosphorylation of the Cardiac Ryanodine Receptor Play Any Role in Adrenergic Regulation of Calcium Handling in Health and Disease?

See related articles, pages 1743–1752 Cardiac myocyte ryanodine receptors (sarcoplasmic reticulum [SR] Ca release channel; cardiac ryanodine receptor [RyR2]) are localized in the junctional SR, in close proximity to L-type Ca channels (LTCCs) embedded in the membranes of the transverse (T)-tubules. This signaling microdomain has been termed the couplon,1 because it is here that excitation–contraction coupling takes place. As the heart fills with blood during diastole, RyR2 is stabilized in a closed state, allowing Ca uptake by the SR Ca ATPase to pump Ca from the cytoplasm into the SR, providing the primary source of Ca to activate the contractile apparatus during the next heart beat (systole). During systole, the cardiac action potential depolarizes the T-tubules and causes the opening of LTCCs. Ca influx through LTCCs elevates the [Ca] within the cytoplasmic space between the T-tubular and SR membrane, which promotes Ca binding to neighboring RyR2s, inducing some to open. Ca then moves out of the SR lumen into the subsarcolemmal, space and the additional elevation of [Ca] induces a regenerative opening of other RyRs in the couplon. The resulting localized Ca signal is called a Ca spark.2 The action potential synchronizes these local Ca release events throughout the cell (termed Ca-induced Ca release) to produce the global cardiac [Ca] transient.3 The stabilized closed state and Ca-dependent opening of RyR2 during diastole and systole are essential for normal cardiac diastolic and systolic function. Destabilizing the behavior of RyR2 would be expected to have a negative …

Stress synchronizes calcium release and promotes SR calcium leak

Stress synchronizes calcium release and promotes SR calcium leak

Aerobic Interval Training Prevents Cardiac Dysfunction and Mortality by Improving Calcium Handling in MI Diabetic Mice

Diabetic patients have greater risk of developing congestive heart failure (HF) after myocardium infarction (MI). Exercise training is an effective strategy for preventing the development of cardiomyopathies and the incidence of cardiovascular morbidity and mortality during diabetes.Aim - To study the effects of aerobic interval training (AIT) on cardiac function and the role of calcium handling in a combined experimental model of MI-induced HF and diabetic cardiomyopathy.Methods and Results - A cohort of male diabetic db/db and age-matched nondiabetic control mice was randomly assigned into untrained and trained sham and MI groups. MI was induced by coronary ligation. Exercise tolerance was evaluated by VO2 max. Standard echocardiography and tissue Doppler imaging were performed by high-resolution in-vivo imaging system, and diastolic sarcoplasmic reticulum (SR) calcium leak was measured in isolated cardiomyocytes using fluorescence microscope. MI diabetic mice displayed higher mortality rate compared to MI nondiabetic and sham mice (55% vs. 25% and 0%, respectively). In addition, exercise intolerance, reduced fractional shortening (FS), and cardiomyocyte dysfunction were observed in MI diabetic mice compared to other groups. AIT increased survival rate and exercise tolerance in MI diabetic to diabetic sham levels, paralleled by increased FS. AIT reestablished contractile function of MI diabetic to diabetic sham levels associated with improved SR calcium release synchronicity, T-tubule density and SR calcium leak.Conclusion - These results provide evidence for improvement of calcium handling by AIT in MI-induced HF during diabetes. Therefore, AIT is a potential therapeutic tool for the management of HF associated with diabetes.

Regulation of Sarcoplasmic Reticulum Calcium Leak by Cytosolic Calcium in Rabbit Ventricular Myocytes

Diastolic sarcoplasmic reticulum (SR) Ca leak determines Ca content and, therefore, the amplitude of action potential-induced global Ca transients in ventricular myocytes. However, the pathways and properties of SR Ca leak have been poorly described. Here, we studied the effects of cytosolic [Ca] ([Ca]i) on SR Ca leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we simultaneously measured intra-SR free Ca ([Ca]SR) with fluo-5N and cytosolic Ca sparks with rhod-2, and monitored SR Ca leak as the change in [Ca]SR over time after complete SERCA inhibition with thapsigargin (10 μM). Increasing [Ca]i from 150 to 250 nM significantly increased SR Ca leak (by ∼30%) over the entire range of [Ca]SR. This increase in SR Ca leak associated with an increase in Ca spark frequency. Further increasing [Ca]i to 350 nM led to rapid [Ca]SR depletion due to the occurrence of spontaneous Ca waves. In contrast, lowering [Ca]i to 50 nM markedly decreased SR Ca leak rate (by ∼60%) and nearly abolished Ca spark activity. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 μM), changes in [Ca]i between 50 and 350 nM did not produce any significant effect on SR Ca leak, showing that changes of [Ca]i over a physiological range alter SR Ca leak solely by regulating RyR activity. However, decreasing [Ca]i to a lower, non-physiologically level (5 nM) activated additional SR Ca leak pathway(s) that were insensitive to RyR or SR Ca ATPase inhibition. In summary, [Ca]i plays an important role in regulating SR Ca leak by activating RyR and preventing Ca leak through unspecified pathways.

Transient Receptor Potential Canonical Type 1 (TRPC1) Operates as a Sarcoplasmic Reticulum Calcium Leak Channel in Skeletal Muscle

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca(2+) stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca(2+) influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca(2+) pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca(2+) pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca(2+) which was not observed in control cells. Ca(2+) measurements indicated that resting Ca(2+) and the rate of Ca(2+) increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca(2+) transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca(2+) leak channel in skeletal muscle.

Increased Sensitivity to Ischemia in an Early Diabetic Cardiomyopathy: The Role of Calcium Handling

Diabetic cardiomyopathy is characterized by early-onset diastolic dysfunction and late-onset systolic dysfunction. However, little is known about the mechanisms underlying the response of the diabetic myocardium to ischemia.Aim - To study the left ventricular (LV) dysfunction and the role of calcium handling in infarcted diabetic mice in an early stage of diabetic cardiomyopathy.Methods and Results - A cohort of male diabetic db/db and age-matched nondiabetic control mice at 10 wk of age was randomly assigned into Sham and myocardial infarction (MI) groups. MI was induced by coronary ligation. Standard echocardiography and tissue Doppler imaging were performed by high-resolution in-vivo imaging system, and diastolic sarcoplasmic reticulum (SR) calcium leak was measured in isolated cardiomyocytes using fluorescence microscope. One month after MI, 75% of the nondiabetic mice survived vs. 55% of the MI diabetic mice (p=0.04). A significant LV dilatation was observed in MI diabetic mice compared to nondiabetic (p=0.03). Peak systolic tissue velocity (Sm) was 28% lower in MI diabetic mice than in nondiabetic group (nondiabetic: 19±1 vs. 17±2mm/s; diabetic: 18±2 vs. 13±2∗mm/s, ∗p=0.05, for Sham and MI, respectively). Peak early diastolic tissue velocity (Em) was decreased in both Sham and MI diabetic mice (17±2∗ and 16±4∗ vs. 25±3 and 25±4 mm/s, ∗p<0.05, respectively). Diastolic SR calcium leak was unchanged in 10-wk diabetic mice compared with nondiabetic mice. However, a significant increase diastolic SR calcium leak was observed in MI diabetic mice relative to MI nondiabetic mice.Conclusion - The altered calcium homeostasis is an important determinant of sensitivity to ischemia and of loss of the ventricular function in the early stage of diabetic heart disease in diabetic mice.

Prevention of endotoxin-induced sarcoplasmic reticulum calcium leak improves mitochondrial and myocardial dysfunction*

Growing evidence suggests that mitochondria function is impaired in sepsis. Here, we tested the hypothesis that lipopolysaccharide would induce mitochondrial Ca2+ overload and oxygen utilization abnormalities as consequences of sarcoplasmic reticulum Ca2+ handling derangements that are typically observed in sepsis. As lipopolysaccharide-induced sarcoplasmic reticulum dysfunction was mainly characterized by reduced sarcoplasmic reticulum Ca2+ uptake and Ca2+ leak, we tested whether dantrolene, a sarco(endo)plasmic reticulum calcium ATPase leak inhibitor, would prevent mitochondrial and cardiac contractile dysfunction. Randomized controlled trial. Experimental laboratory. Male Sprague Dawley rats. Sepsis was induced by injection of endotoxin lipopolysaccharide (10 mg/kg/intravenously). Assessment of contractile function and Ca2+ handling was performed 4 hr after lipopolysaccharide. The relative contribution of the different Ca2+ transporters to relaxation in intact cardiomyocytes was studied during successive electrically evoked twitches and caffeine stimulation. Sarcoplasmic reticulum vesicles and mitochondria from ventricles of rats treated or not with lipopolysaccharide were prepared to evaluate Ca2+ uptake-release and oxygen fluxes, respectively. Effects of dantrolene (10 mg/kg) treatment in rats were evaluated in sarcoplasmic reticulum vesicles, mitochondria, and isolated hearts. Lipopolysaccharide challenge elicited cardiac contractile dysfunction that was accompanied by severe derangements in sarcoplasmic reticulum function, i.e., reduced Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Functional sarcoplasmic reticulum changes were associated with modification in the status of phospholamban phosphorylation whereas SERCA was unchanged. Rises in mitochondrial Ca2+ content observed in lipopolysaccharide-treated rats coincided with derangements in mitochondrial oxygen efficacy, i.e., reduced respiratory control ratio. Administration of dantrolene in lipopolysaccharide-treated rats prevented mitochondrial Ca2+ overload and mitochondrial oxygen utilization abnormalities. Moreover, dantrolene treatment in lipopolysaccharide rats improved heart mitochondrial redox state and myocardial dysfunction. These experiments suggest that sarcoplasmic reticulum Ca2+ handling dysfunction is an early event during endotoxemia that could be responsible for, or contribute to, mitochondrial Ca2+ overload, metabolic failure, and cardiac dysfunction.

Ca 2+ -Calmodulin–Dependent Protein Kinase Phosphorylation of Ryanodine Receptor May Contribute to the β-Adrenergic Regulation of Myocardial Contractility Independently of Increases in Heart Rate

To the Editor: In a recent article, MacDonnell et al1 reported that protein kinase A (PKA) phosphorylation of ryanodine receptor (RyR2) (the Ca2+ release channel of the sarcoplasmic reticulum [SR]) at Ser2808/09 site does not have a major role in the sympathetic nervous system (SNS) regulation of cardiac function. This conclusion was based on comparing the effect of isoproterenol on ventricular performance, in vivo, in isolated hearts and myocytes, in wild-type mice and in a genetically modified mouse in which Ser2808 of RyR2 was replaced by alanine (S2808A) to prevent PKA-mediated phosphorylation at this site. Isoproterenol produced an increase in cardiac function both in vivo and in isolated hearts, as well as an enhancement in the L-type Ca2+ current ( I CaL), the amplitude of the Ca2+ transient and the excitation–contraction coupling (ECC) gain in isolated myocytes, which were not significantly different between wild-type and …

Abstract 1084: Myosin Light Chain Kinase Signaling Regulates Ectopic Atrial Activity

Objective : Determine whether calmodulin (CaM) acts through novel signaling pathways to elicit left atrial ectopic activity. Methods : Rat left atria (n=6 –9 per group) were isolated and superfused at 30°C. Mechanical function was recorded using a Grass recorder. Left atrial proteins were analyzed using Western analysis. Findings : Our rat left atria are quiescent in the absence of external pacing. However, in the presence of 2-aminoethoxydiphenyl borate (20μM; 2APB), a novel sarcoplasmic reticulum (SR) calcium leak inducer, left atria contract spontaneously at 67±12 contractions (min) − 1 in the absence of pacing. Pre-treating left atria with two irreversible CaM inhibitors, phenoxybenzamine and fluphenazine chloroethane (90μM each), decreases spontaneous contraction frequency to 5±3 and 2±1 (min) − 1 , respectively, indicating CaM signaling is activated and required for this ectopic activity. Pre-treating left atria with 20μM KN62 did not decrease the frequency of 2APB-induced ectopic activity (72±8 contractions (min) − 1 ) indicating calmodulin-dependent protein kinase II (CAMKII) is not a downstream target here. In contrast, 20μM ML7, a specific inhibitor of myosin light chain kinase (MLCK), a second cardiac CaM-regulated kinase, decreases 2APB-induced spontaneous contractions to 7±3 min −1 . Western analysis shows left atria contain smooth muscle MLCK. This kinase preferentially phosphorylates non-muscle myosin light chain 2 (nmMLC2). Western analyses also show left atria contain nmMLC2 which is not phosphorylated in untreated muscles. However, nmMLC2 phosphorylation increases significantly in left atria exposed to 2APB. Conclusions : CaM and MLCK inhibitors prevent the ectopic atrial activity induced by 2APB. This suggests a unique SR calcium leak occurs under this condition which activates CaM signaling whose specific downstream target is MLCK, a major myocardial CaM-dependent kinase. MLCK signaling via its sole substrate, nmMLC2, is required for this novel ectopic atrial activity. Hyperphosphorylation of SR ryanodine receptors by CAMKII is known to induce an SR calcium leak that underlies triggered ectopic activity. Our data suggest multiple calcium leaks and CaM signaling pathways regulate the stability of intact atrial muscle.

A pharmacological model for calcium overload-induced tachycardia in isolated rat left atria

Few experimental models produce spontaneous tachycardia in normal left atria to allow the study of the cellular mechanisms underlying this contributor to atrial fibrillation. We reported 2-aminoethoxydiphenyl borate (2-APB) that provokes sporadic spontaneous mechanical activity and calcium leak in isolated rat left atria. Since sarcoplasmic reticulum calcium leak in the presence of high calcium load may trigger tachyarrhythmias, we tested how conditions that increase calcium load affect 2-APB-induced ectopic activity. Exposing superfused rat left atria to (i) 30 nM isoproterenol, (ii) 3 μM forskolin, (iii) 300 nM (−)BayK 8644 ((4 S)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluormethyl)phenyl]-3-pyridinecarboxylic acid methyl ester), (iv) 300 nM FPL-64176 (2,5-Dimethyl-4-[2-(phenylmethyl)benzoyl]-1 H-pyrrole-3-carboxylic acid methyl ester) or (v) 120 μM ouabain increases their force of contraction, evidence of calcium loading, but does not produce ectopic activity. Spontaneous mechanical activity occurs in left atria superfused with 20 μM 2-APB at 47 ± 6 contractions/min in the absence of pacing. Any of these five agents increase rates of 2-APB-induced spontaneous mechanical activity to > 200 contractions/min in the absence of pacing. Washing tachycardic left atria with superfusate lacking 2-APB restores normal function, demonstrating the reversibility of these effects. Decreasing superfusate sodium reverses this tachycardia and two hyperpolarization-activated current (I f) inhibitors blunt this ectopic activity. Thus conditions that increase atrial calcium load increase the frequency of spontaneous mechanical activity. Decreasing extracellular sodium and I f inhibitors suppress this spontaneous tachycardia suggesting forward-mode sodium–calcium exchange and I f-like activities underlie this activity. This model may help define cell pathways that trigger atrial tachyarrhythmias.