Sarcoplasmic Reticulum Ca Content Research Articles

The effects of doxorubicin on calcium handling and contractility in sheep ventricular myocytes; role of oxidative stress

Anthracyclines such as doxorubicin (DOX) are effective chemotherapeutics and contribute to improved cancer survival rates in children and adults. However, anthracyclines exhibit acute and chronic cardiotoxicity which can produce heart failure in cancer survivors. While the cellular basis remains unclear, previous studies show anthracyclines perturb aspects of excitation-contraction coupling (ECC), though to our knowledge this has yet to be demonstrated in a large animal model. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced reactive oxygen species (ROS) production / oxidative stress remains ambiguous. This is important given the key role of oxidative stress in other cardiotoxic processes. Therefore, we aimed to (1) characterise the effects of DOX on Ca handling and contractility in sheep ventricular myocytes and (2) quantify the dependence of any effect on oxidative stress.Sheep myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986. Cells were loaded with fura-2 (0.1 μM) then field stimulated at 0.5 Hz. Fura-2 was excited sequentially at 340 nm and 380 nm (200 Hz) and changes of cytoplasmic Ca inferred from the ratio of emitted light. Sarcomere length was measured simultaneously using a Myocam-S CCD camera and SarcLen acquisition module (Ion Optix, USA). Changes to sarcoplasmic reticulum (SR) Ca content were estimated by rapid application of caffeine (10 mM). Two-way ANOVA was used for statistical analysis.1 nM DOX reduced the amplitude of systolic Ca and shortening by 39 ± 3 % (n=49, p<0.001) and 42 ± 4 % (n=45, p<0.001) respectively. The amplitude of the caffeine-evoked Ca transient decreased by 33 ± 5 % (n=24, p<0.001). The rate of decay of the systolic Ca transient ( k sys ) represents the combined activity of SERCA and NCX and was reduced by 17 ± 3 % (n=46, p<0.001). The rate of decay of the caffeine-evoked Ca transient ( k caff ) represents NCX alone and was increased by 64 ± 26 % (n=23, p=0.002).Subtraction of k caff from k sys indicates SERCA activity ( k SERCA ), which was decreased by 34 ± 8 % (n=20, p<0.001). When the experiment was repeated in the presence of the antioxidant N-acetylcysteine (NAC), the effect of DOX on systolic Ca, systolic shortening and the caffeine-evoked Ca transient was attenuated by 52 % (n=29, p<0.001), 58 % (n=29, p<0.001) and 45 % (n=15, p=0.003) respectively. Furthermore, NAC prevented the effect of DOX on k sys , k caff and k SERCA .These data suggest DOX increases NCX activity and reduces SERCA activity which decreases SR Ca content thence systolic Ca and contractility. The effect of DOX on NCX and SERCA appears to be entirely ROS / oxidative stress dependent. However, other non-oxidative mechanisms likely contribute to net DOX-mediated negative cellular inotropy. Our next experiments will investigate this and the sources of anthracycline-induced oxidative stress. This work was funded by Kidscan childrens cancer research, UK. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulating mitochondrial phosphate transport shapes intracellular Ca signaling to impact arrhythmogenesis.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondria Ca uptake and buffering. Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knock out (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. An inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Our results suggest that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce Ca waves and cardiac arrhythmias in CPVT.

Abstract 13377: Modulating Mitochondrial Phosphate Transport Shapes Intracellular Ca Signaling to Impact Arrhythmogenesis

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. Hypothesis: We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondrial Ca uptake and buffering. Methods: Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knockout (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. Results: In the presence of β agonist isoproterenol, an inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Conclusions: Modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce arrhythmogenic Ca waves and cardiac arrhythmias in CPVT.

Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known. A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators. K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness. CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Loss of CASK Accelerates Heart Failure Development.

[Figure: see text].

Resveratrol Stimulates the Na+-Ca2+ Exchanger on the Plasma Membrane to Reduce Cytosolic Ca2+ in Rat Aortic Smooth Muscle Cells.

Resveratrol is well known to exhibit vascular relaxant and antihypertensive effects. In this study, we determined the effects of resveratrol on the modulation of cytosolic [Ca] level and adenosine 5'-triphosphate-induced Ca release from the sarcoplasmic reticulum (SR) in rat aortic smooth muscle cells (ASMCs) and explored its underlying mechanisms. In this article, cytosolic [Ca] and SR [Ca] in ASMCs were determined by Fluo-4/acetoxymethyl and Mag-Fluo-4/acetoxymethyl respectively. Resveratrol (20, 50, and 100 µM) caused a rapid and substantial reduction in cytosolic [Ca] in ASMCs bathed in normal Hank's Balanced Salt Solution or Ca-free Hank's Balanced Salt Solution. Pretreatment with resveratrol reduced adenosine 5'-triphosphate-induced SR Ca release and SR Ca content. In the cells bathed in Na-free physiological saline, which favors the reverse mode of the Na-Ca exchanger (NCX), resveratrol induced an increase in cytosolic [Ca] and SR [Ca]. However, its effect on cytosolic [Ca] was inhibited by the selective NCX inhibitor, SEA0400. Our findings suggest that resveratrol reduces cytosolic [Ca] and SR [Ca] in ASMCs in normal physiological saline, which might be, at least in part, mediated by the NCX.

The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.

AimsExcessive activation of Ca/calmodulin‐dependent kinase II (CaMKII) is of critical importance in heart failure (HF) and atrial fibrillation. Unfortunately, lack of selectivity, specificity, and bioavailability have slowed down development of inhibitors for clinical use. We investigated a novel CaMKIIδ/CaMKIIɣ‐selective, ATP‐competitive, orally available CaMKII inhibitor (RA608) on right atrial biopsies of 119 patients undergoing heart surgery. Furthermore, we evaluated its oral efficacy to prevent deterioration of HF in mice after transverse aortic constriction (TAC).Methods and resultsIn human atrial cardiomyocytes and trabeculae, respectively, RA608 significantly reduced sarcoplasmic reticulum Ca leak, reduced diastolic tension, and increased sarcoplasmic reticulum Ca content. Patch‐clamp recordings confirmed the safety of RA608 in human cardiomyocytes. C57BL6/J mice were subjected to TAC, and left ventricular function was monitored by echocardiography. Two weeks after TAC, RA608 was administered by oral gavage for 7 days. Oral RA608 treatment prevented deterioration of ejection fraction. At 3 weeks after TAC, ejection fraction was 46.1 ± 3.7% (RA608) vs. 34.9 ± 2.6% (vehicle), n = 9 vs. n = 12, P < 0.05, ANOVA, which correlated with significantly less CaMKII autophosphorylation at threonine 287. Moreover, a single oral dose significantly reduced inducibility of atrial and ventricular arrhythmias in CaMKIIδ transgenic mice 4 h after administration. Atrial fibrillation was induced in 6/6 mice for vehicle vs. 1/7 for RA608, P < 0.05, 'n − 1' χ 2 test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n − 1' χ 2 test.ConclusionsRA608 is the first orally administrable CaMKII inhibitor with potent efficacy in human myocytes. Moreover, oral administration potently inhibits arrhythmogenesis and attenuates HF development in mice in vivo.

Increased SR Calcium Leak is Promoted by O-GlcNAcylation of CaMKII in Diabetes and Hyperglycemia

Abnormal calcium (Ca) leak via cardiac ryanodine receptor 2 (RyR2) is enhanced by high extracellular [glucose] via O-GlcNAcylation of CaMKII and may be important in diabetic cardiomyopathy (PMID: 24077098). Indeed, CaMKII O-GlcNAcylation and activity are elevated in humans and animal diabetic models. Here, we tested whether CaMKIIδ and the site identified as an O-GlcNAcylation target (Ser280) are required for promoting SR Ca leak. We measured Ca sparks, Ca transients, and sarcoplasmic reticulum (SR) Ca content in isolated mouse ventricular cardiomyocytes.

Exploring Impaired SERCA Pump-Caused Alternation Occurrence in Ischemia.

Impaired sarcoplasmic reticulum (SR) calcium transport ATPase (SERCA) gives rise to Ca2+ alternans and changes of the Ca2+release amount. These changes in Ca2+ release amount can reveal the mechanism underlying how the interaction between Ca2+ release and Ca2+ uptake induces Ca2+ alternans. This study of alternans by calculating the values of Ca2+ release properties with impaired SERCA has not been explored before. Here, we induced Ca2+ alternans by using an impaired SERCA pump under ischemic conditions. The results showed that the recruitment and refractoriness of the Ca2+ release increased as Ca2+ alternans occurred. This indicates triggering Ca waves. As the propagation of Ca waves is linked to the occurrence of Ca2+ alternans, the “threshold” for Ca waves reflects the key factor in Ca2+ alternans development, and it is still controversial nowadays. We proposed the ratio between the diastolic network SR (NSR) Ca content (Cansr) and the cytoplasmic Ca content (Cai) (Cansr/Cai) as the “threshold” of Ca waves and Ca2+ alternans. Diastolic Cansr, Cai, and their ratio were recorded at the onset of Ca2+ alternans. Compared with certain Cansr and Cai, the “threshold” of the ratio can better explain the comprehensive effects of the Ca2+ release and the Ca2+ uptake on Ca2+ alternans onset. In addition, these ratios are related with the function of SERCA pumps, which vary with different ischemic conditions. Thus, values of these ratios could be used to differentiate Ca2+ alternans from different ischemic cases. This agrees with some experimental results. Therefore, the certain value of diastolic Cansr/Cai can be the better “threshold” for Ca waves and Ca2+ alternans.

Cellular mechanisms of metabolic syndrome-related atrial decompensation in a rat model of HFpEF

Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about 50% of HF patients. Atrial remodeling is common in HFpEF and associated with increased mortality. We postulate that atrial remodeling is associated with atrial dysfunction in vivo related to alterations in cardiomyocyte Calcium (Ca) signaling and remodeling. We examined atrial function in vivo and Ca transients (CaT) (Fluo4-AM, field stim) in atrial cardiomyocytes of ZSF-1 rats without (Ln; lean hypertensive) and with metabolic syndrome (Ob; obese, hypertensive, diabetic) and HFpEF. ResultsAt 21weeks Ln showed an increased left ventricular (LV) mass and left ventricular end-diastolic pressure (LVEDP), but unchanged left atrial (LA) size and preserved atrial ejection fraction vs. wild-type (WT). CaT amplitude in atrial cardiomyocytes was increased in Ln (2.9±0.2 vs. 2.3±0.2F/F0 in WT; n=22 cells/group; p<0.05). Studying subcellular Ca release in more detail, we found that local central cytosolic CaT amplitude was increased, while subsarcolemmal CaT amplitudes remained unchanged. Moreover, Sarcoplasmic reticulum (SR) Ca content (caffeine) was preserved while Ca spark frequency and tetracaine-dependent SR Ca leak were significantly increased in Ln. Ob mice developed a HFpEF phenotype in vivo, LA area was significantly increased and atrial in vivo function was impaired, despite increased atrial CaT amplitudes in vitro (2.8±0.2; p<0.05 vs. WT). Ob cells showed alterations of the tubular network possibly contributing to the observed phenotype. CaT kinetics as well as SR Ca in Ob were not significantly different from WT, but SR Ca leak remained increased. Angiotensin II (Ang II) reduced in vitro cytosolic CaT amplitudes and let to active nuclear Ca release in Ob but not in Ln or WT. SummaryIn hypertensive ZSF-1 rats, a possibly compensatory increase of cytosolic CaT amplitude and increased SR Ca leak precede atrial remodeling and HFpEF. Atrial remodeling in ZSF-1 HFpEF is associated with an altered tubular network in-vitro and atrial contractile dysfunction in vivo, indicating insufficient compensation. Atrial cardiomyocyte dysfunction in vitro is induced by the addition of angiotensin II.

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes.

Key points Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart.Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur.We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly.We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria.The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age‐associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via I Ca‐L but decreased peak I Ca‐L. Decreased peak I Ca‐L was found to be responsible for the age‐associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak I Ca‐L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

Arrhythmogenic Delayed Afterdepolarizations Are Promoted by Severe Hypothermia But Not Therapeutic Hypothermia.

Severe hypothermia (SH) is known to be arrhythmogenic, but the effect of therapeutic hypothermia (TH) on arrhythmias is unclear. It is hypothesized that susceptibility to Ca-mediated arrhythmia triggers would be increased only by SH.Methods and Results:Spontaneous Ca release (SCR) and resultant delayed afterdepolarizations (DADs) were evaluated by optical mapping in canine wedge preparations during normothermia (N, 36℃), TH (32℃) or SH (28℃; n=8 each). The slope (amplitude/rise time) of multicellular SCR (mSCR) events, a determinant of triggered activity, was suppressed in TH (24.4±3.4%/s vs. N: 41.5±6.0%/s), but significantly higher in SH (96.3±8.1%/s) producing higher amplitude DADs in SH (35.7±1.6%) and smaller in TH (5.3±1.0% vs. N: 10.0±1.1%, all P<0.05). Triggered activity was only observed in SH. In isolated myocytes, sarcoplasmic reticulum (SR) Ca release kinetics slowed in a temperature-dependent manner, prolonging Ca transient rise time [33±3 (N) vs. 50±6 (TH) vs. 88±12 ms (SH), P<0.05], which can explain the decreased mSCR slope and DAD amplitude in TH. Although the SR Ca content was similar in TH and SH, Ca spark frequency was markedly increased only in SH, suggesting that increased ryanodine receptor open probability could explain the increased triggered activity during SH. Temperature dependence of Ca release can explain susceptibility to Ca-mediated arrhythmia triggers in SH. This may therefore explain the increased risk of lethal arrhythmia in SH, but not during TH.

Multiscale Determinants of Delayed Afterdepolarization Amplitude in Cardiac Tissue

Spontaneous calcium (Ca) waves in cardiac myocytes underlie delayed afterdepolarizations (DADs) that trigger cardiac arrhythmias. How these subcellular/cellular events overcome source-sink factors in cardiac tissue to generate DADs of sufficient amplitude to trigger action potentials is not fully understood. Here, we evaluate quantitatively how factors at the subcellular scale (number of Ca wave initiation sites), cellular scale (sarcoplasmic reticulum (SR) Ca load), and tissue scale (synchrony of Ca release in populations of myocytes) determine DAD features in cardiac tissue using a combined experimental and computational modeling approach. Isolated patch-clamped rabbit ventricular myocytes loaded with Fluo-4 to image intracellular Ca were rapidly paced during exposure to elevated extracellular Ca (2.7 mmol/L) and isoproterenol (0.25 μmol/L) to induce diastolic Ca waves and subthreshold DADs. As the number of paced beats increased from 1 to 5, SR Ca content (assessed with caffeine pulses) increased, the number of Ca wave initiation sites increased, integrated Ca transients and DADs became larger and shorter in duration, and the latency period to the onset of Ca waves shortened with reduced variance. In silico analysis using a computer model of ventricular tissue incorporating these experimental measurements revealed that whereas all of these factors promoted larger DADs with higher probability of generating triggered activity, the latency period variance and SR Ca load had the greatest influences. Therefore, incorporating quantitative experimental data into tissue level simulations reveals that increased intracellular Ca promotes DAD-mediated triggered activity in tissue predominantly by increasing both the synchrony (decreasing latency variance) of Ca waves in nearby myocytes and SR Ca load, whereas the number of Ca wave initiation sites per myocyte is less important.

Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis.

Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca loss that mediate altered Ca handling and contractile dysfunction associated with sepsis. Randomized controlled trial. Research laboratorySUBJECTS:: Male wild type and transgenic miceINTERVENTIONS:: We induced sepsis in mice using the colon ascendens stent peritonitis model. Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca transient amplitude and sarcoplasmic reticulum Ca content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 µM of KN93 prevented the decrease in cell shortening, Ca transient amplitude, and sarcoplasmic reticulum Ca content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23). Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca content, Ca transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging.

Maintenance of steady-state calcium (Ca(2+)) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca(2+) content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca(2+) transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca(2+)-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca(2+) indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca(2+) store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca(2+). This tool is useful for investigation into the nuanced changes in SR Ca(2+) that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca(2+) content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca(2+) indicator, D1SR, is used to detect Ca(2+) fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca(2+) levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca(2+) movement.

Latrunculin B modulates electrophysiological characteristics and arrhythmogenesis in pulmonary vein cardiomyocytes.

AF (atrial fibrillation) is the most common sustained arrhythmia, and the PVs (pulmonary veins) play a critical role in triggering AF. Stretch causes structural remodelling, including cytoskeleton rearrangement, which may play a role in the genesis of AF. Lat-B (latrunculin B), an inhibitor of actin polymerization, is involved in Ca(2+) regulation. However, it is unclear whether Lat-B directly modulates the electrophysiological characteristics and Ca(2+) homoeostasis of the PVs. Conventional microelectrodes, whole-cell patch-clamp, and the fluo-3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca(2+) within isolated rabbit PV preparations, or within isolated single PV cardiomyocytes, before and after administration of Lat-B (100 nM). Langendorff-perfused rabbit hearts were exposed to acute and continuous atrial stretch, and we studied PV electrical activity. Lat-B (100 nM) decreased the spontaneous electrical activity by 16±4% in PV preparations. Lat-B (100 nM) decreased the late Na(+) current, L-type Ca(2+) current, Na(+)/Ca(2+) exchanger current, and stretch-activated BKCa current, but did not affect the Na(+) current in PV cardiomyocytes. Lat-B reduced the transient outward K(+) current and ultra-rapid delayed rectifier K(+) current, but increased the delayed rectifier K(+) current in isolated PV cardiomyocytes. In addition, Lat-B (100 nM) decreased intracellular Ca(2+) transient and sarcoplasmic reticulum Ca(2+) content in PV cardiomyocytes. Moreover, Lat-B attenuated stretch-induced increased spontaneous electrical activity and trigger activity. The effects of Lat-B on the PV spontaneous electrical activity were attenuated in the presence of Y-27632 [10 μM, a ROCK (Rho-associated kinase) inhibitor] and cytochalasin D (10 μM, an actin polymerization inhibitor). In conclusion, Lat-B regulates PV electrophysiological characteristics and attenuates stretch-induced arrhythmogenesis.

A Single Protein Kinase A or Calmodulin Kinase II Site Does Not Control the Cardiac Pacemaker Ca2+ Clock.

Fight or flight heart rate (HR) increases depend on protein kinase A (PKA)- and calmodulin kinase II (CaMKII)-mediated enhancement of Ca(2+) uptake and release from sarcoplasmic reticulum (SR) in sinoatrial nodal cells (SANC). However, the impact of specific PKA and CaMKII phosphorylation sites on HR is unknown. We systematically evaluated validated PKA and CaMKII target sites on phospholamban and the ryanodine receptor using genetically modified mice. We found that knockin alanine replacement of ryanodine receptor PKA (S2808) or CaMKII (S2814) target sites failed to affect HR responses to isoproterenol or spontaneous activity in vivo or in SANC. Similarly, selective mutation of phospholamban amino acids critical for enhancing SR Ca(2+) uptake by PKA (S16) or CaMKII (T17) to alanines did not affect HR in vivo or in SANC. In contrast, CaMKII inhibition by expression of AC3-I has been shown to slow SANC rate responses to isoproterenol and decrease SR Ca(2+) content. Phospholamban deficiency rescued SR Ca(2+) content and SANC rate responses to isoproterenol in mice with AC3-I expression, suggesting that CaMKII affects HR by modulation of SR Ca(2+) content. Consistent with this, mice expressing a superinhibitory phospholamban mutant had low SR Ca(2+) content and slow HR in vivo and in SANC. SR Ca(2+) depletion reduces HR and SR Ca(2+) repletion restores physiological SANC rate responses, despite CaMKII inhibition. PKA and CaMKII do not affect HR by a unique target site governing SR Ca(2+) uptake or release. HR acceleration may require an SR Ca(2+) content threshold.

Altered Na/Ca exchange distribution in ventricular myocytes from failing hearts.

In mammalian cardiac ventricular myocytes, Ca efflux via Na/Ca exchange (NCX) occurs predominantly at T tubules. Heart failure is associated with disrupted t-tubular structure, but its effect on t-tubular function is less clear. We therefore investigated t-tubular NCX activity in ventricular myocytes isolated from rat hearts ∼18 wk after coronary artery ligation (CAL) or corresponding sham operation (Sham). NCX current (INCX) and l-type Ca current (ICa) were recorded using the whole cell, voltage-clamp technique in intact and detubulated (DT) myocytes; intracellular free Ca concentration ([Ca]i) was monitored simultaneously using fluo-4. INCX was activated and measured during application of caffeine to release Ca from sarcoplasmic reticulum (SR). Whole cell INCX was not significantly different in Sham and CAL myocytes and occurred predominantly in the T tubules in Sham myocytes. CAL was associated with redistribution of INCX and ICa away from the T tubules to the cell surface and an increase in t-tubular INCX/ICa density from 0.12 in Sham to 0.30 in CAL myocytes. The decrease in t-tubular INCX in CAL myocytes was accompanied by an increase in the fraction of Ca sequestered by SR. However, SR Ca content was not significantly different in Sham, Sham DT, and CAL myocytes but was significantly increased by DT of CAL myocytes. In Sham myocytes, there was hysteresis between INCX and [Ca]i, which was absent in DT Sham but present in CAL and DT CAL myocytes. These data suggest altered distribution of NCX in CAL myocytes.

Abstract 11016: Bag3 Regulates Contractility and Ca Homeostasis in Adult Mouse Ventricular Myocytes

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations are associated with familial dilated cardiomyopathy and reduced BAG3 expression was observed in murine transverse aortic constriction and porcine myocardial infarction models. BAG3 is expressed in the sarcolemma and t-tubules in adult myocytes. To simulate reduced BAG3 expression in disease states, we downregulated BAG3 in adult myocytes by shRNA (shBAG3). Despite ~50% reduction in BAG3 by shRNA, expression of α-subunit of L-type Ca channel, SR Ca-ATPase, Na/Ca exchanger, α1- and α2-subunits of Na-K-ATPase, and ryanodine receptor was unchanged. At baseline, BAG3 downregulation had no effect on myocyte contraction and [Ca]i dynamics. After isoproterenol, BAG3 downregulation resulted in reduced myocyte contraction amplitudes, shortening and re-lengthening velocities, lower systolic [Ca]i and [Ca]i transient amplitudes, and prolongation in both APD50 and APD90. L-type Ca current (ICa) and sarcoplasmic reticulum (SR) Ca content but not Na/Ca exchange current or SR Ca uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes. On the other hand, BAG3 overexpression resulted in enhanced myocyte contractility after isoproterenol. In addition, BAG3 overexpression rescued the depressed contraction amplitudes in myocytes overexpressing the HIV protein Tat, and improved myocardial contractility in Tg26 (HIV-1 transgenic) mice. We conclude: (1) BAG3 modulates myocyte contraction, Ca dynamics and action potential duration by regulating ion channels and provides a novel paradigm for contractile dysfunction and increased arrhythmia risks in familial dilated cardiomyopathy; (2) BAG3 is a therapeutic target for cardiomyopathy including HIV-associated cardiomyopathy.