Sarcoplasmic Reticulum Calcium Uptake Research Articles

SERCA2a overexpression improves muscle function in a canine Duchenne muscular dystrophy model

Excessive cytosolic calcium accumulation contributes to muscle degeneration in Duchenne muscular dystrophy (DMD). Sarco/endoplasmic reticulum calcium ATPase (SERCA) is a sarcoplasmic reticulum (SR) calcium pump that actively transports calcium from the cytosol into the SR. We previously showed that adeno-associated virus (AAV)-mediated SERCA2a therapy reduced cytosolic calcium overload and improved muscle and heart function in the murine DMD model. Here we tested whether AAV SERCA2a therapy could ameliorate muscle disease in the canine DMD model. 7.83 x 1013 viral genome particles of the AAV vector were injected into the extensor carpi ulnaris (ECU) muscles of four juvenile-affected dogs. Contralateral ECU muscles received excipient. Three months later, we observed widespread transgene expression and significantly increased SERCA2a levels in the AAV-injected muscles. Treatment improved SR calcium uptake, significantly reduced calpain activity, significantly improved contractile kinetics, and significantly enhanced resistance to eccentric contraction-induced force loss. Nonetheless, muscle histology was not improved. To evaluate the safety of AAV SERCA2a therapy, we delivered the vector to the ECU muscle of adult normal dogs. We achieved strong transgene expression without altering muscle histology and function. Our results suggest that AAV SERCA2a therapy has the potential to improve muscle performance in a dystrophic large mammal.

NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression.

The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase–dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/– mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy.

Contraction and electrophysiological abnormalities in myofilament mutation-positive and mutation-negative human HCM myocardium

Myofilament mutation-positive and mutation-negative hypertrophic cardiomyopathy (HCM) patients show different prognosis and arrhythmic burden that are likely related to the different pathogenesis. For instance, post-translational activation of CaMKII pathway is a specific biomarker of myofilament mutation-positive HCM while depressed sarcoplasmic reticulum calcium uptake is present in both groups. However, the functional consequences of a mutation-driven pathogenesis is still not completely understood.

CAPN3: A muscle‑specific calpain with an important role in the pathogenesis of diseases (Review).

Calpains are a family of Ca2+-dependent cysteine proteases that participate in various cellular processes. Calpain 3 (CAPN3) is a classical calpain with unique N-terminus and insertion sequence 1 and 2 domains that confer characteristics such as rapid autolysis, Ca2+-independent activation and Na+ activation of the protease. CAPN3 is the only muscle-specific calpain that has important roles in the promotion of calcium release from skeletal muscle fibers, calcium uptake of sarcoplasmic reticulum, muscle formation and muscle remodeling. Studies have indicated that recessive mutations in CAPN3 cause limb-girdle muscular dystrophy (MD) type 2A and other types of MD; eosinophilic myositis, melanoma and epilepsy are also closely related to CAPN3. In the present review, the characteristics of CAPN3, its biological functions and roles in the pathogenesis of a number of disorders are discussed.

Effect of Yixinshu Capsules on calcium regulatory protein in cardiac myocytes of rats with heart failure

To explore the molecular mechanism of Yixinshu Capsules(YXS) in restoring cardiac function in rats with heart failure(HF) from the perspective of calmodulin in cardiac myocytes on the basis of determining the therapeutic effect of YXS on HF. The SD rats were subjected to the surgery of ligating the anterior descending branch of the left coronary artery for 4 weeks to established myocardial ischemia-induced heart failure animal model. Then the rats were randomly divided into Sham operation group(Sham, saline), model group(HF, saline), high dose YXS group(HF+YXS-H, 1 600 mg·kg~(-1)·d~(-1)), low dose YXS group(HF+YXS-L, 800 mg·kg~(-1)·d~(-1)) and positive drug valsartan group(HF+VST, 8 mg·kg~(-1)·d~(-1)). After continuous intragastric administration for 6 weeks, the rats were sacrificed and myocardial tissue was collected. Real time quantitative polymerase chain reaction(RT-PCR) and Western blot were used to detect the expression of genes and proteins related to calcium regulation in cardiomyocytes. RESULTS:: showed that as compared with the model group, YXS increased the transcription level of Atp2 a2, Ryr2, CACNA1 C and PRKACA, and increased the expression levels of P-Ryr2, CACNA1 C and SERCA2 a, while decreased the level of NCX1.On the other hand, YXS treatment significantly decreased the RIP3 level and the phosphorylation of its substrate CaMKⅡ protein, and enhanced the phosphorylation expression of PLB. In summary, YXS therapy could regulate the expression of genes and proteins related to calcium regulation in cardiomyocytes, decrease RIP3 and the phosphorylation of CaMKⅡ protein, increase the phosphorylation of PLB at Ser16, and increase the expression of SERCA2 a protein, suggesting that YXS may regulate myocardial calcium homeostasis through CaMKⅡ/PLB/SERCA2 a pathway, to improve the ability of calcium uptake in sarcoplasmic reticulum and stabilize intracellular free Ca~(2+), so as to improve the cardiac function in rats with heart failure. Our study revealed the possible mechanism of YXS in the treatment of heart failure, especially from the perspective of intervention of calmodulin, promoting the clinical application of YXS.

A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice.

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk.

Computational Analysis of Heart-Failure-Related Remodeling of Cytosolic and Nuclear Calcium Handling in the Canine Atrial Cardiomyocyte

Background: Heart failure (HF) results in extensive remodeling of ventricular cytosolic and nucleoplasmic calcium handling (CaCyt / CaNucl) and produces atrial calcium-handling dysregulation, promoting atrial fibrillation. The molecular mechanisms underlying arrhythmogenic HF-related atrial calcium-handling abnormalities, notably for atrial CaNucl, are largely unknown. Computational modeling has emerged as a tool to study arrhythmogenic mechanisms, but no currently available atrial model can simulate CaNucl, which was addressed here.Methods: CaCyt and CaNucl were measured using confocal microscopy in atrial cardiomyocytes from control or ventricular-tachypacing-induced HF dogs loaded with Fluo4 (1 Hz stimulation). We extended the Ramirez et al. 2000 canine atrial cardiomyocyte model with a nuclear compartment with nuclear-pore channel-mediated calcium diffusion from the cytosol, perinuclear calcium-store (periCs) linked to the sarcoplasmic reticulum (SR), and IP3-receptor-mediated calcium release from periCs to nucleus. IP3-receptor expression in control and HF dogs was evaluated using Western blot.Results: Nucleoplasmic calcium fluorescence ratios exceeded cytosolic levels in experiments (+28% diastolic, +9% systolic) and model (+32% diastolic, +9% systolic). HF significantly increased nuclear and cytosolic fluorescence, and IP3-receptor expression (+20%; P<0.05). We simulated the relative contribution of increased IP3-receptor expression and HF-related atrial CaCyt remodeling (−60% ryanodine-receptor expression, increased SR calcium-uptake; Yeh et al. 2008) to CaNucl remodeling. Increased IP3-receptor expression slightly augmented CaNucl (+5% diastolic, +2% systolic), without affecting CaCyt, whereas CaCyt remodeling strongly increased CaNucl (+28% diastolic, +41% systolic) in the model. The combined effect (IP3-receptor+Cacyt remodeling) was overadditive (+38% diastolic, +46% systolic).Conclusion: We developed the first computational model to simulate atrial CaNucl. HF-related CaCyt and CaNucl remodeling synergistically modulate nuclear calcium transients. The model provides new insights into atrial CaNucl determinants and their regulation in HF, and may help uncover the mechanisms of proarrhythmic atrial gene-expression changes.

Rate-dependent force, intracellular calcium, and action potential voltage alternans are modulated by sarcomere length and heart failure induced-remodeling of thin filament regulation in human heart failure: A myocyte modeling study

Microvolt T-wave alternans (MTWA) testing identifies heart failure patients at risk for lethal ventricular arrhythmias at near-resting heart rates (<110 beats per minute). Since pressure alternans occurs simultaneously with MTWA and has a higher signal to noise ratio, it may be a better predictor of arrhythmia, although the mechanism remains unknown. Therefore, we investigated the relationship between force alternans (FORCE-ALT), the cellular manifestation of pressure alternans, and action potential voltage alternans (APV-ALT), the cellular driver of MTWA. Our goal was to uncover the mechanisms linking APV-ALT and FORCE-ALT in failing human myocytes and to investigate how the link between those alternans was affected by pacing rate and by physiological conditions such as sarcomere length and heart failure induced-remodeling of mechanical parameters. To achieve this, a mechanically-based, strongly coupled human electromechanical myocyte model was constructed. Reducing the sarcoplasmic reticulum calcium uptake current (Iup) to 27% was incorporated to simulate abnormal calcium handling in human heart failure. Mechanical remodeling was incorporated to simulate altered thin filament activation and crossbridge (XB) cycling rates. A dynamical pacing protocol was used to investigate the development of intracellular calcium concentration ([Ca]i), voltage, and active force alternans at different pacing rates. FORCE-ALT only occurred in simulations incorporating reduced Iup, demonstrating that alternans in the intracellular calcium concentration (CA-ALT) induced FORCE-ALT. The magnitude of FORCE-ALT was found to be largest at clinically relevant pacing rates (<110 bpm), where APV-ALT was smallest. We found that the magnitudes of FORCE-ALT, CA-ALT and APV-ALT were altered by heart failure induced-remodeling of mechanical parameters and sarcomere length due to the presence of myofilament feedback. These findings provide important insight into the relationship between heart-failure-induced electrical and mechanical alternans and how they are altered by physiological conditions at near-resting heart rates.

Abstract 19960: Human Cardiac Fibroblasts Increase SR-Dependency of Induced Pluripotent Stem Cell-Derived Cardiomyocyte Calcium Handling by Increasing SR Calcium Uptake and SERCA2a Expression via Direct Physical Contact

Introduction: Cardiac fibroblasts can influence cardiomyocyte structure and function through direct physical interaction and/or by the secretion of soluble factors. Here we assess the relative importance of these different modalities of interaction in terms of their effects on cardiomyocyte electrophysiology. Methods: Ventricular fibroblasts from the explanted hearts of dilated cardiomyopathy (DCM) patients (n=4) were seeded directly onto a confluent monolayer of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) at a ratio of 2:1 and cultured for 24 hours. iPS-CMs alone were used as control. Ca2+ transients and action potentials were recorded optically using fluo-4 AM and di-8-ANEPPS. Data are presented as percentage change from control where appropriate. Results: iPS-CM Ca2+ transient duration was significantly reduced by contact with fibroblasts (-8.4±2.2% n=12 p&lt;0.01). Sarcoplasmic reticulum (SR) Ca2+ uptake assessed by caffeine application was highly upregulated (2.07±0.12s-1 vs 0.63±0.07s-1 in control n=26, 23 p&lt;0.001), associated with a significant increase in SR Ca2+ content (F/F0: 3.74±0.11 vs 2.97±0.11 in control n=37, 32 p&lt;0.001) and SERCA2a protein expression (1.02±0.08 vs 0.55±0.01 n=3 p&lt;0.01). iPS-CMs in contact with fibroblasts displayed a greater dependence on SR uptake for Ca2+ extrusion than other mechanisms (SR/NCX/Slow: 60.7/31.9/7.4% vs 23.3/70.9/5.8% in control). Action potential duration (90%) was significantly prolonged in contact with fibroblasts (+18.8±2.5% n=22 p&lt;0.001 vs control). Screening of culture supernatants from the co-culture has identified the presence of a number of cytokines known to affect cardiomyocyte function including IL-6 (680pg/ml), MIF (1.16ng/ml), MCP-1 (1.25ng/ml) and TGF-β. (110pg/ml). Conclusions: DCM fibroblasts influence iPS-CM electrophysiology through different modalities; however physical contact with fibroblasts has effects not seen with soluble mediators alone. This contact-dependent shift towards greater SR-dependency represents a novel effect of fibroblasts on cardiomyocyte Ca2+ handling. Determining whether these effects are mediated through mechanical, electrical or paracrine signalling will be the focus of future work.

Inhibition of miR-25 improves cardiac contractility in the failing heart

Heart failure is characterized by a debilitating decline in cardiac function1, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy2,3. microRNAs (miRs) are dysregulated with heart failure4,5 but whether they control contractility or constitute therapeutic targets remain speculative. Using high throughput, functional screening of the human microRNAome, we identified miRs that suppress intracellular calcium handling in heart muscle by interacting with mRNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a. Of 875 miRs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas AAV9-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 dramatically halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggests that it might be targeted therapeutically to restore function.

Hydrogen Peroxide–Mediated SERCA Cysteine 674 Oxidation Contributes to Impaired Cardiac Myocyte Relaxation in Senescent Mouse Heart

BackgroundA hallmark of aging of the cardiac myocyte is impaired sarcoplasmic reticulum (SR) calcium uptake and relaxation due to decreased SR calcium ATPase (SERCA) activity. We tested the hypothesis that H2O2‐mediated oxidation of SERCA contributes to impaired myocyte relaxation in aging.Methods and ResultsYoung (5‐month‐old) and senescent (21‐month‐old) FVB wild‐type (WT) or transgenic mice with myocyte‐specific overexpression of catalase were studied. In senescent mice, myocyte‐specific overexpression of catalase (1) prevented oxidative modification of SERCA as evidenced by sulfonation at Cys674, (2) preserved SERCA activity, (3) corrected impaired calcium handling and relaxation in isolated cardiac myocytes, and (4) prevented impaired left ventricular relaxation and diastolic dysfunction. Nitroxyl, which activates SERCA via S‐glutathiolation at Cys674, failed to activate SERCA in freshly isolated ventricular myocytes from senescent mice. Finally, in adult rat ventricular myocytes in primary culture, adenoviral overexpression of SERCA in which Cys674 is mutated to serine partially preserved SERCA activity during exposure to H2O2.ConclusionOxidative modification of SERCA at Cys674 contributes to decreased SERCA activity and impaired myocyte relaxation in the senescent heart. Strategies to decrease oxidant levels and/or protect target proteins such as SERCA may be of value to preserve diastolic function in the aging heart.

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Targeted Sarcoplasmic Reticulum Ca 2+ ATPase 2a Gene Delivery to Restore Electrical Stability in the Failing Heart

Background— Recently, we reported that sarcoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a), the pump responsible for reuptake of cytosolic calcium during diastole, plays a central role in the molecular mechanism of cardiac alternans. Heart failure (HF) is associated with impaired myocardial calcium handling, deficient SERCA2a, and increased susceptibility to cardiac alternans. Therefore, we hypothesized that restoring deficient SERCA2a by gene transfer will significantly reduce arrhythmogenic cardiac alternans in the failing heart. Methods and Results— Adult guinea pigs were divided into 3 groups: control, HF, and HF+AAV9.SERCA2a gene transfer. HF resulted in a decrease in left ventricular fractional shortening compared with controls ( P &lt;0.001). As expected, isolated HF myocytes demonstrated slower sarcoplasmic reticulum calcium uptake, decreased Ca 2+ release, and increased diastolic Ca 2+ ( P &lt;0.05) compared with controls. Moreover, SERCA2a, cardiac ryanodine receptor 2, and sodium-calcium exchanger protein expression was decreased in HF compared with control ( P &lt;0.05). As predicted, HF increased susceptibility to cardiac alternans, as evidenced by decreased heart rate thresholds for both V m alternans and Ca alternans compared with controls ( P &lt;0.01). Interestingly, in vivo gene transfer of AAV9.SERCA2a in the failing heart improved left ventricular contractile function ( P &lt;0.01), suppressed cardiac alternans ( P &lt;0.01), and reduced ryanodine receptor 2 P o secondary to reduction of ryanodine receptor 2–P S2814 ( P &lt;0.01). This ultimately resulted in a decreased incidence of inducible ventricular arrhythmias ( P= 0.05). Conclusions— These data show that SERCA2a gene transfer in the failing heart not only improves contractile function but also directly restores electric stability through the amelioration of key arrhythmogenic substrate (ie, cardiac alternans) and triggers (ie, sarcoplasmic reticulum Ca 2+ leak).

Right Ventricular Pacing With Mechanical Dyssynchrony Causes Apoptosis Interruptus and Calcium Mishandling

BackgroundMechanical dyssynchrony associated with rapid pacing induces cardiac cell stress and myocardial apoptotic pathway activation that has been implicated in the pathophysiology of left ventricular (LV) dysfunction. Effects of dyssynchrony per se are not fully understood. The objective of our study was to test whether ventricular dyssynchrony would elicit myocardial alterations in LV calcium handling regulation and cell survival or apoptosis signalling in right ventricular-paced swine. MethodsImplantation of pacemaker was performed under anaesthesia. Endocardial bipolar screw lead was inserted into the right jugular vein and positioned either in the right atrium or at the right ventricular (RV) apex. Swine were paced at 150 beats per minute for 3 weeks. ResultsCompared with right atrial pacing, RV pacing led to abnormal LV sarcoplasmic reticulum calcium uptake (315 ± 65 vs 155 ± 55 nmol/min/mg, P < 0.05) and LV calcium-handling protein expression, ie, 35% reduction in ryanodine receptor 2, 25% decline in sarcoplasmic reticulum Ca2+ ATPase, 70% increase in Na+/Ca2+ exchanger, and 10% increase in phospholamban. RV pacing also elicited activation of LV apoptotic cascades without nuclear apoptosis. So-called interrupted apoptosis was the result of increased expression of X-linked inhibitor of apoptosis protein. Apoptosis and calcium mishandling were documented in absence of depressed heart function (ejection fraction 62 ± 8% vs 57 ± 12%, in right atrial- and RV-paced hearts, respectively, P > 0.05). ConclusionsSlow rate RV pacing causes mechanical dyssynchrony and profound LV alterations in both apoptotic pathways and calcium handling in the early stages of pacing-induced cardiomyopathy.

Modulation of Cardiac Contractility by the Phopholamban/SERCA2a Regulatome

Heart disease remains the leading cause of death and disability in the Western world. Current therapies aim at treating the symptoms rather than the subcellular mechanisms, underlying the etiology and pathological remodeling in heart failure. A universal characteristic, contributing to the decreased contractile performance in human and experimental failing hearts, is impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and phosphorylation of PLN relieves this inhibition. However, the initial simple view of a PLN/SERCA regulatory complex has been modified by our recent identification of SUMO, S100 and the histidine-rich Ca-binding protein as regulators of SERCA activity. In addition, PLN activity is regulated by 2 phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect the overall SERCA-mediated Ca-transport. This review will highlight the regulatory mechanisms of cardiac contractility by the multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues targeting this complex by using small molecules and gene transfer methods.

Direct interaction of Bestrophin 3 channels with Hrc calcium‐handling protein

Bestrophin 3 (Best3) is a calcium‐activated chloride channel expressed in heart and other tissues. We hypothesized that Best3 channels function in a macromolecule complex similar to other cardiac ion channels.Yeast two‐hybrid assays performed on a mouse heart library, identified histidine‐rich calcium binding protein (Hrc) as a novel binding partner of Best3. Hrc is a regulator of sarcoplasmic reticulum (SR) calcium uptake, storage and release. This interaction was further determined to involve Best3 amino acids 541–669 in the cytosolic C‐terminus of the channel and the cysteine‐rich region in mouse Hrc (amino acids 665–738 aa). Specific binding between Best3 channels and Hrc was verified by co‐immunoprecipitation using mouse heart homogenates. Confocal immunofluorescence microscopy further demonstrated a strong co‐localization of Best3 and Hrc in isolated ventricular and atrial myocytes.In this study we report that cardiac Best3 channels interact with Hrc. Calcium‐activated chloride channels play a critical role in excitation‐contraction coupling in the SR by balancing charge movement during calcium release and reuptake. The direct coupling of Best3 with Hrc suggests that Best3 channels may play a role in calcium handling in the heart by functioning as a counterion channel.

Ginkgo biloba extract EGB761 protects against aging-associated diastolic dysfunction in cardiomyocytes of D-galactose-induced aging rat.

The aim of the present study was to make use of the artificially induced aging model cardiomyocytes to further investigate potential anti-aging-associated cellular diastolic dysfunction effects of EGB761 and explore underlying molecular mechanisms. Cultured rat primary cardiomyocytes were treated with either D-galactose or D-galactose combined with EGB761 for 48 h. After treatment, the percentage of cells positive for SA-β-gal, AGEs production, cardiac sarcoplasmic reticulum calcium pump (SERCA) activity, the myocardial sarcoplasmic reticulum calcium uptake, and relative protein levels were measured. Our results demonstrated that in vitro stimulation with D-galactose induced AGEs production. The addition of EGB761 significantly decreased the number of cells positive for SA-β-gal. Furthermore, decreased diastolic [Ca2+]i, curtailment of the time from the maximum concentration of Ca2+ to the baseline level and increased reuptake of Ca2+ stores in the SR were also observed. In addition, the level of p-Ser16-PLN protein as well as SERCA was markedly increased. The study indicated that EGb761 alleviates formation of AGEs products on SERCA2a in order to mitigate myocardial stiffness on one hand; on other hand, improve SERCA2a function through increase the amount of Ser16 sites PLN phosphorylation, which two hands finally led to ameliorate diastolic dysfunction of aging cardiomyocytes.

Detection, properties, and frequency of local calcium release from the sarcoplasmic reticulum in teleost cardiomyocytes.

Calcium release from the sarcoplasmic reticulum (SR) plays a central role in the regulation of cardiac contraction and rhythm in mammals and humans but its role is controversial in teleosts. Since the zebrafish is an emerging model for studies of cardiovascular function and regeneration we here sought to determine if basic features of SR calcium release are phylogenetically conserved. Confocal calcium imaging was used to detect spontaneous calcium release (calcium sparks and waves) from the SR. Calcium sparks were detected in 16 of 38 trout atrial myocytes and 6 of 15 ventricular cells. The spark amplitude was 1.45±0.03 times the baseline fluorescence and the time to half maximal decay of sparks was 27±3 ms. Spark frequency was 0.88 sparks µm−1 min−1 while calcium waves were 8.5 times less frequent. Inhibition of SR calcium uptake reduced the calcium transient (F/F0) from 1.77±0.17 to 1.12±0.18 (p = 0.002) and abolished calcium sparks and waves. Moreover, elevation of extracellular calcium from 2 to 10 mM promoted early and delayed afterdepolarizations (from 0.6±0.3 min−1 to 8.1±2.0 min−1, p = 0.001), demonstrating the ability of SR calcium release to induce afterdepolarizations in the trout heart. Calcium sparks of similar width and duration were also observed in zebrafish ventricular myocytes. In conclusion, this is the first study to consistently report calcium sparks in teleosts and demonstrate that the basic features of calcium release through the ryanodine receptor are conserved, suggesting that teleost cardiac myocytes is a relevant model to study the functional impact of abnormal SR function.

Catecholaminergic-induced arrhythmias in failing cardiomyocytes associated with human HRCS96A variant overexpression

The histidine-rich calcium binding protein (HRC) Ser96Ala polymorphism was shown to correlate with ventricular arrhythmias and sudden death only in dilated cardiomyopathy patients but not in healthy human carriers. In the present study, we assessed the molecular and cellular mechanisms underlying human arrhythmias by adenoviral expression of the human wild-type (HRC(WT)) or mutant HRC (HRC(S96A)) in adult rat ventricular cardiomyocytes. Total HRC protein was increased by ∼50% in both HRC(WT)- and HRC(S96A)-infected cells. The HRC(S96A) mutant exacerbated the inhibitory effects of HRC(WT) on the amplitude of Ca(2+) transients, prolongation of Ca(2+) decay time, and caffeine-induced sarcoplasmic reticulum Ca(2+) release. Consistent with these findings, HRC(S96A) reduced maximal sarcoplasmic reticulum calcium uptake rate to a higher extent than HRC(WT). Furthermore, the frequency of spontaneous Ca(2+) sparks, which was reduced by HRC(WT), was increased by mutant HRC(S96A) under resting conditions although there were no spontaneous Ca(2+) waves under stress conditions. However, expression of the HRC(S96A) genetic variant in cardiomyocytes from a rat model of postmyocardial infarction heart failure induced dramatic disturbances of rhythmic Ca(2+) transients. These findings indicate that the HRC Ser96Ala variant increases the propensity of arrhythmogenic Ca(2+) waves in the stressed failing heart, suggesting a link between this genetic variant and life-threatening ventricular arrhythmias in human carriers.

Adenylyl Cyclase 6 Improves Calcium Uptake and Left Ventricular Function in Aged Hearts

This study tested the hypothesis that activation of adenylyl cyclase 6 (AC6) expression in cardiac myocytes improves calcium uptake and left ventricular (LV) function in aging mice. Aging hearts exhibit impaired β-adrenergic receptor signaling and LV dysfunction. Twenty-month-old mice with cardiac-directed and regulated AC6 expression were randomized into 2 groups, and AC6 expression was activated in 1 group (AC6-On) but not the other (AC6-Off). One month later, LV function and sarcoplasmic reticulum calcium uptake were assessed. AC6 expression was associated with increased LV contractility, as reflected by ejection fraction (p = 0.02), rate of pressure development (p = 0.002), and slope of the LV end-systolic pressure-volume relationship (p = 0.04). No changes in LV weight to tibial length ratio, LV fibrosis, and expression of fetal genes (atrial natriuretic factor, α-skeletal muscle actin, and β-myosin heavy chain) and collagens were observed between AC6-On and AC6-Off groups. However, LV samples from AC6-On mice showed increases in: isoproterenol-stimulated cAMP production (p = 0.04), cAMP-dependent protein kinase activity (p < 0.0004), phosphorylation of phospholamban (at Ser16 site; p = 0.04) and cardiac troponin I (at Ser23/24 sites; p = 0.01), velocity of sarcoplasmic reticulum calcium uptake (p < 0.0001), and sarcoplasmic reticulum calcium-ATPase2a (SERCA2a) affinity for calcium (p < 0.0001). Finally, we found that AC6 expression increased sarcoplasmic reticulum calcium storage in cardiac myocytes isolated from 23-month-old rats. In contrast, AC6 expression in 7-month-old mice did not change LV function and calcium uptake. These results indicate that activation of cardiac AC6 expression improves impaired function of aged hearts through improved calcium uptake.