Decrease In Sarcoplasmic Reticulum Ca2 Research Articles

Glu-rich N-terminal variable region of troponin T functions as a myofilament Ca2+-reservoir to reduce SR Ca2+-cycling.

The long Glu-rich N-terminal segment of avian pectoral muscle troponin T (TnT) and C-terminal extension of insect TnT are multi-valent Ca2+-binding sites with physiologically relevant affinities. Genetic deletion of the Glu-rich C-terminal extension of insect TnT decreases muscle and heart functions but increases the tolerance of cardiomyocytes to Ca2+ overloading, indicating decreased [Ca2+]i. In contrast, cardiomyocytes from gene-edited mice expressing modified cardiac TnT with N-terminal variable region replaced by a C-terminal segment of insect TnT (51E-cTnT) show higher shortening amplitude and contraction and relaxation velocities than that of wild type (WT) control. Isolated 51E-cTnT cardiomyocytes exhibited shorter resting sarcomeres, which can be corrected by inhibiting myosin ATPase, indicating increased resting [Ca2+]i. A similar gain-of-function model,fast skeletal muscle TnTgene-edited mice with the N-terminal variable region replaced by the N-terminal segment of adult turkey pectoral muscle TnT (Tx9-fsTnT), shows normal baseline muscle functions but slower and less rising of contractile force during caffeine-induced Ca2+ release from sarcoplasmic reticulum (SR) than that of WT control, indicating a decreased SR Ca2+ store. Consistently, 51E-cTnT mouse hearts respond to beta-adrenergic stimulation or Ca2+ agonist with higher than WT ventricular peak pressure and diastolic velocity. The data support a hypothesis that the Glu-rich segments of insect and avian flight muscle TnT function as myofilament Ca2+ reservoirs to tune muscle kinetics and reduce the amount of Ca2+ cycled during contraction and relaxation. The functional effects are prominent in heterozygotes of the genetically modified flies and mice whereas the loss-of-function in Drosophila TnT and gain-of-function in 51E-cTnT and Tx9-fsTnT mice both produced homozygote lethality, indicating sensitive dose effects of the myofilament Ca2+ reservoir for use as a potent mechanism to improve energetic efficiency of cardiac and skeletal muscles.

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

PDE5 Inhibition Suppresses Ventricular Arrhythmias by Reducing SR Ca2+ Content.

[Figure: see text].

Cardiac-specific ablation of glutaredoxin 3 leads to cardiac hypertrophy and heart failure.

Growing evidence suggests that redox‐sensitive proteins including glutaredoxins (Grxs) can protect cardiac muscle cells from oxidative stress‐induced damage. Mammalian Grx3 has been shown to be critical in regulating cellular redox states. However, how Grx3 affects cardiac function by modulating reactive oxygen species (ROS) signaling remains unknown. In this study, we found that the expression of Grx3 in the heart is decreased during aging. To assess the physiological role of Grx3 in the heart, we generated mice in which Grx3 was conditionally deleted in cardiomyocytes (Grx3 conditional knockout (CKO) mice). Grx3 CKO mice were viable and grew indistinguishably from their littermates at young age. No difference in cardiac function was found comparing Grx3 CKO mice and littermate controls at this age. However, by the age of 12 months, Grx3 CKO mice exhibited left ventricular hypertrophy with a significant decrease in ejection fraction and fractional shortening along with a significant increase of ROS production in cardiomyocytes compared to controls. Deletion of Grx3 also impaired Ca2+ handling, caused enhanced sarcoplasmic reticulum (SR) calcium (Ca2+) leak, and decreased SR Ca2+ uptake. Furthermore, enhanced ROS production and alteration of Ca2+ handling in cardiomyocytes occurred, prior to cardiac dysfunction in young mice. Therefore, our findings demonstrate that Grx3 is an important factor in regulating cardiac hypertrophy and heart failure by modulating both cellular redox homeostasis and Ca2+ handling in the heart.

Activation of protein phosphatase 1 by a selective phosphatase disrupting peptide reduces sarcoplasmic reticulum Ca2+ leak in human heart failure.

Disruption of Ca2+ homeostasis is a key pathomechanism in heart failure. CaMKII-dependent hyperphosphorylation of ryanodine receptors in the sarcoplasmic reticulum (SR) increases the arrhythmogenic SR Ca2+ leak and depletes SR Ca2+ stores. The contribution of conversely acting serine/threonine phosphatases [protein phosphatase 1 (PP1) and 2A (PP2A)] is largely unknown. Human myocardium from three groups of patients was investigated: (i) healthy controls (non-failing, NF, n = 8), (ii) compensated hypertrophy (Hy, n = 16), and (iii) end-stage heart failure (HF, n = 52). Expression of PP1 was unchanged in Hy but greater in HF compared to NF while its endogenous inhibitor-1 (I-1) was markedly lower expressed in both compared to NF, suggesting increased total PP1 activity. In contrast, PP2A expression was lower in Hy and HF compared to NF. Ca2+ homeostasis was severely disturbed in HF compared to Hy signified by a higher SR Ca2+ leak, lower systolic Ca2+ transients as well as a decreased SR Ca2+ load. Inhibition of PP1/PP2A by okadaic acid increased SR Ca2+ load and systolic Ca2+ transients but severely aggravated diastolic SR Ca2+ leak and cellular arrhythmias in Hy. Conversely, selective activation of PP1 by a PP1-disrupting peptide (PDP3) in HF potently reduced SR Ca2+ leak as well as cellular arrhythmias and, importantly, did not compromise systolic Ca2+ release and SR Ca2+ load. This study is the first to functionally investigate the role of PP1/PP2A for Ca2+ homeostasis in diseased human myocardium. Our data indicate that a modulation of phosphatase activity potently impacts Ca2+ cycling properties. An activation of PP1 counteracts increased kinase activity in heart failure and successfully seals the arrhythmogenic SR Ca2+ leak. It may thus represent a promising future antiarrhythmic therapeutic approach.

Calcium-handling abnormalities underlying atrial arrhythmogenesis in a Fontan operation canine model.

Atrial tachyarrhythmia (AT) is a common complication in patients who have undergone a Fontan operation. In this study, we investigated whether abnormal Ca2+ handling contributes to the Fontan operation-related atrial arrhythmogenic substrate. Mongrel dogs were randomly assigned to sham and Fontan groups. The Fontan operation model was developed by performing an atriopulmonary anastomosis. After 14days, an electrophysiological study was performed to evaluate the AT vulnerability. Ca2+ handlingproperties were measured by loading atrial cardiomyocytes (CMs) with fura-2 AM. The L-type Ca2+ (ICa-L) and Na+-Ca2+exchanger (INCX) currentsof the CMs were recorded by the whole-cell patch-clamp technique. The key Ca2+ handling proteins expression was assessed by Western blotting. The AT inducibility was higher in the Fontan group than in the sham group (85.71 vs. 14.29%, P < 0.05). The Fontan operation resulted in decreased Ca2+ transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca2+ content, but in enhanced diastolic intracellular Ca2+ concentration and SR Ca2+ leak in the atrial CMs. The spontaneous CaT events, triggered ectopic activity and INCX density were increased, but ICa-L density was reduced in CMs from the Fontan atria (all P < 0.05). Additionally, the Fontan operation resulted in decreased SR Ca2+ ATPase expression and Cav1.2 expression, but in increased NCX1 and Ser2814-phosphorylated ryanodine receptor 2. The calmodulin-dependent protein kinase II expression and function were markedly enhanced in the Fontan atria. The Fontan operation caused atrial CM Ca2+ handling abnormalities that produced arrhythmogenic-triggered activity and increased vulnerability to AT in experimental Fontan dogs.

The role of RyR2 oxidation in the blunted frequency-dependent facilitation of Ca2+ transient amplitude in rabbit failing myocytes.

Defective Ca2+ regulation plays a key role in the blunted force-frequency response in heart failure (HF). Since HF is commonly associated with oxidative stress, we studied whether oxidation of ryanodine receptor (RyR2) contributes to this defect. In control ventricular myocytes, oxidative stress induced formation of disulfide bonds between RyR2 subunits: intersubunit cross-linking (XL). Western blot analysis and Ca2+ imaging revealed a strong positive correlation between RyR2 XL and sarcoplasmic reticulum (SR) Ca2+ leak. These results illustrate that RyR2 XL can be used as a sensitive indicator of RyR2 dysfunction during oxidative stress. HF myocytes were in a state of oxidative stress since they exhibited an increase in reactive oxygen species (ROS) level, a decrease in ROS defense and an overall protein oxidation. These myocytes were also characterized by RyR2 XL and increased SR Ca2+ leak. Moreover, the frequency-dependent increase of Ca2+ transient amplitude was suppressed due to the inability of the SR to maintain Ca2+ load at high pacing rates. Because SR Ca2+ load is determined by the balance between SR Ca2+ uptake and leak, the blunted frequency-dependent inotropy in HF can be mediated by ROS-induced SR Ca2+ leak. Preventing RyR2 XL in HF myocytes decreased SR Ca2+ leak and increased Ca2+ transients at high pacing rate. We also studied whether RyR2 oxidation alone can cause the blunted frequency-dependent facilitation of Ca2+ transient amplitude in control myocytes. When RyR2 XL was induced in control myocytes to a similar level seen in HF, an increase of Ca2+ transient amplitude at high pacing rate was significantly suppressed. These results suggest that SR Ca2+ leak induced by RyR2 oxidation can play an important role in the blunted frequency-dependent inotropy of HF.

Impact of RyR2 potentiation on myocardial function.

This perspective attempts to shed light on an old and not yet solved controversy in cardiac physiology, i.e., the impact of increasing ryanodine receptor (RyR)2 open probability on myocardial function. Based on an already proven myocyte model, it was shown that increasing RyR2 open probability results in a purely short-lived increase in Ca2+ transient amplitude, and, therefore, it does not increase cardiac contractility. However, potentiation of RyR2 activity permanently enhances fractional Ca2+ release, shifting the intracellular Ca2+ transient versus sarcoplasmic reticulum (SR) Ca2+ content curve to a new state of higher efficiency. This would allow the heart to maintain a given contractility despite a decrease in SR Ca2+ content, to enhance contractility if SR Ca2+ content is simultaneously preserved or to successfully counteract the effects of a negative inotropic intervention.NEW & NOTEWORTHY Increasing ryanodine receptor (RyR)2 open probability does not increase cardiac contractility. However, RyR2 potentiation shifts the intracellular Ca2+ transient-sarcoplasmic reticulum (SR) Ca2+ content relationship toward an enhanced efficiency state, which may contribute to a positive inotropic effect, preserve contractility despite decreased SR Ca2+ content, or successfully counteract the effects of a negative inotropic action.

A chemical chaperone improves muscle function in mice with a RyR1 mutation

Mutations in the RYR1 gene cause severe myopathies. Mice with an I4895T mutation in the type 1 ryanodine receptor/Ca2+ release channel (RyR1) display muscle weakness and atrophy, but the underlying mechanisms are unclear. Here we show that the I4895T mutation in RyR1 decreases the amplitude of the sarcoplasmic reticulum (SR) Ca2+ transient, resting cytosolic Ca2+ levels, muscle triadin content and calsequestrin (CSQ) localization to the junctional SR, and increases endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and mitochondrial ROS production. Treatment of mice carrying the I4895T mutation with a chemical chaperone, sodium 4-phenylbutyrate (4PBA), reduces ER stress/UPR and improves muscle function, but does not restore SR Ca2+ transients in I4895T fibres to wild type levels, suggesting that decreased SR Ca2+ release is not the major driver of the myopathy. These findings suggest that 4PBA, an FDA-approved drug, has potential as a therapeutic intervention for RyR1 myopathies that are associated with ER stress.

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca(2+) channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca(2+) leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca(2+)]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca(2+)]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca(2+) transient was normal, despite the reduced SR Ca(2+) content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca(2+) transients because they are incapable of properly increasing the amount of Ca(2+) stored in the SR because of a larger SR Ca(2+) leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca(2+) channel current (ICa-L) and SR Ca(2+) content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po and fractional Ca(2+) release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca(2+) content, but cannot compensate for the inability to increase SR Ca(2+) content during inotropic challenge.

A novel human R25C-phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia.

Depressed sarcoplasmic reticulum (SR) Ca(2+) cycling, a universal characteristic of human and experimental heart failure, may be associated with genetic alterations in key Ca(2+)-handling proteins. In this study, we identified a novel PLN mutation (R25C) in dilated cardiomyopathy (DCM) and investigated its functional significance in cardiomyocyte Ca(2+)-handling and contractility. Exome sequencing identified a C73T substitution in the coding region of PLN in a family with DCM. The four heterozygous family members had implantable cardiac defibrillators, and three developed prominent ventricular arrhythmias. Overexpression of R25C-PLN in adult rat cardiomyocytes significantly suppressed the Ca(2+) affinity of SR Ca(2+)-ATPase (SERCA2a), resulting in decreased SR Ca(2+) content, Ca(2+) transients, and impaired contractile function, compared with WT-PLN. These inhibitory effects were associated with enhanced interaction of R25C-PLN with SERCA2, which was prevented by PKA phosphorylation. Accordingly, isoproterenol stimulation relieved the depressive effects of R25C-PLN in cardiomyocytes. However, R25C-PLN also elicited increases in the frequency of Ca(2+) sparks and waves as well as stress-induced aftercontractions. This was accompanied by increased Ca(2+)/calmodulin-dependent protein kinase II activity and hyper-phosphorylation of RyR2 at serine 2814. The findings demonstrate that human R25C-PLN is associated with super-inhibition of SERCA2a and Ca(2+) transport as well as increased SR Ca(2+) leak, promoting arrhythmogenesis under stress conditions. This is the first mechanistic evidence that increased PLN inhibition may impact both SR Ca(2+) uptake and Ca(2+) release activities and suggests that the human R25C-PLN may be a prognostic factor for increased ventricular arrhythmia risk in DCM carriers.

Myocardial Calcium Handling is Up‐Regulated During the Spontaneous Resolution of Endotoxemic Cardiomyopathy in Mice

Sepsis and systemic inflammation induce a specific cardiomyopathy that resolves spontaneously in surviving patients, through mechanisms that are currently unknown. Here we studied cardiomyocyte calcium (Ca2+) handling during endotoxemic cardiomyopathy (EC) resolution in mice.Male C57Bl6 mice were administered lipopolysaccharide (LPS, 7 μg/g, ip). LPS induced an inflammatory shock syndrome, cardiomyopathy and 59% mortality. We measured sarcomere shortening (SS) and Ca2+ transients (ΔCai) in externally paced cardiomyocytes, at 37o C.12h after LPS administration, SS and ΔCai were decreased to 53 ± 10% and 78 ± 5% of baseline, respectively (n &gt;30 cells from 3 mice, 30/3, for each group; p&lt;0.05 for this and the following) in association with a decrease in sarcoplasmic reticulum Ca2+ pump (SERCA) reuptake (to 88 ± 5% of control, n &gt; 19/3). In surviving mice, ΔCai was fully recovered 3 days after LPS administration (Day 3, D3) and reached levels exceeding normal at D6 (124 ± 7 % of baseline, n &gt; 18/3). ΔCai recovery was associated with SERCA supra‐normal activation (138 ± 4% of control, n &gt; 28/3) at D3, due to phospholamban (PLB) down‐regulation (to 35 ± 9% of control, n = 6 hearts each group). At D6, membrane Na+/Ca2+ exchange (NCX) function was 51 ± 4 % of baseline (n &gt; 12/3), contributing to the supra‐normal ΔCai levels.In conclusion, myocardial recovery during the spontaneous resolution of EC is the result of active cellular mechanisms that include down‐regulation of PLB and NCX and lead to supra‐normal ΔCai.

Abstract 18739: Acute Effect of the Na+/ca2+ Exchanger (ncx) Inhibitor Sea0400 on Diastolic Function in vivo and in vitro in a Model of Heart Failure With Preserved Ejection Fraction

Background: Heart failure with preserved ejection fraction (HFPEF) is increasingly common but underlying cellular mechanisms are not well understood. We investigated cardiomyocyte function and the role of SEA0400, in vivo and in vitro in a rat model of diastolic heart failure. Methods: Young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). After 24 weeks in vivo (pressure-volume loops) and in vitro characterization (cardiomyocyte function (Ca2+ transients, sarcoplasmic reticulum (SR) diastolic Ca2+ leak (Ca2+ sparks) and SR Ca2+ content)) as well as NCX function (caffeine-induced Ca2+ transient, TAU) and protein expression) were determined without an with the NCX inhibitor SEA0400 (in vivo: 1 mg/kgKG for 30 minutes/ in vitro: 300nM for 5 minutes incubation). Results: NXT rats showed stable compensated renal impairment and signs and symptoms of HFPEF (hypertrophied left ventricle (LV), left- and upward shift of end diastolic pressure (EDP) volume relationship, increased lung weight/body weight ratio indicating pulmonary congestion and preserved LV systolic function (EF, dP/dt)). In LV cardiomyocytes from NXT Ca2+ transient amplitude was unchanged but time for early (50%) decay was significantly prolonged at 24 weeks and correlated with diastolic dysfunction (EDP) in vivo. TAU of the caffeine transient was significantly prolonged at 24 weeks indicating reduced NCX forward mode activity, while NCX protein expression was up-regulated, suggesting increased NCX reverse mode activity. Ca2+ spark frequency was increased and SR Ca2+ content was decreased in NXT (p&lt;0.05). SEA0400 significantly accelerated Ca2+ transient decay and reduced Ca2+ spark frequency in NXT. Acute in vivo treatment with SEA0400 significantly enhanced active relaxation (isovolumetric relaxation constant) of the LV in NXT. Conclusion: This model of HFPEF is associated with prolonged cytosolic Ca2+ decay and increased diastolic Ca2+ leak in LV cardiomyocytes. Acute treatment with NCX inhibitor SEA0400 normalized cytosolic Ca2+ transients, improved net transsarcolemmal Ca2+ export and decreased SR Ca2+ leak in NXT, in line with a role for reverse mode NCX activity in HFPEF. In vivo active relaxation was improved after acute SEA0400 treatment.

The role of CaMKII regulation of phospholamban activity in heart disease

Phospholamban (PLN) is a phosphoprotein in cardiac sarcoplasmic reticulum (SR) that is a reversible regulator of the Ca2+-ATPase (SERCA2a) activity and cardiac contractility. Dephosphorylated PLN inhibits SERCA2a and PLN phosphorylation, at either Ser16 by PKA or Thr17 by Ca2+-calmodulin-dependent protein kinase (CaMKII), reverses this inhibition. Through this mechanism, PLN is a key modulator of SR Ca2+ uptake, Ca2+ load, contractility, and relaxation. PLN phosphorylation is also the main determinant of β1-adrenergic responses in the heart. Although phosphorylation of Thr17 by CaMKII contributes to this effect, its role is subordinate to the PKA-dependent increase in cytosolic Ca2+, necessary to activate CaMKII. Furthermore, the effects of PLN and its phosphorylation on cardiac function are subject to additional regulation by its interacting partners, the anti-apoptotic HAX-1 protein and Gm or the anchoring unit of protein phosphatase 1. Regulation of PLN activity by this multimeric complex becomes even more important in pathological conditions, characterized by aberrant Ca2+-cycling. In this scenario, CaMKII-dependent PLN phosphorylation has been associated with protective effects in both acidosis and ischemia/reperfusion. However, the beneficial effects of increasing SR Ca2+ uptake through PLN phosphorylation may be lost or even become deleterious, when these occur in association with alterations in SR Ca2+ leak. Moreover, a major characteristic in human and experimental heart failure (HF) is depressed SR Ca2+ uptake, associated with decreased SERCA2a levels and dephosphorylation of PLN, leading to decreased SR Ca2+ load and impaired contractility. Thus, the strategy of altering SERCA2a and/or PLN levels or activity to restore perturbed SR Ca2+ uptake is a potential therapeutic tool for HF treatment. We will review here the role of CaMKII-dependent phosphorylation of PLN at Thr17 on cardiac function under physiological and pathological conditions.

High inborn aerobic capacity does not protect the heart following myocardial infarction

Maximal oxygen uptake (Vo2max) is a strong prognostic marker for morbidity and mortality, but the cardio-protective effect of high inborn Vo2max remains unresolved. We aimed to investigate whether rats with high inborn Vo2max yield cardio-protection after myocardial infarction (MI) compared with rats with low inborn Vo2max. Rats breed for high capacity of running (HCR) or low capacity of running (LCR) were randomized into HCR-SH (sham), HCR-MI, LCR-SH, and LCR-MI. Vo2max was lower in HCR-MI and LCR-MI compared with respective sham (P < 0.01), supported by a loss in global cardiac function, assessed by echocardiography. Fura 2-AM loaded cardiomyocyte experiments revealed that HCR-MI and LCR-MI decreased cardiomyocyte shortening (39%, and 34% reduction, respectively, both P < 0.01), lowered Ca(2+) transient amplitude (37%, P < 0.01, and 20% reduction, respectively), and reduced sarcoplasmic reticulum (SR) Ca(2+) content (both; 20%, P < 0.01) compared with respective sham. Diastolic Ca(2+) cycling was impaired in HCR-MI and LCR-MI evidenced by prolonged time to 50% Ca(2+) decay that was partly explained by the 47% (P < 0.01) and 44% (P < 0.05) decrease in SR Ca(2+)-ATPase Ca(2+) removal, respectively. SR Ca(2+) leak increased by 177% in HCR-MI (P < 0.01) and 67% in LCR-MI (P < 0.01), which was abolished by inhibition of Ca(2+)/calmodulin-dependent protein kinase II. This study demonstrates that the effect of MI in HCR rats was similar or even more pronounced on cardiac- and cardiomyocyte contractile function, as well as on Ca(2+) handling properties compared with observations in LCR. Thus our data do not support a cardio-protective effect of higher inborn aerobic capacity.

Effects of the Inflammatory Cytokines TNF-α and IL-13 on Stromal Interaction Molecule–1 Aggregation in Human Airway Smooth Muscle Intracellular Ca2+ Regulation

Inflammation elevates intracellular Ca(2+) ([Ca(2+)]i) concentrations in airway smooth muscle (ASM). Store-operated Ca(2+) entry (SOCE) is an important source of [Ca(2+)]i mediated by stromal interaction molecule-1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca(2+) depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca(2+) release-activated Ca(2+) channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein-tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca(2+)-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca(2+) depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca(2+) depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca(2+)]i, and decreased SR Ca(2+) content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness.

Searching for Sudden Death SNPs in Calcium Handling Genes

Cardiac arrhythmias are a major clinical problem and lead to more than 250 000 sudden cardiac deaths (SCDs) each year in the United States alone.[1][1] Our ability to predict those at risk for sudden death remains poor. The majority of patients who die suddenly are not identified as being at high

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.

Reperfusion Ca2+ Waves in the Intact Heart: A Possible Trigger for the Generation of Reperfusion Arrhythmias

Abnormal intracellular Ca2+ cycling plays a key role in cardiac dysfunction, particularly during the setting of cardiac ischaemia/reperfusion (I/R). At the onset of reperfusion there is an increase in cytosolic Ca2+, which has been usually associated with an uncontrolled influx from the extracellular space. Ca2+ mishandling has been in turn related to reperfusion arrhythmias. However, the basic mechanism for these arrhythmias is still unresolved. We performed experiments to test the hypothesis that the increase in cytosolic Ca2+ at the onset of reperfusion originates in Ca2+ efflux from the sarcoplasmic reticulum (SR) and propagates through the cell as cytosolic Ca2+ waves serving as a trigger for the initiation of reperfusion arrhythmias. By using a combination of pulsed local-field fluorescence (PLFF) and laser scanning confocal microscopy in mouse intact hearts loaded with Rhod-2 and/or Mag-Fluo-4 (cytosolic and/or SR Ca2+ measurements) or Fluo-4 (Ca2+ sparks and waves) and submitted to global I/R (12/30 min), it was found that ischemia evoked an increase in cytosolic and SR Ca2+. This increase was associated with a significant rise in Ca2+ sparks relative to preischemia: 2.07 ± 0.33 vs. 1.13 ± 0.20 sp/sec/100μm (P<0.05, ANOVA). Reperfusion evoked an increment in cytosolic Ca2+ (Ca2+ bump) that was associated with a significant decrease in SR Ca2+ and in Ca2+ sparks with respect to ischemia and a significant increase in Ca2+ waves relative to ischemia and preischemia: 0.71± 0.14 vs. 0.38± 0.06 and 0.25 ± 0.04 w/sec/100μm. The results show the first direct evidence of an increase in Ca2+ sparks in ischemia that transform in Ca2+ waves during reperfusion. These waves may constitute a main trigger of reperfusion arrhythmias. Supported by NIH R01-HL-084487 and PICT/1903 (FONCYT).

TLR4 as receptor for HMGB1 induced muscle dysfunction in myositis

ObjectivesPolymyositis and dermatomyositis are characterised by muscle weakness and fatigue even in patients with normal muscle histology via unresolved pathogenic mechanisms. In this study, we investigated the mechanisms by which...