Occurrence of early afterdepolarization under healthy or hypertrophic cardiomyopathy conditions in the human ventricular endocardial myocyte: In silico study using 109 torsadogenic or non-torsadogenic compounds.

The goal of the CiPA initiative (Comprehensive in vitro Proarrhythmia Assay) was to assess a more accurate prediction of new drug candidate proarrhythmic severe liabilities such as torsades de pointes, for example. This new CiPA paradigm was partly based on in silico reconstruction of human ventricular cardiomyocyte action potential useful to identify repolarization abnormalities such early afterdepolarization (EAD), for example. Using the ToR-ORd algorithm (Tomek-Rodriguez-O'Hara-Rudy dynamic model), the aim of the present work was (i) to identify intracellular parameters leading to EAD occurrence under healthy and hypertrophic cardiomyopathy (HCM) conditions and (ii) to evaluate the prediction accuracy of compound torsadogenic risk based on EAD occurrence using a large set of 109 torsadogenic and non-torsadogenic compounds under both experimental conditions. In silico results highlighted the crucial involvement of Ca++ handling in the ventricular cardiomyocyte intracellular subspace compartment for the initiation of EAD, demonstrated by a higher amplitude of Ca++ release from junctional sarcoplasmic reticulum to subspace compartments (Jrel) measured at EAD take-off voltage in the presence vs. the absence of EAD initiated either by high IKr inhibition or by high enough concentration of a torsadogenic compound under both experimental conditions. Under healthy or HCM conditions, the prediction accuracy of the torsadogenic risk of compound based on EAD occurrence was observed to be 61 or 92%, respectively. This high accuracy under HCM conditions was discussed regarding its usefulness for cardiac safety pharmacology at least at early drug screening/preclinical stage of the drug development process.

Chrysosplenol-C Increases Contraction by Augmentation of Sarcoplasmic Reticulum Ca2+ Loading and Release via Protein Kinase C in Rat Ventricular Myocytes.

Naturally found chrysosplenol-C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) increases the contractility of cardiac myocytes independent of ß-adrenergic signaling. We investigated the cellular mechanism for chrysosplenol-C-induced positive inotropy. Global and local Ca2+ signals, L-type Ca2+ current (ICa), and contraction were measured from adult rat ventricular myocytes using two-dimensional confocal Ca2+ imaging, the whole-cell patch-clamp technique, and video-edge detection, respectively. Application of chrysosplenol-C reversibly increased Ca2+ transient magnitude with a maximal increase of ∼55% within 2- to 3-minute exposures (EC50 ≅ 21 µM). This chemical did not alter ICa and slightly increased diastolic Ca2+ level. The frequency and size of resting Ca2+ sparks were increased by chrysosplenol-C. Chrysosplenol-C significantly increased sarcoplasmic reticulum (SR) Ca2+ content but not fractional release. Pretreatment of protein kinase C (PKC) inhibitor but not Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor abolished the stimulatory effects of chrysosplenol-C on Ca2+ transients and Ca2+ sparks. Chrysosplenol-C-induced positive inotropy was removed by the inhibition of PKC but not CaMKII or phospholipase C. Western blotting assessment revealed that PKC-δ protein level in the membrane fractions significantly increase within 2 minutes after chrysosplenol-C exposure with a delayed (5-minute) increase in PKC-α levels in insoluble membrane. These results suggest that chrysosplenol-C enhances contractility via PKC (most likely PKC-δ)-dependent enhancement of SR Ca2+ releases in ventricular myocytes. SIGNIFICANCE STATEMENT: Study shows that chrysosplenol-C, a natural flavone showing a positive inotropic effect, increases SR Ca2+ releases on depolarizations and Ca2+ sparks with an increase of SR Ca2+ loading but not L-type Ca2+ current in ventricular myocytes. Chrysosplenol-C-induced enhancement in contraction is eliminated by PKC inhibition, and it is associated with redistributions of PKC to the membrane. These indicate that chrysosplenol-C enhances contraction via PKC-dependent augmentations of SR Ca2+ release and Ca2+ loading during action potentials.

Functional sympatholysis in mouse skeletal muscle involves sarcoplasmic reticulum swelling in arterial smooth muscle cells.

The vasoconstrictive effect of sympathetic activity is attenuated in contracting skeletal muscle (functional sympatholysis), allowing increased blood supply to the working muscle but the underlying mechanisms are incompletely understood. The purpose of this study was to examine α-adrenergic receptor responsiveness in isolated artery segments from non-exercised and exercised mice, using wire myography. Isometric tension recordings performed on femoral artery segments from exercised mice showed decreased α-adrenergic receptor responsiveness compared to non-exercised mice (logEC50 -5.2 ± 0.04 M vs. -5.7 ± 0.08 M, respectively). In contrast, mesenteric artery segments from exercised mice displayed similar α-adrenergic receptor responses compared to non-exercised mice. Responses to the vasoconstrictor serotonin (5-HT) and vasodilator isoprenaline, were similar in femoral artery segments from non-exercised and exercised mice. To study sarcoplasmic reticulum (SR) function, we examined arterial contractions induced by caffeine, which depletes SR Ca2+ and thapsigargin, which inhibits SR Ca2+ -ATPase (SERCA) and SR Ca2+ uptake. Arterial contractions to both caffeine and thapsigargin were increased in femoral artery segment from exercised compared to non-exercised mice. Furthermore, 3D electron microscopy imaging of the arterial wall showed SR volume/length ratio increased 157% in smooth muscle cells of the femoral artery from the exercised mice, whereas there was no difference in SR volume/length ratio in mesenteric artery segments. These results show that in arteries surrounding exercising muscle, the α-adrenergic receptor constrictions are blunted, which can be attributed to swollen smooth muscle cell SR's, likely due to increased Ca2+ content that is possibly reducing free intracellular Ca2+ available for contraction. Overall, this study uncovers a previously unknown mechanism underlying functional sympatholysis.

Abnormal Calcium Handling in Atrial Fibrillation Is Linked to Changes in Cyclic AMP Dependent Signaling.

Both, the decreased L-type Ca2+ current (ICa,L) density and increased spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), have been associated with atrial fibrillation (AF). In this study, we tested the hypothesis that remodeling of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the ß-agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to ß-adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation).

Effects of Bergamot Polyphenols on Mitochondrial Dysfunction and Sarcoplasmic Reticulum Stress in Diabetic Cardiomyopathy.

Cardiovascular disease is the leading cause of death and disability in the Western world. In order to safeguard the structure and the functionality of the myocardium, it is extremely important to adequately support the cardiomyocytes. Two cellular organelles of cardiomyocytes are essential for cell survival and to ensure proper functioning of the myocardium: mitochondria and the sarcoplasmic reticulum. Mitochondria are responsible for the energy metabolism of the myocardium, and regulate the processes that can lead to cell death. The sarcoplasmic reticulum preserves the physiological concentration of the calcium ion, and triggers processes to protect the structural and functional integrity of the proteins. The alterations of these organelles can damage myocardial functioning. A proper nutritional balance regarding the intake of macronutrients and micronutrients leads to a significant improvement in the symptoms and consequences of heart disease. In particular, the Mediterranean diet, characterized by a high consumption of plant-based foods, small quantities of red meat, and high quantities of olive oil, reduces and improves the pathological condition of patients with heart failure. In addition, nutritional support and nutraceutical supplementation in patients who develop heart failure can contribute to the protection of the failing myocardium. Since polyphenols have numerous beneficial properties, including anti-inflammatory and antioxidant properties, this review gathers what is known about the beneficial effects of polyphenol-rich bergamot fruit on the cardiovascular system. In particular, the role of bergamot polyphenols in mitochondrial and sarcoplasmic dysfunctions in diabetic cardiomyopathy is reported.

Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action.

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels.

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

[Figure: see text].

CaMKII inhibition has dual effects on spontaneous Ca2+ release and Ca2+ alternans in ventricular cardiomyocytes from mice with a gain-of-function RyR2 mutation.

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. We studied spontaneous Ca2+ release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+ release events in RyR2-R2474S cardiomyocytes exposed to the ß-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+ release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca2+ release but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca2+ release. Prolonged refractoriness of Ca2+ release leads to arrhythmogenic Ca2+ alternans. CaMKII inhibition promotes Ca2+ alternans by prolonging Ca2+ release refractoriness.

GSK-7975A, an inhibitor of Ca2+ release-activated calcium channels, depresses isometric contraction of mouse aorta.

GSK-7975A is described to inhibit stromal interaction molecule 1(STIM1)-mediated Ca2+ release-activated Ca2+ channels ORAI 1, ORAI 2 and ORAI 3 in different cell types. The present study investigated whether isometric contractions of mouse aortic segments were affected by this selective store-operated calcium channel inhibitor. Depending on the way by which Ca2+ influx pathways were activated during contraction, GSK-7975A inhibited contractility of mouse aortic segments with different affinity. When contractile effects were induced by depolarization as with elevated extracellular K+ and opening of voltage-gated calcium channels, the affinity was approximately 10 times lower than when contraction was elicited with Ca2+ influx via non-selective cation channels. GSK-7975A may repolarize the aortic smooth muscle cells by inhibiting non-selective cation channels, has no effect on IP3-mediated phenylephrine-induced phasic contractions or on refilling of the contractile sarcoplasmic reticulum Ca2+ store, but has significant effects on non-contractile store-operated Ca2+ influx.

Inhibition of lung cancer by vitamin D depends on downregulation of histidine-rich calcium-binding protein.

Introduction: Intrinsic vitamin D affects the proliferation, apoptosis, invasion, metastasis, and tumorigenesis of lung cancer by regulating tumor signaling pathways. Histidine-rich calcium-binding protein (HRC) maintains Ca2+ homeostasis, which plays crucial roles in the occurrence and development of cancer. Objectives: Our study aims to investigate the ability of vitamin D in the regulation of HRC and the role of HRC playing in lung cancer. Methods: We investigated the effects of vitamin D on lung cancer and the underlying mechanisms, by measuring HRC and vitamin D receptor (VDR) expression in lung cancer, paracancer, and normal tissues from patients using immunohistochemistry, western blotting, and real time RT-PCR. We transfected H460 lung cancer cells (supplemented or not with vitamin D) with PX458-HRC and pcDNA3.1-HRC plasmids and injected mice with lung cancer cells harboring pcDNA3.1-vector or pcDNA3.1-HRC plasmids. Results: Vitamin D inhibited HRC expression and H460 cell migration and proliferation, and promoted apoptosis compared with controls. The expression of HRC and VDR was significantly upregulated and downregulated, respectively, in lung cancer versus paracancer or normal tissues. Cell proliferation and migration were reduced, apoptotic cells were more and tumors were smaller in mice treated with vitamin D/cholecalciferol cholesterol emulsion (CCE) than in vitamin D/CCE+HRC+/+ mice. Conclusion: Vitamin D inhibited lung cancer tumor growth, migration, and proliferation by downregulating HRC.

Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle.

The Ragulator protein complex is critical for directing the Rag GTPase proteins and mTORC1 to the lysosome membrane mediating amino acid-stimulated protein synthesis. As there is a lack of evidence on alcohol's effect on the Rag-Ragulator complex as a possible mechanism for the development of alcoholic skeletal muscle wasting, the aim of our study was to examine alterations in various protein-protein complexes in the Rag-Ragulator pathway produced acutely by feeding and how these are altered by alcohol under in vivo conditions. Mice (C57Bl/6; adult males) were fasted, and then provided rodent chow for 30 min ("refed") or remained food-deprived ("fasted"). Mice subsequently received ethanol (3 g/kg ethanol) or saline intraperitoneally, and hindlimb muscles were collected 1 h thereafter for analysis. Refeeding-induced increases in myofibrillar and sarcoplasmic protein synthesis, and mTOR and S6K1 phosphorylation, were prevented by alcohol. This inhibition was not associated with a differential rise in the intracellular leucine concentration or plasma leucine or insulin levels. Alcohol increased the amount of the Sestrin1•GATOR2 complex in the fasted state and prevented the refeeding-induced decrease in Sestrin1•GATOR2 seen in control mice. Alcohol antagonized the increase in the RagA/C•Raptor complex formation seen in the refed state. Alcohol antagonized the increase in Raptor with immunoprecipitated LAMPTOR1 (part of the Ragulator complex) after refeeding and decreased the association of RagC with LAMPTOR1. Finally, alcohol increased the association of the V1 domain of v-ATPase with LAMPTOR1 and prevented the refeeding-induced decrease in v-ATPase V1 with LAMPTOR1. Overall, these data demonstrate that acute alcohol intake disrupts multiple protein-protein complexes within the Rag-Ragulator complex, which are associated with and consistent with the concomitant decline in nutrient-stimulated muscle protein synthesis under in vivo conditions.

Sarcoplasmic reticulum-mitochondria communication; implications for cardiac arrhythmia.

Sudden cardiac death due to ventricular tachyarrhythmias remains the major cause of mortality in the world. Heart failure, diabetic cardiomyopathy, old age-related cardiac dysfunction and inherited disorders are associated with enhanced propensity to malignant cardiac arrhythmias. Both defective mitochondrial function and abnormal intracellular Ca2+ homeostasis have been established as the key contributing factors in the pathophysiology and arrhythmogenesis in these conditions. This article reviews current advances in understanding of bidirectional control of ryanodine receptor-mediated sarcoplasmic reticulum Ca2+ release and mitochondrial function, and how defects in crosstalk between these two organelles increase arrhythmic risk in cardiac disease.

Sarcoplasmic reticular Ca2+-ATPase inhibition paradoxically upregulates murine skeletal muscle Nav1.4 function.

Skeletal muscle Na+ channels possess Ca2+- and calmodulin-binding sites implicated in Nav1.4 current (INa) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased INa. These findings were attributed to actions of consequently altered cytosolic Ca2+, [Ca2+]i, on Nav1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca2+]i, but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase. Loose patch clamping determined Na+ current (INa) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca2+]i perturbations. A bespoke flow system permitted continuous INa comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in INa within 1-4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in INa produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum INa by ~ 30%. It only slightly decreased half-maximal activating voltages (V0.5) and steepness factors (k), by 2 mV and 0.7, in contrast to the V0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca2+]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca2+]TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca2+ depletion.

Osteocytic Connexin43 Channels Regulate Bone-Muscle Crosstalk.

Bone-muscle crosstalk plays an important role in skeletal biomechanical function, the progression of numerous pathological conditions, and the modulation of local and distant cellular environments. Previous work has revealed that the deletion of connexin (Cx) 43 in osteoblasts, and consequently, osteocytes, indirectly compromises skeletal muscle formation and function. However, the respective roles of Cx43-formed gap junction channels (GJs) and hemichannels (HCs) in the bone-muscle crosstalk are poorly understood. To this end, we used two Cx43 osteocyte-specific transgenic mouse models expressing dominant negative mutants, Δ130-136 (GJs and HCs functions are inhibited), and R76W (only GJs function is blocked), to determine the effect of these two types of Cx43 channels on neighboring skeletal muscle. Blockage of osteocyte Cx43 GJs and HCs in Δ130-136 mice decreased fast-twitch muscle mass with reduced muscle protein synthesis and increased muscle protein degradation. Both R76W and Δ130-136 mice exhibited decreased muscle contractile force accompanied by a fast-to-slow fiber transition in typically fast-twitch muscles. In vitro results further showed that myotube formation of C2C12 myoblasts was inhibited after treatment with the primary osteocyte conditioned media (PO CM) from R76W and Δ130-136 mice. Additionally, prostaglandin E2 (PGE2) level was significantly reduced in both the circulation and PO CM of the transgenic mice. Interestingly, the injection of PGE2 to the transgenic mice rescued fast-twitch muscle mass and function; however, this had little effect on protein synthesis and degradation. These findings indicate a channel-specific response: inhibition of osteocytic Cx43 HCs decreases fast-twitch skeletal muscle mass alongside reduced protein synthesis and increased protein degradation. In contrast, blockage of Cx43 GJs results in decreased fast-twitch skeletal muscle contractile force and myogenesis, with PGE2 partially accounting for the measured differences.

Depressed ß-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients' induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+ -handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to ß-adrenergic stimulation. To test the hypothesis, [Ca2+ ]i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca2+ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the ß1-adrenergic receptor (ADRß1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed ß-adrenergic responsiveness.

Orthograde signal of dihydropyridine receptor increases Ca2+ leakage after repeated contractions in rat fast-twitch muscles in vivo.

The purpose of this study was to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers and SR microsomes were prepared from the muscles excised 30 min after repeated high-intensity contractions. The mechanically skinned fibers maintained the interaction between dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), whereas the SR microsomes did not. Interestingly, skinned fibers from the stimulated muscles showed increased SR Ca2+ leakage, whereas Ca2+ leakage decreased in SR microsomes from the stimulated muscles. To enhance the orthograde signal of DHPRs, SR Ca2+ leakage in the skinned fiber was measured 1) under a continuously depolarized condition and 2) in the presence of nifedipine. As a result, in either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, the increased SR Ca2+ leakage from the stimulated fibers was alleviated by treatment with 1 mM tetracaine (Tet) but not by treatment with 3 mM free Mg2+ (3 Mg). Tet exerted a greater inhibitory effect on the DHPR signal to RyR than 3 Mg, although their inhibitory effects on RyR were almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RyRs.

Efavirenz, atazanavir, and ritonavir disrupt sarcoplasmic reticulum Ca2+ homeostasis in skeletal muscles.

While muscle fatigue, pain and weakness are common co-morbidities in HIV-1 infected people, their underlying cause remain poorly defined. To this end, we evaluated whether the common antiretroviral drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) could be a contributing factor by pertubating sarcoplasmic reticulum (SR) Ca2+ cycling. In live-cell imaging, EFV (6.0 µM), ATV (6.0 µM), and RTV (3.0 µM) elicited Ca2+ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca2+ release channel blocker ryanodine (50 µM), slowed the rate and amplitude of Ca2+ release from and reuptake of Ca2+ into the SR. EFV, ATV and RTV (1 nM - 20 µM) potentiated and then displaced [3H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca2+ release channel (RyR1). These drugs at concentrations 0.25-31.2 µM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤5 µM) potentiated and >5µM inhibited the ability of sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA1) to hydrolyze ATP and transport Ca2+. RTV (2.5-31.5 µM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca2+ transport. EFV (0.25-31.5 µM) had no measurable effect on SERCA1's ability to hydrolyze ATP and transport Ca2+. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca2+ homeostasis.

Modulating ALDH2 reveals a differential dependence on ROS for hypertrophy and SR Ca2+ release in aldosterone-treated cardiac myocytes.

Growing evidence links high aldosterone levels with atrial fibrillation and other heart diseases. Here, we have investigated the functional consequences of culturing adult rat atrial myocytes with aldosterone, at the level of cell size, homeostasis of Ca2+, reactive oxygen species (ROS), and nitrogen oxide (NO). The protein levels of NO synthase (NOS), aldehyde dehydrogenase 2 (ALDH2), NADPH oxidase (NOX), and Na+-Ca2+ exchanger (NCX) were also studied. Aldosterone did not alter the expression of these proteins, except for the NCX, which was enhanced by nearly 100%. Additionally, the hormone inhibited and stimulated, respectively, the production of NO and ROS (the effect on ROS appeared after 24 h of treatment and reached a maximum by 4-6 days, with an EC50 of 1.2 nM). These changes in reactive species generation were blunted by tetrahydrobiopterin (BH4, a NOS cofactor), suggesting the involvement of an uncoupled NOS. An activator (Alda-1) and an inhibitor (daidzin) of ALDH2 were used, to determine if this enzyme activity is related to aldosterone effects, through possible modulation of ROS. Aldosterone produced a ∼10% increase in cell size and, remarkably, this hypertrophic effect, along with the corresponding changes in ROS and NO, were all mimicked by daidzin and prevented by Alda-1. Something different happened with SR Ca2+ release. Aldosterone increased both the magnitude of Ca2+ transients and the incidence of spontaneous Ca2+ oscillations, but these actions were not reproduced by daidzin. Moreover, rather than being prevented, they were further promoted by Alda-1, which also increased the rate of SR Ca2+ reuptake. These results suggest that NOS and ALDH2 may prevent some adverse consequences of aldosteronism (in the case of ALDH2, at the expense of exacerbating SR Ca2+ release). Our data also suggest a hierarchical model in which aldosterone promotes: SR Ca2+ release, then ROS production, and finally hypertrophy.

Stretch-induced sarcoplasmic reticulum calcium leak is causatively associated with atrial fibrillation in pressure-overloaded hearts.

AIMS: Despite numerous reports documenting an important role of hypertension in the development of atrial fibrillation (AF), the detailed mechanism underlying the pathological process remains incompletely understood. Here, we aim to test the hypothesis that diastolic sarcoplasmic reticulum (SR) Ca2+ leak in atrial myocytes, induced by mechanical stretch due to elevated pressure in the left atrium (LA), plays an essential role in the AF development in pressure-overloaded hearts. METHODS AND RESULTS: Isolated mouse atrial myocytes subjected to acute axial stretch displayed an immediate elevation of SR Ca2+ leak. Using a mouse model of transverse aortic constriction (TAC), the relation between stretch, SR Ca2+ leak, and AF susceptibility was further tested. At 36 h post-TAC, SR Ca2+ leak in cardiomyocytes from the LA (with haemodynamic stress), but not right atrium (without haemodynamic stress), significantly increased, which was further elevated at 4 weeks post-TAC. Accordingly, AF susceptibility to atrial burst pacing in the 4-week TAC mice were also significantly increased, which was unaffected by inhibition of atrial fibrosis or inflammation via deletion of galectin-3. Western blotting revealed that type 2 ryanodine receptor (RyR2) in left atrial myocytes of TAC mice was oxidized due to activation and up-regulation of Nox2 and Nox4. Direct rescue of dysfunctional RyR2 with dantrolene or rycal S107 reduced diastolic SR Ca2+ leak in left atrial myocytes and prevented atrial burst pacing stimulated AF. CONCLUSION: Our study demonstrated for the first time the increased SR Ca2+ leak mediated by enhanced oxidative stress in left atrial myocytes that is causatively associated with higher AF susceptibility in pressure-overloaded hearts.

Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.