Activation of small conductance Ca2+ -activated K+ channels suppresses Ca2+ transient and action potential alternans in ventricular myocytes.

At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We tested the hypothesis that in single rabbit ventricular myocytes pharmacological modulation of SK channels plays a causative role for the development of pacing-induced CaT and AP duration (APD) alternans. SK channel blockers (apamin, UCL1684) had only a minor effect on AP repolarization. However, SK channel activation by NS309 resulted in significant APD shortening, demonstrating that functional SK channels are well expressed in ventricular myocytes. The effects of NS309 were prevented or reversed by apamin and UCL1684, indicating that NS309 acted on SK channels. SK channel activation abolished or reduced the degree of pacing-induced CaT and APD alternans. Inhibition of KV 7.1 (with HMR1556) and KV 11.1 (with E4031) channels was used to mimic conditions of long QT syndromes type-1 and type-2, respectively. Both HMR1556 and E4031 enhanced CaT alternans that was prevented by SK channel activation. In AP voltage-clamped cells the SK channel activator had no effect on CaT alternans, confirming that suppression of CaT alternans was caused by APD shortening. APD shortening contributed to protection from alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest that SK activation could be a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy for patients with long QT syndrome. KEY POINTS: At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and intracellular Ca2+ release amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We investigated whether pharmacological modulation of SK channels affects the development of cardiac alternans in normal ventricular cells and in cells with drug-induced long QT syndrome (LQTS). While SK channel blockers have only a minor effect on AP morphology, their activation leads to AP shortening and abolishes or reduces the degree of pacing-induced Ca2+ and AP alternans. AP shortening contributed to protection against alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest SK activation as a potential intervention to avert the development of alternans with important ramifications for arrhythmia prevention for patients with LQTS.

Inositol 1,4,5-trisphosphate receptor - reactive oxygen signaling domain regulates excitation-contraction coupling in atrial myocytes.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is up-regulated in patients with atrial fibrillation (AF) and InsP3-induced Ca2+ release (IICR) is linked to pro-arrhythmic spontaneous Ca2+ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP3Rs in atrial muscle is still limited. We hypothesize that InsP3R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP3R agonist affinity and thereby Ca2+ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2-/-) mice and changes in the cytoplasmic Ca2+ concentration ([Ca2+]i; fluo-4/AM, indo-1) or ROS (2',7'-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 µmol/L) significantly increased diastolic [Ca2+]i (F/F0, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca2+ transient (CaT) amplitude (ΔF/F0, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca2+]i. These changes in [Ca2+]i were suppressed by the InsP3R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 µmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 µmol/L). In NOX2-/- AMs, AngII failed to increase diastolic [Ca2+]i, CaT amplitude, and the frequency of spontaneous Ca2+ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP3 was abolished by NOX inhibition with apocynin (1 µM). AngII induced IICR in Nox2-/- AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 µmol/L). In saponin permeabilized AMs InsP3 (5 µmol/L) induced Ca2+ sparks that increased in frequency in the presence of ROS (InsP3: 9.65 ± 1.44 sparks*s-1*(100µm)-1; InsP3 + tBHP: 10.77 ± 1.5 sparks*s-1*(100µm)-1; n = 5; p < 0.05). The combined effect of InsP3 + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP3R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP3R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP3R is under dual control of agonist induced InsP3 and ROS formation and suggest that InsP3 and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP3R/NOX2 signaling domain.

Mitochondrial calcium uniporter complex activation protects against calcium alternans in atrial myocytes.

Cardiac alternans, defined as beat-to-beat alternations in action potential duration, cytosolic Ca transient (CaT) amplitude, and cardiac contraction is associated with atrial fibrillation (AF) and sudden cardiac death. At the cellular level, cardiac alternans is linked to abnormal intracellular calcium handling during excitation-contraction coupling. We investigated how pharmacological activation or inhibition of cytosolic Ca sequestration via mitochondrial Ca uptake and mitochondrial Ca retention affects the occurrence of pacing-induced CaT alternans in isolated rabbit atrial myocytes. Cytosolic CaTs were recorded using Fluo-4 fluorescence microscopy. Alternans was quantified as the alternans ratio (AR = 1 - CaTsmall/CaTlarge, where CaTsmall and CaTlarge are the amplitudes of the small and large CaTs of a pair of alternating CaTs). Inhibition of mitochondrial Ca sequestration via mitochondrial Ca uniporter complex (MCUC) with Ru360 enhanced the severity of CaT alternans (AR increase) and lowered the pacing frequency threshold for alternans. In contrast, stimulation of MCUC mediated mitochondrial Ca uptake with spermine-rescued alternans (AR decrease) and increased the alternans pacing threshold. Direct measurement of mitochondrial [Ca] in membrane permeabilized myocytes with Fluo-4 loaded mitochondria revealed that spermine enhanced and accelerated mitochondrial Ca uptake. Stimulation of mitochondrial Ca retention by preventing mitochondrial Ca efflux through the mitochondrial permeability transition pore with cyclosporin A also protected from alternans and increased the alternans pacing threshold. Pharmacological manipulation of MCUC activity did not affect sarcoplasmic reticulum Ca load. Our results suggest that activation of Ca sequestration by mitochondria protects from CaT alternans and could be a potential therapeutic target for cardiac alternans and AF prevention.NEW & NOTEWORTHY This study provides conclusive evidence that mitochondrial Ca uptake and retention protects from Ca alternans, whereas uptake inhibition enhances Ca alternans. The data suggest pharmacological mitochondrial Ca cycling modulation as a potential therapeutic strategy for alternans-related cardiac arrhythmia prevention.

The role of fibroblast - Cardiomyocyte interaction for atrial dysfunction in HFpEF and hypertensive heart disease.

AIMS: Atrial contractile dysfunction is associated with increased mortality in heart failure (HF). We have shown previously that a metabolic syndrome-based model of HFpEF and a model of hypertensive heart disease (HHD) have impaired left atrial (LA) function in vivo (rat). In this study we postulate, that left atrial cardiomyocyte (CM) and cardiac fibroblast (CF) paracrine interaction related to the inositol 1,4,5-trisphosphate signalling cascade is pivotal for the manifestation of atrial mechanical dysfunction in HF and that quantitative atrial remodeling is highly disease-dependent. METHODS AND RESULTS: Differential remodeling was observed in HHD and HFpEF as indicated by an increase of atrial size in vivo (HFpEF), unchanged fibrosis (HHD and HFpEF) and a decrease of CM size (HHD). Baseline contractile performance of rat CM in vitro was enhanced in HFpEF. Upon treatment with conditioned medium from their respective stretched CF (CM-SF), CM (at 21 weeks) of WT showed increased Ca2+ transient (CaT) amplitudes related to the paracrine activity of the inotrope endothelin (ET-1) and inositol 1,4,5-trisphosphate induced Ca2+ release. Concentration of ET-1 was increased in CM-SF and atrial tissue from WT as compared to HHD and HFpEF. In HHD, CM-SF had no relevant effect on CaT kinetics. However, in HFpEF, CM-SF increased diastolic Ca2+ and slowed Ca2+ removal, potentially contributing to an in-vivo decompensation. During disease progression (i.e. at 27 weeks), HFpEF displayed dysfunctional excitation-contraction-coupling (ECC) due to lower sarcoplasmic-reticulum Ca2+ content unrelated to CF-CM interaction or ET-1, but associated with enhanced nuclear [Ca2+]. In human patients, tissue ET-1 was not related to the presence of arterial hypertension or obesity. CONCLUSIONS: Atrial remodeling is a complex entity that is highly disease and stage dependent. The activity of fibrosis related to paracrine interaction (e.g. ET-1) might contribute to in vitro and in vivo atrial dysfunction. However, during later stages of disease, ECC is impaired unrelated to CF.

Action potential shortening rescues atrial calcium alternans.

KEY POINTS: Cardiac alternans refers to a beat-to-beat alternation in contraction, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and represents a risk factor for cardiac arrhythmia, including atrial fibrillation. We developed strategies to pharmacologically manipulate the AP waveform with the goal to reduce or eliminate the occurrence of CaT and contraction alternans in atrial tissue. With combined patch-clamp and intracellular Ca2+ measurements we investigated the effect of specific ion channel inhibitors and activators on alternans. In single rabbit atrial myocytes, suppression of Ca2+ -activated Cl- channels eliminated AP duration alternans, but prolonged the AP and failed to eliminate CaT alternans. In contrast, activation of K+ currents (IKs and IKr ) shortened the AP and eliminated both AP duration and CaT alternans. As demonstrated also at the whole heart level, activation of K+ conductances represents a promising strategy to suppress alternans, and thus reducing a risk factor for atrial fibrillation. ABSTRACT: At the cellular level alternans is observed as beat-to-beat alternations in contraction, action potential (AP) morphology and magnitude of the Ca2+ transient (CaT). Alternans is a well-established risk factor for cardiac arrhythmia, including atrial fibrillation. This study investigates whether pharmacological manipulation of AP morphology is a viable strategy to reduce the risk of arrhythmogenic CaT alternans. Pacing-induced AP and CaT alternans were studied in rabbit atrial myocytes using combined Ca2+ imaging and electrophysiological measurements. Increased AP duration (APD) and beat-to-beat alternations in AP morphology lowered the pacing frequency threshold and increased the degree of CaT alternans. Inhibition of Ca2+ -activated Cl- channels reduced beat-to-beat AP alternations, but prolonged APD and failed to suppress CaT alternans. In contrast, AP shortening induced by activators of two K+ channels (ML277 for Kv7.1 and NS1643 for Kv11.1) abolished both APD and CaT alternans in field-stimulated and current-clamped myocytes. K+ channel activators had no effect on the degree of Ca2+ alternans in AP voltage-clamped cells, confirming that suppression of Ca2+ alternans was caused by the changes in AP morphology. Finally, activation of Kv11.1 channel significantly attenuated or even abolished atrial T-wave alternans in isolated Langendorff perfused hearts. In summary, AP shortening suppressed or completely eliminated both CaT and APD alternans in single atrial myocytes and atrial T-wave alternans at the whole heart level. Therefore, we suggest that AP shortening is a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy.

Membrane potential determines calcium alternans through modulation of SR Ca2+ load and L-type Ca2+ current.

Alternans is a risk factor for cardiac arrhythmia, including atrial fibrillation. At the cellular level alternans is observed as beat-to-beat alternations in contraction, action potential (AP) morphology and magnitude of the Ca2+ transient (CaT). It is widely accepted that the bi-directional interplay between membrane voltage and Ca2+ is crucial for the development of alternans, however recently the attention has shifted to instabilities in cellular Ca2+ handling, while the role of AP alternation remains poorly understood. This study provides new insights how beat- to-beat alternation in AP morphology affects occurrence of CaT alternans in atrial myocytes. Pacing-induced AP and CaT alternans were studied in rabbit atrial myocytes using combined Ca2+ imaging and electrophysiological measurements. To determine the role of AP morphology for the generation of CaT alternans, trains of two voltage commands in form of APs recorded during large and small alternans CaTs were applied to voltage-clamped cells. APs of longer duration (as observed during small amplitude alternans CaT) and especially beat-to-beat alternations in AP morphology (AP alternans) reduced the pacing frequency threshold and increased the degree of CaT alternans. AP morphology contributes to the development of CaT alternans by two mechanisms. First, the AP waveform observed during small alternans CaTs coincided with higher end-diastolic sarcoplasmic reticulum Ca2+ levels ([Ca2+]SR), and AP alternans resulted in beat-to-beat alternations in end-diastolic [Ca2+]SR. Second, L-type Ca2+ current was significantly affected by AP morphology, where the AP waveform observed during large CaT elicited L-type Ca2+ currents of higher magnitude and faster kinetics, resulting in more efficient triggering of SR Ca2+ release. In conclusion, alternation in AP morphology plays a significant role in the development and stabilization of atrial alternans. The demonstration that CaT alternans can be controlled or even prevented by modulating AP morphology has important ramifications for arrhythmia prevention and therapy strategies.

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes.

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2+] and intra-SR [Ca2+]SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10µM) increased Ca2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10µM) abolished the cAMP-dependent increase of SR Ca2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2+ leak.

A novel mechanism of tandem activation of ryanodine receptors by cytosolic and SR luminal Ca2+ during excitation-contraction coupling in atrial myocytes.

KEY POINTS: In atrial myocytes excitation-contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca2+ release starts in the cell periphery and propagates towards the cell centre by Ca2+ -induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store. The cytosolic Ca2+ sensitivity of the ryanodine receptor (RyRs) Ca2+ release channel is low and it is unclear how Ca2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra-SR calcium revealed a transient elevation of store Ca2+ that we termed 'Ca2+ sensitization signal'. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca2+ through a 'fire-diffuse-uptake-fire' (or FDUF) mechanism: Ca2+ uptake by SR Ca2+ pumps at the propagation front elevates Ca2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca2+ activation threshold. ABSTRACT: In atrial myocytes Ca2+ release during excitation-contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca2+ store into the peripheral subsarcolemmnal junctional (j-SR) and the much more abundant central non-junctional (nj-SR) SR. Action potential (AP)-induced Ca2+ entry activates Ca2+ -induced Ca2+ release (CICR) from j-SR ryanodine receptor (RyR) Ca2+ release channels. Peripheral elevation of [Ca2+ ]i initiates CICR from nj-SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca2+ ]i ; with the fluorescent Ca2+ indicator rhod-2) and intra-SR ([Ca2+ ]SR ; fluo-5N) Ca2+ in rabbit atrial myocytes revealed that Ca2+ release from j-SR resulted in a cytosolic Ca2+ transient of higher amplitude compared to release from nj-SR; however, the degree of depletion of j-SR [Ca2+ ]SR was smaller than nj-SR [Ca2+ ]SR . Similarly, Ca2+ signals from individual release sites of the j-SR showed a larger cytosolic amplitude (Ca2+ sparks) but smaller depletion (Ca2+ blinks) than release from nj-SR. During AP-induced Ca2+ release the rise of [Ca2+ ]i detected at individual release sites of the nj-SR preceded the depletion of [Ca2+ ]SR , and during this latency period a transient elevation of [Ca2+ ]SR occurred. We propose that Ca2+ release from nj-SR is activated by cytosolic and luminal Ca2+ (tandem RyR activation) via a novel 'fire-diffuse-uptake-fire' (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca2+ uptake by sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) at the propagation front elevates local [Ca2+ ]SR , leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR.

The mechanisms of calcium cycling and action potential dynamics in cardiac alternans.

RATIONALE: Alternans is a risk factor for cardiac arrhythmia, including atrial fibrillation. At the cellular level alternans manifests as beat-to-beat alternations in contraction, action potential duration (APD), and magnitude of the Ca(2+) transient (CaT). Electromechanical and CaT alternans are highly correlated, however, it has remained controversial whether the primary cause of alternans is a disturbance of cellular Ca(2+) signaling or electrical membrane properties. OBJECTIVE: To determine whether a primary failure of intracellular Ca(2+) regulation or disturbances in membrane potential and AP regulation are responsible for the occurrence of alternans in atrial myocytes. METHODS AND RESULTS: Pacing-induced APD and CaT alternans were studied in single rabbit atrial and ventricular myocytes using combined [Ca(2+)]i and electrophysiological measurements. In current-clamp experiments, APD and CaT alternans strongly correlated in time and magnitude. CaT alternans was observed without alternation in L-type Ca(2+) current, however, elimination of intracellular Ca(2+) release abolished APD alternans, indicating that [Ca(2+)]i dynamics have a profound effect on the occurrence of CaT alternans. Trains of 2 distinctive voltage commands in form of APs recorded during large and small alternans CaTs were applied to voltage-clamped cells. CaT alternans was observed with and without alternation in the voltage command shape. During alternans AP-clamp large CaTs coincided with both long and short AP waveforms, indicating that CaT alternans develop irrespective of AP dynamics. CONCLUSIONS: The primary mechanism underlying alternans in atrial cells, similarly to ventricular cells, resides in a disturbance of Ca(2+) signaling, whereas APD alternans are a secondary consequence, mediated by Ca(2+)-dependent AP modulation.

Harnessing the Power of Integrated Mitochondrial Biology and Physiology: A Special Report on the NHLBI Mitochondria in Heart Diseases Initiative.

Mitochondrial biology is the sum of diverse phenomena from molecular profiles to physiological functions. A mechanistic understanding of mitochondria in disease development, and hence the future prospect of clinical translations, relies on a systems-level integration of expertise from multiple fields of investigation. Upon the successful conclusion of a recent National Institutes of Health, National Heart, Lung, and Blood Institute initiative on integrative mitochondrial biology in cardiovascular diseases, we reflect on the accomplishments made possible by this unique interdisciplinary collaboration effort and exciting new fronts on the study of these remarkable organelles.

Tissue Specificity: SOCE: Implications for Ca2+ Handling in Endothelial Cells.

Many cellular functions of the vascular endothelium are regulated by fine-tuned global and local, microdomain-confined changes of cytosolic free Ca2+ ([Ca2+]i). Vasoactive agonist-induced stimulation of vascular endothelial cells (VECs) typically induces Ca2+ release through IP3 receptor Ca2+ release channels embedded in the membrane of the endoplasmic reticulum (ER) Ca2+ store, followed by Ca2+ entry from the extracellular space elicited by Ca2+ store depletion and referred to as capacitative or store-operated Ca2+ entry (SOCE). In vascular endothelial cells, SOCE is graded with the degree of store depletion and controlled locally in the subcellular microdomain where depletion occurs. SOCE provides distinct Ca2+ signals that selectively control specific endothelial functions: in calf pulmonary artery endothelial cells, the SOCE Ca2+ signal drives nitric oxide (an endothelium-derived relaxing factor of the vascular smooth muscle) production and controls activation and nuclear translocation of the transcription factor NFAT. Both cellular events are not affected by Ca2+ signals of comparable magnitude arising directly from Ca2+ release from intracellular stores, clearly indicating that SOCE regulates specific Ca2+-dependent cellular tasks by a unique and exclusive mechanism. This review discusses the mechanisms of intracellular Ca2+ regulation in vascular endothelial cells and the role of store-operated Ca2+ entry for endothelium-dependent smooth muscle relaxation and nitric oxide signaling, endothelial oxidative stress response, and excitation-transcription coupling in the vascular endothelium.

Alternans in atria: Mechanisms and clinical relevance.

Atrial fibrillation is the most common sustained arrhythmia and its prevalence is rapidly rising with the aging of the population. Cardiac alternans, defined as cyclic beat-to-beat alternations in contraction force, action potential (AP) duration and intracellular Ca2+ release at constant stimulation rate, has been associated with the development of ventricular arrhythmias. Recent clinical data also provide strong evidence that alternans plays a central role in arrhythmogenesis in atria. The aim of this article is to review the mechanisms that are responsible for repolarization alternans and contribute to the transition from spatially concordant alternans to the more arrhythmogenic spatially discordant alternans in atria.

Calcium-activated chloride current determines action potential morphology during calcium alternans in atrial myocytes.

KEY POINTS: Cardiac alternans--periodic beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic calcium transient (CaT) amplitude--is a high risk indicator for cardiac arrhythmias and sudden cardiac death. However, it remains an unresolved issue whether beat-to-beat alternations in intracellular Ca(2+) ([Ca(2+)]i ) or AP morphology are the primary cause of pro-arrhythmic alternans. Here we show that in atria AP alternans occurs secondary to CaT alternans. CaT alternans leads to complex beat-to-beat changes in Ca(2+)-regulated ion currents that determine alternans of AP morphology. We report the novel finding that alternans of AP morphology is largely sustained by the activity of Ca(2+)-activated Cl(-) channels (CaCCs). Suppression of the CaCCs significantly reduces AP alternans, while CaT alternans remains unaffected. The demonstration of a major role of CaCCs in the development of AP alternans opens new possibilities for atrial alternans and arrhythmia prevention. Cardiac alternans, described as periodic beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias and sudden cardiac death. We investigated mechanisms of cardiac alternans in single rabbit atrial myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. Beat-to-beat alternations of AP morphology and CaT amplitude revealed a strong quantitative correlation. Application of voltage clamp protocols in the form of pre-recorded APs (AP-clamp) during pacing-induced CaT alternans revealed a Ca(2+)-dependent current consisting of a large outward component (4.78 ± 0.58 pA pF(-1) in amplitude) coinciding with AP phases 1 and 2 that was followed by an inward current (-0.42 ± 0.03 pA pF(-1); n = 21) during AP repolarization. Approximately 90% of the initial outward current was blocked by substitution of Cl(-) ions or application of the Cl(-) channel blocker DIDS identifying it as a Ca(2+)-activated Cl(-) current (ICaCC). The prominent AP prolongation at action potential duration at 30% repolarization level during the small alternans CaT was due to reduced ICaCC. Inhibition of Cl(-) currents abolished AP alternans, but failed to affect CaT alternans, indicating that disturbances in Ca(2+) signalling were the primary event leading to alternans, and ICaCC played a decisive role in shaping the beat-to-beat alternations in AP morphology observed during alternans.

Inositol-1,4,5-trisphosphate induced Ca2+ release and excitation-contraction coupling in atrial myocytes from normal and failing hearts.

KEY POINTS: Impaired calcium (Ca(2+)) signalling is the main contributor to depressed ventricular contractile function and occurrence of arrhythmia in heart failure (HF). Here we report that in atrial cells of a rabbit HF model, Ca(2+) signalling is enhanced and we identified the underlying cellular mechanisms. Enhanced Ca(2+) transients (CaTs) are due to upregulation of inositol-1,4,5-trisphosphate receptor induced Ca(2+) release (IICR) and decreased mitochondrial Ca(2+) sequestration. Enhanced IICR, however, together with an increased activity of the sodium-calcium exchange mechanism, also facilitates spontaneous Ca(2+) release in form of arrhythmogenic Ca(2+) waves and spontaneous action potentials, thus enhancing the arrhythmogenic potential of atrial cells. Our data show that enhanced Ca(2+) signalling in HF provides atrial cells with a mechanism to improve ventricular filling and to maintain cardiac output, but also increases the susceptibility to develop atrial arrhythmias facilitated by spontaneous Ca(2+) release. ABSTRACT: We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca(2+) release in normal and heart failure (HF) rabbit atrial cells. Left ventricular HF was induced by combined volume and pressure overload. In HF atrial myocytes diastolic [Ca(2+)]i was increased, action potential (AP)-induced Ca(2+) transients (CaTs) were larger in amplitude, primarily due to enhanced Ca(2+) release from central non-junctional sarcoplasmic reticulum (SR) and centripetal propagation of activation was accelerated, whereas HF ventricular CaTs were depressed. The larger CaTs were due to enhanced IP3 receptor-induced Ca(2+) release (IICR) and reduced mitochondrial Ca(2+) buffering, consistent with a reduced mitochondrial density and Ca(2+) uptake capacity in HF. Elementary IP3 receptor-mediated Ca(2+) release events (Ca(2+) puffs) were more frequent in HF atrial myoctes and were detected more often in central regions of the non-junctional SR compared to normal cells. HF cells had an overall higher frequency of spontaneous Ca(2+) waves and a larger fraction of waves (termed arrhythmogenic Ca(2+) waves) triggered APs and global CaTs. The higher propensity of arrhythmogenic Ca(2+) waves resulted from the combined action of enhanced IICR and increased activity of sarcolemmal Na(+)-Ca(2+) exchange depolarizing the cell membrane. In conclusion, the data support the hypothesis that in atrial myocytes from hearts with left ventricular failure, enhanced CaTs during ECC exert positive inotropic effects on atrial contractility which facilitates ventricular filling and contributes to maintaining cardiac output. However, HF atrial cells were also more susceptible to developing arrhythmogenic Ca(2+) waves which might form the substrate for atrial rhythm disorders frequently encountered in HF.

NFAT transcription factor regulation by urocortin II in cardiac myocytes and heart failure.

Urocortin II (UcnII), a cardioactive peptide with beneficial effects in normal and failing hearts, is also arrhythmogenic and prohypertrophic. We demonstrated that cardiac effects are mediated by a phosphatidylinositol-3 kinase (PI3K)/Akt kinase (Akt)/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathways. Nuclear factor of activated T-cells (NFAT) transcription factors play a key role in the regulation of gene expression in cardiac development, maintenance of an adult differentiated cardiac phenotype, and remodeling processes in cardiac hypertrophy and heart failure (HF). We tested the hypothesis that UcnII differentially regulates NFAT activity in cardiac myocytes from both normal and failing hearts through the PI3K/Akt/eNOS/NO pathway. Isoforms NFATc1 and NFATc3 revealed different basal subcellular distribution in normal and HF rabbit ventricular myocytes with a nuclear NFATc1 and a cytosolic localization of NFATc3. However, in HF, the nuclear localization of NFATc1 was less pronounced, whereas the nuclear occupancy of NFATc3 was increased. In normal myocytes, UcnII induced nuclear export of NFATc1 and attenuated NFAT-dependent transcriptional activity but did not affect the distribution of NFATc3. In HF UcnII facilitated nuclear export of both isoforms and reduced transcriptional activity. NFAT regulation was mediated by a PI3K/Akt/eNOS/NO signaling cascade that converged on the activation of several kinases, including glycogen synthase kinase-3ß (GSK3ß), c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated kinase (p38), and PKG, resulting in phosphorylation, deactivation, and nuclear export of NFAT. In conclusion, while NFATc1 and NFATc3 reveal distinct subcellular distribution patterns, both are regulated by the UcnII-PI3K/Akt/eNOS/NO pathway that converges on the activation of NFAT kinases and NFAT inactivation. The data reconcile cardioprotective and prohypertrophic UcnII effects mediated by different NFAT isoforms.

Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.

Cardiac alternans and intracellular calcium cycling.

Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca(2+) transient amplitude (Ca(2+) alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (Vm ) and intracellular calcium ([Ca(2+) ]i ). Bidirectional coupling refers to the fact that, in cardiac cells, Vm depolarization and the generation of action potentials cause the elevation of [Ca(2+) ]i that is required for contraction (a process referred to as excitation-contraction coupling); conversely, changes of [Ca(2+) ]i control Vm because important membrane currents are Ca(2+) dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca(2+) signalling. Herein we review how two key factors of cardiac cellular Ca(2+) cycling, namely the release of Ca(2+) from internal stores and the capability of clearing the cytosol from Ca(2+) after each beat, determine the conditions under which alternans occurs. The contributions from key Ca(2+) -handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca(2+) release channels) are discussed.

Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes.

Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternations in CaT amplitude and is causally linked to atrial fibrillation (AF). Mitochondria play a significant role in cardiac excitation-contraction coupling and Ca signaling through redox environment regulation. In isolated rabbit atrial myocytes, ROS production is enhanced during CaT alternans, measured by fluorescence microscopy. Exogenous ROS (tert-butyl hydroperoxide) enhanced CaT alternans, whereas ROS scavengers (dithiothreitol, MnTBAP, quercetin, tempol) alleviated CaT alternans. While the inhibition of cellular NADPH oxidases had no effect on CaT alternans, interference with mitochondrial ROS (ROSm) production had profound effects: (1) the superoxide dismutase mimetic MitoTempo diminished CaT alternans and shifted the pacing threshold to higher frequencies; (2) the inhibition of cyt c peroxidase by SS-31, and inhibitors of ROSm production by complexes of the electron transport chain S1QEL1.1 and S3QEL2, decreased the severity of CaT alternans; however (3) the impairment of mitochondrial antioxidant defense by the inhibition of nicotinamide nucleotide transhydrogenase with NBD-Cl and thioredoxin reductase-2 with auranofin enhanced CaT alternans. Our results suggest that intact mitochondrial antioxidant defense provides crucial protection against pro-arrhythmic CaT alternans. Thus, modulating the mitochondrial redox state represents a potential therapeutic approach for alternans-associated arrhythmias, including AF.

Calcium signaling in cardiac mitochondria.

Mitochondrial Ca signaling contributes to the regulation of cellular energy metabolism, and mitochondria participate in cardiac excitation-contraction coupling (ECC) through their ability to store Ca, shape the cytosolic Ca signals and generate ATP required for contraction. The mitochondrial inner membrane is equipped with an elaborate system of channels and transporters for Ca uptake and extrusion that allows for the decoding of cytosolic Ca signals, and the storage of Ca in the mitochondrial matrix compartment. Controversy, however remains whether the fast cytosolic Ca transients underlying ECC in the beating heart are transmitted rapidly into the matrix compartment or slowly integrated by the mitochondrial Ca transport machinery. This review summarizes established and novel findings on cardiac mitochondrial Ca transport and buffering, and discusses the evidence either supporting or arguing against the idea that Ca can be taken up rapidly by mitochondria during ECC.

Mitochondria-mediated cardioprotection by trimetazidine in rabbit heart failure.

Trimetazidine (TMZ) is used successfully for treatment of ischemic cardiomyopathy, however its therapeutic potential in heart failure (HF) remains to be established. While the cardioprotective action of TMZ has been linked to inhibition of free fatty acid oxidation (FAO) via 3-ketoacyl CoA thiolase (3-KAT), additional mechanisms have been suggested. The aim of this study was to evaluate systematically the effects of TMZ on calcium signaling and mitochondrial function in a rabbit model of non-ischemic HF and to determine the cellular mechanisms of the cardioprotective action of TMZ. TMZ protected HF ventricular myocytes from cytosolic Ca(2+) overload and subsequent hypercontracture, induced by electrical and ß-adrenergic (isoproterenol) stimulation. This effect was mediated by the ability of TMZ to protect HF myocytes against mitochondrial permeability transition pore (mPTP) opening via attenuation of reactive oxygen species (ROS) generation by the mitochondrial electron transport chain (ETC) and uncoupled mitochondrial nitric oxide synthase (mtNOS). The majority of ROS generated by the ETC in HF arose from enhanced complex II-mediated electron leak. TMZ inhibited the elevated electron leak at the level of mitochondrial ETC complex II and improved impaired activity of mitochondrial complex I, thereby restoring redox balance and mitochondrial membrane potential in HF. While TMZ decreased FAO by ~15%, the 3-KAT inhibitor 4-bromotiglic acid did not provide protection against palmitic acid-induced mPTP opening, indicating that TMZ effects were 3-KAT independent. Thus, the beneficial effect of TMZ in rabbit HF was not linked to FAO inhibition, but rather associated with reduced complex II- and uncoupled mtNOS-mediated oxidative stress and decreased propensity for mPTP opening.