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In spontaneously hypertensive rats (SHR) atrial remodeling has been shown to involve increase in endothelin (ET) signaling. Furthermore, inflammatory processes may further contribute to tissue remodeling. The aimed of this study was to investigate whether an endothelin receptor antagonist, macitentan, could reduce left atrial (LA) remodeling in arterial hypertension.Molecular characterization of atria was performed in SHR at the age of 8 months and their age-matched normotensive control rats (WKY). SHR were treated with macitentan and, for comparison with a blood pressure reducing drug, with doxazosin. After two months of treatment, molecules involved in endocardial inflammation and atrial calcium handling were assessed. The molecular changes provoked by rapid-pacing (RAP) were analyzed in atrial tissue slices.Doxazosin reduced the systolic blood pressure compared with the untreated SHR (159 ± 26 vs. 176 ± 17;Macitentan reversed pro-inflammatory remodeling in hypertensive atria in a blood pressure-independent manner, which might prevent endocardial dysfunction and thereby, thrombogenesis in arterial hypertension.

Background: Cardiac-specific JDP2 overexpression provokes ventricular dysfunction and atrial dilatation in mice. We performed in vivo studies on JDP2-overexpressing mice to investigate the impact of JDP2 on the predisposition to spontaneous atrial fibrillation (AF). Methods: JDP2-overexpression was started by withdrawal of a doxycycline diet in 4-week-old mice. The spontaneous onset of AF was documented by ECG within 4 to 5 weeks of JDP2 overexpression. Gene expression was analyzed by real-time RT-PCR and Western blots. Results: In atrial tissue of JDP2 mice, besides the 3.6-fold increase of JDP2 mRNA, no changes could be detected within one week of JDP2 overexpression. Atrial dilatation and hypertrophy, combined with elongated cardiomyocytes and fibrosis, became evident after 5 weeks of JDP2 overexpression. Electrocardiogram (ECG) recordings revealed prolonged PQ-intervals and broadened P-waves and QRS-complexes, as well as AV-blocks and paroxysmal AF. Furthermore, reductions were found in the atrial mRNA and protein level of the calcium-handling proteins NCX, Cav1.2 and RyR2, as well as of connexin40 mRNA. mRNA of the hypertrophic marker gene ANP, pro-inflammatory MCP1, as well as markers of immune cell infiltration (CD68, CD20) were increased in JDP2 mice. Conclusion: JDP2 is an important regulator of atrial calcium and immune homeostasis and is involved in the development of atrial conduction defects and arrhythmogenic substrates preceding paroxysmal AF.

Cardiac arrhythmias remain a common challenge and are associated with significant morbidity and mortality. Effective and safe rhythm control strategies are a primary, yet unmet need in everyday clinical practice. Despite significant pharmacological and technological advances, including catheter ablation and device-based therapies, the development of more effective alternatives is of significant interest to increase quality of life and to reduce symptom burden, hospitalizations and mortality. The mechanistic understanding of pathophysiological pathways underlying cardiac arrhythmias has advanced profoundly, opening up novel avenues for mechanism-based therapeutic approaches. Current management of arrhythmias, however, is primarily guided by clinical and demographic characteristics of patient groups as opposed to individual, patient-specific mechanisms and pheno-/genotyping. With this state-of-the-art paper, the Working Group on Cellular Electrophysiology of the German Cardiac Society aims to close the gap between advanced molecular understanding and clinical decision-making in cardiac electrophysiology. The significance of cellular electrophysiological findings for clinical arrhythmia management constitutes the main focus of this document. Clinically relevant knowledge of pathophysiological pathways of arrhythmias and cellular mechanisms of antiarrhythmic interventions are summarized. Furthermore, the specific molecular background for the initiation and perpetuation of atrial and ventricular arrhythmias and mechanism-based strategies for therapeutic interventions are highlighted. Current "hot topics" in atrial fibrillation are critically appraised. Finally, the establishment and support of cellular and translational electrophysiology programs in clinical rhythmology departments is called for to improve basic-science-guided patient management.

In continuously beating cells like cardiac myocytes, there are rapid alterations of cytosolic Ca2+ levels. We therefore hypothesize that decoding Ca2+ signals for hypertrophic signaling requires intracellular Ca2+ microdomains that are partly independent from cytosolic Ca2+. Furthermore, there is a need for a Ca2+ sensor within these microdomains that translates Ca2+ signals into hypertrophic signaling. Recent evidence suggested that the nucleus of cardiac myocytes might be a Ca2+ microdomain and that calcineurin, once translocated into the nucleus, could act as a nuclear Ca2+ sensor. We demonstrate that nuclear calcineurin was able to act as a nuclear Ca2+ sensor detecting local Ca2+ release from the nuclear envelope via IP3R. Nuclear calcineurin mutants defective for Ca2+ binding failed to activate NFAT-dependent transcription. Under hypertrophic conditions Ca2+ transients in the nuclear microdomain were significantly higher than in the cytosol providing a basis for sustained calcineurin/NFAT-mediated signaling uncoupled from cytosolic Ca2+. Measurements of nuclear and cytosolic Ca2+ transients in IP3 sponge mice showed no increase of Ca2+ levels during diastole as we detected in wild-type mice. Nuclei, isolated from ventricular myocytes of mice after chronic Ang II treatment, showed an elevation of IP3R2 expression which was dependent on calcineurin/NFAT signaling and persisted for 3 weeks after removal of the Ang II stimulus. These data provide an explanation how Ca2+ and calcineurin might regulate transcription in cardiomyocytes in response to neurohumoral signals independently from their role in cardiac contraction control. • Calcineurin acts as an intranuclear Ca2+ sensor to promote NFAT activity. • Nuclear Ca2+ in cardiac myocytes increases via IP3R2 upon Ang II stimulation. • IP3R2 expression is directly dependent on calcineurin/NFAT.

Hypertensive heart disease (HHD) can cause left ventricular (LV) hypertrophy and heart failure (HF). It is unclear, though, which factors may contribute to the transition from compensated LV hypertrophy to HF in HHD. We hypothesized that maladaptive atrial remodeling with impaired atrial myocyte function would occur in advanced HHD and may be associated with the emergence of HF. Experiments were performed on atrial myocytes and tissue from old (15-25months) normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) with advanced HHD. Based on the absence or presence of elevated lung weight, a sign of lung congestion and heart failure, SHR were divided into a non-failing (SHR-NF) and failing (SHR-HF) group. Compared with WKY, SHR exhibited elevated blood pressure, LV hypertrophy and left atrial (LA) hypertrophy with increased LA expression of markers of hypertrophy and fibrosis. SHR-HF were distinguished from SHR-NF by aggravated hypertrophy and fibrosis. SHR-HF atrial myocytes exhibited reduced contractility and impaired SR Ca2+ handling. Moreover, in SHR the expression and phosphorylation of SR Ca2+-regulating proteins (SERCA2a, calsequestrin, RyR2 and phospholamban) showed negative correlation with increasing lung weight. Increasing stimulation frequency (1-2-4Hz) of atrial myocytes caused a progressive increase in arrhythmogenic Ca2+ release (including alternans), which was observed most frequently in SHR-HF. Thus, in old SHR with advanced HHD there is profound structural and functional atrial remodeling. The occurrence of HF in SHR is associated with LA and RA hypertrophy, increased atrial fibrosis, impaired atrial myocyte contractility and SR Ca2+ handling and increased propensity for arrhythmogenic Ca2+ release. Therefore, functional remodeling intrinsic to atrial myocytes may contribute to the transition from compensated LV hypertrophy to HF in advanced HHD and an increased propensity of atrial arrhythmias in HF.

In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch‐activated K2P potassium channel TREK‐1 ( KCNK2 or K2P2.1). This mutation introduces abnormal sodium permeability to TREK‐1. In addition, mutant channels exhibit a hypersensitivity to stretch‐activation, suggesting that the selectivity filter is directly involved in stretch‐induced activation and desensitization. Increased sodium permeability and stretch‐sensitivity of mutant TREK‐1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT. We present a pharmacological strategy to rescue the selectivity defect of the TREK‐1 pore. Our findings provide important insights for future studies of K2P channel stretch‐activation and the role of TREK‐1 in mechano‐electrical feedback in the heart. ![][1] A point mutation in the selectivity filter of the stretch‐activated K2P potassium channel TREK‐1 was identified in a patient with right ventricular outflow tract tachycardia. The mutation most likely causes arrhythmias through abnormal sodium permeability and hypersensitivity to stretch‐activation. [1]: /embed/graphic-1.gif

Arterial hypertension causes left ventricular (LV) myocyte hypertrophy. Alterations in nuclear Ca2+ may be involved in regulation of histone acetylation, transcription and hypertrophy. Regulation of nuclear Ca2+ in hypertension, however, is unknown. Therefore, we elucidated cellular mechanisms underlying nuclear Ca2+ regulation in LV myocytes from hypertensive versus normotensive rats and evaluated possible consequences for Ca2+-dependent regulation of histone acetylation. LV myocytes and myocyte nuclei were isolated from young spontaneously hypertensive rats (SHR) shortly after development of hypertension. Normotensive Wistar-Kyoto rats (WKY) served as controls. Cytoplasmic and nucleoplasmic Ca2+ transients (CaTs) were imaged simultaneously using linescan confocal microscopy and Fluo-4. LV myocytes and nuclei from SHR exhibited hypertrophy. Cytoplasmic and nucleoplasmic CaTs were increased in SHR. The increase in nucleoplasmic Ca2+, however, exceeded the increase in cytoplasmic Ca2+, indicating enhanced nuclear Ca2+ signaling in SHR. Ca2+ load of sarcoplasmic reticulum and perinuclear Ca2+ stores was also increased in SHR, while fractional release from both stores remained unchanged. Intranuclear Ca2+ propagation was accelerated in SHR, associated with preserved density of nuclear envelope invaginations and elevated nuclear expression of nucleoporins and SR-Ca2+-ATPase, SERCA2a. Nuclear Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) expression was elevated and histone deacetylases exhibited redistribution from nucleus to cytosol associated with increased histone acetylation in SHR. Thus, in early hypertension, there is remodeling of nuclear Ca2+ handling resulting in enhanced nuclear Ca2+ signaling. Enhanced nuclear Ca2+ signaling, in turn, increases nuclear localization and activity of CaMKIIδ driving nuclear export of histone deacetylases and increased histone acetylation.

Left ventricular (LV) hypertrophy in response to hypertension and increased afterload frequently progresses to heart failure. It is under debate whether the loss of cardiomyocytes contributes to this transition. To address this question, female C57BL/6 wild‐type mice were subjected to transverse aortic constriction (TAC) and developed compensated LV hypertrophy after 1 week, which progressed to heart failure characterized by reduced ejection fraction and pulmonary congestion 4 weeks post‐TAC. Quantitative, design‐based stereology methods were used to estimate number and mean volume of LV cardiomyocytes. DNA strand breaks were visualized using the TUNEL method 6 weeks post‐TAC to quantify the number of apoptotic cell nuclei. The volume of the LV myocardium as well as the cardiomyocyte mean volume increased progressively after TAC. In contrast, the number of LV cardiomyocytes remained constant 1 and 4 weeks post‐TAC in comparison to sham‐operated mice. Moreover, there was no significant difference in the number of cardiomyocyte nuclei stained for DNA strand breaks at 6 weeks post‐TAC. It was concluded that the loss of cardiomyocytes is not required for the transition from compensated hypertrophy to heart failure induced by TAC in the female murine heart.

In cardiomyocytes, nuclear calcium is involved in regulation of transcription and, thus, remodeling. The cellular mechanisms regulating nuclear calcium, however, remain elusive. Therefore, the aim of this study was to identify and characterize the factors that regulate nuclear calcium in cardiomyocytes. We focused on the roles of (1) the cytoplasmic calcium transient (CaT), (2) the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and (3) intracellular calcium stores for nuclear calcium handling. Experiments were performed on rat ventricular myocytes loaded with Fluo-4/AM. Subcellularly resolved CaTs were visualized using confocal microscopy. The cytoplasmic CaT was varied by reducing extracellular calcium (from 1.5 to 0.3 mM) or by exposure to isoprenaline (ISO, 10 nM). SERCA was blocked by thapsigargin (5 μM). There was a strict linear dependence of the nucleoplasmic CaT on the cytoplasmic CaT over a wide range of calcium concentrations. Increasing SERCA activity impaired, whereas decreasing SERCA activity augmented the systolic calcium increase in the nucleus. Perinuclear calcium store load, on the other hand, did not change with either 0.3 mM calcium or ISO and was not a decisive factor for the nucleoplasmic CaT. The results indicate, that the nucleoplasmic CaT is determined largely by the cytoplasmic CaT via diffusion of calcium through nuclear pores. They identify perinuclear SERCA activity, which limits the systolic calcium increase in the nucleus, as a novel regulator of the nuclear CaT in cardiac myocytes.

High quality cardiomyocyte isolation is of critical importance for successful studies of myocardial function at the cellular and molecular level. Although previous work has established isolation procedures for various species, it still remains challenging to produce consistently a high yield of viable and healthy cardiomyocytes. The basis for the most successful and reproducible isolation of cardiomyocytes from intact hearts is the Langendorff retrograde perfusion technique. Here, we will illustrate in detail all practical aspects of the enzyme-based Langendorff isolation of rat atrial and ventricular cardiomyocytes. This includes a series of obligatory steps starting from quick aortic cannulation to rinse the heart from blood, short perfusion of the heart with Ca

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this network.

High quality cardiomyocyte isolation is of critical importance for successful studies of myocardial function at the cellular and molecular level. Although previous work has established isolation procedures for various species, it still remains challenging to produce consistently a high yield of viable and healthy cardiomyocytes. The basis for the most successful and reproducible isolation of cardiomyocytes from intact hearts is the Langendorff retrograde perfusion technique. Here, we will illustrate in detail all practical aspects of the enzyme-based Langendorff isolation of rat atrial and ventricular cardiomyocytes. This includes a series of obligatory steps starting from quick aortic cannulation to rinse the heart from blood, short perfusion of the heart with Ca2+-free solution to dissociate cells at the level of intercalated discs, followed by longer perfusion with low Ca2+-containing enzyme solution in order to disrupt the extracellular matrix network, extraction of the released cardiomyocytes and gentle Ca2+ reintroduction to allow cells to return gradually to normal cytosolic Ca2+ levels. The average yield of intact viable ventricular myocytes that can be achieved with our protocol is ≈70% (range ≈50-90%). For atrial myocytes, in general, it is slightly (≈10%) lower than for ventricular myocytes. The yield depends on the age of the rat and the degree of cardiac remodeling such that digestion of older and more remodeled hearts (more fibrosis) usually results in lower yields. Isolated atrial and ventricular cardiomyocytes may be employed for studies of cardiomyocyte function (e.g., shortening/contraction, intracellular [Ca2+] transients) as well as for biochemical and molecular biological studies (e.g., immunoblotting, PCR).

In continuously beating cells like cardiac myocytes, there are rapid alterations of cytosolic Ca

The initiation and perpetuation of atrial fibrillation (AF) can be regarded as a complication of a progressive transformation of atrial structure and function. This transformation is the result of complex changes at the molecular, cellular and organ levels, leading to the profibrillation arrhythmic mechanisms in AF. Numerous individual and environmental factors are probably involved in this process. Therefore, progress in the diagnosis, prevention and treatment of AF requires highly integrative research from the benchtop to bedside and from specific signaling pathways and electrophysiological mechanisms to population-based studies. The European Network for Translational Research in Atrial Fibrillation was formed to provide this variety of expertise and has identified central research objectives for improvements in AF prevention and therapy.

BACKGROUND AND PURPOSE Ca2+ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca2+ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca2+ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms. EXPERIMENTAL APPROACH SR Ca2+ leak was induced by ouabain in murine cardiomyocytes. [Ca2+]-transients, SR Ca2+ load and RyR2-mediated Ca2+ leak (sparks/waves) were quantified, with or without JTV519 (1 µmol·L−1). Contribution of Ca2+-/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser2814 phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae. KEY RESULTS Ouabain increased systolic and diastolic cytosolic [Ca2+]i, SR [Ca2+], and SR Ca2+ leak (Ca2+ spark (SparkF) and Ca2+ wave frequency), independently of CaMKII and RyR-Ser2814 phosphorylation. JTV519 decreased SparkF but also SR Ca2+ load. At matched SR [Ca2+], Ca2+ leak was significantly reduced by JTV519, but it had no effect on fractional Ca2+ release or Ca2+ wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function. CONCLUSIONS AND IMPLICATIONS JTV519 was effective in reducing SR Ca2+ leak by specifically regulating RyR2 opening at diastolic [Ca2+]i in the absence of increased RyR2 phosphorylation at Ser2814, extending the potential use of JTV519 to conditions of acute cellular Ca2+ overload.

Quantification of subcellularly resolved Ca2+ signals in cardiomyocytes is essential for understanding Ca2+ fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca2+-dependent fluorescence changes into [Ca2+] changes. Therefore, we determined the in situ characteristics of a frequently used Ca2+ indicator, Fluo-4, and a ratiometric Ca2+ indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca2+ signals in isolated cardiomyocytes. Ca2+ calibration curves revealed significant differences in the apparent Ca2+ dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. These parameters were used for transformation of fluorescence into nucleoplasmic and cytoplasmic [Ca2+]. Resting and diastolic [Ca2+] were always higher in the nucleoplasm. Systolic [Ca2+] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca2+] in the nucleoplasm. Ca2+ store depletion or blockade of Ca2+ leak pathways eliminated the resting [Ca2+] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest the presence of significant nucleoplasmic-to-cytoplasmic [Ca2+] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca2+] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca2+ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca2+ stores.

Introduction Fundamental differences in Ca2+ homeostasis between mice and larger mammals require the validation of the mechanisms of arrhythmogenesis before translation into human pathophysiology. The purpose of this study was to create transgenic rabbits that express defective human cardiac ryanodine receptor (hRyR2) with a mutation (R4497C) causing a clinically relevant arrhythmogenic syndrome. Methods The construct pcDNA3-EGFP-hRyR2-R4497C with the CMV promoter was used to generate transgenic rabbits. The founder animals were created by microinjection and identified by PCR with specific primers for the EGFP sequence. The copy number of the transgene was quantified by real-time PCR using genomic DNA from blood cells. mRNA expression of EGFP-hRyR2-R4497C was quantified using RT-PCR with specific primers for the RyR2 and EGFP sequence. Protein expression of the transgene in heart and non-cardiac tissues was determined using immunoblots with antibodies directed against EGFP and RyR2. Results Real-time PCR in peripheral blood cells identified several rabbit lines with the construct integrated into their genome. Transcription levels of the transgene were low (Ct > 30). On the protein level, neither EGFP nor hRyR2 R4497C was detected in either cardiac or non-cardiac tissue. A truncated gene product (3′ end and central part of hRyR2 R4497C, but not EGFP) could be detected at the mRNA level in the heart. Discussion Lack of significant protein expression of the EGFP-RyR2 R4497C gene construct despite successful incorporation into the genomic DNA is due to combination of at least two factors: low mRNA expression, and truncation of the transgene on the mRNA level. Our results suggest that the CMV promoter may not be well suited for creating transgenic rabbits.

BACKGROUND AND PURPOSE Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms. EXPERIMENTAL APPROACH Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca2+]i transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16. KEY RESULTS Urocortin 2 elicited time- and concentration-dependent positive inotropic and lusitropic effects (EC50: 19 nM) that were abolished by antisauvagine-30 (10 nM, n= 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF2 receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca2+]i transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca2+]i transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca2+]i increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93. CONCLUSIONS AND IMPLICATIONS Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF2 receptors in a cAMP/PKA- and Ca2+/CaMKII-dependent manner. This enhancement was accompanied by Ca2+-dependent arrhythmogenic effects mediated by PKA and CaMKII.

Corticotropin-releasing factor (CRF), a 41 amino acid peptide, was discovered as a key signal in mediating neuroendocrine, autonomic, and behavioral responses to stress. It was revealed later that there exist additional CRF-like peptides, termed urocortins. The CRF receptor subtype 1 (CRF1 receptor) is predominant in the brain whereas subtype 2 (CRF2 receptor) is highly expressed in the brain and the heart. Both centrally and peripherally administered CRF and urocortins produce significant hemodynamic effects via activation of CRF receptors in the brain and the heart. CRF and urocortins are important neural and cardioactive hormones, and are potentially useful therapy for heart failure.

Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive.Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events.The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Background— RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. Here, we report on targeted RNAi for the treatment of heart failure, an important disorder in humans that results from multiple causes. Successful treatment of heart failure is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban, a key regulator of cardiac Ca 2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy uses regulatory RNAs to achieve its effect. Methods and Results— We describe structural requirements to obtain high RNAi activity from adenoviral and adeno-associated virus (AAV9) vectors and show that an adenoviral short hairpin RNA vector (AdV-shRNA) silenced phospholamban in cardiomyocytes (primary neonatal rat cardiomyocytes) and improved hemodynamics in heart-failure rats 1 month after aortic root injection. For simplified long-term therapy, we developed a dimeric cardiotropic adeno-associated virus vector (rAAV9-shPLB) to deliver RNAi activity to the heart via intravenous injection. Cardiac phospholamban protein was reduced to 25%, and suppression of sacroplasmic reticulum Ca 2+ ATPase in the HF groups was rescued. In contrast to traditional vectors, rAAV9 showed high affinity for myocardium but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (left ventricular end-diastolic pressure, dp/dt min , and τ) and systolic (fractional shortening) functional parameters to normal ranges. The massive cardiac dilation was normalized, and cardiac hypertrophy, cardiomyocyte diameter, and cardiac fibrosis were reduced significantly. Importantly, no evidence was found of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusions— Our data show for the first time the high efficacy of an RNAi therapeutic strategy in a cardiac disease.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. Indirect evidence from clinical trials demonstrates that chronic inhibition of the renin–angiotensin-system (RAS) significantly reduces the incidence of AF. Since mechanisms of this protective effect of RAS-blockade are poorly understood, we directly tested proarrhythmic effects of angiotensin II (Ang II) in human atrial myocardium. Methods: Isolated trabeculae from human atrial appendages (n = 80) were electrically stimulated. We assessed isometric force and incidence of arrhythmic extra contractions (AECs) with and without increasing concentrations of Ang II (1–1000 nmol/L) in the absence or presence of receptor-blockade by saralasin (non-specific ATR-antagonist), irbesartan (AT1R-antagonist) or PD123319 (AT2R-antagonist). Results: Twitch force and AECs concentration-dependently increased with Ang II. Effects became significant at concentrations >1 nmol/L Ang II and were maximal at 1000 nmol/L (increase in twitch force to 157±14% and AECs from 0 to 80%) saralasin and irbesartan partially prevented the inotropic effect of 100 nmol/L Ang II (by 4±12% and 68±6%; p

Mechanical load is an important regulator of cardiac force. Stretching human atrial and ventricular trabeculae elicited a biphasic force increase: an immediate increase (Frank-Starling mechanism) followed by a further slow increase (slow force response, SFR). In ventricle, the SFR was unaffected by AT- and ET-receptor antagonism, by inhibition of protein-kinase-C, PI-3-kinase, and NO-synthase, but attenuated by inhibition of Na+/H+- (NHE) and Na+/Ca2+-exchange (NCX). In atrium, however, neither NHE- nor NCX-inhibition affected the SFR. Stretch elicited a large NHE-dependent [Na+]i increase in ventricle but only a small, NHE-independent [Na+]i increase in atrium. Stretch-activated non-selective cation channels contributed to basal force development in atrium but not ventricle and were not involved in the SFR in either tissue. Interestingly, inhibition of AT-receptors or pre-application of angiotensin II or endothelin-1 reduced the atrial SFR. Furthermore, stretch increased phosphorylation of atrial myosin light chain 2 (MLC2) and inhibition of myosin light chain kinase (MLCK) attenuated the SFR in atrium and ventricle. Thus, in human heart both atrial and ventricular myocardium exhibit a stretch-dependent SFR that might serve to adjust cardiac output to increased workload. In ventricle, there is a robust NHE-dependent (but angiotensin II- and endothelin-1-independent) [Na+]i increase that is translated into a [Ca2+]i and force increase via NCX. In atrium, on the other hand, there is an angiotensin II- and endothelin-dependent (but NHE- and NCX-independent) force increase. Increased myofilament Ca2+ sensitivity through MLCK-induced phosphorylation of MLC2 is a novel mechanism contributing to the SFR in both atrium and ventricle.

Die chronische Herzinsuffizienz ist mit einer Dysfunktion der Herzmuskelzellen und einer gestorten intrazellularen Ionenhomoostase verbunden. Charakteristische Veranderungen sind eine Verringerung der systolischen intrazellularen Ca2+-Freisetzung, ein Anstieg des diastolischen zytosolischen Ca2+ und ein erhohtes intrazellulares Na+. Dieser Beitrag beschreibt die Zusammenhange zwischen gestortem intrazellularem Ionengleichgewicht, kontraktiler Dysfunktion, Arrhythmogenese und myokardialem Remodeling nach derzeitigem Kenntnisstand. Der Einfluss auf die intrazellulare Ionenhomoostase begrundet sowohl den Erfolg als auch die Grenzen der gegenwartigen Herzinsuffizienztherapie. Neue Therapieansatze mit klinischem Potenzial werden diskutiert.

Nuclear Ca2+ plays a key role in the regulation of gene expression. Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3)] might be an important regulator of nuclear Ca2+ but its contribution to nuclear Ca2+ signalling in adult cardiomyocytes remains elusive. We tested the hypothesis that endothelin-1 enhances nuclear Ca2+ concentration transients (CaTs) in rabbit atrial myocytes through Ins(1,4,5)P3-induced Ca2+ release from perinuclear stores. Cytoplasmic and nuclear CaTs were measured simultaneously in electrically stimulated atrial myocytes using confocal Ca2+ imaging. Nuclear CaTs were significantly slower than cytoplasmic CaTs, indicative of compartmentalisation of intracellular Ca2+ signalling. Endothelin-1 elicited a preferential (10 nM) or a selective (0.1 nM) increase in nuclear versus cytoplasmic CaTs. This effect was abolished by inhibition of endothelin-1 receptors, phospholipase C and Ins(1,4,5)P3 receptors. Fractional Ca2+ release from the sarcoplasmic reticulum and perinuclear stores was increased by endothelin-1 at an otherwise unaltered Ca2+ load. Comparable increases of cytoplasmic CaTs induced by β-adrenoceptor stimulation or elevation of extracellular Ca2+ could not mimic the endothelin-1 effects on nuclear CaTs, suggesting that endothelin-1 specifically modulates nuclear Ca2+ signalling. Thus, endothelin-1 enhances nuclear CaTs in atrial myocytes by increasing fractional Ca2+ release from perinuclear stores. This effect is mediated by the coupling of endothelin receptor A to PLC-Ins(1,4,5)P3 signalling and might contribute to excitation-transcription coupling.

Cardiomyocytes are electrically excitable cells built to contract. The cellular processes linking electrical excitation, i.e. the sarcolemmal action potential, with contraction are referred to as excitation–contraction coupling. An increase in intracellular Ca2+ concentration ([Ca2+]i) is the key mediator of excitation–contraction coupling. Cardiac action potentials are characterised by a long plateau phase carried by Ca2+ influx through L-type Ca2+ channels. L-type Ca2+ influx triggers Ca2+ release from the sarcoplasmic reticulum (SR) through Ca2+ release channels or ryanodine receptors. This Ca2+-induced Ca2+ release causes a large increase in [Ca2+]i from ≈ 100 nM in diastole to ≈ 1 μM in systole. Ca2+ binding to the myofilaments causes contraction. Ca2+ removal from the cytosol by the SR-Ca2+-ATPase (SERCA) and the sarcolemmal Na+–Ca2+ exchanger (NCX) mediates relaxation. The amplitude of the [Ca2+]i transient (CaT) decides about the strength of contraction. Increases in L-type Ca2+ current, SERCA activity, SR Ca2+ load and fractional release, IP3 signalling and [Na+]i all increase CaT amplitude. Under conditions of Ca2+ overload, SR Ca2+ release also occurs spontaneously, i.e. in the absence of an action potential, and may elicit life-threatening arrhythmias via activation of electrogenic NCX and subsequent membrane depolarisation. Atrial and ventricular myocytes share these basic principles of excitation–contraction coupling and [Ca2+]i regulation. However, there are important differences between these types of cardiomyocytes regarding action potential configuration, sarcolemmal structure (transverse tubules) and subcellular Ca2+ regulation. Remodelling of excitation–contraction coupling occurs in cardiac disease such as heart failure and atrial fibrillation and represents a potential therapeutic target.

Background Cardiac glycosides are characterized by a narrow therapeutic range with Ca2+-overload and arrhythmias occurring at higher concentrations. Data on cardiac glycosides in isolated failing human myocardium are scarce and the frequency-dependent actions and toxicity of Strophanthidin have not yet been characterized. Aims To determine inotropic responses and toxicity of Strophanthidin in failing human myocardium. Methods and results Experiments were performed in trabeculae from 64 end-stage failing hearts. Developed force, and intracellular [Ca2+]i and [Na+]i were recorded with Strophanthidin (0.01 to 1 μmol/L; 37°C, 1 Hz) and compared to interventions with distinct mechanisms of action (elevated [Ca2+]o, Isoproterenol, and EMD57033). The effects of Strophanthidin on force–frequency behaviour were also assessed. Strophanthidin exerted concentration-dependent positive inotropic effects. These were paralleled by increases in intracellular [Na+] as well as increasing [Ca2+]i-transients and SR-Ca2+-load. At high concentrations (>0.5 μmol/L), Strophanthidin caused afterglimmers and aftercontractions, with declining developed force despite further increasing [Ca2+]i-transients. The force–frequency-relationship and diastolic function at higher pacing rates was worsened by Strophanthidin in a concentration-dependent manner. Conclusions Strophanthidin toxicity was dependent on concentration, calcium load, beating rate and β-adrenergic receptor activation. Our data support the view that low doses, heart rate control and additional β-adrenergic receptor blockade are essential in the use of cardiac glycosides in heart failure.

Background— Proton pump inhibitors are used extensively for acid-related gastrointestinal diseases. Their effect on cardiac contractility has not been assessed directly. Methods and Results— Under physiological conditions (37°C, pH 7.35, 1.25 mmol/L Ca 2+ ), there was a dose-dependent decrease in contractile force in ventricular trabeculae isolated from end-stage failing human hearts superfused with pantoprazole. The concentration leading to 50% maximal response was 17.3±1.3 μg/mL. Similar observations were made in trabeculae from human atria, normal rabbit ventricles, and isolated rabbit ventricular myocytes. Real-time polymerase chain reaction demonstrated the expression of gastric H + /K + –adenosine triphosphatase in human and rabbit myocardium. However, measurements with BCECF-loaded rabbit trabeculae did not reveal any significant pantoprazole-dependent changes of pH i . Ca 2+ transients recorded from field-stimulated fluo 3–loaded myocytes (F/F 0 ) were significantly depressed by 10.4±2.1% at 40 μg/mL. Intracellular Ca 2+ fluxes were assessed in fura 2–loaded, voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 μg/mL) caused an increase in diastolic [Ca 2+ ] i by 33±12%, but peak systolic [Ca 2+ ] i was unchanged, resulting in a decreased Ca 2+ transient amplitude by 25±8%. The amplitude of the L-type Ca 2+ current ( I Ca,L ) was reduced by 35±5%, and sarcoplasmic reticulum Ca 2+ content was reduced by 18±6%. Measurements of oxalate-supported sarcoplasmic reticulum Ca 2+ uptake in permeabilized cardiomyocytes indicated that pantoprazole decreased Ca 2+ sensitivity (K d ) of sarcoplasmic reticulum Ca 2+ adenosine triphosphatase: control, K d =358±15 nmol/L; 40 μg/mL pantoprazole, K d =395±12 nmol/L ( P 2+ -activated force. Conclusions— Pantoprazole depresses cardiac contractility in vitro by depression of Ca 2+ signaling and myofilament activity. In view of the extensive use of this agent, the effects should be evaluated in vivo.

Arterial hypertension causes left ventricular (LV) myocyte hypertrophy. Alterations in nuclear Ca

Aims Hypertension is a major risk factor for atrial fibrillation. We hypothesized that arterial hypertension would alter atrial myocyte calcium (Ca2+) handling and that these alterations would serve to trigger atrial tachyarrhythmias. Methods and results Left atria or left atrial (LA) myocytes were isolated from spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) controls. Early after the onset of hypertension, at 3 months of age, there were no differences in Ca2+ transients (CaTs) or expression and phosphorylation of Ca2+ handling proteins between SHR and WKY. At 7 months of age, when left ventricular (LV) hypertrophy had progressed and markers of fibrosis were increased in left atrium, CaTs (at 1 Hz stimulation) were still unchanged. Subcellular alterations in Ca2+ handling were observed, however, in SHR atrial myocytes including (i) reduced expression of the alpha 1C subunit of and reduced Ca2+ influx through L-type Ca2+ channels, (ii) reduced expression of ryanodine receptors with increased phosphorylation at Ser2808, (iii) decreased activity of the Na+/Ca2+ exchanger (at unaltered intracellular Na+ concentration), and (iv) increased SR Ca2+ load with reduced fractional release. These changes were associated with an increased propensity of SHR atrial myocytes to develop frequency-dependent, arrhythmogenic Ca2+ alternans. Conclusions In SHR, hypertension induces early subcellular LA myocyte Ca2+ remodelling during compensated LV hypertrophy. In basal conditions, atrial myocyte CaTs are not changed. At increased stimulation frequency, however, SHR atrial myocytes become moreprone to arrhythmogenic Ca2+ alternans, suggesting a link between hypertension, atrial Ca2+ homeostasis, and development of atrial tachyarrhythmias.

Localization of glycolytic enzymes in close proximity to Ca2+ transport systems of the sarcoplasmic reticulum (SR) in cardiac cells suggests an important functional role for glycolysis in intracellular [Ca2+] regulation and, consequently, excitation–contraction coupling. Here, we investigated the mechanisms of regulation of SR Ca2+ release by glycolytic sugar phosphate intermediates in cat ventricular myocytes. Experiments with permeabilized myocytes revealed that with normal cytosolic energy reserves (mm: ATP 5, ADP 0.01, phosphocreatine (CrP) 10) fructose-1,6-bisphosphate (FBP; 1 mm) and fructose-6-phosphate (F6P; 1 mm) caused a transient increase of Ca2+ spark frequency by 62 and 42%, respectively. This effect of sugar phosphates was associated with a 13% decrease in SR Ca2+ load. Pretreatment of the cells with an inhibitor of glycolysis, iodoacetate (IAA; 0.5 mm), did not prevent the effects of FBP and F6P on Ca2+ sparks. Recording of single ryanodine receptor (RyR) channel activity indicated that FBP and F6P significantly increased RyR open probability. Reduction of cytosolic energy reserves decreased Ca2+ spark activity. Increasing [ADP] to 0.4 mm or removal of CrP ([ATP] was kept constant) caused a slowly developing decrease of Ca2+ spark frequency by 29 and 42%, respectively. Changing [ADP] and [CrP] simultaneously decreased Ca2+ spark frequency by 66%. This inhibition of Ca2+ sparks was associated with a 40% decrease in SR Ca2+ load. The subsequent addition of FBP (1 mm) partially restored Ca2+ spark frequency and SR Ca2+ load. This recovery of Ca2+ sparks was blocked completely by IAA. These data suggest that at physiological ATP, ADP and CrP levels accumulation of sugar phosphates from glycolysis can stimulate SR Ca2+ release. This effect does not require the activity of downstream glycolytic enzymes, but rather is the result of direct activation of RyRs. However, under conditions associated with depletion of cellular energy reserves (e.g. myocardial ischaemia), ATP generated from glycolysis may play an important role in maintaining myocardial Ca2+ homeostasis by improving SR Ca2+ uptake.

Rabbit ventricular myocardium is characterized by a biphasic response to stretch with an initial, rapid increase in force followed by a delayed, slow increase in force (slow force response, SFR). The initial phase is attributed to increased myofilament Ca(2+) sensitivity, but the mechanisms of the delayed phase are only incompletely understood. We tested whether stretch-dependent stimulation of Na(+)/H(+) exchange (NHE1) and consecutive changes in pH(i) and/or [Na(+)](i) may underlie the SFR.Isometric contractions of rabbit ventricular muscles were recorded in bicarbonate-containing Tyrode's (Tyrode) or bicarbonate-free HEPES-buffered solution (HEPES). Muscles were loaded with the Ca(2+) indicator aequorin, the pH indicator BCECF, or the Na(+) indicator SBFI and rapidly stretched from 88% (L(88)) to 98% (L(98)) of optimal length. The resulting immediate and slow increases in twitch force (1st phase and SFR) as well as changes in [Ca(2+)](i), [Na(+)](i), or pH(i) were quantified before and after inhibition of NHE1 by HOE 642 (3 microM) or reverse-mode Na(+)/Ca(2+) exchange (NCX) by KB-R 7943 (5 microM).In both Tyrode (n=21) and HEPES (n=22), developed force increased to approximately 160% during the 1st phase followed by a further increase to approximately 205% during the SFR. The SFR was accompanied by a 21% increase of the aequorin light transient (n=4; normalized to the 1st phase) and a approximately 3 mM increase in [Na(+)](i) (n=4-7). The SFR was also associated with an increase in pH(i). However, this increase was delayed and was significant only after the SFR had reached its maximum. The delayed pH(i) increase was larger in HEPES than in Tyrode. HOE 642 and/or KB-R 7943 reduced the SFR by approximately 30-40%. In addition, HOE 642 diminished the stretch-mediated elevation of [Na(+)](i) by 72% and the delayed alkalinization.The data are consistent with the hypothesis that SFR results from increases in [Ca(2+)](i) secondary to altered flux via NCX in part resulting from increases in [Na(+)](i) mediated by NHE1.

In cardiac myocytes, glycolysis and excitation–contraction (E–C) coupling are functionally coupled. We studied the effects of inhibitors (2-deoxy-d-glucose (2-DG), iodoacetate (IAA)), intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP)) and products (pyruvate, l-lactate) of glycolysis on sarcoplasmic reticulum (SR) Ca2+ release and uptake in intact and permeabilized cat atrial myocytes. In field-stimulated (0.5–0.7 Hz) intact myocytes, 2-DG (10 mm) and IAA (1 mm) caused elevation of diastolic [Ca2+]i and [Ca2+]i transient alternans (Ca2+ alternans) followed by a decrease of the amplitude of the [Ca2+]i transient. Focal application of 2-DG resulted in local Ca2+ alternans that was confined to the region of exposure. 2-DG and IAA slowed the decay kinetics of the [Ca2+]i transient and delayed its recovery (positive staircase) after complete SR depletion, suggesting impaired activity of the SR Ca2+-ATPase (SERCA). 2-DG and IAA reduced the rate of reuptake of Ca2+ into the SR which was accompanied by a 15–20% decrease of SR Ca2+ load. Major changes of mitochondrial redox state (measured as FAD autofluorescence) were not observed after inhibition of glycolysis. Pyruvate (10 mm) and l-lactate (10 mm) elicited similar changes of the [Ca2+]i transient. Pyruvate, l-lactate and IAA – but not 2-DG – induced intracellular acidosis. Recording of single channel activity of ryanodine receptors (RyRs) incorporated into lipid bilayers revealed complex modulation by glycolytic intermediates and products (1 mm each): some were without effect (G6P, PEP, l-lactate) while others either increased (F6P, +40%; FBP, +265%) or decreased (pyruvate, −58%) the open probability of the RyR. Consistent with these findings, spontaneous SR Ca2+ release (Ca2+ sparks) in permeabilized myocytes was facilitated by FBP and inhibited by pyruvate. The results indicate that in atrial myocytes glycolysis regulates Ca2+ release from the SR by multiple mechanisms including direct modulation of RyR activity by intermediates and products of glycolysis and modulation of SERCA activity through local changes of glycolytically derived ATP.

Palytoxin is a coral toxin that seriously impairs heart function, but its effects on excitation-contraction (E-C) coupling have remained elusive. Therefore, we studied the effects of palytoxin on mechanisms involved in atrial E-C coupling. In field-stimulated cat atrial myocytes, palytoxin caused elevation of diastolic intracellular Ca2+ concentration ([Ca2+]i), a decrease in [Ca2+]i transient amplitude, Ca2+ alternans followed by [Ca2+]i waves, and failures of Ca2+ release. The decrease in [Ca2+]i transient amplitude occurred despite high sarcoplasmic reticulum (SR) Ca2+ load. In voltage-clamped myocytes, palytoxin induced a current with a linear current-voltage relationship (reversal potential ∼5 mV) that was blocked by ouabain. Whole cell Ca2+ current and ryanodine receptor Ca2+ release channel function remained unaffected by the toxin. However, palytoxin significantly reduced Ca2+ pumping of isolated SR vesicles. In current-clamped myocytes stimulated at 1 Hz, palytoxin induced a depolarization of the resting membrane potential that was accompanied by delayed afterdepolarizations. No major changes of action potential configuration were observed. The results demonstrate that palytoxin interferes with the function of the sarcolemmal Na+-K+ pump and the SR Ca2+ pump. The suggested mode of palytoxin toxicity in the atrium involves the conversion of Na+-K+ pumps into nonselective cation channels as a primary event followed by depolarization, Na+ accumulation, and Ca2+ overload, which, in turn, causes arrhythmogenic [Ca2+]i waves and delayed afterdepolarizations.

Via the Na/Ca and Na/H exchange, intracellular Na concentration ([Na](i)) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na](i) might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na](i) in rat ventricular myocytes. In vivo calibration yielded an apparent K(d) of 27 +/- 2 mM Na. Similar resting [Na](i) was found using two-photon or single-photon ratiometric measurements with SBFI (10.8 +/- 0.7 vs. 11.1 +/- 0.7 mM). To assess longitudinal [Na](i) gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na](i) at sites downstream of the pipette (where Na enters the myocyte and Na/K pumps are blocked). [Na](i) rise was smaller at upstream sites. This resulted in sustained [Na](i) gradients (up to approximately 17 mM/120 microm cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10-12 microm(2)/s, significantly lower than in aqueous solutions.

Subcellular Ca(2+) signalling during normal excitation-contraction (E-C) coupling and during Ca(2+) alternans was studied in atrial myocytes using fast confocal microscopy and measurement of Ca(2+) currents (I(Ca)). Ca(2+) alternans, a beat-to-beat alternation in the amplitude of the [Ca(2+)](i) transient, causes electromechanical alternans, which has been implicated in the generation of cardiac fibrillation and sudden cardiac death. Cat atrial myocytes lack transverse tubules and contain sarcoplasmic reticulum (SR) of the junctional (j-SR) and non-junctional (nj-SR) types, both of which have ryanodine-receptor calcium release channels. During E-C coupling, Ca(2+) entering through voltage-gated membrane Ca(2+) channels (I(Ca)) triggers Ca(2+) release at discrete peripheral j-SR release sites. The discrete Ca(2+) spark-like increases of [Ca(2+)](i) then fuse into a peripheral 'ring' of elevated [Ca(2+)](i), followed by propagation (via calcium-induced Ca(2+) release, CICR) to the cell centre, resulting in contraction. Interrupting I(Ca) instantaneously terminates j-SR Ca(2+) release, whereas nj-SR Ca(2+) release continues. Increasing the stimulation frequency or inhibition of glycolysis elicits Ca(2+) alternans. The spatiotemporal [Ca(2+)](i) pattern during alternans shows marked subcellular heterogeneities including longitudinal and transverse gradients of [Ca(2+)](i) and neighbouring subcellular regions alternating out of phase. Moreover, focal inhibition of glycolysis causes spatially restricted Ca(2+) alternans, further emphasising the local character of this phenomenon. When two adjacent regions within a myocyte alternate out of phase, delayed propagating Ca(2+) waves develop at their border. In conclusion, the results demonstrate that (1) during normal E-C coupling the atrial [Ca(2+)](i) transient is the result of the spatiotemporal summation of Ca(2+) release from individual release sites of the peripheral j-SR and the central nj-SR, activated in a centripetal fashion by CICR via I(Ca) and Ca(2+) release from j-SR, respectively, (2) Ca(2+) alternans is caused by subcellular alterations of SR Ca(2+) release mediated, at least in part, by local inhibition of energy metabolism, and (3) the generation of arrhythmogenic Ca(2+) waves resulting from heterogeneities in subcellular Ca(2+) alternans may constitute a novel mechanism for the development of cardiac dysrhythmias.

Ca(2+) alternans is a potentially arrhythmogenic beat-to-beat alternation of the amplitude of the action potential-induced [Ca(2+)](i) transient in cardiac myocytes. Despite its pathophysiological significance the cellular mechanisms underlying Ca(2+) alternans are poorly understood. Recent evidence, however, points to the modulation of Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) by localized alterations in energy metabolism as an important determinant of Ca(2+) alternans. We therefore studied the subcellular properties of Ca(2+) alternans in field-stimulated cat atrial myocytes employing fast two-dimensional fluorescence confocal microscopy. Ca(2+) alternans was elicited by an increase in stimulation frequency or by metabolic interventions targeting glycolysis. Marked subcellular variations in the time of onset, the magnitude, and the phase of alternans were observed. Longitudinal and transverse gradients of Ca(2+) alternans were found as well as neighbouring subcellular regions alternating out-of-phase. Moreover, focal inhibition of glycolysis resulted in spatially restricted Ca(2+) alternans. When two adjacent regions within a myocyte alternated out-of-phase, steep [Ca(2+)](i) gradients developed at their border giving rise to delayed propagating Ca(2+) waves. The results demonstrate that Ca(2+) alternans is a subcellular phenomenon caused by modulation of SR Ca(2+) release, which is mediated, at least in part, by local inhibition of energy metabolism. The generation of arrhythmogenic Ca(2+) waves by subcellular variations in the phase of Ca(2+) alternans represents a novel mechanism for the development of atrial disrhythmias.

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca2+-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]iand cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]iand 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.

Fast two-dimensional confocal microscopy and the Ca(2+) indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca(2+) concentration ([Ca(2+)](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced approximately 2 microm apart. The amplitude and the latency of Ca(2+) release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca(2+)](i), which actively propagated to the center of the cells via Ca(2+)-induced Ca(2+) release. Resting myocytes exhibited spontaneous Ca(2+) release events, including Ca(2+) sparks and local (microscopic) or global (macroscopic) [Ca(2+)](i) waves. The microscopic [Ca(2+)](i) waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca(2+) sparks) revealing the sequential activation of Ca(2+) release sites of the j-SR. Moreover, during global [Ca(2+)](i) waves, Ca(2+) release was evident from individual nj-SR sites. Ca(2+) release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca(2+) release channels. The longitudinal and transverse distances between individual Ca(2+) release sites were both approximately 2 microm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca(2+)](i) transient is the spatio-temporal summation of Ca(2+) release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca(2+) channels. Subsequently, nj-SR sites are activated by Ca(2+)-induced Ca(2+) release propagating from the periphery.

See related article, pages 398–406 Excitation-contraction coupling in the heart relies on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). Ca2+ influx via L-type Ca2+ channels during an action potential triggers Ca2+ release from the SR via Ca2+ release channels, or ryanodine receptors (RyR2). Fine tuning of RyR2-mediated SR Ca2+ release is central to cardiac function. When RyR2-mediated Ca2+ release increases, the resulting augmentation of the [Ca2+]i transient causes increased contraction. Uncontrolled openings of RyR2 during diastole, on the other hand, may elicit delayed afterdepolarizations and arrhythmias. Dysfunction of RyR2 may occur under certain pathological conditions, eg, excess sympathetic stimulation, and may contribute to such cardiac diseases as heart failure1 or atrial fibrillation.2 Furthermore, mutations in RyR2 can cause stress-induced ventricular tachycardias and sudden death in otherwise healthy individuals.3 Thus, proper regulation and function of RyR2 is essential for adequate cardiac function. Not surprisingly, because of its crucial role in cardiac excitation-contraction coupling, RyR2 activity is highly regulated.4 Substances involved in regulation of RyR2 activity include Ca2+, Mg2+, H+, adenine nucleotides, calmodulin, NAD+/NADH, nitric oxide, and glycolytic intermediates, to name but a few. The list of molecules regulating RyR2 activity is far from complete and will undoubtedly grow longer as research continues. In addition, RyR2 is regulated by phosphorylation. The protein forms a macromolecular complex with regulatory proteins (notably FKBP12.6), cytoskeletal proteins, adapter proteins, kinases, and phosphatases.1,5 This allows for tight control and localized regulation of RyR2 activity in the microenvironment of the channel. Regulation of RyR2 activity by phosphorylation is not only important from a physiological point of view to adjust SR Ca2+ release and, ultimately, cardiac output to the varying demands …

Background— A hallmark of heart failure is impaired cytoplasmic Ca 2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca 2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results— Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca 2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca 2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca 2+ transporters. These changes were associated with altered nucleoplasmic Ca 2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca 2+ levels with diminished and prolonged nuclear Ca 2+ transients and slowed intranuclear Ca 2+ diffusion. Altered nucleoplasmic Ca 2+ levels were translated to higher activation of nuclear Ca 2+ /calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca 2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca 2+ changes that are typical of heart failure. Conclusions— During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca 2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca 2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.

The Na/K pump current I(p) of single HEK293 cells either untransfected (endogenous I(p)) or transfected with the alpha1 subunit of the rat Na/K pump (exogenous I(p)) was investigated in Na-containing solution by means of whole-cell recording at 30 degrees C. The endogenous I(p) was irreversibly blocked by 10(-4) M ouabain or 2 x 10(-4) M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF(-1) at 0 mV and 5.4 mM K(o). It was half maximally activated at 1.5 mM K(o) and increased linearly with depolarization over the entire voltage range studied (-80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant alpha1 subunit of the rat Na/K pump showed an I(p) in the presence of 10(-4) M ouabain and 2 x 10(-4) M DHO, respectively. This exogenous I(p) was reversibly blocked by 10(-2) M ouabain. Half maximal activation of the exogenous I(p) occurred at 1.7 mM K(o). Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I(p) of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I(p) in HEK293 cells is generated by the alpha1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.

In 1872 Traube1 first described pulsus alternans, a regular beat-to-beat alternation of the strength of the heartbeat. Since then, cardiologists and physiologists have learned that cardiac alternans can come in many flavors: as mechanical, electrical, or [Ca2+]i transient alternans (Ca2+ alternans).2 They also had to realize that alternans is a life-threatening condition, less so because of impaired cardiac output but because it can lead to ventricular fibrillation (VF) and sudden cardiac death. How exactly electromechanical alternations of the heartbeat can cause VF has long been an open question. A leap forward came recently with an elegant study on electrical (T-wave) alternans in guinea-pig hearts.3 It was shown that during alternans, neighboring regions within the heart started to alternate out-of-phase with each other (discordant alternans). Such discordant electrical alternans, if sufficient in magnitude, led to unidirectional block and reentry, thereby causing VF. Despite recent advances in our understanding of the mechanisms linking electromechanical alternans to VF, the crucial question still remains: how does alternans develop in the first place? The study of cellular Ca2+ alternans might help answer this question because Ca2+ alternans lies at the heart of the problem. It causes both mechanical alternans (by activation of the myofilaments) and electrical alternans (by modulation of Ca2+-dependent membrane currents). Experimental interventions aimed at disabling sarcoplasmic reticulum (SR) Ca2+ release abolish electromechanical alternans.4 Furthermore, enhancement of sarcolemmal Ca2+ influx and/or SR Ca2+ load and release can reverse alternans.5,6⇓ Thus, modulation of the SR Ca2+ release process is somehow critically involved in the generation of alternans and only a detailed study of this process can help elucidate the underlying mechanisms. In the last 20 years, scientists have learned a tremendous deal about the subcellular and …