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BackgroundCardiac alternans are proarrhythmic and mechanistically link cardiac mechanical dysfunction and sudden cardiac death. Beat-to-beat alternans occur when beats with large Ca(2+) transients and long action potential duration (APD) alternate with the converse. APD alternans are typically driven by Ca(2+) alternans and sarcoplasmic reticulum (SR) Ca(2+) release alternans. But the effect of intercellular communication via gap junctions (GJ) on alternans in the intact heart remains unknown.ObjectiveWe assessed the effects of cell-to-cell coupling on local alternans in intact Langendorff-perfused mouse hearts, measuring single myocyte [Ca(2+)] alternans synchronization among neighboring cells, and effects of β-adrenergic receptor (β-AR) activation and reduced GJ coupling.Methods and resultsMouse hearts (C57BL/6) were retrogradely perfused and loaded with Fluo8-AM to record cardiac myocyte [Ca(2+)] in situ with confocal microscopy. Single cell resolution allowed analysis of alternans within the intact organ during alternans induction. Carbenoxolone (25 μM), a GJ inhibitor, significantly increased the occurrence and amplitude of alternans in single cells within the intact heart. Alternans were concordant between neighboring cells throughout the field of view, except transiently during onset. β-AR stimulation only reduced Ca(2+) alternans in tissue that had reduced GJ coupling, matching effects seen in isolated myocytes.ConclusionsCa(2+) alternans among neighboring myocytes is predominantly concordant, likely because of electrical coupling between cells. Consistent with this, partial GJ uncoupling increased propensity and amplitude of Ca(2+) alternans, and made them more sensitive to reversal by β-AR activation, as in isolated myocytes. Electrical coupling between myocytes may thus limit the alternans initiation, but also allow alternans to be more stable once established.

Calcium (Ca) is a universal second messenger involved in the regulation of various cellular processes, including electrical signaling, contraction, secretion, memory, gene transcription, and cell death. In heart, Ca governs cardiomyocyte contraction, is central in electrophysiological properties, and controls major signaling pathway implicated in gene transcription. How cardiomyocytes decode Ca signal to regulate gene expression without interfering with, or being controlled by, "contractile" Ca that floods the entire cytosol during each heartbeat is still elusive. In this review, we summarize recent findings on nuclear Ca regulation and its downstream signaling in cardiomyocytes. We will address difficulties in reliable quantification of nuclear Ca fluxes and discuss its role in the development and progression of cardiac hypertrophy and heart failure. We also point out key open questions to stimulate future work.

BackgroundA 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 resultsUsing 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.ConclusionsDuring 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.

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

Aims The sarcoplasmic reticulum ( SR) Ca2+ leak is an important pathomechanism in heart failure ( HF). It has been suggested that Ca2+/calmodulin-dependent protein kinase II ( CaMKII) is only relevant for the induction of the SR Ca2+ leak in non-ischaemic but not in ischaemic HF. Therefore, we investigated CaMKII and its targets as well as the functional effects of CaMKII inhibition in human ischaemic cardiomyopathy ( ICM, n = 37) and dilated cardiomyopathy ( DCM, n = 40). Methods and results Western blots showed a significantly increased expression (by 54 ± 9%) and autophosphorylation at Thr286 (by 129 ± 29%, P < 0.05 each) of CaMKII in HF compared with healthy myocardium. However, no significant difference could be detected in ICM compared with DCM as to the expression and autophosphorylation of CaMKII nor the phosphorylation of the target sites ryanodine receptor 2 ( RyR2)- S2809, RyR2-S2815, and phospholamban-Thr17. Isolated human cardiomyocytes ( CMs) of patients with DCM and ICM showed a similar frequency of diastolic Ca2+ sparks (confocal microscopy) as well as of major arrhythmic events (Ca2+ waves, spontaneous Ca2+ transients). Despite a slightly smaller size of Ca2+ sparks in DCM ( P < 0.01), the calculated SR Ca2+ leak [Ca2+ spark frequecy ( CaSpF) × amplitude × width × duration] did not differ between CMs of ICM vs. DCM. Importantly, CaMKII inhibition by autocamide-2-related inhibitory peptide ( AIP, 1 µmol/L) reduced the SR Ca2+ leak by ∼80% in both aetiologies ( P < 0.05 each) and effectively decreased the ratio of arrhythmic cells ( P < 0.05). Conclusion Functional and molecular measures of the SR Ca2+ leak are comparable in human ICM and DCM. CaMKII is equally responsible for the induction of the ' RyR2 leakiness' in both pathologies. Thus, CaMKII inhibition as a therapeutic measure may not be restricted to patients suffering from DCM but rather may be beneficial for the majority of HF patients. [ABSTRACT FROM AUTHOR]

Background--A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ 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 Ca2+ 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 Ca2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+ calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions--During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling. [ABSTRACT FROM AUTHOR]

Objectives: This study sought to explore whether subclinical alterations of sarcoplasmic reticulum (SR) Ca(2+) release through cardiac ryanodine receptors (RyR2) aggravate cardiac remodeling in mice carrying a human RyR2(R4496C+/-) gain-of-function mutation in response to pressure overload.Background: RyR2 dysfunction causes increased diastolic SR Ca(2+) release associated with arrhythmias and contractile dysfunction in inherited and acquired cardiac diseases, such as catecholaminergic polymorphic ventricular tachycardia and heart failure (HF).Methods: Functional and structural properties of wild-type and catecholaminergic polymorphic ventricular tachycardia-associated RyR2(R4496C+/-) hearts were characterized under conditions of pressure overload induced by transverse aortic constriction (TAC).Results: Wild-type and RyR2(R4496C+/-) hearts had comparable structural and functional properties at baseline. After TAC, RyR2(R4496C+/-) hearts responded with eccentric hypertrophy, substantial fibrosis, ventricular dilation, and reduced fractional shortening, ultimately resulting in overt HF. RyR2(R4496C+/-)-TAC cardiomyocytes showed increased incidence of spontaneous SR Ca(2+) release events, reduced Ca(2+) transient peak amplitude, and SR Ca(2+) content as well as reduced SR Ca(2+)-ATPase 2a and increased Na(+)/Ca(2+)-exchanger protein expression. HF phenotype in RyR2(R4496C+/-)-TAC mice was associated with increased mortality due to pump failure but not tachyarrhythmic events. RyR2-stabilizer K201 markedly reduced Ca(2+) spark frequency in RyR2(R4496C+/-)-TAC cardiomyocytes. Mini-osmotic pump infusion of K201 prevented deleterious remodeling and improved survival in RyR2(R4496C+/-)-TAC mice.Conclusions: The combination of subclinical congenital alteration of SR Ca(2+) release and pressure overload promoted eccentric remodeling and HF death in RyR2(R4496C+/-) mice, and pharmacological RyR2 stabilization prevented this deleterious interaction. These findings suggest potential clinical relevance for patients with acquired or inherited gain-of-function of RyR2-mediated SR Ca(2+) release. [ABSTRACT FROM AUTHOR]

Aims: The sarcoplasmic reticulum (SR) Ca(2+) leak is an important pathomechanism in heart failure (HF). It has been suggested that Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is only relevant for the induction of the SR Ca(2+) leak in non-ischaemic but not in ischaemic HF. Therefore, we investigated CaMKII and its targets as well as the functional effects of CaMKII inhibition in human ischaemic cardiomyopathy (ICM, n = 37) and dilated cardiomyopathy (DCM, n = 40)., Methods and Results: Western blots showed a significantly increased expression (by 54 ± 9%) and autophosphorylation at Thr286 (by 129 ± 29%, P < 0.05 each) of CaMKII in HF compared with healthy myocardium. However, no significant difference could be detected in ICM compared with DCM as to the expression and autophosphorylation of CaMKII nor the phosphorylation of the target sites ryanodine receptor 2 (RyR2)-S2809, RyR2-S2815, and phospholamban-Thr17. Isolated human cardiomyocytes (CMs) of patients with DCM and ICM showed a similar frequency of diastolic Ca(2+) sparks (confocal microscopy) as well as of major arrhythmic events (Ca(2+) waves, spontaneous Ca(2+) transients). Despite a slightly smaller size of Ca(2+) sparks in DCM (P < 0.01), the calculated SR Ca(2+) leak [Ca(2+) spark frequecy (CaSpF) × amplitude × width × duration] did not differ between CMs of ICM vs. DCM. Importantly, CaMKII inhibition by autocamide-2-related inhibitory peptide (AIP, 1 µmol/L) reduced the SR Ca(2+) leak by ∼80% in both aetiologies (P < 0.05 each) and effectively decreased the ratio of arrhythmic cells (P < 0.05)., Conclusion: Functional and molecular measures of the SR Ca(2+) leak are comparable in human ICM and DCM. CaMKII is equally responsible for the induction of the 'RyR2 leakiness' in both pathologies. Thus, CaMKII inhibition as a therapeutic measure may not be restricted to patients suffering from DCM but rather may be beneficial for the majority of HF patients., (© 2014 The Authors. European Journal of Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)