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We analyzed the effect of conditional, [alpha]MHC-dependent genetic [beta]-catenin depletion and stabilization on cardiac remodeling following experimental infarct. [beta]-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: [CT.sup.[delta]ex3-6]: 24 [+ or -] 1.9%; [beta]-[cat.sup.[delta]ex3-6]; 30.2 [+ or -] 1.6%, P < 0.001). [beta]-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: [beta]-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small [cTnT.sup.pos] cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by [alpha]MHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage ([alpha]MHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-[kit.sup.pos] and stem cell antigen-1 [(Sca-1).sup.pos] precursor cell population but not with the Islet-[1.sup.pos] precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-[1.sup.pos] cardiac precursor cells from [beta]-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward [alpha]-[actin.sup.pos] and [cTnT.sup.pos] cardiomyocytes after 10 days ([CT.sup.[delta]ex3-6]: 38.0 [+ or -] 1.0% [alpha]-[actin.sup.pos] [beta]-[cat.sup.[delta]ex3-6]: 49.9 [+ or -] 2.4% [alpha]-[actin.sup.pos], P < 0.001). We conclude that [beta]-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of [GATA4.sup.pos]/Sca-[1.sup.pos] resident cardiac progenitor cells.

Abnormalities in intracellular calcium release and reuptake are responsible for decreased contractility in heart failure (HF). We have previously shown that cardiac ryanodine receptors (RyRs) are protein kinase A-hyperphosphorylated and depleted of the regulatory subunit calstabin-2 in HF. Moreover, similar alterations in skeletal muscle RyR have been linked to increased fatigability in HF. To determine whether restoration of calstabin binding to RyR may ameliorate cardiac and skeletal muscle dysfunction in HF, we treated WT and calstabin-[2.sup.-/-] mice subjected to myocardial infarction (MI) with JTV519. JTV519, a 1,4-benzothiazepine, is a member of a class of drugs known as calcium channel stabilizers, previously shown to increase calstabin binding to RyR. Echocardiography at 21 days after MI demonstrated a significant increase in ejection fraction in WT mice treated with JTV519 (45.8 [+ or -] 5.1%) compared with placebo (31.1 [+ or -] 3.1%; P < 0.05). Coimmunoprecipitation experiments revealed increased amounts of calstabin-2 bound to the RyR2 channel in JTV519-treated WT mice. However, JTV519 did not show any of these beneficial effects in calstabin-[2.sup.-/-] mice with MI. Additionally, JTV519 improved skeletal muscle fatigue in WT and calstabin-[2.sup.-/-] mice with HF by increasing the binding of calstabin-1 to RyR1. The observation that treatment with JTV519 improved cardiac function in WT but not calstabin-[2.sup.-/-] mice indicates that calstabin-2 binding to RyR2 is required for the beneficial effects in failing hearts. We conclude that JTV519 may provide a specific way to treat the cardiac and skeletal muscle myopathy in HF by increasing calstabin binding to RyR. calcium | FKBP12.6 | myocardial infarction | contractility

Aim: In healthy hearts, ventricular gap junctions are mainly composed by connexin43 (Cx43) and localize in the intercalated disc, enabling appropriate electrical coupling. In diseased hearts, Cx43 is heterogeneously down-regulated, whereas activity of calmodulin/calcium-calmodulin protein kinase II (CaM/CaMKII) signalling increases. It is unclear if CaM/CaMKII affects Cx43 expression/localization or impulse propagation. We analysed different models to assess this. Methods and results: AC3-I mice with CaMKII genetically inhibited were subjected to pressure overload (16 weeks, TAC vs. sham). Optical and epicardial mapping was performed on Langendorff-perfused rabbit and AC3-I hearts, respectively. Cx43 subcellular distribution from rabbit/mouse ventricles was evaluated by immunoblot after Triton X-100-based fractionation. In mice with constitutively reduced CaMKII activity (AC3-I), conduction velocity (CV) was augmented (n = 11, P, 0.01 vs. WT); in AC3-I, CV was preserved after TAC, in contrast to a reduction seen in TAC-WT mice (220%). Cx43 expression was preserved after TAC in AC3-I mice, though arrhythmias and fibrosis were still present. In rabbits, W7 (CaM inhibitor, 10 mM) increased CV (6-13%, n = 6, P, 0.05), while susceptibility to arrhythmias decreased. Immunoconfocal microscopy revealed enlarged Cx43 cluster sizes at intercalated discs of those hearts. Total Cx43 did not change by W7 (n = 4), whereas Triton X-100 insoluble Cx43 increased (+21%, n = 4, P, 0.01). Similar findings were obtained in AC3-I mouse hearts when compared with control, and in cultured dog cardiomyocytes. Functional implication was shown through increased intercellular coupling in cultured neonatal rat cardiomyocytes. Conclusion: Both acute and chronic CaM/CaMKII inhibition improves conduction characteristics and enhances localization of Cx43 in the intercalated disc. In the absence of fibrosis, this reduced the susceptibility for arrhythmias. [ABSTRACT FROM AUTHOR]

In mice, the calcium-dependent phosphatase calcineurin A (CnA) induces a transcriptional pathway leading to pathological cardiac hypertrophy. Interestingly, induction of CnA has been frequently noticed in human hypertrophic and failing hearts. Independently, the arrhythmia vulnerability of such hearts has been regularly associated with remodeling of parameters determining electrical conduction (expression level of connexin43 (Cx43) and NaV1.5, connective tissue architecture), for which the precise molecular basis and sequence of events is still unknown. Recently, we observed reduced Cx43 and NaV1.5 expression in 4-week old mouse hearts, overexpressing a constitutively active form of CnA (MHC-CnA model), but the order of events is still unknown. Therefore, three key parameters of conduction (Cx43, NaV1.5 and connective tissue expression) were characterized in MHC-CnA ventricles versus wild-type (WT) during postnatal development on a weekly basis. At postnatal week 1, CnA overexpression induced cardiac hypertrophy in MHC-CnA. Moreover, protein and RNA levels of both Cx43 and NaV1.5 were reduced by at least 50% as compared to WT. Cx43 immunoreactive signal was reduced at week 2 in MHC-CnA. At postnatal week 3, Cx43 was less phosphorylated and RNA level of Cx43 normalized to WT values, although the protein level was still reduced. Additionally, MHC-CnA hearts displayed substantial fibrosis relative to WT, which was accompanied by increased RNA levels for genes previously associated with fibrosis such as Col1a1, Col1a2, Col3a1, Tgfb1, Ctgf, Timp1 and microRNA miR-21. In MHC-CnA, reduction in Cx43 and NaV1.5 expression thus coincided with overexpression of CnA and hypertrophy development and preceded significant presence of fibrosis. At postnatal week 4 the alterations in conductional parameters observed in the MHC-CnA model lead to abnormal conduction and arrhythmias, similar to those observed in cardiac remodeling in heart failure patients. The MHC-CnA model, therefore, provides for a unique model to resolve the molecular origin of conductional remodeling in detail. [ABSTRACT FROM AUTHOR]

Abstract: Alterations in expression levels of Nav1.5, Cx43 and Cx40 have been frequently reported in cardiac disease and are associated with the development of arrhythmias, but little is known about the underlying molecular mechanisms. In this study we investigated electrical conduction and expression of Nav1.5, Cx43 and Cx40 in hearts of transgenic mice overexpressing a constitutively active form of calcineurin (MHC-CnA). ECG recordings showed that atrial, atrioventricular and ventricular activation were significantly prolonged in MHC-CnA hearts as compared to wildtype (WT) littermates. Epicardial activation and arrhythmia susceptibility analysis revealed increased ventricular activation thresholds and arrhythmia vulnerability. Moreover, epicardial ventricular activation patterns in MHC-CnA mice were highly discontinuous with multiple areas of block. These impaired conduction properties were associated with severe reductions in Nav1.5, Cx43 and Cx40 protein expression in MHC-CnA hearts as visualized by immunohistochemistry and immunoblotting. Real-time RT-PCR demonstrated that the decreased protein levels for Nav1.5 and Cx40, but not for Cx43, were accompanied by corresponding reductions at the RNA level. Cx43 RNA isoform analysis indicated that the reduction in Cx43 protein expression is caused by a post-transcriptional mechanism rather than by RNA isoform switching. In contrast, RNA isoform analysis for Cx40 and Nav1.5 provided additional evidence that in calcineurin-induced hypertrophy the downregulation of these proteins originates at the transcriptional level. These results provide the molecular rationale for Nav1.5, Cx43 and Cx40 downregulation in this model of hypertrophy and failure and the development of the pro-arrhythmic substrate. [Copyright &y& Elsevier]