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Kontogeorgis A, Li X, Kang EY, Feig JE, Ponzio M, Kang G, Kaba RA, Wit AL, Fisher EA, Morley GE, Peters NS, Coetzee WA, Gutstein DE. Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents. Am J Physiol Heart Circ Physiol 295: H1905-H1916, 2008. First published August 29, 2008; doi:10.1152/ajpheart.590.2008.-Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that shortterm pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43[sup+/-]; 66% mean reduction in Cx43) mice for 6 h at 10-15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43[sup+/-] myocytes had significantly shorter action potential durations (APD) and increased steady-state (I[subss]) and inward rectifier (I[subκ1]) potassium currents compared with those of wild-type littermate cells. In Cx43[sup+/-] hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of 1[subss] compared with unpaced Cx43[sup+/-] hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart. [ABSTRACT FROM AUTHOR]

Mutations in human KCNE2, which encodes the MiRP1 potassium channel ancillary subunit, associate with long QT syndrome (LQTS), a defect in ventricular repolarization. The precise cardiac role of MiRP1 remains controversial, in part, because it has marked functional promiscuity in vitro. Here, we disrupted the murine kcne2 gene to define the role of MiRP1 in murine ventricles. kcne2 disruption prolonged ventricular action potential duration (APD), suggestive of reduced repolarization capacity. Accordingly, kcne2 (-/-) ventricles exhibited a 50% reduction in IK,slow1, generated by Kv1.5-a previously unknown partner for MiRP1.Ito,f, generated by Kv4 α subunits, was also diminished, by ~25%. Ventricular MiRP1 protein coimmunoprecipitated with native Kv1.5 and Kv4.2 but not Kv1.4 or Kv4.3. Unexpectedly, kcne2 (-/-) ventricular membrane fractions exhibited 50% less mature Kv1.5 protein than wild type, and disruption of Kv1.5 trafficking to the intercalated discs. Consistent with the reduction in ventricular K+ currents and prolonged ventricular APD, kcne2 deletion lengthened the QTc under sevoflurane anesthesia. Thus, targeted disruption of kcne2 has revealed a novel cardiac partner for MiRP1, a novel role for MiRPs in α subunit targeting in vivo, and a role for MiRP1 in murine ventricular repolarization with parallels to that proposed for the human heart. [ABSTRACT FROM AUTHOR]

Mutations in human KCNE2, which encodes the MiRP1 potassium channel ancillary subunit, associate with long QT syndrome (LQTS), a defect in ventricular repolarization. The precise cardiac role of MiRP1 remains controversial, in part, because it has marked functional promiscuity in vitro. Here, we disrupted the murine kcne2 gene to define the role of MiRP1 in murine ventricles. kcne2 disruption prolonged ventricular action potential duration (APD), suggestive of reduced repolarization capacity. Accordingly, kcne2 (-/-) ventricles exhibited a 50% reduction in IK,slow1, generated by Kv1.5-a previously unknown partner for MiRP1.Ito,f, generated by Kv4 α subunits, was also diminished, by ~25%. Ventricular MiRP1 protein coimmunoprecipitated with native Kv1.5 and Kv4.2 but not Kv1.4 or Kv4.3. Unexpectedly, kcne2 (-/-) ventricular membrane fractions exhibited 50% less mature Kv1.5 protein than wild type, and disruption of Kv1.5 trafficking to the intercalated discs. Consistent with the reduction in ventricular K+ currents and prolonged ventricular APD, kcne2 deletion lengthened the QTc under sevoflurane anesthesia. Thus, targeted disruption of kcne2 has revealed a novel cardiac partner for MiRP1, a novel role for MiRPs in α subunit targeting in vivo, and a role for MiRP1 in murine ventricular repolarization with parallels to that proposed for the human heart. [ABSTRACT FROM AUTHOR]

Background: Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time. Results: The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10-15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias. Conclusion: Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease. [ABSTRACT FROM AUTHOR]

KATP channels are involved in regulating coronary function, but the contribution of endothelial KATP channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial KATP channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothelium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine-induced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The KATP channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the KATP channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin-1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin-1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin-1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of KATP channels led to elevated endothelin-1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin-1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial KATP channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothelin-1. [ABSTRACT FROM AUTHOR]

ATP-sensitive K+(KATP) channels couple cellular energy status to membrane excitability. Although it is established that KATP channels regulate coronary blood vessel tone, the specific role of endothelial KATP channels is unknown. We transgenically targeted endothelial KATP channels by expressing a dominant-negative Kir6.1 subunit under the control of the Tek promoter. Histologically, the coronary endothelium was preserved in transgenic (TG) mice. Ergonovine did not induce coronary vasospasm and the mean blood pressure was not detectably different in these mice. The nitric oxide pathway was intact since bradykinin decreased the coronary perfusion pressure (CPP). Levcromakalim also decreased the CPP, most likely by acting on smooth muscle KATP channels. In contrast, the CPP of isolated hearts was substantially elevated in TG mice. Pre-treatment of mice with bosentan [an endothelin-1 (ET-1) receptor antagonist] normalized the CPP, suggestive of an enhanced ET-1 release. Indeed, ET-1 levels (measured by RIA) were elevated in the coronary effluent of TG hearts. KATP channel blockers (e.g. tolbutamide) similarly elevated ET-1 release; both from isolated rat hearts and 'from cultured human coronary artery endothelial cells. In conclusion, our data suggest a novel role for endothelial KATP channels in regulating coronary vessel tone by controlling regulated exocytosis of the vasoconstrictor ET-1. [ABSTRACT FROM AUTHOR]