Cardiac inward rectifier K+ currents (IK1) play an important role in maintaining resting membrane potential and contribute to late phase repolarization. Members of the Kir2.x channel family appear to encode for IK1. The purpose of this study was to determine the molecular composition of cardiac IK1 in rabbit ventricle. Western blots revealed that Kir2.1 and Kir2.2, but not Kir2.3, are expressed in rabbit ventricle. Culturing rabbit myocytes resulted in a ∼50 % reduction of IK1 density after 48 or 72 h in culture which was associated with an 80 % reduction in Kir2.1, but no change in Kir2.2, protein expression. Dominant‐negative (DN) constructs of Kir2.1, Kir2.2 and Kir2.3 were generated and tested in tsA201 cells. Adenovirus‐mediated over‐expression of Kir2.1dn, Kir2.2dn or Kir2.1dn plus Kir2.2dn in cultured rabbit ventricular myocytes reduced IK1 density equally by 70 % 72 h post‐infection, while AdKir2.3dn had no effect, compared to green fluorescent protein (GFP)‐infected myocytes. Previous studies indicate that the [Ba2+] required for half‐maximum block (IC50) differs significantly between Kir2.1, Kir2.2 and Kir2.3 channels. The dependence of IK1 on [Ba2+] revealed a single binding isotherm which did not change with time in culture. The IC50 for block of IK1 was also unaffected by expression of the different DN genes after 72 h in culture. Taken together, these results demonstrate functional expression of Kir2.1 and Kir2.2 in rabbit ventricular myocytes and suggest that macroscopic IK1 is predominantly composed of Kir2.1 and Kir2.2 heterotetramers.

Abstract-Prolonged action potential duration (APD) and decreased transient outward K ϩ current (I to ) as a result of decreased expression of K v4.2 and K v4.3 genes are commonly observed in heart disease. We found that treatment of cultured neonatal rat ventricular myocytes with Heteropoda Toxin 3 , a blocker of cardiac I to , induced hypertrophy as measured using cell membrane capacitance and 3 H-leucine uptake. To dissect the role of specific I to -encoding genes in hypertrophy, I to was selectively reduced by overexpressing mutant dominant-negative (DN) transgenes. I to amplitude was reduced equally (by about 50%) by overexpression of DN K v1.4 (K v1.4 O ne prominent feature of diseased cardiac muscle is prolongation of action potential duration (APD). Although changes in many ionic currents have been reported in heart disease, 1 reductions in transient outward K ϩ current (I to ) have consistently been observed in heart disease regardless of species. 2 In mammalian hearts, I to has been shown to be encoded by K v1.4 , K v4.2 , and K v4.3 potassium channel genes, although the relative contribution of these genes varies between species. 3,4 While a number of changes in cardiac function and gene expression occur in diseased hearts, decreased expression of K v4.2 and K v4.3 genes and associated changes in both I to density and AP profile are commonly observed in myocytes from many animal models of heart disease 2,5,6 and human heart failure. 7 Moreover, the magnitude of I to and the level of expression of K v4.2 and K v4.3 channels are also reduced by both acute and long-term activation of various receptor-mediated pathways in response to neurohumoral factors known to be involved in initiating cardiac hypertrophy, such as angiotensin II and phenylephrine. 8 -10 Despite the correlation between reduced K v4.2/3 expression and heart disease, the link between reductions in I to , K v4.2/3 expression, and cardiac hypertrophy is still unclear, although reductions in I to density and K v4.2/3 expression occur very early after myocardial infarction in rats. 6 APD prolongation following I to reduction can increase Ca 2ϩ influx through voltagedependent L-type Ca 2ϩ channels (I Ca,L ), thereby elevating [Ca 2ϩ ] i . 2,11 Because Ca 2ϩ is an essential cofactor for several hypertrophy signaling pathways, including calcineurin, mitogenactivated protein kinases (MAPK), and protein kinase C, 12,13 it is conceivable that increased Ca 2ϩ influx by I to reduction might modulate hypertrophy signaling in myocardium. One particularly attractive candidate pathway, linking reductions in I to to hypertrophy, is the Ca 2ϩ /calmodulin-activated cytoplasmic serine/threonine phosphatase calcineurin that dephosphorylates NFAT3 leading to nuclear translocation and transcriptional activation of numerous hypertrophy genes. 14 Moreover, calcineurin has been shown to play an important role in triggering hypertrophy signaling. 13 In this study, the connection between I to reduction and hypertrophy was investigated in cultured neonatal r...

Background-Cardiac-targeted expression of truncated K v 4.2 subunit (K v 4.2N) reduces transient outward current (I to ) density, prolongs action potentials (APs), and enhances contractility in 3-to 4-week-old transgenic mice. By 13 to 15 weeks of age, these mice develop severely impaired cardiac function and signs of heart failure. In this study, we examined whether augmented contractility in K v 4.2N mice results from elevations in intracellular calcium ([Ca 2ϩ ] i ) secondary to AP prolongation and investigated the putative roles of calcineurin activation in heart disease development of K v 4.2N mice. Methods and Results-At 3 to 4 weeks of age, L-type Ca 2ϩ influx and peak [Ca 2ϩ ] i were significantly elevated in K v 4.2N myocytes compared with control because of AP prolongation. Cardiac calcineurin activity was also significantly elevated in K v 4.2N mice by 5 weeks of age relative to controls and increased progressively as heart disease developed. This was associated with activation of protein kinase C (PKC)-␣ and PKC-but not PKC-⑀, as well as increases in ␤-myosin heavy chain (␤-MHC) and reductions in sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA)-2a expression. Treatment with either cyclosporin A or verapamil prevented increases in heart weight to body weight ratios, interstitial fibrosis, impaired contractility, PKC activation, and changes in the expression patterns of ␤-MHC and SERCA2a.Conclusions-Our results demonstrate that AP prolongation caused by I to reduction results in enhanced Ca 2ϩ cycling and hypercontractility in mice and suggests that elevations in [Ca 2ϩ ] i via I Ca,L and activation of calcineurin play a central role in disease development after I to reduction using the K v 4