Transgenic Expression of Kir6.1 Gain-of-Function Mutations in the Mouse Heart Result in Increased L-Type Calcium Current in Ventricular Myocytes

Abstract

Background The role of sarcolemmal K ATP current (I KATP ) in myocardial excitability remains unclear. Previously, mice with gain-of-function (GOF) 6.2 mutations (Kir6.2 GOF) were engineered to help elucidate I KATP function in the heart. Surprisingly, L-type calcium channel current (LTCCC) was increased in Kir6.2 GOF ventricular myocytes (VM), suggesting that increased I KATP secondarily leads to increased LTCCC. Methods and Results To further test this hypothesis, Kir6.1 GOF mice expressing a Kir6.1 GOF mutation (G343D) under αMHC promoter control have been generated. Patch-clamp data demonstrate reduced sensitivity of K ATP to inhibition by ATP in VM (EC 50 = 43.5 ± 6.1 µM in GOF, 8.3 ± 3.1 µM in wild type (WT); P = .0007). As with Kir6.2 GOF VM, Kir6.1 GOF VM showed markedly increased LTCCC density (pA/pF), comparable to that of WT VM treated with isoproterenol (ISO): (@ –15 mV (pA/pF): WT: –9.6 ± 1.1, n=12; WT+iso: –18.7 ± 2.7, n=4; Kir 6.1 GOF: –17.7 ± 3.6, n=15; Kir 6.1+ISO: –25.0 ± 8.3, n=9, analysis of variance [ANOVA], P P = NS). LTCCC was also recorded from VM expressing dominant-negative (DN) Kir6.1 [AAA] subunits (Kir6.1 DN). In contrast to Kir6.1 GOF VM, LTCCC was not increased in Kir6.1 DN VM: (@–15 mV (pA/pF): WT: –9.6 ± 1.1, n=12; WT+ISO: –18.7 ± 2.7, n=4; Kir 6.1DN: –14.4 ± 1.5, n=10; Kir 6.1 DN+ISO: –8.4 ± 3.8, n=4, ANOVA, P P = significant), suggesting that increased LTCCC in the former results from increased I KATP and not from increased protein per se. Slow-release minoxidil (MIN, a K ATP channel opener) pellets or placebo (P) pellets were also implanted in WT mice for 3 weeks. LTCCC was subsequently recorded to test whether pharmacologically increased I KATP results in increased LTCCC. As with the genetic models, the MIN model also showed increased LTCCC similar to WT+ISO VM: (@–15 mV (pA/pF): WT+P: –11.9 ± 1.1, n=6; WT+P+ISO: –25.4 ± 1.7, n=3; WT+MIN: –22.0 ± 7.6, n=7; WT+MIN+ISO: –35.4 ± 12.3, n=6, ANOVA, P P = NS). Conclusions The results support the idea that K ATP GOF leads to constitutive upregulation of LTCCC and suggest a potential mechanism underlying ventricular arrhythmias in Kir6.1-associated Brugada and early repolarization syndromes.