ASSA14-02-13 CaMKII inhibition influences IKATPchannel's function through SR Ca2+feedback regulation

Abstract

<h3>Objectives</h3> We thought to investigate whether SR Ca²⁺ content can regulate I_KATP channels function using the cross-breeding mice of CaMKII inhibition (Inh) and phospholamban (PLN) gene knockout mice (PLN^-/-). <h3>Methods</h3> Mice were sent from Dr. Anderson’s Lab in USA to Animal Centre of Tonji Medical College in China. I_KATP from ventricular myocytes was recorded using inside-out patch-clamp configuration of the patch-clamp technique. Steady-state dependence of membrane current on [ATP] was obtained by calculating the relative current indexed to zero ATP (<i>I</i>rel). The data were fitted using the Hill equation: <i>I</i>rel = 100/(1 + ([ATP]/K_1/2)^H, where <i>I</i>rel is the relative current, K_1/2 is the concentration causing half-maximum blockade, and H is the Hill coefficient. Single channel I_KATP recordings were obtained from inside out patches using fire-polished pipettes (resistance ∼9–10 Ohm). The threshold for judging the open state of I_KATP channels was set at half of the single channel amplitude. The <i>nP</i>o, where <i>n </i>represents the number of channels in the patch and <i>P</i>o the probability of each channel to open, was assessed using Clampfit-10 software. Using qRTPCR, we measured mRNA expression levels of genes encoding SUR1 (ABCC8), SUR2 (ABCC9), Kir6.1 (KCNJ8) and Kir6.2 (KCNJ11), the subunits reconstituting functional I_KATP channels. <h3>Results</h3> Consistent with our previous study, I_KATP was significantly increased, while the negative regulatory dose-dependence of ATP was unchanged in Inh mice compared to wild type (WT) and CaMKII inhibition control (Con) mice, suggesting that CaMKII inhibition has feedback regulation of the functional I_KATP channels. Furthermore, I_KATP channel opening probability was equivalent in cell membrane patches from Inh, WT and Con ventricular myocytes. We found that CaMKII inhibition does not affect mRNA levels for I_KATP encoding genes, indicating that CaMKII mediated increases in I_KATP are independent of augmented transcription of I_KATP channel subunit genes. Breeding Inh mice with PLN^-/- mice returned I_KATP and I_KATP channel opening probability to control levels and equalised the APD and QT intervals in Inh mice to Con and WT levels. Dialysis of CaMKII inhibitory peptide into WT cells did not result in increased IKATP, suggesting that enhanced I_KATP in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity. <h3>Conclusions</h3> These findings provide novel evidence that CaMKII links intracellular Ca²⁺ to cardiac I_KATP and suggest that PLN is a critical CaMKII target for feedback regulation of I_KATP in ventricular myocytes.