Objectives We thought to investigate whether SR Ca2+ content can regulate IKATP channels function using the cross-breeding mice of CaMKII inhibition (Inh) and phospholamban (PLN) gene knockout mice (PLN-/-).
Methods Mice were sent from Dr. Anderson’s Lab in USA to Animal Centre of Tonji Medical College in China. IKATP 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 (Irel). The data were fitted using the Hill equation: Irel = 100/(1 + ([ATP]/K1/2)H, where Irel is the relative current, K1/2 is the concentration causing half-maximum blockade, and H is the Hill coefficient. Single channel IKATP recordings were obtained from inside out patches using fire-polished pipettes (resistance ∼9–10 Ohm). The threshold for judging the open state of IKATP channels was set at half of the single channel amplitude. The nPo, where n represents the number of channels in the patch and Po 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 IKATP channels.
Results Consistent with our previous study, IKATP 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 IKATP channels. Furthermore, IKATP 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 IKATP encoding genes, indicating that CaMKII mediated increases in IKATP are independent of augmented transcription of IKATP channel subunit genes. Breeding Inh mice with PLN-/- mice returned IKATP and IKATP 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 IKATP in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity.
Conclusions These findings provide novel evidence that CaMKII links intracellular Ca2+ to cardiac IKATP and suggest that PLN is a critical CaMKII target for feedback regulation of IKATP in ventricular myocytes.
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