Fig. 2
γ2 AMPK activation lowers intrinsic HR by downregulating SA cell I f and Ca2+ clock pacemaker mechanisms. a HR in beats per minute (bpm) of R299Q γ2 and WT mice under anesthesia (n = 7–12). b HR during ex vivo-isolated cardiac perfusion (n = 6–11). c Representative action potentials from SA cells isolated from R299Q γ2 and WT mice. d Mean beating rate of SA cells from groups illustrated in c (n = 17 cells). e qRT-PCR validation of differentially expressed genes on SA node microarray (n = 3). FC fold-change. f, g Representative western blot (f) and analysis (g) of HCN4 levels in SA nodes from R299Q γ2 and WT mice. h Representative SA cell I f traces during steps to −125 mV. i Mean fully activated I/V curves (I f density plotted against membrane voltage) recorded in WT and R299Q γ2 SA cells. Linear data fitting yielded significant differences (P < 0.0001) in I f slope conductance (648 and 333 pS/pF for WT and homozygous R299Q γ2 SA cells, respectively) (n = 8–10 cells/2–6 mice). j Mean voltage dependence of I f activation of WT and R299Q γ2 SA cells (n = 6 per genotype). Half-activation voltages (V 1/2, mV) and inverse-slope factors (s, mV) depicted. k Representative confocal line-scan images and Ca2+ transients of isolated, single, permeabilized WT, and homozygous R299Q γ2 SA node cells bathed in 50 nmol/L free [Ca2+]. l–n Mean spontaneous local Ca2+ release (LCR) amplitude (l) expressed as peak value (F) normalized to minimal (F 0) fluorescence, size (m), and duration (n). o, p Ca2+ signal of individual LCR (o) and LCR ensembl (p) (n = 15–17 cells/3 mice per genotype). Uncropped western blots are shown in Supplementary Fig. 10. a, b, d One-way ANOVA followed by Holm–Sidak’s multiple comparisons test was performed. i Comparison of the slopes of linear regression lines was performed. e, g, l–p Student’s t-test was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. a, b, d, e, g, l–p Data are shown as means ± s.e.m.
