Fig. 1
Generation of the R299Q γ2 AMPK knock-in mouse and enrichment of γ2 AMPK in WT SA nodes. a Mean 24-h heart rate (HR) of human heterozygous R302Q γ2 mutation carriers (age 41.2 ± 2.8 years) vs genotype-negative sibling controls (age 38.9 ± 2.3 years) (n = 10–15). All subjects had any anti-arrhythmic drugs or β-adrenoceptor blockers discontinued for 5 days prior to ECG and none were on amiodarone. b Schematic of gene-targeting strategy to generate the R299Q γ2 AMPK knock-in. Neo, neomycin selection cassette; FRT, Flp recombinase recognition target; red asterisk denotes mutation in exon 7 of Prkag2. c γ2 AMPK-specific activity of freeze-clamped ex vivo perfused hearts measured by SAMS peptide phophorylation assay in the absence or presence of AMP (n = 18–22). d Representative western blot of whole heart tissue from R299Q γ2 and WT mice for phospho-(p) ACC (n = 11–15). e–g Cine MRI analysis of left ventricular (LV) mass (e), end-diastolic volume (EDV) (f) and ejection fraction (EF) (g) in R299Q γ2 and WT mice aged 2 months (n = 8–19). h Cardiac tissue glycogen content from 12 month R299Q γ2 and WT mice together with a positive control heart from a homozygous Gaa (encoding acid α-glucosidase) knockout mouse (n = 12–15). i, j Periodic acid-Schiff (PAS) staining (i) (scale bar, 5 µm) and quantification of glycogen content (j) (as %PAS-positive cells/field) of SA node (SAN) sections (n = 12). k, l Quantitative real-time PCR (qRT-PCR) of γ2 and γ1 AMPK isoform relative gene expression levels (normalized to β-actin) from normal murine SA node and LV (SAN, n = 4; LV, n = 10). m–o Western blot (m) and densitometry (n, o) of γ2 and γ1 AMPK in normal murine SA node and LV, together with SA node positive (HCN4) and loading (GAPDH) controls (n = 6–8). Uncropped western blots are shown in Supplementary Fig. 10. a, k, l, n, o Student’s t-test was performed. c Kruskal–Wallis test followed by Dunn’s multiple comparisons test was performed. e–h, j One-way analysis of variance (ANOVA) followed by Holm–Sidak’s multiple comparisons test was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. RE relative expression, AU arbitrary units. a, c, e–h, j–l, n, o Data are shown as means ± s.e.m.
