An ALPK3 truncation variant causing autosomal dominant hypertrophic cardiomyopathy is partially rescued by mavacamten

Figures

• 

  Fig. 1. Phenotype of the novel Alpk3 K201X mouse model. Cardiac structure and function were assessed via echocardiography in wildtype (WT), heterozygous (Het) and homozygous (Hom) Alpk3 K201X animals at 3 months. WT and Het animals were also assessed at 6 months. Fractional shortening using the short axis view (FS SAX, A), peak systolic velocity (PSV, B), calculated left ventricular mass (LVM (c), C), heart weight (HW) to tibia length (TL) ratio (D), and left ventricular internal diameter in diastole (LVID: d, E) and systole (LVID: s, F) are shown. Values are presented as mean ± SEM. Kruskal-Wallis test indicates significant changes in Hom mice; **p < 0.01, ****p < 0.0001 versus WT 3 months. To compare WT and Het mice at 6 months, unpaired Student’s t-test was performed and no significant changes were observed. For a full set of echocardiographic parameters and n numbers per parameter see (Table S3).

• 

  Fig. 2. Chronic adrenergic challenge aggravates the hypertrophic response in heterozygous Alpk3 K201X. (A) The heart weight (HW) to tibia length (TL) ratio increased in both wildtype (WT) and heterozygous (Het) animals upon isoprenaline and phenylephrine (Iso/PE) treatment compared to their control groups (sham) treated with saline, indicating cardiac hypertrophy. There was a stronger response in Iso/PE treated Het compared to WT animals. WT sham n = 12; WT Iso/PE n = 11; Het sham n = 10; Het Iso/PE n = 10. (B) Het Alpk3 K201X mice displayed an aggravated hypertrophic response upon chronic adrenergic challenge as evidenced by calculated left ventricular mass (LVM). (C) Left ventricular anterior wall thickness in diastole (LVAW: d) increased in challenged Het animals only. WT sham n = 12; WT Iso/PE n = 10; Het sham n = 8; Het Iso/PE n = 12 (for B and C). Values are presented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 (2-way-ANOVA), otherwise considered not significant. For a full set of echocardiographic parameters please refer to (Table S5).

• 

  Fig. 3. Isolated left ventricular cardiomyocytes from Alpk3 K201X mice in the absence of the Ca2+ indicator fura2 displayed reduced diastolic sarcomere length, which is partially rescued by mavacamten treatment. (A) Average sarcomere length traces from heterozygous Alpk3 K201X (Het, red) and homozygous Alpk3 K201X cardiomyocytes (Hom, grey) decreased compared to wild type (WT) cardiomyocytes (blue). Contractile traces following treatment with 0.5 µM mavacamten (M) are shown in (B). Dot plots for selected extracted parameters: basal sarcomere length, fractional shortening, T90% contraction and T90% relaxation are given in (C–F) respectively. Data presented as median ± 95% confidence interval. Statistical differences comparing WT to Het and Hom are given by *, WT + M to Het + M and Hom + M are given by §, and control versus M for each genotype are given by #. They were calculated using a Kruskal-Wallis test with Dunns test for multiple comparisons., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Individual n numbers (cells per group of animals) are shown in (A) and (B) and all extracted parameters are tabulated in (Supplementary Table S6).

• 

  Fig. 4. Depleted PKA dependent protein phosphorylation in Alpk3 K201X mice Western blots of isolated cardiomyocyte preparations showed the phosphorylation of cTnI at Ser23/24 compared to total cTnI and GAPDH loading control (A), Densitometry analysis in (B) showed the degree of phosphorylation in wild type (WT), heterozygous (Het) and homozygous (Hom) mice (n = 5 mice per group). All PKA dependent phosphorylation targets were detected using an anti-phospho-RRXS/T antibody (C top), red stars denote an overexposed phospho-cTnI band, which was manually removed from the subsequent densitometric analysis. The loading control was cardiac actin, taken from the 42 kDa band on Coomassie stained control gels. (D) shows examples of lane densitometry measurements used to calculate cumulative band intensity across all detected proteins for WT, Het and Hom mice. The ratio of PKA target densitometry versus cardiac actin from Coomassie stained gels run in parallel is plotted in (E) (n = 6 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001 using a Kruskal-Wallis test with Dunn’s test for multiple comparisons. Uncropped blots are shown in (Fig. S9).

• 

  Fig. 5. Isolated left ventricular cardiomyocytes from Alpk3 K201X mice have elevated diastolic intracellular calcium levels and prolonged calcium reuptake, which is fully rescued by mavacamten treatment. (A) Average calibrated calcium transients from wild type (WT, blue), heterozygous Alpk3 K201X (Het, red), homozygous Alpk3 K201X cardiomyocytes (Hom, grey). (B) Calcium transients following treatment with 0.5 µM mavacamten (M) were similar between all genotypes. Dot plots for selected extracted parameters: Diastolic intracellular calcium levels [Ca2+]i (C), calcium transient amplitude (D), time to 90% (T90%) calcium release (E) and time to 50% (T50%) calcium reuptake (F). Black lines represent median ± 95% confidence interval. Statistical differences comparing WT to Het and Hom are given by *, WT + M to Het + M and Hom + M are given by §, and control versus M for each genotype are given by #. They were calculated using a Kruskal-Wallis test with Dunns test for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Individual n numbers (cells per group of animals) are shown in (A) and (B) and all extracted parameters are tabulated in (Supplementary Table S6).