Loss of CASK Accelerates Heart Failure Development

Abstract

[Figure: see text].

Keywords: calmodulin; heart failure; myocardium; neurons; sarcoplasmic reticulum.

Figure 1:. CASK expression is regulated in hypertrophy and HF

A) Western blots (left panels) and mean analysis (right panel) of CASK expression in human left ventricular (LV) myocardium from patients with end-stage heart failure (HF) or patients with aortic stenosis (AS) in comparison to LV samples from healthy organ donor hearts (NF). CASK expression is increased in AS compared to HF and NF. Data are normally distributed (Shapiro-Wilk-test). *One way ANOVA p=0.0017, Holm-Sidak post-test (p in figure, n=mice). B) Original blots analyzing CASK (upper panel) and CaMKII (lower panel) expression in immunoprecipitated proteins CASK (anti-CASK), NaV1.5 (anti-NaV1.5) and CaMKII (anti-CaMKII) from homogenates of HF vs. NF.

Figure 2:. CASK-KO mice have no cardiac structural or functional abnormalities

A) Original M-mode acquisitions (left panel, parasternal long axis view) from anesthetized CASK-CTRL (CTRL), CASK-Flox (Flox) and CASK-KO (KO) mice. Upper right panel shows mean data of left ventricular ejection fraction and left ventricular end diastolic diameter. Data follows normal distribution (Shapiro-Wilk-test). No significant differences could be detected (One way ANOVA with Tukey’s post-test). EF: ANOVA p=0.8668. LVEDD: ANOVA p=0.9447. B) Kaplan-Meier survival analysis for CASK-CTRL, CASK-Flox, and CASK-KO mice shows no increased mortality. Log-rank (Mantel-Cox) p=0.6462. n=mice at risk. C) Western blots and mean data of CaMKII T286-phosphorylation (normalized to CaMKII-expression; data are normally distributed [Kolmogorov-Smirnov-test], one way ANOVA *p=0.0293), phospholamban T17-phosphorylation (normal distribution [D’Agostino-Pearson test], one way ANOVA *p=0.0052), and CaMKII T305-phosphorylation (normal distribution [Shapiro-Wilk-test], one way ANOVA p=0.2758) in ventricular homogenates. For multiple comparisons within each data set, Holm-Sidak post-tests were applied (p in figures). For pT286/CaMKII ratio, one extreme statistical outlier was removed from the CASK-Flox group after statistical outlier analysis.

Figure 3:. CASK-KO mice show increased Ca leak

A) Western blots and mean data of CaMKII T286 (pT286) phosphorylation (normalized to CaMKII-expression), and CaMKII T305-phosphorylation in ventricular homogenates from mice that were harvested 30 min after intraperitoneal injection of ISO (2 mg/kg body weight). Left-panel: data are not normally distributed (D’Agostino-Pearson test) and Mann-Whitney-Test was applied; right panel: data are normally distributed (Shapiro-Wilk-test) and Student’s unpaired t-test was applied. n=mice. B) Western blots and mean data of RyR2 serine 2814 (pS2814) phosphorylation (normalized to RyR2-expression) in ventricular homogenates from mice that were harvested 30 min after intraperitoneal injection of ISO (2 mg/kg body weight). Data are normally distributed (Shapiro-Wilk test) and unpaired t-test applied, n=mice. C) Left panel: original line scans analyzing elementary SR Ca release events (Ca sparks) in resting Fluo-4-loaded ventricular myocytes. Center panel: violin plot of SR Ca spark frequency. n=cells. Data are not normally distributed (D’Agostino-Pearson test), and Kruskal-Wallis-test (p<0.0001) with Dunn’s post-test (p in graph) was applied. Right panel: mean data for Caffeine transient amplitude. Data are normally distributed (Shapiro-Wilk-test) and two-way ANOVA was applied (p for substance p<0.0001 and for genotype p<0.0243). Multiple comparisons were done by Newman-Keuls post-test (p in graph). n=cells. D) Ca transient amplitude at 1 Hz before and after exposure to ISO (10-7 mol/l) in Fluo-4 –loaded isolated ventricular myocytes. Data are not normally distributed (D’Agostino-Pearson test) and *Kruskal-Wallis-test (p<0.0001) was applied. Multiple comparison by Dunn’s post-test (p in graph). n=cells.

Figure 4:. CASK-KO mice show disturbed Ca handling and ventricular arrhythmias in vivo

A) Original trace of Fluo-4 fluorescence in an isolated ventricular myocyte of a CASK KO mouse. Arrows indicate electrical field-stimulation. The left panel displays a burst of Ca release events followed by a single delayed non-stimulated event (DNSE). In the right panel, data for scored arrhythmias normalized to observation time are shown. Data were normally distributed (D’Agostino-Pearson) and analyzed after Box-Cox transformation. Two-way repeated measured mixed-effects analysis with two-stage linear step-up post-test procedure of Benjamini, Krieger and Yekutieli was performed (n=cells). In the lower left panel, the proportions of cells showing DNSE upon isoproterenol are shown. Lower mid panel shows the proportions of cells with Ca alternans or runs upon isoproterenol. Lower right panel shows the proportions of cells with severe arrhythmias upon isoproterenol (defined as either doublets, triplets, runs, alternans or sustained premature contractions, Fisher’s exact tests, n=cells). B) Original surface ECG acquisitions (lead II) in mice subjected to burst stimulation at the right ventricular apex in vivo. C) Mean data of the proportion of mice showing arrhythmias (Fisher’s exact test, n=mice). Label information: CTRL=CASK-CTRL, Flox=CASK-Flox, KO=CASK-KO

Figure 5:. CASK-KO accelerates heart failure progression and increases mortality upon TAC

A) Original traces of echocardiographic M-mode acquisitions (parasternal long axis view) in CASK-CTRL or CASK-KO mice before TAC, and at two weeks after TAC operation. B) Mean data of left ventricular ejection fraction (EF). Data follows normally distribution (Shapiro-Wilk-test, *two-way repeated measured mixed-effects analysis p for TAC=0.0459 with Holm-Sidak post-test (p values in graph, n=mice). C) Kaplan-Meier survival analysis for CASK-CTRL, CASK-Flox and CASK-KO mice upon TAC. Data were tested by Log-rank (Mantel-Cox) with p in graph. n=mice at risk. D) Mean densitometric expression of CASK (normalized to GAPDH, western blot) in left ventricular homogenates of mice after TAC (vs. Sham-operated mice). Data are normally distributed (Shapiro-Wilk-test) and tested by unpaired t-test (p in graph, n=mice. E) Original registrations (western blot) and mean densitometric data for CaMKII expression and T286 autophosphorylation in ventricular homogenates from CASK-CTRL and KO mice 2 weeks (2w) after TAC. Left panel: Data are not normally distributed (Shapiro-Wilk) and tested by *Kruskall-Wallis p=0.0089. Multiple comparison were done by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (p in graph, n=mice. Right panel: Data are normally distributed (Shapiro-Wilk-test) and tested by *ANOVA p=0.0132 with Holm-Sidak post-test (p in graph, n=mice). F) Mean densitometric data for T305-autophosphorylation shows that compared to CASK-CTRL, T305 autophosphorylation was significantly reduced in CASK KO upon TAC. Data are normally distributed (Shapiro-Wilk-test) and compared by unpaired t-test (p in graph, n=mice). G) Mean densitometric analysis of CaMKII-oxidation relative to CaMKII expression indicates enhanced CaMKII oxidation in CASK KO upon TAC. Data are normally distributed (Shapiro-Wilk-test) and compared by unpaired t-test (p in graph, n=mice. Label information: CASK-CTRL = CTRL, CASK-Flox = Flox, CASK-KO = KO

Figure 6:. CASK expression is increased upon GLP1-receptor agonist treatment

A) Original Western blots and mean densitometric data of CASK-expression in isolated ventricular cardiomyocytes from CASK-CTRL or CASK-KO mice that were harvested 24 h after culture with either vehicle control (DMSO) or 100 nmol/L of the GLP1-receptor agonist (GLP1-RA) exenatide. Data follows normal distribution (D’Agostino-Pearson-test) and was compared by paired t-test (p in graph, n=mice). B) Left panel: original Western blots and mean densitometric data of T305-phosphorylation (original registration contrast-enhanced equally across all lanes). Data are normally distributed (D’Agostino-Pearson test) and tested by two-way mixed-effects analysis (substance *p=0.0278, genotype *p=0.0115, interaction *p=0.0295) with Sidak post-tests (p in graph). Right panel: CASK-expression correlates strongly with T305-phosphorylation (linear regression p<0.05, R²=0.45). C) Original Western blots and mean densitometric data of CaMKII T286-phosphorylation. Data are normally distributed (D’Agostino-Pearson test) and tested by two-way ANOVA (genotype *p=0.0274, substance p=0.8033, interaction *p=0.0339). n=mice) D) Mean data of SR Ca spark frequency (CaSpF) as a marker of SR Ca leak in isolated ventricular cardiomyocytes from CASK-CTRL (left panel) or CASK-KO (right panel) mice that were harvested 24 h after culture with either vehicle control (DMSO) or 100 nmol/L of GLP1-RA and exposed to 10⁻⁷ mol/l isoproterenol. Data are normally distributed (D’Agostino-Pearson test) and compated by paired t-test (p in graph, n=mice) E) Mean data of Caffeine transient amplitude in Fluo-4 –loaded isolated ventricular myocytes that were harvested 24 h after culture with either vehicle control (DMSO) or 100 nmol/L of GLP1-RA and exposed to 10-7 mol/l isoproterenol. Left panel: CASK-CTRL. Data are normally distributed (Shapiro-Wilk-test) and compared by paired t-test (p in graph, n=mice). Right panel: CASK-KO. Data are not normally distributed (Shapiro-Wilk-test) and tested by Wilcoxon test (p in graph, n=mice).

Figure 7:. GLP-1 RA inhibits CaMKII activation upon TAC

A) Original confocal line scans (upper panel) and mean data of SR Ca²⁺ spark frequency (CaSpF) as a marker of SR Ca²⁺⁻leak (lower left panel), Caffeine transient amplitude (lower mid panel), and Ca²⁺ transient amplitude (lower right panel) in isolated ventricular cardiomyocytes from mice exposed to 5 weeks of transverse aortic constriction (TAC) that were harvested 24 h after culture with either vehicle control (DMSO) or 100 μmol/l of the GLP1-receptor agonist (GLP1-RA) exenatide. Lower left: Data are not normally distributed (D’Agostino-Pearson test) and tested by Mann-Whitney-test (p in graph, n=cells/mice). Lower mid panel: Data are normally distributed (D’Agostino-Pearson) and tested by unpaired t-test (p in graph, n=cells/mice). Lower right panel: Data are normally distributed (D’Agostino-Pearson) and tested by two-way-ANOVA (p in graph, n=cells/mice). B) Original Western blots and mean densitometric data of CaMKII T287-phosphorylation and CaMKII expression in hearts at 5 weeks after TAC and in vivo treatment with either vehicle or the GLP1-RA peptide 7-36. Data are normally distributed (Shapiro-Wilk-test) and tested by unpaired t-test for both panels (p in graph, n=mice).