Targeting calpain-2-mediated junctophilin-2 cleavage delays heart failure progression following myocardial infarction

Abstract

Coronary heart disease (CHD) is a prevalent cardiac disease that causes over 370,000 deaths annually in the USA. In CHD, occlusion of a coronary artery causes ischemia of the cardiac muscle, which results in myocardial infarction (MI). Junctophilin-2 (JPH2) is a membrane protein that ensures efficient calcium handling and proper excitation-contraction coupling. Studies have identified loss of JPH2 due to calpain-mediated proteolysis as a key pathogenic event in ischemia-induced heart failure (HF). Our findings show that calpain-2-mediated JPH2 cleavage yields increased levels of a C-terminal cleaved peptide (JPH2-CTP) in patients with ischemic cardiomyopathy and mice with experimental MI. We created a novel knock-in mouse model by removing residues 479-SPAGTPPQ-486 to prevent calpain-2-mediated cleavage at this site. Functional and molecular assessment of cardiac function post-MI in cleavage site deletion (CSD) mice showed preserved cardiac contractility and reduced dilation, reduced JPH2-CTP levels, attenuated adverse remodeling, improved T-tubular structure, and normalized SR Ca²⁺-handling. Adenovirus mediated calpain-2 knockdown in mice exhibited similar findings. Pulldown of CTP followed by proteomic analysis revealed valosin-containing protein (VCP) and BAG family molecular chaperone regulator 3 (BAG3) as novel binding partners of JPH2. Together, our findings suggest that blocking calpain-2-mediated JPH2 cleavage may be a promising new strategy for delaying the development of HF following MI.

Keywords: Calpain; Heart failure; Ischemia; Junctophilin-2; Myocardial infarction.

Fig. 1.. JPH2 cleavage is significantly increased in myocardial ischemia.

(A) Representative western blots with anti-JPH2 and GAPDH antibodies of left ventricular samples from patients with non-ischemic control (NIC) heart disease and patients with ischemic heart disease (IHD). (B) Quantification of full-length JPH2 (FL) and (C) JPH2 C-terminal fragment (CTP) normalized to GAPDH showing reduced FL but increased CTP protein levels in patients with IHD. n, number of patients per group. (D) Representative western blots with anti-JPH2 and GAPDH antibodies of left ventricular tissues from mice subjected to myocardial infarction (MI) or sham surgery. (E) Quantification of JPH2-FL and (F) JPH2-CTP normalized to GAPDH showing reduced FL but increased CTP protein levels in mice with MI. n, number of mice per group. Statistical analysis was performed using Mann-Whitney test for Fig. 1A–C and unpaired t-test for Fig. 1D–F.

Fig. 2.. Characterization of JPH2-CSD knock-in mice.

(A) Schematic diagram of mouse JPH2 protein showing the calpain-cleavage site (marked in red) that was deleted using CRISPR/Cas9 in the JPH2-CSD knock-in mouse. Yellow circles indicate MORN domains at the N terminus, and the blue box indicates the trans-membrane domain at the C-terminus. (B) Using primers around the cleavage motif, genotyping confirmed the 24 bp deletion (i.e., 8 amino acid residues) in the CSD heterozygous (het) and homozygous (homo) knock-in mice. (C-E) Echocardiography showed unaltered ejection fraction (EF), end-diastolic diameter (EDD), and end-systolic diameter (ESD) in 16-month-old male (m) and female (f) JPH2-CSD homo mice compared to WT littermates. (F) Representative western blots with anti-JPH2 and total protein staining from the hearts of JPH2-CSD homo mice and WT littermates after MI or sham surgery. (G) Quantification of JPH2 C-terminal fragment (CTP) and full length (FL) normalized to total protein comparing hearts from WT and CSD mice post-MI or sham surgery showing prevention of CTP formation in CSD mice after MI. n, number of mice per group. Statistical analysis was performed using unpaired t-test for C, D and E. Two-way ANOVA test was with Tukey’s multiple comparison test was performed for panel G between 4 groups.

Fig. 3.. Blocking CTP cleavage from JPH2 delays HF progression following myocardial ischemia.

(A) Representative M-mode echocardiographic tracings from CSD homozygous mice and WT littermates at baseline and 4 weeks after MI or sham surgery. Quantification of echocardiographic tracings showing (B) ejection fraction (EF), (C) end-diastolic diameter (EDD), and (D) end-systolic diameter (ESD) at baseline, 2-weeks, and 4-weeks post-MI or sham procedure. n, number of mice per group. Two-way ANOVA multiple comparison test was performed to assess statistical significance between 4 groups at different timepoints. The P values shown for the comparison between WT-MI vs CSD-MI groups, were measured using Tukey’s multiple comparison test after two-way ANOVA test.

Fig. 4.. Blocking CTP cleavage from JPH2 protects from cardiac remodeling after MI.

(A) Heart weight (HW) to tibia length (TL) ratio (HW/TL) and (B) lung weight (LW)-to-tibia length (TL) ratio (LW/TL) from CSD homozygous mice and WT littermates at 4-weeks after MI or sham surgery. (C) Representative Masson’s trichrome stained hearts from CSD homozygous mice and WT littermates at 4-weeks after MI or sham surgery. Light blue colour indicates collagen disposition and fibrosis. (D) Quantification of fibrosis in the left ventricles from CSD homozygous mice and WT littermates at 4-weeks after MI or sham surgery. n, number of mice per group. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparison test.

Fig. 5.. Blocking JPH2-CTP formation attenuates T-tubule structure and SR Ca²⁺-mishandling after MI.

(A) T-tubule visualized with di-4-ANEPPS in ventricular cardiomyocytes isolated from WT and CSD mice 2-weeks after MI surgery; magnified areas in white boxes showed below. (B) Quantification of total T-tubular fraction and (C) total cell area, comparing WT and CSD after 2-weeks of MI. (D) Representative confocal line scan images showing Ca²⁺-sparks in cardiomyocytes isolated from WT and CSD mice 2-weeks after MI. (E) Quantification of Ca²⁺-spark frequency (CaSpF) in cardiomyocytes from WT and CSD mice 2-weeks after MI. n, numbers of mice and cells per group, respectively. Nested analysis was performed followed by two-way ANOVA test using Tukey’s multiple comparison test to assess significance between groups.

Fig. 6.. Reducing calpain-2 expression delays HF progression post-MI.

(A) Representative M-mode echo images from C57BL/6J mice injected with either AAV9-shCapn2 or saline controls at baseline and 4 weeks after sham or MI surgery. Quantification of echocardiographic findings showing (B) ejection fraction (EF), (C) end-systolic volume (ESV), and (D) cardiac output (CO) at baseline, 2-weeks, and 4-weeks post sham and MI surgeries. (E) Representative western blots of JPH2 and GAPDH in hearts from control and shCapn2-injected mice 4-weeks after sham or MI surgery. Quantification of JPH2 C-terminal fragment (CTP) (F), normalized to GAPDH showing reduced CTP levels in shCapn2 injected mice at 4-weeks after MI surgery. (G) Representative western blots for calpain-2 and GAPDH loading controls from hearts of C57BL/6J mice injected with either AAV9-shCapn2 or saline controls at 4 weeks after MI surgery or sham procedure. (H) Quantification of calpain-2 levels normalized to GAPDH, showing lower calpain-2 levels in shCapn2 treated mice. (I) Representative western blots for calpain-1 and GAPDH loading controls from hearts of C57BL/6J mice injected with either AAV9-shCapn2 or saline controls at 4 weeks after MI surgery or sham procedure. (J) Quantification of calpain-1 levels normalized to GAPDH, showing unaltered calpain-1 levels in shCapn2 treated mice. n, number of mice per group. Two-way ANOVA with Tukey’s multiple comparison test was performed to assess statistical significance between 4 groups at different timepoints.

Fig. 7.. Reducing calpain-2 expression protects from cardiac remodeling in MI.

(A) Heart weight (HW) to tibia length (TL) ratio (HW/TL) and (B) lung weight (LW)-to-tibia length (TL) ratio (LW/TL) from C57BL/6J mice injected with either AAV9-shCapn2 or saline controls at 4 weeks after sham or MI surgery. (C) Representative Masson’s trichrome stained hearts from control and shCapn2 injected mice at 4-weeks after sham or MI surgery. Light blue colour indicates collagen disposition and fibrosis. (D) Quantification of the fibrosis in the left ventricles from control and shCapn2 injected mice at 4-weeks after sham or MI surgery. n, number of mice per group. Two-way ANOVA with Tukey’s multiple comparison test was performed to assess the statistical significance.

Fig. 8.. JPH2-CTP binds to BAG3 and induces cell death.

(A) Western blotting with JPH2-Ab2 [12] of WT mouse hearts obtained 4 weeks after MI surgery revealed pull-down of JPH2-CTP levels after sequential preclearing with JPH2-Ab1 to remove FL JPH2. The red box indicates samples used for MS analysis. Blotting with Ab2 selectively pulled down CTP compared with IgG negative control antibody (n = 3). (B) Label-free quantification (LFQ) interactome volcano plot showing CTP-binding proteins in hearts from WT mice 4-weeks post-MI (n = 3) compared to IgG negative controls. The vertical grey line indicates the cutoff for binding partners with a 2-fold increase in binding. The red dots indicate the highly significant binding partners of JPH2-CTP. (C) Western blots with JPH2 and GAPDH antibodies of mouse embryonic fibroblasts (MEF) transfected with empty vector, full-length (FL) JPH2, or JPH2-CTP encoding-plasmids. (D) Quantification of cell death percentage (% of cells positive for propidium iodide stain) in MEFs transfected with empty vector, full-length (FL) JPH2, or JPH2-CTP encoding-plasmids for 48 h. n, number of transfected cell wells per group. An unpaired t-test was performed to assess the significance of panel B and one-way ANOVA was performed to assess the significance of panel D.