Targeting Calpain for Heart Failure Therapy: Implications From Multiple Murine Models

Abstract

Heart failure remains a major cause of morbidity and mortality in developed countries. There is still a strong need to devise new mechanism-based treatments for heart failure. Numerous studies have suggested the importance of the Ca²⁺-dependent protease calpain in cardiac physiology and pathology. However, no drugs are currently under development or testing in human patients to target calpain for heart failure treatment. Herein the data demonstrate that inhibition of calpain activity protects against deleterious ultrastructural remodeling and cardiac dysfunction in multiple rodent models of heart failure, providing compelling evidence that calpain inhibition is a promising therapeutic strategy for heart failure treatment.

Keywords: CAPN1-OE, calpain-1 overexpressing; E-C coupling, excitation-contraction coupling; EF, ejection fraction; IP, intraperitoneally; ISO, isoproterenol; JP2, junctophilin-2; JP2-OE, junctophilin-2 overexpressing; LV, left ventricle/ventricular; MI, myocardial infarction; RV, right ventricular; SR, sarcoplasmic reticulum; T-tubule, transverse tubule; T-tubules; TAB, transverse aortic banding; TTpower, strength of regularity of the T-tubule system; WT, wild-type; calcium; calpain; excitation-contraction coupling; heart failure.

Graphical abstract

Figure 1

Calpain Activity Is Increased in Human Heart Failure (A) Calpain activity assay in myocardium from healthy donors (n = 4, 45.0 ± 5.3 years of age), ICM patients (n = 4, 47.0 ± 4.7 years of age), and DCM patients (n = 6, 44.0 ± 7.5 years of age). (B–E) Representative images of Western blots (B) and quantitation of calpain-1 (C), calpastatin (D), JP2 (E), and cleaved form of fodrin (F) in left ventricular lysates from samples of A. GAPDH was used as the loading control. *p < 0.05, **p < 0.01 versus healthy donor. DCM = dilated cardiomyopathy; ICM = ischemic cardiomyopathy; n.s. = not significant.

Figure 2

MDL-28170 Protects Against Cardiac Stress–Induced Heart Failure (A) Calpain activity was assessed in LV lysates after TAB, MI, or ISO minipump infusion. (B and C) Average heart weight/body weight (B) and lung weight/body weight (C) ratios at 5 weeks after surgery or 2 weeks after ISO minipump implantation. (D to F) End-systolic volume (ESV) (D), end-diastolic volume (EDV) (E), and ejection fraction (EF) (F) were assessed by echocardiography. n = 7 to 11 mice per group. For A, **p < 0.01 as indicated. For B to F, *p < 0.05, **p < 0.01 versus sham; †p < 0.05, ††p < 0.01 versus saline control. Control = the saline control beginning 3 days after surgery; HW/BW = heart weight/body weight; ISO = isoproterenol; LV = left ventricular; LW/BW = lung weight/body weight; MDL = MDL-28170 beginning 3 days after surgery; MI = myocardial infarction; Sham = sham surgery; TAB = transverse aortic banding.

Figure 3

Calpain Inhibitor MDL-28170 Ameliorates T-Tubule Remodeling in Response to Cardiac Stress (A, C, E) Representative in situ LV T-tubule confocal images after staining with lipophilic marker MM 4–64. (A, C) LV cardiomyocytes after TAB (A) or ISO infusion (C). (E) Representative images of the border and remote zones relative to the infarct zone in the MI model. (B, D, F) Mean values of TT_power. n = 4 to 5 mice per group. **p < 0.01 versus sham; †p < 0.05 versus saline control. T-tubule = transverse tubule; TT_power = strength of regularity of the T-tubule system; other abbreviations as in Figure 2.

Figure 4

Calpain Inhibition Blunts Stress-Induced JP2 Dysregulation (A to F) Western blots (A, C, E) and quantitation (B, D, F) of JP2 expression in heart lysates after TAB or MI at 5 weeks or ISO infusion at 2 weeks. GAPDH was used as the loading control. **p < 0.01 versus sham; †p < 0.05 versus saline control. n = 3 to 4 mice per group. JP2 = junctophilin-2; other abbreviations as in Figure 2.

Figure 5

Effects of JP2 Overexpression on Survival and Cardiac Function in CAPN1-OE Mice (A) Kaplan-Meier survival curve for WT, CAPN1-OE, and CAPN1-OExJP2-OE mice (n = 32, 36, and 33, respectively). (B to D) Ejection fraction (B), ESV (C), and EDV (D) were assessed by echocardiography. (E, F) Average HW/BW (E) and LW/BW (F) ratios. n = 7 to 11 per group. *p < 0.05, **p < 0.01 versus age-matched WT; †p < 0.05 versus age-matched CAPN1-OE. CAPN1-OE = calpain-1 overexpressing; CAPN1-OExJP2-OE = calpain-1 overexpressing junctophilin-2 overexpressing double-transgenic cross; WT = wild-type; other abbreviations as in Figures 1, 2, and 4.

Figure 6

JP2 Expression Progressively Declines in CAPN1-OExJP2-OE Mice (A) Calpain activity assay in heart lysates from WT, CAPN1-OE, and CAPN1-OExJP2-OE mice at different ages. (B, C) Representative Western blot (B) and summary data (C) of JP2 expression in heart lysates WT, CAPN1-OE, and CAPN1-OExJP2-OE mice. n = 6 to 11 per group. *p < 0.05, **p < 0.01 versus age-matched WT; †p < 0.05, ††p < 0.01 versus age-matched CAPN1-OE. Abbreviations as in Figure 5.

Figure 7

JP2 Overexpression Initially Preserves T-Tubule Integrity in CAPN1-OE Mice (A and B) Representative in situ confocal images of T-tubules in LV and RV myocytes from WT, CAPN1-OE, and CAPN1-OExJP2-OE mice at 3 weeks (A) and 5 weeks (B) of age. (C, D) Average TT_power in LV and RV myocytes from WT, CAPN1-OE and CAPN1-OExJP2-OE mice at 3 weeks (C) and 5 weeks (D) of age. n = 4 to 6 hearts for each group. **p < 0.01 versus age-matched WT; †p < 0.05 versus age-matched CAPN1-OE. RV = right ventricle; other abbreviations as in Figures 4 and 5.

Figure 8

JP2 Overexpression Initially Alleviates Impaired Ca²⁺ Handling Dysfunction Induced by Excessive Calpain Activation (A, B) Representative examples of in situ confocal Ca²⁺ images in WT, CAPN1-OE, and CAPN1-OExJP2-OE hearts at 3 weeks (A) and 5 weeks (B) of age under autonomous beating. Bottom traces under each image are profiles of Ca²⁺ transients. (C, D) Summary data of the amplitude (left) and time to peak (right) of Ca²⁺ transients under autonomous beating at 3 weeks (C) and 5 weeks (D) after birth. n = 250 to 300 cells from 4 to 5 hearts per group. **p < 0.01 versus age-matched WT; †p < 0.05, ††P < 0.01 versus age-matched CAPN1-OE. Abbreviations as in Figures 4 and 5.