Fig 2: 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.

Fig 3: Knockdown of the regulatory subunit CAPNS1 and overexpression of CAPN1 and CAPN2(A and B) Ea.Hy-926 cells were transfected with siRNA against CAPNS1 or an OFF-Target siRNA. (A) Twenty-four hours later cells were treated with 30 nM BTZ for 16 h, total cell lysates were analysed by immunoblot, representative image from n=3, (B) RNA was isolated and used for qRT-PCR to analyse expression of PSMA3, PSMB6 and PSMC4, values were calcuated using the ΔΔCT method with HPRT-1 as housekeeping gene and the normalised to samples without BTZ treatment (set to 1), mean ± SEM, n=3. (C) Ea.Hy-926 cells were transfected with flag-tagged constructs for the large subunits of calpain-1 (CAPN1) and -2 (CAPN2). The following day, the cells were treated with 30 nM BTZ for 16 h, total cell lysates were analysed by immunoblot, representative image from n=3.

Fig 4: 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.

Fig 5: 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 TTpower 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.