CaMKII-δ9 Induces Cardiomyocyte Death to Promote Cardiomyopathy and Heart Failure

Figures

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  Figure 1. KN-93 treatment prevents CaMKII-δ9-induced cardiomyopathy and heart failure. (A–C), Cell viability assayed by caspase 3/7 activity (A) and representative western blots and statistical data showing the levels of γH2AX (B) and UBE2T (C) in NRVMs infected with Ad-β-gal or Ad-CaMKII-δ9 with or without KN-93 treatment (5 μM) (n = 5). (D) Experimental protocol for in vivo KN-93 treatment in CaMKII-d9 tg mice. The mice were treated with KN-93 (10 μmol/kg, i.p.) every other day from 5 to 12 weeks of age. (E) Kaplan-Meier survival curves of wt and CaMKII-δ9 tg mice with or without KN-93 treatment (n = 8 for wt vehicle, n = 7 for wt KN-93, n = 14 for CaMKII-δ9 tg vehicle, n = 10 for CaMKII-δ9 tg KN-93). (F–J) Gross morphology of the hearts (F), statistical data of the left ventricle echocardiography (G), ratio of heart weight to body weight (H), and statistical data of TUNEL (I) and γH2AX (J) staining of the hearts from CaMKII-δ9 tg mice with or without KN-93 treatment. EF, ejection fraction; FS, fractional shortening; LVIDd and LVIDs, diastolic and systolic left ventricular internal diameter; LVPWd and LVPWs, diastolic and systolic left ventricular posterior wall thickness. Scale bar, 2…

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  Figure 2. Inhibition of cardiomyocyte death alleviates CaMKII-δ9-induced cardiomyopathy and heart failure. (A) Experimental protocol for in vivo z-VAD treatment in CaMKII-δ9 tg mice. The mice were treated with z-VAD (0.5 mg/kg, i.p.) twice a week from 5 to 12 weeks of age. (B) Kaplan-Meier survival curves of wt and CaMKII-δ9 tg mice with or without z-VAD treatment (n = 6 for wt vehicle, n = 5 for wt z-VAD, n = 11 for CaMKII-δ9 tg vehicle, n = 7 for CaMKII-δ9 tg z-VAD). (C–E) Gross morphology of the hearts (C), statistical data of the left ventricle echocardiography (D), and statistical data of TUNEL staining of the hearts (E) from CaMKII-δ9 tg mice with or without z-VAD treatment. Scale bar, 2 mm (n = 5 for vehicle, n = 6 for z-VAD). Data are mean ± S.E.M. NS, not significant; *P < 0.05, **P < 0.01; log-rank (Mantel-Cox) test (B), or Student's t-test (D,E).

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  Figure 3. CaMKII-δ9 does not directly regulate cardiac hypertrophy. (A) Representative western blots and statistical data showing the levels of CaMKII-δ in the hearts of the mice 2 weeks after sham or transverse aortic constriction (TAC) surgery. n = 6. (B) Averaged data of mRNA levels of ANP and BNP assayed by real-time PCR in NRVMs infected with Ad-β-gal or Ad-CaMKII-δ9 (50 MOI, 48 h; Ad-β-gal, n = 12; Ad-CaMKII-δ9, n = 13). The NRVMs treated with isoproterenol (ISO, 10 mM, 24 h) were used as positive control (n = 9). (C,D) Averaged data of mRNA levels of ANP (C) and BNP (D) assayed by real-time PCR in NRVMs transfected with scrambled or CaMKII-δ9 siRNA with or without ISO treatment (10 μM, 24 h; Vehicle scrambled, n = 12; other groups, n = 13). Data are mean ± S.E.M. *P < 0.05, **P < 0.01; NS, not significant; Student's t-test (A,B), or two-way ANOVA (C,D).

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  Figure 4. CaMKII-δ9 is abundant in the adult hearts of different species. (A) Representative western blots and statistical data showing the levels of CaMKII-δ9 in the hearts of rhesus monkey and human, neonatal rat ventricular myocytes (NRVMs), adult mouse together with rat (n = 4). Data are mean ± S.E.M. **P < 0.01; one-way ANOVA. (B) Amino acid sequences of the peptides encoded by the exon 16 of CaMKII-δ of human, mouse, rhesus monkey and rat.

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  Figure 5. CaMKII-δ9 mediates DNA damage and cell death in adult cardiomyocytes. (A–C) Representative western blots and statistical data showing the levels of cleaved caspase 3 (A) γH2AX (B) and UBE2T (C) in adult rat ventricular myocytes (ARVMs) infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (100 MOI, 24 h) (n = 4). (D–F) Representative western blots and statistical data showing the levels of cleaved caspase 3 (D), γH2AX (E) and UBE2T (F) in ARVMs infected with scrambled or CaMKII-δ9 siRNA with or without Dox treatment (1 mM, 16 h) (n = 4). Data are mean ± S.E.M. *P < 0.05, **P < 0.01; one-way ANOVA (A–C) or two-way ANOVA (D–F).

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  Figure 6. No gender difference in the CaMKII-δ9-induced cardiac pathology. (A,B) Representative western blots and statistical data showing the levels of CaMKII-δ9 from the hearts of wt (A, n = 7) and CaMKII-δ9 tg mice (B, n = 8). (C) Kaplan-Meier survival curves of male and female wt and CaMKII-δ9 tg mice (n = 20 for wt male, n = 22 for CaMKII-δ9 tg male, n = 16 for wt female and CaMKII-δ9 tg female). (D) Statistical data of the left ventricle echocardiography of male and female wt and CaMKII-δ9 tg mice at 10 weeks of age (n = 15 for wt male, n = 9 for CaMKII-δ9 tg male, n = 8 for wt female and CaMKII-δ9 tg female). EF, ejection fraction; FS, fractional shortening; LVIDd and LVIDs, diastolic and systolic left entricular internal diameter; LVPWd and LVPWs, diastolic and systolic left ventricular posterior wall thickness. (E,F) Statistical data of TUNEL (E, n = 5) and γH2AX (F, n = 6) staining of the hearts of male and female wt and CaMKII-δ9 tg mice at the age of 10 weeks (n = 5). Data are mean ± S.E.M. **P < 0.01; Student's t-test (A,B), log-rank (Mantel-Cox) test (C), or two-way ANOVA (D–F).