Overexpression of Heat Shock Protein 70 Improves Cardiac Remodeling and Survival in Protein Phosphatase 2A-Expressing Transgenic Mice with Chronic Heart Failure

Figures

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  Figure 1. HSP70 alleviated PP2CA-induced cardiac dysfunction and subsequent death. (A) The serial changes in the ejection fraction (EF) of transgenic mice were measured. Note that the abrupt decrease in EF was significantly improved in double transgenic mice expressing both HSP70 and PP2CA (dTg). (B,C) Cardiac function of individual mice was measured at 20 weeks postnatal using echocardiography. Both EF (B) and fractional shortening (FS) were dramatically reduced in TgPP2CA mice (blue squares), whereas these decreases in contractile function were retarded in dTg mice (green rotated squares). (D) Heart failure caused by PP2CA was improved in HSP70-expressing mice. The EF of many TgPP2CA mice was below 40%, which indicates systolic heart failure. In contrast, the proportion of mice with heart failure was markedly reduced among dTg mice. (A–D) Every dot indicates a single mouse. Cohort size, nTg: 18, TgHSP70: 27, TgPP2CA: 16, dTg: 23. (E) Kaplan–Meier survival curves of transgenic mice. During the 6-month cohort study, sudden death of the TgPP2CA mice was observed after 15 weeks, which was correlated with the deterioration of cardiac function, as shown in (A). Survival was significantly…

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  Figure 2. Cardiac geometry in echocardiogram measurements. (A) Representative M-mode echocardiograms from a mouse in each group. (B,C) Ventricular enlargement. Left ventricular (LV) internal diameter at diastole (LVIDd, B) and systole (LVIDs, C), which represents the LV chamber size at the papillary muscle level. LV cavity enlargement was predominant in TgPP2CA mice but was attenuated in dTg mice. (D,E) Global remodeling. End-diastolic volume (D) and end-systolic volume (E) in each group of transgenic mice. Fatal LV remodeling observed in TgPP2CA mice was ameliorated in dTg mice. (B–E) Cohort size, nTg: 18, TgHSP70: 27, TgPP2CA: 16, dTg: 23. (F) Representative heart morphology. Scale bars = 1 mm. Statistical significance was assessed using ANOVA with LSD post hoc test. Asterisks indicate statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 3. Overexpression of HSP70 prevents PP2CA-driven dilated cardiomyopathy. (A,B) Echocardiograms revealed that the LV free wall of TgPP2CA mice was significantly thinner than that of their non-Tg littermates, and this change was reversed in dTg mice. Cohort size, nTg: 18, TgHSP70: 27, TgPP2CA: 16, dTg: 23. (C) Overall increase in heart weight was observed in TgPP2CA mice, without any changes in body weight, while it was attenuated in dTg mice. Cohort size, nTg: 33, TgHSP70: 42, TgPP2CA: 27, dTg: 25. (D) Representative images of the LV free wall. Scale bars = 5 μm. (E) Measurement of the cross-sectional area. Mild cardiac hypertrophy was observed in TgPP2CA mice, and HSP70 suppressed this change. Cell counts were assessed from 5 individual mice in each group, nTg: 145, TgHSP70: 197, TgPP2CA: 93, dTg: 110. Data were analyzed using ANOVA with LSD post hoc testing. Asterisks indicate statistical significance: * p < 0.05; *** p < 0.001.

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  Figure 4. Relative changes in cardiac marker gene expression. (A,B) Fetal gene reprogramming: alpha myosin heavy chain (A) and beta myosin heavy chain (B) are indicators of the hypertrophic response. (C,D) Natriuretic peptides: atrial (C) and B-type (D) natriuretic peptides are indicators of LV wall stress. (E,F) Fibrosis markers: smooth muscle alpha-actin (E) and type 1 collagen (F). HSP70 failed to reverse the changes in marker gene expression. Cohort size, nTg: 19, TgHSP70: 11, TgPP2CA: 15, dTg: 17. ANOVA and LSD post hoc testing were performed. Asterisks indicate statistical significance: **, p < 0.01; ***, p < 0.001; ns, not significant.

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  Figure 5. HSP70 modulates post-translational modifications. (A–C) Protein modifications in heart lysates obtained from TgHSP70 mice were compared to those in lysates from non-Tg littermates; phospho-serine (pSer, A), methyl-lysine (MeK, B), and acetyl-lysine (AcK, C). Both serine phosphorylation and lysine acetylation were significantly increased in TgHSP70 hearts. Phosphorylation was predominantly changed. The numbers at the bottom of each figure indicate the results of densitometer analysis.

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  Figure 6. Phosphorylation levels of select proteins in hearts from each group of transgenic mice at 6 months of age. (A,B) Calcium handling factors. Known PP2CA substrates related to the failing heart phenotype were assessed. Phosphorylation of cardiac troponin I (Ser23/24) (A) and phospholamban (Ser16/Thr17) (B) was significantly reduced in TgPP2CA mice, and expression of HSP70 did not affect these changes in phosphorylation. Known factors associated with HSP70 were also assessed, and the phosphorylation levels of protein kinase B (Ser473) (AKT1, C). (D) HSP90, an important binding partner of HSP70, was measured. HSP90 levels were significantly increased in transgenic mice. Western blotting (E) and real-time PCR (G,H) also confirmed transgenic overexpression of PP2CA and HSP70 in the relevant groups. (F) Individual dot plots beside Western blot depict densitometer analysis. Transgenic expression of PP2CA attenuated SERCA2 mRNA levels in the heart, whereas transgenic HSP70 expression partially restored it. (F–H) Cohort size, nTg: 19, TgHSP70: 11, TgPP2CA: 15, dTg: 17. One-way ANOVA and LSD post hoc testing were performed. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.…