Alpha B-crystallin suppresses pressure overload cardiac hypertrophy

Abstract

AlphaB-crystallin (CryAB) is the most abundant small heat shock protein (HSP) constitutively expressed in cardiomyocytes. Gain- and loss-of-function studies demonstrated that CryAB can protect against myocardial ischemia/reperfusion injury. However, the role of CryAB or any HSPs in cardiac responses to mechanical overload is unknown. This study addresses this issue. Nontransgenic mice and mice with cardiomyocyte-restricted transgenic overexpression of CryAB or with germ-line ablation of the CryAB/HSPB2 genes were subjected to transverse aortic constriction or sham surgery. Two weeks later, cardiac responses were analyzed by fetal gene expression profiling, cardiac function analyses, and morphometry. Comparison among the 3 sham surgery groups reveals that CryAB overexpression is benign, whereas the knockout is detrimental to the heart as reflected by cardiac hypertrophy and malfunction at 10 weeks of age. Compared to nontransgenic mice, transgenic mouse hearts showed significantly reduced NFAT transactivation and attenuated cardiac hypertrophic responses to transverse aortic constriction but unchanged cardiac function, whereas NFAT transactivation was significantly increased in cardiac and skeletal muscle of the knockout mice at baseline, and they developed cardiac insufficiency at 2 weeks after transverse aortic constriction. CryAB overexpression in cultured neonatal rat cardiomyocytes significantly attenuated adrenergic stimulation-induced NFAT transactivation and hypertrophic growth. We conclude that CryAB suppresses cardiac hypertrophic responses likely through attenuating NFAT signaling and that CryAB and/or HSPB2 are essential for normal cardiac function.

Figure 1

CryAB protein expression in the left ventricle (LV). Age-matched male FVB/N mice with the indicated genotype were subjected to TAC or sham surgery. LV myocardium was collected at 1 or 2 weeks (wk) after TAC for protein analysis. A, A representative western blot image (upper part) and a bar graph summarizing densitometry data (lower part). Compared with sham and 1wk TAC, *: p<0.01. B, Representative western blot images for CryAB in the LV of the indicated groups at 2 wk after TAC. α-Actinin was probed as loading controls.

Figure 2

Reactivation of the fetal gene program. Total RNA isolated from LV myocardium at 2 weeks after TAC or sham surgery was used for quantitative RNA dot blot analysis of the transcript levels of atrial natriuretic factor (ANF), β-myosin heavy chain (β-MyHC), skeletal α-actin (s-Actin), α-MyHC, sarcoplasmic endoplasmic reticulum calcium ATPase 2A (SERCA), phospholamban (PLN), and GAPDH with ³²P-labeled transcript-specific oligonucleotide probes. The composed RNA dot blot images are shown in panel A. Each dot represents an individual animal. After normalized to the corresponding GAPDH signal, the mean intensity value of the NTG sham group was set at 100 arbitrary units (AU). The intensity signal of each individual dot was then normalized to the mean of the NTG sham. Comparison among the three sham control groups is presented in the online supplementary Figure 1. ANF, s-Actin, and β-MyHC transcripts were significantly increased whereas α-MyHC, SERCA, and PLN were significantly decreased in the NTG TAC (p<0.01). Compared with the NTG TAC group, ANF and β-MyHC up-regulation and the down-regulation of α-MyHC, SERCA, and PLN were substantially attenuated in the TG group (B) whereas the up-regulation of ANF and β-MyHC was significantly enhanced in the KO group (C).

Figure 3

Gravimetric analyses of cardiac hypertrophy at 2 weeks after TAC. A, Changes in the heart weight (HW)/body weight (BW) ratio. B, Changes in the ventricular weight (VW)/BW ratio. C, Changes in the Lung/BW ratio. Compared with the sham control of the same genotype, *: p<0.01.

Figure 4

Echocardiography analysis of LV morphometry and function at 2 weeks after TAC or sham surgery. LVPWd: LV diastolic posterior wall thickness; EF: the ejection fraction; FS: fractional shortening; compared to the sham control of the same genotype, *: p<0.01.

Figure 5

Changes in LV pressure at 2 weeks after TAC or sham surgery. A, Changes in the LV systolic peak pressure (LVSP). B, Changes in the LV end diastolic pressure (LVEDP). C and D, Changes in the LV pressure rising (+dP/dt₄₀, C) and declining velocities (-dP/dt₄₀, D) at the LV pressure being 40mmHg. Compared with the same genotype sham control, *: p<0.05, 0.01.

Figure 6

CryAB overexpression suppresses hypertrophy of cultured neonatal rat cardiomyocytes (NRCMs). Twenty-four hours after being plated, NRCMs were infected with Ad-CryAB or Ad-Empty for 24 hours and then treated with isoproterenol (ISO), norepinephrine (NE), phenylepinephrine (PE), or vehicle control (CTR, ascorbic acid)), in combination with or without cyclosporine A (CsA) for 48 hours in serum-free media. A, Western blot analysis of CryAB overexpression at 48 hours after Ad-CryAB infection. The transgenic CryAB contains a HA tag (HA-CryAB) and therefore migrates slower than the endogenous CryAB. Changes in myocyte profile area are shown in panel B and C. The cells in chamber slides were fixed and double-stained with Alexa Fluor-568 conjugated phalloidin (red) and DAPI (blue). Digital images were captured at the same settings for subsequent profile area measurements. B, Representative fluorescence micrographs of NRCMs treated with: (a) CTR + Ad-Empty; (b) CTR + Ad-CryAB; (c) PE + Ad-Empty; (d) PE + Ad-CryAB; (e) PE + Ad-Empty + CsA; (f) PE + Ad-CryAB + CsA. C, A summary of NRCMs profile area changes after the indicated treatments. Approximately 60 cells evenly from 3 duplicates per group were measured. Mean + SE are presented. Compared to the CTR+Ad-Empty group, *: p < 0.01.

Figure 7

CryAB suppresses NFAT transactivation in mice. A, NFAT-Luc transgenic mice were cross-bred with the KO (CryAB-null) mice and the luciferase (Luc) activities in myocardium and the soleus muscle of CryAB-null and wild type littermates (WT) were measured. Compared with WT, **: p < 0.01. B, Western blot analyses of NFATc4 in the nuclear and the cytoplasmic (Cytopla.) fractions of myocardium from WT and KO mice. A representative set of images are shown at the top and the nuclear to cytoplasmic NFAT ratios derived from the densitometry of the Western blot images are summarized in the bar graph. C, RT-PCR analyses of myocardial MCIP1.4 expression in KO mice. *: p < 0.05, KO vs WT. D, The NFAT-Luc mice were cross-bred with the CryAB TG mice and the resulting littermate mice with the indicated genotypes were subjected to TAC or sham surgery at 12 weeks. LV myocardial luciferase activities were assessed at 2 weeks after the surgery. N(-): NFAT-Luc Ntg; N(+): NFAT-Luc Tg; C(-): CryAB Ntg; C(+): CryAB Tg. Mean+SD; n = 4 mice/group; compared with either sham groups, **: p < 0.01.

Figure 8

CryAB overexpression suppresses NFAT transactivation in cultured NRCMs. Overexpression of NFAT-GFP, HA-tagged CryAB, and CnAΔ (CnA) was mediated by respective adenoviral infection. Ad-Empty (Empty) or Ad-β-Gal was used as control viral infection. A, Representative micrographs of NRCMs to show NFAT-GFP distribution. B, A summary of changes in NFAT-GFP nuclear translocation which is semi-quantitatively reflected by the percentage of NRCMs with nuclear NFAT-GFP over all cardiomyocytes with NFAT-GFP expression. C and D, RT-PCR analyses of MCIP1.4. Cultured NRCMs infected with the indicated adenoviruses for 48 hours were treated with ISO (+ISO) or vehicle control (-ISO) for 24 hours in serum-free media. Total RNA was then isolated from the cells for RT-PCR. A representative gel image is shown in panel C. The MCIP1.4 signal of each sample is normalized to its GAPDH signal and the mean of each vehicle control group is set as 100%. The semi-quantitative data are summarized in panel D. P<0.05, +ISO vs -ISO.