Constitutively active calcineurin induces cardiac endoplasmic reticulum stress and protects against apoptosis that is mediated by alpha-crystallin-B

Abstract

Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.

Fig. 1.

Hierarchical clustering and statistical analysis of cardiac proteins in WT and CNA hearts. (A) A heat map demonstrating the clustering of proteins based on their expression intensities in the cytosolic (Cyto), microsomal (Micro), mitochondrial matrix (mitoI), and mitochondrial membrane (MitoII) fractions for WT and CNA mice. To the right of the heat map are selected annotations found to be enriched in the protein clusters shown. (B) A volcano plot showing the CNA/WT protein expression ratio (Log₂ scale) of proteins against their P value (−Log₂). Red diamonds indicate the 290 significantly altered proteins.

Fig. 2.

Immunoblot and RT-PCR expression analysis of significantly altered genes. Protein expression levels were analyzed by immunoblot (A) or RT-PCR (B). Numbers on the right of the panels indicate the fold-change of the respective proteins in the proteomic analysis and arrows indicate up- or down-regulation.

Fig. 3.

ER stress and cardiac apoptosis. (A) Immunoblots demonstrating increased Grp78, Pdia1, and phosphorylated eIF2α in adult CNA hearts, compared with their WT littermates and a PCR gel demonstrating increased ratio of spliced (active) Xbp1 (sXbp1) to unspliced Xbp1 (uXbp1) in CNA hearts, compared with WT hearts. (B) Photomicrographs demonstrating negligible TUNEL labeling of a WT heart section; a CNA heart section demonstrating negligible TUNEL labeling, a heart section from the PLN^R9C mouse model of dilated cardiomyocyte demonstrating a high degree of TUNEL labeling (bright spots) and, the absence of TUNEL labeling in a negative control section in which the labeling enzyme was omitted.

Fig. 4.

Cytoprotective candidate selection, knockdown and Cryab mediated cyto-protection. (A) Immunoblot validating the significantly increased Cryab protein levels in NCMs (1 wk old) and in CNA mouse hearts (14 wk old) compared with their WT littermates. (B) Bar graph demonstrating significantly decreased Cryab mRNA expression in WT NCMs following transduction with lentivectors expressing shRNAs targeting Cryab. (C and E) Photomicrograph (630× magnification) of WT NCMs immunofluorescently probed with anti-Cryab antibody 4 and 7 d after transduction with the control lentivectors expressing the scrambled shRNA. (D and F) Photomicrograph of WT NCMs immunofluorescently probed with anti-Cryab antibody 4 and 7 d after transduction with lentivectors expressing the Cryab targeting shRNAs. (G) Bar graph demonstrating the significant increase in viability in CNA NCMs, compared with WT NCMs (both transduced with the control lentivectors expressing the scrambled shRNAs). Both cultures were incubated with serum-free media and 200 μM H₂O₂ for 24 and 72 h. Abrogation of the protective effect in CNA NCMs incubated with serum-free media and 200 μmol/L H₂O₂ for 24 and 72 h, compared with WT NCMs, was observed when Cryab was silenced (Cryab KD).