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. 2006 Sep;291(3):H1411-20.
doi: 10.1152/ajpheart.01378.2005. Epub 2006 Apr 14.

Activation of endoplasmic reticulum stress response during the development of ischemic heart disease

Asim Azfer  1 , Jianli Niu, Linda M Rogers, Frances M Adamski, Pappachan E Kolattukudy
Affiliations

Activation of endoplasmic reticulum stress response during the development of ischemic heart disease

Asim Azfer et al. Am J Physiol Heart Circ Physiol. 2006 Sep.

Abstract

Endoplasmic reticulum (ER) stress has been found to be associated with neurodegenerative diseases and diabetes mellitus. Whether ER stress is involved in the development of heart disease is not known. Cardiac-specific expression of monocyte chemoattractant protein-1 (MCP-1) in mice causes the development of ischemic heart disease. Here we report that microarray analysis of gene expression changes in the heart of these transgenic mice revealed that a cluster of ER stress-related genes was transcriptionally activated in the heart during the development of ischemic heart disease. The gene array results were verified by quantitative real-time PCR that showed highly elevated transcript levels of genes involved in unfolded protein response such as ER and cytoplasmic chaperones, oxidoreductases, protein disulfide isomerase (PDI) family, and ER-associated degradation system such as ubiquitin. Immunoblot analysis confirmed the expression of chaperones, PDI, and ubiquitin. Immunohistochemical analyses showed that ER stress proteins were associated mainly with the degenerating cardiomyocytes. A novel ubiquitin fold modifier (Ufm1) that has not been previously associated with ER stress and not found to be induced under any condition was also found to be upregulated in the hearts of MCP mice (transgenic mice that express MCP-1 specifically in the heart). The present results strongly suggest that activation of ER stress response is involved in the development of ischemic heart disease in this murine model.

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Figures

Fig. 1
Fig. 1
Elevated levels of transcripts of endoplasmic reticulum (ER) stress-associated genes in the hearts of transgenic mice that express monocyte chemoattractant protein-1 (MCP-1) specifically in the heart (MCP mice). Relative mRNA levels of ER stress-associated genes Bip/Grp78 (A), Grp94 (B), protein disulfide isomerase (PDI; C), ribophorin (D), asparagine synthetase (E), Cai (F), endoplasmic reticulum oxidoreductase (ERO1; G), C/EBP homologous protein (CHOP; H), ATF6 (I) and tribbles-related protein 3 (TRB3; J) were measured by quantitative real-time PCR. A total of 5 samples for each time point was used, and each sample was run in triplicate for real-time PCR. Expression level of all the samples was normalized by β-actin. *Significantly different from wild-type controls (P < 0.05).
Fig. 2
Fig. 2
Elevated levels of transcripts of cytosolic chaperones in the hearts of MCP mice. Relative mRNA levels of cytosolic chaperones, 25-kDa heat shock protein (HSP25; A), 40-kDa heat shock protein (HSP40; B), and 70-kDa heat shock protein (HSP70; C), were measured by quantitative real-time PCR. A total of 5 samples for each time point was used, and each sample was run in triplicate for real-time PCR. The expression level of all the samples was normalized by β-actin. *Significantly different from wild-type controls (P < 0.05).
Fig. 3
Fig. 3
Immunoblot analysis of ER resident chaperones and cytosolic chaperones in the hearts expressing MCP-1. A: Western blots showing levels of BiP/Grp78, Grp94, PDI, HSP25, and HSP40 proteins in the hearts of 2-, 4-, and 6-mo-old wild-type controls and MCP mice. The expression level of all the samples was normalized to GADPH (B–F). Quantitative analyses of levels of Bip/Grp78, Grp94, PDI, HSP25, and HSP40 proteins by densitometry. *Significantly different from wild-type controls (P < 0.05).
Fig. 4
Fig. 4
Immunohistochemical staining of ER-stress associated proteins Bip/Grp78, Grp94, and PDI in the hearts of wild-type controls and MCP mice of 6 mo of age. A–C: immunoreactivity (brown) in myocardium from wild-type controls. Note the absence or very weak cytoplasmic staining in cardiomyocytes and positive staining of Grp78 or Grp94 in interstitial infiltrating cells (arrow). D–F: immunoreactivity (brown) in myocardium from MCP mice. Note the strong staining in cardiomyocytes undergoing degenerative changes such as vacuolation (arrow) and the weaker immunoreactivity in cardiomyocytes with normal appearance (arrowheads). Original magnification, × 400.
Fig. 5
Fig. 5
Immunohistochemical staining of heat-shock proteins HSP25, HSP40, and HSP70 in the hearts of wild-type controls and MCP mice of 6 mo of age. A–C: immunore-activity (brown) in myocardium from wild-type controls. Note the absence or very weak cytoplasmic staining in cardiomyocytes. D–F: immunoreactivity (brown) in myocardium from MCP mice. Note the strong staining in cardiomyocytes undergoing degenerative changes such as vacuolation (arrow) and the weaker immunoreactivity in cardiomyocytes with less degeneration (arrowheads). Original magnification, ×400.
Fig. 6
Fig. 6
Accumulation of ubiquitinylated proteins in the hearts of wild-type and MCP mice. Immunohistochemical staining of ubiquitin in the hearts of wild-type controls (A) and MCP (B) mice of 6 mo of age. Note the absence or weaker cytoplasmic staining (brown) in cardiomyocytes of wild-type controls and the strong staining in cardiomyocytes undergoing degenerative changes such as vacuolation (arrow). Original magnification, ×400. C: Western blotting of heart homogenates from wild-type controls and MCP mice of 2, 4, and 6 mo of age showing marked accumulation of ubiquitinylated proteins in the hearts of MCP mice. D: elevated expression of ubiquitin fold modifier 1 (Ufm1) in the hearts of MCP mice determined by quantitative real-time PCR. *Significantly different from wild-type controls (P < 0.05).
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