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. 2010 Feb;48(2):360-6.
doi: 10.1016/j.yjmcc.2009.11.009. Epub 2009 Nov 27.

Regulation of heat shock protein 60 and 72 expression in the failing heart

Y Wang  1 , L Chen, N Hagiwara, A A Knowlton
Affiliations

Regulation of heat shock protein 60 and 72 expression in the failing heart

Y Wang et al. J Mol Cell Cardiol. 2010 Feb.

Abstract

Heart failure, a progressive, fatal disease of the heart muscle, is a state of chronic inflammation and injury. Heat shock protein (HSP) 72, a ubiquitous protective protein that is well-established as cardioprotective, is not increased in heart failure. In contrast, HSP60 levels are doubled in the failing heart. We hypothesized that HSF-1 is not activated in heart failure and that the increased expression of HSP60 was driven by NFkappaB activation. To test this hypothesis, we measured levels of heat shock factor (HSF) -1 and -2, the transcription factors controlling HSP expression, which were increased in heart failure. There was no increased phosphorylation of serine 230 or serine 303/307 in HSF-1, which are thought to regulate its activity; EMSA showed no increase in HSF binding activity with heart failure. Nonetheless, mRNA was increased for HSP60, but not HSP72. In contrast to HSF, NFkappaB activity was increased in heart failure. HSP60, but not HSP72, contained NFkappaB binding elements. ChIP assay demonstrated increased binding of NFkappaB to both of the NFkappaB binding elements in the heart failure HSP60 gene. TNFalpha treatment was used to test the role of NFkappaB activation in HSP60 expression in a cardiac cell line. TNFalpha increased HSP60 expression, and this could be prevented by pretreatment with siRNA inhibiting p65 expression. In conclusion, HSP72 is not increased in heart failure because HSF activity is not changed; increased expression of HSP60 may be driven by NFkappaB activation.

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Figures

Fig. 1
Fig. 1
(A) Real-time PCR for HSP60 normalized to GAPDH. (B) Real-time PCR for HSP72 normalized to GAPDH. (C) Graph summarizes relative expression of HSF-1 by Western normalized to beta actin. Lower panel, western showing expression of HSF-1 and beta actin on the same blot. (D) Relative expression of HSF-2 by Western normalized to beta actin. Lower panels, Western showing expression of HSF-2 and GAPDH on same blot. *p<0.001 vs. C, +p<0.05 vs. C; n=4–6/group.
Fig. 2
Fig. 2
Phosphorylation of HSF-1 at serine 230 (thought to be activating) and serine 303/307 (inhibitory) by Western. (A) Graph summarizes relative expression of phospho-serine230 and phospho-serine303/307 each normalized to total HSF-1. (B) Westerns for phospho-serine 230, phospho-serine303/307 and HSF-1 on same blot. HSF-1 was immunoprecipitated for these experiments. C, control; HF, heart failure. n=6–14/group.
Fig. 3
Fig. 3
(A) Graph summarizes relative expression of ERK by Western normalized to beta actin. (B) Blot showing total ERK and beta actin from same blot. (C) Graph summarizes phospho-ERK by western normalized to beta actin. (D) Western for phospho-ERK and total ERK on same blot. *p<0.05 vs. C. n=4/group.
Fig. 4
Fig. 4
(A) Graph summarizes relative expression of GSK by Western normalized to beta actin. (B) Western for total GSK and beta actin on the same blot. (C) Graph summarizes phospho-GSK normalized to total GSK by Western. (D) Western showing expression of phospho-GSK and total GSK done on same blot. p=ns. n=4/group.
Fig. 5
Fig. 5
HSF and NFκB activation. (A) EMSA shows HSF activation in both controls and CHF heart samples with loading of a large amount of nuclear extract. CC, cold compete. (B) Supershift for HSF-1, faint supershift seen (arrow) for all lanes. (C) Graph summarizes p65 binding activity in control nuclear extracts vs. CHF. n=5–7/group. *p<0.05. (D) Representative EMSA for NFκB. CC (cold compete), sham and CHF samples, respectively. (E) Representative results for ChIP assay for NFκB binding sites 1 (300 bp) and 2 (150 bp). Input is total DNA for each sample (starting material), and IgG control lanes are shown on far right for control and CHF, respectively.
Fig. 6
Fig. 6
NFκB and HSP60 expression. (A) Effect of siRNA on p65 protein levels in H9c2 cells. Control siRNA(siRNA-C) shown on left. (B) Representative western blot showing effect of TNFα on HSP60 expression in H9c2 cells and effect of p65 knockdown on HSP60. (C) Graph summarizes results of multiple experiments. TNFα treatment status shown at bottom. *p<0.05 vs. all.
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