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. 2009 Dec 4;105(12):1223-31.
doi: 10.1161/CIRCRESAHA.109.200378. Epub 2009 Oct 22.

Blockade of Hsp20 phosphorylation exacerbates cardiac ischemia/reperfusion injury by suppressed autophagy and increased cell death

Jiang Qian  1 , Xiaoping Ren, Xiaohong Wang, Pengyuan Zhang, W Keith Jones, Jeffery D Molkentin, Guo-Chang Fan, Evangelia G Kranias
Affiliations

Blockade of Hsp20 phosphorylation exacerbates cardiac ischemia/reperfusion injury by suppressed autophagy and increased cell death

Jiang Qian et al. Circ Res. .

Abstract

Rationale: The levels of a small heat shock protein (Hsp)20 and its phosphorylation are increased on ischemic insults, and overexpression of Hsp20 protects the heart against ischemia/reperfusion injury. However, the mechanism underlying cardioprotection of Hsp20 and especially the role of its phosphorylation in regulating ischemia/reperfusion-induced autophagy, apoptosis, and necrosis remain to be clarified.

Objective: Herein, we generated a cardiac-specific overexpression model, carrying nonphosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20(S16A)). By subjecting this model to ischemia/reperfusion, we addressed whether: (1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; (2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and (3) the aggregation pattern of Hsp20 is altered by its phosphorylation.

Methods and results: Our results demonstrated that Hsp20(S16A) hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis, compared with non-TG hearts. Interestingly, autophagy was activated in non-TG hearts but significantly inhibited in Hsp20(S16A) hearts following ischemia/reperfusion. Accordingly, pretreatment of Hsp20(S16A) hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20(S16A) hearts. Furthermore, on ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20(S16A) hearts.

Conclusions: Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be attributable to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.

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Figures

Figure 1
Figure 1
Phosphorylation of Hsp20 in ex vivo ischemia/reperfusion injured wild-type mouse hearts and failing human hearts. The ratio of phospho-Ser16-Hsp20/total Hsp20 was increased in post-ischemia/reperfusion myocardium (Figure 1A, n=6, *: P<0.01, Post vs. Pre) and in failing human hearts (Figure 1B, n=5 for HF, n=4 for Donor, *: P<0.01, HF vs. Donor,). CSQ: calsequestrin (loading control). HF: heart failure.
Figure 2
Figure 2
Generation of Hsp20S16A transgenic mouse models. (A) Diagram of Hsp20S16A TG constructs. The mutant mouse Hsp20 cDNA, in which Serine 16 encoded by codon TCA was mutated into GCA (encoding alanine), was driven by the α-myosin heavy chain promoter (a -MHCp). (B) DNA sequencing of PCR products from Hsp20S16A mice genome confirmed the TCA to GCA mutation. (C) Quantitative immunoblotting analysis showed that in Hsp20S16A TG hearts there was a 7-fold increase in total Hsp20 levels relative to non-TG hearts (NTG). (D) 2-D gel electrophoresis identified one non-modified Hsp20 spot (PI=5.5) in WT hearts, while there was another S16A modified spot in Hsp20S16A hearts (PI=5.2). (E) The amino acid sequence of S16A-modified Hsp20 spot was indentified by Mass Spectrometry.
Figure 2
Figure 2
Generation of Hsp20S16A transgenic mouse models. (A) Diagram of Hsp20S16A TG constructs. The mutant mouse Hsp20 cDNA, in which Serine 16 encoded by codon TCA was mutated into GCA (encoding alanine), was driven by the α-myosin heavy chain promoter (a -MHCp). (B) DNA sequencing of PCR products from Hsp20S16A mice genome confirmed the TCA to GCA mutation. (C) Quantitative immunoblotting analysis showed that in Hsp20S16A TG hearts there was a 7-fold increase in total Hsp20 levels relative to non-TG hearts (NTG). (D) 2-D gel electrophoresis identified one non-modified Hsp20 spot (PI=5.5) in WT hearts, while there was another S16A modified spot in Hsp20S16A hearts (PI=5.2). (E) The amino acid sequence of S16A-modified Hsp20 spot was indentified by Mass Spectrometry.
Figure 2
Figure 2
Generation of Hsp20S16A transgenic mouse models. (A) Diagram of Hsp20S16A TG constructs. The mutant mouse Hsp20 cDNA, in which Serine 16 encoded by codon TCA was mutated into GCA (encoding alanine), was driven by the α-myosin heavy chain promoter (a -MHCp). (B) DNA sequencing of PCR products from Hsp20S16A mice genome confirmed the TCA to GCA mutation. (C) Quantitative immunoblotting analysis showed that in Hsp20S16A TG hearts there was a 7-fold increase in total Hsp20 levels relative to non-TG hearts (NTG). (D) 2-D gel electrophoresis identified one non-modified Hsp20 spot (PI=5.5) in WT hearts, while there was another S16A modified spot in Hsp20S16A hearts (PI=5.2). (E) The amino acid sequence of S16A-modified Hsp20 spot was indentified by Mass Spectrometry.
Figure 3
Figure 3
Overexpression of Hsp20S16A increased susceptibility to ex vivo ischemia/reperfusion injury. During reperfusion, (A, B) recovery of ±dP/dt was significantly lower in Hsp20S16A hearts compared with non-TGs, (C) LVDP recovery was lower in Hsp20S16A TG hearts compared with non-TGs, (D) the increase of EDP in Hsp20S16A hearts was higher than non-TGs, (non-TGs: n=10, Hsp20S16A: n=8; * : P<0.01, Hsp20S16A vs. non-TG).
Figure 4
Figure 4
Hsp20S16A overexpression increased ischemia/reperfusion-induced necrosis and apoptosis. At basal level, there is no significant differences in LDH release (A), DNA fragmentation (B), TUNEL positive nuclei (C) and caspase-3 activity (D), between Hsp20S16A and non-transgenic hearts (non-TGs: n=6, Hsp20S16A: n=6, P>0.05). Hsp20S16A hearts, subjected to no-flow ischemia followed by reperfusion, exhibited significantly increased total LDH release (A), DNA fragmentation (B), TUNEL-positive nuclei (C) and caspase-3 activity (D), compared to non-TGs (non-TGs: n=6, Hsp20S16A: n=6, *: P <0.01 vs. non-TG).
Figure 5
Figure 5
Infarct areas were increased in Hsp20S16A TG hearts after prolonged ischemia/reperfusion injury. (A) In vivo infarction was induced by 30 minutes occlusion of the left anterior descending artery. The mice were euthanized after 24 hours of reperfusion and infarct size was determined as described previously. (B) There were no significant differences in size of the risk region in transgenic vs. non-TG groups. (C) Infarct size, expressed as a percentage of the region at risk, was 2-fold larger in Hsp20S16A hearts, compared to non-TGs (Hsp20S16A: 53.5±0.8%; non-TG: 22.0±3.0%; n=6, P<0.005).
Figure 6
Figure 6
Autophagy was inhibited in Hsp20S16A hearts in response to ischemia/reperfusion injury. Cardiac homogenates from Hsp20S16A and non-TG mice were subjected to immunoblot analysis for detection of LC3-II/LC3-I and Beclin1 (A). Quantitative analysis results of LC3-II/-LC3-I ratio (B) and Beclin 1 level (C) are from 3 different preparations (six hearts for each group,* : P<0.05, Post- vs. Pre-ischemic group). Pretreatment with the autophagy inducer rapamycin in Hsp20S16A hearts increased the LC3-II/LC3-1 ratio (D) and improved recovery of left ventricular developed pressure (E) and decreased EDP (F) following ischemia/reperfusion, compared with saline-treated controls. (non-TG: n=6, Hsp20S16A: n=6, *: P<0.01, rapamycin vs. saline group).
Figure 6
Figure 6
Autophagy was inhibited in Hsp20S16A hearts in response to ischemia/reperfusion injury. Cardiac homogenates from Hsp20S16A and non-TG mice were subjected to immunoblot analysis for detection of LC3-II/LC3-I and Beclin1 (A). Quantitative analysis results of LC3-II/-LC3-I ratio (B) and Beclin 1 level (C) are from 3 different preparations (six hearts for each group,* : P<0.05, Post- vs. Pre-ischemic group). Pretreatment with the autophagy inducer rapamycin in Hsp20S16A hearts increased the LC3-II/LC3-1 ratio (D) and improved recovery of left ventricular developed pressure (E) and decreased EDP (F) following ischemia/reperfusion, compared with saline-treated controls. (non-TG: n=6, Hsp20S16A: n=6, *: P<0.01, rapamycin vs. saline group).
Figure 6
Figure 6
Autophagy was inhibited in Hsp20S16A hearts in response to ischemia/reperfusion injury. Cardiac homogenates from Hsp20S16A and non-TG mice were subjected to immunoblot analysis for detection of LC3-II/LC3-I and Beclin1 (A). Quantitative analysis results of LC3-II/-LC3-I ratio (B) and Beclin 1 level (C) are from 3 different preparations (six hearts for each group,* : P<0.05, Post- vs. Pre-ischemic group). Pretreatment with the autophagy inducer rapamycin in Hsp20S16A hearts increased the LC3-II/LC3-1 ratio (D) and improved recovery of left ventricular developed pressure (E) and decreased EDP (F) following ischemia/reperfusion, compared with saline-treated controls. (non-TG: n=6, Hsp20S16A: n=6, *: P<0.01, rapamycin vs. saline group).
Figure 7
Figure 7
Sucrose-gradient centrifugation analysis of Hsp20 proteins. Cardiac lysates (100µg total protein) from pre- and post-ischemic/reperfused Hsp20S16A and non-TG hearts were layered on the top of 5%-40% sucrose gradients. After centrifugation, as described in Methods, fractions of the gradients (labeled 1–18 from top to bottom) were collected, and resolved by electrophoresis. A blot of the gel was probed with anti-Hsp20 antibody. Relative protein levels in each fraction were calculated by densitometric scans of each immunoreactive band/total Hsp20. Mutant Hsp20S16A promoted a shift of Hsp20 oligomers to a larger complex profile after ischemia/reperfusion, compared with non-TG controls. Arrows indicate standards: alcohol dehydrogenase (ADH) (150 kDa), β-amylase (250 kDa), and thyroglobulin (690 kDa). Other abbreviations are as defined in the text.
Figure 8
Figure 8
Proposed scheme for phosphorylation of Ser16-Hsp20 protecting against cardiac ischemia/reperfusion injury. Upon phosphorylation, Hsp20 tends to form small oligomers which increase autophagy activity and decrease cell death, therefore preventing cardiac injury during ischemia/reperfusion. When phosphorylation of the Ser16 site is blocked (Hsp20S16A hearts), during ischemia/reperfusion injury, Hsp20 forms large oligomers, suppresses autophagy activity and increases cell death, which leads to cardiac injury.
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