CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice

Abstract

The abundantly expressed small molecular weight proteins, CRYAB and HSPB2, have been implicated in cardioprotection ex vivo. However, the biological roles of CRYAB/HSPB2 coexpression for either ischemic preconditioning and/or protection in situ remain poorly defined. Wild-type (WT) and age-matched ( approximately 5-9 mo) CRYAB/HSPB2 double knockout (DKO) mice were subjected either to 30 min of coronary occlusion and 24 h of reperfusion in situ or preconditioned with a 4-min coronary occlusion/4-min reperfusion x 6, before similar ischemic challenge (ischemic preconditioning). Additionally, WT and DKO mice were subjected to 30 min of global ischemia in isolated hearts ex vivo. All experimental groups were assessed for area at risk and infarct size. Mitochondrial respiration was analyzed in isolated permeabilized cardiac skinned fibers. As a result, DKO mice modestly altered heat shock protein expression. Surprisingly, infarct size in situ was reduced by 35% in hearts of DKO compared with WT mice (38.8 +/- 17.9 vs. 59.8 +/- 10.6% area at risk, P < 0.05). In DKO mice, ischemic preconditioning was additive to its infarct-sparing phenotype. Similarly, infarct size after ischemia and reperfusion ex vivo was decreased and the production of superoxide and creatine kinase release was decreased in DKO compared with WT mice (P < 0.05). In permeabilized fibers, ADP-stimulated respiration rates were modestly reduced and calcium-dependent ATP synthesis was abrogated in DKO compared with WT mice. In conclusion, contrary to expectation, our findings demonstrate that CRYAB and HSPB2 deficiency induces profound adaptations that are related to 1) a reduction in calcium-dependent metabolism/respiration, including ATP production, and 2) decreased superoxide production during reperfusion. We discuss the implications of these disparate results in the context of phenotypic responses reported for CRYAB/HSPB2-deficient mice to different ischemic challenges.

Fig. 1

In situ analysis. A: indirect immunofluorescence analysis of heart sections stained with anti-CryAB detected by FITC-conjugated secondary antibodies shows CRYAB (green) in cardiomyocytes of wild-type (WT) but not in double knockout (DKO) hearts. No histomorphological abnormalities are seen for either WT or DKO hearts at 6 mo. B: protocols for acute myocardial infarction (AMI) without and with early preconditioning (EPC). Group I (WT; n = 10) and group II (DKO; n = 11) animals were subjected to 30 min occlusion followed by 24 h reperfusion. Preconditioning (PC) consisted of 6 cycles of 4 min of coronary occlusion (O) and 4 min of reperfusion (R), followed 10 min later, before prolonged 30 min occlusion and 24 h reperfusion. Group III (WT, early PC; n = 11) and group IV (DKO, early PC; n = 10) Infarct size was determined with triphenyltetrazolium (TTC) staining and phthalo blue. TTC staining distinguishes the infracted (white) from viable (red) myocardial regions. Infarct size is qualitatively smaller in DKO and after early PC. C: myocardial infarct sizes in groups I–IV are quantitatively expressed as percentage of the risk region at risk. Data are means ± SE. *P < 0.05 vs. group I.

Fig. 2

Ex vivo analysis. A: infarct size was similarly determined after reperfusion with TTC staining. The hearts of 4-mo-old animals were subjected to 30 min of global ischemia followed by 15 min (superoxide production) or 120 min (infarct size) of reperfusion. *P < 0.05; n = 4 WT and n = 5 DKO. B: creatine kinase (CK), a biomarker of myocardial injury, was assessed directly from the coronary effluent before (Pre) and following (Post) ischemia. *P < 0.05 vs. Pre value; †P < 0.05 vs. WT Post; n = 7 WT and DKO. C: measurements of superoxide production were determined during and after reperfusion as described in materials and methods. *P < 0.05; n = 5 WT and n = 8 DKO. Data are means ± SE.

Fig. 3

Western Blot. A: representative Western blot probed with anti-HSP25 antibody of either the soluble [supernatant (S/N)] or insoluble (pellet) fractions isolated from hearts of 6-mo-old WT and DKO animals. DKO is associated with modest downregulation of heat shock protein (HSP)25 in the cytosolic fraction and equivalent translocation into the cytoskeletal/nuclear fraction assessed by densitometry (B). Each lane represents an individual animal. Western blot analysis (C) and densitometry (D) were performed on cardiac homogenates of WT and CRYAB/HSPB2-deficient (DKO) for HSP60, HSP70, and HSP90. Each lane represents an individual animal in either the WT or DKO experimental groups (n = 3/group). *P < 0.05.

Fig. 4

Respiration in cardiac fibers. Mitochondrial respiratory parameters were obtained from permeabilized fibers incubated with glutamate-malate (A) and palmitoyl carnitine-malate (B) as substrates. Gray bars, fibers obtained from the glucose-perfused WT hearts (n = 6); white bars, fibers from CRYAB/HSPB2-deficient (DKO) hearts (n = 6). Mitochondria was harvested from hearts of 6–8-wk-old animals. State 2, respiration in the absence of ADP; state 3, respiration ADP (1 mmol/l)-stimulated respiration; state 4, oligomycin (1 μg/ml)-inhibited respiration; RC, respiratory control ratio. Values are shown as means ± SE. *P < 0.05. State 3 respiratory was significantly lower with both substrates in DKO compared with WT. Mgdw, milligrams of dry weight.

Fig. 5

ATP in cardiac fibers. ATP rates and ATP-to-O ratios (ATP/O) obtained from permeabilized fibers, where O refers to oxygen consumption under state 3 conditions. ATP production was measured by a bioluminescence assay initiated by the addition of 1 mM ADP to the skinned fiber preparation. ATP synthesis rates were significantly lower in DKO fibers from hearts of 6–8-wk-old animals compared with WT. Values are shown as means ± SE.