Reduced HtrA2 protein levels in mitochondria but elevated levels in cytosol of left ventricular myocardium of dogs with chronic heart failure

Background: The high-temperature requirement serine peptidase 2 (HtrA2) is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent programmed cell death. When HtrA2 protein is tightly associated with mitochondria (MITO), its protease activity is masked. In contrast, once the protein is released from MITO into the cytosol (CS), its protease activity is exposed. Once active HtrA2 activates caspase-3, a pro-apoptotic enzyme, by degrading the caspase-3 inhibitor XIAP (x-linked inhibitor of apoptosis protein. Recent studies have shown that translocation of HtrA2 protein from the MITO to the CS occurs in mice hearts during experimental myocardial ischemic and reperfusion. Hypothesis: In this study we examined if translocation of HtrA2 from MITO to CS occurs in LV myocardium of dogs with chronic stable heart failure (HF). Methods: LV tissue from 7 dogs with chronic HF produced by multiple sequential intracoronary microembolizations (LV ejection fraction \u3c35%) and 7 normal (NL) dogs was used to prepare crude homogenate (HG) and to isolate MITO and CS fractions. Using primary antibodies, HtrA2 protein was determined in HG, MITO and CS fractions by Western blotting coupled with chemiluminescence. GAPDH was used as an internal loading control for both HG and CS fractions. Porin was used as an internal loading control for MITO fractions. HtrA2 band intensity was normalized to GAPDH for HG and CS and to porin for MITO. Results: GAPDH and porin protein levels were unchanged between NL and HF dogs. Compared to NL, HtrA2 protein levels in dogs with HF were unchanged in HG (0.76 ± 0.04 vs. 0.73 ± 0.08), significantly reduced in MITO (0.17 ± 0.02 vs. 0.40 ± 0.05, p\u3c0.05) and significantly increased in CS (0.90 ± 0.07 vs. 0.36 ± 0.05, p\u3c0.05). Conclusions: Compared to NL dogs, HtrA2 protein levels are significantly reduced in MITO but increased in cytosol fractions from LV myocardium of dogs with chronic stable HF; a maladaptations that promotes cardiomyocyte apoptosis and progressive LV dysfunction. Inhibiting the translocation of HtrA2 from MITO to CS may have therapeutic potential for the preventing progressive dysfunction of the failing heart