Response of caspase-independent apoptotic factors to high salt diet-induced heart failure

Abstract

The role of caspase-independent apoptotic events in heart failure is largely unknown. The present study examined the response of apoptotic factors, which can function independently of caspase machinery including AIF, EndoG, and HtrA2/Omi to high salt diet-induced pathologic heart failure and exercise-induced physiologic cardiac hypertrophy. Following approximately 4 months of a daily diet containing 6% salt, animals developed clinical evidence of heart failure accompanied by changes in AIF, EndoG, and HtrA2/Omi. Assessment of the mitochondria-free cytosolic fraction revealed cytosolic accumulations of AIF and processed HtrA2/Omi in the failed ventricle muscles. The subcellular translocation of AIF from mitochondria to cytosol and nuclei was supported by immunofluorescent analysis using confocal microscopy. However, according to our RT-PCR analyses, AIF and EndoG mRNA were decreased, rather than elevated, in the failed heart relative to control heart. No difference in any of the measured parameters of AIF, EndoG, and HtrA2/Omi was found in the ventricle muscle of either exercise-trained or 6 weeks high salt diet fed animals compared to controls. These findings are consistent with the hypothesis that caspase-independent events are involved in cardiac apoptosis during the late remodeling stage of pathologic heart failure.

Figure 1. Endogenous level of AIF, EndoG and HtrA2/Omi in different organs

The representative Western blot of AIF, EndoG, and HtrA2/Omi from rats. Sm, skeletal muscle; Hr, heart; Br, brain; Lu, lung; Li, liver; Sp, spleen.

Figure 2. Subcellular localization of AIF, EndoG, and HtrA2/Omi

A. Verification of the protein fraction purity. The purity of the extracted protein fractions was examined by immunoblot of mitochondrial protein (MP) and cytosolic protein fractions (CP) probed with anti-COXIV and anti-GAPDH antibodies. The mitochondrial and cytosolic protein fractions were extracted from a same ventricle muscle and equal amount of protein was employed in the immunoblot. B. Mitochondrial protein content of AIF, EndoG, and HtrA2/Omi. The protein content of AIF, EndoG, and HtrA2/Omi was measured by Western immunoblot in the mitochondria fraction. The insets show representative blots. Con, control; Ex, exercise; HS, high salt; Fail, failed heart. C. Representative Western immunoblot of AIF, EndoG, and HtrA2/Omi in the mitochondria-free cytosolic fraction. GAPDH was used as an internal control. D. Quantification of cytosolic protein content of AIF, EndoG, and HtrA2/Omi. The data are presented as means ± SE. *P < 0.05, data are significantly different from control animals.

Figure 3. AIF immunofluorescent staining

Immunofluorescent labeling of AIF was performed simultaneously with TUNEL staining. AIF (red) stained with secondary rhodamin, TUNEL (green) stained with fluorescein, and nuclei (blue) labeled using To-Pro-3. Image was obtained using ×2000 magnification.

Figure 4. mRNA content of AIF, EndoG, and HtrA2/Omi

A. Representative semi-quantitative RT-PCR of AIF, EndoG, and HtrA2/Omi. 18S RNA is shown for internal control. B. Quantification of the mRNA expression of AIF, EndoG, and HtrA2/Omi. The data are presented as means ± SE. *P < 0.05, data are significantly different from control animals.