Reduced Apaf-1 levels in cardiomyocytes engage strict regulation of apoptosis by endogenous XIAP

Abstract

Overexpression studies have identified X-linked inhibitor of apoptosis protein (XIAP) as a potent inhibitor of caspases. However, the exact function of endogenous XIAP in regulating mammalian apoptosis is less clear. Endogenous XIAP strictly regulates cytochrome c-dependent caspase activation in sympathetic neurons but not in many mitotic cells. We report that postmitotic cardiomyocytes, unlike fibroblasts, are remarkably resistant to cytosolic microinjection of cytochrome c. The cardiomyocyte resistance to cytochrome c is mediated by endogenous XIAP, as XIAP-deficient cardiomyocytes die rapidly with cytosolic cytochrome c alone. Importantly, we found that cardiomyocytes, like neurons, have markedly reduced Apaf-1 levels and that this decrease in Apaf-1 is directly linked to the tight regulation of caspase activation by XIAP. These data identify an important function of XIAP in cardiomyocytes and point to a striking similarity in the regulation of apoptosis in postmitotic cells.

Figure 1.

Cytosolic microinjection of cytochrome c induces death in fibroblasts but not cardiomyocytes. Neonatal rat cardiomyocytes and dermal fibroblasts were microinjected with 25 μg/μl of bovine or yeast cytochrome c, and cell survival (using morphological criteria) was assessed at multiple times after the injections. (A) Phase-contrast and fluorescence photographs of the cells 3 h after the injections of cytochrome c. The injected cells (arrows) were identified by the presence of rhodamine dextran coinjected with the cytochrome c. (B) Quantitation of cell survival. Data shown are the mean ± SEM of three independent experiments.

Figure 2.

Cardiomyocyte resistance to cytochrome c can be overcome with the exogenous addition of the IAP inhibitor Smac. (A) Western blots showing that the rat cardiomyocyte cultures express the core apoptotic components (Apaf-1, caspase-9, and caspase-3) and various IAPs (XIAP, cIAP-1, and cIAP-2). LDH and Troponin I are shown as controls. (B) Rat cardiomyocytes were microinjected with 25 μg/μl of bovine cytochrome c, 1 μg/μl of wild-type mature Smac, or both, and cell survival was assessed at multiple times after the injections. (C) Rat cardiomyocytes were injected with 25 μg/μl of bovine cytochrome c and 1 μg/μl of either mature wild-type AVPI-Smac or mature mutant MVPI-Smac in the presence or absence of 50 μM of the pan caspase inhibitor zVAD-FMK. Cell survival was assessed at multiple times after the injections. Data shown are the mean ± SEM of three independent experiments.

Figure 3.

XIAP-deficient cardiomyocytes die with injection of cytochrome c . Cardiomyocytes isolated from XIAP-deficient (−/−) or wild-type (+/+) littermate mice were microinjected with 25 μg/μl of bovine cytochrome c. As a control, XIAP-deficient cardiomyocytes were also injected with 25 μg/μl of yeast cytochrome c. (A) Phase-contrast and fluorescence photographs of representative cells. Arrows mark the injected cells, identified by coinjection of rhodamine dextran along with cytochrome c. (B) Quantitation of cell survival at multiple times after the injections. Data shown are the mean ± SEM of three independent experiments.

Figure 4.

Apaf-1 but not caspase-9 levels are markedly reduced in cardiomyocytes in comparison to fibroblasts. (A) Western blots showing levels of Apaf-1 and caspase-9 proteins (and LDH and Troponin I as controls) in cultures of rat dermal fibroblasts and cardiomyocytes. (B) Quantitation of the data in which Apaf-1 and caspase-9 protein levels detected in cardiomyocyte cultures are expressed as a percentage of the levels (normalized to LDH) seen in fibroblast cultures. Data shown are the mean ± SEM of three independent experiments.

Figure 5.

Restoring Apaf-1 levels eliminates the strict control of XIAP and permits cytochrome c to induce apoptosis in cardiomyocytes. Rat cardiomyocytes were transfected with plasmids expressing GFP alone (Vector/GFP), Apaf-1 and GFP (Apaf-1/GFP), or procaspase-9 and GFP (caspase-9/GFP). After 24 h, the transfected cells (identifiable by GFP expression) were microinjected with 25 μg/μl of bovine or yeast cytochrome c and rhodamine dextran (Cyt c/Rhod). (A) Fluorescence photographs (for the GFP- or Rhodamine-selective channels) of representative cells taken 2 h after the cytochrome c injections. Arrows point to the GFP-positive cells that were injected with cytochrome c and rhodamine. (B) Quantitation of cell survival (2 h after the injections) for the various conditions. Data shown are the mean ± SEM of three independent experiments.