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

Potts MB, Vaughn AE, McDonough H, Patterson C, Deshmukh M - J. Cell Biol. (2005)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Center, Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

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.

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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.
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fig2: 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.

Mentions: Western analysis indicated that the neonatal cardiomyocytes expressed Apaf-1, caspase-9, and caspase-3 (Fig. 2 A). This result argues against the possibility that cytochrome c was unable to induce death in cardiomyocytes because of a deficiency of the core components of the apoptotic machinery. In sympathetic neurons, cytochrome c–induced caspase activation is tightly regulated by endogenous IAPs. Addition of exogenous Smac, which inhibits IAPs, overcomes the resistance to cytochrome c and permits apoptosis to occur in neurons (Deshmukh et al., 2002; Potts et al., 2003). To examine whether the cardiomyocyte resistance to cytochrome c was also attributable to a stringent regulation by endogenous IAPs, we examined whether injection of excess Smac permitted cytochrome c to activate apoptosis in cardiomyocytes. Coinjection of cytochrome c and mature Smac, but neither alone, was remarkably effective in inducing death in cardiomyocytes (Fig. 2 B). To confirm that exogenous Smac permitted cytochrome c to induce apoptosis by inhibiting IAPs in cardiomyocytes, we examined whether a mutant Smac protein that cannot inhibit IAPs (Chai et al., 2000) was capable of permitting cytochrome c to induce death. We found that unlike wild-type Ala-Val-Pro-Ile (AVPI)–Smac, the mutant Met-Val-Pro-Ile (MVPI)–Smac was incapable of cooperating with cytochrome c to induce death in cardiomyocytes (Fig. 2 C). We also confirmed that the death induced by injection of cytochrome c and AVPI-Smac occurred as a consequence of caspase activation. Addition of the pan caspase inhibitor zVAD-FMK completely blocked the cardiomyocyte death induced by cytochrome c and Smac (Fig. 2 C). Together, these results show that the components of the apoptosome are present and functional in cardiomyocytes but only become permissive for apoptosis if the activity of IAPs is blocked.


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

Potts MB, Vaughn AE, McDonough H, Patterson C, Deshmukh M - J. Cell Biol. (2005)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171313&req=5

fig2: 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.
Mentions: Western analysis indicated that the neonatal cardiomyocytes expressed Apaf-1, caspase-9, and caspase-3 (Fig. 2 A). This result argues against the possibility that cytochrome c was unable to induce death in cardiomyocytes because of a deficiency of the core components of the apoptotic machinery. In sympathetic neurons, cytochrome c–induced caspase activation is tightly regulated by endogenous IAPs. Addition of exogenous Smac, which inhibits IAPs, overcomes the resistance to cytochrome c and permits apoptosis to occur in neurons (Deshmukh et al., 2002; Potts et al., 2003). To examine whether the cardiomyocyte resistance to cytochrome c was also attributable to a stringent regulation by endogenous IAPs, we examined whether injection of excess Smac permitted cytochrome c to activate apoptosis in cardiomyocytes. Coinjection of cytochrome c and mature Smac, but neither alone, was remarkably effective in inducing death in cardiomyocytes (Fig. 2 B). To confirm that exogenous Smac permitted cytochrome c to induce apoptosis by inhibiting IAPs in cardiomyocytes, we examined whether a mutant Smac protein that cannot inhibit IAPs (Chai et al., 2000) was capable of permitting cytochrome c to induce death. We found that unlike wild-type Ala-Val-Pro-Ile (AVPI)–Smac, the mutant Met-Val-Pro-Ile (MVPI)–Smac was incapable of cooperating with cytochrome c to induce death in cardiomyocytes (Fig. 2 C). We also confirmed that the death induced by injection of cytochrome c and AVPI-Smac occurred as a consequence of caspase activation. Addition of the pan caspase inhibitor zVAD-FMK completely blocked the cardiomyocyte death induced by cytochrome c and Smac (Fig. 2 C). Together, these results show that the components of the apoptosome are present and functional in cardiomyocytes but only become permissive for apoptosis if the activity of IAPs is blocked.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Center, Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

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.

Show MeSH
Related in: MedlinePlus