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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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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.
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fig5: 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.

Mentions: To determine whether the low Apaf-1 expression in cardiomyocytes was indeed responsible for the strict regulation of caspase activation by XIAP, we examined whether elevating the levels of Apaf-1 in these cells rendered XIAP ineffective. Cardiomyocytes were transfected with plasmids expressing GFP and either Apaf-1 or vector alone. After 24 h to allow for expression, the transfected cardiomyocytes (GFP positive) were injected with cytochrome c to examine the ability of cytosolic cytochrome c to induce apoptosis. Increasing Apaf-1 levels was highly effective in permitting cytochrome c alone to induce cell death in cardiomyocytes (Fig. 5). Apaf-1 overexpression alone did not affect survival, as >90% of Apaf-1–expressing cardiomyocytes remained alive when injected with the control, yeast cytochrome c. Also, this effect was selective for Apaf-1, as overexpression of procaspase-9, the other apoptosome component, did not overcome XIAP inhibition (Fig. 5). Thus, the ability of endogenous XIAP to strictly regulate apoptosis in cardiomyocytes was coupled to the reduced Apaf-1 levels.


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)

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.
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Related In: Results  -  Collection

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fig5: 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.
Mentions: To determine whether the low Apaf-1 expression in cardiomyocytes was indeed responsible for the strict regulation of caspase activation by XIAP, we examined whether elevating the levels of Apaf-1 in these cells rendered XIAP ineffective. Cardiomyocytes were transfected with plasmids expressing GFP and either Apaf-1 or vector alone. After 24 h to allow for expression, the transfected cardiomyocytes (GFP positive) were injected with cytochrome c to examine the ability of cytosolic cytochrome c to induce apoptosis. Increasing Apaf-1 levels was highly effective in permitting cytochrome c alone to induce cell death in cardiomyocytes (Fig. 5). Apaf-1 overexpression alone did not affect survival, as >90% of Apaf-1–expressing cardiomyocytes remained alive when injected with the control, yeast cytochrome c. Also, this effect was selective for Apaf-1, as overexpression of procaspase-9, the other apoptosome component, did not overcome XIAP inhibition (Fig. 5). Thus, the ability of endogenous XIAP to strictly regulate apoptosis in cardiomyocytes was coupled to the reduced Apaf-1 levels.

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