Deficiency of the Bax gene attenuates denervation-induced apoptosis

Abstract

Apoptosis has been implicated in mediating denervation-induced muscle wasting. In this study we determined the effect of interference of apoptosis on muscle wasting during denervation by using mice genetically deficient in pro-apoptotic Bax. After denervation, muscle wasting was evident in both wild-type and Bax(-/-) muscles but reduction of muscle weight was attenuated in Bax(-/-) mice. Apoptotic DNA fragmentation increased in wild-type denervated muscles whereas there was no statistical increase in DNA fragmentation in denervated muscles from Bax(-/-) mice. Mitochondrial AIF and Smac/DIABLO releases and Bcl-2, p53 and HSP27 increased whereas XIAP and MnSOD decreased to a similar extent in muscles from wild-type and Bax(-/-) mice following denervation. Mitochondrial cytochrome c release was elevated in denervated muscles from wild-type mice but the increase was suppressed in muscles from Bax(-/-) mice. Increases in caspase-3 and -9 activities and oxidative stress markers H(2)O(2), MDA/4-HAE and nitrotyrosine were all evident in denervated muscles from wild-type mice but these changes were absent in muscles from Bax(-/-) mice. Moreover, ARC increased exclusively in denervated Bax(-/-) muscle. Our data indicate that under conditions of denervation, pro-apoptotic signalling is suppressed and muscle wasting is attenuated when the Bax gene is lacking. These findings suggest that interventions targeting apoptosis may be valuable in ameliorating denervation-associated pathologic muscle wasting in certain neuromuscular disorders that involve partial or full denervation.

Figure 1

Muscle weight. The extent of muscle loss following denervation was estimated by examining the gastrocnemius muscle wet weight loss normalized to the animal’s bodyweight between the denervated and the contralateral control sides. The data are expressed as means ± standard error of mean (SE). ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain.

Figure 2

Apoptotic DNA fragmentation. The extent of apoptotic DNA fragmentation was estimated by measuring the cytosolic mono- and oligonucleosomes. The OD₄₀₅ is normalized to the total milligrams protein content of the sample used in the assay. The normalized data are expressed as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain;^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition.

Figure 3

(A) Bax protein content. The Bax protein content was examined by Western immunoblot. The insets show representative blots for the Bax protein. The data are expressed as means ± SE of the arbitrary unit. ^*P < 0.05, denervated muscle was significant different from the contralateral control muscle under the same animal strain. (B) Bcl-2 protein content. The Bcl-2 protein content was examined by Western immunoblots. The insets show representative blots for the Bcl-2 protein. The data are expressed as means ± SE of the arbitrary unit. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain.

Figure 4

Mitochondrial cytochrome c release. The release of mitochondrial cytochrome c was estimated by measuring the protein content of cytochrome c in the mitochondria-free cytosolic fraction using an ELISA. The OD₄₅₀ is normalized to the total milligrams protein content of the sample used in the assay. The normalized data are expressed as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain;^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition.

Figure 5

(A) Caspase-3 protease activity. The caspase-3 activity was assessed by a fluorometric caspase activity assay. The fluorescence unit (FU) is normalized to the total milligrams protein content of the sample used in the assay. The normalized data are expressed as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain; ^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition. (B) Caspase-9 protease activity. The caspase-9 activity was assessed by a fluorometric caspase activity assay. The fluorescence unit (FU) is normalized to the total milligrams protein content of the sample used in the assay. The normalized data are expressed as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain; ^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition.

Figure 6

(A) Mitochondrial Smac/DIABLO release. The protein content of Smac/DIABLO was measured by Western immunoblot in the mitochondria-free cytosolic fraction in order to estimate the mitochondrial Smac/DIABLO release. The insets show representative blots. The data are expressed as means ± SE of the arbitrary unit.^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain. (B) XIAP protein content. The protein content of XIAP was measured by Western immunoblots in the total cytosolic fraction. The insets show representative blots. The data are expressed as means ± SE of the arbitrary unit.^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain. (C) ARC protein content. The protein content of ARC was measured by Western immunoblots in the total cytosolic fraction. The insets show representative blots. The data are expressed as means ± SE of the arbitrary unit. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain; ^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition.

Figure 7

(A) Mitochondrial AIF release. The protein content of AIF was measured by Western immunoblots in the mitochondria-free cytosolic fraction in order to estimate the mitochondrial AIF release. The insets show representative blots. The data are expressed as means ± SE of the arbitrary unit. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain. (B) AIF nuclear translocation. The protein content of AIF was measured by Western immunoblots in the nuclear fraction in order to estimate the nuclear translocation of AIF. The insets show representative blots. The data are expressed as means ± SE of the arbitrary unit.

Figure 8

(A) Nuclear p53 protein content. The protein content of p53 was measured by Western immunoblots in the nuclear fraction. The insets show representative blots. The data are expressed as means ± SE of the arbitrary unit. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain. (B) Cytosolic p53 protein content. The protein content of p53 was measured by Western immunoblots in the total cytosolic fraction. The insets show representative blots. The data are expressed as means ± SE of the arbitrary unit.^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain.

Figure 9

(A) HSP70 protein content. The protein content of HSP70 was measured by Western immunoblots in the total cytosolic fraction. The insets show representative blots. The data are presented as means ± SE of the arbitrary unit. (B) HSP27 protein content. The protein content of HSP27 was measured by Western immunoblots in the total cytosolic fraction. The insets show representative blots. The data are presented as means ± SE of the arbitrary unit. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain. (C) HSP60 protein content. The protein content of HSP60 was measured by Western immunoblots in the total cytosolic fraction. The insets show representative blots. The data are presented as means ± SE of the arbitrary unit.

Figure 10

(A) H₂O₂ content. The H₂O₂ content was determined by a fluorometric assay. The fluorescence unit (FU) is normalized to the total milligrams protein content of the sample used in the assay. The normalized data are presented as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain;^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition. (B) MDA/4-HAE content. The level of lipid peroxidation was estimated by measuring the content of MDA/4-HAE. The OD₅₈₆ is normalized to the total milligrams protein content of the sample used in the assay. The normalized data are presented as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain;^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition. (C) Nitrotyrosine content. The content of nitrotyrosine was measured in the cytosolic fraction by an immunodot blot. The data are expressed as OD x resulting band area, and expressed in arbitrary units. The insets show representative blots for nitrotyrosine. The data are presented as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain; ^**P < 0.05, Bax^−/− mice were significantly different from the wild-type mice under the same experimental condition.

Figure 11

(A) MnSOD protein content. The protein content of Mn-SOD was assessed in cytosolic fraction by immunoblot analysis. The data are expressed as OD x resulting band area, and expressed in arbitrary units. The insets show representative blots for MnSOD. The data are presented as means ± SE. ^*P < 0.05, denervated muscle was significantly different from the contralateral control muscle under the same animal strain. (B) CuZnSOD protein content. The protein content of CuZnSOD was assessed in cytosolic fraction by immunoblot analysis. The data are expressed as OD x resulting band area, and expressed in arbitrary units. The insets show representative blots for MnSOD. The data are presented as means ± SE.