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Mechanisms of hyperhomocysteinemia induced skeletal muscle myopathy after ischemia in the CBS-/+ mouse model.

Veeranki S, Tyagi SC - Int J Mol Sci (2015)

Bottom Line: Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy.Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia.Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, University of Louisville, Louisville, KY 40202, USA. s0veer02@louisville.edu.

ABSTRACT
Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

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Attenuated anti-oxidant capacity in CBS−/+ mice. (A) Representative confocal images were obtained from the normal and ischemic gastrocnemius tissue sections of WT and CBS−/+ mice. Blue fluorescence represents nuclei and green fluorescence represents glutathione levels; (B) ImageJ quantification of glutathione levels from the confocal images obtained from three different mice is presented in the bar graph. ** indicates p < 0.01, and * indicates p < 0.05 and (C) Q-PCR data showing the levels of hemoxygenase-1 mRNA in normal and ischemic muscle tissue of WT and CBS−/+ mice is presented in the bar graph. * indicates p < 0.05.
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ijms-16-01252-f003: Attenuated anti-oxidant capacity in CBS−/+ mice. (A) Representative confocal images were obtained from the normal and ischemic gastrocnemius tissue sections of WT and CBS−/+ mice. Blue fluorescence represents nuclei and green fluorescence represents glutathione levels; (B) ImageJ quantification of glutathione levels from the confocal images obtained from three different mice is presented in the bar graph. ** indicates p < 0.01, and * indicates p < 0.05 and (C) Q-PCR data showing the levels of hemoxygenase-1 mRNA in normal and ischemic muscle tissue of WT and CBS−/+ mice is presented in the bar graph. * indicates p < 0.05.

Mentions: To determine the levels of homocysteine in WT and CBS−/+ mouse skeletal muscles before and after ischemia, we performed immunohistochemical staining using the anti-Hcy (homocysteine) antibody. As observed in Figure 2A,B, CBS−/+ skeletal muscles exhibited relatively higher levels of Hcy. Next, to test if the anti-oxidant capacity in skeletal muscles is compromised during HHcy in addition to lack of H2S (a known anti-oxidant) production capability, we first enumerated the levels of key anti-oxidant glutathione in normal as well as ischemic skeletal muscle sections. As depicted in Figure 3A,B, the glutathione levels were significantly attenuated in both the normal and ischemic CBS−/+ mouse tissue sections when compared to that of the WT muscle sections. In addition, we also determined the levels of another key anti-oxidant enzyme Hemoxygenase-1 (HO-1) in the same set of tissue samples through Q-PCR. As presented in Figure 3C, the levels of HO-1 are not significantly different in normal tissue sections between WT and CBS−/+ mice. However, the HO-1 level induction was significantly decreased after ischemia in CBS−/+ skeletal muscles when compared to that of the WT muscles. Together, all these results indicate a heightened propensity for ROS accumulation, especially during ischemic conditions.


Mechanisms of hyperhomocysteinemia induced skeletal muscle myopathy after ischemia in the CBS-/+ mouse model.

Veeranki S, Tyagi SC - Int J Mol Sci (2015)

Attenuated anti-oxidant capacity in CBS−/+ mice. (A) Representative confocal images were obtained from the normal and ischemic gastrocnemius tissue sections of WT and CBS−/+ mice. Blue fluorescence represents nuclei and green fluorescence represents glutathione levels; (B) ImageJ quantification of glutathione levels from the confocal images obtained from three different mice is presented in the bar graph. ** indicates p < 0.01, and * indicates p < 0.05 and (C) Q-PCR data showing the levels of hemoxygenase-1 mRNA in normal and ischemic muscle tissue of WT and CBS−/+ mice is presented in the bar graph. * indicates p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307302&req=5

ijms-16-01252-f003: Attenuated anti-oxidant capacity in CBS−/+ mice. (A) Representative confocal images were obtained from the normal and ischemic gastrocnemius tissue sections of WT and CBS−/+ mice. Blue fluorescence represents nuclei and green fluorescence represents glutathione levels; (B) ImageJ quantification of glutathione levels from the confocal images obtained from three different mice is presented in the bar graph. ** indicates p < 0.01, and * indicates p < 0.05 and (C) Q-PCR data showing the levels of hemoxygenase-1 mRNA in normal and ischemic muscle tissue of WT and CBS−/+ mice is presented in the bar graph. * indicates p < 0.05.
Mentions: To determine the levels of homocysteine in WT and CBS−/+ mouse skeletal muscles before and after ischemia, we performed immunohistochemical staining using the anti-Hcy (homocysteine) antibody. As observed in Figure 2A,B, CBS−/+ skeletal muscles exhibited relatively higher levels of Hcy. Next, to test if the anti-oxidant capacity in skeletal muscles is compromised during HHcy in addition to lack of H2S (a known anti-oxidant) production capability, we first enumerated the levels of key anti-oxidant glutathione in normal as well as ischemic skeletal muscle sections. As depicted in Figure 3A,B, the glutathione levels were significantly attenuated in both the normal and ischemic CBS−/+ mouse tissue sections when compared to that of the WT muscle sections. In addition, we also determined the levels of another key anti-oxidant enzyme Hemoxygenase-1 (HO-1) in the same set of tissue samples through Q-PCR. As presented in Figure 3C, the levels of HO-1 are not significantly different in normal tissue sections between WT and CBS−/+ mice. However, the HO-1 level induction was significantly decreased after ischemia in CBS−/+ skeletal muscles when compared to that of the WT muscles. Together, all these results indicate a heightened propensity for ROS accumulation, especially during ischemic conditions.

Bottom Line: Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy.Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia.Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, University of Louisville, Louisville, KY 40202, USA. s0veer02@louisville.edu.

ABSTRACT
Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

Show MeSH
Related in: MedlinePlus