Date on Master's Thesis/Doctoral Dissertation

12-2018

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Tyagi, Suresh C.

Committee Co-Chair (if applicable)

Clark, Barbara J.

Committee Member

Clark, Barbara J.

Committee Member

Maldonado, Claudio

Committee Member

Cole, Marsha

Committee Member

Watson, Corey

Author's Keywords

homocysteine; cellular stress; muscle atrophy; reactive oxygen species; angiogenesis; ER-stress

Abstract

Although hyperhomocysteinemia (HHcy) occurs due to a deficiency in cystathionine-β-synthase (CBS), causing skeletal muscle myopathy, it is still unclear whether this effect is mediated through oxidative, endoplasmic reticulum (ER) stress, both or something else. Cystathionine γ-lyase (CSE) and CBS are the main H2S producing enzymes, which produce Hydrogen sulfide (H2S) from Hcy in the transsulfuration reaction. H2S is an anti-oxidant compound and patients with a CBS deficiency cannot produce enough H2S, thus rendering them vulnerable to excess Hcy-mediated damage. Both in-vitro (C2C12 cells) and in-vivo (CBS+/- mice) models were used to study HHcy-mediated muscle myopathy, and they were treated with sodium hydrogen sulfide (NaHS, an H2S donor) to mitigate the effect(s) of HHcy in skeletal muscle. To examine whether muscle myopathy is due to poor angiogenesis, we created a hind limb femoral artery ligation (FAL) in CBS+/- mouse model and treated them with GYY4137 (a long-acting H2S donor compound). Results showed that oxidative stress was reversed by NaHS (H2S donor) in Hcy-treated C2C12 cells. ER stress markers (GRP78, ATF6, and pIRE1α) were elevated both in-vivo and in-vitro, and NaHS mitigated these effects. JNK-phosphorylation was upregulated in C2C12 cells after Hcy treatment, but NaHS could not reduce this effect. Apoptosis was induced after Hcy treatment, and it was mitigated by the NaHS supplementation in C2C12 cells. HIF1-α, VEGF, PPAR-γ and p-eNOS expression levels were attenuated in skeletal muscle of CBS+/- mice after 21 days of FAL in comparison to WT mice and were improved via the GYY4137 (another H2S donor compound) treatment. Collateral vessel density and blood flow were significantly reduced in post-FAL CBS+/- mice compared to WT mice, and these effects were ameliorated by the GYY4137 treatment. Plasma nitrite levels were decreased in post-FAL CBS+/- mice compared to WT mice, and these were improved by GYY4137 supplementation. I also noticed FOXO1-mediated MuRF-1 upregulation, which further degrades MHC-I in CBS+/- mice compared to WT mice, and these effects were improved by NaHS intervention. Functional studies revealed that NaHS administration improved muscle fatigability in CBS+/- mice. This work provides evidence that H2S is beneficial in mitigating HHcy-mediated skeletal muscle injury incited by oxidative or ER stress responses and H2S might be employed as a potential therapeutic to alleviate the harmful metabolic effects of HHcy conditions.

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