Date on Master's Thesis/Doctoral Dissertation

8-2015

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Degree Program

Physiology and Biophysics, PhD

Committee Chair

Tyagi, Suresh

Committee Co-Chair (if applicable)

Schuschke, Dale

Committee Member

Joshua, Irving

Committee Member

Stremel, Richard

Committee Member

Bratcher, Adrienne

Subject

Homocysteine; Exercise--Physiological aspects; Exercise therapy

Abstract

Background: Hyperhomocysteinemia (HHcy) is heavily implicated in diabetes and cardiovascular disease. HHcy is known for causing inflammation and vascular remodeling, particularly through production of reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9). Although its impact on skeletal muscle is rather unclear, HHcy is known to cause skeletal muscle weakness and functional impairment. The impact of HHcy on inflammation makes it seem likely that HHcy causes skeletal muscle fibrosis through induction of inflammatory factors and destructive macrophages. Exercise has been shown to reduce homocysteine levels and therefore, could serve as a promising intervention for HHcy. The purpose of this study was to investigate whether HHcy causes skeletal muscle fibrosis through induction of inflammation and determine if exercise can mitigate these effects. Methods: For these experiments we used J774A.1 and Raw 264.7 macrophages in cell culture and C57, CBS+/-, FVB, MMP-9 -/-, and CBS/MMP-9 double KO mice for animal models. A 6 week treadmill exercise protocol was used as intervention for HHcy. Arterial blood pressure measures were taken through tail-cuff method. Hind limb perfusion was measure via laser Doppler. Plasma Hcy estimations were measured thorugh dot blot. Measurement of skeletal muscle or macrophage protein expression occurred through the use of western blot, immunocytochemistry or immunohistochemistry. Levels of skeletal muscle mRNA expression were determined through PCR and qPCR. Collagen deposition in the skeletal muscle was measured using Masson’s trichrome staining. ROS production in macrophages was measured using a DCFDA assay. Results: In CBS+/- mice, increased plasma Hcy levels were associated with decreased body weight and muscle mass, femoral artery perfusion, femoral artery lumen diameter and oxidative metabolism. These mice displayed increased wall to lumen ratio, mean arterial blood pressure, collagen deposition and elevated myostatin protein expression. It was determined that CBS/MMP-9 mice did not display any of these conditions. Exercise was capable of mitigating all of these effects in CBS +/- mice. Skeletal muscle from CBS+/- mice had elevated markers of inflammation and hypoxia including VEGF, iNOS, EMMPRIN, MMP-9, and IL-1β. Cell culture studies determined that Hcy caused macrophages to shift towards a destructive, M1 phenotype as indicated by elevated CD40, ROS, EMMPRIN and MMP-9 production. EMMPRIN inhibition prevented induction of CD40 and ROS. In CBS +/- mice, it was determined that HHcy causes increased elevations in F4/80 and TNF-α expression, which are indicative of M1 macrophages. Exercise was capable of reducing the M1 macrophage population. Conclusions: We conclude that HHcy causes skeletal muscle fibrosis through induction of MMP-9 and M1 macrophages. Exercise is capable of mitigating the pathologies associated with HHcy.

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