Date on Master's Thesis/Doctoral Dissertation

8-2017

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

Schuschke, Dale

Committee Member

Maldonado, Claudio

Committee Member

Sen, Utpal

Committee Member

Borchman, Douglas

Author's Keywords

heart failure; TFAM, cardiac remodeling; NFAT; serca2a

Abstract

Background: An essential mitochondrial component, Mitochondrial Transcription Factor A (TFAM) is reduced within the failing heart. TFAM maintains regulatory functions of cardiomyocyte physiology and stability. Mitochondrial Transcription Factor A forms a mitochondrial nucleoid anti-oxidant structure and reduces pathophysiological abnormalities to structural and contractile proteins. Overall, this functions to physically stabilize mtDNA. Significant molecular changes such as a loss of TFAM drive pathophysiological concentrations of cytoplasmic Ca2+ and reactive oxygen species. Both of these factors induce proteolytic enzymes to degrade the physiological cardiomyocyte. In TFAMs absence, heart function deteriorates and hypertrophic expansion ensues: thereby changing the physiological myocardium to a pathological state. This alteration activates a remodeling cascade leading to cardiomyocyte decline. These maladaptive factors are major players in the pathological and time dependent formation of a failing myocardium. Heart failure is a leading cause of death in the United States and a multitude of molecular therapeutics individually target Ca2+ transporters or inhibit oxidative enzymes. However, the role of TFAM in reducing pathological cardiac remodeling vi maladaptive factors remains unclear. Furthermore, the purpose of this study is to assess TFAM’s role in inhibiting ROS and Ca2+ driven cardiomyocyte remodeling factors. Methods: In the first set of experiments, we studied the effect of TFAM overexpression vector and Crispr-cas9 knockdown on HL-1 cardiomyocytes. The cells were subjected to oxidative stress through hydrogen peroxide treatment and the analysis was compared to a standard control as well as control treatment groups; Lipofectamine and an empty vector. Molecular analysis of cardiomyocte remodeling factors Calpain1, MMP9 and NFAT4 and Ca2+ transporter SERCA2a was assessed via western blotting and immunocytochemistry. Statistical analysis was performed on graphpad prism software. In the second set of experiments, we studied the effects of aortic banding on TFAM transgenic mice. Trans-aortic constriction surgery with a 271/2g needle was performed on 8-10 week old mice. Prior to surgery, echocardiography ultrasound and blood pressure (coda tail cuff method) functional assays were performed and served as a baseline for post-surgical assessment. Eight weeks post-banding, ultrasound of aortic arch perfusion and turbulence were used to assess banding procedure. Cardiac hypertrophy, fibrosis and morphology were assessed via heart weight/body weight ratio, Mason Trichrome and Hematoxylin & Eosin Staining respectively. Molecular vii analysis of cardiac remodeling factors; Calpain1, MMP9, NFAT4 and Ca2+ transporter SERCA2a were assessed via western blotting and immunohistochemistry. Results: In vitro analysis of remodeling factors reveals that TFAM knockdown induces significant increases in maladaptive factors MMP9, Calpain1 and NFAT4. In HL-1 cardiomyocytes subjected to pathological concentrations of H2O2 (mimicking heart failure in vivo studies), TFAM over-expression mitigated hypertrophic stimulator and ROS inducer NFAT4 and decreased MMP9 expression. The TFAM transgenic animal model has reduced cardiac hypertrophy and morphology when compared to it’s WT surgical counterpart. Molecular analysis shows that TFAM over-expression reduces ROS propagated proteases (MMP9) and hypertrophic factors (NFAT4) in cardiac remodeling. Additionally, it was observed that TFAM over-expression does not increase SERCA2a protein expression and Calpain1 remains high in the TFAMTG TAC model. Conclusion: This study reveals that TFAM over-expression plays a vital role in pressure overload heart failure by inhibiting NFAT4 and MMP9. The Ca2+ induced Calpain1 pathway is not mitigated by TFAM over-expression in the TAC pressure overload model, but additional Ca2+ transporters may be affected by TFAM over-expression. TFAM loss or over-expression shifts the homeostatic balance held within the physiological myocardium.

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