Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Avadhesh C Sharma, PharmD, PhD

Second Advisor

Rangaiah Shashidharamurthy, PhD

Third Advisor

Srujana Rayalam, BVSc, MVSc, Ph.D

Fourth Advisor

Richard White, PhD

Fifth Advisor

Brian Matayoshi, PhD

Abstract

Cardiovascular disease (CVD) is the leading cause of death in the United States and has been for the past 80 years. Development of novel therapeutic agents to address the large number of CVD deaths requires an in depth understanding of the structural and functional properties of human cardiomyocytes. Over the last few years we have been developing an in vitro paradigm to assess molecular cardiodynamics in Primary Human Cardiomyocyte in culture (PHCC). We tested the hypothesis whether endotoxemia would exhibit a marked decrease in contractile proteins and cause apoptosis in PHCC. In the current series of experiments, we induced endotoxemia using E. coli lipopolysaccharide (LPS) in PHCC. We investigated cell viability, induction of apoptosis and the level of contractile proteins using immunoblotting, confocal microscopy and flow cytometry. Four treatment groups with varying concentrations of LPS (0, 1, 10 and 100μg/ml) were added to wells containing one million primary human cardiomyocytes and viability was tested at 24, 48 and 72 hours post treatment using Countess automated cell counter. No significant change in the viability of PHCC was observed in both LPS and control groups. In addition, we did not find any significant alterations in the levels of TNF-alpha and Annexin V staining (a marker for early detection of apoptosis). These data indicate that the PHCC are resistant to LPS-induced induction of apoptosis and cell death as reported in other cell lines. Given that endotoxemia is associated with impairment of cardiomyocyte contraction, we hypothesized that LPS treatment would reduce the expression of contractile proteins in PHCC. Norepinephrine (NE) was used as positive control for contractile protein expression. The treatment groups included: Control; LPS, 100μg/ml; NE 10μM; LPS, 100μg/ml + NE 10μM. NE produced a significant increase in the protein levels of troponin I, tropomyosin, and myosin light chain proteins compared to the untreated control group. In contrast, LPS produced a significant decrease in troponin I, tropomyosin, and myosin light chain proteins. In addition, NE-induced increase in protein levels were significantly decreased by LPS in the LPS+NE combination treatment group compared to control groups. The fluorescence intensity of the contractile proteins was less in the LPS treated cells compared to the other treatment groups. The results gathered from confocal microscopy further strengthened immunoblot data suggesting that LPS reduced contractile protein expression. Collectively, these data suggest that LPS affect the expression of contractile proteins in PHCC without affecting the cell viability. The PHCC cell line is not sensitive to LPS-induced activation of cytokines and induction of apoptosis. It appears that LPS induced decrease in protein levels of myofibrillar and contractile proteins in PHCC might not be due to induction of apoptosis.

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