Project Title

Long-Term Cardioprotective Potential of Exogenous Ubiquitin in the Treatment of Post-Myocardial Ischemia/Reperfusion Injury of the Heart

Authors' Affiliations

Dr. Suman Dalal, Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN. Center of Excellence for Inflammation, Infectious Disease, and Immunity, East Tennessee State University, Johnson City, TN. Department of Heath Sciences, College of Public Health, East Tennessee State University, Johnson City, TN. Dr. Mahipal Singh, Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN. Dr. Krishna Singh, Department of Biomedical Sciences, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN. Center of Excellence for Inflammation, Infectious Disease, and Immunity, East Tennessee State University, Johnson City, TN. James H Quillen Veterans Affairs Medical Center East Tennessee State University Johnson City, TN.

Faculty Sponsor’s Department

Biomedical Sciences

Additional Sponsors

Dr. Suman Dalal Dr. Mahipal Singh

Type

Oral Competitive

Classification of First Author

Graduate Student-Doctoral

Project's Category

Physiology

Abstract Text

Background: Heart attack or myocardial infarction (MI) is a major cause of death worldwide. MI is generally attributed to the detrimental effects of myocardial ischemia/reperfusion (I/R) injury. I/R injury induces cell death and reduces heart function. To compensate, the heart remodels with an associated increase in cell death, fibrosis, and hypertrophy, which can further compromise heart function. Ubiquitin (UB) is an evolutionarily conserved protein. Our lab has shown that pre-I/R injury treatment with exogenous UB preserves heart function and reduces fibrosis 3-days post-I/R in mice. A major objective of this study is to analyze the long-term cardioprotective potential of UB post-I/R injury. Here the UB treatment was continued until 28 days post-I/R to include the entire remodeling period. To enhance the clinical applicability, UB treatment was started at the time of reperfusion. Methods: C57BL/6 mice (aged ~3 months) underwent myocardial I/R surgery. Mice were anesthetized and the left anterior descending coronary artery (LAD) was ligated for 45 minutes. The ligature was then removed for reperfusion. Mice were treated with UB (1µg/g body weight; intraperitoneal (IP) injection) or saline at the time of reperfusion; followed by 3-days of saline or UB IP treatment twice per day. The mice were then implanted with micro-osmotic pumps containing UB (1 μg·g−1·h−1) or saline to continue treatment 28-days post I/R. Mice were sacrificed at 28-days post I/R injury. Sham animals underwent the same surgery without LAD ligation. Heart functional parameters (percent ejection fraction and fractional shortening) were analyzed by echocardiography in a time-dependent manner (3, 7, 14 and 28 days post-I/R). Extracted hearts were embedded in paraffin. Heart sections (5µm) were stained with Mason’s Trichrome to measure fibrosis, TUNEL to measure apoptosis, and fluorescein-conjugated wheat germ agglutinin to measure hypertrophy. Index of fibrosis was quantified as a percentage of total left ventricular area, apoptosis was quantified as a percentage of the total number of nuclei, and hypertrophy was quantified by measuring the myocyte cross-sectional area. Major findings: 1) I/R+saline exhibited a significant decrease in the functional parameters of the heart at 3, 7, 14 and 28 days post-I/R vs sham (n=4-12). No significant decrease in heart function observed between I/R+UB vs sham, and heart function was significantly lower in I/R+saline compared to UB+I/R (n=7-12); 2) I/R surgery significantly increased fibrosis in the myocardium of I/R+saline vs sham. No significant difference was observed between UB+I/R and sham, and fibrosis was significantly lower in UB+I/R vs I/R+saline (n=4-6); 3) Apoptosis was significantly higher in I/R+saline vs sham (p4) Myocyte hypertrophy was significantly higher in I/R+saline vs sham (pConclusion: Long-term UB treatment has the potential to preserve heart function with effects on myocardial fibrosis, myocyte apoptosis, and hypertrophy following myocardial I/R injury.

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Long-Term Cardioprotective Potential of Exogenous Ubiquitin in the Treatment of Post-Myocardial Ischemia/Reperfusion Injury of the Heart

Background: Heart attack or myocardial infarction (MI) is a major cause of death worldwide. MI is generally attributed to the detrimental effects of myocardial ischemia/reperfusion (I/R) injury. I/R injury induces cell death and reduces heart function. To compensate, the heart remodels with an associated increase in cell death, fibrosis, and hypertrophy, which can further compromise heart function. Ubiquitin (UB) is an evolutionarily conserved protein. Our lab has shown that pre-I/R injury treatment with exogenous UB preserves heart function and reduces fibrosis 3-days post-I/R in mice. A major objective of this study is to analyze the long-term cardioprotective potential of UB post-I/R injury. Here the UB treatment was continued until 28 days post-I/R to include the entire remodeling period. To enhance the clinical applicability, UB treatment was started at the time of reperfusion. Methods: C57BL/6 mice (aged ~3 months) underwent myocardial I/R surgery. Mice were anesthetized and the left anterior descending coronary artery (LAD) was ligated for 45 minutes. The ligature was then removed for reperfusion. Mice were treated with UB (1µg/g body weight; intraperitoneal (IP) injection) or saline at the time of reperfusion; followed by 3-days of saline or UB IP treatment twice per day. The mice were then implanted with micro-osmotic pumps containing UB (1 μg·g−1·h−1) or saline to continue treatment 28-days post I/R. Mice were sacrificed at 28-days post I/R injury. Sham animals underwent the same surgery without LAD ligation. Heart functional parameters (percent ejection fraction and fractional shortening) were analyzed by echocardiography in a time-dependent manner (3, 7, 14 and 28 days post-I/R). Extracted hearts were embedded in paraffin. Heart sections (5µm) were stained with Mason’s Trichrome to measure fibrosis, TUNEL to measure apoptosis, and fluorescein-conjugated wheat germ agglutinin to measure hypertrophy. Index of fibrosis was quantified as a percentage of total left ventricular area, apoptosis was quantified as a percentage of the total number of nuclei, and hypertrophy was quantified by measuring the myocyte cross-sectional area. Major findings: 1) I/R+saline exhibited a significant decrease in the functional parameters of the heart at 3, 7, 14 and 28 days post-I/R vs sham (n=4-12). No significant decrease in heart function observed between I/R+UB vs sham, and heart function was significantly lower in I/R+saline compared to UB+I/R (n=7-12); 2) I/R surgery significantly increased fibrosis in the myocardium of I/R+saline vs sham. No significant difference was observed between UB+I/R and sham, and fibrosis was significantly lower in UB+I/R vs I/R+saline (n=4-6); 3) Apoptosis was significantly higher in I/R+saline vs sham (p4) Myocyte hypertrophy was significantly higher in I/R+saline vs sham (pConclusion: Long-term UB treatment has the potential to preserve heart function with effects on myocardial fibrosis, myocyte apoptosis, and hypertrophy following myocardial I/R injury.

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