Heart failure is associated with a high mortality rate and is one of the most prevalent diseases worldwide whereby susceptibility increases with age. The development of heart failure occurs over an extensive period of time in which arrhythmias and hypertrophy are both very prevalent manifestations throughout this progression. Arrhythmias are defined as an irregular rhythm originating from intracellular calcium dysregulation, which can be fatal. Cardiac hypertrophy is a compensatory condition induced by increased workload involving augmented cardiomyocyte growth accompanied by myocardial remodelling. However, under prolonged periods of increased stress this compensatory mechanism can lead to cardiac dysfunction. The current treatments for heart failure are mainly aimed at relieving symptoms or itself possess proarrhythmic ability. Therefore it is fundamental to elucidate the pathways involved in arrhythmias and hypertrophy for the development of more effective treatment. p21 activated protein kinase (Pak1) is a novel gene involved in the regulation of cardiac function, however, the mechanisms involved remain inconclusive. This study has demonstrated Pak1 to be both antiarrhythmic and antihypertrophic, emphasizing Pak1 as a credible therapeutic target for simultaneously treating both manifestations. The antiarrhythmic properties of Pak1 were demonstrated through cardiomyocyte-specific Pak1 knockout (Pak1cko) mouse model which underwent Isoproterenol (ISO) stimulation for 2 weeks. Compared with ISO treated control group, the Pak1cko group had increased calcium irregularities and particularly a prolongation in sarcoplasmic reticulum (SR) calcium refill. The absence of Pak1 abrogated the transcriptional up-regulation of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) under stressed conditions. Further analysis in neonatal rat cardiomyocytes (NRCMs) revealed this regulation to be through activation of the transcription factor, SRF. The antihypertrophic effects of Pak1 were further illustrated through cardiomyocyte-specific overexpressed constitutively-active Pak1 (Pak1cTG) mice which were subjected to transverse aortic constriction (TAC) for 3 weeks. Compared to TAC control group, Pak1cTG mice had improved cardiac performance accompanied with diminished fibrosis. Further analysis led to the discovery of a novel antihypertrophic pathway of Pak1 involving positive regulation of the E3ligase, Fbxo32 through activation of Smad3. This pathway is vital in the prevention of calcineurin (PP2B) accretion. Berberine administration in TAC treated mice corroborated that Fbxo32 up-regulation is sufficient in the prevention of hypertrophy. In conclusion, my study has demonstrated that Pak1 conveys antiarrhythmic influence through the up-regulation of SERCA2a. In the prevention of pathological hypertrophy, Pak1 inhibits PP2B through positive regulation of Fbxo32. Overall, my thesis has advanced the knowledge about cardioprotective pathways initiated by Pak1 under stressed conditions, presenting Pak1 as a promising therapeutic target.

The sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) is responsible for calcium transport in the heart and its dysfunction in heart disease and diabetes make it a target for treatment strategies. SERCA2a structure can be modified post-translationally by the addition of an acetyl protein via acetylation. Sirtuin3 (SIRT) is a deacetylase, which may interact with SERCA2a to reverse its acetylation. The aim of this study was to determine if SERCA2a function is altered by acetylation, if this occurs in the diabetic heart, and if SIRT3 influences SERCA2a acetylation. Our data indicates that modification to three SERCA2a acetylation sites impairs its activity in a cell culture model and that SERCA2a acetylation occurs at higher levels in the diabetic heart. Furthermore, SERCA2a is acetylated at higher levels in absence of SIRT3, suggesting that SIRT3 activity influences SERCA2a. Our data identifies possible therapeutic strategies to target and reduce SERCA2a acetylation in the diabetic heart. / February 2016

No description available.

The Ca-ATPase is an integral membrane enzyme which translocates two calcium ions from the cytoplasm of the cell to the sarcoplasmic reticulum lumen utilizing ATP breakdown as its energy source, in order to promote muscle relaxation. The focus of this research is the cardiac isoform of the Ca-ATPase which undergoes allosteric regulation by the phosphoprotein phospholamban (PLN). The Ca-ATPase is thought to be a target for nitrative stress and is affected by several chronic diseases of the heart. In the heart, age-based nitration of the Ca-ATPase inhibits Ca²⁺ transport activity but the physical mechanism by which nitration inhibits Ca-ATPase activity is not understood. Conversely, nitroxyl (HNO), a new candidate for drug therapy for congestive heart failure (CHF), improves overall cardiovascular function by increasing Ca-ATPase activity in the heart. However, the physical mechanism for this activation is unknown. Therefore, we have used enzyme kinetics, fluorescence spectroscopy, and EPR spectroscopy studies to determine the effects of ONOO⁻ and HNO on the Ca-ATPase and the physical regulation of the Ca-ATPase by PLN. Treatment of Ca-ATPase with a nitrating agent, ONOO⁻, inhibited Ca-ATPase activity, and the [ONOO⁻]-dependent inhibition of the Ca-ATPase was more effective in the presence of PLN. ONOO⁻ did not affect the [Ca²]-dependence of Ca-ATPase activity either in the presence or absence of PLN. ONOO⁻ had no effect on Ca-ATPase rotational mobility or oligomeric interactions, as affected by PLN, but ONOO⁻ decreased the amplitude of the Ca²⁺-dependent E2 to E1•Ca2 conformational change, both in the absence and presence of PLN. Treatment with HNO had no affect on the [Ca²⁺]-dependence of Ca-ATPase activity in the absence of PLN; however in the presence of PLN, the [Ca²⁺]-dependent activity was shifted to lower Ca²⁺ levels and corresponded to the uncoupling of PLN from the Ca-ATPase. HNO decreased Ca-ATPase rotational mobility and increased the Ca-ATPase Ca²⁺-dependent conformational transition, consistent with uncoupling PLN from the Ca-ATPase. Taken together, these results suggest that ONOO⁻ inactivates a fraction of enzyme units to lower overall enzyme activity, whereas HNO uncouples PLN from the Ca-ATPase with increases in Ca-ATPase conformational flexibility and Ca-ATPase activity. / Ph. D.

Trichloroethylene (TCE) is among the most common water contaminant in the United States and around the world. It is estimated that between 9% and 34% of all drinking water sources contain some TCE. The EPA set a drinking water standard for TCE at 5 parts per billion (ppb) in 1989, however since this date, many studies have shown TCE is dangerous to the health of adults and unborn children, even at low-level exposures. These studies reveal exposure to TCE can cause multi-organ damage, especially for the kidney, liver, reproductive and development systems. We investigated how TCE can effect embryonic heart development by identifing possible target mechanisms changing after exposure. Acute and chronic exposure to rat cardiomyocytes produced altered calcium flow and significant changes with TCE doses as low as 10ppb. Embryonic carcinoma cells, rat cardiomyocytes and fetal heart tissue all showed global changes in gene expression after low-dose TCE exposure, including critical ion channels that drive calcium flux. High levels of folic acid supplementation in combination with 10ppb TCE exposure in maternal diets caused significant genetic modifications in mRNA expression levels of Day 10 embryonic mouse cardiac tissue. We also found both high and low folate maternal diets leads to similar phenotypic outcomes in embryo development.

The Sarco-endoplasmic Ca2+ ATPase (SERCA2a) plays a crucial role in sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR/ER) and is an important regulator of muscle contraction and relaxation. Recent findings suggest that a novel CAMKIIα splice variant, αKAP, that plays the role of a CAMKII anchoring protein in the myocardium, also directly interacts with SERCA2a. We examined the effects of αKAP on SERCA2a activity using transfection of HEK-293T cells as well as lentiviral infection of primary neonatal mouse cardiomyocytes (NMCM). Our data showed that αKAP reduced Ca2+ ATPase activity, and downregulated SERCA2a expression in both HEK-293T cells coexpressing αKAP and SERCA2a, as well as NMCM overexpressing αKAP. Interestingly in a rat model of myocardial infarction, αKAP expression was found to be elevated, alongside elevated CaMKIIδ, and depressed SERCA2a expression. These data suggest that αKAP may be a unique regulator of SERCA2a activity and cardiac function.

The Sarco-endoplasmic Ca2+ ATPase (SERCA2a) plays a crucial role in sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR/ER) and is an important regulator of muscle contraction and relaxation. Recent findings suggest that a novel CAMKIIα splice variant, αKAP, that plays the role of a CAMKII anchoring protein in the myocardium, also directly interacts with SERCA2a. We examined the effects of αKAP on SERCA2a activity using transfection of HEK-293T cells as well as lentiviral infection of primary neonatal mouse cardiomyocytes (NMCM). Our data showed that αKAP reduced Ca2+ ATPase activity, and downregulated SERCA2a expression in both HEK-293T cells coexpressing αKAP and SERCA2a, as well as NMCM overexpressing αKAP. Interestingly in a rat model of myocardial infarction, αKAP expression was found to be elevated, alongside elevated CaMKIIδ, and depressed SERCA2a expression. These data suggest that αKAP may be a unique regulator of SERCA2a activity and cardiac function.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

Integrin-linked kinase (ILK) is an important mechanoreceptor that mediates many cellular signaling pathways. Its dysregulation causes dilated cardiomyopathy and other complications in the heart. Restoration of ILK improves cardiac function and survival, but the exact mechanism is unknown. Recent studies in our lab suggest that the cardioprotective properties of ILK may be related to its regulation of sarco/endoplasmic reticulum calcium ATPase (SERCA2a). The protein expressions of ILK and SERCA2a are positively correlated based on adenoviral transduction of ILK and siRNA targeting ILK in human induced pluripotent stem cell-derived cardiomyocytes. From analysis of their calcium transients, ILK transduction resulted in increased beat rate and faster calcium clearance while siRNA knockdown produced the opposite effect. The use of SERCA-specific inhibitor thapsigargin nullified the observed effects of ILK transduction. Based on these results, we conclude that ILK’s cardioprotective properties are partly related to improving calcium handling in cardiomyocytes through the regulation of SERCA2a.