Browsing by Author "Tune, Johnathan"

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    Mitochondrial protein acetylation and left ventricular function in a model of hypertrophic cardiomyopathy and heart failure
    (2017-05-23) Stram, Amanda R.; Payne, R. Mark; Brustovetsky, Nickolay; Herring, B. Paul; Tune, Johnathan
    Rationale: The childhood heart disease of Friedreich’s Ataxia (FRDA) is characterized by hypertrophy and failure. It is caused by loss of frataxin (FXN), a mitochondrial protein involved in energy homeostasis. FRDA model hearts have increased mitochondrial protein acetylation and impaired sirtuin 3 (SIRT3) deacetylase activity. Protein acetylation is an important regulator of cardiac metabolism and SIRT3 is protective in heart disease. The underlying pathophysiology of heart failure in FRDA is unclear. I suspect that increased acetylation in FRDA heart mitochondria damages cardiac energy homeostasis by inhibiting activity of key enzymes involved in heart metabolism. Objective: My project tested the hypothesis that altered acetylation of mitochondrial proteins contributes to the cardiomyopathy of FRDA. Methods: Conditional mouse models of FRDA cardiomyopathy with ablation of FXN (FXN KO) or FXN and SIRT3 (FXN/SIRT3 DKO) in the heart were compared to healthy controls. Hearts were evaluated using echocardiography, cardiac catheterization, histology, protein acetylation and expression. FXN KO mice were treated with NAD+ replacement therapy with nicotinamide riboside (NR), and FXN/SIRT3 DKO mice were treated with FXN protein replacement therapy. Results: Acetylation was temporally progressive and paralleled evolution of heart failure in the FXN KO model. High levels of acetylation were associated with cardiac fibrosis, mitochondrial damage, impaired fat metabolism, and diastolic and systolic dysfunction. Acetylation correlated strongly with worse heart function, and loss of SIRT3 in the FXN KO mouse resulted in significant decrease in ejection fraction and fractional shortening. Treatment of the FXN/SIRT3 DKO with FXN protein therapy reduced acetylation but was not sufficient to fully rescue heart function. Increasing NAD+ with NR-treatment in the FXN KO lead to increased mitochondrial protein acetylation and did not improve cardiac outcome. Conclusion: I found a strong negative correlation between heart function and mitochondrial protein acetylation. My findings also provide evidence that absence of SIRT3 expression in the FXN KO heart exacerbates features of heart failure, and that SIRT3 expression is necessary to rescue the FXN KO heart. These results suggest that SIRT3 inactivation and abnormal acetylation contribute to the pathophysiology of heart disease in FRDA.
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    Obesity alters global response to ischemia and GLP-1 agonism
    (2016-05-13) Sassoon, Daniel Jay; Tune, Johnathan; Mather, Kieren
    Glucagon-like peptide 1 (GLP-1) receptor agonists are a class of incretin based therapeutics which aid in blood glucose management in Type II diabetes mellitus (T2DM). Recent studies have demonstrated direct cardiovascular benefits conferred by these agents including protection in ischemia and heart failure. Despite these observations, human clinical trials fail to support improvements in cardiovascular outcomes independent of glucose lowering effects in the T2DM populations. Prior data from our lab demonstrate that obesity impairs GLP-1 associated increases in myocardial glucose uptake. However, the reasons for this impairment/resistance to cardiac effects of GLP-1 in the setting of obesity remain ill defined. This investigation tested the hypothesis that underlying differences in the cardiac proteome and microRNA (miR) transcriptome could contribute to distinct cardiac responses to ischemia and activation of GLP-1 signaling in the setting of obesity. To identify whether obesity modulated cardiac functional responses to GLP 1 related drugs, we first examined the effects of obesity on cardiac function, miR transcriptome, and proteome in response to short duration ischemia-reperfusion (I/R). We observed divergent physiologic responses (e.g. increased diastolic volume and systolic pressure in lean, decreased diastolic volumes in obese) to regional I/R in obese vs lean hearts that were associated with significant molecular changes as detected by protein mass spectrometry and miR microarray. Molecular changes were related to myocardial calcium handling (SERCA2a, histidine-rich Ca2+ binding protein), myocardial structure and function (titin), and miRs relating to cardiac metabolism, hypertrophy, and cell death, including miR-15, miR-30, miR-199a, miR-214. Importantly, these effects were modified differently by GLP-1 agonism in lean vs obese swine. Additional studies investigated the functional effects of 30 days of treatment with the GLP-1 analogue liraglutide on a model of slowly-developing, unrelieved coronary ischemia. Liraglutide failed to reduce infarct size or collagen deposition. However, analysis of left ventricular pressure-volume relationships support that liraglutide improved diastolic relaxation/filling, load-dependent indices of cardiac function, and cardiac efficiency in response to sympathetic stimulation in obese swine. Taken together, these findings support that miR and proteomic differences underlie distinct changes in functional cardiac responses to I/R and pharmacologic activation of GLP-1 signaling in the setting of obesity.
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    Regulation of endoplasmic reticulum calcium homeostasis in pancreatic β cells
    (2016-06-21) Tong, Xin; Evans-Molina, Carmella; Day, Richard; Tune, Johnathan; Fueger, Patrick T.; Dong, X. Charlie
    Diabetes mellitus is a group of metabolic diseases characterized by disordered insulin secretion from the pancreatic β cell and chronic hyperglycemia. In order to maintain adequate levels of insulin secretion, the β cell relies on a highly developed and active endoplasmic reticulum (ER). Calcium localized in this compartment serves as a cofactor for key proteins and enzymes involved in insulin production and maturation and is critical for ER health and function. The ER Ca2+ pool is maintained largely through activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) pump, which pumps two Ca2+ ions into the ER during each catalytic cycle. The goal of our research is to understand the molecular mechanisms through which SERCA2 maintains β cell function and whole body glucose metabolism. Our previous work has revealed marked dysregulation of β cell SERCA2 expression and activity under diabetic conditions. Using a mixture of pro-inflammatory cytokines to model the diabetic milieu, we found that SERCA2 activity and protein stability were decreased through nitric oxide and AMP-activated protein kinase (AMPK)mediated signaling pathways. Moreover, SERCA2 expression, intracellular Ca2+ storage, and β cell death under diabetic conditions were rescued by pharmacologic or genetic inhibition of AMPK. These findings provided novel insight into pathways leading to altered β cell Ca2+ homeostasis and reduced β cell survival in diabetes. To next define the role of SERCA2 in the regulation of whole body glucose homeostasis, SERCA2 heterozygous mice (S2HET) were challenged with high fat diet (HFD). Compare to wild-type controls, S2HET mice had lower serum insulin and significantly reduced glucose tolerance with similar adiposity and systemic and tissue specific insulin sensitivity, suggesting an impairment in insulin secretion rather than insulin action. Consistent with this, S2HET mice exhibited reduced β cell mass, decreased β cell proliferation, increased ER stress, and impaired insulin production and processing. Furthermore, S2HET islets displayed impaired cytosolic Ca2+ oscillations and reduced glucose-stimulated insulin secretion, while a small molecule SERCA2 activator was able to rescue these defects. In aggregate, these data suggest a critical role for SERCA2 and the maintenance of ER Ca2+ stores in the β cell compensatory response to diet induced obesity.
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    Small-Group Activity to Reinforce the Impact of Valvular Defects and Heart Failure on Cardiac Pressure-Volume Relationships
    (Association of American Medical Colleges, 2018-02-06) Hopper, Mari; Tune, Johnathan; Klabunde, Richard; Cellular and Integrative Physiology, School of Medicine
    Introduction: An important topic in cardiac physiology is the relationship between changes in intracardiac pressures and volumes during the cardiac cycle. This topic lends itself well to utilizing active learning principles to facilitate student understanding of pressure and volume changes in normal cardiac physiology and in the pathophysiology of valve disease and heart failure. We describe an active learning exercise regarding this topic that engages and facilitates student learning in a small-group setting. Methods: Following an overview lecture on the normal cardiac physiology, small groups of students under the guidance of a facilitator were provided with a worksheet consisting of questions related to background knowledge of cardiac physiology. Additional questions related to five valve disease and heart failure cases were also provided to promote the application of basic physiology principles to clinically relevant problems. The facilitator was provided with a guide to help facilitate the student interactions. Following the group worksheet activity, an animated slide presentation was shown to further engage student learning through active discussion of their worksheet answers. Results: Students were assessed by written examination, and were found to have a higher performance on the subset of questions related to this learning activity compared to the overall exam. Of the 175 students completing the exercise, 23 voluntarily provided feedback via a survey. Student surveys provided overwhelmingly positive feedback on the benefits of this active learning exercise. Discussion: Small group, active learning exercises benefited student learning by providing a framework for analysis, synthesis, and application of clinically relevant cardiac physiology concepts.
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    Stress-activated MIG6 compromises hepatic metabolism during diet-induced obesity
    (2016-07-25) Lutkewitte, Andrew John; Fueger, Patrick T.; Considine, Robert; Evans-Molina, Carmella; Tune, Johnathan; Elmendorf, Jeffrey
    Obesity-induced hepatic fat accumulation or nonalcoholic fatty liver disease (NAFLD) is the leading cause of liver disease in the United States. Unfortunately, NALFD patients are at higher risk of cardiovascular disease and mortality. The development of hepatic steatosis is multi-factorial and leads to a variety of pathologies. Yet, the molecular mechanisms behind liver disease during hepatic fat accumulation remain unclear. Here, we describe novel mechanisms of impaired liver function in the context of obesity-induced hepatic stress. Using chemical- and fatty acid-induced endoplasmic reticulum (ER) stress, we discovered ER stress decreases the activation of the pro-growth, pro survival, receptor tyrosine kinase, epidermal growth factor receptor (EGFR) in vitro. Importantly, EGFR was inhibited during these stress conditions by the induction and stabilization of mitogen inducible gene 6 (Mig6). Furthermore, Mig6 knockdown in vitro enhanced EGFR signaling and promoted survival. We demonstrated that mice fed a high fat diet have decreased EGFR activation and increased Mig6 protein expression, likely due to obesity-induced ER stress. To determine the functional consequences of increased Mig6 expression, we generated Mig6 liver-specific knockout mice (Mig6 LKO) and subjected them to high fat feeding. During diet-induced obesity, Mig6 LKO mice had improved hepatic glucose tolerance despite no improvements in whole-body insulin sensitivity or insulin secretion. Hepatic insulin signaling, measured by AKT activation, was similar between Mig6 LKO and littermate controls. However, several insulin-sensitive genes involved in gluconeogenesis were altered in Mig6 LKO mice compared to controls. In addition, Mig6 LKO mice had higher plasma high density lipoproteins and triglycerides despite similar liver lipid content. Using RNA sequencing we discovered Mig6 regulates several metabolic pathways in liver. These findings indicated Mig6 not only controls hepatic growth and survival but also regulates metabolism. This work will help us to better understand how augmented growth factor signaling impacts metabolic regulation during pathological obesity.
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    Understanding the Integrated Pathophysiological Role of a Moonlighting Protein in Lung Development
    (2019-08) Lee, Dong Il; Schwarz, Margaret; Tune, Johnathan; Kaplan, Mark; Basile, David
    Sensing, integrating, and relaying signals from the environment through proteins, metabolites, and lipids to the lung are critical for proper development. Moonlighting proteins, such as AIMP1, are a unique subset that serves at least two independent physiological functions. Encoded by gene AIMP1, AIMP1 has two known functions: (1) C-terminus EMAP II domain of full-length AIMP1 can be secreted out of the cell to chemoattract myeloid cells; (2) intracellular full-length protein interacts with tRNA synthetases in protein translation. However, despite the linkage of protein expression levels of with several lung pathologies such as bronchopulmonary dysplasia (BPD), effectively targeting the protein encoded by AIMP1 has been a challenge due to poorly understood mechanisms. This thesis explores physiological, signaling, and immunological moonlighting mechanisms of first, the extracellular EMAP II then the intracellular AIMP1. Experiments utilize both in vitro and in vivo models, including a murine model of BPD and Cre-mediated exon-deletion knockout. Experimental results provide evidence that in the BPD model, EMAP II levels are elevated and sustained – first in bronchial epithelial cells then in macrophages. Mice exposed to sustained and elevated EMAP II protein levels resemble the BPD phenotype while neutralization partially rescued the phenotype, implying EMAP II as a potential therapeutic target against BPD. Results from studies exploring EMAP II’s signaling mechanism identify transient stimulation of JAK-STAT3 phosphorylation, commonly found in inflammation-resolving macrophages. In contrast, it induces unique transcriptional changes that are reversible both by JAK-STAT inhibitor and siRNA-mediated knockdown of Stat3. Studies using AIMP1 knockout mouse reveal a novel function for the intracellular AIMP1. AIMP1 knockout mice exhibited neonatal lethality with a respiratory distress phenotype, decreased type I alveolar cell expression, and disorganized bronchial epithelium, suggesting a role in lung maturation. In vitro experiments suggest that a portion of AIMP1 residing in the cell’s membrane interacts with various phosphatidylinositols and contributes toward F-actin deposition and assembly. Data from these experimental studies provide insight into how the various functions of the promiscuous AIMP1 gene affect lung development. These studies exemplify not only characterize novel moonlighting mechanisms of AIMP1, but also highlight the importance of characterizing moonlighting proteins to promote therapeutic preventions.