MECHANISMS UNDERLYING CARDIOVASCULAR BENEFITS OF SODIUM GLUCOSE CO-TRANSPORTER-2 INHIBITORS: MYOCARDIAL SUBSTRATE OR SODIUM/HYDROGEN EXCHANGER? Hana Elisabeth Baker Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Cellular and Integrative Physiology, Indiana University January 2020 Accepted by the Graduate Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Doctoral Committee ______________________________________ Johnathan D. Tune, Ph.D., Chair ______________________________________ David Basile, PhD December 2, 2019 ______________________________________ Adam Goodwill, PhD ______________________________________ Mark Kowala, PhD ______________________________________ Kieren Mather, MD ______________________________________ Mervyn (Dod) Michael, PhD ii © 2020 Hana Elisabeth Baker iii DEDICATION This work is dedicated to my husband, Mike, and our daughters, Audrey and Margret. Without their love, help, patience, and encouragement, this would have never been possible. iv ACKNOWLEDGEMENT The author would like to express her sincere gratitude to her advisor, Dr. Johnathan Tune, for the valuable discussions, encouragement, advice, guidance, and the freedom to set the directions of her research. Also, the author is grateful for the guidance provided by her thesis committee members including Drs. David Basile, Adam Goodwill, Mark Kowala, Kieren Mather and Dod Michael. Additionally, the author would like to thank her collaborator at the University of Alberta, Dr. Larry Fliegel for his help with NHE-1 activity assay in wild type NHE-1 transfected AP-1 cells and her collaborators at Eli Lilly and Company, Ajit Regmi and Dr. William Roell for their help with mRNA expression of SGLT-1 vs. SGLT-2 and to Fang Li for her help with the NHE-1 activity assay in iCell™ human IPSc-derived cardiomyocytes. v Hana Elisabeth Baker MECHANISMS UNDERLYING CARDIOVASCULAR BENEFITS OF SODIUM GLUCOSE CO-TRANSPORTER-2 INHIBITORS: MYOCARDIAL SUBSTRATE OR SODIUM/HYDROGEN EXCHANGER? Recent clinical outcome studies demonstrate that Sodium glucose cotransporter 2 inhibitors (SGLT2i) significantly reduce major adverse cardiovascular events and heart failure outcomes in subjects with type 2 diabetes mellitus. At present, several hypotheses have been proposed to explain the observed cardiovascular benefit of SGLT2i, however, the mechanisms responsible remain to be elucidated. This investigation tested the hypothesis that SGLT2i improves cardiac function and efficiency during acute, regional ischemia/reperfusion injury via preferential shifts in myocardial substrate selection and/or inhibition of cardiac sodium/hydrogen exchanger-1 (NHE-1). Our initial investigation evaluated the effects of 24 hour pretreatment of the SGLT2i canagliflozin on cardiac contractile function, substrate utilization, and efficiency before and during regional myocardial ischemia/reperfusion injury in healthy swine. At the onset of ischemia, canagliflozin increased left ventricular end diastolic and systolic volumes which returned to baseline with reperfusion. This increased end diastolic volume was directly associated with increased stroke volume and stroke work relative to controls during ischemia. Canagliflozin also increased cardiac work efficiency during ischemia relative to control swine. No differences in myocardial substrate uptake of glucose, lactate, fatty acids or ketones were detected between groups. In separate experiments using a longer 60 min coronary occlusion, canagliflozin significantly diminished myocardial infarct size. Subsequent studies investigated the effect of an acute administration (15-30 min pre-treatment) of canagliflozin and the NHE-1i cariporide on cardiac contractile function vi efficiency in response to myocardial ischemia/reperfusion injury. Similar to our initial studies, canagliflozin increased diastolic filling, stroke work and improved cardiac work efficiency relative to untreated control hearts during the ischemic period. In contrast, cariporide did not alter ventricular filling volume, cardiac output or work efficiency at any time point. Additional examination of AP-1 cells transfected with wild-type NHE-1 showed dose-dependent inhibition of NHE-1 activity by cariporide, while canagliflozin had minimal effect on overall activity. This investigation demonstrates that SGLT2i improves cardiac function and efficiency during acute, regional ischemia in healthy swine. However, the present data fail to support the hypothesis that these SGLT2i-mediated improvements involve either preferential alterations in myocardial substrate utilization or the inhibition of NHE-1 activity. Johnathan Tune, PhD, Chair vii TABLE OF CONTENTS List of Tables .................................................................................................................. xi List of Figures ................................................................................................................ xii List of Abbreviations ..................................................................................................... xvi Chapter 1: Introduction ................................................................................................... 1 Population at risk for cardiovascular disease ............................................................. 1 Cardiovascular disease in obesity and diabetes ........................................................ 3 Anti-hyperglycemic therapies and cardiovascular risk ................................................ 5 Sodium glucose Transporter 2 ................................................................................... 9 SGLT2i and cardiovascular events .......................................................................... 11 SGLT2 expression ................................................................................................... 13 Proposed mechanisms of cardiovascular protection ................................................ 14 Thrifty fuel hypothesis .............................................................................................. 23 Sodium hypothesis .................................................................................................. 25 Summary and proposed experimental aims ............................................................. 28 Chapter 2: Inhibition of Sodium Glucose Cotransporter-2 Preserves Cardiac Function During Regional Myocardial Ischemia Independent of Alterations in Myocardial Substrate Utilization..................................................................................... 32 Introduction .............................................................................................................. 32 Methods ................................................................................................................... 33 Animal model and surgical preparation .............................................................. 33 Left Circumflex Ischemia Protocol ...................................................................... 34 Metabolic Analysis ............................................................................................. 35 Infarct protocol ................................................................................................... 36 Infarct Quantification .......................................................................................... 36 RNA Isolation and cDNA synthesis .................................................................... 37 viii RT PCR ............................................................................................................. 37 Statistical Analyses ............................................................................................ 38 Results .................................................................................................................... 39 mRNA expression of SGLT-1 vs. SGLT-2 in swine heart and kidney ................. 39 Systemic effects of SGLT-2i during ischemia/reperfusion injury of the LCX ....... 39 Effects of SGLT2i on cardiac contractile function during ischemia/reperfusion injury of the LCX ................................................................................................ 40 Effects of SGLT2i on myocardial substrate selection during ischemia/reperfusion injury of the LCX ............................................................... 43 Effect of SGLT2i on myocardial infarct size and cardiac function in response to 60-minute LAD occlusion ............................................................................... 44 Discussion ............................................................................................................... 46 Cardiac effects of SGLT2i .................................................................................. 46 Potential mechanisms underlying cardiac effects of SGLT2i .............................. 48 Conclusion ............................................................................................................... 50 Chapter 3: Inhibition of Sodium Glucose Cotransporter-2 Improves Cardiac Efficiency During Regional Myocardial Ischemia Independent of Sodium/Hydrogen Exchanger-1 ............................................................................................................... 55 Introduction .............................................................................................................