Short-term rapamycin persistently improves cardiac function after cessation of treatment in aged male and female mice. Ellen Quarles A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2017 Reading Committee: Peter Rabinovitch, Chair Michael MacCoss David Marcinek Program Authorized to Offer Degree: Pathology © Copyright 2017 Ellen Quarles University of Washington Abstract Short-term rapamycin persistently improves cardiac function after cessation of treatment in aged male and female mice. Ellen Quarles Chair of the Supervisory Committee: Peter Rabinovitch, Professor and Vice Chair of Research Department of Pathology Cardiac aging is an intrinsic process that results in impaired cardiac function and dysregulation of cellular and molecular quality control mechanisms. These effects are evident in the decline of diastolic function, increase in left ventricular hypertrophy, metabolic substrate shifts, and alterations to the cardiac proteome. This thesis covers the quality control mechanisms that are associated with cardiac aging, results from an anti-aging intervention in aged mice, and a review of mitochondrial dysfunction in the heart. Chapter one is a review of the quality control mechanisms in aging myocardium. Chapter two consists of the results of several mouse experiments that compare the cardiac function, proteomes, and metabolomes of aged and young controls, along with rapamycin treated aged mice. The novelty of this study comes from the inclusion of a group of animals treated only transiently with the drug, then followed for eight weeks post-drug-removal. This persistence cohort may hold clues to deriving long-lasting benefits of rapamycin with only transient treatment. Chapter three includes more results from the cohorts used in chapter two, from work done by our collaborators in two laboratories at the University of Washington. Finally, chapter four is a review of the mechanisms and phenotypes of mitochondrial dysfunction in the aging heart. The goal of my thesis work is to test the persistence of the improvement of cardiac function by rapamycin treatment, and use the correlating changes in the cardiac proteome and metabolome to discover a novel mechanism of functional improvement of the heart in aged animals. TABLE OF CONTENTS List of Figures ................................................................................................................................. v List of Tables ................................................................................................................................. vi Chapter 1. Introduction – Quality control systems in cardiac aging............................................... 1 1.1 Abstract ........................................................................................................................... 1 1.2 Introduction ..................................................................................................................... 1 1.3 Overview of cardiac aging .............................................................................................. 1 1.3.1 Human cardiac aging ..................................................................................................... 1 1.3.2 Large mammal models of cardiac aging ........................................................................ 3 1.3.3 Rodent models of cardiac aging ..................................................................................... 3 1.4 Mechanisms of cardiac aging.......................................................................................... 4 1.4.1 Metabolic changes in cardiac aging ............................................................................... 4 1.4.2 Age-related contractility changes................................................................................... 5 1.5 Quality control mechanisms in cardiac aging ................................................................. 7 1.5.1 mTOR Signaling in regulation of protein homeostasis .................................................. 7 1.5.2 mTORC1 ........................................................................................................................ 7 1.5.3 Proteostasis .................................................................................................................... 8 1.5.4 Autophagy ...................................................................................................................... 9 1.5.5 Ubiquitin-mediated turnover ........................................................................................ 11 1.5.6 Apoptosis ..................................................................................................................... 12 1.5.7 Mitochondrial quality control ...................................................................................... 12 i 1.5.8 Mitochondria fusion and fission .................................................................................. 14 1.5.9 Cardiac matrix homeostasis: matrix metalloproteases ................................................. 15 1.6 Cardiac aging interventions .......................................................................................... 16 1.6.1 Caloric/dietary restriction and mimetics ...................................................................... 16 1.6.2 Mitochondrial antioxidants .......................................................................................... 19 1.6.3 Cardiolipin-targeted therapeutics ................................................................................. 19 1.7 Conclusion .................................................................................................................... 20 Chapter 2. Short term rapamycin persistently improves cardiac function after cessation of treatment in aged male and female mice. ...................................................................................... 21 2.1 Abstract ......................................................................................................................... 21 2.2 Introduction ................................................................................................................... 21 2.3 Results ........................................................................................................................... 22 2.3.1 Rapamycin persistently improves diastolic function. .................................................. 22 2.3.2 Rapamycin dramatically alters proteome abundances in both sexes, however the persistence of these changes varies by sex............................................................................ 25 2.3.3 Metabolome differences seen at 8 weeks of rapamycin treatment are mainly not persistent after a further eight weeks with or without the drug. ........................................... 28 2.3.4 Passive stiffness of the left ventricle ............................................................................ 32 2.3.5 Sarcomeric protein phosphorylation may contribute to reduced passive stiffness and improved diastolic function after rapamycin. ....................................................................... 33 2.3.6 Rapamycin effects on age-related fibrosis of the myocardium.................................... 35 2.3.7 Rapamycin differentially alters respiratory chain complex activity by sex. ................ 36 ii 2.3.8 Do markers of cellular senescence change after rapamycin treatment and if so, are they persistent? ............................................................................................................................. 39 2.4 Discussion ..................................................................................................................... 39 2.4.1 Echocardiography ........................................................................................................ 40 2.4.2 Proteomics and ETC activity ....................................................................................... 40 2.4.3 Metabolomics ............................................................................................................... 41 2.4.4 Senescence ................................................................................................................... 42 2.5 Conclusion .................................................................................................................... 42 2.6 Materials and Methods .................................................................................................. 42 Chapter 3. Additional observations from the rapamycin-persistence animal cohorts .................. 47 3.1 Abstract ......................................................................................................................... 47 3.2 Rapamycin treatment reverses alveolar bone loss in aged mice ................................... 47 3.3 Rapamycin reduces occurrence of tissue lesions in aged mice ..................................... 49 Chapter 4. Mitochondrial dysfunction in cardiac aging................................................................ 51 4.1 Abstract ......................................................................................................................... 51 4.2 Introduction ................................................................................................................... 51 4.3 Mitochondrial energetics in cardiac aging ...................................................................