title: Mechanisms &amp; Properties of Exercise-Induced Cardioprotection
Creators: name: Budiono, Boris
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Cardioprotection
subject: Inactivity physiology
subject: Myocardial infarction

description: Regular physical activity, or exercise, is beneficial in the maintenance of health and wellbeing of the human body. With respect to the cardiovascular system, the potential benefits of physical activity in the prevention, reduction of frequency and severity, and enhanced rehabilitation, of heart disease are clear. However, many questions remain regarding factors such as the mode, duration, and intensity of physical activity and the cardiac impacts of these variables. Moreover, underlying mechanisms remain to be detailed. The current narrative from health organisations and governments worldwide has been the mantra of ‘exercise is medicine’, with a two-pronged approach: to enable the population to meet the minimum standards of daily physical activity, and to limit the amount of extended sedentariness. What is clear is that very few meet these standards, and heart disease, along with other associated chronic diseases attributed to a lack of activity and suboptimal diet, remain top of the list for all-cause mortality and morbidity. The issues pertaining to this problem are as broad as the benefits of exercise itself, and they include behavioral and societal issues beyond the scope of this thesis. The experiments in this thesis aim to specifically investigate the molecular underpinnings of the cardiac and systemic changes that occur in the exercised body. With this knowledge, it is possible to proceed further with potential solutions, such as: personalised exercise protocols for a specific health outcome, determination of short-term, long-term, and lifelong effects of activity or inactivity, and development of agents that may mimic or promote some of the benefits of exercise, especially for those unable to undertake physical exertion. The first experimental studies in this thesis investigated whether short-term voluntary wheelrunning in 8-week old mice beneficially modulates myocardial ischemic tolerance, signaling kinases, and gene expression patterns (Chapter 3). Transcriptome analysis can provide insight into molecular mechanisms. Prior animal studies have utilised forced activity measures, such as treadmill running, expecting increases in heat shock protein or any other canonical stress responses. However, we employed voluntary wheel-running as a ‘low-stress’ method to assess responses to ischaemia-reperfusion insult. Phenotypic results showed that 1 week of wheelrunning improved left ventricular developed pressure recovery from 25 min ischaemia/45 min reperfusion (by 47%) and reduced diastolic dysfunction (by 30%). Analysis of known pro-survival proteins showed limited changes, with only a 30% increase in cytosolic ERK1/2, whilst there were no differences in total Akt, GSK3β and phospho-Akt, -GSK3β and -ERK1/2. Microarray interrogation identified significant changes (!1.3 fold expression change, "5% FDR) in 142 known genes, the majority of which (92%) were repressed. Significantly modified pathways/networks related to inflammatory/immune function (particularly interferon-dependent), together with cell movement, growth, and death signalling. Of only 14 induced transcripts, 3 encoded interrelated sarcomeric proteins titin, #-actinin, and myomesin-2. The next series of studies were designed to investigate the sensitivity of cardiac ischaemictolerance to both activity and inactivity. Studies assessed cardiac effects of voluntary activity (14 days) in running-naïve mice (Active), 7 days of subsequent inactivity (Inactive), and brief (3 day) restoration of running (Re-Active); and tested whether 'cardiac:activity coupling' reflects common modulation of pro-survival (AKT, AMPK, ERK1/2, HSP27, EGFR) and -injury (GSK3$) proteins implicated in ischaemic preconditioning/calorie restriction responses (Chapter 4). Active mice increased running speed and distance by 75-150% over 14 days and their hearts were significantly resistant to post-ischemic dysfunction following 25 min ischaemia (40-50% improvements in functional outcomes). This protection was accompanied by ~2-fold elevations in AKT, AMPK, phosphorylation, and EGFR expression. Ischaemic tolerance was reversed in Inactive hearts, and ERK1/2 phosphorylation (with AKT, AMPK, HSP27 phosphorylation unaltered). Running characteristics, cardioprotection, EGFR expression and GSK3$ all returned to Active levels within 1-3 days of restored activity (with no significant changes in AKT, AMPK, HSP27 phosphorylation). These data reveal sensitive coupling of myocardial stress-resistance to activity level, and show that only initial activity in running-naïve animals induces a molecular profile characteristic of and EGFR modulation more consistently parallel both activity- and inactivity-dependent shifts in myocardial stress resistance. The findings also suggest a particular importance of recent activity level in governing ischaemic tolerance of the heart. After initial identification and characterisation of a cardioprotective phenotype of voluntary wheelrunning, and analysis of responses to activity/inactivity transitions, a time-course analysis was undertaken to determine whether dose-dependence existed in terms of wheel running, cardiac protection and expression of survival and stress kinases. Interestingly, studying the effects at 2-, 7- , 14-, and 28-days of running revealed maximal protection with only 7 days of VWR, and no further improvements with longer running periods. Improvements in ischaemic tolerance were associated with increased cardiac expression of phospho-AKT, phospho-GSK3$, phospho-ERK and to a lesser degree, phospho-AMPK. Running also differentially modified gene transcription, with potentially beneficial up-regulation of determinants of contractile function (Ttn), caveolar signalling (Cav3) and mitochondrial biogenesis (Pgc1a), vs. repression of injurious/degradative Mmp2 (and mitochondrial ketone metabolism - Bdh1). Intriguingly, I-R tolerance was also improved in both 2EX and 2SED groups (i.e. independent of running) vs. mice subjected to longer sedentary periods, suggesting a cardioprotective effect of environment enrichment independent of running activity. Given an absence of impact of running duration on the cardioprotected phenotype, final studies were designed to test effects of differing periods of a more intense (potentially stressful) forced swimming protocol (Chapter 6). Swimming is a well-known inducer of physiological growth/hypertrophy, which itself may promote greater resistance to ischemia-reperfusion injury. After an initial acclimation period, mice were assessed after 1 and 2 weeks of intensive swim training and shown to exhibit modest cardiac hypertrophy (15% and 22% increases in heart:body weight, respectively). Improved contractility and improved functional outcomes from I-R insult was observed at 2 wks, but not 1 wk of swimming, contrasting voluntary running. Molecular analyses in the 2 wk group revealed a reduction in NFkB expression without changes in prosurvival kinase or caveolin-3 expression. Exploratory proteome arrays support differential changes in growth factor signalling, and beneficial shifts in mediators of inflammation and remodelling in other tissues (pancreas, white fat, thymus, lymph nodes, brain), involving a pattern of RANTES, ICAM1 and MMP9 repression vs. LIF and VEGF induction across most tissues. It is likely these cardiac and systemic shifts in growth factor and inflammatory signalling participate in beneficial cardiovascular impacts of exercise. Collectively, the studies within this thesis reveal close coupling between physical activity level (both increased or decreased) and myocardial ischemic tolerance. While many potentially beneficial adaptations in cardiac protein expression and signalling arise, only a handful are consistently linked to cardiac ischemic tolerance including GSK3$ phospho-regulation and expression of the EGFR receptor. Signalling via GSK3$ has been identified as a potentially valuable target, while these are the first data to support a role for altered EGFR signalling in coupling cardiac stress phenotype to physical activity level. Data are also incompatible with the view that exercise represents a physiologic corollary of ischemic preconditioning or calorie restriction responses (except perhaps in exercise naive subjects, in which this physiologic stimulus may present a greater 'stress'). Finally, the studies also reveal a diversity of beneficial changes in inflammatory, RTK and growth factor signalling in the heart and systemically, highlighting the integrative nature of the response to exercise. Unravelling these interactions is a challenge, yet may provide insights not only into normal physiology but management of heart disease and its consequences.