PGC-1α regulates cardiac mitochondrial biogenesis and energy metabolic gene expression, thus transcriptional regulation of PGC-1α gene expression is of great importance in understanding metabolic gene expression in cardiac health and disease. We provide evidence that estrogen related receptor α (ERRα, which also plays a role in cardiac energy metabolism, regulates expression of the PGC-1α gene via direct interaction with the PGC-1α gene promoter. In the presence of an inverse agonist to ERRα PGC-1α gene expression was significantly decreased, while over-expression of ERRα increased PGC-1α gene expression. We have also demonstrated that expression of PGC-1α was down regulated in hypoxic cardiomyocytes due to histone deacetylation. Our data identify ERRα as a novel regulator of cardiac PGC-1α gene expression, and suggests that promoter deacetylation in hypoxia plays a role in reduced PGC-1α expression. These results reveal a new mechanism that may contribute to energetic derangement in the heart during ischemia and/or failure.

PGC-1α regulates cardiac mitochondrial biogenesis and energy metabolic gene expression, thus transcriptional regulation of PGC-1α gene expression is of great importance in understanding metabolic gene expression in cardiac health and disease. We provide evidence that estrogen related receptor α (ERRα, which also plays a role in cardiac energy metabolism, regulates expression of the PGC-1α gene via direct interaction with the PGC-1α gene promoter. In the presence of an inverse agonist to ERRα PGC-1α gene expression was significantly decreased, while over-expression of ERRα increased PGC-1α gene expression. We have also demonstrated that expression of PGC-1α was down regulated in hypoxic cardiomyocytes due to histone deacetylation. Our data identify ERRα as a novel regulator of cardiac PGC-1α gene expression, and suggests that promoter deacetylation in hypoxia plays a role in reduced PGC-1α expression. These results reveal a new mechanism that may contribute to energetic derangement in the heart during ischemia and/or failure.

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. Zebrafish offer a number of advantages as a model in longitudinal toxicology studies and are quite sensitive physiologically and behaviorally to cocaine. In this study, we have used zebrafish to model the effects of embryonic pre-exposure to cocaine on development and on subsequent cardiovascular physiology and cocaine-induced conditioned place preference (CPP) in longitudinal adults. Larval fish showed a progressive decrease in telencephalic size with increased doses of cocaine. These treated larvae also showed a dose dependent response in heart rate that persisted 24 h after drug cessation. Embryonic cocaine exposure had little effect on overall health of longitudinal adults, but subtle changes in cardiovascular physiology were seen including decreased sensitivity to isoproterenol and increased sensitivity to cocaine. These longitudinal adult fish also showed an embryonic dose-dependent change in CPP behavior, suggesting an increased sensitivity. These studies clearly show that pre-exposure during embryonic development affects subsequent cocaine sensitivity in longitudinal adults.

This study tested the hypotheses that 1) 28h head-down bed-rest (HDBR) would result in significant hypovolemia and cardiovascular deconditioning, and that 2) NASA’s fluid loading protocol (ingestion of 15 ml/kg water with a 1g NaCl for every 125ml of water consumed) would restore normovolemia and prevent cardiovascular deconditioning resulting from 28h HDBR. Nine healthy men were tested in 5 testing scenarios, with a progressive lower body negative pressure (LBNP) protocol performed before and after each scenario to measure the subjects’ cardiovascular responses to orthostasis. Subjects were tested in two 28h HDBR conditions, without fluid loading (NFL) and with fluid loading (FL), as well as in three 4-hour control conditions to isolate the effects of circadian rhythm, HDBR, and fluid loading. After 28h NFL HDBR, plasma volume was reduced by 8%. There were no symptoms of syncope during orthostatic testing following 28h NFL HDBR, however cardiovascular deconditioning was apparent as there were significant increases in heart rate, reductions in central venous pressure, and reductions in portal vein diameter during LBNP testing. There were no changes in stroke volume, cardiac output, systemic vasoconstriction, cardiac measures, and arterial and cardiopulmonary baroreflex responses, and no evidence of splanchnic or venous pooling. This study also found that NASA’s fluid loading protocol was ineffective at restoring normovolemia after 28h HDBR, as there were no differences in plasma volume between 28h FL HDBR post and 28h NFL HDBR post tests (p=0.22). Cardiovascular deconditioning was not prevented by fluid loading as the heart rate response remained elevated and central venous pressure remained reduced after 28h FL HDBR. In addition, four of the nine subjects experienced nausea during administration of the fluid loading protocol prescription and two subjects vomited, further evidence that NASA’s fluid loading protocol is not effective at preventing orthostatic hypotension. Investigation of control models verified that deconditioning was the result of HDBR. It was also concluded that circadian rhythm did not affect the measured cardiovascular responses and the fluid loading protocol was ineffective at increasing blood volume in the absence of HDBR.

This study tested the hypotheses that 1) 28h head-down bed-rest (HDBR) would result in significant hypovolemia and cardiovascular deconditioning, and that 2) NASA’s fluid loading protocol (ingestion of 15 ml/kg water with a 1g NaCl for every 125ml of water consumed) would restore normovolemia and prevent cardiovascular deconditioning resulting from 28h HDBR. Nine healthy men were tested in 5 testing scenarios, with a progressive lower body negative pressure (LBNP) protocol performed before and after each scenario to measure the subjects’ cardiovascular responses to orthostasis. Subjects were tested in two 28h HDBR conditions, without fluid loading (NFL) and with fluid loading (FL), as well as in three 4-hour control conditions to isolate the effects of circadian rhythm, HDBR, and fluid loading. After 28h NFL HDBR, plasma volume was reduced by 8%. There were no symptoms of syncope during orthostatic testing following 28h NFL HDBR, however cardiovascular deconditioning was apparent as there were significant increases in heart rate, reductions in central venous pressure, and reductions in portal vein diameter during LBNP testing. There were no changes in stroke volume, cardiac output, systemic vasoconstriction, cardiac measures, and arterial and cardiopulmonary baroreflex responses, and no evidence of splanchnic or venous pooling. This study also found that NASA’s fluid loading protocol was ineffective at restoring normovolemia after 28h HDBR, as there were no differences in plasma volume between 28h FL HDBR post and 28h NFL HDBR post tests (p=0.22). Cardiovascular deconditioning was not prevented by fluid loading as the heart rate response remained elevated and central venous pressure remained reduced after 28h FL HDBR. In addition, four of the nine subjects experienced nausea during administration of the fluid loading protocol prescription and two subjects vomited, further evidence that NASA’s fluid loading protocol is not effective at preventing orthostatic hypotension. Investigation of control models verified that deconditioning was the result of HDBR. It was also concluded that circadian rhythm did not affect the measured cardiovascular responses and the fluid loading protocol was ineffective at increasing blood volume in the absence of HDBR.

Photoplethysmography (PPG) is a simple means of measuring the pulse wave in humans, exploitable for the purposes of timing the arrival of the pulse at a particular point in the arterial tree, and for pulse contour analysis. This thesis describes a methodology for measuring arterial pulse transit time (PTT) from cardiac ejection to pulse arrival at the finger. It describes the effect on PTT of drug and exercise induced changes in BP. The nature of the relationship between the PPG and arterial pressure is also examined, and the PTT technique extended to assessment of conduit vessel pulse wave velocity (PWV) during exercise. PTT measured from ECG R-wave to PPG finger wave (rPTT) had a negative correlation (R2=0.39) with systolic BP (SBP), unaffected by vasoactive drugs in some but not all persons. rPTT showed similar beat-to-beat variability to SBP, unaffected by drugs. rPTT correlated weakly with diastolic (DBP) and mean (MAP) pressure. Cardiac pre-ejection period (PEP) formed a substantial and variable part of rPTT (12% to 35%). Transit time adjusted for PEP (pPTT) correlated better with DBP (R2=0.41) and MAP (R2=0.45), than with SBP. The PPG wave tracked changes in the peripheral pressure wave. Drugs had little effect on the generalised transfer function (GTF) describing the association between arterial and PPG waves. Strenuous exercise induced a large decrease in rPTT, mainly accounted for by decreases in PEP (53% of the total change in rPTT) and in transit time from aorta to distal brachial artery (33%). In contrast, minimal change in transit time from wrist to finger tip occurred with exercise. Simultaneous ear-finger PPG signals were used to measure conduit artery PWV during exercise. Ear-finger PWV (PWVef) overestimated carotid-radial PWV throughout exertion (overall bias 0.81±1.05ms-1, p<0.001), but the degree of difference remained constant. The increase in PWVef with exercise, was greater (1.18±0.54ms-1, p=0.035) in healthy subjects with a positive cardiovascular family history compared to those without. PPG enables analysis of the pulse contour during exercise, but estimation of the radial pressure wave from finger PPG by use of a GTF derived at rest, resulted in inaccuracy following exertion. These effects were variable and relatively short-lived. Furthermore, a resting GTF used to determine central pressure from the peripheral wave, resulted in underestimation of SBP (-5.9±2.1mmHg) and central pressure augmentation index (-8.3±2.9%), which persisted for 10 minutes post-exercise. rPTT had a negative linear association with SBP (R2=0.94) during strenuous exercise, slightly stronger than during recovery (R2=0.85). Differences existed in area-undercurve of the rPTT/SBP relationship between exercise and recovery, due to discrepancies in rate and degree of recovery of SBP and PEP. The linear relationship between the rPTT/SBP during exercise was affected by aerobic capacity, and the regression slope was less in the anaerobic compared to aerobic phase of exercise due to minimal change in PEP during anaerobic exertion. The correlation between rPTT/SBP did not change with prolonged aerobic exercise. Finally, measures of baroreflex sensitivity during exercise, were not significantly different between actual beat-to-beat SBP and SBP estimated using rPTT. In conclusion, absolute BP cannot be reliably estimated by measurement of rPTT following administration of drugs and during exercise. However, rPTT may have a role in measuring BP variability and in the assessing exercise capacity. PPG may also be useful in determining the effects of exercise on arterial stiffness, and for estimating the pressure wave contour, although its use during exercise for the latter purpose must be treated with caution.

An increased prevalence and earlier onset of cardiovascular disease (CVD) occurs in persons with spinal cord injury (SCI); the higher risk may be explained by novel CVD risk factors of aerobic capacity and peripheral vascular structure and function. Physical inactivity likely contributes to the basis of increased CVD risk after SCI, however evidence on the effectiveness of exercise programs in attenuating CVD risk in SCI is insufficient. The present thesis evaluated novel CVD risk factors in a cohort of individuals with chronic SCI, and examined the effects of a single bout of exercise and exercise training on CVD risk. The first study demonstrated dramatic decreases in body composition, aerobic capacity, and sublesional endothelial function via flow-mediated dilation (FMD) in adults with chronic SCI vs. able-bodied (AB) controls. The second, third, and fourth studies assessed the role of shear rate (SR) patterns on FMD. Elevated retrograde SR had a detrimental effect on brachial and superficial-femoral-artery (SFA) FMD in both SCI and AB, but elevated anterograde SR had a favorable effect on SFA FMD in AB only. The fifth study demonstrated that sublesional vasculature does not respond to a 4-month combination aerobic and resistance-training program using the recently released physical activity guidelines for adults with SCI (PAG). The results of this thesis highlight the multilayered regulation of sublesional vasculature, and that it may respond differently to a single bout of exercise and exercise training when compared to an AB population. This information is crucial when designing strategies to combat impaired vascular structure and function after SCI. The results from this thesis also indicate the potential for the PAG to improve aspects of anthropometrics, body composition, and carotid vascular health in adults with SCI. Further investigations are necessary to delineate the effects of SCI itself, and of exercise, on CVD risk in this population. / Dissertation / Doctor of Science (PhD)

Heat stress and dehydration pose a severe challenge to physiological function and the capability to perform physical work. There is, however, limited knowledge on the regional haemodynamic and metabolic responses to strenuous exercise in environmentally stressful conditions. The primary aim of this thesis was to examine whether dehydration and heat stress compromise brain, muscle and systemic blood flow and metabolism, and whether depressed brain and muscle oxygen delivery underpin reduced exercise capacity during graded incremental and prolonged exercise. This thesis makes an original contribution to the knowledge by showing for the first time that dehydration markedly accelerates the decline in cerebral blood flow during maximal incremental (Chapter 4) and prolonged sub-maximal exercise (Chapter 5) in the heat. Cerebral metabolism, however, is preserved by compensatory increases in substrate extraction. Falling carbon dioxide tension underpinned the decline in CBF. However, a distinct regional distribution of blood flow across the head was observed, suggesting that different mechanisms are responsible for the regulation of regional blood flow within the head. A reduced cerebral metabolism is therefore an unlikely factor explaining the compromised exercise capacity in physiologically stressful hot environments. Rather, restrictions in active muscle blood flow and oxygen supply, which are not apparent during sub-maximal exercise, may explain the reduced maximal aerobic power in heat stressed conditions. For the first time we have manipulated skin and core temperature to show that combined internal and skin hyperthermia reduces maximal aerobic power in association with restrictions in limb, brain and systemic blood flow and skeletal muscle metabolism (Chapter 6). Overall, the findings of the present thesis provide novel information on how circulatory limitations across contracting skeletal muscle, brain and systemic tissues and organs might underpin the impairment in exercise capacity in physiologically taxing environments evoking significant dehydration and hyperthermia.

The Langendorff perfused murine heart has become an increasingly important research model in cardiovascular physiology and pharmacology. However, the model remains relatively poorly characterised when compared with the widely employed rat preparation. The purpose of the research within this thesis was initially two-fold: 1) to characterise the functional and substrate-dependent properties of the murine model; and 2) to characterise the relationships between glycolysis, ischaemic tolerance and adenosine-mediated cardioprotection in the mouse. Initial studies, confirmed by simultaneous/subsequent work in other laboratories, revealed the frequent occurrence of regular cyclic oscillations in contractile function and coronary flow in glucose-perfused isovolumically contracting hearts. This phenomenon (labelled 'cycling') was unaltered by inhibition of ?-adrenergic receptors, prostaglandins, and nitric oxide synthase. However, A1/A2 adenosine receptor agonism did abolish the oscillations in flow and reduced contractile oscillations by 50%. Importantly, cycling was eliminated by addition of 50 IU/l insulin to perfusion fluid, or provision of 5 mM pyruvate as a co-substrate with glucose. These data suggest that functional 'cycling' in glucose-perfused murine hearts likely occurs as a result of a mismatch between substrate metabolism (energy supply) and myocardial energy demand. It may be that glycolysis with exogenous glucose is insufficient to ensure appropriate matching of myocardial energy supply and demand. For this reason, it is advisable to employ a co-substrate such as pyruvate in studies of murine hearts. Further studies performed within this thesis generally employ this co-substrate addition. Addition of pyruvate as co-substrate removes 'cycling' but is also known to inhibit/modify glycolysis, which may affect ischaemic tolerance and/or cardioprotection mediated by adenosine. Experiments throughout this thesis demonstrated that pyruvate-perfusion improved tolerance to both ischaemia (delayed time to onset of ischaemic contracture; TOC) and reperfusion (reduced diastolic dysfunction and cell death). The delay in TOC as a result of pyruvate-perfusion also suggests that contracture is not solely influenced by anaerobic glycolysis (as outlined in current paradigms). To test the relevance of glycolysis to ischaemic injury hearts were subjected to various forms of glycolytic inhibition. Glycolysis was inhibited by use of 10 mM pyruvate, (iodoacetic acid) IAA treatment, and glycogen depletion by pre-ischaemic substrate-free perfusion (all groups employing pyruvate as sole-substrate). Each form of glycolytic modification resulted in significant delays in TOC, in complete contrast to findings from other models and species. Glycogen depletion also reduced the peak level of contracture. These findings indicate that the mouse is either unique in terms of substrate metabolism and mechanisms of contracture (an unlikely possibility), or raise serious questions regarding current models of contracture development during ischaemia (theorised to be delayed by prolonging anaerobic glycolysis). Modification of glycolysis also altered post-ischaemic outcome, with pyruvate perfusion and glycogen depletion both enhancing functional recoveries. However, IAA treated hearts, despite near-identical ischaemic tolerance (ie contracture development) to pyruvate-perfused hearts, displayed very poor functional recovery, which was below that for all other groups. These data clearly reveal that blocking glycolysis improves tolerance to ischaemia (as evidenced by reduced contracture), provide evidence of dissociation of ischaemic injury or contracture from post-ischaemic recovery, and confirm the key importance of glycolysis in enhancing recovery from ischaemia. Since tolerance to ischaemia/reperfusion was shown to be glycolysis dependent, and since it has been theorised that adenosine protects hearts through modulating glycolysis, the relationships between glycolytic inhibition and adenosine-mediated cardioprotection was tested. In a number of studies, exogenously applied adenosine was shown to protect both glucose- and pyruvate-perfused hearts (supporting no dependence of adenosinergic protection on glycolysis). However, to more equivocally test the role of glycolysis effects of IAA were studied and were shown to markedly limit protection with adenosine. The effects of adenosine during ischaemia were abolished by IAA treatment, and effects on post-ischaemic recovery were reduced (but not eliminated). Similar results were acquired for protection with endogenous adenosine (using iodotubercidin to block adenosine phosphorylation). Collectively, these data reveal that adenosinergic protection during ischaemia depends entirely upon glycolysis while protection during reperfusion likely involves glycolysis dependent and independent processes. However, glycolysis is required for full recovery of function during reperfusion. Further studies assessed the involvement of glycolysis in cardioprotection afforded by transgenic A1 adenosine receptor (A1AR) overexpression. It was found that pyruvate-perfusion provided the same protection as A1AR overexpression, and the two responses (to pyruvate and A1AR overexpression) were not additive. Thus, it is probable that common mechanisms are targeted in both responses (likely glycolysis). Finally, the effects of adenosine and pyruvate on oxidant injury were studied, testing whether interactions between adenosine and pyruvate observed in prior work within this thesis could be explained by alterations in anti-oxidant responses. It was found that adenosine has quite profound anti-oxidant responses in glucose-perfused hearts, with very selective effects on markers of damage. Pyruvate also had some anti-oxidant effects but interestingly it reduced the anti-oxidant effects of adenosine. In conclusion, the work entailed within this thesis demonstrates that the isolated mouse heart model may possess unique properties and should be further characterised by potential users in order to improve its utility, and the reliability of experimental findings (chiefly when studying ischaemia-reperfusion). Other work within thesis demonstrates that modification of glycolysis is important in dictating recovery from ischaemia-reperfusion, and also impacts on adenosine-mediated protection (principally but not exclusively during ischaemia itself). The manner in which glycolysis is modified and contributes to protection remains unclear.

This thesis investigated whether highly concentrated sodium solutions ([HS] 126-164 mmol.L-1 NaCl) could provide viable strategies before during and after exercise in the heat to improve cardiovascular and thermoregulatory functioning and exercise performance. To do this it also examined the gustatory responses to HS drinks before, during and after exercise. All studies compared HS with a low sodium control ([LS] 10-27 mmol.L-1). Chapter 4 found that during 3 h recovery from dehydration, ingestion of 120 % body mass losses of HS restored fluid balance to a greater extent (121 vs. 84 %) than LS. Chapter 7 was the first to investigate the effects of ingesting HS during exercise in the heat and in an untrained population. HS attenuated the decline in stroke volume [SVDrift] and increase in heart rate [HRDrift], but did not affect rectal temperature [TRec], cardiac output, or oxygen uptake during the second of two consecutive 45 min bouts at 55% . In Chapters 8 and 9 untrained participants ingested either HS or LS during 30-45 min pre-exercise rest. HS reduced HRDrift and SVDrift but did not affect TRec during 45-60 min exercise at 10% of the difference between and gas exchange threshold [∆]. HS also increased both time to exhaustion and exercise toleranceduring subsequent exercise bouts at 60-70% ∆. Chapters 5 and 6 found that taste perceptions act as physiological regulators, in this case, one reflecting the priority to restore hyperosmolality over hypovolemia. Exercise-induced dehydration increased the palatability of water, and decreased the palatability HS, when measured before, immediately after and during 3 h recovery. The changes were highly correlated with physiological indicators of fluid balance. The ingestion of highly concentrated sodium solutions can be both an efficient and acceptable means to improve hydration, reduce cardiovascular stress, and improve exercise performance in the heat. Whilst highly effective, caution should apply since the unpleasant taste evoked by these solutions persists for at least three hours post exercise.