ROLE OF SECOND MESSENGER SIGNALING PATHWAYS IN THE REGULATION OF SARCOPLASMIC RETICULUM CALCIUM-HANDLING PROPERTIES IN THE LEFT VENTRICLE AND SKELETAL MUSCLES OF DIFFERENT FIBRE TYPE COMPOSITION

The overall objective of this thesis was to examine mechanisms involved in the acute regulation of sarcoplasmic reticulum (SR) Ca2+-handling properties by second messenger signaling pathways in skeletal and cardiac muscle. The aim of the first study (Chapter Two) was to characterize changes in the kinetic properties of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA) proteins in cardiac and skeletal muscles in response to b-adrenergic, Ca2+-dependent calmodulin kinase II (CaMKII) and protein kinase C (PKC) signaling. The aim of the second study (Chapter Three) was to determine if insulin signaling could acutely regulate SERCA kinetic properties in cardiac and skeletal muscle. The aim of the final study (Chapter Four) was to determine if alterations in plasma glucose, epinephrine and insulin concentrations during exercise are able to influence SR Ca2+-handling properties in contracting human skeletal muscle. Data collected in Chapter Two and Chapter Three were obtained using tissue prepared from a group of 28 male Sprague-Dawley rats (9 weeks of age; mass = 280 ± 4 g: X ± S.E). Crude muscle homogenates (11:1 dilution) were prepared from selected hind limb muscles (soleus, SOL; extensor digitorum longus, EDL; the red portion of gastrocnemius, RG; and the white portion of gastrocnemius, WG) and the left ventricle (LV). Enriched SR membrane fractions, prepared from WG and LV, were also analyzed. A spectrophotometric assay was used to measure kinetic properties of SERCA, namely, maximal SERCA activity (Vmax), and Ca2+-sensitivity was characterized by both the Ca50, which is defined as the free Ca2+-concentration needed to elicit 50% Vmax, and the Hill coefficient (nH), which is defined as the relationship between SERCA activity and Ca2+f for 10 to 90% Vmax. The observations made in Chapter Two indicated that b-adrenergic signaling, activated by epinephrine, increased (P<0.05) Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50), without altering Vmax in LV and SOL but had no effect (P<0.05) on EDL, RG, or WG. Further analysis using a combination of cAMP, the PKA activator forskolin, and/or the PKA inhibitor KT5270 indicated that the reduced Ca50 in LV was activated by cAMP- and PKA-signaling mechanisms. However, although the reduced Ca50 in SOL was cAMP-dependent, it was not influenced by a PKA-dependent mechanism. In contrast to the effects of b-adrenergic signaling, CaMKII activation increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 and increased nh, without altering SERCA Vmax in LV but was without effect in any of the skeletal muscles examined. The PKC activator PMA significantly reduced SERCA Ca2+-sensitivity, by inducing a right-shift in Ca50 and decreased nH in the LV and all skeletal muscles examined. PKC activation also reduced Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG and WG), but did not alter Vmax in LV or SOL. The results of Chapter Three indicated that insulin signaling increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50) and an increased nH, without altering SERCA Vmax in crude muscle homogenates prepared from LV, SOL, EDL, RG, and WG. An increase in SERCA Ca2+-sensitivity was also observed in enriched SERCA1a and SERCA2a vesicles when an activated form of the insulin receptor (A-INS-R) was included during biochemical analyses. Co-immunoprecipitation experiments were conducted and indicated that IRS-1 and IRS-2 proteins bind SERCA1a and SERCA2a in an insulin-dependent manner. However, the binding of IRS proteins with SERCA does not appear to alter the structural integrity of the SERCA Ca2+-binding site since no changes in NCD-4 fluorescence were observed in response to insulin or A-INS-R. Moreover, the increase in SERCA Ca2+-sensitivity due to insulin signaling was not associated with changes in the phosphorylation status of phospholamban (PLN) since Ser16 or Thr17 phosphorylation was not altered by insulin or A-INS-R in LV tissue. The data described in Chapter Four was collected from 15 untrained human participants (peak O2 consumption, VO2peak= 3.45 ± 0.17 L/min) who completed a standardized cycle test (~60% VO2peak) on two occasions during which they were provided either an artificially sweetened placebo (PLAC) or a 6% glucose (GLUC) beverage (~1.00 g CHO per kg body mass). Muscle biopsies were collected from the vastus lateralis at rest, after 30 min and 90 min of exercise and at fatigue in both conditions to allow assessment of metabolic and SR data. Glucose supplementation increased exercise ride time by ~19% (137 ± 7 min) compared to PLAC (115 ± 6 min). This performance increase was associated with elevated plasma glucose and insulin concentrations and reduced catecholamine concentrations during GLUC compared to PLAC. Prolonged exercise reduced (p<0.05) SR Ca2+-uptake, Vmax, Phase 1 and Phase 2 Ca2+-release rates during both PLAC and GLUC. However, no differences in SR Ca2+-handling properties were observed between conditions when direct comparisons were made at matched time points between PLAC and GLUC. In summary, the results of the first study (Chapter Two) indicate that b-adrenergic and CaMKII signaling increases SERCA Ca2+-sensitivity in the LV and SOL; while PKC signaling reduces SERCA Ca2+-sensitivity in all tissues. PKC activation also reduces Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG, and WG) but has no effect on Vmax in the LV and SOL. The results of the second study (Chapter Three) indicate that insulin signaling acutely increases the Ca2+-sensitivity of SERCA1a and SERCA2a in all tissues examined, without altering the Vmax. Based on our observations, it appears that the increase in SERCA Ca2+-sensitivity may be regulated, in part, through the interaction of IRS proteins with SERCA1a and SERCA2a. The results of the final study (Chapter Four) indicate that alterations in plasma glucose, epinephrine and insulin concentrations associated with glucose supplementation during exercise, do not alter the time course or magnitude of reductions in SERCA or Ca2+-release channel (CRC) function in working human skeletal muscle. Although glucose supplementation did increase exercise ride time to fatigue in this study, our data does not reveal an association with SR Ca2+-cycling measured in vitro. It is possible that the strength of exercise signal overrides the hormonal influences observed in resting muscles. Additionally, these data do not rule out the possibility that glucose supplementation may influence E-C coupling processes or SR Ca2+-cycling properties in vivo

The Effects of Acute Anaerobic Exercise on the Cardiovascular and Metabolic Response to the Cold Pressor Test in Healthy Adult Males

International Journal of Exercise Science 13(3): 1729-1740, 2020. Little is known about the physiological response to the cold pressor test (CPT) when in a clinically-induced state of autonomic nervous system (ANS) imbalance, despite its utility in various disease- and injury-states. To date, research in this area is limited to acute aerobic and isometric exercise, with a paucity of research investigating the effects of anaerobic exercise on the physiological response to the CPT. Therefore, the purpose of our study was to assess the effects of the Wingate anaerobic cycle test (WAT) on cardiovascular (CV) and metabolic recovery following the CPT in a group of healthy adult males. A pre-post intervention study was conducted, whereby 10 healthy adult males (age = 29 ± 4 years, height = 182 ± 7 cm, mass = 83 ± 9 kg) completed a baseline cold pressor test (CPT-only) and a follow-up cold pressor test preceded by a Wingate anaerobic exercise test (WAT+CPT). Recovery slopes for various CV and metabolic variables, including heart rate (HR), blood pressure (BP), and relative oxygen consumption (O2) were analyzed using single-subject analysis, with celeration line slopes calculated for all participants in the CPT-only and WAT+CPT testing sessions. Celeration line slopes were compared between testing sessions using paired t-tests. No differences were identified for recovery slopes for HR (p = .295), diastolic BP (p = .300), and relative O2 (p= .176) when comparing CPT-only and WAT+CPT testing sessions. Our results suggest that the CPT elicits a CV and metabolic response beyond that elicited solely by an acute bout of anaerobic exercise. As such, the CPT may be able to serve as a surrogate test for anaerobic exercise for individuals where high-intensity exercise may be contraindicated. Future research is warranted however, as the specific physiological mechanisms governing the observed responses have yet to be elucidated

Zero- vs. one-dimensional, parametric vs. non-parametric, and confidence interval vs. hypothesis testing procedures in one-dimensional biomechanical trajectory analysis.

Biomechanical processes are often manifested as one-dimensional (1D) trajectories. It has been shown that 1D confidence intervals (CIs) are biased when based on 0D statistical procedures, and the non-parametric 1D bootstrap CI has emerged in the Biomechanics literature as a viable solution. The primary purpose of this paper was to clarify that, for 1D biomechanics datasets, the distinction between 0D and 1D methods is much more important than the distinction between parametric and non-parametric procedures. A secondary purpose was to demonstrate that a parametric equivalent to the 1D bootstrap exists in the form of a random field theory (RFT) correction for multiple comparisons. To emphasize these points we analyzed six datasets consisting of force and kinematic trajectories in one-sample, paired, two-sample and regression designs. Results showed, first, that the 1D bootstrap and other 1D non-parametric CIs were qualitatively identical to RFT CIs, and all were very different from 0D CIs. Second, 1D parametric and 1D non-parametric hypothesis testing results were qualitatively identical for all six datasets. Last, we highlight the limitations of 1D CIs by demonstrating that they are complex, design-dependent, and thus non-generalizable. These results suggest that (i) analyses of 1D data based on 0D models of randomness are generally biased unless one explicitly identifies 0D variables before the experiment, and (ii) parametric and non-parametric 1D hypothesis testing provide an unambiguous framework for analysis when one׳s hypothesis explicitly or implicitly pertains to whole 1D trajectories

Being Barbie: The Size of One’s Own Body Determines the Perceived Size of the World

A classical question in philosophy and psychology is if the sense of one's body influences how one visually perceives the world. Several theoreticians have suggested that our own body serves as a fundamental reference in visual perception of sizes and distances, although compelling experimental evidence for this hypothesis is lacking. In contrast, modern textbooks typically explain the perception of object size and distance by the combination of information from different visual cues. Here, we describe full body illusions in which subjects experience the ownership of a doll's body (80 cm or 30 cm) and a giant's body (400 cm) and use these as tools to demonstrate that the size of one's sensed own body directly influences the perception of object size and distance. These effects were quantified in ten separate experiments with complementary verbal, questionnaire, manual, walking, and physiological measures. When participants experienced the tiny body as their own, they perceived objects to be larger and farther away, and when they experienced the large-body illusion, they perceived objects to be smaller and nearer. Importantly, despite identical retinal input, this “body size effect” was greater when the participants experienced a sense of ownership of the artificial bodies compared to a control condition in which ownership was disrupted. These findings are fundamentally important as they suggest a causal relationship between the representations of body space and external space. Thus, our own body size affects how we perceive the world

Physical activity supports provided by healthcare providers to patients with type 2 diabetes

Introduction There is limited evidence around the physical activity support delivered by different types of healthcare providers (HCPs) to patients with type 2 diabetes mellitus. The primary purpose of this study was to determine the type of physical activity support delivered by 3 HCP types, and to compare this support to that perceived by patients with type 2 diabetes. A secondary aim was to describe awareness, knowledge and use of the Canadian Diabetes Association (CDA) physical activity guidelines and Canada's Physical Activity Guide (CPAG). Method The HCP and the Patient Physical Activity Support Questionnaire were answered by 48 HCPs and 26 patients from 8 interprofessional primary care clinics specializing in diabetes education. Results There was no difference in the type of physical activity support between HCP types, however, there was a difference between overall HCP and patient report of physical activity support (5.29 vs. 2.04 unprompted; 9.90 vs. 6.07 prompted, p<0.001). Approximately half of HCPs used the CDA guidelines and the CPAG in practice. Conclusion Physical activity support is similar between all HCP types in interprofessional primary care settings, but this support is perceived differently by patients. Strategies to increase certain types of physical activity support would allow for optimal counselling in primary care

Mechanisms for the transition from physiological to pathological cardiac hypertrophy

The heart is capable of responding to stressful situations by increasing muscle mass, which is broadly defined as cardiac hypertrophy. This phenomenon minimizes ventricular wall stress for the heart undergoing a greater than normal workload. At initial stages, cardiac hypertrophy is associated with normal or enhanced cardiac function and is considered to be adaptive or physiological; however, at later stages, if the stimulus is not removed, it is associated with contractile dysfunction and is termed as pathological cardiac hypertrophy. It is during physiological cardiac hypertrophy where the function of subcellular organelles, including the sarcolemma, sarcoplasmic reticulum, mitochondria, and myofibrils, may be upregulated, while pathological cardiac hypertrophy is associated with downregulation of these subcellular activities. The transition of physiological cardiac hypertrophy to pathological cardiac hypertrophy may be due to the reduction in blood supply to hypertrophied myocardium as a consequence of reduced capillary density. Oxidative stress, inflammatory processes, Ca2+-handling abnormalities, and apoptosis in cardiomyocytes are suggested to play a critical role in the depression of contractile function during the development of pathological hypertrophy.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Sirtuin 3 overexpression preserves maximal sarco(endo)plasmic reticulum calcium ATPase activity in the skeletal muscle of mice subjected to high fat-high sucrose-feeding

Sarco(endo)plasmic reticulum calcium (CaThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Future directions for patient engagement in research: a participatory workshop with Canadian patient partners and academic researchers

Abstract Background Patient engagement in research (also commonly referred to as patient or patient and public involvement in research) strives to transform health research wherein patients (including caregivers and the public) are regularly and actively engaged as multidisciplinary research team members (i.e. patient partners) working jointly towards improved health outcomes and an enhanced healthcare system. To support its mindful evolution into a staple of health research, this participatory study aimed to identify future directions for Canadian patient engagement in research and discusses its findings in the context of the international literature. Methods The study met its aim through a multi-meeting pan-Canadian virtual workshop. Participants (n = 30) included Strategy for Patient-Oriented Research-funded academic researchers and patient partners identified through a publicly available database, personal and professional networks and social media. All spoke English, could access the workshop virtually, and provided written informed consent. The workshop was composed of four, 1.5–3-h virtual meetings wherein participants discussed the current and preferred future states of Canadian patient engagement in research. Workshop discussions (i.e. data) were video and audio recorded. Themes were generated through an iterative process of inductive thematic analysis that occurred concurrently with the multi-week workshop. Results Our participatory and iterative process identified 10 targetable areas of focus for the future of Canadian patient engagement in research. Five were categorized as system-level (systemic integration; academic culture; engagement networks; funding models; compensation models), one as researcher-level (engagement processes), and four crossed both levels (awareness; diversity and recruitment; training, tools and education; evaluation and impact). System level targetable areas called for reshaping the patient engagement ecosystem to create a legitimized and supportive space for patient engagement to be a staple component of a learning health system. Researcher level targetable areas called for academic researchers and patient partners to collaboratively generate evidence and apply knowledge to inform values and behaviours necessary to foster and sustain supportive health research spaces that are accessible to all. Conclusions Future directions for Canadian patient engagement in research span 10 interconnected targetable areas that require strong leadership and joint action between patient partners, academic researchers, and health and research institutions if patient engagement is to become a ubiquitous component of a learning health system

Protocol for the WARM Hearts study: examining cardiovascular disease risk in middle-aged and older women - a prospective, observational cohort study

Introduction Cardiovascular disease (CVD) is a leading cause of death in women. Novel approaches to detect early signs of elevated CVD risk in women are needed. Enhancement of traditional CVD risk assessment approaches through the addition of procedures to assess physical function or frailty as well as novel biomarkers of cardiovascular, gut and muscle health could improve early identification. The Women’s Advanced Risk-assessment in Manitoba (WARM) Hearts study will examine the use of novel non-invasive assessments and biomarkers to identify women who are at elevated risk for adverse cardiovascular events.Methods and analysis One thousand women 55 years of age or older will be recruited and screened by the WARM Hearts observational, cohort study. The two screening appointments will include assessments of medical history, gender variables, body composition, cognition, frailty status, functional fitness, physical activity levels, nutritional status, quality of life questionnaires, sleep behaviour, resting blood pressure (BP), BP response to moderate-intensity exercise, a non-invasive measure of arterial stiffness and heart rate variability. Blood sample analysis will be used to assess lipid and novel biomarker profiles and stool samples will support the characterisation of gut microbiota. The incidence of the adverse cardiovascular outcomes will be assessed 5 years after screening to compare WARM Hearts approaches to the Framingham Risk Score, the current clinical standard of assessing CVD risk in Canada.Ethics and dissemination The University of Manitoba Health Research Ethics Board (7 October 2019) and the St Boniface Hospital Research Review Committee (7 October 2019) approved the trial (Ethics Number HS22576 (H2019:063)). Recruitment started 10 October 2020. Data gathered from the WARM Hearts study will be published in peer-reviewed journals and presented at national and international conferences. Knowledge translation strategies will be created to share our findings with stakeholders who are positioned to implement evidence-informed CVD risk assessment programming.Trial registration number NCT03938155