A study of the techniques necessary to assess the role of the SR Ca2+ -ATPase in mediating the beneficial effects of exercise training after heart failure

The work described in this thesis has examined the hypothesis that exercise training restores Ca2+ handling and metabolic abnormalities induced by heart failure (HF) in skeletal muscle and left ventricle (LV), with emphasis on sarcoplasmic reticulum Ca2+ ATPase (SERCA) function and energy systems. For this purpose, distinct experimental conditions (ATP+CrP, ADP+CrP, ADP without CrP and ADP without CrP plus azide) were used to assess aspects of mitochondrial-SR interaction. The aims of this study consisted of 1) develop valid and reproducible protocol to retrospectively examine SR function in frozen muscle biopsies and compare uptake responses to the functionality of fresh tissue; 2) determine the ability of high-intensity exercise training to restore Ca2+ handling central (heart) and peripheral (skeletal) dysfunction when implemented as described in the present programme; 3) investigate SERCA mediated Ca2+ uptake and its dependence on ATP or ADP supply (ATP/ADP ratio) and CrP withdrawal; 4) examine the local regulation of SERCA by CK and mitochondria in situ in a rat model of HF; 5) address the effects of exercise training skeletal muscle systems for energy production and transfer in HF; and, 6) investigate the expression of Ca2+-regulatory proteins in failing striated muscle from a rat model of HF. A rat model of post-MI HF was obtained by permanently ligating the left coronary artery during thoracotomy under anaesthesia (Kemi et al. 2007; Wisloff et al. 2002; Kemi et al. 2006). This model mimics important aspects of human HF, including 40% reduction in exercise capacity, pulmonary congestion, diastolic and systolic dilatation, increased diastolic and reduced systolic pressures, reduced pump capacity, and pathologic growth of the heart, as reduced contraction, relaxation, ECC, and Ca2+ handling, abnormal energetics, and pathologic growth of the cardiomyocyte. Intensity-controlled aerobic treadmill running was used as exercise training. Rats with and without post-MI HF and accustomed to the treadmill were tested for VO2max by continuously measuring air flow, O2, and CO2 during progressive running in a metabolic chamber. Exercise training sessions initiated 4 weeks month post-MI consisted of a 10-min warm-up at 50% of VO2max, whereupon 4-min intervals of running at 90% of VO2max are continued for 60-90-min, each interval interspersed by 3-min low-intensity recovery running. This was repeated 5 times per week and continued for 2 months. SERCA function in freshly harvested fibres of EDL and SOL was demonstrated to be significantly different (p=0.0429). This divergence in uptake rates translates into physiological differences in SERCA isoforms, consistent with a higher turnover SERCA rate in fast compared to slow fibres Nonetheless, although SERCA is sequestering Ca2+ at different rates, Ca2+ binding capacity was found to be essentially the same for both muscle groups (p=0.2245); it means that, binding properties were similar regardless of the fibre composition. A comparative study of the uptake capacities in fresh versus frozen specimens has revealed Ca2+ uptake rate displayed by frozen specimens is equivalent, in functionality, to Ca2+ transportation of fresh tissue. This response was observed in both oxidative and glycolytic muscle. Current data support the assumption that SERCA function is maintained after thawing upon equilibration of fibres in appropriate media The present data do not indicate an improved Ca2+ uptake rate in SOL after the implementation of an exercise training regimen. Similar uptake rates observed in SOL fibres across the experimental groups are consistent with previous studies suggesting SERCA2 activity is constant in HF. EDL fibres were tested in ATP and ADP+CrP media, generating as in SOL, comparable results amongst groups. Similarly, investigation of LV indicated no relevant differences amongst experimental groups. It was found that muscle from trained animals have exhibited similar Ca2+ uptake rates compared to sedentary rats under conditions of ATP+CrP, ADP with and without CrP. In summary, data have supported that exercised animals exhibited SERCA rates comparable to the control condition, which suggests there is normalisation (in some degree) of the oxidative function induced by exercise. Investigation of the SR function in muscular tissue under various conditions has provided information on the cellular bioenergetics of a rat model of HF. The present results show that slow-twitch fibres treated with azide (SOL and heart muscle) did not change their Ca2+ uptake after freezing compared to control samples; whereas the glycolytic fibres from frozen EDL greatly varied their uptake rates. This demonstrates a correlation between fibre type composition and survival of mitochondrial function post-thawing. To address how physical training would modulate the energetic state of a dysfunctional muscle, citrate synthase (CS) activity was investigated. Present data evidenced that CS activity was 2.5 fold higher in HF trained rats than in healthy animals, after the implementation of the HIIT protocol. After, energy transfer systems were examined. Adenylate kinase (AK)-mediated phosphotransfer reactions were two-fold higher in post-MI exercised animals than in control group. Increased AK levels were found to be metabolically more efficient in HF trained animals in comparison to control (p=0.02); or HF sedentary rats (p=0.02), suggesting that this enzyme has contributed to a higher cellular ATP turnover in exercised animals. This study has found that the enzymes involved in energy transfer systems in the EDL muscle are increased after the implementation of a HIIT protocol. These alterations occur in parallel with increased aerobic capacity. Biochemical studies on Ca2+ modulatory proteins have shown that the four targets quantified (SERCA, PLB, RyR and CaMKII) in LV lysates displayed minimal differences in protein expression comparing control to HF animals, either sedentary or trained. The small changes in SERCA (decreased by 11% in trained rats and 5% in sedentary infarcted animals) were not statistically or functionally significant, supporting the conclusion that SERCA2 expression is unchanged in sedentary or trained animals. However, the data was gained from samples that were frozen at -80°C for prolonged periods, and degenerative process may add variability to the WB signal. PLB expression in exercised ventricles was not functionally changed compared to control animals. HF sedentary animals displayed increased RyR2 levels by approximately 33% compared to control rats; and the trained group exhibited augmented expression of RyR2 by 14% against control. No major differences could be found in CaMKII expression in the current study. SOL muscle exhibited decreased SERCA2 in the trained group by around 30% in relation to the control group. In this scenario, expression of SERCA2 in this type of fibre needs further examination. Overall, the outcome of these studies contributes to the knowledge concerning SERCA expression in skeletal muscle in HF and, after modulation by exercise training. PLB expression in SOL did not exhibit further significance changes across groups. Decreased CaMKII expression (by 31%) in post-MI trained SOL animals compared to the control group was evident. Expression of fast SERCA1 levels was likewise found to be unchanged in EDL fibres. This indicates the degree of HF in the present animal model did not cause derangements in skeletal muscle SERCA function that could be linked to reduction in SERCA1 expression associated with fibre type shift.