Although it is generally accepted that endurance training improves cardiac function after myocardial infarction the sub-cellular mechanisms are uncertain. The present study reports the effects of aerobic endurance training on myocardial mass, myocyte dimensions, contractile function, Ca2+ handling, and myofilament responsiveness to Ca2+ in cardiomyocytes from healthy and failing rat hearts. Adult female Sprague-Dawley rats ran on a treadmill 1.5 h/day, 5 days a week for 8 weeks. Exercise intervals alternated between 8 min at 85-90% of V(O(2max)) and 2 min at 50-60%. Training started 4 weeks after ligation of the left coronary artery (TR-INF, n=11) or sham operation (TR-SHAM, n=6). Sedentary animals (SED-SHAM, n=6; SED-INF, n=13) were controls. After 6 weeks V(O(2max)) in TR-INF and TR-SHAM leveled off 65% above sedentary controls. In TR-SHAM, left and right ventricle weights were approximately 25% higher than in SED-SHAM, myocytes were approximately 13% longer; width remained unchanged. At physiological stimulation frequencies, relative myocyte shortening was markedly higher whereas peak systolic [Ca2+] and t(1/2) of Ca2+ transient decay were 10-20% lower, indicating higher Ca2+ sensitivity in cardiomyocytes from trained rats, compared to respective controls. In TR-INF the left and right ventricular weights, and myocyte length and width were 15, 23, 12, and 20% less than in SED-INF. Endurance training significantly increased the myocardial SR Ca2+ pump (SERCA-2) and sarcolemmal Na+-Ca2+-exchanger (NCX) protein levels to the extent that TR-INF did not differ from SED-SHAM. This is the first study to show that aerobic endurance training attenuates the ventricular and cellular hypertrophy in failing hearts. Furthermore, training consistently restores contractile function, intracellular Ca2+ handling, and Ca2+-sensitivity in cardiomyocytes from rats with myocardial infarction.

Regular exercise enhances cardiac function and modulates myocyte growth in healthy individuals. The purpose of the present study was to assess contractile function and expression of selected genes associated with intracellular Ca2+ regulation after intensity controlled aerobic endurance training in the rat. Female Sprague-Dawley rats were randomly assigned to sedentary control (SED) or treadmill running (TR) 2 h per day, 5 days per week for 2, 4 or 13 weeks. Rats ran 8-min intervals at 85-90% of VO2max separated by 2 min at 50-60%. Myocyte length, intracellular Ca2+ (Fura-2), and intracellular pH (BCECF) were measured in dissociated cells in response to electrical stimulation at a range of stimulation rates. The increase in VO2max plateaued after 6-8 weeks, 60% above SED. After 13 weeks, left and right ventricular weights were 39 and 36% higher than in SED. Left ventricular myocytes were 13% longer, whereas width remained unchanged. After 4 weeks training, myocyte contractility was approximately 20% higher in TR. Peak systolic intracellular Ca2+ and time for the decay from systole were 20-35 and 12-17% lower, respectively. These results suggest that increased myofilament Ca2+ sensitivity is the dominant effect responsible for enhanced myocyte contractility in TR. Intracellular pH progressively decreased as stimulation frequency was increased in the SED group. This decrease was markedly attenuated in TR and the intracellular pH was significantly higher in the TR group at a stimulation rate of 5-10 Hz. This effect may contribute to the increased contractility observed at the higher stimulation frequencies in TR. A higher intrinsic myofilament Ca2+ sensitivity was observed in permeabilised myocytes from the TR group under conditions of constant pH and [Ca2+]. Western blot analysis indicated 21 and 46% higher myocardial SERCA-2 and phospholamban, but unaltered Na+/Ca(2+)-exchanger levels. Competitive RT-PCR revealed that TR significantly increased Na+/H(+)-exchanger mRNA. Intensity controlled interval training increases cardiomyocyte contractility. Higher myofilament Ca(2+)-sensitivity, and enhanced Ca(2+)-handling and pH-regulation are putative mechanisms. Our results suggest that physical exercise induces adaptive hypertrophy in cardiac myocytes with improved contractile function.

Cardiac ryanodine receptors (RyR2s) play a critical role in excitation–contraction coupling by providing a pathway for the release of Ca²⁺ from the sarcoplasmic reticulum into the cytosol. RyR2s exist as macromolecular complexes that are regulated via binding of Ca²⁺ and protein phosphorylation/dephosphorylation. The present study examined the association of endogenous CaMKII (calcium/calmodulin-dependent protein kinase II) with the RyR2 complex and whether this enzyme could modulate RyR2 function in isolated rabbit ventricular myocardium. Endogenous phosphorylation of RyR2 was verified using phosphorylation site-specific antibodies. Co-immunoprecipitation studies established that RyR2 was physically associated with CaMKIIδ. Quantitative assessment of RyR2 protein was performed by [³H]ryanodine binding to RyR2 immunoprecipitates. Parallel kinase assays allowed the endogenous CaMKII activity associated with these immunoprecipitates to be expressed relative to the amount of RyR2. The activity of RyR2 in isolated cardiac myocytes was measured in two ways: (i) RyR2-mediated Ca²⁺ release (Ca²⁺ sparks) using confocal microscopy and (ii) Ca²⁺-sensitive [³H]ryanodine binding. These studies were performed in the presence and absence of AIP (autocamtide-2-related inhibitory peptide), a highly specific inhibitor of CaMKII. At 1 µM AIP Ca²⁺ spark duration, frequency and width were decreased significantly. Similarly, 1 µM AIP decreased [³H]ryanodine binding. At 5 µM AIP, a more profound inhibition of Ca²⁺ sparks and a decrease in [³H]ryanodine binding was observed. Separate measurements showed that AIP (1–5 µM) did not affect sarcoplasmic reticulum Ca²⁺-ATPase-mediated Ca²⁺ uptake. These results suggest the existence of an endogenous CaMKIIδ that associates directly with RyR2 and specifically modulates RyR2 activity.

To assess the phosphorylation of myocardial phospholamban (PLB) and quantify protein levels of PLB and sarco/endoplasmic reticulum Ca2+ ATPase type 2 (SERCA 2) in a rabbit model of heart failure. Furthermore, to correlate these parameters with the rate of Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles. Heart failure in the rabbit was indicated by the pronounced ventricular contractile dysfunction accompanied by post-mortem evidence of lung and liver congestion 8 weeks after a coronary artery ligation procedure. Phosphorylation of PLB was measured by reduced mobility of the phosphorylated forms on Tris-glycine gels. Phosphoserine and phosphothreonine-specific antibodies against PLB were used to determine the phosphorylated residues. Immunoblotting combined with densitometry was used to assess PLB and SERCA 2 levels. Finally, oxalate-supported Ca2+ uptake into SR vesicles was studied using the fluorescent indicator Fura-2. The phosphorylation state of PLB was significantly higher in myocardium isolated from left ventricles of heart failure rabbits (8.3 +/- 0.42 P-PLB) when compared with sham-operated animals (4.0 +/- 1.7 P-PLB). The kinase activity associated with SR vesicles isolated from animals with heart failure was a factor of 1.58 +/- 0.21-times higher than sham hearts, as assessed by the initial rate of phosphorylation of PLB. This higher kinase activity observed in heart failure was not completely abolished by inhibitors of either A-kinase, C-kinase or Ca2+/calmodulin-dependent protein kinase (CaM-kinase). Abundance of SERCA in heart failure myocardial homogenates was significantly less than sham values (0.68 +/- 0.11 vs. 1.74 +/- 0.27) as was PLB (0.41 +/- 0.08 vs. 0.69 +/- 0.13), similar reductions were seen in vesicle preparations. The rate constant of Ca2+ uptake into the isolated SR vesicles was lower in preparations from heart failure myocardium than from sham myocardium (2.50 +/- 0.23 ms vs. 4.43 +/- 0.3 ms). The higher level of phosphorylation of PLB observed in the left ventricle of rabbits with heart failure is associated with a higher intrinsic kinase activity of the SR. However, the abundance of both of SERCA 2 and PLB proteins are lower in heart failure. The net effect of these changes appears to be a reduced rate of Ca2+ uptake by the SR in heart failure.