1. Maximal lactate steady state (MLSS) corresponds to the highest blood lactate concentration (MLSSc) and workload (MLSSw) that can be maintained over time without continual blood lactate accumulation and is considered an important marker of endurance exercise capacity. The present study was undertaken to determine MLSSw and MLSSc in running mice. In addition, we provide an exercise training protocol for mice based on MLSSw. 2. Maximal lactate steady state was determined by blood sampling during multiple sessions of constant-load exercise varying from 9 to 21 m/min in adult male C57BL/6J mice. The constant-load test lasted at least 21 min. The blood lactate concentration was analysed at rest and then at 7 min intervals during exercise. 3. The MLSSw was found to be 15.1 +/- 0.7 m/min and corresponded to 60 +/- 2% of maximal speed achieved during the incremental exercise testing. Intra- and interobserver variability of MLSSc showed reproducible findings. Exercise training was performed at MLSSw over a period of 8 weeks for 1 h/day and 5 days/week. Exercise training led to resting bradycardia (21%) and increased running performance (28%). Of interest, the MLSSw of trained mice was significantly higher than that in sedentary littermates (19.0 +/- 0.5 vs 14.2 +/- 0.5 m/min; P = 0.05), whereas MLSSc remained unchanged (3.0 mmol/L). 4. Altogether, we provide a valid and reliable protocol to improve endurance exercise capacity in mice performed at highest workload with predominant aerobic metabolism based on MLSS assessment.

Rationale: In the present study we explored the mechanisms behind excitation-contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db 1 This has severe implications, because cardiovascular mortality is Ϸ2-to 4-fold higher in diabetic compared to nondiabetic patients 2 and accounts for Ϸ80% of the mortality in type 2 diabetes, 3 of which Ϸ50% die of sudden cardiac death. 4 Furthermore, diabetics are 2.5 times more likely to develop congestive heart failure compared to nondiabetics. 5 The db/db diabetic mouse model develops cardiomyopathy in a similar manner as type 2 diabetes in humans, 6 and presents with reduced whole-heart 7 and isolated cardiomyocyte 8 11,12 In contrast, exercise training in healthy mice increases the level of phosphorylated cytosolic CaMKII␦ and in so doing increases CaMKII␦ activity. Under these circumstances, increased phosphorylation of CaMKII␦ was associated with an increased cardiac performance. 13 Alongside increased SR Ca 2ϩ leak, reduced transverse (T)-tubule structure leading to less synchronous SR Ca 2ϩ release contributes further to the depressed EC coupling in models of cardiac dysfunction. 14 The mechanism for increased SR Ca 2ϩ leak, and whether T-tubule structure in diabetic cardiomyopathy is conserved, has currently not been studied.In the present study, we explored the mechanisms behind the impaired cardiomyocyte function and increased SR Ca 2ϩ leak in db/db cardiomyocytes, and then reexamined the same parameters in the db/db mice after an aerobic interval exercise training program. Because the activities of both CaMKII␦ and PKA are associated with both pathological and physiological remodeling, we also investigated the contributions of CaMKII␦ and PKA for the observed exercise training-induced changes. MethodsFor a detailed description, see the data supplement (available online at http://circres.ahajournals.org). Mouse Model of Diabetes and Exercise TrainingThe db/db mice model has been proven to be a suitable model to study the consequences of diabetes on the heart. Here we studied the male diabetic (BKS.Cg-m ϩ/ϩ Lepdb/Bom Tac; 20 exercised and 20 sedentary mice) and sedentary (nϭ23) and exercise trained (nϭ6) nondiabetic healthy heterozygote (BKS.Cg-m ϩ/ϩ Lepdb/ϩ lean); all age-matched (7 weeks at study start). To determine maximal oxygen uptake (VO 2max ), mice ran until exhaustion on a customized treadmill in a metabolic chamber, and high-intensity aerobic interval training was performed as uphill running, alternating between 4 minutes at 85% to 90% of VO 2max and 2 minutes at 50% of VO 2max for 80 minutes/day, 5 days/wk, for 13 weeks. 15 Cardiomyocyte Isolation and Ca 2؉ MeasurementsLeft ventricular myocytes were isolated as previously described. 15 Fura-2/AM-loaded cardiomyocytes were stimulated by bipolar electric pulses for Ca 2ϩ handling measurements including SR Ca 2ϩ leak. CaMKII inhibitor and PKA inhibitor were used to determine the influence of the 2 kinases. Contractility was recorded by video-based sarcomere spacing. ...

Rationale Increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in heart failure (HF) development and associated diastolic sarcoplasmic reticulum (SR) Ca2+ leak is unclear. Objective Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with non-ischemic and ischemic forms of HF. Methods and Results Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with non-ischemic but not with ischemic HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development following transverse aortic constriction (TAC) compared to wildtype (WT) littermates. After TAC, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor (ANF). Cardiomyocytes from S2814A mice exhibited significantly lower SR Ca2+ leak and improved SR Ca2+ loading compared to WT mice after TAC. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice following experimental myocardial infarction. Conclusions Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological SR Ca2+ leak and heart failure development in non-ischemic forms of HF such as transverse aortic constriction in mice.

Our findings are the first to demonstrate that HFpEF induces significant molecular, mitochondrial, histological, and functional alterations in the diaphragm and soleus, which were attenuated by exercise training. These data therefore reveal novel mechanisms and potential therapeutic treatments of exercise intolerance in HFpEF.

Rationale Low aerobic exercise capacity is a powerful predictor of premature morbidity and mortality for healthy adults as well as those with cardiovascular disease For aged populations, poor performance on treadmill or extended walking tests indicates closer proximity to future health declines. Together, these findings suggest a fundamental connection between aerobic capacity and longevity. Objectives Through artificial selective breeding, we developed an animal model system to prospectively test the association between aerobic exercise capacity and survivability (aerobic hypothesis). Methods and Results Laboratory rats of widely diverse genetic backgrounds (N:NIH stock) were selectively bred for low or high intrinsic (inborn) treadmill running capacity. Cohorts of male and female rats from generations 14, 15 and 17 of selection were followed for survivability and assessed for age-related declines in cardiovascular fitness including maximal oxygen uptake (VO2max), myocardial function, endurance performance, and change in body mass. Median lifespan for low exercise capacity rats was 28-45% shorter than high capacity rats (hazard ratio, 0.06; P<.001). VO2max, measured across adulthood was a reliable predictor of lifespan (P<.001). During progression from adult to old age, left ventricular myocardial and cardiomyocyte morphology, contractility, and intracellular Ca2+ handling in both systole and diastole, as well as mean blood pressure, were more compromised in rats bred for low aerobic capacity. Physical activity levels, energy expenditure (VO2), and lean body mass were all better sustained with age in rats bred for high aerobic capacity. Conclusions These data obtained from a contrasting heterogeneous model system provide strong evidence that genetic segregation for aerobic exercise capacity can be linked with longevity and useful for deeper mechanistic exploration.

The molecular basis of the beneficial effects associated with exercise training (ET) on overall ventricular function (VF) in heart failure (HF) remains unclear. We investigated potential Ca(2+) handling abnormalities and whether ET would improve VF of mice lacking alpha(2A)- and alpha(2C)-adrenoceptors (alpha(2A)/alpha(2C)ARKO) that have sympathetic hyperactivity-induced HF. A cohort of male wild-type (WT) and congenic alpha(2A)/alpha(2C)ARKO mice in a C57BL/J genetic background (5-7 mo of age) was randomly assigned into untrained and trained groups. VF was assessed by two-dimensional guided M-mode echocardiography. Cardiac myocyte width and ventricular fibrosis were evaluated with a computer-assisted morphometric system. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), phospholamban (PLN), phospho-Ser(16)-PLN, phospho-Thr(17)-PLN, phosphatase 1 (PP1), and Na(+)-Ca(2+) exchanger (NCX) were analyzed by Western blotting. ET consisted of 8-wk running sessions of 60 min, 5 days/wk. alpha(2A)/alpha(2C)ARKO mice displayed exercise intolerance, systolic dysfunction, increased cardiac myocyte width, and ventricular fibrosis paralleled by decreased SERCA2 and increased NCX expression levels. ET in alpha(2A)/alpha(2C)ARKO mice improved exercise tolerance and systolic function. ET slightly reduced cardiac myocyte width, but unchanged ventricular fibrosis in alpha(2A)/alpha(2C)ARKO mice. ET significantly increased the expression of SERCA2 (20%) and phospho-Ser(16)-PLN (63%), phospho-Thr(17)-PLN (211%) in alpha(2A)/alpha(2C)ARKO mice. Furthermore, ET restored NCX and PP1 expression in alpha(2A)/alpha(2C)ARKO to untrained WT mice levels. Thus, we provide evidence that Ca(2+) handling is impaired in this HF model and that overall VF improved upon ET, which was associated to changes in the net balance of cardiac Ca(2+) handling proteins.

Coherent plane wave compounding is a promising technique for achieving very high frame rate imaging without compromising image quality or penetration. However, this approach relies on the hypothesis that the imaged object is not moving during the compounded scan sequence, which is not the case in cardiovascular imaging. This work investigates the effect of tissue motion on retrospective transmit focusing in coherent compounded plane wave imaging (PWI). Two compound scan sequences were studied based on a linear and alternating sequence of tilted plane waves, with different timing characteristics. Simulation studies revealed potentially severe degradations in the retrospective focusing process, where both radial and lateral resolution was reduced, lateral shifts of the imaged medium were introduced, and losses in signal-to-noise ratio (SNR) were inferred. For myocardial imaging, physiological tissue displacements were on the order of half a wavelength, leading to SNR losses up to 35 dB, and reductions of contrast by 40 dB. No significant difference was observed between the different tilt sequences. A motion compensation technique based on cross-correlation was introduced, which significantly recovered the losses in SNR and contrast for physiological tissue velocities. Worst case losses in SNR and contrast were recovered by 35 dB and 27-35 dB, respectively. The effects of motion were demonstrated in vivo when imaging a rat heart. Using PWI, very high frame rates up to 463 fps were achieved at high image quality, but a motion correction scheme was then required.

Exercise training (ET) is a coadjuvant therapy in preventive cardiology. It delays cardiac dysfunction and exercise intolerance in heart failure (HF); however, the molecular mechanisms underlying its cardioprotection are poorly understood. We tested the hypothesis that ET would prevent Ca(2+) handling abnormalities and ventricular dysfunction in sympathetic hyperactivity-induced HF mice. A cohort of male wild-type (WT) and congenic alpha(2A)/alpha(2C)-adrenoceptor knockout (alpha(2A)/alpha(2C)ARKO) mice with C57BL6/J genetic background (3-5 mo of age) were randomly assigned into untrained and exercise-trained groups. ET consisted of 8-wk swimming session, 60 min, 5 days/wk. Fractional shortening (FS) was assessed by two-dimensional guided M-mode echocardiography. The protein expression of ryanodine receptor (RyR), phospho-Ser(2809)-RyR, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), Na(+)/Ca(2+) exchanger (NCX), phospholamban (PLN), phospho-Ser(16)-PLN, and phospho-Thr(17)-PLN were analyzed by Western blotting. At 3 mo of age, no significant difference in FS and exercise tolerance was observed between WT and alpha(2A)/alpha(2C)ARKO mice. At 5 mo, when cardiac dysfunction is associated with lung edema and increased plasma norepinephrine levels, alpha(2A)/alpha(2C)ARKO mice presented reduced FS paralleled by decreased SERCA2 (26%) and NCX (34%). Conversely, alpha(2A)/alpha(2C)ARKO mice displayed increased phospho-Ser(16)-PLN (76%) and phospho-Ser(2809)-RyR (49%). ET in alpha(2A)/alpha(2C)ARKO mice prevented exercise intolerance, ventricular dysfunction, and decreased plasma norepinephrine. ET significantly increased the expression of SERCA2 (58%) and phospho-Ser(16)-PLN (30%) while it restored the expression of phospho-Ser(2809)-RyR to WT levels. Collectively, we provide evidence that improved net balance of Ca(2+) handling proteins paralleled by a decreased sympathetic activity on ET are, at least in part, compensatory mechanisms against deteriorating ventricular function in HF.