Corrigendum: Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy.

[This corrects the article DOI: 10.3389/fphys.2018.01454.].

Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy.

Background: The origin of electrical behavior in post-myocardial infarction scar tissue is still under debate. This study aims to examine the extent and nature of the residual electrical activity within a stabilized ventricular infarct scar. Methods and Results: An apical infarct was induced in the left ventricle of Wistar rats by coronary artery occlusion. Five weeks post-procedure, hearts were Langendorff-perfused, and optically mapped using di-4-ANEPPS. Widefield imaging of optical action potentials (APs) on the left ventricular epicardial surface revealed uniform areas of electrical activity in both normal zone (NZ) and infarct border zone (BZ), but only limited areas of low-amplitude signals in the infarct zone (IZ). 2-photon (2P) excitation of di-4-ANEPPS and Fura-2/AM at discrete layers in the NZ revealed APs and Ca2+ transients (CaTs) to 500-600 µm below the epicardial surface. 2P imaging in the BZ revealed superficial connective tissue structures lacking APs or CaTs. At depths greater than approximately 300 µm, myocardial structures were evident that supported normal APs and CaTs. In the IZ, although 2P imaging did not reveal clear myocardial structures, low-amplitude AP signals were recorded at discrete layers. No discernible Ca2+ signals could be detected in the IZ. AP rise times in BZ were slower than NZ (3.50 ± 0.50 ms vs. 2.23 ± 0.28 ms) and further slowed in IZ (9.13 ± 0.56 ms). Widefield measurements of activation delay between NZ and BZ showed negligible difference (3.37 ± 1.55 ms), while delay values in IZ showed large variation (11.88 ± 9.43 ms). Conclusion: These AP measurements indicate that BZ consists of an electrically inert scar above relatively normal myocardium. Discrete areas/layers of IZ displayed entrained APs with altered electrophysiology, but the structure of this tissue remains to be elucidated.

2-Photon excitation fluorescence microscopy enables deeper high-resolution imaging of voltage and Ca(2+) in intact mice, rat, and rabbit hearts.

We describe a novel two-photon (2P) laser scanning microscopy (2PLSM) protocol that provides ratiometric transmural measurements of membrane voltage (Vm ) via Di-4-ANEPPS in intact mouse, rat and rabbit hearts with subcellular resolution. The same cells were then imaged with Fura-2/AM for intracellular Ca(2+) recordings. Action potentials (APs) were accurately characterized by 2PLSM vs. microelectrodes, albeit fast events (<1 ms) were sub-optimally acquired by 2PLSM due to limited sampling frequencies (2.6 kHz). The slower Ca(2+) transient (CaT) time course (>1ms) could be accurately described by 2PLSM. In conclusion, Vm - and Ca(2+) -sensitive dyes can be 2P excited within the cardiac muscle wall to provide AP and Ca(2+) signals to ∼400 µm.

Exercise training and losartan improve endothelial function in heart failure rats by different mechanisms.

OBJECTIVES: To investigate the mechanisms of losartan- and exercise training-induced improvements on endothelial dysfunction in heart failure. DESIGN: Sprague-Dawley rats subjected to left coronary artery ligation inducing myocardial infarction and heart failure were randomized to losartan treatment, high-intensity exercise training, or both. RESULTS: Losartan, but not exercise training, reduced the heart failure-associated elevation in left ventricular end-diastolic pressure (26 ± 2 mmHg vs. 19 ± 1 mmHg after losartan). In contrast, both exercise training and losartan improved exercise capacity, by 40% and 20%, respectively; no additional effects were observed when exercise training and losartan were combined. Aortic segments were mounted on a force transducer to determine vasorelaxation. Heart failure impaired endothelium-dependent vasorelaxation, observed as a 1.9-fold reduced response to acetylcholine (EC50). Exercise and losartan improved acetylcholine-mediated vasorelaxation to the same extent, but by different mechanisms. Exercise training upregulated the nitric oxide pathway, whereas losartan upregulated a non-nitric oxide or -prostacyclin pathway; possibly involving the endothelium-dependent hyperpolarizing factor. CONCLUSIONS: Both losartan and exercise training reversed endothelial dysfunction in heart failure; exercise training via nitric oxide-dependent vasorelaxation, and losartan via an unknown mechanism that may involve endothelium-dependent hyperpolarizing factor. Thus, the combined treatment activated an additional nitric oxide- independent mechanism that contributed to reduce endothelial dysfunction.

Confocal and multiphoton imaging of intracellular Ca(2+).

This chapter compares the imaging capabilities of a range of systems including multiphoton microscopy in regard to measurements of intracellular Ca(2+) within living cells. In particular, the excitation spectra of popular fluorescent Ca(2+) indicators are shown during 1P and 2P excitation. The strengths and limitations of the current indicators are discussed along with error analysis which highlights the value of matching the Ca(2+) affinity of the dye to a particular aspect of Ca(2+) signaling. Finally, the combined emission spectra of Ca(2+) and voltage sensitive dyes are compared to allow the choice of the optimum combination to allow simultaneous intracellular Ca(2+) and membrane voltage measurement.

High-intensity aerobic exercise training improves the heart in health and disease.

Regular exercise training confers beneficial effects to the heart as well as to the entire body. This occurs partly because exercise training improves skeletal muscle work capacity and reduces resistance, thus increasing conductance in the peripheral circulation. More directly, exercise training also alters extrinsic modulation of the heart and improves the intrinsic pump capacity of the heart. Together, these effects allow for improved exercise capacity. Accumulating evidence suggests that the magnitude of these benefits increases proportionally with the intensity of individual exercise training sessions constituting the exercise training program. It has emerged that regular exercise training also confers beneficial effects to patients at risk for, or who have, established heart dysfunction and disease and, moreover, that exercise training may reduce the dysfunction of the heart itself and, at least, partly restore its ability to effectively function as a pump. The most recent studies in patients with established heart disease suggest that a high relative, yet aerobic, intensity of the exercise training improves the intrinsic pump capacity of the myocardium, an effect not previously believed to occur with exercise training. However, more and larger studies are needed to establish the safety and efficacy of such exercise training in patients with heart disease. Here, we consider the nature of the intensity dependence of exercise training and the causes of the improved heart function.

Pathological and physiological hypertrophies are regulated by distinct gene programs.

BACKGROUND: This study aims to investigate changes that occur during progression and establishment of physiological and pathological cardiac hypertrophy, by microarray technology and functional annotations. DESIGN AND METHODS: Myocardial infarction leading to heart failure was induced in rats, with animals killed 1, 3, 7, 14, 42, and 92 days after coronary artery ligation. A second group was subjected to daily treadmill exercise and killed 1, 4, 24, and 48 h after a single exercise bout, or after 28 or 56 days of exercise training. RESULTS: Physiological hypertrophy was associated with less transcriptional alternation than pathological hypertrophy, indicating that posttranscriptional and translational regulation may be more important. The main difference between the two types of hypertrophy was that myocardial infarction was associated with downregulation of genes related to fatty acid metabolism, whereas no such change occurred after exercise training. Thus, fatty acid metabolism may distinguish adverse maladaptive hypertrophy from beneficial adaptive hypertrophy. CONCLUSION: This study points to specific genes and gene classes related to biological processes that may be important in these well-characterized rat models of physiological and pathological cardiac hypertrophy.

Interval training normalizes cardiomyocyte function, diastolic Ca2+ control, and SR Ca2+ release synchronicity in a mouse model of diabetic cardiomyopathy.

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). OBJECTIVE: We determined whether 13 weeks of aerobic interval training could restore cardiomyocyte Ca(2+) cycling and EC coupling. METHODS AND RESULTS: Reduced contractility in cardiomyocytes isolated from sedentary db/db was associated with increased diastolic sarcoplasmic reticulum (SR)-Ca(2+) leak, reduced synchrony of Ca(2+) release, reduced transverse (T)-tubule density, and lower peak systolic and diastolic Ca(2+) and caffeine-induced Ca(2+) release. Additionally, the rate of SR Ca(2+) ATPase-mediated Ca(2+) uptake during diastole was reduced, whereas a faster recovery from caffeine-induced Ca(2+) release indicated increased Na(+)/Ca(2+)-exchanger activity. The increased SR-Ca(2+) leak was attributed to increased Ca(2+)-calmodulin-dependent protein kinase (CaMKIIdelta) phosphorylation, supported by the normalization of SR-Ca(2+) leak on inhibition of CaMKIIdelta (AIP). Exercise training restored contractile function associated with restored SR Ca(2+) release synchronicity, T-tubule density, twitch Ca(2+) amplitude, SR Ca(2+) ATPase and Na(+)/Ca(2+)-exchanger activities, and SR-Ca(2+) leak. The latter was associated with reduced phosphorylation of cytosolic CaMKIIdelta. Despite normal contractile function and Ca(2+) handling after the training period, phospholamban was hyperphosphorylated at Serine-16. Protein kinase A inhibition (H-89) in cardiomyocytes from the exercised db/db group abolished the differences in SR-Ca(2+) load when compared with the sedentary db/db mice. EC coupling changes were observed without changes in serum insulin or glucose levels, suggesting that the exercise training-induced effects are not via normalization of the diabetic condition. CONCLUSIONS: These data demonstrate that aerobic interval training almost completely restored the contractile function of the diabetic cardiomyocyte to levels close to sedentary wild type.

Gene expression profiling of skeletal muscle in exercise-trained and sedentary rats with inborn high and low VO2max.

The relationship between inborn maximal oxygen uptake (VO(2max)) and skeletal muscle gene expression is unknown. Since low VO(2max) is a strong predictor of cardiovascular mortality, genes related to low VO(2max) might also be involved in cardiovascular disease. To establish the relationship between inborn VO(2max) and gene expression, we performed microarray analysis of the soleus muscle of rats artificially selected for high- and low running capacity (HCR and LCR, respectively). In LCR, a low VO(2max) was accompanied by aggregation of cardiovascular risk factors similar to the metabolic syndrome. Although sedentary HCR were able to maintain a 120% higher running speed at VO(2max) than sedentary LCR, only three transcripts were differentially expressed (FDR

Myocardial sarcoplasmic reticulum Ca2+ ATPase function is increased by aerobic interval training.

OBJECTIVE: Reduced activity of the sarcoplasmic reticulum Ca ATPase-2a (SERCA-2a) contributes to myocardial dysfunction. Exercise training improves myocardial Ca-handling, but SERCA-2a function is uncertain. We assessed SERCA-2a activity after exercise training. METHODS: SERCA-2a function was assessed by sarcoplasmic reticulum Ca uptake in cardiomyocytes with other Ca uptake mechanisms blocked, in mice after aerobic interval training versus sedentary controls. RESULTS: We established protocols to assess SERCA-2a function, and show that aerobic interval training increases the maximal rate of Ca uptake by 30%. This is at least partly explained by reduced phospholamban-to-SERCA-2a ratio. CONCLUSION: Aerobic interval training improves myocardial SERCA-2a performance, explaining at least partly why myocardial Ca-handling improves after exercise training.

Unilateral arm strength training improves contralateral peak force and rate of force development.

Neural adaptation following maximal strength training improves the ability to rapidly develop force. Unilateral strength training also leads to contralateral strength improvement, due to cross-over effects. However, adaptations in the rate of force development and peak force in the contralateral untrained arm after one-arm training have not been determined. Therefore, we aimed to detect contralateral effects of unilateral maximal strength training on rate of force development and peak force. Ten adult females enrolled in a 2-month strength training program focusing of maximal mobilization of force against near-maximal load in one arm, by attempting to move the given load as fast as possible. The other arm remained untrained. The training program did not induce any observable hypertrophy of any arms, as measured by anthropometry. Nevertheless, rate of force development improved in the trained arm during contractions against both submaximal and maximal loads by 40-60%. The untrained arm also improved rate of force development by the same magnitude. Peak force only improved during a maximal isometric contraction by 37% in the trained arm and 35% in the untrained arm. One repetition maximum improved by 79% in the trained arm and 9% in the untrained arm. Therefore, one-arm maximal strength training focusing on maximal mobilization of force increased rapid force development and one repetition maximal strength in the contralateral untrained arm. This suggests an increased central drive that also crosses over to the contralateral side.

Aerobic capacity-dependent differences in cardiac gene expression.

Aerobic capacity is a strong predictor of cardiovascular mortality. To determine the relationship between inborn aerobic capacity and cardiac gene expression we examined genome-wide gene expression in hearts of rats artificially selected for high and low running capacity (HCR and LCR, respectively) over 16 generations. The artificial selection of LCR caused accumulation of risk factors of cardiovascular disease similar to the metabolic syndrome seen in human, whereas HCR had markedly better cardiac function. We also studied alterations in gene expression in response to exercise training in these animals. Left ventricle gene expression of both sedentary and exercise-trained HCR and LCR was characterized by microarray and gene ontology analysis. Out of 28,000 screened genes, 1,540 were differentially expressed between sedentary HCR and LCR. Only one gene was found differentially expressed by exercise training, but this gene had unknown name and function. Sedentary HCR expressed higher amounts of genes involved in lipid metabolism, whereas sedentary LCR expressed higher amounts of the genes involved in glucose metabolism. This suggests a switch in cardiac energy substrate utilization from normal mitochondrial fatty acid beta-oxidation in HCR to carbohydrate metabolism in LCR, an event that often occurs in diseased hearts. LCR were also associated with pathological growth signaling and cellular stress. Hypoxic conditions seemed to be a common source for several of these observations, triggering hypoxia-induced alterations of transcription. In conclusion, inborn high vs. low aerobic capacity was associated with differences in cardiac energy substrate, growth signaling, and cellular stress.

Activation or inactivation of cardiac Akt/mTOR signaling diverges physiological from pathological hypertrophy.

Cardiomyocyte hypertrophy differs according to the stress exerted on the myocardium. While pressure overload-induced cardiomyocyte hypertrophy is associated with depressed contractile function, physiological hypertrophy after exercise training associates with preserved or increased inotropy. We determined the activation state of myocardial Akt signaling with downstream substrates and fetal gene reactivation in exercise-induced physiological and pressure overload-induced pathological hypertrophies. C57BL/6J mice were either treadmill trained for 6 weeks, 5 days/week, at 85-90% of maximal oxygen uptake (VO(2max)), or underwent transverse aortic constriction (TAC) for 1 or 8 weeks. Total and phosphorylated protein levels were determined with SDS-PAGE, and fetal genes by real-time RT-PCR. In the physiologically hypertrophied heart after exercise training, total Akt protein level was unchanged, but Akt was chronically hyperphosphorylated at serine 473. This was accompanied by activation of the mammalian target of rapamycin (mTOR), measured as phosphorylation of its two substrates: the ribosomal protein S6 kinase-1 (S6K1) and the eukaryotic translation initiation factor-4E binding protein-1 (4E-BP1). Exercise training did not reactivate the fetal gene program (beta-myosin heavy chain, atrial natriuretic factor, skeletal muscle actin). In contrast, pressure overload after TAC reactivated fetal genes already after 1 week, and partially inactivated the Akt/mTOR pathway and downstream substrates after 8 weeks. In conclusion, changes in opposite directions of the myocardial Akt/mTOR signal pathway appears to distinguish between physiological and pathological hypertrophies; exercise training associating with activation and pressure overload associating with inactivation of the Akt/mTOR pathway.

Exercise training restores aerobic capacity and energy transfer systems in heart failure treated with losartan.

OBJECTIVE: Clinical and experimental studies demonstrate that exercise training improves aerobic capacity and cardiac function in heart failure, even in patients on optimal treatment with angiotensin inhibitors and beta-blockers, but the cellular mechanisms are incompletely understood. Since myocardial dysfunction is frequently associated with impaired energy status, the aim of this study was to assess the effects of exercise training and losartan on myocardial systems for energy production and transfer in heart failure. METHODS: Maximal oxygen uptake, cardiac function and energy metabolism were assessed in heart failure after a myocardial infarction induced by coronary artery ligation in female Sprague-Dawley rats. Losartan was initiated one week after infarction and exercise training after four weeks, either as single interventions or combined. Animals were sacrificed 12 weeks after surgery. RESULTS: Heart failure, confirmed by left ventricular diastolic pressure >15 mmHg and by >20 mmHg drop in peak systolic pressure, was associated with 40% lower aerobic capacity and significant reductions in enzymes involved in energy metabolism. Combined treatment yielded best improvement of aerobic capacity and ventricular pressure characteristics. Exercise training completely restored aerobic capacity and partly or fully restored creatine and adenylate kinases, whereas losartan alone further reduced these enzymes. In contrast, losartan reduced left ventricle diastolic pressure, whereas exercise training had a neutral effect. CONCLUSION: Exercise training markedly improves aerobic capacity and cardiac function after myocardial infarction, either alone or in combination with angiotensin inhibition. The two interventions appear to act by complementary mechanisms; whereas exercise training restores cardiac energy metabolism, mainly at the level of energy transfer, losartan unloads the heart by lowering filling pressure and afterload.

Time-course of endothelial adaptation following acute and regular exercise.

BACKGROUND: Regular exercise training has emerged as a powerful tool to improve endothelium-dependent vasorelaxation. However, little is known about the magnitude of change and the permanence of exercise-induced adaptations in endothelial function. DESIGN: Rats were randomized to either 6 weeks of regular exercise or one bout of exercise. Rats were then sacrificed 0, 6, 12, 24, 48, 96 or 192 h post-exercise, and vascular responsiveness to acetylcholine was determined. METHODS: Endothelium-dependent dilation was assessed by exposure to accumulating doses of acetylcholine in ring segments of the abdominal aorta from female Sprague-Dawley rats that either exercised regularly for 6 weeks or performed a single bout of exercise. RESULTS: A single exercise session improved endothelium-dependent vasodilatation for about 48 h. Six weeks of regular exercise induced a significantly larger improvement that lasted for about 192 h. Sensitivity to acetylcholine was twofold higher in chronically trained animals than in those exposed to a single bout of exercise. The decay after a single bout of exercise was about eightfold faster than that after 6 weeks of training. CONCLUSION: The present data extend our concept of exercise-induced adaptation of endothelium-dependent vasodilatation in two regards: (1) a single bout of exercise improves endothelium-dependent dilation for about 2 days, with peak effect after 12-24 h; (2) regular exercise further improves adaptation and increases the sensitivity to acetylcholine approximately fourfold, which slowly returns to sedentary levels within a week of detraining.

Aerobic fitness is associated with cardiomyocyte contractile capacity and endothelial function in exercise training and detraining.

BACKGROUND: Physical fitness and level of regular exercise are closely related to cardiovascular health. A regimen of regular intensity-controlled treadmill exercise was implemented and withdrawn to identify cellular mechanisms associated with exercise capacity and maximal oxygen uptake (VO2max). METHODS AND RESULTS: Time-dependent associations between cardiomyocyte dimensions, contractile capacity, and VO2max were assessed in adult rats after high-level intensity-controlled treadmill running for 2, 4, 8, and 13 weeks and detraining for 2 and 4 weeks. With training, cardiomyocyte length, relaxation, shortening, Ca2+ decay, and estimated cell volume correlated with increased VO2max (r=0.92, -0.92, 0.88, -0.84, 0.73; P<0.01). Multiple regression analysis identified cell length, relaxation, and Ca2+ decay as the main explanatory variables for VO2max (R2=0.87, P<0.02). When training stopped, exercise-gained VO2max decreased 50% within 2 weeks and stabilized at 5% above sedentary controls after 4 weeks. Cardiomyocyte size regressed in parallel with VO2max and remained (9%) above sedentary after 4 weeks, whereas cardiomyocyte shortening, contraction/relaxation- and Ca2+-transient time courses, and endothelium-dependent vasorelaxation regressed completely within 2 to 4 weeks of detraining. Cardiomyocyte length, estimated cell volume, width, shortening, and Ca2+ decay and endothelium-dependent arterial relaxation all correlated with VO2max (r=0.85, 0.84, 0.75, 0.63, -0.54, -0.37; P<0.01). Multiple regression identified cardiomyocyte length and vasorelaxation as the main determinants for regressed VO2max during detraining (R2=0.76, P=0.02). CONCLUSIONS: Cardiovascular adaptation to regular exercise is highly dynamic. On detraining, most of the exercise-gained aerobic fitness acquired over 2 to 3 months is lost within 2 to 4 weeks. The close association between cardiomyocyte dimensions, contractile capacity, arterial relaxation, and aerobic fitness suggests cellular mechanisms underlying these changes.

Intensity-controlled treadmill running in mice: cardiac and skeletal muscle hypertrophy.

Whereas novel pathways of pathological heart enlargement have been unveiled by thoracic aorta constriction in genetically modified mice, the molecular mechanisms of adaptive cardiac hypertrophy remain virtually unexplored and call for an effective and well-characterized model of physiological mechanical loading. Experimental procedures of maximal oxygen consumption (VO(2 max)) and intensity-controlled treadmill running were established in 40 female and 36 male C57BL/6J mice. An inclination-dependent VO(2 max) with 0.98 test-retest correlation was found at 25 degrees treadmill grade. Running for 2 h/day, 5 days/wk, in intervals of 8 min at 85-90% of VO(2 max) and 2 min at 50% (adjusted to weekly VO(2 max) testing) increased VO(2 max) to a plateau 49% above sedentary females and 29% in males. Running economy improved in both sexes, and echocardiography indicated significantly increased left ventricle posterior wall thickness. Ventricular weights increased by 19-29 and 12-17% in females and males, respectively, whereas cardiomyocyte dimensions increased by 20-32, and 17-23% in females and males, respectively; skeletal muscle mass increased by 12-18%. Thus the model mimics human responses to exercise and can be used in future studies of molecular mechanisms underlying these adaptations.