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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.

Background Skeletal muscle stem cells (satellite cells) are well known to participate in regeneration and maintenance of the tissue over time. Studies have shown increases in the number of satellite cells after exercise, but their functional role in endurance training remains unexplored. Methods Young adult mice were submitted to endurance exercise training and the function, differentiation, and metabolic characteristics of satellite cells were investigated in vivo and in vitro. Results We found that injured muscles from endurance‐exercised mice display improved regenerative capacity, demonstrated through higher densities of newly formed myofibres compared with controls (evidenced by an increase in embryonic myosin heavy chain expression), as well as lower inflammation (evidenced by quantifying CD68‐marked macrophages), and reduced fibrosis. Enhanced myogenic function was accompanied by an increased fraction of satellite cells expressing self‐renewal markers, while control satellite cells had morphologies suggestive of early differentiation. The beneficial effects of endurance exercise were associated with satellite cell metabolic reprogramming, including reduced mitochondrial respiration (O2 consumption) under resting conditions (absence of muscle injury) and increased stemness. During proliferation or activated states (3 days after injury), O2 consumption was equal in control and exercised cells, while exercise enhanced myogenic colony formation. Surprisingly, inhibition of mitochondrial O2 consumption was sufficient to enhance muscle stem cell self‐renewal characteristics in vitro. Moreover, transplanted muscle satellite cells from exercised mice or cells with reduced mitochondrial respiration promoted a significant reduction in inflammation compared with controls. Conclusions Our results indicate that endurance exercise promotes self‐renewal and inhibits differentiation in satellite cells, an effect promoted by metabolic reprogramming and respiratory inhibition, which is associated with a more favourable muscular response to injury.

Calorie restriction is known to promote healthy aging, which includes prevention of muscle loss. We investigated the effect of rodent calorie restriction on mitochondrial respiration and clonogenic capacity of muscle satellite stem cells, since metabolic alterations are known to regulate stem cell activity. Surprisingly, short or long-term calorie restriction do not change mitochondrial or glycolytic function. Nevertheless, both short- and long-term calorie restriction enhance myogenic colony formation. Overall, our results show that not all changes in satellite stem cell function are accompanied by metabolic remodeling.

Activation, proliferation, and differentiation of satellite cells can be influenced by extracellular factors, such as adiponectin. This adipokine has been proposed as a regulator of in vitro myogenesis, but its action on in vivo regeneration is not still elucidated. We used C57BL/6 (wild-type [WT]) and adiponectin knockout (AdKO) mice injured with barium chloride at periods of 3, 7, and 14 days after injury. The AdKO presented a higher number of centralized nuclei after 7 days, and a reduction in myogenic genes was observed after 3 days. Moreover, these mice presented an increase in anti-inflammatory cytokines after 3 and 7 days, and an increase in the M2 gene marker and proinflammatory cytokines after 7 days. The WT demonstrated an increase in adiponectin messenger RNA after 7 days. These results demonstrate that adiponectin is important in tissue remodeling during regeneration and that its deficiency does not compromise the maturation of muscle fibers, due to an increase in anti-inflammatory response; however, there is a possible impairment in proinflammatory response and an increase in centralized myonuclei.

Skeletal muscle stem cells (satellite cells) are well known to participate in regeneration and maintenance of the tissue over time. Studies have shown increases in the number of satellite cells after exercise, but their functional role in endurance training remains unexplored. Here, we found that injured muscles from endurance-exercised mice display improved regenerative capacity, demonstrated through higher densities of newly formed myofibers compared to controls, as well as lower inflammation and fibrosis. Enhanced myogenic function was accompanied by an increased fraction of satellite cells expressing self-renewal markers. Control satellite cells had morphologies suggestive of early differentiation, while endurance exercise enhanced myogenic colony formation. The beneficial effects of endurance exercise were associated with satellite cell metabolic reprogramming, including reduced mitochondrial respiration (O2consumption) under resting conditions (absence of muscle injury) and increased stemness. During proliferation or activated states (three days after injury), O2consumption was equal in control and exercised cells. Surprisingly, inhibition of mitochondrial O2consumption was sufficient to enhance muscle stem cell self-renewal characteristicsin vitro. Moreover, transplanted muscle satellite cells from exercised mice or cells with reduced mitochondrial respiration promoted a significant reduction in inflammation compared to controls. We propose that endurance exercise promotes self-renewal and inhibits differentiation in satellite cells, an effect promoted by metabolic reprogramming and respiratory inhibition, and which is associated with a more favorable muscular response to injury.

Changes in mitochondrial size and shape have been implicated in several physiologic processes, but their role in mitochondrial Ca(2+) uptake regulation and overall cellular Ca(2+) homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics toward increased fusion through expression of a dominant negative (DN) form of the fission protein [dynamin‐related protein 1 (DRP1)] markedly increased both mitochondrial Ca(2+) retention capacity and Ca(2+) uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion‐promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin (MFN)‐2 strongly reduced the mitochondrial Ca(2+) uptake speed and capacity in these cells. These changes were not dependent on modifications in mitochondrial calcium uniporter expression, inner membrane potentials, or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as indicated by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium‐replenishing process of store‐operated calcium entry was impaired in MFN2 knockdown cells, whereas DRP1‐DN‐promoted fusion resulted in faster cytosolic Ca(2+) increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca(2+) uptake, which impacts cellular Ca(2+) homeostasis.—Kowaltowski, A. J., Menezes‐Filho, S. L., Assali, E. A., Gonçalves, I. G., Cabral‐Costa, J. V., Abreu, P., Miller, N., Nolasco, P., Laurindo, F. R. M., Bruni‐Cardoso, A., Shirihai, O. Mitochondrial morphology regulates organellar Ca(2+) uptake and changes cellular Ca(2+) homeostasis. FASEB J. 33, 13176–13188 (2019). www.fasebj.org

Changes in mitochondrial size and shape have been implicated in several physiological processes, but their role in mitochondrial Ca2+uptake regulation and overall cellular Ca2+homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics towards increased fusion through expression of a dominant negative form of the fission protein DRP1 (DRP1‐DN) markedly increased both mitochondrial Ca2+retention capacity and Ca2+uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion‐promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin 2 (MFN2) strongly reduced mitochondrial Ca2+uptake speed and capacity in these cells. These changes were not dependent on modifications in inner membrane potentials or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as measured by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium‐replenishing process of store‐operated calcium entry (SOCE) was impaired in MFN2 knockdown cells, while DRP1‐DN‐promoted fusion resulted in faster cytosolic Ca2+increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca2+uptake, which impacts on cellular Ca2+homeostasis.

L-Glutamine (L-Gln) supplementation has been pointed out as an anticatabolic intervention, but its effects on protein synthesis and degradation signaling in skeketal muscle are still poorly known. The effects of L-Gln pretreatment (1 g kg-1 day-1 body weight for 10 days) on muscle fiber cross-sectional area (CSA), amino acid composition (measured by LC-MS/MS) and protein synthesis (Akt-mTOR) and degradation (ubiquitin ligases) signaling in soleus and extensor digitorum longus (EDL) muscles in 24-h-fasted mice were investigated. The fiber CSA of EDL muscle was not different between the L-Gln-fasted and L-Gln-fed groups. This finding was associated with reduced contents of L-Leu and L-Iso and activation of protein synthesis signaling (p-RPS6Ser240/244 and Akt-mTOR). The spectrum of soleus muscle fiber CSA distribution was larger in L-Gln-fasted as compared with placebo-fasted mice. This effect of L-Gln pretreatment was associated with changes in red fibers L-Gln metabolism as indicated by increased intracellular L-glutamine/L-glutamate ratio, L-aspartate and GABA levels. L-Gln supplementation reduced fasting-induced mass loss in tibialis anterior and gastrocnemius muscles. Evidence is presented that pretreatment with L-glutamine attenuates skeletal muscle atrophy induced by 24-h fasting through mechanisms that vary with the muscle fiber type.

New Findings What is the central question of this study? Oleic and linoleic acids modulate fibroblast proliferation and myogenic differentiation in vitro. However, their in vivo effects on muscle regeneration have not yet been examined. We investigated the effects of either oleic or linoleic acid on a well-established model of muscle regeneration after severe laceration. What is the main finding and its importance? We found that linoleic acid increases fibrous tissue deposition and impairs muscle regeneration and recovery of contractile function, whereas oleic acid has the opposite effects in severely injured gastrocnemius muscle, suggesting that linoleic acid has a harmful effect and oleic acid a potential therapeutic effect on muscle regeneration. Oleic and linoleic acids control fibroblast proliferation and myogenic differentiation in vitro; however, there was no study in skeletal muscle in vivo. The aim of this study was to evaluate the effects of either oleic or linoleic acid on the fibrous tissue content (collagen deposition) of muscle and recovery of contractile function in rat gastrocnemius muscle after being severely injured by laceration. Rats were supplemented with either oleic or linoleic acid for 4 weeks after laceration [0.44 g (kg body weight)−1 day−1]. Muscle injury led to an increase in oleic-to-stearic acid and palmitoleic-to-palmitic acid ratios, suggesting an increase in Δ9 desaturase activity. Increased fibrous tissue deposition and reduced isotonic and tetanic specific forces and resistance to fatigue were observed in the injured muscle. Supplementation with linoleic acid increased the content of eicosadienoic (20:2, n−6) and arachidonic (20:4, n−6) acids, reduced muscle mass and fibre cross-sectional areas, increased fibrous tissue deposition and further reduced the isotonic and tetanic specific forces and resistance to fatigue induced by laceration. Supplementation with oleic acid increased the content of docosahexaenoic acid (22:6, n−3) and abolished the increase in fibrous tissue area and the decrease in isotonic and tetanic specific forces and resistance to fatigue induced by muscle injury. We concluded that supplementation with linoleic acid impairs muscle regeneration and increases fibrous tissue deposition, resulting in impaired recovery of contractile function. Oleic acid supplementation reduced fibrous tissue deposition and improved recovery of contractile function, attenuating the tissue damage caused by muscle injury.

Several studies have generated numerous terms in the field of exercise training prescription and performance assessment that often do not match the information previously demonstrated by many other works, generating much debate and resulting in an immense pool of scientific results. Several protocols in exercise training prescription and performance assessment have been proposed for these purposes by many reasons. In the field of exercise science, the protocol must be thoroughly investigated and provide real tools to be reproducible. Many laboratories have been adapting and developing evaluation protocols and testing on physical training of rodents in different experimental conditions. In this context, mice, rats and rabbits are preferentially chosen due to easy manipulation and good response to exercise, and comparable at results obtained with humans in compatible effort intensities. But, the exercise training programs and aerobic-anaerobic transition assessment proposed for animal models vary extensively, depending on the species, gender, age, type of stimulus, type of exercise, type of method and also on the specific objectives of the program. This short review demonstrates the need in offering tools performed by invasive measurement to assess the anaerobic threshold by blood lactate employed on evolution of aerobic-anaerobic parameters of rodents. The objective of this short review was to present and to discuss physical evaluation protocols applications to rodents. The table submitted may give a basis for anaerobic threshold employed on exercise training prescription and performance assessment for laboratory rodents in future research.

Muscle stem cells or satellite cells are crucial for muscle maintenance and repair. These cells are mitotically quiescent and uniformly express the transcription factor Pax7, intermittently entering the cell cycle to give rise to daughter myogenic precursors cells and fuse with neighboring myofibers or self-renew, replenishing the stem cell pool in adult skeletal muscle. Pivotal roles of muscle stem cells in muscle repair have been uncovered, but it still remains unclear how muscle stem cell self-renewal is molecularly regulated and how muscle stem cells maintain muscle tissue homeostasis. Defects in muscle stem cell regulation to maintain/return to quiescence and self-renew are observed in degenerative conditions such as aging and neuromuscular disease. Recent works has suggested the existence of metabolic regulation and mitochondrial alterations in muscle stem cells, influencing the self-renewal commitment and function. Here I present a brief overview of recent understanding of how metabolic reprogramming governs self-renewal commitment, which is essential for conservation of muscle satellite cell pools throughout life, as well as the implications for regenerative medicine.

This is a modified experimental model previously developed in mouse to study skeletal muscle laceration in rats. All experimental procedures are performed during the light period, including anesthesia and surgery. The animals are randomly distributed into control and injured groups prior to the procedure. This experimental model can be used to investigate skeletal muscle laceration repair.

This is a modified experimental model previously developed in mouse to study skeletal muscle laceration in rats. All experimental procedures are performed during the light period, including anesthesia and surgery. The animals are randomly distributed into control and injured groups prior to the procedure. This experimental model can be used to investigate skeletal muscle laceration repair.

Summary The skeletal muscle tissue has a remarkable ability to alter its plastic structural and functional properties after a harmful stimulus, regulating the expression of proteins in complex events such as muscle regeneration. In this context, considering that potential therapeutic agents have been widely studied, nutritional strategies have been investigated in order to improve the regenerative capacity of skeletal muscle. There is evidence of the modulatory action of fatty acids, such that oleic and linoleic acids, that are abundant in Western diets, on muscle function and trophism. Thus, fatty acids appear to be potential candidates to promote or impair the recovery of muscle mass and function during regeneration, since they modulate intracellular pathways that regulate myogenesis. This study is the first to describe and discuss the effect of fatty acids on muscle plasticity and trophism, with emphasis on skeletal muscle regeneration and in vitro differentiation of muscle cells. Resumo O tecido muscular esquelético possui a notável capacidade plástica de alterar suas propriedades estruturais e funcionais após um estímulo lesivo, regulando a expressão de proteínas durante eventos complexos como a regeneração muscular. Nesse contexto, considerando que possíveis agentes terapêuticos vêm sendo amplamente estudados, estratégias nutricionais têm sido investigadas na perspectiva de melhorar a capacidade regenerativa do músculo esquelético. Há evidências da ação modulatória dos ácidos graxos, como os ácidos oleico e linoleico, que são abundantes nas dietas ocidentais, sobre a função muscular e o trofismo. Nesse sentido, os ácidos graxos parecem ser potenciais candidatos para promover ou prejudicar a recuperação da massa e a função muscular durante a regeneração, uma vez que modulam vias intracelulares reguladoras da miogênese. Este trabalho é o primeiro a descrever e discutir o efeito dos ácidos graxos sobre a plasticidade e o trofismo muscular, com ênfase na regeneração do músculo esquelético e na diferenciação de células musculares in vitro.

The aim of this research was to investigate the effects of endurance training on reduction of plasma glucose during high intensity constant and incremental speed tests in Wistar rats. We hypothesized that plasma glucose might be decreased in the exercised group during heavy (more intense) exercise. Twenty-four 10-week-old male Wistar rats were randomly assigned to sedentary and exercised groups. The prescription of endurance exercise training intensity was determined as 60% of the maximum intensity reached at the incremental speed test. The animals were trained by running on a motorized treadmill, five days/week for a total period of 67 weeks. Plasma glucose during the constant speed test in the exercised group at 20 m/min was reduced at the 14th, 21st and 28th min compared to the sedentary group, as well at 25 m/min at the 21st and 28th min. Plasma glucose during the incremental speed test was decreased in the exercised group at the moment of exhaustion (48th min) compared to the sedentary group (27th min). Endurance training positively modulates the mitochondrial activity and capacity of substrate oxidation in muscle and liver. Thus, in contrast to other studies on high load of exercise, the effects of endurance training on the decrease of plasma glucose during constant and incremental speed tests was significantly higher in exercised than in sedentary rats and associated with improved muscle and hepatic oxidative capacity, constituting an important non-pharmacological intervention tool for the prevention of insulin resistance, including type 2 diabetes mellitus.

The effects of either eicosapentaenoic (EPA)‐ or docosahexaenoic (DHA)‐rich fish oils on hindlimb suspension (HS)‐induced muscle disuse atrophy were compared. Daily oral supplementations (0.3 mL/100 g b.w.) with mineral oil (MO) or high EPA or high DHA fish oils were performed in adult rats. After 2 weeks, the animals were subjected to HS for further 2 weeks. The treatments were maintained alongside HS. At the end of 4 weeks, we evaluated: body weight gain, muscle mass and fat depots, composition of fatty acids, cross‐sectional areas (CSA) of the soleus muscle and soleus muscle fibers, activities of cathepsin L and 26S proteasome, and content of carbonylated proteins in the soleus muscle. Signaling pathway activities associated with protein synthesis (Akt, p70S6K, S6, 4EBP1, and GSK3‐beta) and protein degradation (atrogin‐1/MAFbx, and MuRF1) were evaluated. HS decreased muscle mass, CSA of soleus muscle and soleus muscle fibers, and altered signaling associated with protein synthesis (decreased) and protein degradation (increased). The treatment with either fish oil decreased the ratio of omega‐6/omega‐3 fatty acids and changed protein synthesis‐associated signaling. EPA‐rich fish oil attenuated the changes induced by HS on 26S proteasome activity, CSA of soleus muscle fibers, and levels of p‐Akt, total p70S6K, p‐p70S6K/total p70S6K, p‐4EBP1, p‐GSK3‐beta, p‐ERK2, and total ERK 1/2 proteins. DHA‐rich fish oil attenuated the changes induced by HS on p‐4EBP1 and total ERK1 levels. The effects of EPA‐rich fish oil on protein synthesis signaling were more pronounced. Both EPA‐ and DHA‐rich fish oils did not impact skeletal muscle mass loss induced by non‐inflammatory HS.

RESUMO Os benefícios para a saúde e as adaptações fisiológicas ao exercício regular são amplamente conhecidos e, com o advento das ciências ômicas e moleculares, revelou-se uma complexa rede de vias de sinalização e moléculas reguladoras que coordenam a resposta adaptativa do músculo esquelético ao exercício. As mudanças orgânicas transientes, porém, são cumulativas no pós-exercício. Elas incluem, de forma principal, a transcrição de genes relacionados aos fatores regulatórios da miogênese, ao metabolismo de carboidratos, à mobilização de gorduras, ao transporte e oxidação de substratos, ao metabolismo mitocondrial através da fosforilação oxidativa e, por fim, à regulação transcricional de genes envolvidos na biogênese mitocondrial. Tendo em vista o grande impacto científico, resumiram-se neste trabalho, além de algumas das principais respostas moleculares sofridas pelo músculo esquelético com o exercício físico, fatores que coordenam a plasticidade muscular para o ganho de desempenho. Foram citadas dezenas de biomarcadores ligados a alguns aspectos moleculares das adaptações do músculo esquelético ao exercício físico, algumas principais vias sinalizadoras e o papel mitocondrial, revelando alguns novos paradigmas para o entendimento desta área científica. ABSTRACT The health benefits and physiological adaptations to regular physical exercise are widely known, and with the advent of the omic and molecular sciences, a complex network of signaling pathways and regulatory molecules that coordinate the adaptive response of skeletal muscle to exercise has been revealed. Transient organic changes, however, are cumulative in the post-exercise period. They mainly include transcription of genes related to regulatory factors of myogenesis, carbohydrate metabolism, fat mobilization, transport and oxidation of substrates, mitochondrial metabolism through oxidative phosphorylation, and finally, the transcriptional regulation of genes involved in mitochondrial biogenesis. Given their great scientific impact, in addition to some of the main molecular responses experienced by the skeletal muscle to exercise, factors that coordinate muscle plasticity for performance gain were summarized in this work. This review mentioned dozens of biomarkers linked to some molecular aspects of skeletal muscle adaptations to physical exercise, some major signaling pathways and the role of mitochondria, revealing some new paradigms for the understanding of this field of science. RESUMEN Los beneficios para la salud y las adaptaciones fisiológicas al ejercicio regular son ampliamente conocidos y, con el advenimiento de las ciencias ómicas y moleculares, se ha revelado una compleja red de vías de señalización y moléculas reguladoras que coordinan la respuesta adaptativa del músculo esquelético al ejercicio físico. Los cambios orgánicos transitorios, sin embargo, son acumulativos después del ejercicio. Ellos incluyen principalmente la trascripción de genes relacionados a los factores de regulación de la miogénesis, metabolismo de carbohidratos, movilización de grasas, transporte y oxidación de substratos, metabolismo mitocondrial por la fosforilación oxidativa y, en última instancia, la regulación transcripcional de genes involucrados en la biogénesis mitocondrial. Teniendo en cuenta el gran impacto científico, se resumió en este trabajo, además de algunas de las principales respuestas moleculares experimentadas por el músculo esquelético con el ejercicio físico, factores que coordinan la plasticidad muscular en la ganancia del rendimiento. Se citaron decenas de biomarcadores relacionados con algunos aspectos moleculares de las adaptaciones del músculo esquelético al ejercicio físico, algunas de las principales vías de señalización y el papel mitocondrial, revelando algunos nuevos paradigmas para el entendimiento de esta área científica.