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

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.

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.

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.