Intracellular Calcium Dynamics in Primary Human Adrenocortical Cells Deciphered with a Novel Pipeline.

INTRODUCTION: The fluctuations of the intracellular Ca2+ concentration ([Ca2+]i) are key physiological signals for cell function under normal conditions and can undergo profound alterations in disease states, as high blood pressure due to endocrine disorders like primary aldosteronism (PA). However, when assessing such fluctuations several parameters in the Ca2+ signal dynamics need to be considered, which renders their assessment challenging. AIM: Aim to develop an observer-independent custom-made pipeline to analyze Ca2+ dynamics in terms of frequency and peak parameters, as amplitude, full width at half maximum (FWHM) and area under the curve (AUC). METHODS: We applied a custom-made methodology to aldosterone-producing adenoma (APA) and APA adjacent cells (AAC) and found this pipeline to be suitable for monitoring and processing a wide-range of [Ca2+]i events in these cell types delivering reproducible results. CONCLUSION: The designed pipeline can provide a useful tool for [Ca2+]i signal analysis that allows comparisons of Ca2+ dynamics not only in PA, but in other cell phenotypes that are relevant for the regulation of blood pressure.

Endocrine disruptors and arterial hypertension: A developing story.

Endocrine disrupting Chemicals (EDCs) are substances that interfere with hormones by several mechanisms including receptor activation or antagonism, changes in gene and protein expression, modification of signal transduction, and/or epigenetic modifications in hormone-producing cells. A survey conducted by the European Union in a Northern Italian region led to the discovery of a large environmental contamination of drinking water by perfluoroalkyl substances (PFAS). As the exposed population showed a high prevalence of arterial hypertension and cardiovascular disease, we decided to investigate if PFAS could enhance the biosynthesis of aldosterone. To this aim, we exposed human adrenocortical carcinoma HAC15 cells to PFAS and found that PFAS markedly increased aldosterone synthase (CYP11B2) gene expression and aldosterone secretion. Moreover, we found that they promoted reactive oxygen species (ROS) production in mitochondria, the organelles where aldosterone biosynthesis takes place. PFAS also enhanced the effects of the aldosterone secretagogue angiotensin II (Ang II) on CYP11B2 gene expression and aldosterone secretion. We also found that not only PFAS but also polychlorinated biphenyl 126 (PCB126), a chemical compound belonging to a different category of EDCs, can increase CYP11B2 gene expression and aldosterone secretion in adrenocortical cells. This novel information needs to be considered in the context of a widespread exposure to the most common EDC, that is excess Na+ intake, whose detrimental effects on human health occur in the setting of aldosterone production exceeding the physiological needs and lead to high blood pressure, congestion, and cardiovascular and renal damage.

Aldosterone Biosynthesis Is Potently Stimulated by Perfluoroalkyl Acids: A Link between Common Environmental Pollutants and Arterial Hypertension.

The large environmental contamination of drinking water by perfluoroalkyl substances (PFAS) markedly increased the plasma levels of pentadecafluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in a Northern Italy population with a high prevalence of arterial hypertension and cardiovascular disease. As the link between PFAS and arterial hypertension is unknown, we investigated if they enhance the biosynthesis of the well-known pressor hormone aldosterone. We found that PFAS increased aldosterone synthase (CYP11B2) gene expression by three-fold and doubled aldosterone secretion and cell and mitochondria reactive oxygen species (ROS) production over controls (p < 0.01 for all) in human adrenocortical carcinoma cells HAC15. They also enhanced the effects of Ang II on CYP11B2 mRNA and aldosterone secretion (p < 0.01 for all). Moreover, when added 1 h before, the ROS scavenger tempol abolished the effect of PFAS on CYP11B2 gene expression. These results indicate that at concentrations mimicking those found in human plasma of exposed individuals, PFAS are potent disruptors of human adrenocortical cell function, and might act as causative factors of human arterial hypertension via increased aldosterone production.

Heterogeneity of the endoplasmic reticulum Ca2+ store determines colocalization with mitochondria.

Contact sites between the endoplasmic reticulum (ER) and mitochondria play a pivotal role in cell signaling, and the interaction between these organelles is dynamic and finely regulated. We have studied the role of ER Ca2+ concentration ([Ca2+]ER) in modulating this association in HeLa and HEK293 cells and human fibroblasts. According to Manders' coefficient, ER-mitochondria colocalization varied depending on the ER marker; it was the highest with ER-Tracker and the lowest with ER Ca2+ indicators (Mag-Fluo-4, erGAP3, and G-CEPIA1er) in both HeLa cells and human fibroblasts. Only GEM-CEPIA1er displayed a high colocalization with elongated mitochondria in HeLa cells, this ER Ca2+ indicator reveals low Ca2+ regions because this ion quenches its fluorescence. On the contrary, the typical rounded and fragmented mitochondria of HEK293 cells colocalized with Mag-Fluo-4 and, to a lesser extent, with GEM-CEPIA1er. The ablation of the three IP3R isoforms in HEK293 cells increased mitochondria-GEM-CEPIA1er colocalization. This pattern of colocalization was inversely correlated with the rate of ER Ca2+ leak evoked by thapsigargin (Tg). Moreover, Tg and Histamine in the absence of external Ca2+ increased mitochondria-ER colocalization. On the contrary, in the presence of external Ca2+, both Bafilomycin A1 and Tg reduced the mitochondria-ER interaction. Notably, knocking down MCU decreased mitochondria-ER colocalization. Overall, our data suggest that the [Ca2+] is not homogenous within the ER lumen and that mitochondria-ER interaction is modulated by the ER Ca2+ leak and the [Ca2+]i.

From the Identification to the Dissection of the Physiological Role of the Mitochondrial Calcium Uniporter: An Ongoing Story.

The notion of mitochondria being involved in the decoding and shaping of intracellular Ca2+ signals has been circulating since the end of the 19th century. Despite that, the molecular identity of the channel that mediates Ca2+ ion transport into mitochondria remained elusive for several years. Only in the last decade, the genes and pathways responsible for the mitochondrial uptake of Ca2+ began to be cloned and characterized. The gene coding for the pore-forming unit of the mitochondrial channel was discovered exactly 10 years ago, and its product was called mitochondrial Ca2+ uniporter or MCU. Before that, only one of its regulators, the mitochondria Ca2+ uptake regulator 1, MICU1, has been described in 2010. However, in the following years, the scientific interest in mitochondrial Ca2+ signaling regulation and physiological role has increased. This shortly led to the identification of many of its components, to the description of their 3D structure, and the characterization of the uniporter contribution to tissue physiology and pathology. In this review, we will summarize the most relevant achievements in the history of mitochondrial Ca2+ studies, presenting a chronological overview of the most relevant and landmarking discoveries. Finally, we will explore the impact of mitochondrial Ca2+ signaling in the context of muscle physiology, highlighting the recent advances in understanding the role of the MCU complex in the control of muscle trophism and metabolism.

Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity.

Casein kinase 2 (CK2) is a tetrameric protein kinase composed of 2 catalytic (α and α') and 2 regulatory ß subunits. Our study provides the first molecular and cellular characterization of the different CK2 subunits, highlighting their individual roles in skeletal muscle specification and differentiation. Analysis of C2C12 cell knockout for each CK2 subunit reveals that: 1) CK2ß is mandatory for the expression of the muscle master regulator myogenic differentiation 1 in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle-specific gene expression and myotube formation; 2) CK2α is involved in the activation of the muscle-specific gene program; and 3) CK2α' activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. Accordingly, CK2α' overexpression in C2C12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. Consistent with these results, pharmacological inhibition of CK2 activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro in C2C12 and primary myoblasts and in vivo in mouse regenerating muscle and zebrafish development. Overall, our work describes the specific and coordinated functions of CK2 subunits in orchestrating muscle differentiation and fusogenic activity, highlighting CK2 relevance in the physiopathology of skeletal muscle tissue.-Salizzato, V., Zanin, S., Borgo, C., Lidron, E., Salvi, M., Rizzuto, R., Pallafacchina, G., Donella-Deana, A. Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity.

Aldosterone Stimulates Its Biosynthesis Via a Novel GPER-Mediated Mechanism.

CONTEXT: The G protein-coupled estrogen receptor (GPER) mediates an aldosterone secretagogue effect of 17ß-estradiol in human HAC15 adrenocortical cells after estrogen receptor ß blockade. Because GPER mediates mineralocorticoid receptor-independent aldosterone effects in other cell types, we hypothesized that aldosterone could modulate its own synthesis via GPER activation. METHODS: HAC15 cells were exposed to aldosterone in the presence or absence of canrenone, a mineralocorticoid receptor antagonist, and/or of the selective GPER antagonist G36. Aldosterone synthase (CYP11B2) mRNA and protein levels changes were the study end points. Similar experiments were repeated in strips obtained ex vivo from aldosterone-producing adenoma (APA) and in GPER-silenced HAC15 cells. RESULTS: Aldosterone markedly increased CYP11B2 mRNA and protein expression (vs untreated samples, P < 0.001) in both models by acting via GPER, because these effects were abolished by G36 (P < 0.01) and not by canrenone. GPER-silencing (P < 0.01) abolished the aldosterone-induced increase of CYP11B2, thus proving that aldosterone acts via GPER to augment the step-limiting mitochondrial enzyme (CYP11B2) of its synthesis. Angiotensin II potentiated the GPER-mediated effect of aldosterone on CYP11B2. Coimmunoprecipitation studies provided evidence for GPER-angiotensin type-1 receptor heterodimerization. CONCLUSION: We propose that this autocrine-paracrine mechanism could enhance aldosterone biosynthesis under conditions of immediate physiological need in which the renin-angiotensin-aldosterone system is stimulated as, for example, hypovolemia. Moreover, as APA overexpresses GPER this mechanism could contribute to the aldosterone excess that occurs in primary aldosteronism in a seemingly autonomous fashion from angiotensin II.

Methods to Measure Intracellular Ca2+ Concentration Using Ca2+-Sensitive Dyes.

Ca2+ ion is universally considered the most versatile second messenger responsible for decoding and regulating the majority of the signaling pathways within the cell. The study of intracellular Ca2+ concentration ([Ca2+]i) dynamics is consequently of primary importance for the interpretation of cellular biology. This chapter will present a relatively simple, largely diffused, and nevertheless robust method to measure variations of [Ca2+]i by the use of the Ca2+-sensitive chemical dye Fura-2. A general protocol for the assessment of [Ca2+]i in adherent cells, applicable to a variety of cell systems, will be first presented. Then, the implementation of Fura-2 to detect [Ca2+]i in two specific cell types, namely, human adrenocortical cells and primary skin fibroblasts, will be discussed in more particulars. Finally, the procedure to monitor Ca2+ influx through the plasma membrane using Fura-2 will be described.

Recent advances in the molecular mechanism of mitochondrial calcium uptake.

In the last few decades, a large body of experimental evidence has highlighted the complex role for mitochondria in eukaryotic cells: they are not only the site of aerobic metabolism (thus providing most of the ATP supply for endergonic processes) but also a crucial checkpoint of cell death processes (both necrosis and apoptosis) and autophagy. For this purpose, mitochondria must receive and decode the wide variety of physiological and pathological stimuli impacting on the cell. The "old" notion that mitochondria possess a sophisticated machinery for accumulating and releasing Ca 2+, the most common and versatile second messenger of eukaryotic cells, is thus no surprise. What may be surprising is that the identification of the molecules involved in mitochondrial Ca 2+ transport occurred only in the last decade for both the influx (the mitochondrial Ca 2+ uniporter, MCU) and the efflux (the sodium calcium exchanger, NCX) pathways. In this review, we will focus on the description of the amazing molecular complexity of the MCU complex, highlighting the numerous functional implications of the tissue-specific expression of the variants of the channel pore components (MCU/MCUb) and of the associated proteins (MICU 1, 2, and 3, EMRE, and MCUR1).

The role of Pitx2 and Pitx3 in muscle stem cells gives new insights into P38α MAP kinase and redox regulation of muscle regeneration.

Skeletal muscle regeneration depends on satellite cells. After injury these muscle stem cells exit quiescence, proliferate and differentiate to regenerate damaged fibres. We show that this progression is accompanied by metabolic changes leading to increased production of reactive oxygen species (ROS). Using Pitx2/3 single and double mutant mice that provide genetic models of deregulated redox states, we demonstrate that moderate overproduction of ROS results in premature differentiation of satellite cells while high levels lead to their senescence and regenerative failure. Using the ROS scavenger, N-Acetyl-Cysteine (NAC), in primary cultures we show that a physiological increase in ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38α MAP kinase. Subjecting cultured satellite cells to transient inhibition of P38α MAP kinase in conjunction with NAC treatment leads to their rapid expansion, with striking improvement of their regenerative potential in grafting experiments.

Role of p66shc in skeletal muscle function.

p66shc is a growth factor adaptor protein that contributes to mitochondrial ROS production. p66shc is involved in insulin signaling and its deletion exerts a protective effect against diet-induced obesity. In light of the role of skeletal muscle activity in the control of systemic metabolism and obesity, we investigated which is the contribution of p66shc in regulating muscle structure and function. Here, we show that p66shc-/- muscles are undistinguishable from controls in terms of size, resistance to denervation-induced atrophy, and force. However, p66shc-/- mice perform slightly better than wild type animals during repetitive downhill running. Analysis of the effects after placing mice on a high fat diet (HFD) regimen demonstrated that running distance is greatly reduced in obese wild type animals, but not in overweight-resistant p66shc-/- mice. In addition, muscle force measured after exercise decreases upon HFD in wild type mice while p66shc-/- animals are protected. Our data indicate that p66shc affect the response to damage of adult muscle in chow diet, and it determines the maintenance of muscle force and exercise performance upon a HFD regimen.

Loss-of-function mutations in the SIGMAR1 gene cause distal hereditary motor neuropathy by impairing ER-mitochondria tethering and Ca2+ signalling.

Distal hereditary motor neuropathies (dHMNs) are clinically and genetically heterogeneous neurological conditions characterized by degeneration of the lower motor neurons. So far, 18 dHMN genes have been identified, however, about 80% of dHMN cases remain without a molecular diagnosis. By a combination of autozygosity mapping, identity-by-descent segment detection and whole-exome sequencing approaches, we identified two novel homozygous mutations in the SIGMAR1 gene (p.E138Q and p.E150K) in two distinct Italian families affected by an autosomal recessive form of HMN. Functional analyses in several neuronal cell lines strongly support the pathogenicity of the mutations and provide insights into the underlying pathomechanisms involving the regulation of ER-mitochondria tethering, Ca2+ homeostasis and autophagy. Indeed, in vitro, both mutations reduce cell viability, the formation of abnormal protein aggregates preventing the correct targeting of sigma-1R protein to the mitochondria-associated ER membrane (MAM) and thus impinging on the global Ca2+ signalling. Our data definitively demonstrate the involvement of SIGMAR1 in motor neuron maintenance and survival by correlating, for the first time in the Caucasian population, mutations in this gene to distal motor dysfunction and highlight the chaperone activity of sigma-1R at the MAM as a critical aspect in dHMN pathology.

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle.

OBJECTIVE: Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. METHODS: We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. RESULTS: We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. CONCLUSIONS: We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca(2+)-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.

The mitochondrial calcium uniporter controls skeletal muscle trophism in vivo.

Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca(2+)], which control aerobic metabolism, cell death, and survival pathways. We investigated in vivo the effects of mitochondrial Ca(2+) homeostasis in skeletal muscle function and trophism by overexpressing or silencing the mitochondrial calcium uniporter (MCU). The results demonstrate that in both developing and adult muscles, MCU-dependent mitochondrial Ca(2+) uptake has a marked trophic effect that does not depend on aerobic control but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-Akt/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca(2+)-dependent organelle-to-nucleus signaling route that links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss.

The mitochondrial calcium uniporter (MCU): molecular identity and physiological roles.

The direct measurement of mitochondrial [Ca(2+)] with highly specific probes demonstrated that major swings in organellar [Ca(2+)] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca(2+) uniporter, the channel allowing rapid Ca(2+) accumulation across the inner mitochondrial membrane.

Characterization of Pax3-expressing cells from adult blood vessels.

We report expression of Pax3, an important regulator of skeletal muscle stem cell behaviour, in the brachial and femoral arteries of adult mice. In these contractile arteries of the limb, but not in the elastic arteries of the trunk, bands of GFP-positive cells were observed in Pax3(GFP/+) mice. Histological and biochemical examination of the vessels, together with clonal analysis after purification of Pax3-GFP-positive cells by flow cytometry, established their vascular smooth muscle identity. These blood-vessel-derived cells do not respond to inducers of other mesodermal cell types, such as bone, however, they can contribute to muscle fibre formation when co-cultured with skeletal muscle cells. This myogenic conversion depends on the expression of Pax3, but is rare and non-cell autonomous as it requires cell fusion. Myocardin, which promotes acquisition of a mature smooth muscle phenotype in these Pax3-GFP-positive cells, antagonises their potential for skeletal muscle differentiation. Genetic manipulation shows that myocardin is, however, positively regulated by Pax3, unlike genes for other myocardin-related factors, MRTFA, MRTFB or SRF. Expression of Pax3 overlaps with that reported for Msx2, which is required for smooth muscle differentiation of blood vessel-derived multipotent mesoangioblasts. These observations are discussed with respect to the origin and function of Pax3-expressing cells in blood vessels, and more general questions of cell fate determination and adult cell plasticity and reprogramming.

An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells.

The satellite cell of skeletal muscle provides a paradigm for quiescent and activated tissue stem cell states. We have carried out transcriptome analyses on satellite cells purified by flow cytometry from Pax3(GFP/+) mice. We compared samples from adult skeletal muscles where satellite cells are mainly quiescent, with samples from growing muscles or regenerating (mdx) muscles, where they are activated. Analysis of regulation that is shared by both activated states avoids other effects due to immature or pathological conditions. This in vivo profile differs from that of previously analyzed satellite cells activated after cell culture. It reveals how the satellite cell protects itself from damage and maintains quiescence, while being primed for activation on receipt of the appropriate signal. This is illustrated by manipulation of the corepressor Dach1, and by the demonstration that quiescent satellite cells are better protected from oxidative stress than those from mdx or 1-week-old muscles. The quiescent versus in vivo activated comparison also gives new insights into how the satellite cell controls its niche on the muscle fiber through cell adhesion and matrix remodeling. The latter also potentiates growth factor activity through proteoglycan modification. Dismantling the extracellular matrix is important for satellite cell activation when the expression of proteinases is up-regulated, whereas transcripts for their inhibitors are high in quiescent cells. In keeping with this, we demonstrate that metalloproteinase function is required for efficient regeneration in vivo.

Autocrine and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal.

Mechanisms governing muscle satellite cell withdrawal from cell cycle to enter into quiescence remain poorly understood. We studied the role of angiopoietin 1 (Ang1) and its receptor Tie-2 in the regulation of myogenic precursor cell (mpc) fate. In human and mouse, Tie-2 was preferentially expressed by quiescent satellite cells in vivo and reserve cells (RCs) in vitro. Ang1/Tie-2 signaling, through ERK1/2 pathway, decreased mpc proliferation and differentiation, increased the number of cells in G0, increased expression of RC-associated markers (p130, Pax7, Myf-5, M-cadherin), and downregulated expression of differentiation-associated markers. Silencing Tie-2 had opposite effects. Cells located in the satellite cell neighborhood (smooth muscle cells, fibroblasts) upregulated RC-associated markers by secreting Ang1 in vitro. In vivo, Tie-2 blockade and Ang1 overexpression increased the number of cycling and quiescent satellite cells, respectively. We propose that Ang1/Tie-2 signaling regulates mpc self-renewal by controlling the return to quiescence of a subset of satellite cells.

Muscle stem cell behavior is modified by microRNA-27 regulation of Pax3 expression.

Skeletal muscle stem cells are regulated by Pax3/7. During development, Pax3 is required for the maintenance of these cells in the somite and their migration to sites of myogenesis; high levels of Pax3 interfere with muscle cell differentiation, both in the embryo and in the adult. Quantitative fine-tuning of Pax3 is critical, and microRNAs provide a potential mechanism. We identify microRNA-27b (miR-27b), which directly targets the 3'-UTR of Pax3 mRNA, as such a regulator. miR-27b is expressed in the differentiating skeletal muscle of the embryonic myotome and in activated satellite cells of adult muscle. In vivo overexpression of a miR-27b transgene in Pax3-positive cells in the embryo leads to down-regulation of Pax3, resulting in interference with progenitor cell migration and in premature differentiation. In a complementary experiment, miR-27b inhibitors were transfected into cultures of adult muscle satellite cells that normally express miR-27b at the onset of differentiation, when Pax3 protein levels undergo rapid down-regulation. Interference with miR-27b function results in continuing Pax3 expression leading to more proliferation and a delay in the onset of differentiation. Pax7 levels are not affected. Introduction of miR-27b antagomirs at a site of muscle injury in vivo also affects Pax3 expression and regeneration in vivo. We therefore conclude that miR-27b regulates Pax3 protein levels and this down-regulation ensures rapid and robust entry into the myogenic differentiation program.

NFAT isoforms control activity-dependent muscle fiber type specification.

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle.