Elisa Calabria

Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy

Skeletal muscle atrophy is a debilitating response to fasting, disuse, cancer, and other systemic diseases. In atrophying muscles, the ubiquitin ligase, atrogin-1 (MAFbx), is dramatically induced, and this response is necessary for rapid atrophy. Here, we show that in cultured myotubes undergoing atrophy, the activity of the PI3K/AKT pathway decreases, leading to activation of Foxo transcription factors and atrogin-1 induction. IGF-1 treatment or AKT overexpression inhibits Foxo and atrogin-1 expression. Moreover, constitutively active Foxo3 acts on the atrogin-1 promoter to cause atrogin-1 transcription and dramatic atrophy of myotubes and muscle fibers. When Foxo activation is blocked by a dominant-negative construct in myotubes or by RNAi in mouse muscles in vivo, atrogin-1 induction during starvation and atrophy of myotubes induced by glucocorticoids are prevented. Thus, forkhead factor(s) play a critical role in the development of muscle atrophy, and inhibition of Foxo factors is an attractive approach to combat muscle wasting.

A protein kinase B-dependent and rapamycin- sensitive pathway controls skeletal muscle growth but not fiber type specification

Nerve activity controls fiber size and fiber type in skeletal muscle, but the underlying molecular mechanisms remain largely unknown. We have previously shown that Ras–mitogen-activated protein kinase and calcineurin control fiber type but not fiber size in regenerating rat skeletal muscle. Here we report that constitutively active protein kinase B (PKB), also known as Akt, increases fiber size and prevents denervation atrophy in regenerating and adult rat muscles but does not affect fiber type profile. The coexistence of hypertrophic muscle fibers overexpressing activated PKB with normal-size untransfected fibers within the same muscle points to a cell-autonomous control of muscle growth by PKB. The physiological role of this pathway is confirmed by the finding that PKB kinase activity and phosphorylation status are significantly increased in innervated compared with denervated regenerating muscles in parallel with muscle growth. Muscle fiber hypertrophy induced by activated PKB and by a Ras double mutant (RasV12C40) that activates selectively the phosphoinositide 3-kinase–PKB pathway is completely blocked by rapamycin, showing that the mammalian target of rapamycin kinase is the major downstream effector of this pathway in the control of muscle fiber size. On the other hand, nerve activity-dependent growth of regenerating muscle is only partially inhibited by dominant negative PKB and rapamycin, suggesting that other nerve-dependent signaling pathways are involved in muscle growth. The present results support the notion that fiber size and fiber type are regulated by nerve activity through different mechanisms.

Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.

Ras is involved in nerve-activity-dependent regulation of muscle genes

Gene expression in skeletal muscle is regulated by the firing pattern of motor neurons, but the signalling systems involved in excitation–transcription coupling are unknown. Here, using in vivo transfection in regenerating muscle, we show that constitutively active Ras and a Ras mutant that selectively activates the MAPK(ERK) pathway are able to mimic the effects of slow motor neurons on expression of myosin genes. Conversely, the effect of slow motor neurons is inhibited by a dominant-negative Ras mutant. MAPK(ERK) activity is increased by innervation and by low-frequency electrical stimulation. These results indicate that Ras–MAPK signalling is involved in promoting nerve-activity-dependent differentiation of slow muscle fibres in vivo.

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.

‘Fast’ and ‘slow’ muscle fibres in hindlimb muscles of adult rats regenerate from intrinsically different satellite cells

Myosin heavy chain (MyHC) expression was examined in regenerating fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of adult rats. Myotoxic bupivacaine was injected into SOL and EDL and the muscles were either denervated or neuromuscularly blocked by tetrodotoxin (TTX) on the sciatic nerve. Three to 10 or 30 days later, denervated SOL or EDL, or innervated but neuromuscularly blocked EDL received a slow 20 Hz stimulus pattern through electrodes implanted on the muscles or along the fibular nerve to EDL below the TTX block. In addition, denervated SOL and EDL received a fast 100 Hz stimulus pattern. Denervated EDL and SOL stimulated with the same slow stimulus pattern expressed different amounts of type 1 MyHC protein (8%versus 35% at 10 days, 13%versus 87% at 30 days). Stimulated denervated and stimulated innervated (TTX blocked) EDL expressed the same amounts of type 1, 2A, 2X and 2B MyHC proteins. Cross‐sections treated for in situ hybridization and immunocytochemistry showed expression of type 1 MyHC in all SOL fibres but only in some scattered single or smaller groups of fibres in EDL. The results suggest that muscle fibres regenerate from intrinsically different satellite cells in EDL and SOL and within EDL. However, induction by different extrinsic factors arising in extracellular matrix or from muscle position and usage in the limb has not been excluded. No evidence for nerve‐derived trophic influences was obtained.

Combinatorial cis-Acting Elements Control Tissue-specific Activation of the Cardiac Troponin I Gene in Vitro and in Vivo

The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (−230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the −230/+126 base pair promoter express β-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome.

Combinatorial cis-acting elements regulate tissue-specific expression of the cardiac troponin I gene in vitro and in vivo

The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (−230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the −230/+126 base pair promoter express β-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome.

How is muscle phenotype controlled by nerve activity

Abstract Motor neurons are known to affect muscle growth and fiber type profile (fast/slow, oxidative/glycolytic) by regulating muscle gene expression. However, the mechanism by which the information contained in specific action potential patterns is decoded by the transcriptional machinery of muscle fiber nuclei remains to be established. This is a basic issue in nerve/muscle biology, which has major implications in neurology, sport medicine and aging. We describe here a general strategy aimed at identifying the signal transduction pathways mediating the effects of nerve activity. This approach is based on the overexpression of constitutively active or dominant negative transduction factors in regenerating skeletal muscle.

Determination of maximal lactate steady state in healthy adults: can NIRS help?

PURPOSE We tested the hypothesis that the maximal lactate steady state (MLSS) can be accurately determined in healthy subjects based on measures of deoxygenated hemoglobin (deoxyHb), an index of oxygen extraction measured noninvasively by near-infrared spectroscopy (NIRS). METHODS Thirty-two healthy men (mean ± SD age = 48 ± 17 yr, range = 23-74 yr) performed an incremental cycling test to exhaustion and square wave tests for MLSS determination. Cardiorespiratory variables were measured bbb and deoxyHb was monitored noninvasively on the right vastus lateralis with a quantitative NIRS device. The individual values of V˙O2 and HR corresponding to the MLSS were calculated and compared to the NIRS-derived MLSS (NIRSMLSS) that was, in turn, determined by double linear function fitting of deoxyHb during the incremental exercise. RESULTS V˙O2 and HR at MLSS were 2.25 ± 0.54 L·min (76% ± 9% V˙O2max) and 133 ± 14 bpm (81% ± 7% HRmax), respectively. Muscle O2 extraction increased as a function of exercise intensity up to a deflection point, NIRSMLSS, at which V˙O2 and HR were 2.23 ± 0.59 L·min (76% ± 9% V˙O2max) and 136 ± 17 bpm (82% ± 8% HRmax), respectively. For both V˙O2 and HR, the difference of NIRSMLSS from MLSS values was not significant and the measures were highly correlated (r = 0.81 and r = 0.76). The Bland-Altman analysis confirmed a nonsignificant bias for V˙O2 and HR (-0.015 L·min and 3 bpm, respectively) and a small imprecision of 0.26 L·min and 8 bpm. CONCLUSIONS A plateau in muscle O2 extraction was demonstrated in coincidence with MLSS during an incremental cycling exercise, confirming the hypothesis that this functional parameter can be accurately estimated with a quantitative NIRS device. The main advantages of NIRSMLSS over lactate-based techniques are the noninvasiveness and the time/cost efficiency.