Marta Canato

Inducible activation of Akt increases skeletal muscle mass and force without satellite cell activation

A better understanding of the signaling pathways that control muscle growth is required to identify appropriate countermeasures to prevent or reverse the loss of muscle mass and force induced by aging, disuse, or neuromuscular diseases. However, two major issues in this field have not yet been fully addressed. The first concerns the pathways involved in leading to physiological changes in muscle size. Muscle hypertrophy based on perturbations of specific signaling pathways is either characterized by impaired force generation, e.g., myostatin knockout, or incompletely studied from the physiological point of view, e.g., IGF‐1 overexpression. A second issue is whether satellite cell proliferation and incorporation into growing muscle fibers is required for a functional hypertrophy. To address these issues, we used an inducible transgenic model of muscle hypertrophy by short‐term Akt activation in adult skeletal muscle. In this model, Akt activation for 3 wk was followed by marked hypertrophy (̃50% of muscle mass) and by increased force generation, as determined in vivo by ankle plantar flexor stimulation, ex vivo in intact isolated diaphragm strips, and in single‐skinned muscle fibers. No changes in fiber‐type distribution and resistance to fatigue were detectable. Bromodeoxyuridine incorporation experiments showed that Akt‐dependent muscle hypertrophy was accompanied by proliferation of interstitial cells but not by satellite cell activation and new myonuclei incorporation, pointing to an increase in myonuclear domain size. We can conclude that during a fast hyper‐trophic growth myonuclear domain can increase without compromising muscle performance.—Blaauw, B., Canato, M., Agatea, L., Toniolo, L., Mammucari, C., Masiero, E., Abraham, R., Sandri, M., Schiaffino, S., Reggiani, C. Inducible activation of Akt increases skeletal muscle mass and force without satellite cell activation. FASEBJ. 23, 3896‐3905 (2009). www.fasebj.org

Reorganized stores and impaired calcium handling in skeletal muscle of mice lacking calsequestrin-1

Calsequestrin (CS), the major Ca2+‐binding protein in the sarcoplasmic reticulum (SR), is thought to play a dual role in excitation–contraction coupling: buffering free Ca2+ increasing SR capacity, and modulating the activity of the Ca2+ release channels (RyRs). In this study, we generated and characterized the first murine model lacking the skeletal CS isoform (CS1). CS1‐null mice are viable and fertile, even though skeletal muscles appear slightly atrophic compared to the control mice. No compensatory increase of the cardiac isoform CS2 is detectable in any type of skeletal muscle. CS1‐null muscle fibres are characterized by structural and functional changes, which are much more evident in fast‐twitch muscles (EDL) in which most fibres express only CS1, than in slow‐twitch muscles (soleus), where CS2 is expressed in about 50% of the fibres. In isolated EDL muscle, force development is preserved, but characterized by prolonged time‐to‐peak and half‐relaxation time, probably related to impaired calcium release from and re‐uptake by the SR. Ca2+‐imaging studies show that the amount of Ca2+ released from the SR and the amplitude of the Ca2+ transient are significantly reduced. The lack of CS1 also causes significant ultrastructural changes, which include: (i) striking proliferation of SR junctional domains; (ii) increased density of Ca2+‐release channels (confirmed also by 3H‐ryanodine binding); (iii) decreased SR terminal cisternae volume; (iv) higher density of mitochondria. Taken together these results demonstrate that CS1 is essential for the normal development of the SR and its calcium release units and for the storage and release of appropriate amounts of SR Ca2+.

Anesthetic- and heat-induced sudden death in calsequestrin-1-knockout mice

CaIsequestrin‐1 (CASQ1) is a moderate‐affinity, high‐capacity Ca2+‐binding protein in the sarcoplasmic reticulum (SR) terminal cisternae of skeletal muscle. CASQ1 functions as both a Ca2+‐binding protein and a luminal regulator of ryanodine receptor (RYR1)‐mediated Ca2+ release. Mice lacking skeletal CASQ1 are viable but exhibit reduced levels of releasable Ca2+ and altered contractile properties. Here we report that CASQ1‐null mice exhibit increased spontaneous mortality and susceptibility to heat‐and anesthetic‐induced sudden death. Exposure of CASQ1‐null mice to either 2% halo‐thane or heat stress triggers lethal episodes characterized by whole‐body contractures, elevated core temperature, and severe rhabdomyolysis, which are prevented by prior dantrolene administration. The characteristics of these events are remarkably similar to analogous episodes observed in humans with malignant hyperthermia (MH) and animal models of MH and environmental heat stroke (EHS). In vitro studies indicate that CASQ1‐null muscle exhibits increased contractile sensitivity to temperature and caffeine, temperature‐dependent increases in resting Ca2+, and an increase in the magnitude of depolarization‐induced Ca2+ release. These results demonstrate that CASQ1 deficiency alters proper control of RYR1 function and suggest CASQ1 as a potential candidate gene for linkage analysis in families with MH/EHS where mutations in the RYR1 gene are excluded.—Dainese, M.,Quarta, M., Lyfenko, A.D., Paolini, C., Canato, M., Reggiani, C., Dirksen, R.T., Protasi, F. Anesthetic‐and heat‐induced sudden death incalsequestrin‐1‐knockout mice. FASEB J. 23, 1710–1720 (2009)

Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe.

The cytosolic free Ca2+ transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca2+] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca2+ dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca2+] in enzymatically dissociated flexor digitorum brevis muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca2+] did not differ between WT, CSQ-KO, and CSQ-DKO fibers. During sustained contractions, changes were rather small in WT, reflecting powerful buffering of CSQ, whereas in CSQ-KO fibers, significant drops in SR [Ca2+] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca2+, both in CSQ-KO and CSQ-DKO fibers. In CSQ-KO fibers, cytosolic free calcium detected with Fura-2 declined during repetitive stimulation, indicating that SR calcium content was insufficient for sustained contractile activity. SR Ca2+ reuptake during and after stimulation trains appeared to be governed by three temporally distinct processes with rate constants of 50, 1–5, and 0.3 s−1 (at 26 °C), reflecting activity of the SR Ca2+ pump and interplay of luminal and cytosolic Ca2+ buffers and pointing to store-operated calcium entry (SOCE). SOCE might play an essential role during muscle contractures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.

Eccentric contractions lead to myofibrillar dysfunction in muscular dystrophy

It is commonly accepted that skeletal muscles from dystrophin-deficient mdx mice are more susceptible than those from wild-type mice to damage from eccentric contractions. However, the downstream mechanisms involved in this enhanced force drop remain controversial. We studied the reduction of contractile force induced by eccentric contractions elicited in vivo in the gastrocnemius muscle of wild-type mice and three distinct models of muscle dystrophy: mdx, alpha-sarcoglycan (Sgca)-null, and collagen 6A1 (Col6a1)-null mice. In mdx and Sgca-null mice, force decreased 35% compared with 14% in wild-type mice. Drop of force in Col6a1-null mice was comparable to that in wild-type mice. To identify the determinants of the force drop, we measured force generation in permeabilized fibers dissected from gastrocnemius muscle that had been exposed in vivo to eccentric contractions and from the contralateral unstimulated muscle. A force loss in skinned fibers after in vivo eccentric contractions was detectable in fibers from mdx and Sgca-null, but not wild-type and Col6a1-null, mice. The enhanced force reduction in mdx and Sgca-null mice was observed only when eccentric contractions were elicited in vivo, since eccentric contractions elicited in vitro had identical effects in wild-type and dystrophic skinned fibers. These results suggest that 1) the enhanced force loss is due to a myofibrillar impairment that is present in all fibers, and not to individual fiber degeneration, and 2) the mechanism causing the enhanced force reduction is active in vivo and is lost after fiber permeabilization.

Lessons from calsequestrin-1 ablation in vivo: much more than a Ca2+ buffer after all

Calsequestrin type-1 (CASQ1), the main sarcoplasmic reticulum (SR) Ca2+ binding protein, plays a dual role in skeletal fibers: a) it provides a large pool of rapidly-releasable Ca2+ during excitation–contraction (EC) coupling; and b) it modulates the activity of ryanodine receptors (RYRs), the SR Ca2+ release channels. We have generated a mouse lacking CASQ1 in order to further characterize the role of CASQ1 in skeletal muscle. Contrary to initial expectations, CASQ1 ablation is compatible with normal motor activity, in spite of moderate muscle atrophy. However, CASQ1 deficiency results in profound remodeling of the EC coupling apparatus: shrinkage of junctional SR lumen; proliferation of SR/transverse-tubule contacts; and increased density of RYRs. While force development during a twitch is preserved, it is nevertheless characterized by a prolonged time course, likely reflecting impaired Ca2+ re-uptake by the SR. Finally, lack of CASQ1 also results in increased rate of SR Ca2+ depletion and inability of muscle to sustain tension during a prolonged tetani. All modifications are more pronounced (or only found) in fast-twitch extensor digitorum longus muscle compared to slow-twitch soleus muscle, likely because the latter expresses higher amounts of calsequestrin type-2 (CASQ2). Surprisingly, male CASQ1-null mice also exhibit a marked increased rate of spontaneous mortality suggestive of a stress-induced phenotype. Consistent with this idea, CASQ1-null mice exhibit an increased susceptibility to undergo a hypermetabolic syndrome characterized by whole body contractures, rhabdomyolysis, hyperthermia and sudden death in response to halothane- and heat-exposure, a phenotype remarkably similar to human malignant hyperthermia and environmental heat-stroke. The latter findings validate the CASQ1 gene as a candidate for linkage analysis in human muscle disorders.

Calsequestrin (CASQ1) rescues function and structure of calcium release units in skeletal muscles of CASQ1-null mice.

Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.

Hypertrophy and transcriptional regulation induced in myogenic cell line L6-C5 by an increase of extracellular calcium

Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6‐C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003 ). Exposure of L6‐C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin‐positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA‐2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin‐dominant negative protein, suggesting the involvement in this process of the Ca2+ responsive phosphatase calcineurin. Furthermore, transient exposure of L6‐C5 cells to high [Ca2+]o increased the expression of luciferase reporter driven by myoglobin (Mb) and β‐MHC promoters but not IIB‐MHC and MCK promoters. Luciferase transcription driven by CK promoter was, instead, enhanced by mobilizing Ca2+ from the intracellular stores. These data indicate that a transient increase of [Ca2+]i due to plasma‐membrane influx is sufficient to induce a hypertrophic phenotype and an increased expression of slow‐fiber genes but not fast‐fiber genes.

Mechanical and Electrophysiological Properties of the Sarcolemma of Muscle Fibers in Two Murine Models of Muscle Dystrophy: Col6a1−/− and Mdx

This study aimed to analyse the sarcolemma of Col6a1−/− fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1−/− and increased in mdx fibers. In the membrane of Col6a1−/− fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane conductance and capacitance. In contrast, in mdx intact FDB muscle resting conductance was increased while resting potential and ap depolarization rate were reduced, likely indicating the presence of a consistent population of severely altered fibers which disappear during the culture preparation.

Nutrition and Acne: Therapeutic Potential of Ketogenic Diets

The influence of nutrition on skin health is a growing research area but the findings of various studies on the effect of diet on the development of acne have often been contradictory. The general opinion among researchers has oscillated between two different, opposing positions: that diet either is or is not a key factor for acne development. This review examines the evidence supporting an influence of various dietary components on the development of acne particularly focusing on the role played by carbohydrates. The physiological and biochemical effects of the ketogenic diet are examined from this perspective and mechanisms will be proposed via which this type of diet could have a role in the treatment of acne.