Myonecrosis Induction by Intramuscular Injection of CTX.

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress. Graphical abstract.

The dominant-negative mitochondrial calcium uniporter subunit MCUb drives macrophage polarization during skeletal muscle regeneration.

Damaged skeletal muscle can regenerate because of the coordinated action of immune cells with muscle stem cells, called satellite cells. Proinflammatory macrophages infiltrate skeletal muscle soon after injury to sustain the proliferation of satellite cells. These macrophages later acquire the anti-inflammatory phenotype and promote the differentiation and fusion of satellite cells. Here, we showed that MCUb, the dominant-negative subunit of the mitochondrial calcium uniporter (MCU) complex, promotes muscle regeneration by controlling macrophage responses. Macrophages lacking MCUb lost the ability to efficiently acquire the anti-inflammatory profile, and mice with MCUb-deficient macrophages showed delayed regeneration through exhaustion of the satellite cell pool. MCUb ablation altered macrophage metabolism by promoting glycolysis and the accumulation of TCA cycle intermediates, which was accompanied by the stabilization of HIF-1α, the master transcriptional regulator of the macrophage proinflammatory program. Together, these data demonstrate that MCUb abundance is tightly controlled in macrophages to enable satellite cell functional differentiation and recovery of tissue homeostasis after damage.

Excessive Accumulation of Ca2 + in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

Mice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca2+ leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca2+ uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca2+ concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca2+ release from SR during repetitive electrical stimulation or caffeine administration. Also mitochondrial ROS production associated with contractile activity (detected with Mitosox probe) was much higher in YS fibers than in WT. Importantly, the inhibition of mitochondrial Ca2+ uptake achieved by silencing MCU reduced ROS accumulation in the matrix and Ca2+ release from SR. Finally, inhibition of mitochondrial ROS accumulation using Mitotempo reduced SR Ca2+ release in YS fibers exposed to caffeine. The present results support the view that mitochondria take up larger amounts of Ca2+ in YS than in WT fibers and that mitochondrial ROS production substantially contributes to the increased caffeine-sensitivity and to the enhanced Ca2+ release from SR in YS fibers.

High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels.

Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 µM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.