Cytochrome c oxidase deficiency associated with the first stop-codon point mutation in human mtDNA.

We have identified the first stop-codon point mutation in mtDNA to be reported in association with human disease. A 36-year-old woman experienced episodes of encephalopathy accompanied by lactic acidemia and had exercise intolerance and proximal myopathy. Histochemical analysis showed that 90% of muscle fibers exhibited decreased or absent cytochrome c oxidase (COX) activity. Biochemical studies confirmed a severe isolated reduction in COX activity. Muscle immunocytochemistry revealed a pattern suggestive of a primary mtDNA defect in the COX-deficient fibers and was consistent with either reduced stability or impaired assembly of the holoenzyme. Sequence analysis of mtDNA identified a novel heteroplasmic G-->A point mutation at position 9952 in the patient's skeletal muscle, which was not detected in her leukocyte mtDNA or in that of 120 healthy controls or 60 additional patients with mitochondrial disease. This point mutation is located in the 3' end of the gene for subunit III of COX and is predicted to result in the loss of the last 13 amino acids of the highly conserved C-terminal region of this subunit. It was not detected in mtDNA extracted from leukocytes, skeletal muscle, or myoblasts of the patient's mother or her two sons, indicating that this mutation is not maternally transmitted. Single-fiber PCR studies provided direct evidence for an association between this point mutation and COX deficiency and indicated that the proportion of mutant mtDNA required to induce COX deficiency is lower than that reported for tRNA-gene point mutations. The findings reported here represent only the second case of isolated COX deficiency to be defined at the molecular genetic level and reveal a new mutational mechanism in mitochondrial disease.     

An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy

A novel heteroplasmic 7587T-->C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T-->C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%-65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation.     

Mitochondrial myopathy in a child with a muscle-restricted mutation in the mitochondrial transfer RNA gene

We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNA^Asn, MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient’s muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNA^Asn steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.     

Slow fibers in the frog cruralis muscle

Extrafusal slow fibers have been identified by electron microscopy in a muscle fiber bundle isolated from the anterior head (m. cruralis) of the triceps femoris of Rana pipiens. Light microscopic examination of this muscle revealed two equally numerous fiber types, one Fibrillenstruktur-like (twitch) and the other structureless (slow). A comparison of force developed by tetanic stimulation and by K⁺ depolarization also suggested approximately equal numbers of twitch and slow fibers.     

Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav_1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm⁻¹) and SEN (469±13 MNs×mm⁻¹). Nav_1.5 positive fibers (1548±70 μm²) were 35% smaller than Nav_1.5 negative fibers (2367±78 μm²; P<0.05) in SEN muscle, whereas Nav_1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm²; P<0.05) where no Nav_1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav_1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.     

Alterations at the Cross-Bridge Level Are Associated with a Paradoxical Gain of Muscle Function In Vivo in a Mouse Model of Nemaline Myopathy

Nemaline myopathy is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. The first disease causing mutation (Met9Arg) was identified in the gene encoding α-tropomyosin_slow gene (TPM3). Considering the conflicting findings of the previous studies on the transgenic (Tg) mice carrying the TPM3 ^Met9Arg mutation, we investigated carefully the effect of the Met9Arg mutation in 8–9 month-old Tg(TPM3)^Met9Arg mice on muscle function using a multiscale methodological approach including skinned muscle fibers analysis and in vivo investigations by magnetic resonance imaging and 31-phosphorus magnetic resonance spectroscopy. While in vitro maximal force production was reduced in Tg(TPM3)^Met9Arg mice as compared to controls, in vivo measurements revealed an improved mechanical performance in the transgenic mice as compared to the former. The reduced in vitro muscle force might be related to alterations occuring at the cross-bridges level with muscle-specific underlying mechanisms. In vivo muscle improvement was not associated with any changes in either muscle volume or energy metabolism. Our findings indicate that TPM3(Met9Arg) mutation leads to a mild muscle weakness in vitro related to an alteration at the cross-bridges level and a paradoxical gain of muscle function in vivo. These results clearly point out that in vitro alterations are muscle-dependent and do not necessarily translate into similar changes in vivo.     

The MELAS mutation m.3243A>G promotes reactivation of fetal cardiac genes and an epithelial-mesenchymal transition-like program via dysregulation of miRNAs

The pathomechanisms underlying oxidative phosphorylation (OXPHOS) diseases are not well-understood, but they involve maladaptive changes in mitochondria-nucleus communication. Many studies on the mitochondria-nucleus cross-talk triggered by mitochondrial dysfunction have focused on the role played by regulatory proteins, while the participation of miRNAs remains poorly explored. MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is mostly caused by mutation m.3243A>G in mitochondrial tRNA^Leu(UUR) gene. Adverse cardiac and neurological events are the commonest causes of early death in m.3243A>G patients. Notably, the incidence of major clinical features associated with this mutation has been correlated to the level of m.3243A>G mutant mitochondrial DNA (heteroplasmy) in skeletal muscle. In this work, we used a transmitochondrial cybrid model of MELAS (100% m.3243A>G mutant mitochondrial DNA) to investigate the participation of miRNAs in the mitochondria-nucleus cross-talk associated with OXPHOS dysfunction. High-throughput analysis of small-RNA-Seq data indicated that expression of 246 miRNAs was significantly altered in MELAS cybrids. Validation of selected miRNAs, including miR-4775 and miR-218-5p, in patient muscle samples revealed miRNAs whose expression declined with high levels of mutant heteroplasmy. We show that miR-218-5p and miR-4775 are direct regulators of fetal cardiac genes such as NODAL, RHOA, ISL1 and RXRB, which are up-regulated in MELAS cybrids and in patient muscle samples with heteroplasmy above 60%. Our data clearly indicate that TGF-β superfamily signaling and an epithelial-mesenchymal transition-like program are activated in MELAS cybrids, and suggest that down-regulation of miRNAs regulating fetal cardiac genes is a risk marker of heart failure in patients with OXPHOS diseases.     

Differences in flux control and reserve capacity of cytochrome c oxidase (COX) in human skeletal muscle and brain suggest different metabolic effects of mild COX deficiencies

To evaluate tissue specific control of oxidative phosphorylation by cytochrome c oxidase (COX) we determined the flux control coefficient and the metabolic reserve capacity of this enzyme in human saponin-permeabilised muscle fibers and digitonin-treated parahippocampal homogenates. In these tissue preparations it is possible to investigate mitochondrial function under conditions which are close to the in vivo situation. In the presence of NAD-dependent substrates we observed, under active state conditions, a flux control coefficient of COX over oxidative phosphorylation of 0.24±0.07 and a 1.9±0.2-fold excess capacity in human skeletal muscle fibers. In human parahippocampal gyrus we determined, under similar conditions, a flux control coefficient of COX of 0.12±0.05 and a 3.9±0.6-fold excess capacity of the enzyme. The observed difference in metabolic control can be attributed to activity differences of COX in human brain and muscle mitochondria. Our results predict stronger metabolic effects of mild COX activity deficits in human skeletal muscle than in brain tissue.     

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

Wing-hearts in Mecoptera (insecta)

The accessory pulsatile organs for hemolymph circulation in the wings of 7 Mecoptera species were investigated by means of serial semi-thin sections, SEM and TEM. The wing-hearts are located in the dorsal meso- and metathorax, and have no connection to the aorta. Each wing-heart consists of a small hemolymph chamber formed above by the convex scutellum, and below by a horizontal muscular diaphragm. The chamber is connected to the posterior wing veins by a cuticular tube on each side of the body. The diaphragm (10–15 μm thick) is convex in cross-section and consists of transversely extended muscle fibers. Their ultrastructure reveals typical characters of myocardial and other visceral muscle fibers. The diaphragm muscle is innervated by a pair of thin nerves originating from the thoracic ganglion of each corresponding segment. The diaphragm is held in a convex position by numerous elastic strands (2 μm in diameter), which extend through the wing-heart lumen between the scutellum and the diaphragm. The diastolic phase of the wing-heart is caused by contraction of the diaphragm muscle fibers. Thus, the diaphragm flattens and hemolymph is drawn from the posterior wing veins. The systolic phase is caused by the elasticity of the suspending strands after relaxation of the muscle fibers. The elastic strands pull the diaphragm back into convex position and hemolymph is expelled out of the scutellum lumen into the thorax cavity through a valvular opening on the anterior side. The hemolymph flow from the posterior wing base to the scutellum lumen, was visualized by staining the hemolymph. In Panorpa communis the volume of the wing-heart lumen measures 1.6 × 10⁻² mm³ in the mesothorax, and 1.2 × 10⁻² mm³ in the metathorax. Each heartbeat transports a maximum of 65% of these volumes. The pumping frequency was 78 ± 20 beats per min, registered with a non-invasive photo-optical method in restrained animals. Corresponding pulsating movements occur as a passive phenomenon of wing-heart activity in a distinct area of the wing base. Only minor differences were found in the construction of wing-hearts among the investigated species, except for Boreus hyemalis, which lacks these accessory circulatory organs. The functional morphology of the wing-hearts in Mecoptera is compared with that of other Holometabola and aspects of the evolution of these organs are discussed.     

Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca²⁺ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca²⁺ channel that conducts L-type Ca²⁺ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca²⁺ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα_1S subunit abolishing Ca²⁺ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn²⁺ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca²⁺ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca²⁺ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca²⁺, Mn²⁺ entry and subsequent quenching did occur because Mn²⁺ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba²⁺ and Ba²⁺ currents were strongly blocked by external Ca²⁺. Ba²⁺ permeation was smaller, current kinetics slower and Ca²⁺ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca²⁺ impeding in this way permeation. Because Ba²⁺ binds with lower affinity to D, Ba²⁺ currents occur, but with reduced amplitudes as compared to Ba²⁺ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.     

Normal levels of wild-type mitochondrial DNA maintain cytochrome c oxidase activity for two pathogenic mitochondrial DNA mutations but not for m.3243A-->G

Mitochondrial DNA (mtDNA) mutations are a common cause of human disease and accumulate as part of normal ageing and in common neurodegenerative disorders. Cells express a biochemical defect only when the proportion of mutated mtDNA exceeds a critical threshold, but it is not clear whether the actual cause of this defect is a loss of wild-type mtDNA, an excess of mutated mtDNA, or a combination of the two. Here, we show that segments of human skeletal muscle fibers harboring two pathogenic mtDNA mutations retain normal cytochrome c oxidase (COX) activity by maintaining a minimum amount of wild-type mtDNA. For these mutations, direct measurements of mutated and wild-type mtDNA molecules within the same skeletal muscle fiber are consistent with the "maintenance of wild type" hypothesis, which predicts that there is nonselective proliferation of mutated and wild-type mtDNA in response to the molecular defect. However, for the m.3243A-->G mutation, a superabundance of wild-type mtDNA was found in many muscle-fiber sections with negligible COX activity, indicating that the pathogenic mechanism for this particular mutation involves interference with the function of the wild-type mtDNA or wild-type gene products.     

Clinical and cellular consequences of the mutation m.12300G>A in the mitochondrial tRNA(Leu(CUN)) gene

We report, for the first time, a patient with an overlap MERRF-NARP syndrome who carries the mutation m.12300G>A in the mitochondrial tRNA(Leu(CUN)) gene. The mutation was heteroplamic and more abundant in her muscle and fibroblast than in blood from her oligosymptomatic mother. Single muscle fiber analysis revealed that the proportion of mutant mtDNA in ragged red fibers was higher than that in normal fibers. Combined defects of mitochondrial respiratory chain complexes were detected in muscle, fibroblasts and transmitochondrial hybrid cells. Significant reduction of total ATP and mitochondrial membrane potential and an increased production of reactive oxygen species were observed.     

Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes

Mg²⁺-selective microelectrodes have been used to measure the intracellular free Mg²⁺ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 μm were backfilled with the Mg²⁺ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg²⁺. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18–24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements. In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than ?82 mV, the intracellular free Mg²⁺ concentration was 3.8±0.41 (S.E.) mM (n=58) at 22°C. No significant change in the intracellular free Mg²⁺ was observed following extensive (approx. 6 h) incubation in Mg²⁺-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg²⁺]_i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na⁺-free solutions. In depolarized muscle fibers (?23±2.7 mV) treated with 100 mM K⁺, the myoplasmic [Mg²⁺] was 3.7±0.45 (S.E.) mM, n=6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg²⁺. These results point out that [Mg²⁺]_i is not modified under these three different experimental conditions.     

A novel MT-CO1 m.6498C>A variation associated with the m.7444G>A mutation in the mitochondrial COI/tRNASer(UCN) genes in a patient with hearing impairment,diabetes and congenital visual loss

Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. Sensorineural hearing loss (SNHL) has been described in association to different mitochondrial multisystem syndromes, often involving the central nervous system, neuromuscular, or endocrine organs. In this study, we described a Tunisian young girl with hearing impairment, congenital visual loss and maternally inherited diabetes. No mutation was found in the mitochondrial tRNA^Leu(UUR) and the 12S rRNA genes. However, we detected the m.7444G>A mutation in the mitochondrial COI/tRNA^Ser(UCN) genes. This mutation eliminates the termination codon of the MT-CO1 gene and extends the COI polypeptide by three amino acids (Lys–Gln–Lys) to the C-terminal. The whole mitochondrial genome screening revealed the presence of a novel mutation m.6498C>A (L199I) in the mitochondrial DNA-encoded subunit I of the cytochrome c oxidase (COX). This “probably damaging” transversion affects a highly conserved domain and it was absent in 200 Tunisian controls. The studied patient was classified under the haplogroup H2a.     

Mutation Types and Aging Differently Affect Revertant Fiber Expansion in Dystrophic Mdx and Mdx52 Mice

Duchenne muscular dystrophy (DMD), one of the most common and lethal genetic disorders, and the mdx mouse myopathies are caused by a lack of dystrophin protein. These dystrophic muscles contain sporadic clusters of dystrophin-expressing revertant fibers (RFs), as detected by immunohistochemistry. RFs are known to arise from muscle precursor cells with spontaneous exon skipping (alternative splicing) and clonally expand in size with increasing age through the process of muscle degeneration/regeneration. The expansion of revertant clusters is thought to represent the cumulative history of muscle regeneration and proliferation of such precursor cells. However, the precise mechanisms by which RFs arise and expand are poorly understood. Here, to test the effects of mutation types and aging on RF expansion and muscle regeneration, we examined the number of RFs in mdx mice (containing a nonsense mutation in exon 23) and mdx52 mice (containing deletion mutation of exon 52) with the same C57BL/6 background at 2, 6, 12, and 18months of age. Mdx mice displayed a significantly higher number of RFs compared to mdx52 mice in all age groups, suggesting that revertant fiber expansion largely depends on the type of mutation and/or location in the gene. A significant increase in the expression and clustering levels of RFs was found beginning at 6months of age in mdx mice compared with mdx52 mice. In contrast to the significant expansion of RFs with increasing age, the number of centrally nucleated fibers and embryonic myosin heavy chain-positive fibers (indicative of cumulative and current muscle regeneration, respectively) decreased with age in both mouse strains. These results suggest that mutation types and aging differently affect revertant fiber expansion in mdx and mdx52 mice.     

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit,Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca²⁺ release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490–524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490–508) is sufficient to reproduce activating effects of β1a490–524. Here we examined the effects of β1a490–508 on Ca²⁺ release and Ca²⁺ currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490–508 (25 nM) increased the peak Ca²⁺ release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490–508 in fibers. β1a490–508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490–508 peptide was used as negative control and produced negligible effects on Ca²⁺ release flux and RyR1 activity. Our results show that the β1a490–508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.     

MERRF syndrome without ragged-red fibers: the need for molecular diagnosis

We report a patient with myoclonic epilepsy who underwent muscle biopsy for suspected mitochondrial disease (myoclonic epilepsy with ragged-red fibers, MERRF). In spite of normal histochemical studies and of the absence of a severe COX deficiency, the molecular analysis showed the common MERRF mutation (A8344G) in the tRNA(Lys) gene on mitochondrial DNA. The case serves to illustrate the importance of pursuing the proposed mitochondrial genetic abnormality, even in patients with normal biopsy findings.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

The effects of low levels of dystrophin on mouse muscle function and pathology

Duchenne muscular dystrophy (DMD) is a severe progressive muscular disorder caused by reading frame disrupting mutations in the DMD gene, preventing the synthesis of functional dystrophin. As dystrophin provides muscle fiber stability during contractions, dystrophin negative fibers are prone to exercise-induced damage. Upon exhaustion of the regenerative capacity, fibers will be replaced by fibrotic and fat tissue resulting in a progressive loss of function eventually leading to death in the early thirties. With several promising approaches for the treatment of DMD aiming at dystrophin restoration in clinical trials, there is an increasing need to determine more precisely which dystrophin levels are sufficient to restore muscle fiber integrity, protect against muscle damage and improve muscle function.To address this we generated a new mouse model (mdx-Xist ^Δhs) with varying, low dystrophin levels (3–47%, mean 22.7%, stdev 12.1, n = 24) due to skewed X-inactivation. Longitudinal sections revealed that within individual fibers, some nuclei did and some did not express dystrophin, resulting in a random, mosaic pattern of dystrophin expression within fibers. Mdx-Xist ^Δhs, mdx and wild type females underwent a 12 week functional test regime consisting of different tests to assess muscle function at base line, or after chronic treadmill running exercise. Overall, mdx-Xist ^Δhs mice with 3–14% dystrophin outperformed mdx mice in the functional tests. Improved histopathology was observed in mice with 15–29% dystrophin and these levels also resulted in normalized expression of pro-inflammatory biomarker genes, while for other parameters >30% of dystrophin was needed. Chronic exercise clearly worsened pathology, which needed dystrophin levels >20% for protection. Based on these findings, we conclude that while even dystrophin levels below 15% can improve pathology and performance, levels of >20% are needed to fully protect muscle fibers from exercise-induced damage.