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Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutation in the dystrophin gene. Currently there is no cure for DMD. We introduced a novel human Dystrophin Expressing Chimeric (DEC) cell therapy of myoblast origin and confirmed the safety and efficacy of DEC in the mdx mouse models of DMD. In this study, we assessed histological and morphological changes in the cardiac, diaphragm, and gastrocnemius muscles of the mdx/scid mice after the transplantation of human DEC therapy via the systemic-intraosseous route. The efficacy of different DEC doses was evaluated at 90 days (0.5 × 106 and 1 × 106 DEC cells) and 180 days (1 × 106 and 5 × 106 DEC cells) after administration. The evaluation of Hematoxylin & Eosin (H&E)-stained sectional slices of cardiac, diaphragm, and gastrocnemius muscles included assessment of muscle fiber size by minimal Feret's diameter method using ImageJ software. The overall improvement in muscle morphology was observed in DMD-affected target muscles in both studies, as evidenced by a shift in fiber size distribution toward the wild type (WT) phenotype and by an increase in the mean Feret's diameter compared to the vehicle-injected controls. These findings confirm the long-term efficacy of human DEC therapy in the improvement of overall morphological pathology in the muscles affected by DMD and introduce DEC as a novel therapeutic approach for DMD patients.

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Small heat shock proteins are the well-known regulators of the cytoskeleton integrity, yet their complexes with actin-binding proteins are underexplored. Filamin C, a dimeric 560 kDa protein, abundant in cardiac and skeletal muscles, crosslinks actin filaments and contributes to Z-disc formation and membrane-cytoskeleton attachment. Here, we analyzed the interaction of a human filamin C fragment containing immunoglobulin-like domains 22-24 (FLNC22-24) with five small heat shock proteins (HspB1, HspB5, HspB6, HspB7, HspB8) and their α-crystallin domains. On size-exclusion chromatography, only HspB7 or its α-crystallin domain formed complexes with FLNC22-24. Despite similar isoelectric points of the small heat shock proteins analyzed, only HspB7 and its α-crystallin domain interacted with FLNC22-24 on native gel electrophoresis. Crosslinking with glutaraldehyde confirmed the formation of complexes between HspB7 (or its α-crystallin domain) and the filamin С fragment, inhibiting intersubunit FLNC crosslinking. These data are consistent with the structure modeling using Alphafold. Thus, the C-terminal fragment (immunoglobulin-like domains 22-24) of filamin C contains the site for HspB7 (or its α-crystallin domain) interaction, which competes with FLNC22-24 dimerization and its probable interaction with different target proteins.

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Impaired exercise capacity is the key symptom of heart failure (HF) and is associated with reduced quality of life and higher mortality rates. Unfortunately, current therapies, although generally lifesaving, have only small or marginal effects on exercise capacity. Specific strategies to alleviate exercise intolerance may improve quality of life, while possibly improving prognosis as well. There is overwhelming evidence that physical exercise improves performance in cardiac and skeletal muscles in health and disease. Unravelling the mechanistic underpinnings of exercise-induced improvements in muscle function could provide targets that will allow us to boost exercise performance in HF. With the current review we discuss: (i) recently discovered signalling pathways that govern physiological muscle growth as well as mitochondrial quality control mechanisms that underlie metabolic adaptations to exercise; (ii) the mechanistic underpinnings of exercise intolerance in HF and the benefits of exercise in HF patients on molecular, functional and prognostic levels; and (iii) potential molecular therapeutics to improve exercise performance in HF. We propose that novel molecular therapies to boost adaptive muscle growth and mitochondrial quality control in HF should always be combined with some form of exercise training.

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Erythropoietin (EPO) is a haematopoietic hormone that regulates erythropoiesis, but the EPO-receptor (EpoR) is also expressed in non-haematopoietic tissues. Stimulation of the EpoR in cardiac and skeletal muscle provides protection from various forms of pathological stress, but its relevance for normal muscle physiology remains unclear. We aimed to determine the contribution of the tissue-specific EpoR to exercise-induced remodelling of cardiac and skeletal muscle. Baseline phenotyping was performed on left ventricle and m. gastrocnemius of mice that only express the EpoR in haematopoietic tissues (EpoR-tKO). Subsequently, mice were caged in the presence or absence of a running wheel for 4 weeks and exercise performance, cardiac function and histological and molecular markers for physiological adaptation were assessed. While gross morphology of both muscles was normal in EpoR-tKO mice, mitochondrial content in skeletal muscle was decreased by 50%, associated with similar reductions in mitochondrial biogenesis, while mitophagy was unaltered. When subjected to exercise, EpoR-tKO mice ran slower and covered less distance than wild-type (WT) mice (5.5 ± 0.6 vs. 8.0 ± 0.4 km/day, p < 0.01). The impaired exercise performance was paralleled by reductions in myocyte growth and angiogenesis in both muscle types. Our findings indicate that the endogenous EPO-EpoR system controls mitochondrial biogenesis in skeletal muscle. The reductions in mitochondrial content were associated with reduced exercise capacity in response to voluntary exercise, supporting a critical role for the extra-haematopoietic EpoR in exercise performance.

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Sprague-Dawley rats (n = 32) underwent 8-weeks of creatine monohydrate (CM) supplementation (0, 2.5, 5, and 10 g/L). Total creatine (TCr) concentrations in female white fibre-dominant gastrocnemius (WGAS) and cardiac muscle (HRT) were significantly higher compared with males (p < 0.05). CM supplementation increased TCr concentrations in female WGAS (p < 0.05) and HRT (p < 0.01) and in male red fibre-dominant gastrocnemius muscle (RGAS) (p < 0.05). Future research should further investigate sex-differences in basal levels of TCr and the response to CM supplementation. Novelty: There is a sex- and tissue-dependant response to CM supplementation in rats.

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AIMS: Our aim was to characterise the actions of novel BIT compounds with structures based on peptides and toxins that bind to significant regulatory sites on ryanodine receptor (RyR) Ca2+ release channels. RyRs, located in sarcoplasmic reticulum (SR) Ca2+ store membranes of striated muscle, are essential for muscle contraction. Although severe sometimes-deadly myopathies occur when the channels become hyperactive following genetic or acquired changes, specific inhibitors of RyRs are rare. MAIN METHODS: The effect of BIT compounds was determined by spectrophotometric analysis of Ca2+ release from isolated SR vesicles, analysis of single RyR channel activity in artificial lipid bilayers and contraction of intact and skinned skeletal muscle fibres. KEY FINDINGS: The inhibitory compounds reduced: (a) Ca2+ release from SR vesicles with IC50s of 1.1-2.5 µM, competing with activation by parent peptides and toxins; (b) single RyR ion channel activity with IC50s of 0.5-1.5 µM; (c) skinned fibre contraction. In contrast, activating BIT compounds increased Ca2+ release with an IC50 of 5.0 µM and channel activity with AC50s of 2 to 12 nM and enhanced skinned fibre contraction. Sub-conductance activity dominated channel activity with both inhibitors and activators. Effects of all compounds on skeletal and cardiac RyRs were similar and reversible. Competition experiments suggest that the BIT compounds bind to the regulatory helical domains of the RyRs that impact on channel gating mechanisms through long-range allosteric interactions. SIGNIFICANCE: The BIT compounds are strong modulators of RyR activity and provide structural templates for novel research tools and drugs to combat muscle disease.

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Pompe disease is an autosomal recessive lysosomal storage disorder caused by disease-associated variants in the acid alpha-glucosidase (GAA) gene. The current Pompe mutation database provides a severity rating of GAA variants based on in silico predictions and expression studies. Here, we extended the database with clinical information of reported phenotypes. We added additional in silico predictions for effects on splicing and protein function and for cross reactive immunologic material (CRIM) status, minor allele frequencies, and molecular analyses. We analyzed 867 patients and 562 GAA variants. Based on their combination with a GAA null allele (i.e., complete deficiency of GAA enzyme activity), 49% of the 422 disease-associated variants could be linked to classic infantile, childhood, or adult phenotypes. Predictions and immunoblot analyses identified 131 CRIM negative and 216 CRIM positive variants. While disease-associated missense variants were found throughout the GAA protein, they were enriched up to seven-fold in the catalytic site. Fifteen percent of disease-associated missense variants were predicted to affect splicing. This should be confirmed using splicing assays. Inclusion of clinical severity rating in the Pompe mutation database provides an invaluable tool for diagnosis, prognosis of disease progression, treatment regimens, and the future development of personalized medicine for Pompe disease.

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Troponin I (TnI) is the inhibitory subunit of the troponin complex in the sarcomeric thin filament of striated muscle and plays a central role in the calcium regulation of contraction and relaxation. Vertebrate TnI has evolved into three isoforms encoded by three homologous genes: TNNI1 for slow skeletal muscle TnI, TNNI2 for fast skeletal muscle TnI and TNNI3 for cardiac TnI, which are expressed under muscle type-specific and developmental regulations. To summarize the current knowledge on the TnI isoform genes and products, this review focuses on the evolution, gene regulation, posttranslational modifications, and structure-function relationship of TnI isoform proteins. Their physiological and medical significances are also discussed.

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The Cre-loxP recombination system has been used to promote DNA recombination both in vitro and in vivo. For in vivo delivery, Cre expression is commonly achieved through the use of tissue/cell type-specific promoters, viral infection, or drug inducible transcription and protein translocation to promote targeted DNA excision. The development of cell permeable (or penetrating) peptide tagged proteins has facilitated the delivery of Cre recombinase protein into cells in culture, organotypic slide culture, or in living animals. In this report, we generated bacterially expressed, his-tagged Cre protein with either a cardiac targeting peptide or an antennapedia peptide at the C-terminus and demonstrated efficient uptake and recombination in both cell culture and mice. To facilitate delivery to cardiac and skeletal muscle, we mixed proteins with pluronic F-127 hydrogel and delivered Cre protein into reporter Rosa26mTmG mouse skeletal muscle or Rosa26LacZ cardiac muscle via ultrasound guided injection. Activation of reporter gene expression indicated that these Cre proteins were enzymatically active. Recombination events were detected only in the vicinity of injection areas. In conclusion, we have developed a method to deliver enzymatically active Cre protein locally to skeletal muscle and cardiac muscle that may be adapted for use with other proteins.

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BACKGROUND AND AIMS: The fundamental mechanisms involved in the genesis and progression of heart failure are not clearly understood. The present study was conducted to analyze the cardiac mitochondrial involvement in heart failure, the possible parallelism between cardiac and skeletal muscle and if there is a link between clinical symptoms and mitochondrial damage. METHODS: Left ventricle and pectoral biopsies were obtained from patients with heart failure (n: 21) and patients with inter-auricular communication as the unique diagnosis for surgery (n: 6). Mitochondria were isolated from these tissues and studied through electron microscopy, spectrophotometry to measure the activity of respiratory complex III and immunohistochemistry to determine the presence of reactive oxygen species. RESULTS: More than 90% of cardiac and skeletal muscle mitochondria presented structural and functional alterations in relation to an increment in the reactive oxygen species production, even in patients without the presence of any clinical Framingham criteria. CONCLUSIONS: We demonstrated some parallelism between cardiac and skeletal muscle mitochondrial alterations in patients with heart failure and that these alterations begin before the major clinical Framingham criteria are installed, pointing to mitochondria as one of the possibly responsible factors for the evolution of cardiac disease.

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Irisin converts white adipose tissue (WAT) into brown adipose tissue (BAT), as regulated by energy expenditure. The relationship between irisin concentrations after exercise in rats compared humans after exercise remains controversial. We therefore: (1) measured irisin expression in cardiac and skeletal muscle, liver, kidney, peripheral nerve sheath and skin tissues, as also serum irisin level in 10 week-old rats without exercise, and (2) measured tissue supernatant irisin levels in cardiac and skeletal muscle, and in response to exercise in young and old rats to establishing which tissues produced most irisin. Young (12 months) and old rats (24 months) with or without 10min exercise (water floating) and healthy 10 week-old Sprague-Dawley rats without exercise were used. Irisin was absent from sections of skeletal muscle of unexercised rats, the only part being stained being the perimysium. In contrast, cardiac muscle tissue, peripheral myelin sheath, liver, kidneys, and skin dermis and hypodermis were strongly immunoreactivity. No irisin was seen in skeletal muscle of unexercised young and old rats, but a slight amount was detected after exercise. Strong immunoreactivity occurred in cardiac muscle of young and old rats with or without exercise, notably in pericardial connective tissue. Serum irisin increased after exercise, being higher in younger than older rats. Irisin in tissue supernatants (cardiac and skeletal muscle) was high with or without exercise. High supernatant irisin could come from connective tissues around skeletal muscle, especially nerve sheaths located within it. Skeletal muscle is probably not a main irisin source.

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