Slow Skeletal Muscle Research Articles

The sodium/ascorbic acid co-transporter SVCT2 distributes in a striated membrane-enriched domain at the M-band level in slow-twitch skeletal muscle fibers

BackgroundVitamin C plays key roles in cellular homeostasis, functioning as a potent antioxidant and a positive regulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. SVCT2 is up-regulated during the early fusion of primary myoblasts and decreases during initial myotube growth, indicating the relevance of vitamin C uptake via SVCT2 for early skeletal muscle differentiation and fiber-type definition. However, our understanding of SVCT2 expression and function in adult skeletal muscles is still limited.ResultsIn this study, we demonstrate that SVCT2 exhibits an intracellular distribution in chicken slow skeletal muscles, following a highly organized striated pattern. A similar distribution was observed in human muscle samples, chicken cultured myotubes, and isolated mouse myofibers. Immunohistochemical analyses, combined with biochemical cell fractionation experiments, reveal a strong co-localization of SVCT2 with intracellular detergent-soluble membrane fractions at the central sarcomeric M-band, where it co-solubilizes with sarcoplasmic reticulum proteins. Remarkably, electrical stimulation of cultured myofibers induces the redistribution of SVCT2 into a vesicular pattern.ConclusionsOur results provide novel insights into the dynamic roles of SVCT2 in different intracellular compartments in response to functional demands.

Preclinical Multi-Omic Assessment of Pioglitazone in Skeletal Muscles of Mice Implanted with Human HER2/neu Overexpressing Breast Cancer Xenografts.

Background/Objectives: Breast cancer (BC) is the second most commonly diagnosed cancer worldwide and is accompanied by fatigue during both active disease and remission in the majority of cases. Our lab has measured fatigue in isolated muscles from treatment-naive BC patient-derived orthotopic xenograft (BC-PDOX) mice. Here, we conducted a preclinical trial of pioglitazone in BC-PDOX mice to determine its efficacy in ameliorating BC-induced muscle fatigue, as well as its effects on transcriptomic, metabolomic, and lipidomic profiles in skeletal muscle. Methods: The pioglitazone and vehicle groups were treated orally for 4 weeks upon reaching a tumor volume of 600 mm3. Whole-animal indirect calorimetry was used to evaluate systemic metabolic states. The transcriptome was profiled using short-read bulk RNA sequencing (RNA-seq). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to profile the metabolome and lipidome. Fast and slow skeletal muscle function were evaluated using isolated ex vivo testing. Results: Pioglitazone was associated with a 16.634% lower average O2 consumption (mL∙h-1, p = 0.035), 16.309% lower average CO2 production (mL∙h-1, p = 0.022), and 16.4% lower cumulative energy expenditure (EE) (kcal∙h-1, p = 0.035), with no changes in substrate utilization. RNA-seq supported the downstream effects of pioglitazone on target genes and displayed considerable upregulation of mitochondrial bioenergetic pathways. K-means cluster 5 showed enrichment of the PPAR signaling pathway (adj. p < 0.05, Log2FC = 2.58). Skeletal muscle metabolomic and lipidomic profiles exhibited dysregulation in response to BC, which was partially restored in pioglitazone-treated mice compared to vehicle-treated BC-PDOX mice. In particular, the overall abundance of total ceramide levels was significantly lower in the PioTx group (-46.327%, p = 0.048). Despite molecular support for pioglitazone's efficacy, isolated muscle function was not affected by pioglitazone treatment. No significant difference in the area under the fatigue curve (AUC) was found between the pioglitazone and vehicle groups (p = 0.596). Conclusions: BC induces multi-omic dysregulation in skeletal muscle, which pioglitazone partially ameliorates. Future research should focus on profiling systemic metabolic dysfunction, identifying molecular biomarkers of fatigue, and testing alternative pioglitazone treatment regimens.

Abstract Mo121: Sarcomere activation biosensor reveals key functional differences in live cell active states between cardiac and skeletal muscle

The sarcomere is the functional unit of striated muscle contraction. Previous studies have provided valuable information about the sarcomeric proteins and modulators that are necessary for activation of contraction through inter-myofilament signaling. This work has revealed key differences in contraction kinetics and paradigms between cardiac, slow skeletal, and fast skeletal muscle. We seek here to expand these studies through the use of myofilament incorporated Förster Resonance Energy Transfer (FRET) fluorescent biosensor in live, intact muscle. This biosensor fuses fluorescent donor and acceptor proteins to the N- and C-terminals of fast skeletal and cardiac (slow) troponin C, respectively, and allows for the monitoring in real-time of the stepwise physiological mechanism of muscle sarcomere activation in an intact system which preserves significant features of the muscle, including excitation-contraction coupling and load. This biosensor has been designed and validated in both cardiac and skeletal muscle and using intact muscles from transgenic animals we have demonstrated FRET fluorescence in cardiac papillaries, extensor digitorum longus, and soleus muscles. This biosensor has shown unique sarcomere activation properties between cardiac and skeletal muscle, notably the emergence of a prolonged return to baseline of the FRET signal in loaded fast and slow skeletal muscle, which is evidence of a “primed state” of myofilament activation unique to skeletal muscles under load. Elucidation of the “primed state” of sarcomere activation is evidence of a transient memory of recent activity enabling the enhanced contraction properties of skeletal muscle. This finding advances previous understanding of skeletal muscle activation, including summation and tetanic contraction, which is not possible in cardiac muscle. The implementation of this biosensor provides a new approach to interrogate the essential physiological features of muscle sarcomere activation and contraction and adds a valuable new tool for small molecule and gene-based discoveries for muscle disease remediation.

Differential Mitochondrial Adaptation of the Slow and Fast Skeletal Muscles by Endurance Running Exercise in Streptozotocin-Induced Diabetic Mice.

The skeletal muscle is the main organ responsible for insulin action, and glucose disposal and metabolism. Endurance and/or resistance training raises the number of mitochondria in diabetic muscles. The details of these adaptations, including mitochondrial adaptations of the slow and fast muscles in diabetes, are unclear. This study aimed to determine whether exercise training in streptozotocin (STZ)-induced mice leads to differential adaptations in the slow and fast muscles, and improving glucose clearance. Eight-week-old mice were randomly distributed into normal control (CON), diabetes (DM), and diabetes and exercise (DM+Ex) groups. In the DM and DM+Ex groups, mice received a freshly prepared STZ (100 mg/kg) intraperitoneal injection on two consecutive days. Two weeks after the injection, the mice in the groups ran on a treadmill for 60 min at 20 m/min for a week and subsequently at 25 m/min for 5 weeks (5 days/week). The analyses indicated that running training at low speed (25 m/min) enhanced mitochondrial enzyme activity and expression of lactate and glucose transporters in the plantaris (low-oxidative) muscle that improved whole-body glucose metabolism in STZ-induced diabetic mice. There were no differences in glucose transporter expression levels in the soleus (high-oxidative) muscle. The endurance running exercise at 20-25 m/min was sufficient to induce mitochondrial adaptation in the low-oxidative muscles, but not in the high-oxidative muscles, of diabetic mice. In conclusion, the present study indicated that running training at 25 m/min improved glucose metabolism by increasing the mitochondrial enzyme activity and glucose transporter 4 and monocarboxylate transporter 4 protein contents in the low-oxidative muscles in STZ-induced diabetic mice.

Effects of Alpha Lipoic Acid Supplementation on Slow Skeletal Muscle Mass and Contractile Functions in Type 2 Diabetic Male Sprague Dawley Rats

Objective: To see the Effects of Alpha-Lipoic Acid (ALA) supplementation on slow skeletal muscle mass andcontractile functions in type 2 diabetic male Sprague Dawley rats.Study Design: Quasi-experimental study.Place and Duration of Study: The study was carried out at the Physiology Research Lab, Army MedicalCollege, Rawalpindi, Pakistan from June 2019 to April 2021 in collaboration with the National Institute of Health(NIH) Islamabad, Pakistan.Methods: Sixty adult SD rats were divided into three equal groups. Rats were fed on a standard diet as per NIHprotocols. After 2 weeks type 2 Diabetes mellitus (T2DM) was induced in groups 2 and 3 by injecting low dose35mg of streptozotocin (STZ) in the abdomen intraperitoneally. T2DM was successfully developed andconfirmed by measuring glucose levels through a glucometer. Group 3 was injected with Alpha Lipoic acid30mg/kg/day at the lower abdomen for the next two weeks. After 04 weeks, soleus muscles were dissected.The animal data acquisition unit (iWorx) was used for assessing the contractile functions of soleus.Results: Alpha Lipoic acid group showed improvement in muscle mass, muscle tension strength, and recoveryfrom fatigue after applying fatigue protocol as compared to group 2.Conclusion: Alpha Lipoic acid supplementation improves contractile force and delays fatigue in the soleusmuscles of diabetic rats. How to cite this: Khan BU, Yousef I, Arshad S, Ikram F, Ali A, Ullah I. Effects of Alpha Lipoic Acid Supplementation on Slow Skeletal Muscle Mass and Contractile Functions in Type 2 Diabetic Male Sprague Dawley Rats. Life and Science. 2024; 5(2): 216-221. doi: http://doi.org/10.37185/LnS.1.1.408

A possible physiological role of sclerostin in aging-associated skeletal muscle atrophy

Sarcopenia has a significant impact on quality of life by inhibiting the ability of daily living. Further, sarcopenia is a well-known risk factor for fall-associated fracture and frailty. Therefore, prevention of sarcopenia is an important issue for healthy life. Recently, it has been revealed that sclerostin (Sost) is a novel target for the treatment of aging-associated bone weakness, so-called osteoporosis, However, the physiological role of Sost in skeletal muscle is unknown. The purpose of this study was to investigate the effect of Sost for skeletal muscle mass. In order to investigate the effect of Sost on myogenic differentiation, we carried out cell culture experiment using mouse myoblast-derived cell line C2C12 cells. During differentiation, Sost (0, 10, 100, 1000 ng/ml) was administrated to the cell culture medium. Effects of anti-sclerostin antibody romosozumab (0, 2, 20, 200 μg/ml) on myotube formation of C2C12 was also investigated. Myotube diameter and the fusion index (FI) and evaluated. Furthermore, we also investigated the expression level of Sost in skeletal muscles of young (10-week old) and aged (100-week old) male mice (C57BL/6J). The effects of hindlimb unloading on the expression level of Sost in skeletal muscle was also investigated. All animal experimental procedures were carried out in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences by Japan Physiological Society, and approved by the Animal Use Committee of Toyohashi SOZO University. Soleus muscle (SOL) and plantaris muscle (PLA) were dissected before and after 2 weeks of hindlimb suspension. Expression level of Sost was evaluated by Western blotting. The relationship between Sost expression level in each muscle and muscle wet weight was analyzed using Pearson correlation coeffcient. The differences between groups were considered statistically significant at p &lt; 0.05. Sost suppressed myogenic differentiation of C2C12 cells in a dose-dependent manner. On the other hand, romosozumab stimulated C2C12 differentiation in a dose-dependent manner. Expression level of Sost in SOL and PLA of aged mice was significantly higher than that in young animals. In SOL and PLA, a negative correlation was observed between the expression level of Sost and muscle weight was observed. Unloading-associated up-regulation of Sost was observed in PLA of aged mice. We demonstrated aging-associated up-regulation of Sost in mouse slow and fast skeletal muscles. Further, skeletal muscle weight was negatively correlated with the expression level of Sost in skeletal muscle. Therefore, it is suggested that skeletal muscle-derived Sost may play a role in aging-associated skeletal muscle atrophy. Sost may be a novel therapeutic target for sarcopenia. This work was supported by KAKENHI (18H03160, K.G.; 19K22825, K.G.; 19KK0254, K.G.;22H03474, K.G.; 22K19722, K.G.; 22H03319, K.G.; 22K18413, K.G.; 20K11221, T.K) from the Japan Society for the Promotion of Science, and a grant from DAIKO FOUNDATION (K.G.), Graduate School of Health Science, Toyohashi SOZO University (K.G.), and a research grant from Toyohashi SOZO university (K.G.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Force and kinetics of fast and slow muscle myosin determined with a synthetic sarcomere–like nanomachine

Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP–driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform–dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self–consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin–attached motors and the rate of transition through the attachment–detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.

Mechanical interaction of myosin and native thin filament in the disused rat soleus muscle

The disuse of skeletal limb muscles occurs in a variety of conditions, yet our comprehension of the molecular mechanisms involved in adaptation to disuse remains incomplete. We studied the mechanical characteristics of actin-myosin interaction using an in vitro motility assay and isoform composition of myosin heavy and light chains by dint of SDS-PAGE in soleus muscle of both control and hindlimb-unloaded rats. 14 days of hindlimb unloading led to the increased maximum sliding velocity of actin, reconstituted, and native thin filaments over rat soleus muscle myosin by 24 %, 19 %, and 20 %, respectively. The calcium sensitivity of the “pCa-velocity” relationship decreased. There was a 26 % increase in fast myosin heavy chain IIa (MHC IIa), a 22 % increase in fast myosin light chain 2 (MLC 2f), and a 13 % increase in fast MLC 1f content. The content of MLC 1s/v, typical for slow skeletal muscles and cardiac ventricles did not change. At the same time, MLC 1s, typical only for slow skeletal muscles, disappeared. The maximum velocity of soleus muscle native thin filaments was 24 % higher compared to control ones sliding over the same rabbit myosin. Therefore, both myosin and native thin filament kinetics could influence the mechanical characteristics of the soleus muscle. Additionally, the MLC 1s and MLC 1s/v ratio may contribute to the mechanical characteristics of slow skeletal muscle, along with MHC, MLC 2, and MLC 1 slow/fast isoforms ratio.

Mechanism of post-tetanic depression of slow muscle fibres.

A brief tetanic stimulation has a very different effect on the subsequent isometric twitch force of fast and slow skeletal muscles. Fast muscle responds with an enhanced twitch force which doubles that of the pre-tetanic value, whereas slow muscle depresses the post-tetanic twitch by about 20%. Twitch potentiation of fast muscle has long been known to be due to myosin light chain 2 phosphorylation. It is proposed that post-tetanic twitch depression in slow muscle is due to the dephosphorylation of the slow isoform of the thick filament protein, myosin-binding protein-C, by Ca2+/calmodulin-activated phosphatase calcineurin, whilst its phosphorylation underlies the force enhancement due to β-adrenergic stimulation in slow and fast muscle.

Phenotypes of mice expressing a myopathic exon 8 deletion mutant of slow skeletal muscle troponin T reveal novel pathogenic and pathophysiological mechanism

Phenotypes of mice expressing a myopathic exon 8 deletion mutant of slow skeletal muscle troponin T reveal novel pathogenic and pathophysiological mechanism

Linking the Glu150Ala mutation in slow skeletal muscle Tpm3.12 to congenital myopathy

Linking the Glu150Ala mutation in slow skeletal muscle Tpm3.12 to congenital myopathy

Myopathy-causing mutation R91P in the TPM3 gene drastically impairs structural and functional properties of slow skeletal muscle tropomyosin γβ-heterodimer

Tropomyosin (Tpm) is a regulatory actin-binding protein involved in Ca2+ activation of contraction of striated muscle. In human slow skeletal muscles, two distinct Tpm isoforms, γ and β, are present. They interact to form three types of dimeric Tpm molecules: γγ-homodimers, γβ-heterodimers, or ββ-homodimers, and a majority of the molecules are present as γβ-Tpm heterodimers. Point mutation R91P within the TPM3 gene encoding γ-Tpm is linked to the condition known as congenital fiber-type disproportion (CFTD), which is characterized by severe muscle weakness. Here, we investigated the influence of the R91P mutation in the γ-chain on the properties of the γβ-Tpm heterodimer. We found that the R91P mutation impairs the functional properties of γβ-Tpm heterodimer more severely than those of earlier studied γγ-Tpm homodimer carrying this mutation in both γ-chains. Since a significant part of Tpm molecules in slow skeletal muscle is present as γβ-heterodimers, our results explain why this mutation leads to muscle weakness in CFTD.

The Effects of Antioxidants and Pulsed Magnetic Fields on Slow and Fast Skeletal Muscle Atrophy Induced by Streptozotocin: A Preclinical Study.

Our findings suggest that antioxidants and PMF may alleviate impaired protein synthesis and degradation pathways in skeletal muscle atrophy. PTS showed a positive effect on the anabolic pathway, while RSV and PMF demonstrated potential for ameliorating the catabolic pathway. Notably, the combination therapy of antioxidants and PMF exhibited a stronger ameliorative effect on skeletal muscle atrophy than either intervention alone. The present results highlight the benefits of employing a multimodal approach, involving both antioxidant and PMF therapy, for the management of muscle-wasting conditions. These treatments may have potential therapeutic implications for skeletal muscle atrophy.

A Novel Variant in TPM3 Causing Muscle Weakness and Concomitant Hypercontractile Phenotype.

A novel variant of unknown significance c.8A > G (p.Glu3Gly) in TPM3 was detected in two unrelated families. TPM3 encodes the transcript variant Tpm3.12 (NM_152263.4), the tropomyosin isoform specifically expressed in slow skeletal muscle fibers. The patients presented with slowly progressive muscle weakness associated with Achilles tendon contractures of early childhood onset. Histopathology revealed features consistent with a nemaline rod myopathy. Biochemical in vitro assays performed with reconstituted thin filaments revealed defects in the assembly of the thin filament and regulation of actin-myosin interactions. The substitution p.Glu3Gly increased polymerization of Tpm3.12, but did not significantly change its affinity to actin alone. Affinity of Tpm3.12 to actin in the presence of troponin ± Ca2+ was decreased by the mutation, which was due to reduced interactions with troponin. Altered molecular interactions affected Ca2+-dependent regulation of the thin filament interactions with myosin, resulting in increased Ca2+ sensitivity and decreased relaxation of the actin-activated myosin ATPase activity. The hypercontractile molecular phenotype probably explains the distal joint contractions observed in the patients, but additional research is needed to explain the relatively mild severity of the contractures. The slowly progressive muscle weakness is most likely caused by the lack of relaxation and prolonged contractions which cause muscle wasting. This work provides evidence for the pathogenicity of the TPM3 c.8A > G variant, which allows for its classification as (likely) pathogenic.

Moderate intensity continuous versus high intensity interval training: Metabolic responses of slow and fast skeletal muscles in rat.

The healthy benefits of regular physical exercise are mainly mediated by the stimulation of oxidative and antioxidant capacities in skeletal muscle. Our understanding of the cellular and molecular responses involved in these processes remain often uncomplete particularly regarding muscle typology. The main aim of the present study was to compare the effects of two types of exercise training protocol: a moderate-intensity continuous training (MICT) and a high-intensity interval training (HIIT) on metabolic processes in two muscles with different typologies: soleus and extensor digitorum longus (EDL). Training effects in male Wistar rats were studied from whole organism level (maximal aerobic speed, morphometric and systemic parameters) to muscle level (transcripts, protein contents and enzymatic activities involved in antioxidant defences, aerobic and anaerobic metabolisms). Wistar rats were randomly divided into three groups: untrained (UNTR), n = 7; MICT, n = 8; and HIIT, n = 8. Rats of the MICT and HIIT groups ran five times a week for six weeks at moderate and high intensity, respectively. HIIT improved more than MICT the endurance performance (a trend to increased maximal aerobic speed, p = 0.07) and oxidative capacities in both muscles, as determined through protein and transcript assays (AMPK-PGC-1α signalling pathway, antioxidant defences, mitochondrial functioning and dynamics). Whatever the training protocol, the genes involved in these processes were largely more significantly upregulated in soleus (slow-twitch fibres) than in EDL (fast-twitch fibres). Solely on the basis of the transcript changes, we conclude that the training protocols tested here lead to specific muscular responses.

Distinct and shared endothermic strategies in the heat producing tissues of tuna and other teleosts.

Although most fishes are ectothermic, some, including tuna and billfish, achieve endothermy through specialized heat producing tissues that are modified muscles. How these heat producing tissues evolved, and whether they share convergent molecular mechanisms, remain unresolved. Here, we generated a high-quality genome from the mackerel tuna (Euthynnus affinis) and investigated the heat producing tissues of this fish by single-nucleus and bulk RNA sequencing. Compared with other teleosts, tuna-specific genetic variation is strongly associated with muscle differentiation. Single-nucleus RNA-seq revealed a high proportion of specific slow skeletal muscle cell subtypes in the heat producing tissues of tuna. Marker genes of this cell subtype are associated with the relative sliding of actin and myosin, suggesting that tuna endothermy is mainly based on shivering thermogenesis. In contrast, cross-species transcriptome analysis indicated that endothermy in billfish relies mainly on non-shivering thermogenesis. Nevertheless, the heat producing tissues of the different species do share some tissue-specific genes, including vascular-related and mitochondrial genes. Overall, although tunas and billfishes differ in their thermogenic strategies, they share similar expression patterns in some respects, highlighting the complexity of convergent evolution.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 μM.

Slow myosin heavy chain 1 is required for slow myofibril and muscle fibre growth but not for myofibril initiation

Slow myosin heavy chain 1 (Smyhc1) is the major sarcomeric myosin driving early contraction by slow skeletal muscle fibres in zebrafish. New mutant alleles lacking a functional smyhc1 gene move poorly, but recover motility as the later-formed fast muscle fibres of the segmental myotomes mature, and are adult viable. By motility analysis and inhibiting fast muscle contraction pharmacologically, we show that a slow muscle motility defect persists in mutants until about 1 month of age. Breeding onto a genetic background marking slow muscle fibres with EGFP revealed that mutant slow fibres undergo terminal differentiation, migration and fibre formation indistinguishable from wild type but fail to generate large myofibrils and maintain cellular orientation and attachments. In mutants, initial myofibrillar structures with 1.67 ​μm periodic actin bands fail to mature into the 1.96 ​μm sarcomeres observed in wild type, despite the presence of alternative myosin heavy chain molecules. The poorly-contractile mutant slow muscle cells generate numerous cytoplasmic organelles, but fail to grow and bundle myofibrils or to increase in cytoplasmic volume despite passive movements imposed by fast muscle. The data show that both slow myofibril maturation and cellular volume increase depend on the function of a specific myosin isoform and suggest that appropriate force production regulates muscle fibre growth.

PROPERTIES OF FATIGUE BETWEEN SLOW AND FAST MUSCLES OF MALE AND FEMALE DIABETIC RATS

Background: The metabolic disorder of Type 2 Diabetes Mellitus (T2DM) has deleterious effects on skeletal muscle contractile functions. This study aimed to compare parameters of fatigue between slow and fast skeletal muscles of male and female type 2 diabetic Sprague-Dawley rats. Methodology: This was a quasi experimental study conducted at Army Medical College Rawalpindi and National Institute of Health, Islamabad. Forty Sprague Dawley rats in good health were split into groups I (Control) and II (Diabetics) of 20 rats each. Each group had 10 male and 10 female rats. Diabetic groups were given intra-peritoneal streptozotocin (STZ), 35 mg/Kg body weight on 15th day. Control groups were fed a normal diet and diabetic rats were given diet with high fat. On 21st day body weight, blood glucose and triglyceride/high density lipoprotein ratio were estimated to confirm extraction of type 2 diabetes mellitus. Intact Soleus and Extensor Digitorium Longus muscles were removed and fixed in organ bath system having Krebs-Ringer buffer solution and connected to data acquisition unit (iWorx®) to study their fatigability parameters. Independent samples t-test was used for statistical analysis. Results: Slow and fast skeletal muscles in diabetic rats showed reduction in resistance to and recovery from fatigue. Fatigue parameters between male and female diabetic rats revealed no significant differences. Conclusion: Fatigue parameters do not differ between male and female diabetic rats. Pak J Physiol 2023;19(1):7?10