Muscle Creatine Kinase Research Articles

Serum creatine kinase elevation following tyrosine kinase inhibitor treatment in cancer patients: Symptoms, mechanism, and clinical management.

Molecular targeted tyrosine kinase inhibitors (TKIs) have produced unprecedented treatment response in cancer therapy for patients harboring specific oncogenic mutations. While the TKIs are mostly well tolerated, they were reported to increase serum levels of creatine kinase (CK) and cause muscle metabolism-related toxicity. CK is an essential enzyme involved in cellular energy metabolism and muscle function. Elevated serum CK levels can arise from both physiological and pathological factors, as well as triggered by specific drug classes. The incidence of serum CK elevation induced by a few approved TKIs (brigatinib, binimetinib, cobimetinib-vemurafenib combination [Food and Drug Administration, United States]; aumolertinib, and sunvozertinib [only approved by National Medical Products Administration, China]) were over 35%. CK elevation-related symptoms include myopathy, myositis, inclusion body myositis (IBM), cardiotoxicity, rhabdomyolysis, rash, and acneiform dermatitis. High-level or severe symptomatic CK elevation may necessitate dose reduction and indirectly dampen TKI efficacy. This review presents an updated summary about the prevalence rate and recent research about mechanisms leading to TKI-induced serum CK elevation in cancer patients. The utility of monitoring serum CK levels for predicting TKI-induced adverse effects and their management will also be discussed.

Inter-individual differences in muscle damage following a single bout of high-intense whole-body electromyostimulation.

This brief report aimed to characterize inter-individual training responses following a single session of high-intense whole-body electromyostimulation (WB-EMS) using markers of muscle damage over a period of 72 h. Twelve healthy individuals (5 men, 7 women; 32.0 ± 7 years) participated in a single 20-minute high-intensity WB-EMS training session. Markers of muscle damage, creatine kinase (CK) and myoglobin (Mb), were assessed before and immediately after training, as well as at 1.5, 3, 24, 48 and 72 h post-exercise. Lactate levels were determined pre- and post-exercise. Overall, WB-EMS induced significant CK elevations, peaking at 72 h (18.358 ± 21.380 U/L; p < 0.01), and correlating Mb levels peaking at 48 h (1.509 ± 1.394 ng/dl, p < 0.01). Despite significant inter-individual variability in CK levels, both slow (SR) and fast responders (FR) were identified. FR showed significant increases in CK at all time points post WB-EMS (p < 0.05), whereas CK in SR significantly elevated after 48 h. Post-WB-EMS lactate concentration was identified to predict peak CK and Mb levels (r ≥ 0.65, both p < 0.05). High-intensity WB-EMS has the potential to induce severe muscle damage, as indicated by elevated levels of CK and Mb. We identified two distinct groups of individuals, SR and FR, indicating variability in response to WB-EMS. Furthermore, we suggest that individual responses to WB-EMS can be predicted based on post-WB-EMS lactate concentration.

Proteomic analysis of wild boar meat: Effect of storage method and time on muscle protein stability

Oxidation processes affect proteins from various molecular pathways and are crucial for wild boar meat quality, shelf life and human health. This study investigated the effects of different storage methods on the formation and composition of oxygen-induced protein aggregates in the muscles of European wild boar (Sus scrofa scrofa). Vacuum packaging (VAC), modified atmosphere packaging (MAP) and dry-ageing (DA) were compared over a 21-day storage period. The results showed significant differences in protein aggregation depending on the method and storage time. The most intense protein aggregation occurred in the MAP (80 % O2), while air DA (20.9 % O2) resulted in intermediate levels of protein aggregation. Crucial myofibrillar proteins involved in aggregate formation were titin, myosin isoforms (MYH1, MYH2 and MYH7) and nebulin, which were cross-linked with small sarcoplasmic enzymes, such as muscle creatine kinase, isocitrate dehydrogenase and ATPase 1. High‑oxygen storage conditions also promoted the oxidation of ATP synthase, beta-enolase 3, ADP/ATP translocase and myoglobin.

Exonisation of an intronic L1 element in the dystrophin gene associated with X-linked muscular dystrophy in a Border Collie dog.

X-linked recessive dystrophinopathies are the most common muscular dystrophies (MDs) in humans and dogs. To date, 20 breed-specific MD-associated variants are described in the canine dystrophin gene (DMD), including one associated with dystrophin-deficient MD in the Border Collie mixed breed. Here, we report the diagnosis and follow-up of mild dystrophin-deficient MD in a 5-month-old male Border Collie, associated with a novel DMD variant. Diagnosis was based on neurological examination and laboratory evaluations including creatine kinase activity, electromyography and muscle biopsies with immunofluorescent staining. Inspection of the Sashimi plots of the RNA-seq data from the affected muscle biopsy led to the discovery of a 162-bp L1 pseudoexon in DMD intron 63, introducing a frameshift and a premature stop codon (NM_001003343.1: c.9271_9272insN[162] p.(Ala3091fs*21)). Reduced DMD mRNA levels were detected for both the non-pseudoexon (50× less) and pseudoexon (3× less) containing transcripts in the affected muscle, compared with the level of the non-pseudoexon containing transcript in a control muscle, resulting in very low dystrophin protein levels and the upregulation of utrophin. Because the variant was only found in the affected dog, not in the healthy mother and grandmother, or in 108 unrelated Border Collies from the Belgian population (46 males and 62 females), it was considered a de novo variant. Although the prognosis for dystrophinopathy is generally regarded as poor, the dog stabilised at the age of 6 months and is still clinically stable at the age of 2 years.

Upstream and downstream pathways of diacylglycerol kinase : Novel phosphatidylinositol turnover-independent signal transduction pathways

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprise ten isozymes (α–κ) that regulate a wide variety of physiological and pathological events. Recently, we revealed that DGK isozymes use saturated fatty acid (SFA)/monosaturated fatty acid (MUFA)-containing and docosahexaenoic acid (22:6)-containing DG species, but not phosphatidylinositol (PI) turnover-derived 18:0/20:4-DG. For example, DGKδ, which is involved in the pathogenesis of type 2 diabetes, preferentially uses SFA/MUFA-containing DG species, such as 16:0/16:0- and 16:0/18:1-DG species, in high glucose-stimulated skeletal muscle cells. Moreover, DGKδ, which destabilizes the serotonin transporter (SERT) and regulates the serotonergic system in the brain, primarily generates 18:0/22:6-PA. Furthermore, 16:0/16:0-PA is produced by DGKζ in Neuro-2a cells during neuronal differentiation. We searched for SFA/MUFA-PA- and 18:0/22:6-PA-selective binding proteins (candidate downstream targets of DGKδ) and found that SFA/MUFA-PA binds to and activates the creatine kinase muscle type, an energy-metabolizing enzyme, and that 18:0/22:6-PA interacts with and activates Praja-1, an E3 ubiquitin ligase acting on SERT, and synaptojanin-1, a key player in the synaptic vesicle cycle. Next, we searched for SFA/MUFA-DG-generating enzymes upstream of DGKδ. We found that sphingomyelin synthase (SMS)1, SMS2, and SMS-related protein (SMSr) commonly act as phosphatidylcholine (PC)-phospholipase C (PLC) and phosphatidylethanolamine (PE)-PLC, generating SFA/MUFA-DG species, in addition to SMS and ceramide phosphoethanolamine synthase. Moreover, the orphan phosphatase PHOSPHO1 showed PC- and PE-PLC activities that produced SFA/MUFA-DG. Although PC- and PE-PLC activities were first described 70–35 years ago, their proteins and genes were not identified for a long time. We found that DGKδ interacts with SMSr and PHOSPHO1, and that DGKζ binds to SMS1 and SMSr. Taken together, these results strongly suggest that there are previously unrecognized signal transduction pathways that include DGK isozymes and generate and utilize SFA/MUFA-DG/PA or 18:0/22:6-DG/PA but not PI-turnover-derived 18:0/20:4-DG/PA.

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

Gene replacement therapies primarily rely on adeno-associated virus (AAV) vectors for transgene expression. However, episomal expression can decline over time due to vector loss or epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to low invasiveness of intramuscular injections, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two highly expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts and skeletal muscle tissue. We hijacked the muscle creatine kinase (Ckm) and myoglobin (Mb) promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human Factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites.This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.

The effect of early neuromuscular electrical stimulation in intensive care unit-acquired weakness

Background &amp; objective: Intensive Care Unit-Acquired Weakness (ICU-AW) is a weakness found in critically ill patients, and this weakness can persist even after discharge from the Intensive Care Unit (ICU). Various rehabilitation medicine procedures have been shown to be effective in prevention as well as managing the established weakness in this cohort of the patients. We analyzed the effect of Neuromuscular Electrical Stimulation (NMES) therapy on the global muscle strength, quadriceps femoris muscle, and creatine kinase examination in patients known to have ICU-AW. Methodology: The type of study used a pre-experimental one-group pre-posttest, and the study population consisted of 23 patients who experienced ICU-AW. Patients were given NMES therapy at the beginning of treatment in the ICU and then evaluated using the Medical Research Council Scale for Muscle Strength (MRC-SS), Manual Muscle Test (MMT), and creatine kinase levels. Results: NMES therapy provides significant results on increasing muscle strength on the fifth day with MRC-SS 42.78 (24-60) and MMT 3.57 (2-5) (P &lt; 0.001), as well as a significant decrease in creatine kinase levels given therapy at the beginning of ICU admission. Conclusion: NMES therapy increases global muscle strength and quadriceps femoris muscle and decreases creatine kinase levels. Abbreviations: ICU-AW - Intensive Care Unit-Acquired Weakness, NMES - Neuromuscular Electrical Stimulation, ICU - Intensive care unit, MRC-SS - Medical Research Council Scale for Muscle Strength, MMT - Manual Muscle Testing, CK-MM - Creatine kinase muscle specific Key Words: Neuromuscular electrical stimulation; Intensive Care Unit-Acquired Weakness, Creatine kinase Citation: Yustiawan A, Semedi BP, Arfianti L, Maulydia, Edwar PPM, Airlangga PS, Santoso KH, Andriana M. The effect of early neuromuscular electrical stimulation in intensive care unit-acquired weakness. Anaesth. pain intensive care 2024;28(4):706−711; DOI: 10.35975/apic.v28i4.2405 Received: March 04, 2024; Reviewed: April 29, 2024; Accepted: May 10, 2024

Cell-free DNA kinetics in response to muscle-damaging exercise: A drop jump study.

A significant increase in circulating cell-free DNA (cfDNA) occurs with physical exercise, which depends on the type of exertion and the duration. The aims of this study were as follows: (1) to investigate the time course of cfDNA and conventional markers of muscle damage from immediately after to 96h after muscle-damaging exercise; and (2) to investigate the relationship between cfDNA and indicators of primary (low-frequency fatigue and maximal voluntary isometric contraction) and secondary (creatine kinase and delayed-onset muscle soreness) muscle damage in young healthy males. Fourteen participants (age, 22±2years; weight, 84.4±11.2kg; height, 184.0±7.4cm) performed 50 intermittent drop jumps at 20s intervals. We measured cfDNA and creatine kinase concentrations, maximal voluntary isometric contraction torque, low-frequency fatigue and delayed-onset muscle soreness before and at several time points up to 96h after exercise. Plasma cfDNA levels increased from immediately postexercise until 72h postexercise (P<0.01). Elevation of postexercise cfDNA was correlated with both more pronounced low-frequency fatigue (r=-0.52, P=3.4×10-11) and delayed-onset muscle soreness (r=0.32, P= 0.00019). Levels of cfDNA change in response to severe primary and secondary muscle damage after exercise. Levels of cfDNA exhibit a stronger correlation with variables related to primary muscle damage than to secondary muscle damage, suggesting that cfDNA is a more sensitive marker of acute loss of muscle function than of secondary inflammation or damaged muscle fibres.

PCSK9 inhibitors and inclisiran with or without statin therapy on incident muscle symptoms and creatine kinase: a systematic review and network meta-analysis.

Atherosclerotic cardiovascular disease (ASCVD), a leading cause of global fatalities, has inconsistent findings regarding the impact of muscle symptoms despite promising clinical trials involving PCSK9 inhibitors (PCSK9i) and siRNA as potential therapeutic options. The databases EMBASE, PubMed, Web of Science, Cochrane, and ClinicalTrials.gov were thoroughly searched without any restrictions on language. Review Manager 5.3 software was utilized to calculate relative risks with 95% confidence intervals (CIs) for dichotomous data and mean differences or standardized mean differences with 95%CIs for continuous data. To evaluate publication bias, Egger's test was employed using Stata/SE software. This analysis included 26 studies comprising 28 randomized controlled trials (RCTs) involving a total of 100,193 patients, and 4 different lipid-lowering therapy combinations. For events with creatine kinase >3ULN, evolocumab and alirocumab demonstrated significant advantages compared to inclisiran. Evolocumab showed the best results in terms of both new muscle symptom events and creatine kinase >3ULN. Based on this network meta-analysis (NMA) results, evolocumab has emerged as a promising treatment option for patients with hyperlipidemia and muscle disorders compared to other PCSK9 inhibitors and inclisiran. PROSPERO [CRD42023459558].

Pre-exercise cryotherapy reduces myoglobin and creatine kinase levels after eccentric muscle stress in young women.

Introduction: The aim of this study was to investigate the effect of pre-exercise whole-body cryotherapy (WBC) on muscle damage indicators following eccentric treadmill exercise in young women. Methods: Twenty-seven participants underwent two 1-h downhill treadmill runs, replicating 60% of their maximal oxygen uptake, with a 4-week intermission for recovery and treatment application. In this intermission, one group underwent 20 sessions of WBC, delivered five times a week at -120°C for 3min each, while the comparison group received no such treatment. Markers of muscle injury-serum myoglobin concentration, creatine kinase and lactate dehydrogenase activity and also uric acid, and cell-free DNA concentration-were measured before and after downhill runs. Results: The study observed a notable reduction in post-exercise myoglobin and CK levels in the WBC group after the second running session. Discussion: The results suggest that WBC can have a protective effects against muscle damage resulting from eccentric exercise.

High Velocity Passive Stretching Mimics Eccentric Exercise in Cerebral Palsy and May Be Used to Increase Spastic Muscle Fascicle Length.

Muscle fascicles are shorter and stiffer than normal in spastic Cerebral Palsy (CP). Increasing fascicle length (FL) has been attempted in CP, the outcomes of which have been unsatisfactory. In healthy muscles, FL can be increased using eccentric exercise at high velocities (ECC). Three conditions are possibly met during such ECC: muscle micro-damage, positive fascicle strain, and momentary muscle deactivation during lengthening. Participants with and without CP underwent a single bout of passive stretching at (appropriately) high velocities using isokinetic dynamometry, during which we examined muscle and fascicle behaviour. Vastus lateralis (VL) FL change was measured using ultrasonography and showed positive fascicle strain. Measures of muscle creatine kinase were used to establish whether micro-damage occurred in response to stretching, but the results did not confirm damage in either group. Vastus medialis (VM) and biceps femoris muscle activity were measured using electromyography in those with CP. Results supported momentary spastic muscle deactivation during lengthening: all participants experienced at least one epoch (60 ms) of increased activation followed by activation inhibition/deactivation of the VM during knee flexion. We argue that high-velocity passive stretching in CP provides a movement context which mimics ECC and could be used to increase spastic FL with training.

Idiopathic inflammatory myopathies : An interdisciplinary challenge

Idiopathic inflammatory myopathies (IIM) are rare diseases (incidence 1:100,000) with awide range of clinical symptoms and manifestations. Typical indicators of IIM are proximally emphasized muscle weakness and myalgias, which are usually accompanied by elevated creatine kinase levels and muscle atrophy. The autoantibody diagnostics separate IIM into different entities, which are each associated with atypical risk of organ manifestations and the occurrence of tumors. The IIM represents an interdisciplinary challenge and the diagnostics and treatment require the involvement of several disciplines including rheumatology, neurology, neuropathology, dermatology and pneumology. An accurate diagnosis and careful tumor screening are essential because of the association between certain subgroups of IIM and the occurrence of malignant tumors.

P.042 Anti-HMG Coenzyme A reductase antibody (anti-HMGCR) myopathy: case review of two pediatric patients from a single center

Background: Necrotizing anti-HMGCR myopathy is rare in children. Pediatric cases are not typically associated with statin use or malignancy. Methods: Retrospective chart review (January 2009 to December 2023) identified cases of anti-HMGCR myopathy at our hospital. Results: Two patients were identified. Patient A, presented at 8 yo with a 2 year history of proximal muscle weakness. His CK was 4,840 U/L (normal &lt;205 U/L) with a high anti-HMGCR antibody titre. His Childhood Myositis Assessment Scale (CMAS) score was 33/52. Monthly IVIG was started and his muscle strength and CK improved. Two years later, weekly methotrexate was started for persistent mild CK elevation (602 to 869 U/L). At 11 years old, 3 years after diagnosis, his CMAS score was 47 and he could participate in soccer with mild fatigue. Patient B, presented at 8 yo with acute proximal weakness, rash and CMAS 13/52. His CK was 20,185 U/L with elevated anti-HMGCR antibody titre. He received oral corticosteroids, weekly methotrexate and monthly IVIG. At 10 yo, 2 years after diagnosis, he is asymptomatic with CMAS 51. He is maintained on methotrexate monotherapy. Conclusions: Anti-HMGCR antibody myopathy requires prompt diagnosis to obviate muscle necrosis and long-term complications. Our patients showed clinical and CMAS improvement with treatment.

Can pre-exercise photobiomodulation improve muscle endurance and promote recovery from muscle strength and injuries in people with different activity levels? A meta-analysis of randomized controlled trials.

Photobiomodulation therapy (PBMT) was introduced as an ergogenic aid for sport performance in healthy individuals is still controversial. The main aim of this study is to assess the potential enhancements in muscle endurance and recovery from muscle strength and injuries mediated by PBMT among individuals exhibiting diverse activity levels. Randomized controlled trials (RCT) of PBMT interventions for healthy people (both trained and untrained individuals) exercising were searched (up to January 16, 2024) in four electronic databases: Web of Science, PubMed, Scopus and Embase. Primary outcome measures included muscle endurance, muscle strength and creatine kinase (CK) levels; secondary outcome measure included Lactate dehydrogenase (LDH) levels. Subgroup analyses based on physical activity levels were conducted for each outcome measure. Thirty-four RCTs were included based on the article inclusion and exclusion criteria. Statistical results showed that PBMT significantly improved muscle endurance (standardized mean difference [SMD] = 0.31, 95%CI 0.11, 0.51, p < 0.01), indicating a moderate effect size. It also facilitated the recovery of muscle strength (SMD = 0.24, 95%CI 0.10, 0.39, p < 0.01) and CK (mean difference [MD] = -77.56, 95%CI -112.67, -42.44, p < 0.01), indicating moderate and large effect sizes, respectively. Furthermore, pre-application of PBMT significantly improved muscle endurance, recovery of muscle strength and injuries in physically inactive individuals and athletes (p < 0.05), while there was no significant benefit for physically active individuals. Pre-application of PBMT improves muscle endurance and promotes recovery from muscle strength and injury (includes CK and LDH) in athletes and sedentary populations, indicating moderate to large effect sizes, but is ineffective in physically active populations. This may be due to the fact that physically active people engage in more resistance training, which leads to a decrease in the proportion of red muscle fibres, thus affecting photobiomodulation.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged male mice compared with those in aged female mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

Therapeutic efficacy and safety of JAK inhibitors in treating polymyositis/dermatomyositis: a single-arm systemic meta-analysis.

We performed a single-arm meta-analysis to evaluate the efficacy and safety of JAK inhibitors in the treatment of dermatomyositis (DM)/ polymyositis (PM). Relevant studies from four databases were systematically searched until April 25, 2023. The primary endpoint was Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI) and other outcomes were Manual Muscle Testing (MMT) and Creatine Kinase (CK). According to the type of JAK and medication regimen, we conducted subgroup analyses. The registration number in PROSPERO was CRD42023416493. According to the selection criteria, we identified 7 publications with a total of 91 patients. Regarding skin lesions, the CDASI decreased by 17.67 (95% CI: -20.94 ~ -14.41). The CK increased by 8.64 U (95% CI: -28.25 ~ 45.53). About muscle lesions, MMT increased by 10.31 (95% CI: -2.83 ~ 23.46). Subgroup analysis revealed that different types of JAK inhibitors had various degrees of reduction. CDASI in patients treated with RUX had the lowest one [-20.00 (95% CI: -34.9 ~ -5.1)], followed by TOF [-18.29 (95% CI: -21.8 ~ -14.78)] and BAR [-11.2 (95% CI: -21.51 ~ -0.89)]. Additionally, the mean reduction in CDASI in patients treated with TOF alone was 16.16 (95% CI: -21.21 ~ -11.11), in combination with other immunosuppressants was 18.59 (95% CI: -22.74 ~ -14.45). For safety evaluation, one patient developed Orolabial HSV, and two patients developed thromboembolism events. In summary, this meta-analysis demonstrated that JAK inhibitors can potentially treat DM/PM without severe adverse reactions. https://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42023416493, identifier CRD42023416493.

BCAAs as Ergogenic Aids: Exploring Their Impact on Exercise-Induced Muscle Damage

This research delves into the multifaceted realm of Branched-Chain Amino Acids (BCAAs) as potential ergogenic aids, specifically exploring their impact on exercise-induced muscle damage. The investigation spans an extensive literature review of studies conducted in the United States from 2010 to 2023, synthesizing diverse methodologies and outcomes. The study employs a systematic approach, considering randomized controlled trials, observational studies, and intervention studies to provide a comprehensive understanding of the nuanced relationship between BCAAs and exercise-induced muscle damage. Findings reveal varied outcomes, suggesting BCAAs may influence markers such as Creatine Kinase levels, recovery time, and muscle soreness. However, inconsistencies and individual variations underscore the complexity of this interaction. The paper also elucidates the biochemical mechanisms through which BCAAs act, addressing considerations for optimal dosage, timing, and potential side effects. Practical implications are drawn for athletes and fitness enthusiasts, emphasizing personalized approaches within the broader context of sports nutrition. Acknowledging limitations, the study concludes with a call for continued research, refining methodologies and advancing the understanding of BCAAs in optimizing athletic performance and recovery.

Beneficial effects of a novel polyherbal formulation on the skeletal muscle antioxidant status, inflammation, and muscle-signaling proteins in exercised rats.

Exhausting exercise can damage muscle tissue due to free radical interactions. It is hypothesized that the increase in free radicals following muscle injury, either due to oxidative damage to biomolecules or the activation of inflammatory cytokines, may lead to secondary muscle damage. This study investigated the effects of a novel joint health formula (JHF) containing bisdemethoxycurcumin-enriched curcumin, 3-O-Acetyl-11-keto-beta-boswellic acid-enriched Boswellia (AKBA), and Ashwagandha on exhaustion time, grip strength, antioxidant status, and muscle-signaling proteins in exhaustively exercised rats. Twenty-eight rats were divided into four groups: Control (C), exercise (E), E + JHF 100 (100 mg/kg), and E + JHF 200 (200 mg/kg). An increase in time to exhaustion and grip strength was recorded with JHF supplementation in a dose-dependent manner (p < 0.0001). In addition, serum and muscle lactate dehydrogenase, malondialdehyde, myoglobin, creatine kinase, and lactic acid concentrations were decreased in the groups supplemented with JHF, particularly at the high dose of JHF (200 mg/kg) (p < 0.0001 for all). JHF supplementation also increased antioxidant enzyme activities and suppressed the production of inflammatory cytokines compared to the exercise group (p < 0.0001). Moreover, JHF reduced the levels of PGC-1α, p-70S6K1, MAFbx, MuRF1, and p-mTOR proteins in muscle tissue compared to the exercise group (p < 0.05), being more effective at high doses. These findings show that JHF might reduce muscle damage by modulating antiinflammatory, antioxidant, and muscle mass regulatory pathways in exhausted training rats. At the same time, JHF improved exercise performance and grip strength.

Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

Human induced pluripotent stem cells (hiPSCs) offer opportunities to study human biology where primary cell types are limited. CRISPR technology allows forward genetic screens using engineered Cas9-expressing cells. Here, we sought to generate a CRISPR activation (CRISPRa) hiPSC line to activate endogenous genes during pluripotency and differentiation. We first targeted catalytically inactive Cas9 fused to VP64, p65 and Rta activators (dCas9-VPR) regulated by the constitutive CAG promoter to the AAVS1 safe harbor site. These CRISPRa hiPSC lines effectively activate target genes in pluripotency, however the dCas9-VPR transgene expression is silenced after differentiation into cardiomyocytes and endothelial cells. To understand this silencing, we systematically tested different safe harbor sites and different promoters. Targeting to safe harbor sites hROSA26 and CLYBL loci also yielded hiPSCs that expressed dCas9-VPR in pluripotency but silenced during differentiation. Muscle-specific regulatory cassettes, derived from cardiac troponin T or muscle creatine kinase promoters, were also silent after differentiation when dCas9-VPR was introduced. In contrast, in cell lines where the dCas9-VPR sequence was replaced with cDNAs encoding fluorescent proteins, expression persisted during differentiation in all loci and with all promoters. Promoter DNA was hypermethylated in CRISPRa-engineered lines, and demethylation with 5-azacytidine enhanced dCas9-VPR gene expression. In summary, the dCas9-VPR cDNA is readily expressed from multiple loci during pluripotency but induces silencing in a locus- and promoter-independent manner during differentiation to mesoderm derivatives. Researchers intending to use this CRISPRa strategy during stem cell differentiation should pilot their system to ensure it remains active in their population of interest.

FoxP1 Represses MEF2A in Striated Muscle.

Myocyte enhancer factor 2 (MEF2) proteins are involved in multiple developmental, physiological, and pathological processes in vertebrates. Protein-protein interactions underlie the plethora of biological processes impacted by MEF2A, necessitating a detailed characterization of the MEF2A interactome. A nanobody based affinity-purification/mass spectrometry strategy was employed to achieve this goal. Specifically, the MEF2A protein complexes were captured from myogenic lysates using a GFP-tagged MEF2A protein immobilized with a GBP-nanobody followed by LC-MS/MS proteomic analysis to identify MEF2A interactors. After bioinformatic analysis, we further characterized the interaction of MEF2A with a transcriptional repressor, FOXP1. FOXP1 coprecipitated with MEF2A in proliferating myogenic cells which diminished upon differentiation (myotube formation). Ectopic expression of FOXP1 inhibited MEF2A driven myogenic reporter genes (derived from the creatine kinase muscle and myogenin genes) and delayed induction of endogenous myogenin during differentiation. Conversely, FOXP1 depletion enhanced MEF2A transactivation properties and myogenin expression. The FoxP1:MEF2A interaction is also preserved in cardiomyocytes and FoxP1 depletion enhanced cardiomyocyte hypertrophy. FOXP1 prevented MEF2A phosphorylation and activation by the p38MAPK pathway. Overall, these data implicate FOXP1 in restricting MEF2A function in order to avoid premature differentiation in myogenic progenitors and also to possibly prevent re-activation of embryonic gene expression in cardiomyocyte hypertrophy.