Myosin Heavy Chain Research Articles

A pre- and postnatal immune challenge influences muscle growth and metabolism in weaned pigs

Abstract The in-utero environment is key to both fetal and postnatal growth and development. The objective of this study was to determine if administration of an acute low-dose lipopolysaccharide (LPS) to gestating sows during mid to late gestation and post-weaning would alter the offsprings metabolomic profile of the longissimus dorsi (LD) and muscle ultrastructure. Pregnant Camborough sows were randomly assigned to receive LPS (LPS; n= 7) at a dose of 2.5 μg/kg or saline (CON; n = 7) on 78 ± 1.8 d of gestation. At weaning (21 ± 1.3 d of age), barrows (CON n = 17; LPS n = 17) from each treatment were selected to receive a secondary LPS. Barrows were administered the secondary LPS challenge at a dose of 10 μg/kg 7 d post weaning. Twenty-four h after the postnatal LPS dose, barrows (31 ± 1.3 d of age) were euthanized, and each LD was removed. The left LD was utilized for morphometric measurements. Two samples from the medial section of the right LD were preserved for immunohistochemical measurements and metabolomic analyses. Mass spectral data were deconvoluted, aligned, and annotated using MS-DIAL. Univariate and multivariate analyses were conducted using MetaboAnalyst. Pathway analysis was conducted and compared to the Homo sapiens pathway library. Morphometric and immunohistochemical measurements were analyzed using the MIXED procedure of SAS version 9.4. Significance for all analyses was declared at P ≤ 0.05 and tendencies were considered at P ≤ 0.10. Average diameter of myosin heavy chain (MHC) type I and IIB/X fibers was increased (P ≤ 0.048) in LPS offspring compared with CON. Average cross-sectional area was increased (P = 0.030) in MHC IIB/X fibers and tended to be increased (P = 0.080) in MHC I fibers of LPS offspring. There were no differences (P ≥ 0.186) between treatment groups for total nuclei or nuclei positive for MYF5, PAX7, or MYF5 and PAX7 nuclei. Metabolomic analyses identified 14 differentially expressed (P < 0.05) metabolites in the LD between treatment groups. There were 10 metabolites within the LD that tended (P ≤ 0.096) to differ between treatment groups. Thus, this study shows that in-utero immune stimulation using LPS in gestating sows and a subsequent LPS challenge postnatally alters the metabolomic profile and muscle ultrastructure of the LD in weaned pigs.

Myokine BDNF highly expressed in Type I fibers inhibits the differentiation of myotubes into Type II fibers

Abstract Background Myofibers are broadly classified as slow-twitch (Type I) and fast-twitch (Type II) fibers. These two types of myofibers coexist within the same skeletal muscle tissue, determining the contractile and metabolic properties of skeletal muscle tissue by fiber type distribution. Methods and results By examining each fiber type separately, we confirmed that brain-derived neurotrophic factor (BDNF) gene is highly expressed in Type I fibers. When exposed to BDNF, primary myotubes exhibited reduced expression of Myosin Heavy Chain (MyHC) II, a marker protein characteristic of Type II fibers. BDNF overexpression in regenerating muscle tissue led to a decrease in the distribution of Type IIA fibers. Conclusions We suggest that BDNF highly expressed in Type I fibers downregulates MyHC II expression in myotubes, eventually inhibiting Type IIA fiber generation.

Abstract 4135301: Cardiac specific gene therapy to treat diabetic heart failure with preserved ejection fraction

Background: We have previously demonstrated that diabetes mellitus (DM) causes cardiac inflammation leading to heart failure with preserved ejection fraction (HFpEF). Arachidonate 5-lipoxygenase (5-LOX), encoded by the ALOX5 gene, is an enzyme that is highly expressed in leukocytes. 5-LOX synthesizes specialized pro-resolving mediators (SPMs) including Resolvin D1 (RvD1), which reduce inflammation. Hypothesis: We hypothesize that cardiac-specific ALOX5 upregulation could prevent diabetes-associated HFpEF without inducing systemic immunosuppression. Methods: DM was induced by feeding mice with a high fat diet (HFD, 60% fat by kcal) starting from 6 weeks of age for 22-24 weeks. At 24 weeks of age, DM mice were treated with RvD1, by intraperitoneal injection at 100 ng/mouse for 2 weeks. To specifically upregulate 5-LOX in the heart, another cohort of DM mice were injected with either AAV9-plain or AAV9- ALOX5 viral vectors under the control of the α-myosin heavy chain (MHC) at 22 weeks old. At 28 weeks, we performed echocardiographic measurement of diastolic function and heart extraction. Results: HFD-induced DM mice showed lower cardiac 5-LOX expression (0.72 ± 0.05 a.u. vs. 1.09 ± 0.12, p < 0.05) and RvD1 levels (1 ± 0.08 vs. 1.9 ± 0.14 fold change over DM mice, p<0.0001) than the mice with normal diet. RvD1 treatment significantly improved DM-associated diastolic function (19.7 ± 1.4 a.u. vs. 22.9 ± 0.4, p<0.05). With cardiac specific upregulation of 5-LOX, cardiac 5-LOX expression (0.89 ± 0.06 vs. 0.70 ± 0.04 a.u., p<0.05) and RvD1 levels were increased (2.1 ± 0.3 a.u. vs. 1.2 ± 0.2, normalized to AAV9-plain, p<0.05). AAV9- ALOX5 also reduced NLRP3 inflammasome activity (0.80 ± 0.02 a.u. vs. 0.90 ± 0.02, p<0.01) and improved DM-associated diastolic dysfunction (17.0 ± 1.2 a.u. vs. 20.5 ± 1.0, p<0.05) without affecting glucose metabolism and obesity in DM mice. Moreover, no alterations of cardiac macrophage infiltration, inflammatory IL1β signaling, or macrophage phenotype were observed in response to cardiac-specific ALOX5 upregulation. Conclusion: In conclusion, both direct RvD1 treatment and cardiac-specific gene therapy to enhance resolution of inflammation in cardiomyocytes reversed DM-associated HFpEF. Gene therapy avoided off-target immunosuppression. Therefore, AAV-directed cardiac ALOX5 upregulation represents a novel cardiac-specific gene therapy for HFpEF that avoids systemic immunosuppression.

MYH6 Variants Are Associated with Atrial Dysfunction in Neonates with Hypoplastic Left Heart Syndrome

Background: MYH6 variants are the most well-known genetic risk factor (10%) for hypoplastic left heart syndrome (HLHS) and are associated with decreased cardiac transplant-free survival. MYH6 encodes for α-myosin heavy chain (α-MHC), a contractile protein expressed in the neonatal atria. We therefore assessed atrial function in HLHS patients with MYH6 variants. Methods: We performed a retrospective, blinded assessment of pre-stage I atrial function using 2D speckle-tracking echocardiography (2D-STE). Variant carriers were control-matched based on AV valve anatomy, sex, and birth year. Studies were obtained postnatally from awake patients prior to surgical intervention. Right atrial (RA) and right ventricular (RV) strain and strain rate (SR) were measured from the apical four-chamber view. Results: A total of 19 HLHS patients with MYH6 variants had echocardiograms available; 18 were matched to two controls each, and one had a single control. RA active strain (ASct) was decreased in variant carriers (−1.41%, IQR −2.13, −0.25) vs. controls (−3.53%, IQR −5.53, −1.28; p = 0.008). No significant differences were identified in RV strain between the groups. RA reservoir strain (ASr) and conduit strain (AScd) positively correlated with heart rate (HR) in MYH6 variant carriers only (ASr R = 0.499, p = 0.029; AScd R = 0.469, p = 0.043). RV global longitudinal strain (GLS) as well as RV systolic strain (VSs) and strain rate (VSRs) correlated with HR in controls only (GLS R = 0.325, p = 0.050; VSs R = 0.419, p = 0.010; VSRs R = 0.410, p = 0.012). Conclusions: We identified functional consequences associated with MYH6 variants, a known risk factor for poor outcomes in HLHS. MYH6 variant carriers exhibit impaired RA contractility despite there being no differences in RV function between variant carriers and controls. MYH6 variants are also associated with an ineffective RA reservoir and conduit function at high heart rates, despite preserved RV diastolic function. RA dysfunction and reduced atrial “kick” may therefore be a significant contributor to RV failure and worse clinical outcomes in HLHS patients with MYH6 variants.

Dichloroacetate, a pyruvate dehydrogenase activator, alleviates high-fat-induced impairment of myogenic differentiation in skeletal muscles.

Obesity-induced impairment of myogenic differentiation leads to muscle loss and sarcopenia. Pyruvate dehydrogenase (PDH) plays a crucial role in glucose metabolism and is associated with muscle differentiation. However, the effect of dichloroacetate (DCA), a PDH activator, on obesity-induced impairment of myogenic differentiation remains unknown. Here, we evaluated the effects of DCA treatment on high-fat intake-induced impairment of myogenic differentiation in C2C12 cells and C57BL/6 mice. In C2C12 cells, DCA treatment improved PDH activity that was reduced by palmitate (PAL) and decreased the lactate concentrations in the media. Additionally, DCA reversed PAL- and high-fat diet (HFD)-induced decrease in the expression of myoblast determination protein 1 (MyoD), myogenin (MyoG) and myosin heavy chain (MyHC) in C2C12 cells and C57BL/6 mice. To explore the possible mechanism, DCA treatment restored the levels of p-Akt, p-FoxO1, p-FoxO3a and p-p38 MAPK levels in PAL-treated C2C12 cells. Moreover, the protective effects of DCA were reversed by treatment with the Akt inhibitor MK2206 in C2C12 cells. In summary, DCA treatment alleviated high-fat intake-induced impairment of myogenic differentiation via Akt signalling, suggesting its potential in treating obesity-associated muscle loss and sarcopenia.

Evaluation of Guanidinoacetic Acid Supplementation on Finishing Beef Steer Growth Performance, Skeletal Muscle Cellular Response, and Carcass Characteristics.

This study elucidated the effects of dosage-dependent guanidinoacetic acid (GAA) supplementation on growth performance, muscle responses, and carcass characteristics in finishing beef steers. Thirty crossbred Red Angus beef steers (395 ± 28.09kg) were randomly assigned one of three treatments during a 146-day feedlot study: basal diet without GAA supplementation (CONTROL), 1g of GAA per 100kg of BW daily (LOWGAA), and 2g of GAA per 100kg of BW daily (HIGHGAA). Individual feed intake was monitored daily, growth performance parameters were collected every 28 days, and longissimus muscle (LM) biopsies occurred every 56 days. In biopsied LM, greater (P = 0.048) mRNA expression of IGF-1 was observed in LOWGAA steers on d 112 compared to the CONTROL group. LOWGAA steers also exhibited greater expression of myosin heavy chain (MHC) I compared to CONTROL steers (P < 0.05) and MHC IIA compared to both CONTROL and HIGHGAA treatment groups (P < 0.01) on d 112. GAA supplementation resulted in no change in carcass characteristics, serum and LM tissue metabolites, LM composition, and Warner-Bratzler shear force (WBSF) values (P > 0.05). Data collected from this study demonstrate the influence of GAA supplementation on the gene expression of MHC isoforms and their role in skeletal muscle growth, differentiation, and muscle fiber-typing.

USP20 deletion promotes eccentric cardiac remodeling in response to pressure overload and increases mortality.

Left ventricular hypertrophy (LVH) caused by chronic pressure overload with subsequent pathological remodeling is a major cardiovascular risk factor for heart failure and mortality. The role of deubiquitinases in LVH has not been well characterized. To define whether the deubiquitinase ubiquitin-specific peptidase 20 (USP20) regulates LVH, we subjected USP20 knockout (KO) and cognate wild-type (WT) mice to chronic pressure overload by transverse aortic constriction (TAC) and measured changes in cardiac function by serial echocardiography followed by histological and biochemical evaluations. USP20-KO mice showed severe deterioration of systolic function within 4 wk of TAC compared with WT cohorts. Both USP20-KO TAC and WT-TAC cohorts presented cardiac hypertrophy following pressure overload. However, USP20-KO-TAC mice showed an increase in cardiomyocyte length and developed maladaptive eccentric hypertrophy, a phenotype generally observed with volume overload states and decompensated heart failure. In contrast, WT-TAC mice displayed an increase in cardiomyocyte width, producing concentric remodeling that is characteristic of pressure overload. In addition, cardiomyocyte apoptosis, interstitial fibrosis, and mouse mortality were augmented in USP20-KO-TAC compared with WT-TAC mice. Quantitative mass spectrometry of LV tissue revealed that the expression of sarcomeric myosin heavy chain 7 (MYH7), a fetal gene normally upregulated during cardiac remodeling, was significantly reduced in USP20-KO after TAC. Mechanistically, we identified increased degradative lysine-48 polyubiquitination of MYH7 in USP20-KO hearts, indicating that USP20-mediated deubiquitination likely prevents protein degradation of MYH7 during pressure overload. Our findings suggest that USP20-dependent signaling pathways regulate the layering pattern of sarcomeres to suppress maladaptive remodeling during chronic pressure overload and prevent cardiac failure.NEW & NOTEWORTHY We identify ubiquitin-specific peptidase 20 (USP20) as an important enzyme that is required for cardiac homeostasis and function, particularly during myocardial pressure overload. USP20 regulates protein stability of cardiac MYH7, an essential molecular motor protein expressed in sarcomeres; loss-of-function mutations of MYH7 are associated with human hypertrophic cardiomyopathy, cardiac failure, and sudden death. Enhancing USP20 activity could be a potential therapeutic approach to prevent the development of maladaptive state of eccentric hypertrophy and heart failure.

Dominant myosin storage myopathy mutations disrupt striated muscles in Drosophila and the myosin tail-tail interactome of human cardiac thick filaments.

Myosin storage myopathy (MSM) is a rare skeletal muscle disorder caused by mutations in the slow muscle/β-cardiac myosin heavy chain (MHC) gene. MSM missense mutations frequently disrupt the tail's stabilizing heptad repeat motif. Disease hallmarks include subsarcolemmal hyaline-like β-MHC aggregates, muscle weakness and, occasionally, cardiomyopathy. We generated transgenic, heterozygous Drosophila to examine the dominant physiological and structural effects of the L1793P, R1845W, and E1883K MHC MSM mutations on diverse muscles. The MHC variants reduced lifespan and flight and jump abilities. Moreover, confocal and electron microscopy revealed that they provoked indirect flight muscle breaks and myofibrillar disarray/degeneration with filamentous inclusions. Incorporation of GFP-myosin enabled in situ determination of thick filament lengths, which were significantly reduced in all mutants. Semi-automated heartbeat analysis uncovered aberrant cardiac function, which worsened with age. Thus, our fly models phenocopied traits observed among MSM patients. We additionally mapped the mutations onto a recently-determined, 6Å resolution, cryo-EM structure of the human cardiac thick filament. The R1845W mutation replaces a basic arginine with a polar-neutral, bulkier tryptophan, while E1883K reverses charge at critical filament loci. Both would be expected to disrupt the core and the outer shell of the backbone structure. Replacing L1793 with a proline, a potent breaker of alpha-helices, could disturb the coiled-coil of the myosin rod and alter the tail-tail interactome. Hence, all mutations likely destabilize and weaken the filament backbone. This may trigger disease in humans, while potentially analogous perturbations are likely to yield the observed thick filament and muscle disruption in our fly models.

Perindopril erbumine-entrapped ultradeformable liposomes alleviate sarcopenia via effective skin delivery in muscle atrophy mouse model

Sarcopenia is a pertinent challenge in the super-aged societies causing reduced functional performance, poor quality of life and increased morbidity. In this study, the potential of perindopril erbumine-loaded ultradeformable liposomes (PE-UDLs) against sarcopenia was investigated. PE-UDLs were prepared by thin-film hydration and extrusion method using egg yolk phosphatidylcholine (EPC) as a lipid bilayer former and Tween 80 or sodium deoxycholate as an edge activator. Owing to the smallest particle size (75.0 nm) and the highest deformability (54.2) and entrapment efficiency (35.7 %), PE-UDLs with EPC to Tween 80 ratio of 8:2 was selected as the optimized formulation. The optimized PE-UDLs showed substantially higher cumulative amount of drug permeated and permeation rate across the rat skin compared to PE solution (485.7 vs. 50.1 µg and 13.4 vs. 2.3 µg/cm2/h, respectively). Topically applied PE-UDLs successfully ameliorated the effects of lipopolysaccharide (LPS)-induced sarcopenia in mice by improving body weight changes, grip strength and muscle weight. Furthermore, PE-UDLs reduced the shrinkage of muscle fibers as demonstrated by higher cross-sectional area than PE solution. PE-UDLs also increased the expression of myosin heavy chain (MHC) protein and reduced the expression of muscle atrophy F-box (Atrogin-1) and muscle ring-finger protein-1 (MuRF1), thereby improving muscles atrophy. In conclusion, these results demonstrate the therapeutic potential of PE-UDLs against sarcopenia.

Improvement on gel properties of chicken myofibrillar protein with electron beam irradiation: Based on protein structure, gel quality, water state

This study aimed to investigate the effects of electron beam (E-beam) irradiation at different doses (0–15 kGy) on the solubility, rheological properties, emulsification characteristics, and moisture distribution of chicken myofibrillar proteins (MPs). Irradiation treatment notably increased the solubility, surface hydrophobicity, emulsification properties, and apparent viscosity of MPs, based on conformational changes caused by irradiation-induced oxidative denaturation of proteins. However, high doses of irradiation (15 kGy) induced in excessive cross-linking and aggregation of proteins, reducing the solubility, emulsification properties, and shear stress. Degradation of myosin heavy and light chains in irradiated MPs increased the content of β-turns and random coils. Additionally, the initial relaxation times of T21 and T22 in irradiated protein gels were reduced, and the peak value of P21 was increased, which improved the water-capturing ability of protein gels. Altogether, these results findings suggest that electron beam irradiation can be applied as a potential technique for modifying muscle proteins.

Controlling the co-aggregation and gelation of cod-soy binary proteins based on partially/fully denatured soy proteins

Structured binary protein networks offer a pathway for designing gel-based foods with less ecological and economic effects. This study investigated the heat-mediated interaction and gelation kinetics of cod-soy mixed proteins at molecular level. Results indicated that denaturation degree of soy proteins was fundamental in determining the protein-protein interactions during heating. Considerable non-natural β-sheet was generated in the heterogeneous structures to initiate their aggregation behavior. Cod actomyosin comprising myosin heavy chains and actin mainly regulated their aggregation below 70 °C. At higher temperatures, the formation of large-sized and intermediary-sized aggregates was dominated by disulfide bond exchange between cod actomyosin and soy A-B aggregates (≤ 85 °C) or dissociated B subunits (> 85 °C). These aggregates were further evidenced as essential components to form gel networks with higher storage modulus (G'). These findings provide fundamental insights for designing gel-based products with desirable nutritional and textural properties based on plant-animal binary proteins.

Endothelial NMMHC IIA dissociation from PAR1 activates the CREB3/ARF4 signaling in thrombin-mediated intracerebral hemorrhage

IntroductionThere is an urgent need for cerebroprotective interventions to improve the suboptimal outcomes with intracerebral hemorrhage (ICH). Despite the important role of nonmuscle myosin heavy chain IIA (NMMHC IIA) in the blood–brain barrier (BBB), its function in ICH remains unclear. ObjectivesThe objective of this study is to explore how NMMHC IIA functions in ICH and to evaluate the effectiveness of targeting NMMHC IIA as a treatment for ICH. MethodsWe firstly examined the protein expression of NMMHC IIA in clinical patients and animal models with ICH. The function of NNMMHC IIA was then corroborated by using overexpress or knockdown NMMHC IIA specifically in ECs mice and pBMECs. In addition, we explored protein interacts with NMMHC IIA and signaling pathways after ICH by LC-MS/MS and transcriptomics analysis with an emphasis on the function of PAR1 and the CREB3/ARF4 signaling pathway, and validated them in three kind of animal models. To support the clinical translation of our results, we targeted NMMHC IIA to bicalutamide selected from a library of marketed drugs and examined to validate its ameliorative effect on ICH. ResultsWe observed an upregulation of endothelial NMMHC IIA in the brain following the onset of ICH in both patients and mice, while inhibited NMMHC ⅡA improved ICH induced by thrombin, warfarin or tissue plasminogen activator (tPA) after ischemic stroke. Mechanistically, the head domain of NMMHC IIA interacted with protease-activated receptor 1 (PAR1) at the 380–430 aa region and subsequently dissociated and activated the CREB3/ARF4 signaling pathway. We found that bicalutamide and blebbistatin could bind to NMMHC IIA and effectively protect mice from thrombin-mediated ICH. ConclusionThe findings indicated that NMMHC IIA dissociated from PAR1 and activated CREB3/ARF4 pathway, which aggravated BBB damage induced by thrombin. This suggested that NMMHC IIA was a novel potential therapeutic target for BBB-related diseases.

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Titin hyper-phosphorylation in the peripheral skeletal muscle but not the diaphragm of HFpEF: association with muscle atrophy and dysfunction

Abstract Background Titin hypo-phosphorylation is an important regulatory mechanism for myocardial stiffness in heart failure with preserved ejection fraction (HFpEF). In contrast, a hyper-phosphorylation of titin in the peripheral skeletal muscle (SKM) was reported in mouse models exhibiting muscle atrophy. In former studies it, was shown that SKM atrophy occurs in HFpEF and is associated with muscle dysfunction and a fiber type shift from type I, towards type II fibers. However, these alterations were not seen in the diaphragm muscle (Dia) and a fiber type shift towards type I was documented. So far, no information about the phosphorylation status of titin is available for SKM and the Dia and its association with muscle dysfunction in HFpEF. Purpose To assess titin phosphorylation in SKM and Dia of a HFpEF animal model and to relate it to muscle function and atrophy. Methods To confirm the development of HFpEF echocardiography was performed in female ZSF1-lean (con, n=18) and ZSF1-obese rats (HFpEF, n=18) at the age of 32 weeks. Skeletal muscle function (soleus) was measured in an organ bath setting after the animals were sacrificed. Tissue of the tibialis anterior muscle (TA) and the diaphragm (Dia) was collected and fixed in formalin or snap frozen in liquid nitrogen. To detect titin expression homogenized TA and Dia tissue samples were resolved on a vertical agarose gel. Phosphorylated titin was detected by staining the gel with Pro-Q Diamond, whereas total titin was visualized using Sypro Ruby. Cross sectional area (CSA) of TA and Dia was quantified after Hematoxylin/eosin staining of tissue section. Myosin heavy chain (MHC) ubiquitination was assessed by western blot. Results Assessment of SKM function revealed significantly reduced maximal absolute (-12 %) and maximal specific force (-22 %) in HFpEF animals when compared to controls. In the TA a significantly reduced CSA was evident in HFpEF (1911±103 vs. 1394±103 µm2, p=0.002), whereas in the diaphragm an increased CSA was noted (438±11 vs. 560±18 µm2, p&amp;lt;0.001). In TA of HFpEF animals, the proportion of phosphorylated titin was significantly elevated whereas the total amount of titin was reduced (Fig. 1A, C). In addition, a significantly higher proportion of ubiquitinated MHC was detected (Figure 1E). However in the Dia these alterations were not observed, even a higher amount of total titin was seen (Fig. 1B, D, F). Furthermore, a significant negative correlation between phosphorylated titin and absolute force (r=-0.461, p=0.035, Fig. 2A) as well as specific force (r=-0.693, p=0.0005, Fig. 2B) was evident. Conclusion In contrast to the heart, the TA showed a titin hyper-phosphorylation in HFpEF, going along with a degradation of total titin. This hyper-phosphorylation could be correlated with a loss of force in the SKM. Additionally a higher ubiquitination of MHC was prevalent in the TA, which may be connected to molecular remodeling and fiber type shift towards a more glycolytic type.

Comparative study of lysine acetylation in Vesicomyidae clam Archivesica marissinica and the manila clam Ruditapes philippinarum: adaptation mechanisms in cold seep environments

BackgroundThe deep-sea cold seep zone is characterized by high pressure, low temperature, darkness, and oligotrophy. Vesicomyidae clams are the dominant species within this environment, often forming symbiotic relationships with chemosynthetic microbes. Understanding the mechanisms by which Vesicomyidae clams adapt to the cold seep environment is significant. Acetylation modification of lysine is known to play a crucial role in various metabolic processes. Consequently, investigating the role of lysine acetylation in the adaptation of Vesicomyidae clams to deep-sea environments is worthwhile. So, a comparative study of lysine acetylation in cold seep clam Archivesica marissinica and shallow water shellfish Ruditapes philippinarum was conducted.ResultsA total of 539 acetylated proteins were identified with 1634 acetylation sites. Conservative motif enrichment analysis revealed that the motifs -KacR-, -KacT-, and -KacF- were the most conserved. Subsequent gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted on significantly differentially expressed acetylated proteins. The GO enrichment analysis indicated that acetylated proteins are crucial in various biological processes, including cellular response to stimulation, and other cellular processes ( p < 0.05 and false discovery rate (FDR) < 0.25). The results of KEGG enrichment analysis indicated that acetylated proteins are involved in various cellular processes, including tight junction, motor proteins, gap junction, phagosome, cGMP-PKG signaling pathways, endocytosis, glycolysis/gluconeogenesis, among others (p < 0.05 and FDR < 0.25). Notably, a high abundance of lysine acetylation was observed in the glycolysis/glycogenesis pathways, and the acetylation of glyceraldehyde 3-phosphate dehydrogenase might facilitate ATP production. Subsequent investigation into acetylation modifications associated with deep-sea adaptation revealed the specific identification of key acetylated proteins. Among these, the adaptation of cold seep clam hemoglobin and heat shock protein to high hydrostatic pressure and low temperature might involve an increase in acetylation levels. Acetylation of arginine kinase might be related to ATP production and interaction with symbiotic bacteria. Myosin heavy chain (Ama01085) has the most acetylation sites and might improve the actomyosin system stability through acetylation. Further validation is required for the acetylation modification from Vesicomyidae clams.ConclusionA novel comparative analysis was undertaken to investigate the acetylation of lysine in Vesicomyidae clams, yielding novel insights into the regulatory role of lysine acetylation in deep-sea organisms. The findings present many potential proteins for further exploration of acetylation functions in cold seep clams and other deep-sea mollusks.

Sinomenine hydrochloride alleviates autoimmune myocarditis via suppressing Th1 cell induced-M1 macrophage pyroptosis

Abstract Background Myocarditis is typical of the complex inflammatory response in the heart with high morbidity and mortality, accompanied by tissue damage and cardiac fibrosis. Previous studies have demonstrated that CD4+ T cells are crucial to the cardiac autoimmunity of myocarditis, but the role of different CD4+ T cell subsets remains unclear. Recently, sinomenine hydrochloride, a natural compound from the traditional Chinese herb Sinomenium acutum, is reported to inhibit T cell proliferation in some systemic autoimmune diseases such as rheumatoid arthritis. Given that autoimmune myocarditis is an organ-specific autoimmune disease, the effect of sinomenine hydrochloride on autoimmune myocarditis demands further study. Purpose This study aims to elucidate the role of CD4+ T-helper 1 (Th1) cells in regulating inflammatory cell death in autoimmune myocarditis and investigate the pharmacological effect of sinomenine hydrochloride on CD4+ Th1 cells and autoimmune myocarditis. Methods Male Balb/c mice were immunized with myosin heavy chain-α peptides to establish the experimental autoimmune myocarditis (EAM) model, followed by the treatments with sinomenine hydrochloride by gavage. Tohoku Hospital Pediatrics-1 (THP-1) cell line and murine T cells were used for in vitro experiments. Results In the acute phase of EAM, we observed plenty of CD4+ T cells infiltrating the heart tissue. RNA sequencing revealed that among the genes encoding typical transcription factors of CD4+ T cell subset, only Tbx21 and Stat4, both representing Th1 cells, were upregulated, which consisted with immunofluorescence staining. Further studies indicated that Th1 cells not only mediated classical activated macrophage (M1 macrophage) polarization by secreting interferon-γ (IFN-γ) but also associated with pro-inflammatory macrophage pyroptosis in the EAM, with the cleavage of gasdermin D (GSDMD), caspase-1 and IL-1β. Subsequent in vitro experiments confirmed that IFN-γ could dramatically promote macrophage pyroptosis combined with ATP, leading to the release of IL-1β and IL-18(Figure 1). To find an effective method for Th1 cell suppression and myocarditis remission, we identified that sinomenine hydrochloride impeded Th1 cell activation, differentiation and proliferation in vitro. Furthermore, sinomenine hydrochloride inhibited Th1 cell infiltration and M1 macrophage pyroptosis in the EAM heart, In the chronic phase, cardiac remodeling and fibrosis were suppressed, and cardiac systolic function was significantly improved in the treatment group (Figure 2). Conclusion Our study demonstrates that Th1 cells-induced M1 macrophage pyroptosis is an essential pathogenic mechanism of EAM. Sinomenine hydrochloride can alleviate the severity and progression of EAM, which may become a novel therapeutic strategy for treating myocarditis.Figure 1Figure 2

Activation of mitochondrial KATP channels attenuates arrhythmogenesis increased in cardiac-specific connexin43-deficient mice

Abstract Introduction Connexin43 (Cx43) is a connexin that forms gap junctions in the hearts and also exits as hemichannels in the inner mitochondrial membrane (mCx43). In our previous study, deficiency of Cx43 increased mitochondrial Ca2+ (mCa2+). It has not yet been established, however, how mCx43 are involved in arrhythmogenesis. Purpose To examine how mCa2+ changes with deficiency of Cx43 and what roles mitochondrial KATP (mKATP) channels play in the changes in mCa2+ and arrhythmogenesis. Methods Cx43flox/flox mice were crossed with α-myosin heavy chain cre+/- mice, and cardiac-specific Cx43-deficient (Cx43-/-) mice were generated. The resulting offspring, Cx43-/- mice and their littermates (Cx43+/+ mice) were used. Trabeculae were dissected from their right ventricles, and force was measured with a strain gauge. To estimate arrhythmogenesis, the minimal extracellular Ca2+ (Cao), at which arrhythmias were induced by electrical stimulation (0.3-s stimulus intervals for 30 s), was determined (Caomin). In digitonin-skinned trabeculae, mCa2+ was estimated using rhod-2, and mitochondrial membrane potential using JC-1 fluorescence ratio (Red/Green). The spatial distribution of rhod-2 and JC-1 was similar to that of MitoTracker Green, meaning that rhod-2 and JC-1 are mainly loaded within the mitochondria. To investigate the roles of mKATP channels and mCx43, 100 µM diazoxide (DZX), a mKATP channel opener, 200 μM 5-hydroxydecanoic acid (5-HD), a mKATP channel inhibitor, and 5 μM carbenoxolone, a Cx43 inhibitor, were used. Results Cx43 was present in the inner mitochondrial membrane in Cx43+/+ mice but not in Cx43-/- mice. Most of Cx43-/- mice died within 8 weeks (p&amp;lt;0.01). The Cao min in Cx43-/- mice was lower than that in Cx43+/+ mice (n=5, p&amp;lt;0.01) and was increased by DZX (n=4, p&amp;lt;0.05), suggesting that increased arrhythmogenesis in Cx43-/- mice is attenuated by DZX. Rhod-2 fluorescence in Cx43-/- mice was higher than that in Cx43+/+ mice (n=7, p&amp;lt;0.01) and was decreased by DZX at Cao from 0.2 to 6 mM (n=7, p&amp;lt;0.05, 2-way ANOVA). Besides, in digitonin-skinned trabeculae, rhod-2 fluorescence in Cx43-/- mice was higher than that in Cx43+/+ mice (n=5, p&amp;lt;0.05) and was decreased by DZX at Ca2+ from 80 to 2700 nM (n=5, p&amp;lt;0.01, 2-way ANOVA). Conversely, rhod-2 fluorescence in Cx43+/+ mice was increased by CBX and 5-HD in skinned trabeculae at Ca2+ from 80 to 2700 nM (n=7, p&amp;lt;0.05, 2-way ANOVA). In skinned trabeculae, JC-1 fluorescence ratio was increased only in Cx43-/- mice depending on the increase in Ca2+ from 80 to 2700 nM (n=5, p&amp;lt;0.01), and this increase was suppressed by DZX, meaning that mitochondrial membrane potential is hyperpolarized with deficiency of mCx43, and that this hyperpolarization is reversed with activation of mKATP channels. Conclusions In the myocardium with deficiency of Cx43, increased arrhythmogenesis is attenuated by activation of mKATP channels probably through a decrease in mCa2+ and normalization of mitochondrial membrane potential.

The essential role of the miRNA-208a/TARBP2/Sox6 axis in myosin expression in the end stage human heart failure

Abstract Despite diverse aetiologies, failing human hearts (HF) of various origins (coronary artery disease (CAD), dilated cardiomyopathy (DCM), restrictive cardiomyopathy, and hypertrophic cardiomyopathy (HCM)) exhibit impaired contractility and share alterations in MYH6, MYH7, and MYH7B gene expression. Here, we explore the miRNA-208a/TARBP2/Sox6 axis in regulating myosin expression within left ventricles of explanted human hearts. Methods Samples of left ventricles were obtained from explanted hearts of patients with terminal HF (NYHA III-IV) and reduced left ventricle ejection fraction (LVEF &amp;lt;25%) due to CAD (n=10), DCM (n=10), HCM (n=6), and controls (n=5). We analysed the expression of cardiac myosin heavy chains (MYH6, MYH7, MYH7B), cardiac damage markers (ANP, BNP), and heart-related MyoMiRs using quantitative real-time PCR. Results Pre-transplant data for the 31 subjects (25 male, 3 female) included: age 54.4±8.2y, BP 112±18/67±11 mmHg, HR 79±16 min-1, LVEF 27±14%, LVEDD 8.8±15.8mm, RVEDD 32.8±5.1mm, QRS 0.140±0.05s, QT 0.42±0.04s, NT-proBNP 5551±4259 ng/l. In all diseased groups, MYH6 mRNA was downregulated, while MYH7 and MHRT ncRNA were upregulated. We observed strong positive correlations between MYH7, MYH7B, and MHRT transcription, suggesting coordinated expression. The miRNA-208a/miRNA-208b ratio shifted heavily towards miRNA-208b in controls, with a similar effect for miRNA-499 and MYH7B. A strong negative correlation existed between miRNA-499 and MYH7B expression across HF samples. TARBP2 expression decreased slightly in all pathological groups, while Sox6 mRNA increased. Conclusion Elevated Sox6 expression may contribute to MYH6 downregulation and MYH7/MYH7B upregulation. Altered TARBP2 expression could promote cardiomyopathy via Sox6-mediated repression of cardiac slow-twitch myofiber proteins. Changes in miRNA-208a processing leading to Sox6 upregulation may inhibit Sox6-dependent myosin transcript expression.

Evaluation of the Physicochemical Attributes of Beef, Chicken, and Pork Muscles Injected with Microbial Proteases for Designing Senior-Friendly Processed Meat Products

In developed countries, the growing elderly population has increased the demand for senior-friendly processed meat products. This study investigated the effects of four commercial microbial proteases (Alcalase, Flavourzyme, Neutrase, and Protamex) on the general physicochemical attributes of beef top round, chicken breast, and pork loin, which are lean muscle cuts suitable for developing senior-friendly meat products. Muscle samples were injected with microbial protease solutions (0.7% and 1.2% (w/w)), cooked, and used for analysis. The microbial protease injection significantly reduced the hardness of cooked muscles. Despite the evident degradation of the myosin heavy chain in Alcalase treatment, the lowest hardness values were observed in Protamex-treated samples, suggesting that myosin degradation alone does not fully account for tenderness improvement. Unfortunately, microbial protease treatments increased cooking loss in beef and chicken muscles (p &lt; 0.05). The surface color characteristics, including redness and yellowness, remained unaffected by the enzymatic treatments, supporting the practical use of these proteases for meat tenderization without inducing color defects. While microbial proteases demonstrate potential for improving meat tenderness, future research should focus on mitigating cooking loss and ensuring desirable taste and flavor for the commercial production of senior-friendly processed meat products using the microbial proteases.

The effect of biocide chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) mixture on C2C12 muscle cell damage attributed to mitochondrial reactive oxygen species overproduction and autophagy activation

ABSTRACT The mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT) is a biocide widely used as a preservative in various commercial products. This biocide has also been used as an active ingredient in humidifier disinfectants in South Korea, resulting in serious health effects among users. Recent evidence suggests that the underlying mechanism of CMIT/MIT-initiated toxicity might be associated with defects in mitochondrial functions. The aim of this study was to utilize the C2C12 skeletal muscle model to investigate the effects of CMIT/MIT on mitochondrial function and relevant molecular pathways associated with skeletal muscle dysfunction. Data demonstrated that exposure to CMIT/MIT during myogenic differentiation induced significant mitochondrial excess production of reactive oxygen species (ROS) and a decrease in intracellular ATP levels. Notably, CMIT/MIT significantly inhibited mitochondrial oxidative phosphorylation (Oxphos) and reduced mitochondrial mass at a lower concentration than the biocide amount, which diminished the viability of myotubes. CMIT/MIT induced activation of autophagy flux and decreased protein expression levels of myosin heavy chain (MHC). Taken together, CMIT/MIT exposure produced damage in C2C12 myotubes by impairing mitochondrial bioenergetics and activating autophagy. Our findings contribute to an increased understanding of the underlying mechanisms associated with CMIT/MIT-induced adverse skeletal muscle health effects.