Depth of Interbreed Difference in Postmortem Bovine Muscle Determined by CE-FT/MS and LC-FT/MS Metabolomics.

Japanese Brown (JBR) cattle have moderately marbled beef compared to the highly marbled beef of Japanese Black (JBL) cattle; however, their skeletal muscle properties remain poorly characterized. To unveil interbreed metabolic differences over the previous results, we explored the metabolome network changes before and after postmortem 7-day aging in the trapezius muscle of the two cattle breeds by employing a deep and high-coverage metabolomics approach. Using both capillary electrophoresis (CE) and ultra-high-performance liquid chromatography (UHPLC)-Fourier transform mass spectrometry (FT/MS), we detected 522 and 384 annotated peaks, respectively, across all muscle samples. The CE-based results showed that the cattle were clearly separated by breed and postmortem age in multivariate analyses. The metabolism related to glutathione, glycolysis, vitamin K, taurine, and arachidonic acid was enriched with differentially abundant metabolites in aged muscles, in addition to amino acid (AA) metabolisms. The LC-based results showed that the levels of bile-acid-related metabolites, such as tauroursodeoxycholic acid (TUDCA), were high in fresh JBR muscle and that acylcarnitines were enriched in aged JBR muscle, compared to JBL muscle. Postmortem aging resulted in an increase in fatty acids and a decrease in acylcarnitine in the muscles of both cattle breeds. In addition, metabolite set enrichment analysis revealed that JBR muscle was distinctive in metabolisms related to pyruvate, glycerolipid, cardiolipin, and mitochondrial energy production, whereas the metabolisms related to phosphatidylethanolamine, nucleotide triphosphate, and AAs were characteristic of JBL. This suggests that the interbreed differences in postmortem trapezius muscle are associated with carnitine/acylcarnitine transport, ß-oxidation, tricarboxylic acid cycle, and mitochondrial membrane stability, in addition to energy substrate and AA metabolisms. These interbreed differences may characterize beef quality traits such as the flavor intensity and oxidative stability.

Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform-dependent manner.

Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement.

Calpain-3 not only proteolyzes calpain-1 and -2 but also is a substrate for calpain-1 and -2.

Calpain is an intracellular cysteine protease that cleaves its specific substrates in a limited region to modulate cellular function. Calpain-1 (C1) and calpain-2 (C2) are ubiquitously expressed in mammalian cells, but calpain-3 (C3) is a skeletal muscle-specific type. In the course of calpain activation, the N-terminal regions of all three isoforms are clipped off in an intramolecular or intermolecular fashion. C1 proteolyzes C2 to promote further proteolysis, but C2 proteolyzes C1 to suspend C1 proteolysis, indicating the presence of C1-C2 reciprocal proteolysis. However, whether C3 is involved in the calpain proteolysis network is unclear. To address this, we examined whether GFP-tagged C3:C129S (GFP-C3:CS), an inactive protease form of C3, was a substrate for C1 or C2 in HEK cells. Intriguingly, the N-terminal region of C3:CS was cleaved by C1 and C2 at the site identical to that of the C3 autoproteolysis site. Furthermore, the N-terminal clipping of C3:CS by C1 and C2 was observed in mouse skeletal muscle lysates. Meanwhile, C3 preferentially cleaved the N-terminus of C1 over that of C2, and the sizes of these cleaved proteins were identical to their autoproteolysis forms. Our findings suggest an elaborate inter-calpain network to prime and suppress proteolysis of other calpains.

DNA Methylation of Genes Participating in Hepatic Metabolisms and Function in Fetal Calf Liver Is Altered by Maternal Undernutrition during Gestation.

This study aimed to elucidate the effects of maternal undernutrition (MUN) on epigenetic modification of hepatic genes in Japanese Black fetal calves during gestation. Using a previously established experimental design feeding the dams with 60% (LN) or 120% (HN) of their global nutritional requirements during the 8.5-month gestational period, DNA methylation in the fetal liver was analyzed with reduced representation bisulfite sequencing (RRBS). The promoters and gene bodies in the LN fetuses were hypomethylated compared to HN fetuses. Pathway analysis showed that the genes with DMR in the exon/intron in the LN group were associated with pathways involved in Cushing syndrome, gastric acid secretion, and aldosterone synthesis and secretion. Promoter hypomethylation in the LN group was frequently observed in genes participating in various signaling pathways (thyroid hormone, Ras/Rap1, PIK3-Akt, cAMP), fatty acid metabolism, and cholesterol metabolism. The promoter hypomethylated genes ALPL and GNAS were upregulated in the LN group, whereas the promoter hypermethylated genes GRB10 and POR were downregulated. The intron/exon hypomethylated genes IGF2, IGF2R, ACAD8, TAT, RARB, PINK1, and SOAT2 were downregulated, whereas the hypermethylated genes IGF2BP2, NOS3, and NR2F1 were upregulated. Collectively, MUN alters the promoter and gene body methylation of genes associated with hepatic metabolisms (energy, cholesterol, mitochondria) and function, suggesting an impact of altered gene methylation on the dysregulation of gene expression in the fetal liver.

Netrin-4 synthesized in satellite cell-derived myoblasts stimulates autonomous fusion.

Satellite cells are indispensable for skeletal muscle regeneration and hypertrophy by forming nascent myofibers (myotubes). They synthesize multi-potent modulator netrins (secreted subtypes: netrin-1, -3, and -4), originally found as classical neural axon guidance molecules. While netrin-1 and -3 have key roles in myogenic differentiation, the physiological significance of netrin-4 is still unclear. This study examined whether netrin-4 regulates myofiber type commitment and myotube formation. Initially, the expression profiles indicated that satellite cells isolated from the extensor digitorum longus muscle (EDL muscle: fast-twitch myofiber-abundant) expressed slightly more netrin-4 than the soleus muscle (slow-type abundant) cells. As netrin-4 knockdown inhibited both slow- and fast-type myotube formation, netrin-4 may not directly regulate myofiber type commitment. However, netrin-4 knockdown in satellite cell-derived myoblasts reduced the myotube fusion index, while exogenous netrin-4 promoted myotube formation, even though netrin-4 expression level was maximum during the initiation stage of myogenic differentiation. Furthermore, netrin-4 knockdown also inhibited MyoD (a master transcriptional factor of myogenesis) and Myomixer (a myoblast fusogenic molecule) expression. These data suggest that satellite cells synthesize netrin-4 during myogenic differentiation initiation to promote their own fusion, stimulating the MyoD-Myomixer signaling axis.

Metabolomic profiling of postmortem aged muscle in Japanese Brown beef cattle revealed an interbreed difference from Japanese Black beef.

OBJECTIVE: Japanese Brown (JBR) cattle, especially the Kochi (Tosa) pedigree (JBRT), is a local breed of moderately marbled beef. Despite the increasing demand, the interbreed differences in muscle metabolites from the highly marbled Japanese Black (JBL) beef remain poorly understood. We aimed to determine flavor-related metabolites and postmortem metabolisms characteristic to JBRT beef in comparison with JBL beef. METHODS: Lean portions of the longissimus thoracis (loin) muscle from four JBRT cattle were collected at 0, 1, and 14 d postmortem. The muscle metabolomic profiles were analyzed using capillary electrophoresis time-of-flight mass spectrometry. The difference in postmortem metabolisms and aged muscle metabolites were analyzed by statistical and bioinformatic analyses between JBRT (n = 12) and JBL cattle (n = 6). RESULTS: A total of 240 metabolite annotations were obtained from the detected signals of the JBRT muscle samples. Principal component analysis separated the beef samples into three different aging point groups. According to metabolite set enrichment analysis, postmortem metabolic changes were associated with the metabolism of pyrimidine, nicotinate and nicotinamide, purine, pyruvate, thiamine, amino sugar, and fatty acid; citric acid cycle; and pentose phosphate pathway as well as various amino acids and mitochondrial fatty acid metabolism. The aged JBRT beef showed higher ultimate pH and lower lactate content than aged JBL beef, suggesting the lower glycolytic activity in postmortem JBRT muscle. JBRT beef was distinguished from JBL beef by significantly different compounds, including choline, amino acids, uridine monophosphate, inosine 5'-monophosphate, fructose 1,6-diphosphate, and betaine, suggesting interbreed differences in the accumulation of nucleotide monophosphate, glutathione metabolism, and phospholipid metabolism. CONCLUSION: Glycolysis, purine metabolism, fatty acid catabolism, and protein degradation were the most common pathways in beef during postmortem aging. The differentially expressed metabolites and the relevant metabolisms in JBRT beef may contribute to the development of a characteristic flavor.

Endogenous slow and fast myosin dynamics in myofibers isolated from mice expressing GFP-Myh7 and Kusabira Orange-Myh1.

Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single-thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFP-Myh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging.

Maternal Nutrient Restriction Disrupts Gene Expression and Metabolites Associated with Urea Cycle, Steroid Synthesis, Glucose Homeostasis, and Glucuronidation in Fetal Calf Liver.

This study aimed to understand the mechanisms underlying the effects of maternal undernutrition (MUN) on liver growth and metabolism in Japanese Black fetal calves (8.5 months in utero) using an approach that integrates metabolomics and transcriptomics. Dams were fed 60% (low-nutrition; LN) or 120% (high-nutrition; HN) of their overall nutritional requirements during gestation. We found that MUN markedly decreased the body and liver weights of the fetuses; metabolomic analysis revealed that aspartate, glycerol, alanine, gluconate 6-phosphate, and ophthalmate levels were decreased, whereas UDP-glucose, UDP-glucuronate, octanoate, and 2-hydroxybutyrate levels were decreased in the LN fetal liver (p ≤ 0.05). According to metabolite set enrichment analysis, the highly different metabolites were associated with metabolisms including the arginine and proline metabolism, nucleotide and sugar metabolism, propanoate metabolism, glutamate metabolism, porphyrin metabolism, and urea cycle. Transcriptomic and qPCR analyses revealed that MUN upregulated QRFPR and downregulated genes associated with the glucose homeostasis (G6PC, PCK1, DPP4), ketogenesis (HMGCS2), glucuronidation (UGT1A1, UGT1A6, UGT2A1), lipid metabolism (ANGPTL4, APOA5, FADS2), cholesterol and steroid homeostasis (FDPS, HSD11B1, HSD17B6), and urea cycle (CPS1, ASS1, ASL, ARG2). These metabolic pathways were extracted as relevant terms in subsequent gene ontology/pathway analyses. Collectively, these results indicate that the citrate cycle was maintained at the expense of activities of the energy metabolism, glucuronidation, steroid hormone homeostasis, and urea cycle in the liver of MUN fetuses.

Thick filament-associated myosin undergoes frequent replacement at the tip of the thick filament.

Myosin plays a fundamental role in muscle contraction. Approximately 300 myosins form a bipolar thick filament, in which myosin is continuously replaced by protein turnover. However, it is unclear how rapidly this process occurs and whether the myosin exchange rate differs depending on the region of the thick filament. To answer this question, we first measured myosin release and insertion rates over a short period and monitored myotubes expressing a photoconvertible fluorescence protein-tagged myosin, which enabled us to monitor myosin release and insertion simultaneously. About 20% of myosins were replaced within 10 min, while 70% of myosins were exchanged over 10 h with symmetrical and biphasic alteration of myosin release and insertion rates. Next, a fluorescence pulse-chase assay was conducted to investigate whether myosin is incorporated into specific regions in the thick filament. Newly synthesized myosin was located at the tip of the thick filament rather than the center in the first 7 min of pulse-chase labeling and was observed in the remainder of the thick filament by 30 min. These results suggest that the myosin replacement rate differs depending on the regions of the thick filament. We concluded that myosin release and insertion occur concurrently and that myosin is more frequently exchanged at the tip of the thick filament.

Estrogen modulates the skeletal muscle regeneration process and myotube morphogenesis: morphological analysis in mice with a low estrogen status.

The purpose of this study was to elucidate the functions of estrogen and two estrogen receptors (ERs; ERα and ERß) in the myoregeneration process and morphogenesis. Cardiotoxin (CTX) was injected into the tibialis anterior (TA) muscles of ovariectomized (OVX) mice to induce muscle injury, and subsequent myoregeneration was morphologically assessed. The diameter of regenerated myotubes in OVX mice was significantly smaller than that in intact mice at all time points of measurement. OVX mice also showed lower muscle recovery rates and slower speeds than did intact mice. ER protein levels showed a predominance of ERß over ERα in both intact and OVX states. The ERß level was increased significantly at 7 days after CTX injection in OVX mice and remained at a high level until 14 days. In addition, continuous administration of E2 to OVX mice in which muscle injury was induced resulted in a significantly larger diameter of regenerated myotubes than that in mice that did not receive estrogen. The results indicate that estrogen is an essential factor in the myoregeneration process since estrogen depletion delayed myoregeneration in injured muscles and administration of estrogen under the condition of a low estrogen status rescued delayed myoregeneration. The results strongly suggested that ERß may be a factor that promotes myoregeneration more than does ERα.