Protective effects and molecular mechanisms of Litopenaeus vannamei treated with l-arginine/l-lysine against myofibrillar proteins oxidation and quality degradation during freeze-thaw cycles.

The storage and processing of Litopenaeus vannamei are often challenged by the freeze-thaw (F-T) cycle phenomenon. This study delved into the influence of pretreatment with l-arginine (Arg) and l-lysine (Lys) on the myofibrillar proteins oxidation and quality of shrimp subjected to F-T cycles. Arg and Lys pretreatment notably improved water-holding capacity (WHC), textural integrity as well as the myofibrillar structure of the shrimps. A lesser reduction in the amounts of immobile and bound water was found in the amino acid-treated groups, and the oxidation of lipids and proteins were both decelerated. Molecular simulation results indicated that Arg and Lys could form hydrogen and salt-bridge bonds with myosin, enhancing the stability of Litopenaeus vannamei. The study concludes that Arg and Lys are effective in alleviating the adverse effects of F-T cycles on the quality of Litopenaeus vannamei, and provides a new solution for the quality maintenance during storage and processing.

Ultrastructural Features of Left Ventricle Cardiomyocytes in Preterm Newborn Rats.

The structure of left ventricular cardiomyocytes of 1 day preterm newborn rats was studied using transmission electron microscopy. It was shown that the relative area of the nucleus in cardiomyocytes of preterm rats is lower, and the relative area of the cytoplasm is higher than in full-term rats, while the relative areas of myofibrils and mitochondria do not differ. In cardiomyocytes of preterm rats damaged mitochondria, subsegmental myofibrillar contracture, and cytoplasmic swelling were found on the first postnatal day. Preterm birth in rats, in contrast to birth at term, is accompanied by the development of a number of ultrastructural damages in cardiomyocytes.

Genetic excision of the regulatory cardiac troponin I extension in high-heart rate mammal clades.

Mammalian cardiac troponin I (cTnI) contains a highly conserved amino-terminal extension harboring protein kinase A targets [serine-23 and -24 (Ser23/24)] that are phosphorylated during ß-adrenergic stimulation to defend diastolic filling by means of an increased cardiomyocyte relaxation rate. In this work, we show that the Ser23/24-encoding exon 3 of TNNI3 was pseudoexonized multiple times in shrews and moles to mimic Ser23/24 phosphorylation without adrenergic stimulation, facilitating the evolution of exceptionally high resting heart rates (~1000 beats per minute). We further reveal alternative exon 3 splicing in distantly related bat families and confirm that both cTnI splice variants are incorporated into cardiac myofibrils. Because exon 3 of human TNNI3 exhibits a relatively low splice strength score, our findings offer an evolutionarily informed strategy to excise this exon to improve diastolic function during heart failure.

Insight into the effects of ultrasound-assisted intermittent tumbling on the gelation properties of myofibrillar proteins: Conformational modifications, intermolecular interactions, rheological properties and microstructure.

The aim of the present study was to evaluate the effects of ultrasound-assisted intermittent tumbling (UT) at 300 W, 20 kHz and 40 min on the conformation, intermolecular interactions and aggregation of myofibrillar proteins (MPs) and its induced gelation properties at various tumbling times (4 and 6 h). Raman results showed that all tumbling treatments led the helical structure of MPs to unfold. In comparison to the single intermittent tumbling treatment (ST), UT treatment exerted more pronounced effects on strengthening the intermolecular hydrogen bonds and facilitating the formation of an ordered ß-sheet structure. When the tumbling time was the same, UT treatment caused higher surface hydrophobicity, fluorescence intensity and disulfide bond content in the MPs, inducing the occurrence of hydrophobic interaction and disulfide cross-linking between MPs molecules, thus forming the MPs aggregates. Additionally, results from the solubility, particle size, atomic force microscopy and SDS-PAGE further indicated that, relative to the ST treatment, UT treatment was more potent in promoting the polymerization of myosin heavy chain. The MPs aggregates in the UT group were more uniform than those in the ST group. During the gelation process, the pre-formed MPs aggregates in the UT treatment increased the thermal stability of myosin, rendering it more resistant to heat-induced unfolding of the myosin rod region. Furthermore, they improved the protein tail-tail interaction, resulting in the formation of a well-structured gel network with higher gel strength and cooking yield compared to the ST treatment.

Improving the texture of braised pork by gradient-temperature heating and its molecular mechanism.

A novel gradient-temperature heating regime was proposed to improve the texture of braised pork. Compared with one-stage pressure heat treatment of around 107 °C, the gradient-temperature heat regime of preheating at 60 °C, followed by a slow increase of temperature to 107 °C and simmering at 97 °C increased the retention of immobilized water and reduced the shear force of meat. In this cooking regime, preheating treatment at 50-60 °C could promote the dissociation of thin and thick myofilaments, which contributed to a weakened shrinkage of myofibrils during the subsequent high temperature heating process. Pressure-heating treatment with a slow increasing temperature and the medium-temperature simmering significantly reduced (p < 0.05) the oxidation of sulfhydryl groups and the loss of α-helical, which weakened the excessive aggregation of protein and promoted the formation of myofibril network. Both the weakened shrinkage and the formation of myofibril network during gradient-temperature heating contributed to the decreased shear force and an increased immobilized water. Hence, the reduction of the oxidation and aggregation of the proteins is the key to improve the tenderness of the braised meat.

Myosin Isoform-Dependent Effect of Omecamtiv Mecarbil on the Regulation of Force Generation in Human Cardiac Muscle.

Omecamtiv mecarbil (OM) is a small molecule that has been shown to improve the function of the slow human ventricular myosin (MyHC) motor through a complex perturbation of the thin/thick filament regulatory state of the sarcomere mediated by binding to myosin allosteric sites coupled to inorganic phosphate (Pi) release. Here, myofibrils from samples of human left ventricle (ß-slow MyHC-7) and left atrium (α-fast MyHC-6) from healthy donors were used to study the differential effects of µmolar [OM] on isometric force in relaxing conditions (pCa 9.0) and at maximal (pCa 4.5) or half-maximal (pCa 5.75) calcium activation, both under control conditions (15 °C; equimolar DMSO; contaminant inorganic phosphate [Pi] ~170 µM) and in the presence of 5 mM [Pi]. The activation state and OM concentration within the contractile lattice were rapidly altered by fast solution switching, demonstrating that the effect of OM was rapid and fully reversible with dose-dependent and myosin isoform-dependent features. In MyHC-7 ventricular myofibrils, OM increased submaximal and maximal Ca2+-activated isometric force with a complex dose-dependent effect peaking (40% increase) at 0.5 µM, whereas in MyHC-6 atrial myofibrils, it had no effect or-at concentrations above 5 µM-decreased the maximum Ca2+-activated force. In both ventricular and atrial myofibrils, OM strongly depressed the kinetics of force development and relaxation up to 90% at 10 µM [OM] and reduced the inhibition of force by inorganic phosphate. Interestingly, in the ventricle, but not in the atrium, OM induced a large dose-dependent Ca2+-independent force development and an increase in basal ATPase that were abolished by the presence of millimolar inorganic phosphate, consistent with the hypothesis that the widely reported Ca2+-sensitising effect of OM may be coupled to a change in the state of the thick filaments that resembles the on-off regulation of thin filaments by Ca2+. The complexity of this scenario may help to understand the disappointing results of clinical trials testing OM as inotropic support in systolic heart failure compared with currently available inotropic drugs that alter the calcium signalling cascade.

Exploring the Effects of S-Nitrosylation on Caspase-3 Modification and Myofibril Degradation of Beef In Vitro.

This study aimed to explore the effects of S-nitrosylation on caspase-3 modification and its subsequent effects on beef myofibril degradation in vitro. Recombinant caspase-3 was reacted with different concentrations of S-nitrosoglutathione (GSNO, nitric oxide donor) at 37 °C for 30 min and subsequently incubated with purified myofibrillar protein from bovine semimembranosus muscle. Results indicated that the activity of caspase-3 was significantly reduced after GSNO treatments (P < 0.05) and showed a dose-dependent inhibitory effect, which was attributed to the increased S-nitrosylation extent of caspase-3. LC-MS/MS analysis revealed that caspase-3 was S-nitrosylated at cysteine sites 116, 170, 184, 220, and 264. Moreover, the degradation of desmin and troponin-T was notably suppressed by S-nitrosylated caspase-3 (P < 0.05). To conclude, protein S-nitrosylation could modify the cysteine residues of caspase-3, which accounts for the reduced caspase-3 activity and further represses its proteolytic ability on beef myofibrillar protein.

Cyclic Continuous Glycation Enhanced Dispersibility of Myofibrillar Protein: Reaction Efficiency and Sites Modification.

Reaction efficiency in glycation lacks sufficient attention, leading to the waste of process costs. Cyclic continuous glycation (CCG) is an effective approach to accelerate covalent binding between myofibrillar protein (MP) and glucose. This study elucidated that CCG promoted the exposure of reactive glycated sites in MP with full unfolding of secondary and tertiary structures. Notably, the glycation rate was significantly increased by 65.43%. Physicochemical properties indicated that MP-glucose conjugates with high graft degree exhibited favorable solubility, dispersibility, and thermal stability. Furthermore, proteomics was applied to reveal the glycated sites and products in glycoconjugates of MP. Glycation preferentially acted on the tails of the myosin heavy chain. The glucosylation modification on the head region was enhanced by CCG contributing to the inhibition of the head-head interaction. Overall, this study systematically clarifies the mechanism of CCG, providing a theoretical basis for the application of glycation in innovative meat products.

Functional and Mechanistic Dissection of Protein Glutaminase PG3 and Its Rational Engineering for Enhanced Modification of Myofibrillar Proteins.

Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t65 °C1/2. Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.

Understanding the textural enhancement of low-salt myofibrillar protein gels filled with pea protein pre-emulsions through interfacial behavior: Effects of structural modification and oil phase polarity.

This study investigated the effects of pea protein pre-emulsions containing triglyceride- or diglyceride-oil on the emulsifying and gelling properties of low-salt myofibrillar protein (MP). Pea protein isolates treated with pH12-shifting (PPIpH) or ultrasonication (PPIU) demonstrated superior initial interfacial adsorption and higher final interfacial pressure than native pea protein. Within MP/PPI blends, an increased ratio of MP led to a decrease in interfacial pressure, while simultaneously enhancing film elasticity at both polar and non-polar interfaces. Polar diglyceride promoted protein adsorption and fostered interfacial interactions between modified pea proteins and MP, enhancing the cross-linking of transglutaminase (TG) in the composite emulsion gels. Combining diglyceride-type PPIU and PPIpH emulsions with TG increased gel strength to 0.58 N and 0.63 N, respectively, from an initial 0.33 N, yielding a denser protein network with uniformly dispersed oil droplets. Therefore, the utilization of diglyceride and modified PPI can serve as structural enhancers in comminuted meat products.

Influence of NaCl and phosphate on gelation properties of chicken breast myofibrillar protein gels and its application to in vitro digestion model.

To investigate the combination effect of sodium chloride and phosphates on chicken breast myofibrillar proteins, MP gels containing various molarity of NaCl (0.15, 0.30 and 0.45 M) and phosphate (0 and 0.05 M) were prepared, their rheological properties were characterized, and applied to an in vitro digestion model. MP mixture containing 0.45 M NaCl and 0.05 M phosphate had the highest viscosity. The gel strength and cooking yield of MP gels was improved by increasing of molarity of NaCl. As NaCl concentration in MP increased, sulfhydryl levels decreased, while disulfide levels increased. As NaCl and phosphate levels increase, MP gels become denser and porosity decreases, which may reduce protein digestibility. In SDS-PAGE, protein bands from MP gels containing low NaCl levels (≤ 0.30 M) degraded more rapidly during in vitro digestion. These results may support the need for the meat industry to develop low-salt meat products with improved digestibility. KEYWORDS: Chicken, Myofibrillar protein, NaCl, Phosphate, Rheological properties, In vitro digestion.

eEF1α2 is required for actin cytoskeleton homeostasis in the aging muscle.

The translation elongation factor eEF1α (eukaryotic elongation factor 1α) mediates mRNA translation by delivering aminoacyl-tRNAs to ribosomes. eEF1α also has other reported roles, including the regulation of actin dynamics. However, these distinct roles of eEF1α are often challenging to uncouple and remain poorly understood in aging metazoan tissues. The genomes of mammals and Drosophila encode two eEF1α paralogs, with eEF1α1 expressed ubiquitously and eEF1α2 expression more limited to neurons and muscle cells. Here, we report that eEF1α2 plays a unique role in maintaining myofibril homeostasis during aging in Drosophila. Specifically, we generated an eEF1α2 null allele, which was viable and showed two distinct muscle phenotypes. In young flies, the mutants had thinner myofibrils in indirect flight muscles that could be rescued by expressing eEF1α1. With aging, the muscles of the mutant flies began showing abnormal distribution of actin and myosin in muscles, but without a change in actin and myosin protein levels. This age-related phenotype could not be rescued by eEF1α1 overexpression. These findings support an unconventional role of Drosophila eEF1α2 in age-related homeostasis of muscle myofibers.

Localization of EFA6A, an exchange factor for Arf6, in Z-lines and sarcoplasmic reticulum membranes in addition to myofilaments in I-domains of skeletal myofibers of peri-natal mice.

Membrane trafficking and actin-remodeling are critical for well-maintained integrity of the cell organization and activity, and they require Arf6 (ADP ribosylation factor 6) activated by GEF (guanine nucleotide exchange factor) including EFA6 (exchange factor for Arf6). In the present immuno-electron microscopic study following previous immunohistochemical study by these authors (Chomphoo et al., 2020) of in situ skeletal myoblasts and myotubes of pre-and perinatal mice, the immunoreactivity for EFA6A was found to be localized at Z-bands and sarcoplasmic reticulum (SR) membranes in I-domains as well as I-domain myofilaments of skeletal myofibers of perinatal mice. Based on the previous finding that EFA6 anchored on the neuronal postsynaptic density via α-actinin which is known to be shared by muscular Z-bands, the present finding suggests that EFA6A is also anchored on Z-bands via α-actinin and involved in the membrane trafficking and actin-remodeling in skeletal myofibers. The localization of EFA6A-immunoreactivity in I-domain SR suggests a differential function in the membrane traffic between the I- and A-domain intracellular membranes in perinatal skeletal myofibers.

Postprandial cardiac hypertrophy is sustained by mechanics, epigenetic, and metabolic reprogramming in pythons.

Constricting pythons, known for their ability to consume infrequent, massive meals, exhibit rapid and reversible cardiac hypertrophy following feeding. Our primary goal was to investigate how python hearts achieve this adaptive response after feeding. Isolated myofibrils increased force after feeding without changes in sarcomere ultrastructure and without increasing energy cost. Ca2+ transients were prolonged after feeding with no changes in myofibril Ca2+ sensitivity. Feeding reduced titin-based tension, resulting in decreased cardiac tissue stiffness. Feeding also reduced the activity of sirtuins, a metabolically linked class of histone deacetylases, and increased chromatin accessibility. Transcription factor enrichment analysis on transposase-accessible chromatin with sequencing revealed the prominent role of transcription factors Yin Yang1 and NRF1 in postfeeding cardiac adaptation. Gene expression also changed with the enrichment of translation and metabolism. Finally, metabolomics analysis and adenosine triphosphate production demonstrated that cardiac adaptation after feeding not only increased energy demand but also energy production. These findings have broad implications for our understanding of cardiac adaptation across species and hold promise for the development of innovative approaches to address cardiovascular diseases.

Engineering of protein glutaminase for highly efficient modification of fish myofibrillar protein through structure-based and computational-aided strategy.

Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp31, Ser72, Asn121, Asp471, and Glu485 were crucial for maintaining PG5-myosin interaction, with Ser72 and Asn121 making significant energy contributions.

Role of sodium pyrophosphate and catechin in the enzymatic hydrolysis of oxidatively damaged myofibrillar protein gels in vitro: Mechanistic insights.

This study investigated the effects of sodium pyrophosphate (SPP) and catechin (C) on the in vitro enzymatic digestion of oxidatively damaged myofibrillar protein (MP) gel. The results indicated that SPP increased the ß-sheet content and the gastric digestibility of the MP gel, while C hindered the transition from α-helix to ß-sheet structure, leading to decreased digestibility. Notably, neither compound significantly affected intestinal digestibility. Furthermore, SPP and C significantly enhanced the antioxidant activity of MP gel digestion products. Notably, their synergistic hydrolysis products, simulating both gastric and gastrointestinal stages, chelated 91.4 % and 89.1 % of Fe2+ and scavenged 59.4 % and 77.6 % of hydroxyl radicals, respectively. Moreover, the final digestion products of the MP gel treated with SPP and C exhibited the highest content of negatively charged amino acids and absolute Zeta potential values. Overall, this study demonstrated that incorporating SPP and C could positively impact the digestion of oxidatively damaged MP gels.

Insight into the effects of large yellow croaker roe (Larimichthys Crocea) phospholipids on the conformational and functional properties of pork myofibrillar protein.

The large yellow croaker roe phospholipids (LYPLs), rich in polyunsaturated fatty acids, is a potential phospholipid additive for meat products. In this work, the effects of LYPLs on the structural and functional properties of myofibrillar protein (MP) were determined, and compared with egg yolk phospholipids (EYPLs) and soybean phospholipids (SBPLs). The results revealed that LYPLs, similar to SBPLs and EYPLs, induced a transformation in the secondary structure of MP from α-helix to ß-sheets and random coils, while also inhibited the formation of carbonyl and disulfide bonds within MP. All three phospholipids induced MP tertiary structure unfolding, with the greatest degree of unfolding observed in MP containing LYPLs. The MP with LYPLs had the highest surface hydrophobicity, emulsification properties and gel strength. In addition, MP with LYPLs added also demonstrated superior rheological properties and water-holding capacity compared with SBPLs and EYPLs. In conclusion, adding LYPLs endowed MP with improved functional properties.

Effects of ultrasonic treatment on the structure and functional characteristics of myofibrillar proteins from black soldier fly.

In the process of utilizing black soldier fly larvae (BSFL) lipids to develop biodiesel, many by-products will be produced, especially the underutilized protein components. These proteins can be recycled through appropriate treatment and technology, such as the preparation of feed, biofertilizers or other kinds of bio-products, so as to achieve the efficient use of resources and reduce the generation of waste. Myofibrillar protein (MP), as the most important component of protein, is highly susceptible to environmental influences, leading to oxidation and deterioration, which ultimately affects the overall performance of the protein and product quality. For it to be high-quality and fully exploited, in this study, black soldier fly myofibrillar protein (BMP) was extracted and primarily subjected to ultrasonic treatment to investigate the impact of varying ultrasonic powers (300, 500, 700, 900 W) on the structure and functional properties of BMP. The results indicated that as ultrasonic power increased, the sulfhydryl content and turbidity of BMP decreased, leading to a notable improvement in the stability of the protein emulsion system. SEM images corroborated the changes in the microstructure of BMP. Moreover, the enhancement of ultrasound power induced modifications in the intrinsic fluorescence spectra and FTIR spectra of BMP. Additionally, ultrasonic treatment resulted in an increase in carbonyl content and emulsifying activity of BMP, with both peaking at 500 W. It was noteworthy that BMP treated with ultrasound exhibited stronger digestibility compared to the untreated. In summary, 500 W was determined as the optimal ultrasound parameter for this study. Overall, ultrasound modification of insect MPs emerges as a dependable technique capable of altering the structure and functionality of BMP.

Loss of mitochondria long-chain fatty acid oxidation impairs skeletal muscle contractility by disrupting myofibril structure and calcium homeostasis.

OBJECTIVE: Abnormal lipid metabolism in mammalian tissues can be highly deleterious, leading to organ failure. Carnitine Palmitoyltransferase 2 (CPT2) deficiency is an inherited metabolic disorder affecting the liver, heart, and skeletal muscle due to impaired mitochondrial oxidation of long-chain fatty acids (mLCFAO) for energy production. METHODS: However, the basis of tissue damage in mLCFAO disorders is not fully understood. Mice lacking CPT2 in skeletal muscle (Cpt2Sk-/-) were generated to investigate the nexus between mFAO deficiency and myopathy. RESULTS: Compared to controls, ex-vivo contractile force was reduced by 70% in Cpt2Sk-/- oxidative soleus muscle despite the preserved capacity to couple ATP synthesis to mitochondrial respiration on alternative substrates to long-chain fatty acids. Increased mitochondrial biogenesis, lipid accumulation, and the downregulation of 80% of dystrophin-related and contraction-related proteins severely compromised the structure and function of Cpt2Sk-/- soleus. CPT2 deficiency affected oxidative muscles more than glycolytic ones. Exposing isolated sarcoplasmic reticulum to long-chain acylcarnitines (LCACs) inhibited calcium uptake. In agreement, Cpt2Sk-/- soleus had decreased calcium uptake and significant accumulation of palmitoyl-carnitine, suggesting that LCACs and calcium dyshomeostasis are linked in skeletal muscle. CONCLUSIONS: Our data demonstrate that loss of CPT2 and mLCFAO compromise muscle structure and function due to excessive mitochondrial biogenesis, downregulation of the contractile proteome, and disruption of calcium homeostasis.

High-intensity interval training improves cardiomyocyte contractile function and myofilament sensitivity to intracellular Ca2+ in obese rats.

High-intensity interval training (HIIT) has shown significant results in addressing adiposity and risk factors associated with obesity. However, there are no studies that investigate the effects of HIIT on contractility and intracellular Ca2+ handling. The purpose of this study was to explore the impact of HIIT on cardiomyocyte contractile function and intracellular Ca2+ handling in rats in which obesity was induced by a saturated high-fat diet (HFD). Male Wistar rats were initially randomized into a standard diet and a HFD group. The experimental protocol spanned 23 weeks, comprising the induction and maintenance of obesity (15 weeks) followed by HIIT treatment (8 weeks). Performance was assessed using the maximum oxygen consumption test ( V ̇ O 2 max ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{max}}}}$ ). Evaluation encompassed cardiac, adipose and skeletal muscle histology, as well as contractility and intracellular Ca2+ handling. HIIT resulted in a reduction in visceral area, an increase in V ̇ O 2 max ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{max}}}}$ , and an augmentation of gastrocnemius fibre diameter in obese subjects. Additionally, HIIT led to a decrease in collagen fraction, an increase in percentage shortening, and a reduction in systolic Ca2+/percentage shortening and systolic Ca2+/maximum shortening rates. HIIT induces physiological cardiac remodelling, enhancing the contractile function of cardiomyocytes and improving myofilament sensitivity to Ca2+ in the context of obesity. This approach not only enhances cardiorespiratory and physical performance but also reduces visceral area and prevents interstitial fibrosis.