Gastrointestinal digestion behavior and bioavailability of greenly prepared highly loaded myofibrillar-luteolin vehicle.

In this study, the highly loaded myofibrillar protein (MP)-luteolin (Lut) complexes were noncovalently constructed by using green high-pressure homogenization technology (HPH) and high-pressure micro-fluidization technology (HPM), aiming to optimize the encapsulation efficiency of flavonoids in the protein-based vehicle without relying on the organic solvent (i.e. DMSO, ethanol, etc.). The loading efficiency of Lut into MPs could reach 100 % with a concentration of 120 µmol/g protein by using HPH (103 MPa, 2 passes) without ethanol adoption. The in vitro gastrointestinal digestion behavior and antioxidant activity of the complexes were then compared with those of ethanol-assisted groups. During gastrointestinal digestion, the MP digestibility of complexes, reaching more than 70.56 % after thermal treatment, was higher than that of sole protein. The release profile of Lut encapsulated in ethanol-containing and ethanol-free samples both well fitted with the Hixson-Crowell release kinetic model (R2 = 0.92 and 0.94, respectively), and the total phenol content decreased by ≥ 40.02 % and ≥ 62.62 %, respectively. The in vitro antioxidant activity (DPPH, ABTS, and Fe2+) of the digestive products was significantly improved by 23.89 %, 159.69 %, 351.12 % (ethanol groups) and 13.43 %, 125.48 %, 213.95 % (non-ethanol groups). The 3 mg/mL freeze-dried digesta significantly alleviated lipid accumulation and oxidative stress in HepG2 cells. The triglycerides and malondialdehyde contents decreased by at least 57.62 % and 67.74 % after digesta treatment. This study provides an easily approached and environment-friendly strategy to construct a highly loaded protein-flavonoid conjugate, which showed great potential in the formulation of healthier meat products.

Bruno 1/CELF regulates splicing and cytoskeleton dynamics to ensure correct sarcomere assembly in Drosophila flight muscles.

Muscles undergo developmental transitions in gene expression and alternative splicing that are necessary to refine sarcomere structure and contractility. CUG-BP and ETR-3-like (CELF) family RNA-binding proteins are important regulators of RNA processing during myogenesis that are misregulated in diseases such as Myotonic Dystrophy Type I (DM1). Here, we report a conserved function for Bruno 1 (Bru1, Arrest), a CELF1/2 family homolog in Drosophila, during early muscle myogenesis. Loss of Bru1 in flight muscles results in disorganization of the actin cytoskeleton leading to aberrant myofiber compaction and defects in pre-myofibril formation. Temporally restricted rescue and RNAi knockdown demonstrate that early cytoskeletal defects interfere with subsequent steps in sarcomere growth and maturation. Early defects are distinct from a later requirement for bru1 to regulate sarcomere assembly dynamics during myofiber maturation. We identify an imbalance in growth in sarcomere length and width during later stages of development as the mechanism driving abnormal radial growth, myofibril fusion, and the formation of hollow myofibrils in bru1 mutant muscle. Molecularly, we characterize a genome-wide transition from immature to mature sarcomere gene isoform expression in flight muscle development that is blocked in bru1 mutants. We further demonstrate that temporally restricted Bru1 rescue can partially alleviate hypercontraction in late pupal and adult stages, but it cannot restore myofiber function or correct structural deficits. Our results reveal the conserved nature of CELF function in regulating cytoskeletal dynamics in muscle development and demonstrate that defective RNA processing due to misexpression of CELF proteins causes wide-reaching structural defects and progressive malfunction of affected muscles that cannot be rescued by late-stage gene replacement.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Differential utilisation of subcellular skeletal muscle glycogen pools: a comparative analysis between 1 and 15 min of maximal exercise.

In skeletal muscle, glycogen particles are distributed both within and between myofibrils, as well as just beneath the sarcolemma. Their precise localisation may influence their degradation rate. Here, we investigated how exercise at different intensities and durations (1- and 15-min maximal exercise) with known variations in glycogenolytic rate and contribution from anaerobic metabolism affects utilisation of the distinct pools. Furthermore, we investigated how decreased glycogen availability achieved through lowering carbohydrate and energy intake after glycogen-depleting exercise affect the storage of glycogen particles (size, numerical density, localisation). Twenty participants were divided into two groups performing either a 1-min (n = 10) or a 15-min (n = 10) maximal cycling exercise test. In a randomised, counterbalanced, cross-over design, the exercise tests were performed following short-term consumption of two distinct diets with either high or moderate carbohydrate content (10 vs. 4 g kg-1 body mass (BM) day-1) mediating a difference in total energy consumption (240 vs. 138 g kg-1 BM day-1). Muscle biopsies from m. vastus lateralis were obtained before and after the exercise tests. Intermyofibrillar glycogen was preferentially utilised during the 1-min test, whereas intramyofibrillar glycogen was preferentially utilised during the 15-min test. Lowering carbohydrate and energy intake after glycogen-depleting exercise reduced glycogen availability by decreasing particle size across all pools and diminishing numerical density in the intramyofibrillar and subsarcolemmal pools. In conclusion, distinct subcellular glycogen pools were differentially utilised during 1-min and 15-min maximal cycling exercise. Additionally, lowered carbohydrate and energy consumption after glycogen-depleting exercise altered glycogen storage by reducing particle size and numerical density, depending on subcellular localisation. KEY POINTS: In human skeletal muscle, glycogen particles are localised in distinct subcellular compartments, referred to as intermyofibrillar, intramyofibrillar and subsarcolemmal pools. The intermyofibrillar and subsarcolemmal pools are close to mitochondria, while the intramyofibrillar pool is at a distance from mitochondria. We show that 1 min of maximal exercise is associated with a preferential utilisation of intermyofibrillar glycogen, and, on the other hand, that 15 min of maximal exercise is associated with a preferential utilisation of intramyofibrillar glycogen. Furthermore, we demonstrate that reduced glycogen availability achieved through lowering carbohydrate and energy intake after glycogen-depleting exercise is characterised by a decreased glycogen particle size across all compartments, with the numerical density only diminished in the intramyofibrillar and subsarcolemmal compartments. These results suggest that exercise intensity influences the subcellular pools of glycogen differently and that the dietary content of carbohydrates and energy is linked to the size and subcellular distribution of glycogen particles.

Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Myofilament-based physiological regulatory compensation preserves diastolic function in failing hearts with severe Ca2+ handling deficits.

Severe dysfunction in cardiac muscle intracellular Ca2+ handling is a common pathway underlying heart failure. Here we used an inducible genetic model of severe Ca2+ cycling dysfunction by the targeted temporal gene ablation of the cardiac Ca2+ ATPase, SERCA2, in otherwise normal adult mice. In this model, in vivo heart performance was minimally affected initially, even though Serca2a protein was markedly reduced. The mechanism underlying the sustained in vivo heart performance in the weeks prior to complete heart pump failure and death is not clear and is important to understand. Studies were primarily focused on understanding how in vivo diastolic function could be relatively normal under conditions of marked Serca2a deficiency. Interestingly, data show increased cardiac troponin I (cTnI) serine 23/24 phosphorylation content in hearts upon Serca2a ablation in vivo. We report that hearts isolated from the Serca2-deficient mice retained near normal heart pump functional responses to ß-adrenergic stimulation. Unexpectedly, using genetic complementation models, in concert with inducible Serca2 ablation, data show that Serca2a-deficient hearts that also lacked the central ß-adrenergic signaling-dependent Serca2a negative regulator, phospholamban (PLN), had severe diastolic dysfunction that could still be corrected by ß-adrenergic stimulation. Notably, integrating a serines 23/24-to-alanine PKA-refractory sarcomere incorporated cTnI molecular switch complex in mice deficient in Serca2 showed blunting of ß-adrenergic stimulation-mediated enhanced diastolic heart performance. Taken together, these data provide evidence of a compensatory regulatory role of the myofilaments as a critical physiological bridging mechanism to aid in preserving heart diastolic performance in failing hearts with severe Ca2+ handling deficits.

Myofibrillar myopathies due to a novel mutation in exon 8 of the LDB3 gene.

Myofibrillar myopathies (MFMs) are a group of genetically heterogeneous diseases affecting the skeletal and cardiac muscles. Myofibrillar myopathies are characterized by focal lysis of myogenic fibers and integration of degraded myogenic fiber products into inclusion bodies, which are typically rich in desmin and many other proteins. Herein, we report a case of a 54-year-old woman who experienced bilateral thigh weakness for over three years. She was diagnosed with MFMs based on muscle biopsy findings and the presence of a novel mutation in exon 8 of the LDB3 gene. Myofibrillar myopathies caused by a mutation in the LDB3 gene are extremely uncommon and often lack distinct clinical characteristics and typically exhibit a slow disease progression. When considering a diagnosis of MFMs, particularly in complex instances of autosomal dominant myopathies where muscle biopsies do not clearly indicate MFMs, it becomes crucial for clinicians to utilize genetic test as a diagnostic tool.

Lower basal and postprandial muscle protein synthesis after 2 weeks single-leg immobilization in older men: No protective effect of anti-inflammatory medication.

Muscle inactivity may reduce basal and postprandial muscle protein synthesis (MPS) rates in humans. Anti-inflammatory treatment alleviates the MPS impairments in younger individuals. The present study explored the influence of nonsteroidal anti-inflammatory drugs (NSAIDs) upon MPS during a period of inactivity in older humans. Eighteen men (age 60-80 years) were allocated to ibuprofen (1200 mg/day, Ibu) or control (Plc) groups. One lower limb was cast immobilized for 2 weeks. Postabsorptive and postprandial MPS was measured before and after the immobilization by L-[ring-13 C6 ]-phenylalanine infusion. The protein expression of select anabolic signaling molecules was investigated by western blot. Basal (0.038 ± 0.002%/h and 0.039 ± 0.005%/h, Plc and Ibu, respectively) and postprandial (0.064 ± 0.004%/h and 0.067 ± 0.010%/h, Plc and Ibu, respectively) MPS rate were higher pre-immobilization compared to basal (0.019 ± 0.005%/h and 0.020 ± 0.010%/h, Plc and Ibu, respectively) and postprandial (0.033 ± 0.005%/h and 0.037 ± 0.006%/h, Plc and Ibu, respectively) MPS rate post-immobilization (p < 0.001). NSAID treatment did not affect the suppression of MPS (p > 0.05). The anabolic signaling were in general reduced after immobilization (p < 0.05). These changes were unaffected by NSAID treatment (p > 0.05). Basal and postprandial MPS dropped markedly after 2 weeks of lower limb immobilization. NSAID treatment neither influenced the reduction in MPS nor the anabolic signaling after immobilization in healthy older individuals.

Solubilization of fish myofibrillar proteins in NaCl and KCl solutions: A DIA-based proteomics analysis.

Understanding the basic solubilization of fish myofibrillar proteins (MPs) in common monovalent chloride solutions is crucial for muscle food processing. In this study, the differential proteomic profiles of MPs during extraction and solubilization in NaCl and KCl solutions were investigated by using advanced four-dimensional data-independent acquisition (4D DIA) quantitative proteomics for the first time. Compared to routine biochemical analysis, this could provide insights into the solubilization of muscle proteins. We ensure the consistency of the effective ionic strength of NaCl and KCl buffers by adjusting the conductivity. The results showed that NaCl extractor mainly facilitated the solubilization of cytoskeletal proteins, biochemical enzymes, and stromal proteins compared to KCl, such as tubulin, myosin-9, collagen, plectin, protein phosphatase, and cathepsin D. However, no significant difference was observed in the extraction of major sarcomeric proteins, including myosin, actin, troponin C, myosin-binding protein C, M-Protein, α-actinin-3, and tropomyosin.

Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Temporal involvement of phosphatidylinositol 4-phosphate 5-kinase γ in differentiation of Z-bands and myofilament bundles as well as intercalated discs in mouse heart at mid-gestation.

Considering the occurrence of serious heart failure in a gene knockout mouse of PIP5Kγ and in congenital abnormal cases in humans in which the gene was defective as reported by others, the present study attempted to localize PIP5Kγ in the heart during prenatal stages. It was done on the basis of the supposition that phenotypes caused by gene mutation of a given molecule are owed to the functional deterioration of selective cellular sites normally expressing it at significantly higher levels in wild mice. PIP5Kγ-immunoreactivity was the highest in the heart at E10 in contrast to almost non-significant levels of the immunoreactivity in surrounding organs and tissues such as liver. The immunoreactivity gradually weakened in the heart with the prenatal age, and it was at non-significant levels at newborn and postnatal stages. Six patterns in localization of distinct immunoreactivity for PIP5Kγ were recognized in cardiomyocytes: (1) its localization on the plasma membranes and subjacent cytoplasm without association with short myofibrils and (2) its localization on them as well as short myofibrils in association with them in cardiomyocytes of early differentiation at E10; (3) its spot-like localization along long myofibrils in cardiomyocytes of advanced differentiation at E10; (4) rare occurrences of such spot-like localization along long myofibrils in cardiomyocytes of advanced differentiation at E14; (5) its localization at Z-bands of long myofibrils; and (6) its localization at intercellular junctions including the intercalated discs in cardiomyocytes of advanced differentiation at E10 and E14, especially dominant at the latter stage. No distinct localization of PIP5Kγ-immunoreactivity of any patterns was seen in the heart at E18 and P1D. The present finding suggests that sites of PIP5Kγ-appearance and probably of its high activity in cardiomyocytes are shifted from the plasma membranes through short myofibrils subjacent to the plasma membranes and long myofibrils, to Z-bands as well as to the intercalated discs during the mid-term gestation. It is further suggested that PIP5Kγ is involved in the differentiation of myofibrils as well as intercellular junctions including the intercalated discs at later stages of the mid-term gestation. Failures in its involvement in the differentiation of these structural components are thus likely to cause the mid-term gestation lethality of the mutant mice for PIP5Kγ.

Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Troponin I Tyrosine Phosphorylation Beneficially Accelerates Diastolic Function.

BACKGROUND: A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction. METHODS: The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease. RESULTS: Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease. CONCLUSIONS: We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo.

Measuring the Contractile Kinetics of Isolated Myofibrils from Human-Induced Pluripotent Stem Cell Derived Cardiomyocyte (hiPSC-CM) Models of Cardiomyopathy.

Isolated myofibrils provide biomechanical data at the contractile organelle level that are independent of cellular calcium handling and signaling pathways. These myofibrils can be harvested from animal tissue, human muscle biopsies, or stem cell-derived striated muscle. Here we present our myofibril isolation and rapid solution switching protocols, which allow for precise measurements of activation (kinetics and tension generation) and a biphasic relaxation relationship (initial slow isometric relaxation followed by a fast exponential decay in tension). This experiment is generated on a custom-built myofibril apparatus utilizing a two-photodiode array to detect micron level deflection of our forged glass tip force transducers. A complete activation/relaxation curve can be produced from a single myofibril in under 30 minutes.

Enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction in patient-specific induced pluripotent stem cell-derived cardiomyocytes with MYH7 mutation.

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is frequently caused by mutations in the ß-cardiac myosin heavy chain gene (MYH7). Abnormal calcium handling and diastolic dysfunction are archetypical features of HCM caused by MYH7 gene mutations. However, the mechanism of how MYH7 mutations leads to these features remains unclear, which inhibits the development of effective therapies. Initially, cardiomyocytes were generated from induced pluripotent stem cells from an eight-year-old girl diagnosed with HCM carrying a MYH7(C.1063 G>A) heterozygous mutation(mutant-iPSC-CMs) and mutation-corrected isogenic iPSCs(control-iPSC-CMs) in the present study. Next, we compared phenotype of mutant-iPSC-CMs to that of control-iPSC-CMs, by assessing their morphology, hypertrophy-related genes expression, calcium handling, diastolic function and myofilament calcium sensitivity at days 15 and 40 respectively. Finally, to better understand increased myofilament Ca2+ sensitivity as a central mechanism of central pathogenicity in HCM, inhibition of calcium sensitivity with mavacamten can improveed cardiomyocyte hypertrophy. Mutant-iPSC-CMs exhibited enlarged areas, increased sarcomere disarray, enhanced expression of hypertrophy-related genes proteins, abnormal calcium handling, diastolic dysfunction and increased myofilament calcium sensitivity at day 40, but only significant increase in calcium sensitivity and mild diastolic dysfunction at day 15. Increased calcium sensitivity by levosimendan aggravates cardiomyocyte hypertrophy phenotypes such as expression of hypertrophy-related genes, abnormal calcium handling and diastolic dysfunction, while inhibition of calcium sensitivity significantly improves cardiomyocyte hypertrophy phenotypes in mutant-iPSC-CMs, suggesting increased myofilament calcium sensitivity is the primary mechanisms for MYH7 mutations pathogenesis. Our studies have uncovered a pathogenic mechanism of HCM caused by MYH7 gene mutations through which enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction. Correction of the myofilament calcium sensitivity was found to be an effective method for treating the development of HCM phenotype in vitro.

Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp.

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases, including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the 'Z-Bodies" of sarcomere precursors and the 'Z-Lines' of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze 'M-Lines' using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. We found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.

Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

The ACTN2 gene encodes α-actinin 2, located in the Z-disc of the sarcomeres in striated muscle. In this study, we sought to investigate the effects of an ACTN2 missense variant of unknown significance (p.A868T) on cardiac muscle structure and function. Left ventricular free wall samples were obtained at the time of cardiac transplantation from a heart failure patient with the ACTN2 A868T heterozygous variant. This variant is in the EF 3-4 domain known to interact with titin and α-actinin. At the ultrastructural level, ACTN2 A868T cardiac samples presented small structural changes in cardiomyocytes when compared to healthy donor samples. However, contractile mechanics of permeabilized ACTN2 A868T variant cardiac tissue displayed higher myofilament Ca2+ sensitivity of isometric force, reduced sinusoidal stiffness, and faster rates of tension redevelopment at all Ca2+ levels. Small-angle X-ray diffraction indicated increased separation between thick and thin filaments, possibly contributing to changes in muscle kinetics. Molecular dynamics simulations indicated that while the mutation does not significantly impact the structure of α-actinin on its own, it likely alters the conformation associated with titin binding. Our results can be explained by two Z-disc mediated communication pathways: one pathway that involves α-actinin's interaction with actin, affecting thin filament regulation, and the other pathway that involves α-actinin's interaction with titin, affecting thick filament activation. This work establishes the role of α-actinin 2 in modulating cross-bridge kinetics and force development in the human myocardium as well as how it can be involved in the development of cardiac disease.

Distribution and Degradation of Pork Filamin during Postmortem Aging.

The filamin C (FLNC) was hypothesized to be colocalized with its certain binding partners in pork tissues and calpain as well as caspase was assumed responsible for the postmortem degradation of FLNC. Therefore, the specific distribution of pork FLNC and its degradation pattern during postmortem aging were investigated in this study. The longissimus thoracis muscles from 12 pigs were removed from the carcasses and then aged at 4 °C for 1, 6, 12, 24, 72, and 168 h, respectively. The FLNC signals appeared to localize in subsarcolemmal areas by cross-sectional images, while the localization was found surrounding the myofibrils at the level of the Z-discs in longitudinal sections. FLNC displayed a highly overlapped spatial colocalization with actin or integrin. Western blot results showed that the intact 290 kDa FLNC was rapidly degraded to produce an approximately 280 kDa band. An almost overlapped distribution pattern was observed between FLNC and µ-calpain or caspase-3 in porcine skeletal muscle cells. Moreover, both the µ-calpain inhibitor and the caspase-3 inhibitor could inhibit the degradation of FLNC in porcine LT muscles during postmortem aging.

A-Band assembly in avian skeletal muscles observed with super-resolution microscopy.

Myofibrils in vertebrate skeletal muscle are organized in aligned arrays of filaments formed from multiple protein components. Despite considerable information describing individual proteins, how they assemble de novo into mature myofibrils is still a challenge. Studies in our lab of sarcomeric protein localization during myofibril assembly led us to propose a three-step progression: premyofibrils to nascent myofibrils, culminating in mature myofibrils. Premyofibrils, forming at the spreading edges of muscle cells, are composed of minisarcomeres containing small bands of non-muscle myosin II filaments alternating with muscle-specific α-actinin Z-Bodies attached to barbed ends of actin filaments, establishing bipolar F-actin arrangements in sarcomeres. Assembly of nascent myofibrils occurs with addition of muscle-specific myosin II, F-actin, titin, and the alignment of Z-Bodies in adjacent fibrils to form beaded Z-Bands. Muscle-specific myosin II filaments in nascent myofibrils appear in an overlapping arrangement when viewed with wide-field and confocal microscopes. In mature myofibrils, non-muscle myosin II is absent, and M-Band proteins localize to the muscle myosin II filaments, aiding their alignment by cross-linking them into A-Bands. Super-resolution microscopy (SIM and STED) revealed muscle myosin II in mini-A-Bands in nascent myofibrils. In contrast to previous reports that vertebrate muscle myosin thick filaments form at their final 1.6 µm lengths, mini-A-Bands are first detected at a length of about 0.4 µm, and gradually increase four-fold in length to 1.6 µm in mature myofibrils. These new discoveries in avian skeletal muscle cells share a common characteristic with invertebrate muscles where some A-Bands can grow to lengths reaching 25 µm.

Electrically stimulated in vitro heart cell mimic of acute exercise reveals novel immediate cellular responses to exercise: Reduced contractility and metabolism, but maintained calcium cycling and increased myofilament calcium sensitivity.

Cardiac cellular responses to acute exercise remain undescribed. We present a model for mimicking acute aerobic endurance exercise to freshly isolated cardiomyocytes by evoking exercise-like contractions over prolonged periods of time with trains of electrical twitch stimulations. We then investigated immediate contractile, Ca2+ , and metabolic responses to acute exercise in perfused freshly isolated left ventricular rat cardiomyocytes, after a matrix-design optimized protocol and induced a mimic for acute aerobic endurance exercise by trains of prolonged field twitch stimulations. Acute exercise decreased cardiomyocyte fractional shortening 50%-80% (p < .01). This was not explained by changes to intracellular Ca2+ handling (p > .05); rather, we observed a weak insignificant Ca2+ transient increase (p = .11), while myofilament Ca2+ sensitivity increased 20%-70% (p < .05). Acidic pH 6.8 decreased fractional shortening 20%-70% (p < .05) because of 20%-30% decreased Ca2+ transients (p < .05), but no difference occurred between control and acute exercise (p > .05). Addition of 1 or 10 mM La- increased fractional shortening in control (1 mM La- : no difference, p > .05; 10 mM La- : 20%-30%, p < .05) and acute exercise (1 mM La- : 40%-90%, p < .01; 10 mM La- : 50%-100%, p < .01) and rendered acute exercise indifferent from control (p > .05). Intrinsic autofluorescence showed a resting NADstate of 0.59 ± 0.04 and FADstate of 0.17 ± 0.03, while acute exercise decreased NADH/FAD ratio 8% (p < .01), indicating intracellular oxidation. In conclusion, we show a novel approach for studying immediate acute cardiomyocyte responses to aerobic endurance exercise. We find that acute exercise in cardiomyocytes decreases contraction, but Ca2+ handling and myofilament Ca2+ sensitivity compensate for this, while acidosis and reduced energy substrate and mitochondrial ATP generation explain this.