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During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. We sought to elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb after treatment with serum or the α-adrenergic agonist phenylephrine. Depletion of Bmal1 decreased the expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by micrococcal nuclease-quantitative PCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting natriuretic peptide B transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented phenylephrine-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

Introduction: The proportion of animal based foods in daily diet of consumers is constantly increasing, with chicken being highly favored due to its high protein and low fat characteristics. The consumption of chicken around the world is steadily increasing. Intramuscular fat (IMF) is a key indicator affecting meat quality. Object: High IMF content can contribute to improve the quality of chicken meat. The regulatory mechanism of IMF deposition in chicken is poorly understood, so its complete elucidation is essential to improve chicken meat quality. Method: Here, we performed whole genome resequencing on 516 yellow feather chickens and single-cell RNA sequencing on 3 63-day-old female JXY chickens. In addition, transcriptome sequencing techniques were also performed on breast muscle tissue of JXY chickens at different developmental stages. And 13C isotope tracing technique was applied. Results: In this study, a large-scale genetic analysis of an IMF-selected population and a control population identified fatty acid synthase (FASN) as a key gene for improving IMF content. Also, contrary to conventional view, de novo lipogenesis (DNL) was deemed to be an important contributor to IMF deposition. As expected, further analyses by isotope tracing and other techniques, confirmed that DNL mainly occurs in myocytes, contributing about 40% of the total fatty acids through the regulation of FASN, using the available FAs as substrates. Additionally, we also identified a relevant causal mutation in the FASN gene with effects on FA composition. Conclusion: These findings contribute to the understanding of fat metabolism in muscle tissue of poultry, and provide the feasible strategy for the production of high-quality chicken meat.

[Display omitted] The proportion of animal based foods in daily diet of consumers is constantly increasing, with chicken being highly favored due to its high protein and low fat characteristics. The consumption of chicken around the world is steadily increasing. Intramuscular fat (IMF) is a key indicator affecting meat quality. High IMF content can contribute to improve the quality of chicken meat. The regulatory mechanism of IMF deposition in chicken is poorly understood, so its complete elucidation is essential to improve chicken meat quality. Here, we performed whole genome resequencing on 516 yellow feather chickens and single-cell RNA sequencing on 3 63-day-old female JXY chickens. In addition, transcriptome sequencing techniques were also performed on breast muscle tissue of JXY chickens at different developmental stages. And 13C isotope tracing technique was applied. In this study, a large-scale genetic analysis of an IMF-selected population and a control population identified fatty acid synthase (FASN) as a key gene for improving IMF content. Also, contrary to conventional view, de novo lipogenesis (DNL) was deemed to be an important contributor to IMF deposition. As expected, further analyses by isotope tracing and other techniques, confirmed that DNL mainly occurs in myocytes, contributing about 40% of the total fatty acids through the regulation of FASN, using the available FAs as substrates. Additionally, we also identified a relevant causal mutation in the FASN gene with effects on FA composition. These findings contribute to the understanding of fat metabolism in muscle tissue of poultry, and provide the feasible strategy for the production of high-quality chicken meat. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy can be appreciated microscopically by assessing for myocyte hypertrophy. Features of myocyte hypertrophy include increased myocyte diameter, together with increase in nuclear hyperchromicity, size and irregularity, and an increase in binucleated myocytes. There is limited literature to demonstrate whether these features are uniform within the myocardium in the post mortem setting. This study compared the myocyte diameter, myocyte nuclear length, and proportion binucleated myocytes between the free wall of the left ventricle and interventricular septum in 21 post mortem cases. It showed regional variation in myocyte hypertrophy parameters between the free walls of the left ventricle and intraventricular septum. Over 75% of cases showed significant differences in myocyte nuclear length and myocyte diameter (ANOVA one-way, p < 0.05), whereas binucleated myocyte nuclei did not show any significant difference. The result of this study has implication in sampling and assessing myocyte hypertrophy. Further studies are recommended to explore the reasons why this variation occurred and whether it is clinically significant. [ABSTRACT FROM AUTHOR]

SARS-CoV-2, the virus underlying COVID-19, has now been recognized to cause multiorgan disease with a systemic effect on the host. To effectively combat SARS-CoV-2 and the subsequent development of COVID-19, it is critical to detect, monitor, and model viral pathogenesis. In this review, we discuss recent advancements in microfluidics, organ-on-a-chip, and human stem cell-derived models to study SARS-CoV-2 infection in the physiological organ microenvironment, together with their limitations. Microfluidic-based detection methods have greatly enhanced the rapidity, accessibility, and sensitivity of viral detection from patient samples. Engineered organ-on-a-chip models that recapitulate in vivo physiology have been developed for many organ systems to study viral pathology. Human stem cell-derived models have been utilized not only to model viral tropism and pathogenesis in a physiologically relevant context but also to screen for effective therapeutic compounds. The combination of all these platforms, along with future advancements, may aid to identify potential targets and develop novel strategies to counteract COVID-19 pathogenesis.

Gamma-aminobutyric acid (GABA) is commonly considered as a signaling molecule in the synapses of the central nervous system, where it plays the role of the main inhibitory neurotransmitter in the mature brain and participates in the process of neurogenesis. Recently, data have been obtained indicating that GABA may also be involved in the early stages of skeletal muscle tissue development. In the present study, performed on rat myocyte culture, the effect of exogenous GABA on the process of fusion of myocytes into myotubes was investigated by analyzing such a morphometric indicator as the "fusion index". The addition of the amino acid to the culture led to a significant concentration-dependent inhibition (up to a complete cessation) of the formation of myotubes. GABAA receptors and GABA transporters (GAT-2) were considered among possible proteins capable of mediating the effect of amino acids. Evidence of the presence of these proteins on cultured cells was obtained using immunohistochemical methods. The blockade of GABA receptors by gabazine did not affect the fusion index in any way, and GABA continued to exert inhibitory effect in its presence. Inhibition of GABA transporters by nipecotic acid reduced the myocyte fusion index; however, the GABA effect was no longer present under the action of the transporter blocker. The data obtained are consistent with the hypothesis about the participation of the amino acid GABA at the early stages of skeletal muscle development; it suggests that the inhibitory effect of the exogenous amino acid may be due to an increase in its concentration in the sarcoplasm, since both the addition of a GABA transporter blocker and an increase in the extracellular concentration of GABA negatively affect the formation of myotubes. [ABSTRACT FROM AUTHOR]

To discuss the resistance and regeneration effects of long non-coding RNA (lncRNA) GPRC5D-AS1 on the muscle atrophy of myocytes in the mice induced by dexamethasone,and to clarify its mechanism. Methods The C2C12 cells at logarithmic growth phase were selected. The survival rates of the cells after treated with 0,25,50,75,100,200 and 400 mg·L－1 dexamethasone for 24,48 and 72 h were detected,which was used to select the optimal dose and time to induce the muscle atrophy model.The expression level of lncRNA GPRC5D-AS1 in the C2C12 cells was detected by real-time fluroscence quantitative PCR(RT-qPCR) method to validate the cell model. The C2C12 cells were divided into normal group,control group，lncRNA GPRC5D-AS1-NC group，and lncRNA GPRC5D-AS1-OE group. The expression levels of lncRNA GPRC5D-AS1 in the cells in various groups were detected by RT-qPCR method. The levels of reactive oxygen species (ROS),glutathione peroxidase 4 (GPX4),ferrous ion (Fe2+) and malondialdehyde (MDA) in the cells in various groups were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA) method； the ultrastructural changes of mitochondria in the cells in various groups were observed under transmission electron microscope； the expression of myogenic differentiation protein (MyoD) in the cells in various groups was detected by immunofluorescence； the expression levels of ferroptosis-related proteins in the cells in various groups were detected by Western blotting method. Results The best modeling effect was achieved with 100 mg·L-1 dexamethasone at 48 h. Compared with normal C2C12 cells,the expression level of lncRNA GPRC5D-AS1 in the C2C12 cells after treated with 100 mg·L-1dexamethasone for 48 h was decreased (P<0.05),confirming that lncRNA GPRC5D-AS1 had a regulatory role in the muscle atrophy model. The RT-qPCR results showed that compared with normal group,the expression level of lncRNA GRPC5D-AS1 in the cells in model group was decreased (P<0.05)； compared with model group and lncRNA GRPC5D-AS1-NC group,the expression level of lncRNA GRPC5D-AS1 in the cells in lncRNA GRPC5D-AS1-OE group was increased (P<0.05). The flow cytometry results showd that compared with normal group,the ROS level in the cells in model group was increased (P<0.05)； compared with model group and lncRNA GRPC5D-AS1-NC group,the ROS level in the cells in lncRNA GRPC5D-AS1-OE group was decreased (P<0.05). The ELISA results showed that compared with normal group,the levels of Fe2+ and MDA in the cells in model group were increased (P<0.05),and the level of GPX4 was decreased (P<0.05)； compared with model group and lncRNA GRPC5D-AS1-NC group,the levels of Fe2+ and MDA in the cells in lncRNA GRPC5D-AS1-OE group were decreased (P<0.05),and the level of GPX4 was increased (P<0.05). The transmission electron microscope results showed that the cells in normal group were round and normal in size with rich cilia on the membrane,and the mitochondria had a normal morphology； the cells in model group had less mitochondria and granulation in the cytoplasm,with blurred and swollen mitochondrial structures and typical ferroptosis-related manifestations； the number of mitochondria in the cells in lncRNA GRPC5D-AS1-OE group was increased，and the granulation in cytoplasm of the cells was decreased,and showed clearer mitochondrial structures. The immunofluorescence detection results showed that compared with normal group，the expression of MyoD in the cells in model group was decreased and the cells exhibited muscle atrophy； compared with model group and lncRNA GRPC5D-AS1-NC group,the expression of MyoD in the cells in lncRNA GRPC5D-AS1-OE group was increased,and the cells exhibited less muscle atrophy and more regeneration of myocytes. The Western blotting results revealed that compared with the normal group,the expression level of long-chain acyl-coenzyme A synthase (ACSL)4 protein in the cell in model group was increased (P<0.05),and the expression levels of solute carrier family 7 member 11 (SLC7A11) and GPX4 proteins were decreased (P<0.05)； compared with model group and lncRNA GRPC5D-AS1-NC group,the expression level of ACSL4 protein in the cells in lncRNA GRPC5D-AS1-OE group was decreased (P<0.05),and the expression levels of SLC7A11 and GPX4 proteins were increased (P<0.05). Conclusion LncRNA GPRC5D-AS1 can protect the ferroptosis in the myocytes induced by dexamethasone and promote the repairment and regeneration of the cells，and its mechanism is related to regulating the SLC7A11/GPX4/ACSL4 signaling pathway. [ABSTRACT FROM AUTHOR]

Normal contractile function of the myocardium is essential for optimal cardiovascular health. Evaluating drug effects on cardiomyocyte function at the cellular level is difficult for long-term studies. Present culture systems rely on isolated, cardiomyocyte preparations or cardiomyocytes derived from pluripotent stem cells (PSCs), all of which have limitations. Isolated, endogenous cardiomyocytes do not remain contractile in culture long term. While PSC-derived cardiomyocytes show contractile activity for longer periods of time, their phenotype is more embryonic than adult. Here we report that dexamethasone (DEX) treatment of adult mouse atrial tissue can extend its functionality in culture. Normally, cardiac explants cease their capacity as a contractile tissue within the first month, as the tissue flattens and spreads out on the culture substrate, while the cells dedifferentiate and lose their myocardial phenotype. However, with DEX treatment, cardiac explants maintain their contractile function, 3D morphology, and myocyte phenotype for up to 6 months. Moreover, DEX also preserved the contractile phenotype of isolated rat cardiomyocytes. These data with DEX suggest that simple modifications in culture conditions can greatly improve the long-term utility of in vitro model systems for screening drugs and agents that could be employed to alleviate human cardiac disease.

Skeletal muscle anatomy and physiology are sexually dimorphic but molecular underpinnings and muscle-specificity are not well-established. Variances in metabolic health, fitness level, sedentary behavior, genetics, and age make it difficult to discern inherent sex effects in humans. Therefore, mice under well-controlled conditions were used to determine female and male (n = 19/sex) skeletal muscle fiber type/size and capillarity in superficial and deep gastrocnemius (GA-s, GA-d), soleus (SOL), extensor digitorum longus (EDL), and plantaris (PLT), and transcriptome patterns were also determined (GA, SOL). Summed muscle weight strongly correlated with lean body mass (r2  = 0.67, p 6000 (GA) and >4000 (SOL) mRNAs differentially-expressed by sex; only a minority of these were shared across GA and SOL. Pathway analyses revealed differences in ribosome biology, transcription, and RNA processing. Curation of sexually dimorphic muscle transcripts shared in GA and SOL, and literature datasets from mice and humans, identified 11 genes that we propose are canonical to innate sex differences in muscle: Xist, Kdm6a, Grb10, Oas2, Rps4x (higher, females) and Ddx3y, Kdm5d, Irx3, Wwp1, Aldh1a1, Cd24a (higher, males). These genes and those with the highest "sex-biased" expression in our study do not contain estrogen-response elements (exception, Greb1), but a subset are proposed to be regulated through androgen response elements. We hypothesize that innate muscle sexual dimorphism in mice and humans is triggered and then maintained by classic X inactivation (Xist, females) and Y activation (Ddx3y, males), with coincident engagement of X encoded (Kdm6a) and Y encoded (Kdm5d) demethylase epigenetic regulators that are complemented by modulation at some regions of the genome that respond to androgen.

Skeletal muscle anatomy and physiology are sexually dimorphic but molecular underpinnings and muscle-specificity are not well-established. Variances in metabolic health, fitness level, sedentary behavior, genetics, and age make it difficult to discern inherent sex effects in humans. Therefore, mice under well-controlled conditions were used to determine female and male (n = 19/sex) skeletal muscle fiber type/size and capillarity in superficial and deep gastrocnemius (GA-s, GA-d), soleus (SOL), extensor digitorum longus (EDL), and plantaris (PLT), and transcriptome patterns were also determined (GA, SOL). Summed muscle weight strongly correlated with lean body mass (r2  = 0.67, p 6000 (GA) and >4000 (SOL) mRNAs differentially-expressed by sex; only a minority of these were shared across GA and SOL. Pathway analyses revealed differences in ribosome biology, transcription, and RNA processing. Curation of sexually dimorphic muscle transcripts shared in GA and SOL, and literature datasets from mice and humans, identified 11 genes that we propose are canonical to innate sex differences in muscle: Xist, Kdm6a, Grb10, Oas2, Rps4x (higher, females) and Ddx3y, Kdm5d, Irx3, Wwp1, Aldh1a1, Cd24a (higher, males). These genes and those with the highest "sex-biased" expression in our study do not contain estrogen-response elements (exception, Greb1), but a subset are proposed to be regulated through androgen response elements. We hypothesize that innate muscle sexual dimorphism in mice and humans is triggered and then maintained by classic X inactivation (Xist, females) and Y activation (Ddx3y, males), with coincident engagement of X encoded (Kdm6a) and Y encoded (Kdm5d) demethylase epigenetic regulators that are complemented by modulation at some regions of the genome that respond to androgen.

The small conductance Ca2+-activated K+ current (I SK) is a potential therapeutic target for treating atrial fibrillation. To clarify, in rabbit and human atrial cardiomyocytes, the intracellular [Ca2+]-sensitivity of I SK , and its contribution to action potential (AP) repolarisation, under physiological conditions. Whole-cell-patch clamp, fluorescence microscopy: to record ion currents, APs and [Ca2+] i ; 35–37°C. In rabbit atrial myocytes, 0.5 mM Ba2+ (positive control) significantly decreased whole-cell current, from −12.8 to −4.9 pA/pF (P < 0.05, n = 17 cells, 8 rabbits). By contrast, the I SK blocker apamin (100 nM) had no effect on whole-cell current, at any set [Ca2+] i (∼100–450 nM). The I SK blocker ICAGEN (1 μM: ≥2 x IC 50) also had no effect on current over this [Ca2+] i range. In human atrial myocytes, neither 1 μM ICAGEN (at [Ca2+] i ∼ 100–450 nM), nor 100 nM apamin ([Ca2+] i ∼ 250 nM) affected whole-cell current (5–10 cells, 3–5 patients/group). APs were significantly prolonged (at APD 30 and APD 70) by 2 mM 4-aminopyridine (positive control) in rabbit atrial myocytes, but 1 μM ICAGEN had no effect on APDs, versus either pre-ICAGEN or time-matched controls. High concentration (10 μM) ICAGEN (potentially I SK -non-selective) moderately increased APD 70 and APD 90 , by 5 and 26 ms, respectively. In human atrial myocytes, 1 μM ICAGEN had no effect on APD 30–90 , whether stimulated at 1, 2 or 3 Hz (6–9 cells, 2–4 patients/rate). I SK does not flow in human or rabbit atrial cardiomyocytes with [Ca2+] i set within the global average diastolic-systolic range, nor during APs stimulated at physiological or supra-physiological (≤3 Hz) rates. [Display omitted] • Inhibiting the cardiac atrial cell ion current "I SK " can prevent atrial fibrillation. • Whether I SK flows in healthy cells with normal internal Ca2+ levels is under debate. • The I SK blockers apamin and ICAGEN did not affect I SK in rabbit or human atrial cells. • I SK inhibition also did not affect action potentials, even at high stimulation rates. • We add weight to the argument that I SK does not flow in healthy cardiac atrial cells. [ABSTRACT FROM AUTHOR]

Although obese sarcopenia is a major public health problem with increasing prevalence worldwide, the factors that contribute to the development of obese sarcopenia are still obscure. In order to clarify this issue, a high-fat-diet-induced obese sarcopenia mouse model was utilized. After being fed with a high-fat diet for 24 weeks, decreased motor functions and muscle mass ratios were found in the C57BL/6 mice. In addition, the expression of calsarcin-2 was significantly increased in their skeletal muscle, which was determined by a microarray analysis. In order to clarify the role of calsarcin-2 in muscle, lentiviral vectors containing the calsarcin-2 gene or short hairpin RNA targeted to calsarcin-2 were used to manipulate calsarcin-2 expressions in L6 myoblasts. We found that an overexpression of calsarcin-2 facilitated L6 myoblast differentiation, whereas a calsarcin-2 knockdown delayed myoblast differentiation, as determined by the expression of myogenin. However, the calsarcin-2 knockdown showed no significant effects on myoblast proliferation. In addition, to clarify the relationship between serum calsarcin-2 and sarcopenia, the bilateral gastrocnemius muscle mass per body weight in mice and appendicular skeletal muscle mass index in humans were measured. Although calsarcin-2 facilitated myoblast differentiation, the serum calsarcin-2 concentration was negatively related to skeletal muscle mass index in mice and human subjects. Taken together, calsarcin-2 might facilitate myoblast differentiation and appear to play a compensatory role in sarcopenia. [ABSTRACT FROM AUTHOR]

Normal contractile function of the myocardium is essential for optimal cardiovascular health. Evaluating drug effects on cardiomyocyte function at the cellular level is difficult for long-term studies. Present culture systems rely on isolated, cardiomyocyte preparations or cardiomyocytes derived from pluripotent stem cells (PSCs), all of which have limitations. Isolated, endogenous cardiomyocytes do not remain contractile in culture long term. While PSC-derived cardiomyocytes show contractile activity for longer periods of time, their phenotype is more embryonic than adult. Here we report that dexamethasone (DEX) treatment of adult mouse atrial tissue can extend its functionality in culture. Normally, cardiac explants cease their capacity as a contractile tissue within the first month, as the tissue flattens and spreads out on the culture substrate, while the cells dedifferentiate and lose their myocardial phenotype. However, with DEX treatment, cardiac explants maintain their contractile function, 3D morphology, and myocyte phenotype for up to 6 months. Moreover, DEX also preserved the contractile phenotype of isolated rat cardiomyocytes. These data with DEX suggest that simple modifications in culture conditions can greatly improve the long-term utility of in vitro model systems for screening drugs and agents that could be employed to alleviate human cardiac disease. [ABSTRACT FROM AUTHOR]

Aquaculture is the fastest-growing food production sector and nowadays provides more food than extractive fishing. Studies focused on the understanding of how teleost growth is regulated are essential to improve fish production. Cysteamine (CSH) is a novel feed additive that can improve growth through the modulation of the GH/IGF axis; however, the underlying mechanisms and the interaction between tissues are not well understood. This study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg of feed for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea bream (Sparus aurata) fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g). Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4 days on cell viability and GH/IGF axis relative gene expression were evaluated. Results showed that CSH-1.65 improved growth performance by 16% and 26.7% after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks compared to control diet (0 g/kg). However, no significant differences were found between both experimental doses. CSH reduced the plasma levels of GH after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene expression analysis revealed a significant upregulation of the ghr-1, different igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher cell viability and mRNA levels of ghr1, igf-1b, igf-2 and igf-1rb compared to control (0 µM) in a similar way to white muscle. Overall, CSH improves growth and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status through different synergic ways, revealing CSH as a feasible candidate to be included in fish feed. [ABSTRACT FROM AUTHOR]

Intramuscular fat (IMF) is one of the most important indexes of pork taste quality. Diacylglycerol acyltransferase 1 (DGAT1), belonging to the acyl‐coenzyme A: DGAT enzymes family, is a rate‐limiting enzyme responsible for the final step of triglyceride (TG) synthesis. It is involved in TG storage in skeletal muscle; however, the underlying mechanism is not well understood. This study aimed to uncover functional mutations that can influence DGAT1 expression and consequently affect IMF deposition in pork. Two experimental groups containing individuals with high and low IMF content (6.23 ± 0.20 vs. 1.25 ± 0.05, p < 0.01) were formed from 260 Duroc × Large White × Yorkshire (D × L × Y) cross‐bred pigs. A novel SNP c.−379 C>T was uncovered in the DGAT1 gene using comparative sequencing with pool DNA of high‐ and low‐IMF groups. The IMF content of CT genotype individuals (3.19 ± 0.11%) was higher than that of CC genotype individuals (2.86 ± 0.11%) when analyzing 260 D × L × Y pigs (p < 0.05). The DGAT1 expression levels revealed a significant positive correlation with IMF content (r = 0.33, p < 0.01). Luciferase assay revealed that the DGAT1 promoter with the c.−379 T allele has a higher transcription activity than that bearing the C allele in C2C12 myoblast cells, but not in 3T3‐L1 pre‐adipocytes. Online prediction followed by chromatin immunoprecipitation‐polymerase chain reaction assay confirmed that myogenic determination factor 1 (MYOD1) binds to the DGAT1 promoter with the c.−379 C allele but not the T allele. In vitro experiments demonstrated that MYOD1 represses DGAT1 transcription and lipogenesis. As a muscle‐specific transcription factor, MYOD1 can inhibit the transcription of DGAT1 with the c.−379 C allele in muscle cells. However, in the absence of MYOD1 binding to the mutated DGAT1 promoter with the c.−379 T allele, DGAT1 expresses at a higher level in the muscle cells of the c.−379 T genotype, leading to more intramyocellular lipid accumulation than in the muscle cells of the c.−379 C genotype. The SNP c.−379 C>T in the promoter region of the DGAT1 gene provides a promising molecular marker for improving pork IMF content without affecting other fat depots. [ABSTRACT FROM AUTHOR]

Background and objective: In this present research paper, a mathematical model has been developed to study myocyte contraction in the human cardiac muscle, using the Land model. Different parts of the human heart with a focus on the composition of the myocyte cells have been explored numerically to enabling us to determine the interaction of various parameters in the heart muscle. The main objective of the work is to direct the study of the Land model, which has been exploited to simulate the contraction of real human myocytes. Methods: Mathematical models has been developed based on the Hill model and Huxley model. Myocyte contraction for different scenarios, such as in isometric tension and isotonic tension have been studied. Results: It is found that increase in stretch, the peak active tension increases, in line with well-established length-dependent tension generation. Five parameters are selected: [Ca2+]T50, Tref, TRPN50, β0, and β1, which have been varied in between the range of −50%–100%, to examine the isometric effects of each parameter on the behavior of the tension developed in the intact myocyte cells, with the most sensitive parameter being [Ca2+]T50. Conclusion: In conclusion, it is found that the Land model provides a good platform for the analysis of the active contraction of the human cardiac myocyte.

RationaleCardiotoxic β1 adrenergic receptor (β1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome.ObjectiveWe aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure.Methods and resultsThe integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts.ConclusionsThese data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Background: Panax ginseng (ginseng) is a traditional medicine that is reported to have cardioprotective effects; ginsenosides are the major bioactive compounds in the ginseng root. Methods: Magnetic molecularly imprinted polymer (MMIP) nanoparticles might be useful for both the extraction of the targeted (imprinted) molecules, and for the delivery of those molecules to cells. In this work, plant growth regulators were used to enhance the adventitious rooting of ginseng root callus; imprinted polymeric particles were synthesized for the extraction of ginsenoside Rb1 from root extracts, and then employed for subsequent particle-mediated delivery to cardiomyocytes to mitigate hypoxia/reoxygenation injury. Results: These synthesized composite nanoparticles were first characterized by their specific surface area, adsorption capacity, and magnetization, and then used for the extraction of ginsenoside Rb1 from a crude extract of ginseng roots. The ginsenoside-loaded MMIPs were then shown to have protective effects on mitochondrial membrane potential and cellular viability for H9c2 cells treated with CoCl2 to mimic hypoxia injury. The protective effect of the ginsenosides was assessed by staining with JC-1 dye to monitor the mitochondrial membrane potential. Conclusion: MMIPs can play a dual role in both the extraction and cellular delivery of therapeutic ginsenosides.

Background and purpose: While flecainide is now an accepted treatment for arrhythmias associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), its mechanism of action remains controversial. In studies on myocytes from CPVT mice, inhibition of proarrhythmic Ca2+ waves was initially attributed to a novel action on the type-2 ryanodine receptor (RyR2). However, subsequent work on wild type (WT) myocytes questioned the conclusion that flecainide has a direct action on RyR2. In the present study, the effects of flecainide were compared in intact and permeabilized WT myocytes. Experimental approach: Intracellular Ca2+ was measured using confocal microscopy in intact or saponin permeabilized adult rat ventricular myocytes (ARVM). In some experiments on permeabilized cells, flecainide was studied following partial inhibition of the sarcoplasmic reticulum (SR) counter-current. Key results: Flecainide induced sustained changes Ca2+ sparks and waves in permeabilized ARVM, which were comparable to those reported in intact or permeabilized myocytes from CPVT mice. However, a relatively high level of flecainide (25 μM) was required to induce these effects. Inhibition of the SR counter-current potentiated the effects of flecainide on SR Ca2+ waves. In intact field stimulated ARVM, prolonged exposure to 15 μM flecainide decreased wave frequency but RyR2 dependent effects on Ca2+ sparks were absent; higher drug concentrations blocked field stimulation, consistent with inhibition of Nav1.5. Conclusions and implications: In intact ARVM, the absence of effects on Ca2+ sparks suggests that the intracellular flecainide concentration was insufficient to influence RyR2. Wave inhibition in intact ARVM may reflect secondary effects of Nav1.5 inhibition. Potentiation of flecainide’s action by counter-current inhibition can be explained if transient polarization of the SR membrane during SR Ca2+ release facilitates its action on RyR2. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a worldwide prevalent respiratory disease mainly caused by tobacco smoke exposure. COPD is now considered as a systemic disease with several comorbidities. Among them, skeletal muscle dysfunction affects around 20% of COPD patients and is associated with higher morbidity and mortality. Although the histological alterations are well characterized, including myofiber atrophy, a decreased proportion of slow-twitch myofibers, and a decreased capillarization and oxidative phosphorylation capacity, the molecular basis for muscle atrophy is complex and remains partly unknown. Major difficulties lie in patient heterogeneity, accessing patients' samples, and complex multifactorial process including extrinsic mechanisms, such as tobacco smoke or disuse, and intrinsic mechanisms, such as oxidative stress, hypoxia, or systemic inflammation. Muscle wasting is also a highly dynamic process whose investigation is hampered by the differential protein regulation according to the stage of atrophy. In this review, we report and discuss recent data regarding the molecular alterations in COPD leading to impaired muscle mass, including inflammation, hypoxia and hypercapnia, mitochondrial dysfunction, diverse metabolic changes such as oxidative and nitrosative stress and genetic and epigenetic modifications, all leading to an impaired anabolic/catabolic balance in the myocyte. We recapitulate data concerning skeletal muscle dysfunction obtained in the different rodent models of COPD. Finally, we propose several pathways that should be investigated in COPD skeletal muscle dysfunction in the future. [ABSTRACT FROM AUTHOR]

Aquaculture is the fastest-growing food production sector and nowadays provides more food than extractive fishing. Studies focused on the understanding of how teleost growth is regulated are essential to improve fish production. Cysteamine (CSH) is a novel feed additive that can improve growth through the modulation of the GH/IGF axis; however, the underlying mechanisms and the interaction between tissues are not well understood. This study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg of feed for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea bream (Sparus aurata) fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g). Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4 days on cell viability and GH/IGF axis relative gene expression were evaluated. Results showed that CSH-1.65 improved growth performance by 16% and 26.7% after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks compared to control diet (0 g/kg). However, no significant differences were found between both experimental doses. CSH reduced the plasma levels of GH after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene expression analysis revealed a significant upregulation of the ghr-1, different igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher cell viability and mRNA levels of ghr1, igf-1b, igf-2 and igf-1rb compared to control (0 µM) in a similar way to white muscle. Overall, CSH improves growth and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status through different synergic ways, revealing CSH as a feasible candidate to be included in fish feed.

Background and purpose: While flecainide is now an accepted treatment for arrhythmias associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), its mechanism of action remains controversial. In studies on myocytes from CPVT mice, inhibition of proarrhythmic Ca2+ waves was initially attributed to a novel action on the type-2 ryanodine receptor (RyR2). However, subsequent work on wild type (WT) myocytes questioned the conclusion that flecainide has a direct action on RyR2. In the present study, the effects of flecainide were compared in intact and permeabilized WT myocytes.Experimental approach: Intracellular Ca2+ was measured using confocal microscopy in intact or saponin permeabilized adult rat ventricular myocytes (ARVM). In some experiments on permeabilized cells, flecainide was studied following partial inhibition of the sarcoplasmic reticulum (SR) counter-current.Key results: Flecainide induced sustained changes Ca2+ sparks and waves in permeabilized ARVM, which were comparable to those reported in intact or permeabilized myocytes from CPVT mice. However, a relatively high level of flecainide (25 μM) was required to induce these effects. Inhibition of the SR counter-current potentiated the effects of flecainide on SR Ca2+ waves. In intact field stimulated ARVM, prolonged exposure to 15 μM flecainide decreased wave frequency but RyR2 dependent effects on Ca2+ sparks were absent; higher drug concentrations blocked field stimulation, consistent with inhibition of Nav1.5.Conclusions and implications: In intact ARVM, the absence of effects on Ca2+ sparks suggests that the intracellular flecainide concentration was insufficient to influence RyR2. Wave inhibition in intact ARVM may reflect secondary effects of Nav1.5 inhibition. Potentiation of flecainide’s action by counter-current inhibition can be explained if transient polarization of the SR membrane during SR Ca2+ release facilitates its action on RyR2.

Although obese sarcopenia is a major public health problem with increasing prevalence worldwide, the factors that contribute to the development of obese sarcopenia are still obscure. In order to clarify this issue, a high-fat-diet-induced obese sarcopenia mouse model was utilized. After being fed with a high-fat diet for 24 weeks, decreased motor functions and muscle mass ratios were found in the C57BL/6 mice. In addition, the expression of calsarcin-2 was significantly increased in their skeletal muscle, which was determined by a microarray analysis. In order to clarify the role of calsarcin-2 in muscle, lentiviral vectors containing the calsarcin-2 gene or short hairpin RNA targeted to calsarcin-2 were used to manipulate calsarcin-2 expressions in L6 myoblasts. We found that an overexpression of calsarcin-2 facilitated L6 myoblast differentiation, whereas a calsarcin-2 knockdown delayed myoblast differentiation, as determined by the expression of myogenin. However, the calsarcin-2 knockdown showed no significant effects on myoblast proliferation. In addition, to clarify the relationship between serum calsarcin-2 and sarcopenia, the bilateral gastrocnemius muscle mass per body weight in mice and appendicular skeletal muscle mass index in humans were measured. Although calsarcin-2 facilitated myoblast differentiation, the serum calsarcin-2 concentration was negatively related to skeletal muscle mass index in mice and human subjects. Taken together, calsarcin-2 might facilitate myoblast differentiation and appear to play a compensatory role in sarcopenia.

BackgroundRSD921, the R,R enantiomer of the kappa (k) agonist PD117,302, lacks significant activity on opioid receptors.MethodsThe pharmacological and toxicological actions were studied with reference to cardiovascular, cardiac, antiarrhythmic, toxic and local anaesthetic activity.ResultsIn rats, dogs and baboons, RSD921 dose-dependently reduced blood pressure and heart rate. In a manner consistent with sodium channel blockade it prolonged the PR and QRS intervals of the ECG. Furthermore, in rats and NHP, RSD921 increased the threshold currents for induction of extra-systoles and ventricular fibrillation (VFt), and prolonged effective refractory period (ERP). In rats, RSD921 was protective against arrhythmias induced by electrical stimulation and coronary artery occlusion. Application of RSD921 to voltage-clamped rat cardiac myocytes blocked sodium currents. RSD921 also blocked transient (ito) and sustained (IKsus) outward potassium currents, albeit with reduced potency relative to sodium current blockade. Sodium channel blockade due to RSD921 in myocytes and isolated hearts was enhanced under ischaemic conditions (low pH and high extracellular potassium concentration). When tested on the cardiac, neuronal and skeletal muscle forms of sodium channels expressed in Xenopus laevis oocytes, RSD921 produced equipotent tonic block of sodium currents, enhanced channel block at reduced pH (6.4) and marked use-dependent block of the cardiac isoform. RSD921 had limited but quantifiable effects in subacute toxicology studies in rats and dogs. Pharmacokinetic analyses were performed in baboons. Plasma concentrations producing cardiac actions in vivo after intravenous administration of RSD921 were similar to the concentrations effective in the in vitro assays utilized.ConclusionsRSD921 primarily blocks sodium currents, and possesses antiarrhythmic and local anaesthetic activity.

Deng and colleagues highlight the importance of understanding the divergent roles of β2-adrenoceptor (β 2 AR) in high-fat diet-induced heart failure. β 2 AR signaling has beneficial and detrimental effects depending on the context and level of activation. We discuss the importance of these findings and their implications in developing effective and safe therapies. [ABSTRACT FROM AUTHOR]

Objective: Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor. Paradoxically, a decreased risk of abdominal aortic aneurysm (AAA) presence and growth rate is described among patients with T2DM, associated with metformin use. This study aimed to investigate the effect of metformin on AAA patient derived aortic smooth muscle cell (SMC) function., Methods: Aortic biopsies were obtained from patients with AAA (n = 21) and controls (n = 17) during surgery. The SMCs of non-pathological aortic controls, non-diabetic patients with AAA, and diabetic patients with AAA were cultured from explants and treated with or without metformin. The SMC contractility was measured upon ionomycin stimulation, as well as metabolic activity, proliferation, and migration. Then, mRNA and protein expression of markers for contraction, metabolic activity, proliferation, and inflammation were measured., Results: The mRNA expression of KLF4 and GYS1, genes involved in metabolic activity, differed between the SMCs from non-diabetic and diabetic patients with AAA before metformin stimulation (p < .041). However, the effect of metformin on the various SMC functions was similar between non-diabetic and diabetic patients with AAA. Upon stimulation, metformin increased the contractility of AAA patient SMCs (p = .001). The mRNA expression of smoothelin, a marker for the contractile phenotype, increased in the SMCs of patients with AAA after treatment with metformin (p = .006). An increase in metabolic activity (p < .001) and a decrease in proliferation (p < .001) and migration were found in the SMCs of controls and patients with AAA with metformin. Increased mRNA expression of PPARγ, a nuclear receptor involved in mitochondrial biogenesis (p < .009), and a decrease in gene expression of Ki-67, a marker for proliferation (p < .005), were observed. Gene expression of inflammation markers MCP-1 and IL-6, and protein expression of NF-κB p65 decreased after treatment with metformin in patients with AAA., Conclusion: This study found that metformin increases contractility and metabolic activity, and reduces proliferation, migration, and inflammation in aortic SMCs in vitro., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cells actively sense differences in topology, matrix elasticity and protein composition of the extracellular microenvironment and adapt their function and morphology. In this study, we focus on the cross-talk between matrix stiffness and protein coating density that regulates morphology and proliferation dynamics of single myocytes. For this, C2C12 myocytes were monitored on L-DOPA functionalized hydrogels of 22 different elasticity and fibronectin density compositions. Static images were recorded and statistically analyzed to determine morphological differences and to identify the optimized extracellular matrix (ECM). Using that information, selected ECMs were used to study the dynamics before and after cell proliferation by statistical comparison of distinct cell states. We observed a fibronectin-density-independent increase of the projected cell area until 12 kPa. Additionally, changes in fibronectin density led to an area that was optimum at about 2.6 μ g / cm 2 , which was confirmed by independent F-actin analysis, revealing a maximum actin-filament-to-cell-area ratio of 7.5%. Proliferation evaluation showed an opposite correlation between cell spreading duration and speed to matrix elasticity and protein density, which did not affect cell-cycle duration. In summary, we identified an optimized ECM composition and found that independent matrix properties regulate distinct cell characteristics. [ABSTRACT FROM AUTHOR]

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.

Chronic obstructive pulmonary disease (COPD) is a worldwide prevalent respiratory disease mainly caused by tobacco smoke exposure. COPD is now considered as a systemic disease with several comorbidities. Among them, skeletal muscle dysfunction affects around 20% of COPD patients and is associated with higher morbidity and mortality. Although the histological alterations are well characterized, including myofiber atrophy, a decreased proportion of slow-twitch myofibers, and a decreased capillarization and oxidative phosphorylation capacity, the molecular basis for muscle atrophy is complex and remains partly unknown. Major difficulties lie in patient heterogeneity, accessing patients’ samples, and complex multifactorial process including extrinsic mechanisms, such as tobacco smoke or disuse, and intrinsic mechanisms, such as oxidative stress, hypoxia, or systemic inflammation. Muscle wasting is also a highly dynamic process whose investigation is hampered by the differential protein regulation according to the stage of atrophy. In this review, we report and discuss recent data regarding the molecular alterations in COPD leading to impaired muscle mass, including inflammation, hypoxia and hypercapnia, mitochondrial dysfunction, diverse metabolic changes such as oxidative and nitrosative stress and genetic and epigenetic modifications, all leading to an impaired anabolic/catabolic balance in the myocyte. We recapitulate data concerning skeletal muscle dysfunction obtained in the different rodent models of COPD. Finally, we propose several pathways that should be investigated in COPD skeletal muscle dysfunction in the future.

Cross-talk between peripheral tissues is essential to ensure the coordination of nutrient intake with disposition during the feeding period, thereby preventing metabolic disease. This mini-review considers the interactions between the key peripheral tissues that constitute the metabolic clock, each of which is considered in a separate mini-review in this collation of articles published in Endocrinology in 2020 and 2021, by Martchenko et al (Circadian rhythms and the gastrointestinal tract: relationship to metabolism and gut hormones); Alvarez et al (The microbiome as a circadian coordinator of metabolism); Seshadri and Doucette (Circadian regulation of the pancreatic beta cell); McCommis et al (The importance of keeping time in the liver); Oosterman et al (The circadian clock, shift work, and tissue-specific insulin resistance); and Heyde et al (Contributions of white and brown adipose tissues to the circadian regulation of energy metabolism). The use of positive- and negative-feedback signals, both hormonal and metabolic, between these tissues ensures that peripheral metabolic pathways are synchronized with the timing of food intake, thus optimizing nutrient disposition and preventing metabolic disease. Collectively, these articles highlight the critical role played by the circadian clock in maintaining metabolic homeostasis. [ABSTRACT FROM AUTHOR]

Pulsed electrical field (PEF) energy is a promising technique for catheter ablation of cardiac arrhythmias. In this article, the key aspects that need to be considered for safe and effective PEF delivery are reviewed, and their impact on clinical feasibility is discussed. The most important benefit of PEF appears to be the ability to kill cells through mechanisms that do not alter stromal proteins, sparing sensitive structures to improve safety, without sacrificing cardiomyocyte ablation efficacy. Many parameters affect PEF treatment outcomes, including pulse intensity, waveform shape, and number of pulses, as well as electrode configuration and geometry. These physical and electrical characteristics must be titrated carefully to balance target tissue effects with collateral implications (muscle contraction, temperature rise, risk of electrical arcing events). It is important to note that any combination of parameters affecting PEF needs to be tested for clinical efficacy and safety. Applying PEF clinically requires knowledge of the fundamentals of this technology to exploit its opportunities and generate viable, durable health improvements for patients. [ABSTRACT FROM AUTHOR]

Abstract Skeletal muscle anatomy and physiology are sexually dimorphic but molecular underpinnings and muscle‐specificity are not well‐established. Variances in metabolic health, fitness level, sedentary behavior, genetics, and age make it difficult to discern inherent sex effects in humans. Therefore, mice under well‐controlled conditions were used to determine female and male (n = 19/sex) skeletal muscle fiber type/size and capillarity in superficial and deep gastrocnemius (GA‐s, GA‐d), soleus (SOL), extensor digitorum longus (EDL), and plantaris (PLT), and transcriptome patterns were also determined (GA, SOL). Summed muscle weight strongly correlated with lean body mass (r2 = 0.67, p 6000 (GA) and >4000 (SOL) mRNAs differentially‐expressed by sex; only a minority of these were shared across GA and SOL. Pathway analyses revealed differences in ribosome biology, transcription, and RNA processing. Curation of sexually dimorphic muscle transcripts shared in GA and SOL, and literature datasets from mice and humans, identified 11 genes that we propose are canonical to innate sex differences in muscle: Xist, Kdm6a, Grb10, Oas2, Rps4x (higher, females) and Ddx3y, Kdm5d, Irx3, Wwp1, Aldh1a1, Cd24a (higher, males). These genes and those with the highest “sex‐biased” expression in our study do not contain estrogen‐response elements (exception, Greb1), but a subset are proposed to be regulated through androgen response elements. We hypothesize that innate muscle sexual dimorphism in mice and humans is triggered and then maintained by classic X inactivation (Xist, females) and Y activation (Ddx3y, males), with coincident engagement of X encoded (Kdm6a) and Y encoded (Kdm5d) demethylase epigenetic regulators that are complemented by modulation at some regions of the genome that respond to androgen.

β-Adrenergic stimulation differentially modulates different K(+) channels and thus fine-tunes cardiac action potential (AP) repolarization. However, it remains unclear how the proportion of I Ks, I Kr, and I K1 currents in the same cell would be altered by β-adrenergic stimulation, which would change the relative contribution of individual K(+) current to the total repolarization reserve. In this study, we used an innovative AP-clamp sequential dissection technique to directly record the dynamic I Ks, I Kr, and I K1 currents during the AP in guinea pig ventricular myocytes under physiologically relevant conditions. Our data provide quantitative measures of the magnitude and time course of I Ks, I Kr, and I K1 currents in the same cell under its own steady-state AP, in a physiological milieu, and with preserved Ca(2+) homeostasis. We found that isoproterenol treatment significantly enhanced I Ks, moderately increased I K1, but slightly decreased I Kr in a dose-dependent manner. The dominance pattern of the K(+) currents was I Kr > I K1 > I Ks at the control condition, but reversed to I Kr

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) – the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) – a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

Cells actively sense differences in topology, matrix elasticity and protein composition of the extracellular microenvironment and adapt their function and morphology. In this study, we focus on the cross-talk between matrix stiffness and protein coating density that regulates morphology and proliferation dynamics of single myocytes. For this, C2C12 myocytes were monitored on L-DOPA functionalized hydrogels of 22 different elasticity and fibronectin density compositions. Static images were recorded and statistically analyzed to determine morphological differences and to identify the optimized extracellular matrix (ECM). Using that information, selected ECMs were used to study the dynamics before and after cell proliferation by statistical comparison of distinct cell states. We observed a fibronectin-density-independent increase of the projected cell area until 12 kPa. Additionally, changes in fibronectin density led to an area that was optimum at about 2.6 μg/cm2, which was confirmed by independent F-actin analysis, revealing a maximum actin-filament-to-cell-area ratio of 7.5%. Proliferation evaluation showed an opposite correlation between cell spreading duration and speed to matrix elasticity and protein density, which did not affect cell-cycle duration. In summary, we identified an optimized ECM composition and found that independent matrix properties regulate distinct cell characteristics.

The objective of this work is to examine the contribution of late Na(+) current (INa,L) to the cardiac action potential (AP) and arrhythmogenic activities. In spite of the rapidly growing interest toward this current, there is no publication available on experimental recording of the dynamic INa,L current as it flows during AP with Ca(2+) cycling. Also unknown is how the current profile changes when the Ca(2+)-calmodulin dependent protein kinase II (CaMKII) signaling is altered, and how the current contributes to the development of arrhythmias. In this study we use an innovative AP-clamp Sequential Dissection technique to directly record the INa,L current during the AP with Ca(2+) cycling in the guinea pig ventricular myocytes. First, we found that the magnitude of INa,L measured under AP-clamp is substantially larger than earlier studies indicated. CaMKII inhibition using KN-93 significantly reduced the current. Second, we recorded INa,L together with IKs, IKr, and IK1 in the same cell to understand how these currents counterbalance to shape the AP morphology. We found that the amplitude and the total charge carried by INa,L exceed that of IKs. Third, facilitation of INa,L by Anemone toxin II prolonged APD and induced Ca(2+) oscillations that led to early and delayed afterdepolarizations and triggered APs; these arrhythmogenic activities were eliminated by buffering Ca(2+) with BAPTA. In conclusion, INa,L contributes a significantly large inward current that prolongs APD and unbalances the Ca(2+) homeostasis to cause arrhythmogenic APs.

Keywords: cellular proliferation; heart; hypertrophy; myocyte; regeneration EN cellular proliferation heart hypertrophy myocyte regeneration 1635 1638 4 04/22/21 20210420 NES 210420 Preclinical studies have suggested that transplanted human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) grafts expand because of proliferation.[1] This knowledge came from cell cycle activity measurements that cannot discriminate between cytokinesis or DNA synthesis associated with hypertrophy. By examining the generation of newly formed cardiomyocytes, rather than using proxies for cell proliferation, this study distinguished bona fide cardiomyocyte division versus incomplete cell cycle activation. 2 https://www.ahajournals.org/journal/circ References 1 Bargehr J, Ong LP, Colzani M, Davaapil H, Hofsteen P, Bhandari S, Gambardella L, Le Novère N, Iyer D, Sampaziotis F. Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration. Engrafted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Undergo Clonal Expansion In Vivo. [Extracted from the article]

Heat stress, via its effects on muscle intracellular Ca2+ concentrations ([Ca2+]i), has been invoked as a putative therapeutic countermeasure to type 1 diabetes-induced muscle atrophy. Using a circulation- and neurally intact in vivo muscle preparation, we tested the hypothesis that impaired muscle Ca2+ homeostasis in type 1 diabetic rats is due to attenuated heat stress tolerance mediated via transient receptor potential vanilloid 1 (TRPV1). Male Wistar rats were randomly assigned to one of the following four groups: 1) healthy control 30°C (CONT 30°C); 2) CONT 40°C; 3) diabetes 30°C (DIA 30°C); and 4) DIA 40°C. The temperature of 40°C was selected because it exceeds the TRPV1 activation threshold. Spinotrapezius muscles of Wistar rats were exteriorized in vivo and loaded with the fluorescent Ca2+ probe Fura-2 AM. [Ca2+]i was estimated over 20 min using fluorescence microscopy (340/380nm ratio) in quiescent muscle held at the required temperature, using a calibrated heat source applied to the ventral muscle surface. Western blotting was performed to determine the protein expression levels of TRPV1 in spinotrapezius muscle. After 20 min of heat stress, the CONT 40°C condition induced a 12.3 ± 5% [Ca2+]i (P < 0.05) elevation that was markedly absent in the DIA 40°C or other conditions. Thus, no significant differences were found among DIA 40°C, DIA 30°C, and CONT 30°C. TRPV1 protein expression was decreased by 42.0 ± 9% in DIA compared with CONT (P < 0.05) and, unlike CONT, heat stress did not increase TRPV1 phosphorylation. In conclusion, diabetes suppresses TRPV1 protein expression and function and inhibits the elevated myocyte [Ca2+]i evoked normally by heat stress. These results suggest that capsaicin or other therapeutic strategies to increase Ca2+ accumulation via TRPV1 might be more effective than hyperthermic therapy for type 1 diabetic patients. [ABSTRACT FROM AUTHOR]

Activation of AT1 (type 1 Ang) receptors stimulates cardiomyocyte hypertrophy in vitro. Accordingly, it has been suggested that regression of cardiac hypertrophy associated with renin-Ang system blockade is due to inhibition of cellular actions of Ang II in the heart, above and beyond their effects to reduce pressure overload. We generated 2 distinct mouse lines with cell-specific deletion of AT1A receptors, from cardiomyocytes. In the first line (C-SMKO), elimination of AT1A receptors was achieved using a heterologous Cre recombinase transgene under control of the Sm22 promoter, which expresses in cells of smooth muscle lineage including cardiomyocytes and vascular smooth muscle cells of conduit but not resistance vessels. The second line (R-SMKO) utilized a Cre transgene knocked-in to the Sm22 locus, which drives expression in cardiac myocytes and vascular smooth muscle cells in both conduit and resistance arteries. Thus, although both groups lack AT1 receptors in the cardiomyocytes, they are distinguished by presence (C-SMKO) or absence (R-SMKO) of peripheral vascular responses to Ang II. Similar to wild-types, chronic Ang II infusion caused hypertension and cardiac hypertrophy in C-SMKO mice, whereas both hypertension and cardiac hypertrophy were reduced in R-SMKOs. Thus, despite the absence of AT1A receptors in cardiomyocytes, C-SMKOs develop robust cardiac hypertrophy. By contrast, R-SMKOs developed identical levels of hypertrophy in response to pressure overload-induced by transverse aortic banding. Our findings suggest that direct activation of AT1 receptors in cardiac myocytes has minimal influence on cardiac hypertrophy induced by renin-Ang system activation or pressure overload. [ABSTRACT FROM AUTHOR]

Abstract The failure of adult cardiomyocytes to reproduce themselves to repair an injury results in the development of severe cardiac disability leading to death in many cases. The quest for an understanding of the inability of cardiac myocytes to repair an injury has been ongoing for decades with the identification of various factors which have a temporary effect on cell‐cycle activity. Fetal cardiac myocytes are continuously replicating until the time that the developing fetus reaches a stage of maturity sufficient for postnatal life around the time of birth. Recent reports of the ability for early neonatal mice and pigs to completely repair after the severe injury has stimulated further study of the regulators of the cardiomyocyte cell cycle to promote replication for the remuscularization of injured heart. In all mammals just before or after birth, single‐nucleated hyperplastically growing cardiomyocytes, 1X2N, undergo ≥1 additional DNA replications not followed by cytokinesis, resulting in cells with ≥2 nuclei or as in primates, multiple DNA replications (polyploidy) of 1 nucleus, 2X2(+)N or 1X4(+)N. All further growth of the heart is attributable to hypertrophy of cardiomyocytes. Animal studies ranging from zebrafish with 100% 1X2N cells in the adult to some strains of mice with up to 98% 2X2N cells in the adult and other species with variable ratios of 1X2N and 2X2N cells are reviewed relative to the time of conversion. Various structural, physiologic, metabolic, genetic, hormonal, oxygenation, and other factors that play a key role in the inability of post‐neonatal and adult myocytes to undergo additional cytokinesis are also reviewed.