Protective effects of 3, 4-dihydroxybenzoic acid on myocardial infarction induced by isoproterenol in rats.

Despite considerable advances in interventions and treatment, there is a high mortality rate in patients with myocardial infarction (MI). This is the first study to investigate the protective effects of 3, 4-dihydroxybenzoic acid against isoproterenol induced MI in rats. MI was induced by isoproterenol (100-mg/kg body weight) in rats. Then, rats were treated with 3, 4-dihydroxybenzoic acid (16-mg/kg body weight) for 2 weeks. Serum creatine kinase-MB, cardiac troponin-T, cardiac troponin-I, and heart thiobarbituric acid reactive substances were significantly (p < 0.05) increased and heart superoxide dismutase and catalase activities were significantly (p < 0.05) reduced in isoproterenol-induced myocardial infarcted rats. Isoproterenol induction significantly (p < 0.05) elevated the plasma homocysteine and serum high sensitivity-C-reactive protein levels. Furthermore, an enzyme-linked immunosorbent assay, reverse transcription polymerase chain study, and immunohistochemical (IHC) staining revealed significantly (p < 0.05) elevated levels and expression of serum/myocardial nuclear factor-κB, tumor necrosis factor-alpha, interleukin-1 beta, and Interleukin-6 and significantly (p < 0.05) reduced levels/expression of serum/myocardial interleukin-10 in myocardial infarcted rats. Nevertheless, isoproterenol-induced rats treated with 3, 4-dihydroxybenzoic acid considerably (p < 0.05) attenuated all the biochemical, molecular, and IHC parameters investigated and inhibited oxidative stress and inflammation and protected the heart, through its antioxidant and anti-inflammatory mechanisms.

Barbed arrow-like structure membrane with ultra-high rectification coefficient enables ultra-fast, highly-sensitive lateral-flow assay of cTnI.

Acute myocardial infarction (AMI) has become a public health disease threatening public life safety due to its high mortality. The lateral-flow assay (LFA) of a typical cardiac biomarker, troponin I (cTnI), is essential for the timely warnings of AMI. However, it is a challenge to achieve an ultra-fast and highly-sensitive assay for cTnI (hs-cTnI) using current LFA, due to the limited performance of chromatographic membranes. Here, we propose a barbed arrow-like structure membrane (BAS Mem), which enables the unidirectional, fast flow and low-residual of liquid. The liquid is rectified through the forces generated by the sidewalls of the barbed arrow-like grooves. The rectification coefficient of liquid flow on BAS Mem is 14.5 (highest to date). Using BAS Mem to replace the conventional chromatographic membrane, we prepare batches of lateral-flow strips and achieve LFA of cTnI within 240 s, with a limit of detection of 1.97 ng mL-1. The lateral-flow strips exhibit a specificity of 100%, a sensitivity of 93.3% in detecting 25 samples of suspected AMI patients. The lateral-flow strips show great performance in providing reliable results for clinical diagnosis, with the potential to provide early warnings for AMI.

Ckip-1 3'UTR alleviates prolonged sleep deprivation induced cardiac dysfunction by activating CaMKK2/AMPK/cTNI pathway.

Sleep deprivation (SD) has emerged as a critical concern impacting human health, leading to significant damage to the cardiovascular system. However, the underlying mechanisms are still unclear, and the development of targeted drugs is lagging. Here, we used mice to explore the effects of prolonged SD on cardiac structure and function. Echocardiography analysis revealed that cardiac function was significantly decreased in mice after five weeks of SD. Real-time quantitative PCR (RT-q-PCR) and Masson staining analysis showed that cardiac remodeling marker gene Anp (atrial natriuretic peptide) and fibrosis were increased, Elisa assay of serum showed that the levels of creatine kinase (CK), creatine kinase-MB (CK-MB), ANP, brain natriuretic peptide (BNP) and cardiac troponin T (cTn-T) were increased after SD, suggesting that cardiac remodeling and injury occurred. Transcript sequencing analysis indicated that genes involved in the regulation of calcium signaling pathway, dilated cardiomyopathy, and cardiac muscle contraction were changed after SD. Accordingly, Western blotting analysis demonstrated that the cardiac-contraction associated CaMKK2/AMPK/cTNI pathway was inhibited. Since our preliminary research has confirmed the vital role of Casein Kinase-2 -Interacting Protein-1 (CKIP-1, also known as PLEKHO1) in cardiac remodeling regulation. Here, we found the levels of the 3' untranslated region of Ckip-1 (Ckip-1 3'UTR) decreased, while the coding sequence of Ckip-1 (Ckip-1 CDS) remained unchanged after SD. Significantly, adenovirus-mediated overexpression of Ckip-1 3'UTR alleviated SD-induced cardiac dysfunction and remodeling by activating CaMKK2/AMPK/cTNI pathway, which proposed the therapeutic potential of Ckip-1 3'UTR in treating SD-induced heart disease.

Morphine timing-dependent modulation of TRPV1 phosphorylation correlates with differential morphine effects on myocardial ischemia/reperfusion injury.

Opioids are used for pain relief in patients suffering from acute myocardial ischemia or infarction. Clinical and laboratory studies demonstrate that morphine treated patients or the experimental animal model suffering acute myocardial ischemia and reperfusion, may worsen myocardial viability. As transient receptor potential vanilloid 1 (TRPV1) plays important roles in pain sensation and cardio-protection, we query whether opioids may exacerbate myocardial viability via interaction with TRPV1 activity in the pain relief. We found the co-expressions of TRPV1 and opioid µ, δ and κ receptors in adult rat cardiomyocytes. Intravenous injection of morphine (0.3 mg/kg) at 20 min after induction of myocardial ischemia, in the rat model of acute myocardial ischemia and reperfusion, induced significant reduction of phosphorylated TRPV1 (p-TRPV1) in the ventricular myocardium and increase in serum cardiac troponin I (cTnI), compared with the ischemia/reperfusion controls (all P < 0.05). The effects of morphine were completely reversed by selective opioid µ, δ and κ receptor antagonists. While significant upregulation of p-TRPV1 (P < 0.05) and improvement of ±dP/dt max (all P < 0.05) were detected in the animals giving the same dose of morphine before induction of myocardial ischemia. The changes in p-TRPV1 correlate with the alterations of cTnI (r = -0.5840, P = 0.0283) and ±dP/dt max (r = 0.8084, P = 0.0005 and r = -0.8133, P = 0.0004, respectively). The findings of this study may indicate that potentiation and attenuation of TRPV1 sensitivity correlate with the improvement of the cardiac performance and the aggravation of myocardial viability, respectively, by giving morphine before and during myocardial ischemia and reperfusion.

Histidine-Rich Glycoprotein Modulates the Toxic Effects of High-Dose Polyphosphate in Mice.

BACKGROUND: Polyphosphate (polyP), a procoagulant released from platelets, activates coagulation via the contact system and modulates cardiomyocyte viability. High-dose intravenous polyP is lethal in mice, presumably because of thrombosis. Previously, we showed that HRG (histidine-rich glycoprotein) binds polyP and attenuates its procoagulant effects. In this study, we investigated the mechanisms responsible for the lethality of intravenous polyP in mice and the impact of HRG on this process. METHODS: The survival of wild-type or HRG-deficient mice given intravenous synthetic or platelet-derived polyP in doses up to 50 mg/kg or saline was compared. To determine the contribution of thrombosis, the effect of FXII (factor XII) knockdown or enoxaparin on polyP-induced fibrin deposition in the lungs was examined. To assess cardiotoxicity, the ECG was continuously monitored, the levels of troponin I and the myocardial band of creatine kinase were quantified, and the viability of a cultured murine cardiomyocyte cell line exposed to polyP in the absence or presence of HRG was determined. RESULTS: In HRG-deficient mice, polyP was lethal at 30 mg/kg, whereas it was lethal in wild-type mice at 50 mg/kg. Although FXII knockdown or enoxaparin administration attenuated polyP-induced fibrin deposition in the lungs, neither affected mortality. PolyP induced dose-dependent ECG abnormalities, including heart block and ST-segment changes, and increased the levels of troponin and myocardial band of creatine kinase, effects that were more pronounced in HRG-deficient mice than in wild-type mice and were attenuated when HRG-deficient mice were given supplemental HRG. Consistent with its cardiotoxicity, polyP reduced the viability of cultured cardiomyocytes in a dose-dependent manner, an effect attenuated with supplemental HRG. CONCLUSIONS: High-dose intravenous polyP is cardiotoxic in mice, and HRG modulates this effect.

Infliximab Limits Injury in Myocardial Infarction.

BACKGROUND: The purpose of this study was to investigate a therapeutic approach targeting the inflammatory response and consequent remodeling from ischemic myocardial injury. METHODS AND RESULTS: Coronary thrombus aspirates were collected from patients at the time of ST-segment-elevation myocardial infarction and subjected to array-based proteome analysis. Clinically indistinguishable at myocardial infarction (MI), patients were stratified into vulnerable and resilient on the basis of 1-year left ventricular ejection fraction and death. Network analysis from coronary aspirates revealed prioritization of tumor necrosis factor-α signaling in patients with worse clinical outcomes. Infliximab, a tumor necrosis factor-α inhibitor, was infused intravenously at reperfusion in a porcine MI model to assess whether infliximab-mediated immune modulation impacts post-MI injury. At 3 days after MI (n=7), infliximab infusion increased proregenerative M2 macrophages in the myocardial border zone as quantified by immunofluorescence (24.1%±23.3% in infliximab versus 9.29%±8.7% in sham; P<0.01). Concomitantly, immunoassays of coronary sinus samples quantified lower troponin I levels (41.72±7.34 pg/mL versus 58.11±10.75 pg/mL; P<0.05) and secreted protein analysis revealed upregulation of injury-modifying interleukin-2, -4, -10, -12, and -18 cytokines in the infliximab-treated cohort. At 4 weeks (n=12), infliximab treatment resulted in significant protective influence, improving left ventricular ejection fraction (53.9%±5.4% versus 36.2%±5.3%; P<0.001) and reducing scar size (8.31%±10.9% versus 17.41%±12.5%; P<0.05). CONCLUSIONS: Profiling of coronary thrombus aspirates in patients with ST-segment-elevation MI revealed highest association for tumor necrosis factor-α in injury risk. Infliximab-mediated immune modulation offers an actionable pathway to alter MI-induced inflammatory response, preserving contractility and limiting adverse structural remodeling.

Baricitinib protects ICIs-related myocarditis by targeting JAK1/STAT3 to regulate Macrophage polarization.

PURPOSE: The emergence of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, but these drugs can also cause severe immune-related adverse effects (irAEs), including myocarditis. Researchers have become interested in exploring ways to mitigate this side effect, and one promising avenue is the use of baricitinib, a Janus kinase inhibitor known to have anti-inflammatory properties. This study aimed to examine the potential mechanism by which baricitinib in ICIs-related myocarditis. METHODS: To establish an ICIs-related myocarditis model, BALB/c mice were administered murine cardiac troponin I (cTnI) peptide and anti-mouse programmed death 1 (PD-1) antibodies. Subsequently, baricitinib was administered to the mice via intragastric administration. Echocardiography, HE staining, and Masson staining were performed to evaluate myocardial functions, inflammation, and fibrosis. Immunofluorescence was used to detect macrophages in the cardiac tissue of the mice.In vitro experiments utilized raw264.7 cells to induce macrophage polarization using anti-PD-1 antibodies. Different concentrations of baricitinib were applied to assess cell viability, and the release of pro-inflammatory cytokines was measured. The activation of the JAK1/STAT3 signaling pathway was evaluated through western blot analysis. RESULTS: Baricitinib demonstrated its ability to improve cardiac function and reduce cardiac inflammation, as well as fibrosis induced by ICIs. Mechanistically, baricitinib treatment promoted the polarization of macrophages towards the M2 phenotype. In vitro and in vivo experiments showed that anti-PD-1 promoted the release of inflammatory factors. However, treatment with baricitinib significantly inhibited the phosphorylation of JAK1 and STAT3. Additionally, the use of RO8191 reversed the effects of baricitinib, further confirming our findings. CONCLUSION: Baricitinib demonstrated its potential as a protective agent against ICIs-related myocarditis by modulating macrophage polarization. These findings provide a solid theoretical foundation for the development of future treatments for ICIs-related myocarditis.

miR-652-3p Suppressed the Protective Effects of Isoflurane Against Myocardial Injury in Hypoxia/Reoxygenation by Targeting ISL1.

This research is concentrated on investigating the role and mechanism of miR-652-3p in the protective effects of isoflurane (ISO) against myocardial ischemia-reperfusion (I/R) injury. H9c2 cells underwent pretreatment with varying concentrations of ISO, and subsequently, a hypoxia/reoxygenation (H/R) model was constructed. The levels of miR-652-3p, ISL LIM homeobox 1 (ISL1), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were evaluated through reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay was employed to investigate concentrations of myocardial injury markers, such as creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Cell counting kit-8 was used to evaluate cell viability, while flow cytometry was utilized to measure apoptosis. Additionally, a dual luciferase reporter assay was conducted to validate the targeting relationship between ISL1 and miR-652-3p. Herein, we confirmed that the level of miR-652-3p was gradually increased with prolonged hypoxia; nevertheless, this increase was suppressed by ISO pretreatment (P < 0.05). Additionally, ISO pretreatment prevented the decrease in cell viability, increase in apoptosis, and overproduction of IL-6, TNF-α, CK-MB, and cTnI induced by H/R (P < 0.05). However, the inhibitory effects of ISO were counteracted by the increased levels of miR-652-3p (P < 0.05). ISL1 is a potential target of miR-652-3p. H/R induction suppressed ISL1 levels compared to the control, but ISO treatment increased its expression (P < 0.05). Overexpression of ISL1 inhibited the elimination of the protective effect of ISO on myocardial damage induced by the elevation of miR-652-3p (P < 0.05). The findings of this research confirm that miR-652-3p attenuated the protective effect of ISO on cardiomyocytes in myocardial ischemia by targeting ISL1.

The Impact of Normobaric Hypoxia and Intermittent Hypoxic Training on Cardiac Biomarkers in Endurance Athletes: A Pilot Study.

This study explores the effects of normobaric hypoxia and intermittent hypoxic training (IHT) on the physiological condition of the cardiac muscle in swimmers. Hypoxia has been reported to elicit both beneficial and adverse changes in the cardiovascular system, but its impact on the myocardium during acute exercise and altitude/hypoxic training remains less understood. We aimed to determine how a single bout of intense interval exercise and a four-week period of high-intensity endurance training under normobaric hypoxia affect cardiac marker activity in swimmers. Sixteen young male swimmers were divided into two groups: one undergoing training in hypoxia and the other in normoxia. Cardiac markers, including troponin I and T (cTnI and cTnT), heart-type fatty acid-binding protein (H-FABP), creatine kinase-MB isoenzyme (CK-MB), and myoglobin (Mb), were analyzed to assess the myocardium's response. We found no significant differences in the physiological response of the cardiac muscle to intense physical exertion between hypoxia and normoxia. Four weeks of IHT did not alter the resting levels of cTnT, cTnI, and H-FABP, but it resulted in a noteworthy decrease in the resting concentration of CK-MB, suggesting enhanced cardiac muscle adaptation to exercise. In contrast, a reduction in resting Mb levels was observed in the control group training in normoxia. These findings suggest that IHT at moderate altitudes does not adversely affect cardiac muscle condition and may support cardiac muscle adaptation, affirming the safety and efficacy of IHT as a training method for athletes.

The molecular interaction of six single-stranded DNA aptamers to cardiac troponin I revealed by docking and molecular dynamics simulation.

Cardiac troponin I (cTnI) is a cardiac biomarker for diagnosing ischemic heart disease and acute myocardial infarction. Current biochemical assays use antibodies (Abs) due to their high specificity and sensitivity. However, there are some limitations, such as the high-cost production of Abs due to complex instruments, reagents, and steps; the variability of Abs quality from batch to batch; the low stability at high temperatures; and the difficulty of chemical modification. Aptamer overcomes the limitations of antibodies, such as relatively lower cost, high reproducibility, high stability, and ease of being chemically modified. Aptamers are three-dimensional architectures of single-stranded RNA or DNA that bind to targets such as proteins. Six aptamers (Tro1-Tro6) with higher binding affinity than an antibody have been identified, but the molecular interaction has not been studied. In this study, six DNA aptamers were modeled and docked to cTnI protein. Molecular docking revealed that the interaction between all aptamer and cTnI happened in the similar cTnI region. The interaction between aptamer and cTnI involved hydrophobic interaction, hydrogen bonds, π-cation interactions, π-stack interactions, and salt-bridge formation. The calculated binding energy of all complexes was negative, which means that the complex formation was thermodynamically favorable. The electrostatic energy term was the main driving force of the interaction between all aptamer and cTnI. This study could be used to predict the behavior of further modified aptamer to improve aptamer performance.

Decreased intranuclear cardiac troponin I impairs cardiac autophagy through FOS/ATG5 in ageing hearts.

In our previous study, intranuclear cardiac troponin I (cTnI) may function as a co-factor of Yin Yang 1(YY1). Here, we aimed to explore the role of intranuclear cTnI in ageing hearts. Nuclear translocation of cTnI was demonstrated using Western blot and immunofluorescence. The potential nuclear localization sequences (NLSs) of cTnI were predicted by a web server and then verified in 293T cells by putative NLS-eGFP-GST and NLS-mutant transfection. The ratio of Nuclear cTnI/ Total cTnI (Nu/T) decreased significantly in ageing hearts, accompanied with ATG5-decline-related impaired cardiac autophagy. RNA sequencing was performed in cTnI knockout hearts. The differential expressed genes (DEGs) were analysed by overlapping with YY1 ChIP-sequencing data. cTnI gain and loss experiments in vitro determined those filtered DEGs' expression levels. A strong correlation was found between expression patterns cTnI and FOS. Using ChIP-q-PCR, we demonstrated that specific binding DNA sequences of cTnI were enriched in the FOS promoter -299 to -157 region. It was further verified that pcDNA3.1 (-)-cTnI could increase the promoter activity of FOS by using luciferase report assay. At last, we found that FOS can regulate the ATG5 (autophagy-related gene 5) gene by using a luciferase report assay. Taken together, our results indicate that decreased intranuclear cTnI in ageing hearts may cause impaired cardiac autophagy through the FOS/ATG5 pathway.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Dapagliflozin prevents ERK activation and SGLT2-dependent endoglin upregulation in a mechanically provoked cardiac injury model.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are rapidly gaining ground in the treatment of heart failure (HF) with reduced ejection fraction (HFrEF) and acute myocardial infarction (AMI) by an unknown mechanism. Upregulation of Na+/H+ exchanger 1 (NHE1), SGLT1, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the diseased hearts was found to be attenuated by prolonged SGLT2i treatment. Unfortunately, dapagliflozin is not well understood as to how Na+/Ca2+ homeostasis is affected in cardiomyocytes. In this study, we aimed to investigate whether mechanical stretch in cardiomyocytes upregulate SGLT2, resulted to loss of Na+/Ca2+ homeostasis via ERK and eNOS signaling. AMI (+) and AMI (-) serum levels were estimated using ELISA assays of TGFß-1 or endoglin (CD105). Human cardiomyocyte cell line AC16 was subjected to different stresses: 5% mild and 25% aggressive, at 1 Hz for 24 h. Immunofluorescence assays were used to estimate troponin I, CD105, SGLT1/2, eNOSS633, and ERK1/2T202/Y204 levels was performed for 5% (mild), and 25% elongation for 24 h. AMI (+) serum showed increased TGFß1 and CD105 compared to AMI (-) patients. In consistent, troponin I, CD105, SGLT1/2, eNOSS633 and ERK1/2T202/Y204 were upregulated after 25% of 24 h cyclic stretch. Dapagliflozin addition caused SGLT2 inhibition, which significantly decreased troponin I, CD105, SGLT1/2, eNOSS633, and ERK1/2T202/Y204 under 25% cyclic stretching. In summary, SGLT2 may have sensed mechanical stretch in a way similar to cardiac overloading as in vivo. By blocking SGLT2 in stretched cardiomyocytes, the AMI biomarkers (CD105, troponin I and P-ERK) were decreased, potentially to rescue eNOS production to maintain normal cellular function. This discovery of CD105 and SGLT2 increase in mechanically stretched cardiomyocytes suggests that SGLT2 may conceive a novel role in direct or indirect sensing of mechanical stretch, prompting the possibility of an in vitro cardiac overloaded cell model, an alternative to animal heart model.

Impaired Relaxation in Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Pathogenic TNNI3 Mutation of Pediatric Restrictive Cardiomyopathy.

BACKGROUND: Restrictive cardiomyopathy (RCM) is characterized by impaired diastolic function with preserved ventricular contraction. Several pathogenic variants in sarcomere genes, including TNNI3, are reported to cause Ca2+ hypersensitivity in cardiomyocytes in overexpression models; however, the pathophysiology of induced pluripotent stem cell (iPSC)-derived cardiomyocytes specific to a patient with RCM remains unknown. METHODS AND RESULTS: We established an iPSC line from a pediatric patient with RCM and a heterozygous TNNI3 missense variant, c.508C>T (p.Arg170Trp; R170W). We conducted genome editing via CRISPR/Cas9 technology to establish an isogenic correction line harboring wild type TNNI3 as well as a homozygous TNNI3-R170W. iPSCs were then differentiated to cardiomyocytes to compare their cellular physiological, structural, and transcriptomic features. Cardiomyocytes differentiated from heterozygous and homozygous TNNI3-R170W iPSC lines demonstrated impaired diastolic function in cell motion analyses as compared with that in cardiomyocytes derived from isogenic-corrected iPSCs and 3 independent healthy iPSC lines. The intracellular Ca2+ oscillation and immunocytochemistry of troponin I were not significantly affected in RCM-cardiomyocytes with either heterozygous or homozygous TNNI3-R170W. Electron microscopy showed that the myofibril and mitochondrial structures appeared to be unaffected. RNA sequencing revealed that pathways associated with cardiac muscle development and contraction, extracellular matrix-receptor interaction, and transforming growth factor-ß were altered in RCM-iPSC-derived cardiomyocytes. CONCLUSIONS: Patient-specific iPSC-derived cardiomyocytes could effectively represent the diastolic dysfunction of RCM. Myofibril structures including troponin I remained unaffected in the monolayer culture system, although gene expression profiles associated with cardiac muscle functions were altered.

Practical Considerations for Delandistrogene Moxeparvovec Gene Therapy in Patients With Duchenne Muscular Dystrophy.

BACKGROUND: Delandistrogene moxeparvovec is a gene transfer therapy approved in the United States, United Arab Emirates, and Qatar for the treatment of ambulatory patients aged four through five years with a confirmed Duchenne muscular dystrophy (DMD)-causing mutation in the DMD gene. This therapy was developed to address the underlying cause of DMD through targeted skeletal, respiratory, and cardiac muscle expression of delandistrogene moxeparvovec micro-dystrophin, an engineered, functional dystrophin protein. METHODS: Drawing on clinical trial experience from Study 101 (NCT03375164), Study 102 (NCT03769116), and ENDEAVOR (Study 103; NCT04626674), we outline practical considerations for delandistrogene moxeparvovec treatment. RESULTS: Before infusion, the following are recommended: (1) screen for anti-adeno-associated virus rhesus isolate serotype 74 total binding antibody titers <1:400; (2) assess liver function, platelet count, and troponin-I; (3) ensure patients are up to date with vaccinations and avoid vaccine coadministration with infusion; (4) administer additional corticosteroids starting one day preinfusion (for patients already on corticosteroids); and (5) postpone dosing patients with any infection or acute liver disease until event resolution. Postinfusion, the following are recommended: (1) monitor liver function weekly (three months postinfusion) and, if indicated, continue until results are unremarkable; (2) monitor troponin-I levels weekly (first month postinfusion, continuing if indicated); (3) obtain platelet counts weekly (two weeks postinfusion), continuing if indicated; and (4) maintain the corticosteroid regimen for at least 60 days postinfusion, unless earlier tapering is indicated. CONCLUSIONS: Although the clinical safety profile of delandistrogene moxeparvovec has been consistent, monitorable, and manageable, these practical considerations may mitigate potential risks in a real-world clinical practice setting.

Modulation of Structure and Dynamics of Cardiac Troponin by Phosphorylation and Mutations Revealed by Molecular Dynamics Simulations.

Adrenaline acts on ß1 receptors in the heart muscle to enhance contractility, increase the heart rate, and increase the rate of relaxation (lusitropy) via activation of the cyclic AMP-dependent protein kinase, PKA. Phosphorylation of serines 22 and 23 in the N-terminal peptide of cardiac troponin I is responsible for lusitropy. Mutations associated with cardiomyopathy suppress the phosphorylation-dependent change. Key parts of troponin responsible for this modulatory system are disordered and cannot be resolved by conventional structural approaches. We performed all-atom molecular dynamics simulations (5 × 1.5 µs runs) of the troponin core (419 amino acids) in the presence of Ca2+ in the bisphosphorylated and unphosphorylated states for both wild-type troponin and the troponin C (cTnC) G159D mutant. PKA phosphorylation affects troponin dynamics. There is significant rigidification of the structure involving rearrangement of the cTnI(1-33)-cTnC interaction and changes in the distribution of the cTnC helix A/B angle, troponin I (cTnI) switch peptide (149-164) docking, and the angle between the regulatory head and ITC arm domains. The familial dilated cardiomyopathy cTnC G159D mutation whose Ca2+ sensitivity is not modulated by cTnI phosphorylation exhibits a structure inherently more rigid than the wild type, with phosphorylation reversing the direction of all metrics relative to the wild type.

Evaluation of Oxidative Status, Cytokines, Acute Phase Proteins and Cardiac Damage Markers in Sheep Naturally Infected with Babesia ovis.

PURPOSE: Babesiosis is a tick-borne disease caused by protozoon species in the Babesia genus of the Babesiadae family. The systemic inflammatory response induced by infection is considered to be an important feature of the pathophysiology of ovine babesiosis. In this study, it was aimed to determine serum oxidative status, levels of some cytokines, acute phase proteins and heart damage markers in babesiosis infection. MATERIALS AND METHODS: A sample of 40 sheep was used for this purpose, of which 20 were healthy and 20 were infected with Babesia ovis. Babesia infection was diagnosed from Giemsa-stained peripheral blood smears. Infection was also confirmed by the polymerase chain reaction (PCR). Sera from blood samples was tested for oxidative stress parameters (malondialdehyde [MDA], total antioxidant status [TAS], superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPx]), cytokines (interleukins IL-6, IL-1ß, IL-10, tumour necrosis factor α (TNF-α) and interferon-ϒ [IFN-ϒ]), acute-phase proteins (C-reactive protein [CRP], serum amyloid A [SAA] and haptoglobin [Hp]) and specific (troponin I [cTnI], creatine kinase-MB [CK-MB]) and nonspecific (lactate dehydrogenase [LDH], aspartate transaminase [AST]) cardiac damage markers. RESULTS: MDA, SOD, CAT, Hp, TAS, IL-6, IL-10, TNF-α, IL-1ß, INF-γ, AST, LDH, CK-MB mass and troponin I values were higher in the patient group than in the healthy group (P < 0.05). However, there was not found to be a statistical difference between the healthy and patient groups in terms of GPx, SAA and CRP values (P > 0.05). CONCLUSIONS: It can be stated that serum levels of oxidative stress, some acute phase proteins and cardiac damage markers may increase in naturally infected sheep with babesiosis.

Troponin-I-induced tropomyosin pivoting defines thin-filament function in relaxed and active muscle.

Regulation of the crossbridge cycle that drives muscle contraction involves a reconfiguration of the troponin-tropomyosin complex on actin filaments. By comparing atomic models of troponin-tropomyosin fitted to cryo-EM structures of inhibited and Ca2+-activated thin filaments, we find that tropomyosin pivots rather than rolls or slides across actin as generally thought. We propose that pivoting can account for the Ca2+ activation that initiates muscle contraction and then relaxation influenced by troponin-I (TnI). Tropomyosin is well-known to occupy either of three meta-stable configurations on actin, regulating access of myosin motorheads to their actin-binding sites and thus the crossbridge cycle. At low Ca2+ concentrations, tropomyosin is trapped by TnI in an inhibitory B-state that sterically blocks myosin binding to actin, leading to muscle relaxation. Ca2+ binding to TnC draws TnI away from tropomyosin, while tropomyosin moves to a C-state location over actin. This partially relieves the steric inhibition and allows weak binding of myosin heads to actin, which then transition to strong actin-bound configurations, fully activating the thin filament. Nevertheless, the reconfiguration that accompanies the initial Ca2+-sensitive B-state/C-state shift in troponin-tropomyosin on actin remains uncertain and at best is described by moderate-resolution cryo-EM reconstructions. Our recent computational studies indicate that intermolecular residue-to-residue salt-bridge linkage between actin and tropomyosin is indistinguishable in B- and C-state thin filament configurations. We show here that tropomyosin can pivot about relatively fixed points on actin to accompany B-state/C-state structural transitions. We argue that at low Ca2+ concentrations C-terminal TnI domains attract tropomyosin, causing it to bend and then pivot toward the TnI, thus blocking myosin binding and contraction.

Ursodeoxycholic acid induces sarcopenia associated with decreased protein synthesis and autophagic flux.

BACKGROUND: Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation. This event causes muscle mass loss and decreased strength and muscle function, a syndrome known as sarcopenia. Recently, our laboratory described secondary sarcopenia in a chronic cholestatic liver disease (CCLD) mouse model. Interestingly, the administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is an effective therapy for cholestatic hepatic alterations. However, the effect of UDCA on skeletal muscle mass and functionality has never been evaluated, nor the possible involved mechanisms. METHODS: We assessed the ability of UDCA to generate sarcopenia in C57BL6 mice and develop a sarcopenic-like phenotype in C2C12 myotubes and isolated muscle fibers. In mice, we measured muscle strength by a grip strength test, muscle mass by bioimpedance and mass for specific muscles, and physical function by a treadmill test. We also detected the fiber's diameter and content of sarcomeric proteins. In C2C12 myotubes and/or isolated muscle fibers, we determined the diameter and troponin I level to validate the cellular effect. Moreover, to evaluate possible mechanisms, we detected puromycin incorporation, p70S6K, and 4EBP1 to evaluate protein synthesis and ULK1, LC3 I, and II protein levels to determine autophagic flux. The mitophagosome-like structures were detected by transmission electron microscopy. RESULTS: UDCA induced sarcopenia in healthy mice, evidenced by decreased strength, muscle mass, and physical function, with a decline in the fiber's diameter and the troponin I protein levels. In the C2C12 myotubes, we observed that UDCA caused a reduction in the diameter and content of MHC, troponin I, puromycin incorporation, and phosphorylated forms of p70S6K and 4EBP1. Further, we detected increased levels of phosphorylated ULK1, the LC3II/LC3I ratio, and the number of mitophagosome-like structures. These data suggest that UDCA induces a sarcopenic-like phenotype with decreased protein synthesis and autophagic flux. CONCLUSIONS: Our results indicate that UDCA induces sarcopenia in mice and sarcopenic-like features in C2C12 myotubes and/or isolated muscle fibers concomitantly with decreased protein synthesis and alterations in autophagic flux.