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.

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.

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.

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.

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.

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.

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.

Exosomal microRNA­4516, microRNA­203 and SFRP1 are potential biomarkers of acute myocardial infarction.

Acute myocardial infarction (AMI) is a serious disease which threatens public health. Exosomes (exos) contain certain genetic information and are important communication vehicles between cells. In the present study, different exosomal microRNAs (miRs), which exhibit a notable association between expression levels in plasma and AMI were assessed to support the development of new diagnostic and clinical assessment markers of patients with AMI. In total, 93 individuals, including 31 healthy controls and 62 patients with AMI, were recruited for the present study. Data on age, blood pressure, glucose levels, lipid levels and coronary angiography images were collected from the enrolled individuals, and plasma samples were collected. Plasma exos were extracted and verified using ultracentrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting (WB). Exo­miR­4516 and exo­miR­203 in plasma exos were identified by exosomal miRNA sequencing analysis, reverse transcription­quantitative PCR was performed to detect the levels of exo­miR­4516 and exo­miR­203 in plasma exos, and ELISA was performed to detect the levels of secretory frizzled­related protein 1 (SFRP1) in samples. The correlation analysis between exo­miR­4516, exo­miR­203 and SFRP1 in plasma exos and AMI was presented as receiver operating characteristic curves (ROCs) of the SYNTAX score, cardiac troponin I (cTnI), low­density lipoprotein (LDL) and each indicator separately. Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed to predict relevant enrichment pathways. Exos were successfully isolated from plasma by ultracentrifugation, which was confirmed by TEM, NTA and WB. Exo­miR­4516, exo­miR­203 and SFRP1 levels in plasma were significantly higher in the AMI group compared with the healthy control group. ROCs demonstrated that exo­miR­4516, exo­miR­203 and SFRP1 levels had a high diagnostic efficiency in predicting AMI. Exo­miR­4516 was positively correlated with SYNTAX score, and plasma SFRP1 was positively correlated with plasma cTnI and LDL. In conclusion, the data demonstrated that exo­miR­4516, exo­miR­203 and SFRP1 levels could be used in combination to diagnose and assess the severity of AMI. The present study was retrospectively registered (TRN, NCT02123004).

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.

Differential changes in cyclic adenosine 3'-5' monophosphate (cAMP) effectors and major Ca2+ handling proteins during diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is associated with differential and time-specific regulation of ß-adrenergic receptors and cardiac cyclic nucleotide phosphodiesterases with consequences for total cyclic adenosine 3'-5' monophosphate (cAMP) levels. We aimed to investigate whether these changes are associated with downstream impairments in cAMP and Ca2+ signalling in a type 1 diabetes (T1D)-induced DCM model. T1D was induced in adult male rats by streptozotocin (65 mg/kg) injection. DCM was assessed by cardiac structural and molecular remodelling. We delineated sequential changes affecting the exchange protein (Epac1/2), cAMP-dependent protein kinase A (PKA) and Ca2+ /Calmodulin-dependent kinase II (CaMKII) at 4, 8 and 12 weeks following diabetes, by real-time quantitative PCR and western blot. Expression of Ca2+ ATPase pump (SERCA2a), phospholamban (PLB) and Troponin I (TnI) was also examined. Early upregulation of Epac1 transcripts was noted in diabetic hearts at Week 4, followed by increases in Epac2 mRNA, but not protein levels, at Week 12. Expression of PKA subunits (RI, RIIα and Cα) remained unchanged regardless of the disease stage, whereas CaMKII increased at Week 12 in DCM. Moreover, PLB transcripts were upregulated in diabetic hearts, whereas SERCA2a and TnI gene expression was unchanged irrespective of the disease evolution. PLB phosphorylation at threonine-17 was increased in DCM, whereas phosphorylation of both PLB at serine-16 and TnI at serine-23/24 was unchanged. We show for the first time differential and time-specific regulations in cardiac cAMP effectors and Ca2+ handling proteins, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Acyl ghrelin improves cardiac function in heart failure and increases fractional shortening in cardiomyocytes without calcium mobilization.

BACKGROUND AND AIMS: Ghrelin is an endogenous appetite-stimulating peptide hormone with potential cardiovascular benefits. Effects of acylated (activated) ghrelin were assessed in patients with heart failure and reduced ejection fraction (HFrEF) and in ex vivo mouse cardiomyocytes. METHODS AND RESULTS: In a randomized placebo-controlled double-blind trial, 31 patients with chronic HFrEF were randomized to synthetic human acyl ghrelin (0.1 µg/kg/min) or placebo intravenously over 120 min. The primary outcome was change in cardiac output (CO). Isolated mouse cardiomyocytes were treated with acyl ghrelin and fractional shortening and calcium transients were assessed. Acyl ghrelin but not placebo increased cardiac output (acyl ghrelin: 4.08 ± 1.15 to 5.23 ± 1.98 L/min; placebo: 4.26 ± 1.23 to 4.11 ± 1.99 L/min, P < 0.001). Acyl ghrelin caused a significant increase in stroke volume and nominal increases in left ventricular ejection fraction and segmental longitudinal strain and tricuspid annular plane systolic excursion. There were no effects on blood pressure, arrhythmias, or ischaemia. Heart rate decreased nominally (acyl ghrelin: 71 ± 11 to 67 ± 11 b.p.m.; placebo 69 ± 8 to 68 ± 10 b.p.m.). In cardiomyocytes, acyl ghrelin increased fractional shortening, did not affect cellular Ca2+ transients, and reduced troponin I phosphorylation. The increase in fractional shortening and reduction in troponin I phosphorylation was blocked by the acyl ghrelin antagonist D-Lys 3. CONCLUSION: In patients with HFrEF, acyl ghrelin increased cardiac output without causing hypotension, tachycardia, arrhythmia, or ischaemia. In isolated cardiomyocytes, acyl ghrelin increased contractility independently of preload and afterload and without Ca2+ mobilization, which may explain the lack of clinical side effects. Ghrelin treatment should be explored in additional randomized trials. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05277415.

Thin filament regulation of cardiac muscle power output: Implications for targets to improve human failing hearts.

The heart's pumping capacity is determined by myofilament power generation. Power is work done per unit time and measured as the product of force and velocity. At a sarcomere level, these contractile properties are linked to the number of attached cross-bridges and their cycling rate, and many signaling pathways modulate one or both factors. We previously showed that power is increased in rodent permeabilized cardiac myocytes following PKA-mediated phosphorylation of myofibrillar proteins. The current study found that that PKA increased power by ∼30% in permeabilized cardiac myocyte preparations (n = 8) from human failing hearts. To address myofilament molecular specificity of PKA effects, mechanical properties were measured in rat permeabilized slow-twitch skeletal muscle fibers before and after exchange of endogenous slow skeletal troponin with recombinant human Tn complex that contains cardiac (c)TnT, cTnC and either wildtype (WT) cTnI or pseudo-phosphorylated cTnI at sites Ser23/24Asp, Tyr26Glu, or the combinatorial Ser23/24Asp and Tyr26Glu. We found that cTnI Ser23/24Asp, Tyr26Glu, and combinatorial Ser23/24Asp and Tyr26Glu were sufficient to increase power by ∼20%. Next, we determined whether pseudo-phosphorylated cTnI at Ser23/24 was sufficient to increase power in cardiac myocytes from human failing hearts. Following cTn exchange that included cTnI Ser23/24Asp, power output increased ∼20% in permeabilized cardiac myocyte preparations (n = 6) from the left ventricle of human failing hearts. These results implicate cTnI N-terminal phosphorylation as a molecular regulator of myocyte power and could serve as a regional target for small molecule therapy to unmask myocyte power reserve capacity in human failing hearts.

In vivo assessment of inflammatory cytokines induced oxidative stress signalling, and troponin I gene dysregulation in cardiac tissue associated with chronic administration of boldenone and tramadol, alone or in combination.

INTRODUCTION: The risk of cardiotoxicity is associated with the use of anabolic-androgenic steroids and analgesics, several deaths were attributed to such medications. OBJECTIVES: This study investigates the effects of boldenone (BOLD) and tramadol (TRAM) alone or in combination on the heart. MATERIAL AND METHODS: Forty adult male rats were divided into four groups. Normal control group, BOLD (5 mg/kg, i.m.) per week, tramadol Hcl (TRAM) (20 mg/kg, i.p.) daily and a combination of BOLD (5 mg/kg) and TRAM (20 mg/kg), respectively for two months. Serum and cardiac tissue were extracted for determination of serum, aspartate aminotransferase (AST), creatine phosphokinase (CPK) and lipid profiles, tissue malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and histopathological examination. Troponin I gene expression was quantified in cardiac tissue using real-time polymerase chain reaction technique. RESULTS: Groups received BOLD and TRAM alone and in combination showed elevated serum biochemical parameters (AST, CPK) and deviations in lipid profiles, elevation in oxidative and inflammatory parameters (MDA, NO, TNF-α and IL-6), and decrease in GSH and SOD, up-regulated cardiac troponin I as well as distorted cardiac histopathological pictures. CONCLUSION: The current study elucidated the risk of administration of these drugs for sustained periods as well as the marked detrimental effects of using these drugs in combination.

The lack of Troponin I Ser-23/24 phosphorylation is detrimental to in vivo cardiac function and exacerbates cardiac disease.

Troponin I (TnI) is a key regulator of cardiac contraction and relaxation with TnI Ser-23/24 phosphorylation serving as a myofilament mechanism to modulate cardiac function. Basal cardiac TnI Ser-23/24 phosphorylation is high such that both increased and decreased TnI phosphorylation may modulate cardiac function. While the effects of increasing TnI Ser-23/24 phosphorylation on heart function are well established, the effects of decreasing TnI Ser-23/24 phosphorylation are not clear. To understand the in vivo role of decreased TnI Ser-23/24 phosphorylation, mice expressing TnI with Ser-23/24 mutated to alanine (TnI S23/24A) that lack the ability to be phosphorylated at these residues were subjected to echocardiography and pressure-volume hemodynamic measurements in the absence or presence of physiological (pacing increasing heart rate or adrenergic stimulation) or pathological (transverse aortic constriction (TAC)) stress. In the absence of pathological stress, the lack of TnI Ser-23/24 phosphorylation impaired systolic and diastolic function. TnI S23/24A mice also had an impaired systolic and diastolic response upon stimulation increased heart rate and an impaired adrenergic response upon dobutamine infusion. Following pathological cardiac stress induced by TAC, TnI S23/24A mice had a greater increase in ventricular mass, worse diastolic function, and impaired systolic and diastolic function upon increasing heart rate. These findings demonstrate that mice lacking the ability to phosphorylate TnI at Ser-23/24 have impaired in vivo systolic and diastolic cardiac function, a blunted cardiac reserve and a worse response to pathological stress supporting decreased TnI Ser23/24 phosphorylation is a modulator of these processes in vivo.