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Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg 9 and Arg 11 exert off-target effects on cellular Ca 2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca 2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca 2+ leak remained unchanged, indicating that protracted Ca 2+ removal from the cytosol stemmed from inhibition of the SR Ca 2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.

Background: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca 2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators., Methods: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays., Results: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding., Conclusions: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.

Background: In cardiac muscle, the ubiquitously expressed proteoglycan syndecan-4 is involved in the hypertrophic response to pressure overload. Protein kinase Akt signaling, which is known to regulate hypertrophy, has been found to be reduced in the cardiac muscle of exercised male syndecan-4 -/- mice. In contrast, we have recently found that pSer473-Akt signaling is elevated in the skeletal muscle ( tibialis anterior, TA) of female syndecan-4 -/- mice. To determine if the differences seen in Akt signaling are sex specific, we have presently investigated Akt signaling in the cardiac muscle of sedentary and exercised female syndecan-4 -/- mice. To get deeper insight into the female syndecan-4 -/- heart, alterations in cardiomyocyte size, a wide variety of different extracellular matrix components, well-known syndecan-4 binding partners and associated signaling pathways have also been investigated. Methods: Left ventricles (LVs) from sedentary and exercise trained female syndecan-4 -/- and WT mice were analyzed by immunoblotting and real-time PCR. Cardiomyocyte size and phosphorylated Ser473-Akt were analyzed in isolated adult cardiomyocytes from female syndecan-4 -/- and WT mice by confocal imaging. LV and skeletal muscle (TA) from sedentary male syndecan-4 -/- and WT mice were immunoblotted with Akt antibodies for comparison. Glucose levels were measured by a glucometer, and fasting blood serum insulin and C-peptide levels were measured by ELISA. Results: Compared to female WT hearts, sedentary female syndecan-4 -/- LV cardiomyocytes were smaller and hearts had higher levels of pSer473-Akt and its downstream target pSer9-GSK-3β. The pSer473-Akt inhibitory phosphatase PHLPP1/SCOP was lowered, which may be in response to the elevated serum insulin levels found in the female syndecan-4 -/- mice. We also observed lowered levels of pThr308-Akt/Akt and GLUT4 in the female syndecan-4 -/- heart and an increased LRP6 level after exercise. Otherwise, few alterations were found. The pThr308-Akt and pSer473-Akt levels were unaltered in the cardiac and skeletal muscles of sedentary male syndecan-4 -/- mice. Conclusion: Our data indicate smaller cardiomyocytes, an elevated insulin/pSer473-Akt/pSer9-GSK-3β signaling pathway, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels in the female syndecan-4 -/- heart. In contrast, cardiomyocyte size, and Akt signaling were unaltered in both cardiac and skeletal muscles from male syndecan-4 -/- mice, suggesting important sex differences., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor MEP declared a past collaboration with one of the authors CC., (Copyright © 2022 Støle, Lunde, Shen, Martinsen, Lunde, Li, Lockwood, Sjaastad, Louch, Aronsen, Christensen and Carlson.)

Background: The sarcoplasmic reticulum (SR) Ca 2+ -ATPase 2 (SERCA2) mediates Ca 2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca 2+ release from SR and triggers contraction. Ca 2+ /CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca 2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR., Methods: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology., Results: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca 2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca 2+ reuptake by SERCA2 and Ca 2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca 2+ -frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR., Conclusions: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Background and Objective: A number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca 2+ driven by sarco (endo)plasmic reticulum Ca 2+ -ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes)., Methods: In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively., Results: Both acute (4 ​h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase ( CPT ) 1B , pyruvate dehydrogenase kinase ( PDK ) 4, as well as increased AMP-activated protein kinase (AMPK) Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase ( SCD ) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA., Conclusion: Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiao-Rong Peng, Sven Göpel and Gavin O’ Mahony are employees of AstraZeneca. The other authors had no conflict of interest to disclose., (© 2021 The Authors.)

Subjects with functionally univentricular circulation who have completed staged single ventricle palliation, with the final stage culminating in the Fontan procedure, are often living into adulthood. However, high morbidity and mortality remain prevalent in these patients, as diastolic and systolic dysfunction of the single systemic ventricle are linked to Fontan circulatory failure. We presently investigated the effects of probenecid in post-Fontan patients. Used for decades for the treatment of gout, probenecid has been shown in recent years to positively influence cardiac function via effects on the Transient Receptor Potential Vanilloid 2 (TRPV2) channel in cardiomyocytes. Indeed, we observed that probenecid improved cardiac function and exercise performance in patients with a functionally univentricular circulation. This was consistent with our findings from a retrospective cohort of patients with single ventricle physiology where TRPV2 expression was increased. Experiments in isolated cardiomyocytes associated these positive actions to augmentation of diastolic calcium homeostasis.

Background: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca 2+ /calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia., Methods: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca 2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes., Results: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca 2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca 2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca 2+ current., Conclusions: SN production is upregulated in conditions with cardiomyocyte Ca 2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca 2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca 2+ sparks (Ca 2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented 'silent' Ca 2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca 2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca 2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca 2+ leak and slows Ca 2+ release kinetics, leading to weakened contraction in this disease., Competing Interests: TK, Jv, NM, PL, MF, JA, EN, AC, IS, OS, AE, GL, WL No competing interests declared, (© 2018, Kolstad et al.)

Protein O-GlcNAcylation has emerged as an important intracellular signaling system with both physiological and pathophysiological functions, but the role of protein O-GlcNAcylation in skeletal muscle remains elusive. In this study, we tested the hypothesis that protein O-GlcNAcylation is a dynamic signaling system in skeletal muscle in exercise and disease. Immunoblotting showed different protein O-GlcNAcylation pattern in the prototypical slow twitch soleus muscle compared to fast twitch EDL from rats, with greater O-GlcNAcylation level in soleus associated with higher expression of the modulating enzymes O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine fructose-6-phosphate amidotransferase isoforms 1 and 2 (GFAT1, GFAT2). Six weeks of exercise training by treadmill running, but not an acute exercise bout, increased protein O-GlcNAcylation in rat soleus and EDL There was a striking increase in O-GlcNAcylation of cytoplasmic proteins ~50 kDa in size that judged from mass spectrometry analysis could represent O-GlcNAcylation of one or more key metabolic enzymes. This suggests that cytoplasmic O-GlcNAc signaling is part of the training response. In contrast to exercise training, postinfarction heart failure (HF) in rats and humans did not affect skeletal muscle O-GlcNAcylation level, indicating that aberrant O-GlcNAcylation cannot explain the skeletal muscle dysfunction in HF Human skeletal muscle displayed extensive protein O-GlcNAcylation that by large mirrored the fiber-type-related O-GlcNAcylation pattern in rats, suggesting O-GlcNAcylation as an important signaling system also in human skeletal muscle., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims: In pressure overload, left ventricular (LV) dilatation is a key step in transition to heart failure (HF). We recently found that collagen VIII (colVIII), a non-fibrillar collagen and extracellular matrix constituent, was reduced in hearts of mice with HF and correlated to degree of dilatation. A reduction in colVIII might be involved in LV dilatation, and we here examined the role of reduced colVIII in pressure overload-induced remodelling using colVIII knock-out (col8KO) mice., Methods and Results: Col8KO mice exhibited increased mortality 3-9 days after aortic banding (AB) and increased LV dilatation from day one after AB, compared with wild type (WT). LV dilatation remained increased over 56 days. Forty-eight hours after AB, LV expression of main structural collagens (I and III) was three-fold increased in WT mice, but these collagens were unaltered in the LV of col8KO mice together with reduced expression of the pro-fibrotic cytokine TGF-β, SMAD2 signalling, and the myofibroblast markers Pxn, α-SMA, and SM22. Six weeks after AB, LV collagen mRNA expression and protein were increased in col8KO mice, although less pronounced than in WT. In vitro, neonatal cardiac fibroblasts from col8KO mice showed lower expression of TGF-β, Pxn, α-SMA, and SM22 and reduced migratory ability possibly due to increased RhoA activity and reduced MMP2 expression. Stimulation with recombinant colVIIIα1 increased TGF-β expression and fibroblast migration., Conclusion: Lack of colVIII reduces myofibroblast differentiation and fibrosis and promotes early mortality and LV dilatation in response to pressure overload in mice., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Myosin light chain 2 (MLC2) is a small protein in the myosin complex, regulating muscle contractile function by modulating Ca(2+) sensitivity of myofilaments. MLC2 can be modified by phosphorylation and O-GlcNAcylation, two reversible and dynamic posttranslational modifications. The slow isoform of MLC2 (sMLC2) is dephosphorylated in soleus muscle during in situ loaded shortening contractions, which correlates with reduction in shortening capacity. Here, we hypothesize that exhausting in vivo treadmill running induces dephosphorylation of MLC2 in slow twitch soleus, but not in fast twitch EDL muscle, and that there are reciprocal changes in MLC2 O-GlcNAcylation. At rest, both phosphorylation and O-GlcNAcylation of MLC2 were lower in slow than fast twitch muscles. One bout of exhausting treadmill running induced dephosphorylation of sMLC2 in soleus, paralleled by reduced levels of the kinase MLCK2 associated to myofilaments, suggesting that the acute reduction in phosphorylation is mediated by dissociation of MLCK2 from myofilaments. O-GlcNAcylation of MLC2 did not change significantly, and seems of limited importance in the regulation of MLC2 phosphorylation during in vivo running. After 6 weeks of treadmill running, the dephosphorylation of sMLC2 persisted in soleus along with reduction in MLCK2 both in myofilament- and total protein fraction. In EDL on the contrary, phosphorylation of MLC2 was not altered after one exercise bout or after 6 weeks of treadmill running. Thus, in contrast to fast twitch muscle, MLC2 dephosphorylation occurs in slow twitch muscle during in vivo exercise and may be linked to reduced myofilament-associated MLCK2 and reduced shortening capacity., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Cannabidiol (CBD) is abundant in the Cannabis sativa plant and exhibits complex immunomodulatory, anxiolytic, antioxidant, and antiepileptic properties. Several studies suggest that CBD could be used for different purposes in alcohol use disorder (AUD) and alcohol-related injuries to the brain and the liver. In this study, we focused on analyzing transcriptional alterations in human dermal fibroblasts (HDFs) cell line challenged simultaneously with ethanol and CBD as an ethanol-protective agent. We aimed to expose the genes and pathways responsible for at least some of the CBD effects in those cells that can be related to the AUD. Transcriptome analysis was performed using HDFs cell line that expresses both cannabinoid receptors and can metabolize ethanol through alcohol dehydrogenase activity. Fibroblasts are also responsible for the progression of liver fibrosis, a common comorbidity in AUD. With the use of a cellular test, we found that CBD at the lowest applied concentration (0.75 μM) was able to stimulate depressed metabolism and reduce the level of apoptosis of cells treated with different concentrations of ethanol to the level observed in the control cells. Similar observations were made at the transcriptome level, in which cells treated with ethanol and CBD had similar expression profiles to the control cells. CBD also affects several genes connected with extracellular matrix formation (especially its collagen constituent), which can have potential implications for, e.g., fibrosis process. [ABSTRACT FROM AUTHOR]

Background: The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear. Results: We show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte. Conclusions: We discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Thyroid hormone dysfunction is frequently observed in patients with chronic illnesses including heart failure, which increases the risk of adverse events. This study examined the effects of thyroid hormones (THs) on cardiac transverse-tubule (TT) integrity, Ca2+ sparks, and nanoscale organization of ion channels in excitation-contraction (EC) coupling, including L-type calcium channel (CaV1.2), ryanodine receptor type 2 (RyR2), and junctophilin-2 (Jph2). TH deficiency was established in adult female rats by propyl-thiouracil (PTU) ingestion for 8 wk; followed by randomization to continued PTU without or with oral triiodo- l -thyronine (T3; 10 µg/kg/day) for an additional 2 wk (PTU + T3). Confocal microscopy of isolated cardiomyocytes (CMs) showed significant misalignment of TTs and increased Ca2+ sparks in thyroid-deficient CMs. Density-based spatial clustering of applications with noise (DBSCAN) analysis of stochastic optical reconstruction microscopy (STORM) images showed decreased (P < 0.0001) RyR2 cluster number per cell area in PTU CMs compared with euthyroid (EU) control myocytes, and this was normalized by T3 treatment. CaV1.2 channels and Jph2 localized within a 210 nm radius of the RyR2 clusters were significantly reduced in PTU myocytes, and these values were increased with T3 treatment. A significant percentage of the RyR2 clusters in the PTU myocytes had neither CaV1.2 nor Jph2, suggesting fewer functional clusters in EC coupling. Nearest neighbor distances between RyR2 clusters were greater (P < 0.001) in PTU cells compared with EU- and T3-treated CMs that correspond to disarray of TTs at the sarcomere z-discs. These results support a regulatory role of T3 in the nanoscale organization of RyR2 clusters and colocalization of CaV1.2 and Jph2 in optimizing EC coupling. NEW & NOTEWORTHY: Thyroid hormone (TH) dysfunction exacerbates preexisting heart conditions leading to an increased risk of premature morbidity/mortality. Triiodo- l -thyronine (T3) optimizes cardiac excitation-contraction (EC) coupling by maintaining myocardial T-tubule (TT) structures and organization of calcium ion channels. Single-molecule localization microscopy shows T3 effects on the clustering of ryanodine receptors (RyR2) with colocalization of L-type calcium channels (CaV1.2) and junctophilin-2 (Jph2) at TT-SR structures. Heart disease with subclinical hypothyroidism/low T3 syndrome may benefit from TH treatment. [ABSTRACT FROM AUTHOR]

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H+) act to directly and indirectly (via decreased Ca2+ activation) inhibit myosin's force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases. [ABSTRACT FROM AUTHOR]

Thyroid hormones modulate the cardiovascular system. However, the effects of subclinical thyroid dysfunction and euthyroidism on cardiac function remain unclear. We investigated the association between left ventricular (LV) diastolic dysfunction and subclinical thyroid dysfunction or thyroid hormones within the reference range. This cross-sectional study included 26,289 participants (22,197 euthyroid, 3,671 with subclinical hypothyroidism, and 421 with subclinical thyrotoxicosis) who underwent regular health check-ups in the Republic of Korea. Individuals with thyroid stimulating hormone (TSH) levels > 4.2 µIU/mL and normal free thyroxine (FT4, 0.78–1.85 ng/dL) and triiodothyronine (T3, 76–190 ng/dL) levels were defined as having subclinical hypothyroidism. Individuals with serum TSH levels < 0.4 µIU/mL and normal FT4 and T3 levels were defined as having subclinical thyrotoxicosis. The cardiac structure and function were evaluated using echocardiography. LV diastolic dysfunction with normal ejection fraction (EF) was defined as follows: EF of > 50% and (a) E/e' ratio > 15, or (b) E/e' ratio of 8–15 and left atrial volume index ≥ 34 mL/m2. Subclinical hypothyroidism was significantly associated with cardiac indices regarding LV diastolic dysfunction. The odds of having LV diastolic dysfunction was also increased in participants with subclinical hypothyroidism (adjusted odds ratio [AOR] 1.36, 95% confidence interval [CI], 1.01–1.89) compared to euthyroid participants. Subclinical thyrotoxicosis was not associated with LV diastolic dysfunction. Among the thyroid hormones, only serum T3 was significantly and inversely associated with LV diastolic dysfunction even within the normal range. Subclinical hypothyroidism was significantly associated with LV diastolic dysfunction, whereas subclinical thyrotoxicosis was not. Serum T3 is a relatively important contributor to LV diastolic dysfunction compared to TSH or FT4. [ABSTRACT FROM AUTHOR]

Objetive: this study was to determine the relationship between acute febrile illness and bacterial pathogens with zoonotic potential that cause emerging and re-emerging diseases in a central-eastern region of Peru. Results: Out of the 279 samples analyzed, 23 (8.2%) tested positive for infection by Rickettsia spp., while a total of 15 (5.4%) tested positive for Leptospira spp. Women had a higher frequency of infection by Rickettsia spp., with 13 cases (53.3%), while men had a higher frequency of infection by Leptospira spp., with 10 cases (66.7%). The most frequently reported general symptom was headache, with 100.0% (n = 23) of patients with Rickettsia (+) and 86.7% (n = 13) of patients with Leptospira (+) experiencing it. Arthralgia was the second most frequent symptom, reported by 95.6% (n = 22) and 60% (n = 9) of patients with Rickettsia (+) and Leptospira (+), respectively. Myalgia was reported by 91.3% (n = 21) and 66.7% (n = 10) of patients with Rickettsia (+) and Leptospira (+), respectively. Retroocular pain, low back pain, and skin rash were also present, but less frequently. Among the positives, no manifestation of bleeding was recorded, although only one positive case for Leptospira spp. presented a decrease in the number of platelets. [ABSTRACT FROM AUTHOR]

The goal of the present study was to characterize changes in mitochondrial respiration in the maternal heart during pregnancy and after birth. Timed pregnancy studies were performed in 12-wk-old female FVB/NJ mice, and cardiac mitochondria were isolated from the following groups of mice: nonpregnant (NP), midpregnancy (MP), late pregnancy (LP), and 1-wk postbirth (PB). Similar to our previous studies, we observed increased heart size during all stages of pregnancy (e.g., MP and LP) and postbirth (e.g., PB) compared with NP mice. Differential cardiac gene and protein expression analyses revealed changes in several mitochondrial transcripts at LP and PB, including several mitochondrial complex subunits and members of the Slc family, important for mitochondrial substrate transport. Respirometry revealed that pyruvate- and glutamate-supported state 3 respiration was significantly higher in PB vs. LP mitochondria, with respiratory control ratio (RCR) values higher in PB mitochondria. In addition, we found that PB mitochondria respired more avidly when given 3-hydroxybutyrate (3-OHB) than mitochondria from NP, MP, and LP hearts, with no differences in RCR. These increases in respiration in PB hearts occurred independent of changes in mitochondrial yield but were associated with higher abundance of 3-hydroxybutyrate dehydrogenase 1. Collectively, these findings suggest that, after birth, maternal cardiac mitochondria have an increased capacity to use 3-OHB, pyruvate, and glutamate as energy sources; however, increases in mitochondrial efficiency in the postpartum heart appear limited to carbohydrate and amino acid metabolism. NEW & NOTEWORTHY Few studies have detailed the physiological adaptations that occur in the maternal heart. We and others have shown that pregnancy-induced cardiac growth is associated with significant changes in cardiac metabolism. Here, we examined mitochondrial respiration and substrate preference in isolated mitochondria from the maternal heart. We show that following birth, cardiac mitochondria are "primed" to respire on carbohydrate, amino acid, and ketone bodies. However, heightened respiratory efficiency is observed only with carbohydrate and amino acid sources. These results suggest that significant changes in mitochondrial respiration occur in the maternal heart in the postpartum period. [ABSTRACT FROM AUTHOR]

Background and aims: Hyperthyroidism is an endocrine disease with multiple etiologies and manifestations. Heart failure (HF) is a common, costly, and deadly medical condition in clinical practice. Numerous studies have suggested that abnormal thyroid function can induce or aggravate the development of heart disease. However, no study has demonstrated a causal relationship between hyperthyroidism and heart failure. Therefore, the purpose of this study was to explore the causal link between hyperthyroidism and HF. Methods: Summary data for genetically predicted hyperthyroidism were obtained from a genetic association study. The data examined for genetically determined all-cause heart failure came from 218,208 individuals from the FinnGen Consortium. Two-sample Mendelian randomization (MR) analysis was used to estimate the causal link between hyperthyroidism and heart failure. Statistical analyses were conducted using the inverse variance-weighted, weighted median, simple median, weighted mode, MR-PRESSO (number of distribution = 5000), MR-Egger, and leave-one-out. Results: The results of the inverse-variance weighted analysis indicated a causal association between hyperthyroidism and an increased risk of all-cause heart failure (IVW: b=0.048, OR=1.049, 95%CI: [1.013 to 1.087], P=0.007). Similarly, the weighted median approach demonstrated a positive correlation between hyperthyroidism and all-cause heart failure (OR=1.049, [95% CI, 1.001-1.100]; P=0.044). Additionally, no horizontal pleiotropy or heterogeneity was observed. The leave-one-out analysis revealed that the majority of the SNP-driven associations were not influenced by a single genetic marker. Conclusion: Our study observed a causal relationship between hyperthyroidism and all-cause heart failure. Hyperthyroidism may associate with heart failure genetically. [ABSTRACT FROM AUTHOR]

It remains unclear why some patients develop heart failure without evidence of structural damage. One theory relates to impaired myocardial energetics and ventricular-arterial decoupling as the heart works against adverse mechanical load. In this original study, we propose the novel concept of myocardial fatigue to capture this phenomenon and aim to investigate this using human cardiomyocytes subjected to a modern work-loop contractility model that closely mimics in vivo cardiac cycles. This proof-of-concept study (NCT04899635) will use human myocardial tissue samples from patients undergoing cardiac surgery to develop a reproducible protocol to isolate robust calcium-tolerant cardiomyocytes. Thereafter, work-loop contractility experiments will be performed over a range of preload, afterload and cycle frequency as a function of time to elicit any reversible reduction in contractile performance (i.e. fatigue). This will provide novel insight into mechanisms behind heart failure and myocardial recovery and serve as a valuable research platform in translational cardiovascular research. [ABSTRACT FROM AUTHOR]

Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both cyclic nucleotides are critical secondary messengers in the neurohormonal regulation in the cardiovascular system. PDEs precisely control spatiotemporal subcellular distribution of cyclic nucleotides in a cell- and tissue-specific manner, playing critical roles in physiological responses to hormone stimulation in the heart and vessels. Dysregulation of PDEs has been linked to the development of several cardiovascular diseases, such as hypertension, aneurysm, atherosclerosis, arrhythmia, and heart failure. Targeting these enzymes has been proven effective in treating cardiovascular diseases and is an attractive and promising strategy for the development of new drugs. In this review, we discuss the current understanding of the complex regulation of PDE isoforms in cardiovascular function, highlighting the divergent and even opposing roles of PDE isoforms in different pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Lactate is traditionally recognized as a by-product of anaerobic metabolism. However, lactate is a preferred oxidative substrate for stressed myocardium. Exogenous lactate infusion increases cardiac output (CO). The exact mechanism underlying this mechanism has yet to be elucidated. The aim of this study was to investigate the cardiovascular mechanisms underlying the acute haemodynamic effects of exogenous lactate infusion in an experimental model of human-sized pigs. Methods: In this randomised, blinded crossover study in eight 60-kg-pigs, the pigs received infusions with one molar sodium lactate and a control infusion of tonicity matched hypertonic saline in random order. We measured CO and pulmonary pressures using a pulmonary artery catheter. A pressure–volume admittance catheter in the left ventricle was used to measure contractility, afterload, preload and work-related parameters. Results: Lactate infusion increased circulating lactate levels by 9.9 mmol/L (95% confidence interval (CI) 9.1 to 11.0) and CO by 2.0 L/min (95% CI 1.2 to 2.7). Afterload decreased as arterial elastance fell by -1.0 mmHg/ml (95% CI -2.0 to -0.1) and systemic vascular resistance decreased by -548 dynes/s/cm5 (95% CI -261 to -835). Mixed venous saturation increased by 11 percentage points (95% CI 6 to 16), whereas ejection fraction increased by 16.0 percentage points (95% CI 1.1 to 32.0) and heart rate by 21 bpm (95% CI 8 to 33). No significant changes in contractility nor preload were observed. Conclusion: Lactate infusion increased cardiac output by increasing heart rate and lowering afterload. No differences were observed in left ventricular contractility or preload. Lactate holds potential as a treatment in situations with lowered CO and should be investigated in future clinical studies. [ABSTRACT FROM AUTHOR]

Introduction: Thyroid hormones (THs) are known to have various effects on the cardiovascular system. However, the impact of TH levels on preexisting cardiac diseases is still unclear. Pressure overload due to arterial hypertension or aortic stenosis and aging are major risk factors for the development of structural and functional abnormalities and subsequent heart failure. Here, we assessed the sensitivity to altered TH levels in aged mice with maladaptive cardiac hypertrophy and cardiac dysfunction induced by transverse aortic constriction (TAC). Methods: Mice at the age of 12 months underwent TAC and received T4 or antithyroid medication in drinking water over the course of 4 weeks after induction of left ventricular pressure overload. Results: T4 excess or deprivation in older mice had no or only very little impact on cardiac function (fractional shortening), cardiac remodeling (cardiac wall thickness, heart weight, cardiomyocyte size, apoptosis, and interstitial fibrosis), and mortality. This is surprising because T4 excess or deprivation had significantly changed the outcome after TAC in young 8-week-old mice. Comparing the gene expression of deiodinases (Dio) 2 and 3 and TH receptor alpha (TRα) 1 and the dominant-negative acting isoform TRα2 between young and aged mice revealed that aged mice exhibited a higher expression of TRα2 and Dio3, while expression of Dio2 was reduced compared with young mice. These changes in Dio2 and 3 expressions might lead to reduced TH availability in the hearts of 12- month-old mice accompanied by reduced TRα action due to higher TRα2. Discussion: In summary, our study shows that low and high TH availability have little impact on cardiac function and remodeling in older mice with preexisting pressure-induced cardiac damage. This observation seems to be the result of an altered expression of deiodinases and TRα isoforms, thus suggesting that even though cardiovascular risk is increasing with age, the response to TH stress may be dampened in certain conditions. [ABSTRACT FROM AUTHOR]

Background/objectives: Myocardial infarction (MI) frequently leads to cardiac remodeling and failure with impaired life quality, playing an important role in cardiovascular deaths. Although physical exercise is a well-recognized effective non-pharmacological therapy for cardiovascular diseases, the effects of strength training (ST) on the structural and functional aspects of cardiac remodeling need to be further documented. In this study, we aimed to investigate the role of a linear block ST protocol in the rat model of MI. Methods and results: After 6 weeks of MI induction or sham surgery, male adult rats performed ST for the following 12 weeks. The ladder-based ST program was organized in three mesocycles of 4 weeks, with one load increment for each block according to the maximal carrying load test. After 12 weeks, the infarcted-trained rats exhibited an increase in performance, associated with reduced cardiac hypertrophy and pulmonary congestion compared with the untrained group. Despite not changing MI size, the ST program partially prevented cardiac dilatation and ventricular dysfunction assessed by echocardiography and hemodynamics, and interstitial fibrosis evaluated by histology. In addition, isolated cardiac muscles from infarcted-trained rats had improved contractility parameters in a steady state, and in response to calcium or stimuli pauses. Conclusions: The ST in infarcted rats increased the capacity to carry mass, associated with attenuation of cardiac remodeling and pulmonary congestion with improving cardiac function that could be attributed, at least in part, to the improvement of myocardial contractility. [ABSTRACT FROM AUTHOR]

Background: Right ventricle failure (RVF) is a progressive heart disease that has yet to be fully understood at the molecular level. Elevated M-type pyruvate kinase 2 (PKM2) tetramerization alleviates heart failure, but detailed molecular mechanisms remain unclear. Objective: We observed changes in PKM2 tetramerization levels during the progression of right heart failure and in vitro cardiomyocyte hypertrophy and explored the causal relationship between altered PKM2 tetramerization and the imbalance of redox homeostasis in cardiomyocytes, as well as its underlying mechanisms. Ultimately, our goal was to propose rational intervention strategies for the treatment of RVF. Method: We established RVF in Sprague Dawley (SD) rats by intraperitoneal injection of monocrotaline (MCT). The pulmonary artery pressure and right heart function of rats were assessed using transthoracic echocardiography combined with right heart catheterization. TEPP-46 was used both in vivo and in vitro to promote PKM2 tetramerization. Results: We observed that oxidative stress and mitochondrial disorganization were associated with increased apoptosis in the right ventricular tissue of RVF rats. Quantitative proteomics revealed that PKM2 was upregulated during RVF and negatively correlated with the cardiac function. Facilitating PKM2 tetramerization promoted mitochondrial network formation and alleviated oxidative stress and apoptosis during cardiomyocyte hypertrophy. Moreover, enhancing PKM2 tetramer formation improved cardiac mitochondrial morphology, mitigated oxidative stress and alleviated heart failure. Conclusion: Disruption of PKM2 tetramerization contributed to RVF by inducing mitochondrial fragmentation, accumulating ROS, and finally promoted the progression of cardiomyocyte apoptosis. Facilitating PKM2 tetramerization holds potential as a promising therapeutic approach for RVF. [ABSTRACT FROM AUTHOR]

This article presents the findings of a survey conducted in a dermatology department in southern Rajasthan, India. The study aimed to assess the prescription patterns and drug utilization among outdoor patients. The results revealed that fungal infection and dermatitis were the most common disorders, and antihistamines and antifungal drugs were the most frequently prescribed medications. The study concluded that this information could be used to enhance the quality of prescriptions and promote cost-effective treatment. Additionally, the study examined the distribution of patients based on age groups and diseases, finding that the highest number of patients fell within the age groups of 21-30 years and 31-40 years. Fungal infection was the most prevalent disease, followed by dermatitis and viral infection. The majority of patients were prescribed topical medications, with tablets being the most common dosage form. The study suggested that drug utilization studies could encourage rational and economical drug prescribing. [Extracted from the article]

Ca 2+ homeostasis is essential for cell function and survival. As such, the cytosolic Ca 2+ concentration is tightly controlled by a wide number of specialized Ca 2+ handling proteins. One among them is the Na + -Ca 2+ exchanger (NCX), a ubiquitous plasma membrane transporter that exploits the electrochemical gradient of Na + to drive Ca 2+ out of the cell, against its concentration gradient. In this critical role, this secondary transporter guides vital physiological processes such as Ca 2+ homeostasis, muscle contraction, bone formation, and memory to name a few. Herein, we review the progress made in recent years about the structure of the mammalian NCX and how it relates to function. Particular emphasis will be given to the mammalian cardiac isoform, NCX1.1, due to the extensive studies conducted on this protein. Given the degree of conservation among the eukaryotic exchangers, the information highlighted herein will provide a foundation for our understanding of this transporter family. We will discuss gene structure, alternative splicing, topology, regulatory mechanisms, and NCX's functional role on cardiac physiology. Throughout this article, we will attempt to highlight important milestones in the field and controversial topics where future studies are required. © 2021 American Physiological Society. Compr Physiol 12:1-37, 2021., (Copyright © 2022 American Physiological Society. All rights reserved.)

The heart requires a substantial amount of energy to function, utilising various substrates including lipids, glucose and lactate as energy sources. In times of increased stress, lactate becomes the primary energy source of the heart, but persistently elevated lactate levels are linked to poor patient outcomes and increased mortality. Recently, carnosine dipeptidase II (CNDP2) was discovered to catalyse the formation of Lac-Phe, an exercise-induced metabolite derived from lactate, which has been shown to suppress appetite in mice and reduce adipose tissue in humans. This review discusses CNDP2, including its role in lactate clearance, carnosine hydrolysis, oxidative stress regulation, and involvement in metabolite regulation. The association between CNDP2 and cardiometabolic and renal diseases is also explored, and knowledge gaps are highlighted. CNDP2 appears to be a complex participant in human physiological processes and disease, necessitating additional research to unveil its functions and potential therapeutic applications., (© 2024. The Author(s).)

Exercise causes major shifts in multiple ions (e.g., K + , Na + , H + , lactate - , Ca 2+ , and Cl - ) during muscle activity that contributes to development of muscle fatigue. Sarcolemmal processes can be impaired by the trans-sarcolemmal rundown of ion gradients for K + , Na + , and Ca 2+ during fatiguing exercise, while changes in gradients for Cl - and Cl - conductance may exert either protective or detrimental effects on fatigue. Myocellular H + accumulation may also contribute to fatigue development by lowering glycolytic rate and has been shown to act synergistically with inorganic phosphate (Pi) to compromise cross-bridge function. In addition, sarcoplasmic reticulum Ca 2+ release function is severely affected by fatiguing exercise. Skeletal muscle has a multitude of ion transport systems that counter exercise-related ionic shifts of which the Na + /K + -ATPase is of major importance. Metabolic perturbations occurring during exercise can exacerbate trans-sarcolemmal ionic shifts, in particular for K + and Cl - , respectively via metabolic regulation of the ATP-sensitive K + channel (K ATP ) and the chloride channel isoform 1 (ClC-1). Ion transport systems are highly adaptable to exercise training resulting in an enhanced ability to counter ionic disturbances to delay fatigue and improve exercise performance. In this article, we discuss (i) the ionic shifts occurring during exercise, (ii) the role of ion transport systems in skeletal muscle for ionic regulation, (iii) how ionic disturbances affect sarcolemmal processes and muscle fatigue, (iv) how metabolic perturbations exacerbate ionic shifts during exercise, and (v) how pharmacological manipulation and exercise training regulate ion transport systems to influence exercise performance in humans. © 2021 American Physiological Society. Compr Physiol 11:1895-1959, 2021., (Copyright © 2021 American Physiological Society. All rights reserved.)

Aims Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis. Methods and results The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding, rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30% reduction in E/e′ and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a down-regulation of transforming growth factor (TGF)-β target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-β levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-β-binding proteins, i.e. latent-binding protein of TGF-β and extra domain A-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity. Conclusion Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-β availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction. [ABSTRACT FROM AUTHOR]

The effects of reduced glutathione (GSH) on skeletal muscle fatigue were investigated. GSH was depressed by buthionine sulfoximine (BSO) (100 mg/kg body wt/day) treatment for 5 days, which decreased GSH content to ~10%. Male Wistar rats were assigned to the control (N = 18) and BSO groups (N = 17). Twelve hours after BSO treatment, the plantar flexor muscles were subjected to fatiguing stimulation (FS). Eight control and seven BSO rats were rested for 0.5 h (early stage of recovery), and the remaining were rested for 6 h (late stage of recovery). Forces were measured before FS and after rest, and physiological functions were estimated using mechanically skinned fibers. The force at 40 Hz decreased to a similar extent in both groups in the early stage of recovery and was restored in the control but not in the BSO group in the late stage of recovery. In the early stage of recovery, sarcoplasmic reticulum (SR) Ca2+ release was decreased in the control greater than in the BSO group, whereas myofibrillar Ca2+ sensitivity was increased in the control but not in the BSO group. In the late stage of recovery, SR Ca2+ release decreased and SR Ca2+ leakage increased in the BSO group but not in the control group. These results indicate that GSH depression alters the cellular mechanism of muscle fatigue in the early stage and delays force recovery in the late stage of recovery, due at least in part, to the prolonged Ca2+ leakage from the SR. [ABSTRACT FROM AUTHOR]

Background: Syndecan-4 (SDC4) is a member of the heparan sulfate proteoglycan family of cell-surface receptors. We and others previously reported that variation in the SDC4 gene was associated with several components of the metabolic syndrome, including intra-abdominal fat, fasting glucose and triglyceride levels, and hypertension, in human cohorts. Additionally, we demonstrated that high fat diet (HFD)-induced obese female mice with a Sdc4 genetic deletion had higher visceral adiposity and a worse metabolic profile than control mice. Here, we aimed to first investigate whether the mouse Sdc4 null mutation impacts metabolic phenotypes in a sex- and diet-dependent manner. We then tested whether SDC4 polymorphisms are related to the metabolic syndrome (MetS) in humans. Methods: For the mouse experiment, Sdc4-deficient (Sdc4−/−) and wild-type (WT) mice were treated with 14-weeks of low-fat diet (LFD). Body composition, energy balance, and selected metabolic phenotypes were assessed. For the human genetic study, we used logistic regression models to test 11 SDC4 SNPs for association with the MetS and its components in a cohort of 274 (113 with MetS) elderly subjects from southern Italy. Results: Following the dietary intervention in mice, we observed that the effects of the Sdc4 null mutation on several phenotypes were different from those previously reported in the mice kept on an HFD. Nonetheless, LFD-fed female Sdc4−/− mice, but not males, displayed higher levels of triglycerides and lower insulin sensitivity at fasting than WT mice, as seen earlier in the HFD conditions. In the parallel human study, we found that carriers of SDC4 rs2228384 allele C and rs2072785 allele T had reduced risk of MetS. The opposite was true for carriers of the SDC4 rs1981429 allele G. Additionally, the SNPs were found related to fasting triglyceride levels and triglyceride glucose (TyG) index, a reliable indicator of insulin resistance, with sex-stratified analysis detecting the association of rs1981429 with these phenotypes only in females. Conclusions: Altogether, our results suggest that SDC4 is an evolutionary conserved genetic determinant of MetS and that its genetic variation is associated with fasting triglyceride levels in a female-specific manner. [ABSTRACT FROM AUTHOR]

Dysfunction of skeletal muscles and their atrophy under unloading are accompanied by excess calcium accumulation in the myoplasm of muscle fibers. We hypothesized that calcium accumulation may occur, among other reasons, due to inhibition of SERCA activity under muscle unloading. In this case, the use of a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activator would reduce the calcium level in the myoplasm and prevent the consequences of unloading. Male Wistar rats were divided into 3 groups: vivarium control with placebo administration (C, n = 8), 7-day suspension group with placebo administration (7HS, n = 8), and 7-day suspension group with intraperitoneal administration of the SERCA activator CDN1163 (7HS + CDN, 50 mg/kg, n = 8). One m. soleus of each rat was frozen in liquid nitrogen, the second was tested for functional properties. 7HS rats demonstrated an increased m. soleus fatigue in the ex vivo test, a significant increase in mRNA and the number of fast-twitch muscle fibers, an increase in the level of calcium-dependent CaMK II phosphorylation and tropomyosin oxidation, as well as a decrease in mitochondrial DNA and protein levels. All these changes were prevented in the 7HS + CDN group administered with SERCA activator. Conclusion: 7-day SERCA activator administration does not delay m. soleus atrophy but prevents the development of its fatigue, probably due to prevention of a reduction in the number of type I fibers and mitochondrial biogenesis markers. [ABSTRACT FROM AUTHOR]

Repeated, episodic bouts of skeletal muscle contraction undertaken frequently as structured exercise training are a potent stimulus for physiological adaptation in many organs. Specifically, in skeletal muscle, remarkable plasticity is demonstrated by the remodeling of muscle structure and function in terms of muscular size, force, endurance, and contractile velocity as a result of the functional demands induced by various types of exercise training. This plasticity, and the mechanistic basis for adaptations to skeletal muscle in response to exercise training, are underpinned by activation and/or repression of molecular pathways and processes in response to each individual acute exercise session. These pathways include the transduction of signals arising from neuronal, mechanical, metabolic, and hormonal stimuli through complex signal transduction networks, which are linked to a myriad of effector proteins involved in the regulation of pre- and posttranscriptional processes, and protein translation and degradation processes. This review therefore describes acute exercise-induced signal transduction and the molecular responses to acute exercise in skeletal muscle including emerging concepts such as epigenetic pre- and posttranscriptional regulation and the regulation of protein translation and degradation. A critical appraisal of methodological approaches and the current state of knowledge informs a series of recommendations offered as future directions in the field. [ABSTRACT FROM AUTHOR]

Research during the past decades has yielded numerous insights into the presence and function of lactate in the body. Lactate is primarily produced via glycolysis and plays special roles in the regulation of tissues and organs, particularly in the cardiovascular system. In addition to being a net consumer of lactate, the heart is also the organ in the body with the greatest lactate consumption. Furthermore, lactate maintains cardiovascular homeostasis through energy supply and signal regulation under physiological conditions. Lactate also affects the occurrence, development, and prognosis of various cardiovascular diseases. We will highlight how lactate regulates the cardiovascular system under physiological and pathological conditions based on evidence from recent studies. We aim to provide a better understanding of the relationship between lactate and cardiovascular health and provide new ideas for preventing and treating cardiovascular diseases. Additionally, we will summarize current developments in treatments targeting lactate metabolism, transport, and signaling, including their role in cardiovascular diseases. [ABSTRACT FROM AUTHOR]

In the last 30 years, the field of cyclic adenosine 3′,5′-monophosphate (cAMP) signalling has witnessed a transformative development with the realization that cAMP is compartmentalized and that spatial regulation of cAMP is critical for faithful signal propagation and hormonal specificity. This recognition has changed our understanding of cAMP signalling from the canonical model, where a linear pathway connects a plasma membrane receptor to intracellular effectors and their targets, to a model where signal transduction occurs within a complex network of alternative branches and where an individual receptor leads to activation of a limited fraction of the network, enabled by local regulation of independent signalling units, resulting in a specific functional outcome. The cardiac myocyte has served as the cell model for many of the original findings leading to this paradigm. In this review, we cover some of the evidence supporting this new perspective and discuss how this model is providing novel mechanistic insight into cardiac myocyte physiology. With a focus on the regulation of cardiac rhythm, we consider how this model can provide an original framework for the identification of disease mechanisms. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'. [ABSTRACT FROM AUTHOR]

In a recent randomized, double-blind, placebo-controlled trial, we were able to demonstrate the superiority of a dietary supplement composed of essential amino acids (EAAs) over whey protein, in older adults with low physical function. In this paper, we describe the comparative plasma protein expression in the same subject groups of EAAs vs whey. The plasma proteomics data was generated using SOMA scan assay. A total of twenty proteins were found to be differentially expressed in both groups with a 1.5-fold change. Notably, five proteins showed a significantly higher fold change expression in the EAA group which included adenylate kinase isoenzyme 1, casein kinase II 2-alpha, Nascent polypeptide-associated complex subunit alpha, peroxiredoxin-1, and peroxiredoxin-6. These five proteins might have played a significant role in providing energy for the improved cardiac and muscle strength of older adults with LPF. On the other hand, fifteen proteins showed slightly lower fold change expression in the EAA group. Some of these 15 proteins regulate metabolism and were found to be associated with inflammation or other comorbidities. Gene Ontology (GO) enrichment analysis showed the association of these proteins with several biological processes. Furthermore, protein–protein interaction network analysis also showed distinct networks between upregulated and downregulated proteins. In conclusion, the important biological roles of the upregulated proteins plus better physical function of participants in the EAAs vs whey group demonstrated that EAAs have the potential to improve muscle strength and physical function in older adults. This study was registered with ClinicalTrials.gov: NCT03424265 "Nutritional interventions in heart failure." [ABSTRACT FROM AUTHOR]

Background Amyloid plaques and neurofibrillary tangles, the molecular lesions that characterize Alzheimer's disease (AD) and other forms of dementia, are emerging as determinants of proteinopathies 'beyond the brain'. This study aims to establish tau's putative pathophysiological mechanistic roles and potential future therapeutic targeting of tau in heart failure (HF). Methods and results A mouse model of tauopathy and human myocardial and brain tissue from patients with HF, AD, and controls was employed in this study. Tau protein expression was examined together with its distribution, and in vitro tau-related pathophysiological mechanisms were identified using a variety of biochemical, imaging, and functional approaches. A novel tau-targeting immunotherapy was tested to explore tau-targeted therapeutic potential in HF. Tau is expressed in normal and diseased human hearts, in contradistinction to the current oft-cited observation that tau is expressed specifically in the brain. Notably, the main cardiac isoform is high-molecular-weight (HMW) tau (also known as big tau), and hyperphosphorylated tau segregates in aggregates in HF and AD hearts. As previously described for amyloid-beta, the tauopathy phenotype in human myocardium is of diastolic dysfunction. Perturbation in the tubulin code, specifically a loss of tyrosinated microtubules, emerged as a potential mechanism of myocardial tauopathy. Monoclonal anti-tau antibody therapy improved myocardial function and clearance of toxic aggregates in mice, supporting tau as a potential target for novel HF immunotherapy. Conclusion The study presents new mechanistic evidence and potential treatment for the brain–heart tauopathy axis in myocardial and brain degenerative diseases and ageing. [ABSTRACT FROM AUTHOR]

Although opioids are necessary for the treatment of acute pain, cancer pain, and palliative care, opioid abuse is a serious threat to society. Heroin (Diacetylmorphine) is the most commonly abused opioid, and it can have a variety of effects on the body's tissues and organs, including the well-known gastrointestinal depression and respiratory depression; however, there is little known about the effects of diacetylmorphine on cardiac damage. Here, we demonstrate that diacetylmorphine induces abnormal electrocardiographic changes in rats and causes damage to cardiomyocytes in vitro by an underlying mechanism of increased autophosphorylation of CaMKII and concomitant regulation of myocardial contractile protein TPM1 and MYOM2 protein expression. The CaMKII inhibitor KN-93 was first tested to rescue the toxic effects of heroin on cardiomyocytes in vitro and the abnormal ECG changes caused by heroin in SD rats, followed by the TMT relative quantitative protein technique to analyze the proteome changes. Diacetylmorphine causes increased phosphorylation at the CaMKII Thr287 site in myocardium, resulting in increased autophosphorylation of CaMKII and subsequent alterations in myocardial contractile proteins, leading to myocardial rhythm abnormalities. These findings provide a theoretical basis for the treatment and prevention of patients with arrhythmias caused by diacetylmorphine inhalation and injection. [ABSTRACT FROM AUTHOR]

Aging is associated with skeletal muscle strength decline and cardiac diastolic dysfunction. The structural arrangements of the sarcomeric proteins, such as myosin binding protein-C (MyBP-C) are shown to be pivotal in the pathogenesis of diastolic dysfunction. Yet, the role of fast (fMyBP-C) and slow (sMyBP-C) skeletal muscle MyBP-C remains to be elucidated. Herein, we aimed to characterize MyBP-C and its paralogs in the fast tibialis anterior (TA) muscle from adult and old mice. Immunoreactivity preparations showed that the relative abundance of the fMyBP-C paralog was greater in the TA of both adult and old, but no differences were noted between groups. We further found that the expression level of cardiac myosin binding protein-C (cMyBP-C), an important modulator of cardiac output, was lowered by age. Standard SDS-PAGE along with Pro-Q Diamond phosphoprotein staining did not identify age-related changes in phosphorylated MyBP-C proteins from TA and cardiac muscles; however, it revealed that MyBP-C paralogs in fast skeletal and cardiac muscle were highly phosphorylated. Mass spectrometry further identified glycogen phosphorylase, desmin, actin, troponin T, and myosin regulatory light chain 2 as phosphorylated myofilament proteins in both ages. MyBP-C protein-bound carbonyls were determined using anti-DNP immunostaining and found the carbonyl level of fMyBP-C, sMyBP-C, and cMyBP-C to be similar between old and adult animals. In summary, our data showed some differences regarding the MyBP-C paralog expression and identified an age-related reduction of cMyBP-C expression. Future studies are needed to elucidate which are the age-driven post-translational modifications in the MyBP-C paralogs. [ABSTRACT FROM AUTHOR]