Cardiomyocyte Membrane Research Articles

Deletion of nephrocystin 4 exacerbates cardiac function and survival rate in a mouse model of cardio-renal syndrome

Abstract Background Cardiorenal syndrome (CRS), characterized by cardiac and renal dysfunction, is an increasing health problem associated with high mortality rate. Nephrocystin 4 (NPHP4) gene is the major cause of end-stage renal disease in children. We have reported NPHP4 single nucleotide polymorphism is the genetic risk for CRS and subsequent cardiovascular events in general population. However, the functional role of Nphp4 in the development of CRS has never been examined yet. Methods and Results We generated systemic Nphp4 knockout (KO) mice deleting exon 3 to 8 using CRISPR-Cas9 system. RNA sequence analysis of heart and kidney tissues demonstrated that gene-sets related to mitochondrial function were significantly downregulated in Nphp4 KO mice compared to their wild-type (WT) littermates. Nphp4 KO mice showed less cardiac hypertrophy, however, exacerbated cardiac function and survival compared to WT mice in a CRS model induced by thoracic transverse aortic constriction. Nphp4 located in endosome and cell membrane in H9C2 cardiomyocytes. Immunoprecipitation confirmed protein interaction between Nphp4 and HECT-type E3 ligase Itch. Furthermore, we found that Itch targeted Lats2 and insulin-like growth factor-1 receptor in cardiomyocytes. Knockdown of Nphp4 activated Hippo signaling and worsened mitochondrial function in H9C2 cardiomyocytes through the inhibition of Itch-dependent Lats2 degradation. Knockdown of Nphp4 inhibited insulin-like growth factor-1 signaling through the disturbance of Itch-mediated endosome function. Conclusions Deletion of Nphp4 impaired compensatory cardiac hypertrophy and exacerbated cardiac function and survival in a mouse CRS model through mitochondrial dysfunction. Nphp4 mediated Hippo signaling and insulin-like growth factor-1 signaling through the interaction with Itch. NPHP4 could be the novel therapeutic target in CRS.figure1figure2

Salidroside rescues hypoxic cardiomyocytes by regulating the EGLN1/HIF‑1α pathway.

Myocardial infarction is characterized by oxygen deficiency caused by arterial flow restriction. Salidroside (SAL) protects against myocardial damage via antioxidant production and inhibition of apoptosis. The present study aimed to investigate potential rescue mechanism of SAL on hypoxic cardiomyocytes. H9C2 cardiomyocytes were divided into normoxia, hypoxia and hypoxia + SAL groups. The inhibitory rate of hypoxia and the optimal concentration and rescue effect of SAL were determined using Cell Counting Kit-8 assay and flow cytometry. Ca2+ concentration following hypoxia treatment and SAL intervention were detected by Fluo-4/acetoxymethyl. Tandem mass tag (TMT) proteomics was used to analyze the differential expression of hypoxia-associated proteins among the three groups. SAL exerted a protective effect on hypoxia-injured cardiomyocytes by enhancing aerobic metabolism during hypoxia and rescuing cardiomyocytes from hypoxic damage. SAL promoted cell proliferation, decreased apoptosis and increased Ca2+ levels in cell membranes of hypoxic cardiomyocytes. TMT proteomics results showed that the expression levels of intracellular hypoxia inducible factor-1 (HIF)-1α and Egl-9 family HIF 1 (EGLN1) in H9C2 cells were elevated under hypoxic conditions. However, SAL significantly decreased expression levels of HIF-1α and EGLN1. SAL inhibited mitochondrial calcium overload in hypoxic cardiomyocytes and attenuated expression of hypoxia-associated factors. SAL exerted its rescue effect on hypoxic cardiomyocytes through the EGLN1/HIF-1α pathway, thereby suppressing cardiomyocyte apoptosis, improving mitochondrial energy metabolism efficiency and rescuing cardiomyocytes from hypoxic injury.

Accumulated β-catenin is associated with human atrial fibrosis and atrial fibrillation

BackgroundAtrial fibrillation (AF) is associated with increased risk of stroke and mortality. It has been reported that the process of atrial fibrosis was regulated by β-catenin in rats with AF. However, pathophysiological mechanisms of this process in human with AF remain unclear. This study aims to investigate the possible mechanisms of β-catenin in participating in the atrial fibrosis using human right atrial appendage (hRAA) tissues .MethodsWe compared the difference of β-catenin expression in hRAA tissues between the patients with AF and sinus rhythm (SR). The possible function of β-catenin in the development of AF was also explored in mice and primary cells.ResultsFirstly, the space between the membrane of the gap junctions of cardiomyocytes was wider in the AF group. Secondly, the expression of the gap junction function related proteins, Connexin40 and Connexin43, was decreased, while the expression of β-catenin and its binding partner E-cadherin was increased in hRAA and cardiomyocytes of the AF group. Thirdly, β-catenin colocalized with E-cadherin on the plasma membrane of cardiomyocytes in the SR group, while they were dissociated and accumulated intracellularly in the AF group. Furthermore, the expression of glycogen synthase kinase 3β (GSK-3β) and Adenomatous Polyposis Coli (APC), which participated in the degradation of β-catenin, was decreased in hRAA tissues and cardiomyocytes of the AF group. Finally, the development of atrial fibrosis and AF were proved to be prevented after inhibiting β-catenin expression in the AF model mice.ConclusionsBased on human atrial pathological and molecular analyses, our findings provided evidence that β-catenin was associated with atrial fibrosis and AF progression.

Functional consequences of changes in the distribution of Ca2+ extrusion pathways between t-tubular and surface membranes in a model of human ventricular cardiomyocyte

The sarcolemmal Ca2+ efflux pathways, Na+-Ca2+-exchanger (NCX) and Ca2+-ATPase (PMCA), play a crucial role in the regulation of intracellular Ca2+ load and Ca2+ transient in cardiomyocytes. The distribution of these pathways between the t-tubular and surface membrane of ventricular cardiomyocytes varies between species and is not clear in human. Moreover, several studies suggest that this distribution changes during the development and heart diseases. However, the consequences of NCX and PMCA redistribution in human ventricular cardiomyocytes have not yet been elucidated. In this study, we aimed to address this point by using a mathematical model of the human ventricular myocyte incorporating t-tubules, dyadic spaces, and subsarcolemmal spaces. Effects of various combinations of t-tubular fractions of NCX and PMCA were explored, using values between 0.2 and 1 as reported in animal experiments under normal and pathological conditions. Small variations in the action potential duration (≤ 2%), but significant changes in the peak value of cytosolic Ca2+ transient (up to 17%) were observed at stimulation frequencies corresponding to the human heart rate at rest and during activity. The analysis of model results revealed that the changes in Ca2+ transient induced by redistribution of NCX and PMCA were mainly caused by alterations in Ca2+ concentrations in the subsarcolemmal spaces and cytosol during the diastolic phase of the stimulation cycle. The results suggest that redistribution of both transporters between the t-tubular and surface membranes contributes to changes in contractility in human ventricular cardiomyocytes during their development and heart disease and may promote arrhythmogenesis.

Geneless optomodulation of cardiomyocytes membrane potential by sarcolemma-targeted azobenzene photoswitches

Geneless optomodulation of cardiomyocytes membrane potential by sarcolemma-targeted azobenzene photoswitches

YTHDF1 is pivotal for maintenance of cardiac homeostasis

The YTH-domain family (YTHDF) of RNA binding proteins can control gene expression at the post-transcriptional level by regulating mRNAs with N6-methyladenosine (m6A) modifications. Despite the established importance of m6A in the heart, the cardiac role of specific m6A-binding proteins remains unclear. Here, we characterized the function of YTHDF1 in cardiomyocytes using a newly generated cardiac-restricted mouse model. Deletion of YTHDF1 in adult cardiomyocytes led to hypertrophy, fibrosis, and dysfunction. Using mass spectrometry, we identified the necessity of YTHDF1 for the expression of cardiomyocyte membrane raft proteins. Specifically, YTHDF1 bound to m6A-modified Caveolin 1 (Cav1) mRNA and favored its translation. We further demonstrated that YTHDF1 regulates downstream ERK signaling. Altogether, our findings highlight a novel role for YTHDF1 as a post-transcriptional regulator of caveolar proteins which is necessary for the maintenance of cardiac function.

Potential limitations of microdystrophin gene therapy for Duchenne muscular dystrophy.

Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (microdystrophins) are underway for Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with 4 microdystrophin constructs (3 in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using AAV doses comparable with those in clinical trials. We achieved high levels of microdystrophin expression in striated muscles with cardiac expression approximately 10-fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease occurred with 2 of the microdystrophins. The detrimental cardiac effect appears to be caused by variable competition (dependent on microdystrophin design and expression level) between microdystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-microdystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, these findings suggest that microdystrophin treatments need to avoid excessively high levels of expression in the heart and that cardiac function should be carefully monitored in these patients.

MCT4-dependent lactate transport: a novel mechanism for cardiac energy metabolism injury and inflammation in type 2 diabetes mellitus

Diabetic cardiomyopathy (DCM) is a major contributor to mortality in diabetic patients, characterized by a multifaceted pathogenesis and limited therapeutic options. While lactate, a byproduct of glycolysis, is known to be significantly elevated in type 2 diabetes, its specific role in DCM remains uncertain. This study reveals an abnormal upregulation of monocarboxylate transporter 4 (MCT4) on the plasma membrane of cardiomyocytes in type 2 diabetes, leading to excessive lactate efflux from these cells. The disruption in lactate transport homeostasis perturbs the intracellular lactate-pyruvate balance in cardiomyocytes, resulting in oxidative stress and inflammatory responses that exacerbate myocardial damage. Additionally, our findings suggest increased lactate efflux augments histone H4K12 lactylation in macrophages, facilitating inflammatory infiltration within the microenvironment. In vivo experiments have demonstrated that inhibiting MCT4 effectively alleviates myocardial oxidative stress and pathological damage, reduces inflammatory macrophage infiltration, and enhances cardiac function in type 2 diabetic mice. Furthermore, a clinical prediction model has been established, demonstrating a notable association between peripheral blood lactate levels and diastolic dysfunction in individuals with type 2 diabetes. This underscores the potential of lactate as a prognostic biomarker for DCM. Ultimately, our findings highlight the pivotal involvement of MCT4 in the dysregulation of cardiac energy metabolism and macrophage-mediated inflammation in type 2 diabetes. These insights offer novel perspectives on the pathogenesis of DCM and pave the way for the development of targeted therapeutic strategies against this debilitating condition.

Neutrophil extracellular traps mediate cardiomyocyte ferroptosis via the Hippo–Yap pathway to exacerbate doxorubicin-induced cardiotoxicity

Doxorubicin-induced cardiotoxicity (DIC), which is a cardiovascular complication, has become the foremost determinant of decreased quality of life and mortality among survivors of malignant tumors, in addition to recurrence and metastasis. The limited ability to accurately predict the occurrence and severity of doxorubicin-induced injury has greatly hindered the prevention of DIC, but reducing the dose to mitigate side effects may compromise the effective treatment of primary malignancies. This has posed a longstanding clinical challenge for oncologists and cardiologists. Ferroptosis in cardiomyocytes has been shown to be a pivotal mechanism underlying cardiac dysfunction in DIC. Ferroptosis is influenced by multiple factors. The innate immune response, as exemplified by neutrophil extracellular traps (NETs), may play a significant role in the regulation of ferroptosis. Therefore, the objective of this study was to investigate the involvement of NETs in doxorubicin-induced cardiomyocyte ferroptosis and elucidate their regulatory role. This study confirmed the presence of NETs in DIC in vivo. Furthermore, we demonstrated that depleting neutrophils effectively reduced the occurrence of doxorubicin-induced ferroptosis and myocardial injury in DIC. Additionally, our findings showed the pivotal role of high mobility group box 1 (HMGB1) as a critical molecule implicated in DIC and emphasized its involvement in the modulation of ferroptosis subsequent to NETs inhibition. Mechanistically, we obtained preliminary evidence suggesting that doxorubicin-induced NETs could modulate yes-associated protein (YAP) activity by releasing HMGB1, which subsequently bound to toll like receptor 4 (TLR4) on the cardiomyocyte membrane, thereby influencing cardiomyocyte ferroptosis in vitro. Our findings suggest that doxorubicin-induced NETs modulate cardiomyocyte ferroptosis via the HMGB1/TLR4/YAP axis, thereby contributing to myocardial injury. This study offers a novel approach for preventing and alleviating DIC by targeting alterations in the immune microenvironment.

Do calcium channel blockers applied to cardiomyocytes cause increased channel expression resulting in reduced efficacy?

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug’s efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug’s inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug’s overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

ROCK1 deficiency preserves caveolar compartmentalization of signaling molecules and cell membrane integrity.

In this study, we investigated the roles of ROCK1 in regulating structural and functional features of caveolae located at the cell membrane of cardiomyocytes, adipocytes, and mouse embryonic fibroblasts (MEFs) as well as related physiopathological effects. Caveolae are small bulb-shaped cell membrane invaginations, and their roles have been associated with disease conditions. One of the unique features of caveolae is that they are physically linked to the actin cytoskeleton that is well known to be regulated by RhoA/ROCKs pathway. In cardiomyocytes, we observed that ROCK1 deficiency is coincident with an increased caveolar density, clusters, and caveolar proteins including caveolin-1 and -3. In the mouse cardiomyopathy model with transgenic overexpressing Gαq in myocardium, we demonstrated the reduced caveolar density at cell membrane and reduced caveolar protein contents. Interestingly, coexisting ROCK1 deficiency in cardiomyocytes can rescue these defects and preserve caveolar compartmentalization of β-adrenergic signaling molecules including β1-adrenergic receptor and type V/VI adenylyl cyclase. In cardiomyocytes and adipocytes, we detected that ROCK1 deficiency increased insulin signaling with increased insulin receptor activation in caveolae. In MEFs, we identified that ROCK1 deficiency increased caveolar and total levels of caveolin-1 and cell membrane repair ability after mechanical or chemical disruptions. Together, these results demonstrate that ROCK1 can regulate caveolae plasticity and multiple functions including compartmentalization of signaling molecules and cell membrane repair following membrane disruption by mechanical force and oxidative damage. These findings provide possible molecular insights into the beneficial effects of ROCK1 deletion/inhibition in cardiomyocytes, adipocytes, and MEFs under certain diseased conditions.

Physiological stress improves stem cell modeling of dystrophic cardiomyopathy.

Heart failure contributes to Duchenne muscular dystrophy (DMD), which arises from mutations that ablate dystrophin, rendering the plasma membrane prone to disruption. Cardiomyocyte membrane breakdown in patients with DMD yields a serum injury profile similar to other types of myocardial injury with the release of creatine kinase and troponin isoforms. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are highly useful but can be improved. We generated hiPSC-CMs from a patient with DMD and subjected these cells to equibiaxial mechanical strain to mimic in vivo stress. Compared to healthy cells, DMD hiPSC-CMs demonstrated greater susceptibility to equibiaxial strain after 2 h at 10% strain. We generated an aptamer-based profile of proteins released from hiPSC-CMs both at rest and subjected to strain and identified a strong correlation in the mechanical stress-induced proteome from hiPSC-CMs and serum from patients with DMD. We exposed hiPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced biomarker release in DMD and control hiPSC-CMs subjected to strain. Thus, the application of mechanical strain to hiPSC-CMs produces a model that reflects an in vivo injury profile, providing a platform to assess pharmacologic intervention.

Apelin C-terminal fragments: biological properties and therapeutic potential

The creation of bioactive molecules for the treatment of cardiovascular diseases based on natural peptides stimulates intensive experimental research. In recent years, it has been established that the C-terminal fragments of apelin, an endogenous ligand of the APJ receptor, reduce metabolic and functional disorders in experimental heart damage. The review presents literature data and generalized results of our own experiments on the effect of apelin-13, [Pyr]apelin-13, apelin-12, and their chemically modified analogues on the heart in normal conditions and when modeling pathophysiological conditions in vitro and in vivo. It has been shown that the spectrum of action of apelin peptides on the damaged myocardium covers a decrease in the death of cardiomyocytes from necrosis, a decrease in cardiomyocyte membrane damage, an improvement in the metabolic state of the myocardium, and a decrease in the formation of reactive oxygen species and lipid peroxidation products. The mechanisms of the protective action of these peptides associated with the activation of the APJ receptor and the manifestation of antioxidant properties are discussed. The data presented in the review indicate the promise of molecular design of pharmacological APJ receptor agonists, which can serve as the basis for the development of cardioprotectors that affect the processes of free radical oxidation and metabolic adaptation.

Impact of microtubule detyrosination on subcellular distribution of Nav1.5 in mdx cardiomyocytes

Abstract Background Subcellular trafficking and distribution of the cardiac sodium channel Nav1.5 in cardiomyocytes is dependent on the microtubule network as well as Nav1.5 interacting proteins. Duchenne muscular dystrophy (DMD) is associated with loss of dystrophin, a Nav1.5-interacting protein localized at the lateral membrane of cardiomyocytes, leading to sodium current (INa) reduction in this microdomain. Cardiomyocytes of DMD (mdx) mice also display increased microtubule detyrosination, but its impact on INa and Nav1.5 distribution is unclear. Purpose To investigate the effect of reducing microtubule detyrosination by the compound parthenolide (PTL) on INa and subcellular Nav1.5 distribution in mdx cardiomyocytes (CMs). Methods and Results Isolated ventricular CMs from wild type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL or DMSO for 3-5 hours. INa properties were assessed using the patch-clamp technique, confocal microscopy was used to investigate microtubule detyrosination, and stochastic optical reconstruction microscopy (STORM) was employed to assess Nav1.5 cluster density and size, as well as Nav1.5 cluster distribution (crest vs groove) at the cardiomyocyte lateral membrane. Compared to WT. mdx CMs displayed increased levels of detyrosinated tubulin and decreased whole-cell INa. Incubation with PTL decreased the fraction of detyrosinated tubulin and significantly increased whole-cell INa magnitude in mdx CMs, but had no effect in WT CMs. No effect of PTL on INa gating properties were observed, indicating that it increased INa by enhancing Nav1.5 membrane trafficking. Indeed, STORM analysis showed that PTL increased Nav1.5 cluster density at both the lateral membrane and intercalated disc region in mdx CMs while it had no effect on WT CMs. Furthermore, Nav1.5-α-actinin cluster distance analysis showed that PTL increased Nav1.5 clusters specifically at the crest of the lateral membrane in mdx CMs. Conclusions In mdx CMs, reduction of MT detyrosination rescues INa density and increases Nav1.5 cluster density both at the intercalated disc and lateral membrane. Hence, in addition to loss of the Nav1.5-interacting protein dystrophin, MT remodelling contributes to INa reduction in DMD. The PTL-induced increase in Nav1.5 at the crest of the lateral membrane of mdx CMs is likely due to a dystrophin-independent mechanism since dystrophin is normally absent from this microdomain. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution during pathophysiological conditions.

Targeting microtubule dynamics improves cardiac sodium channel function in arrhythmogenic cardiomyopathy

Abstract Background Pathophysiological conditions such as heart failure and cardiomyopathy are associated with alterations in microtubule (MT) dynamics (in particular increased MT detyrosination), which have detrimental effects on cardiac function. Since the MT network is also involved in trafficking of ion channels to the cell membrane, its remodeling may also affect sodium channel (Nav1.5) function. Indeed, sodium current (INa) is typically decreased in the setting of heart failure and (arrhythmogenic) cardiomyopathy. Purpose To investigate the impact of MT detyrosination on Nav1.5 and INa in a mouse model of arrhythmogenic cardiomyopathy (tamoxifen-activated cardiac-specific knockout of plakophilin-2 (PKP2cKO)). Methods and Results Freshly isolated cardiomyocytes (CMs) from PKP2cKO mice were incubated for 2-4 hours with either 10 µM parthenolide (PTL; a compound that decreases the fraction of detyrosinated MTs) or vehicle only (DMSO). Vehicle-treated CMs from PKP2cKO mice displayed significantly increased levels of detyrosinated tubulin as compared to wild type (WT), which was attenuated by PTL treatment. PTL significantly increased whole-cell INa without affecting gating properties in PKP2cKO CMs, while it did not affect INa in WT CMs. Macropatch measurements demonstrated that PTL increased INa at both the cell end (Intercalated disc, ID) and lateral membrane (LM) of PKP2cKO CMs. Accordingly, stochastic optical reconstruction microscopy (STORM), a super-resolution approach, revealed that PTL increased Nav1.5 cluster density at both ID and LM of PKP2cKO CMs. Conclusions Reducing MT detyrosination by PTL increases Nav1.5 cluster density and sodium current magnitude in a mouse model of arrhythmogenic cardiomyopathy. These findings identify restoration of MT dynamics as a potential novel anti-arrhythmic therapeutic in the setting of cardiac pathology.

Abstract 15271: Modification of Cardiomyocyte Lateral Membrane Composition Promotes Cardiac Function by Regulating Connexome Organization

Introduction: Loss of contacts between adjacent cardiomyocytes is detrimental to proper electromechanical function. For instance, connexome remodeling is a common feature of both acquired and inherited cardiac arrhythmias. We have shown that the lateral membrane-specific scaffolding protein CASK regulates the trafficking and targeting of certain connexome components, notably the sodium channel. Question: Does compartmentalization of the cardiomyocyte membrane involves lateral membrane proteins? Our hypothesis is that CASK, due to its association with costamere and its developmental expression gradient, may be involved in a more global control of connexome organization. Aims: To demonstrate that CASK, a lateral membrane-specific protein, is crucial for the development and maintenance of connexome integrity. Method: Viral constructs were designed to inhibit the expression of CASK in vivo (AAV-shCASK) and in vitro (Ad-shCASK) both in murine and human cardiomyocytes. Results: In vivo, echocardiography phenotyping of CASK invalidated rats at the neonatal stage revealed, at the adult stage, eccentric ventricular remodeling associated with increased stroke volume and increased cardiac output suggestive of athlete’s heart phenotype. Hemodynamic measurements (pressure-volume loops) confirmed that CASK invalidation increases both myocardium contractility and compliance. Proteomic analysis showed that CASK invalidation in vitro strongly modify immature cardiomyocyte proteome, notably junction signaling. Immunostaining experiments on cultured neonatal rat cardiomyocytes and adult rat hearts confirmed that CASK is required for the correct organization of myofibrils and favors connexome organization. Remarkably, CASK invalidation in a human in vitro model of arrhythmogenic cardiomyopathy (hiPSC-CM PKP2 +/- ) rescues desmosome assembly. Conclusion: CASK regulates the organization of myofibrils and connexome, with major functional consequences for cardiac performance. Our results suggest for the first time the existence of a crosstalk between lateral membrane and connexome proteins. Therefore, targeting CASK to promote electromechanical coupling of cardiomyocytes could be promising in the context of cardiac remodeling.

MicroRNAs in extracellular vesicles released from epicardial adipose tissue promote arrhythmogenic conduction slowing

Patients with excess epicardial adipose tissue (EAT) are at increased risk of developing cardiac arrhythmias. EAT promotes arrhythmias by depolarizing the resting membrane of cardiomyocytes, which slows down conduction and facilitates re-entrant arrhythmias. We hypothesized that EAT slows conduction by secreting extracellular vesicles (EVs) and their microRNA (miRNA) cargo. We aimed to determine the role of EAT-derived EVs and their miRNA cargo in conduction slowing. EAT and subcutaneous adipose tissue (SAT) were collected from patients with atrial fibrillation. Adipose tissue explants were incubated in culture medium and secretome was collected. The numbers of EVs in the EAT and SAT secretome were measured by calibrated flow cytometry. EVs in the EAT secretome were isolated by size exclusion chromatography and miRNAs were sequenced. Pathway analysis was performed to predict candidates involved in cardiac electrophysiology. The candidates were validated in the EAT and SAT by quantitative real-time polymerase chain reaction. Finally, miRNA candidates were overexpressed in neonatal rat ventricular myocytes. The EV concentration was higher in the EAT secretome than in the SAT and control secretomes. miRNA sequencing of EAT-derived EVs detected a total of 824 miRNAs. Pathway analysis led to the identification of 7 miRNAs potentially involved in regulation of cardiac resting membrane potential. Validation of those miRNA candidates showed that they were all expressed in EAT, and that miR-1-3p and miR-133a-3p were upregulated in EAT in comparison with SAT. Overexpression of miR-1-3p and miR-133a-3p in neonatal rat ventricular myocytes led to conduction slowing and reduced Kcnj2 and Kcnj12 expression. miR-1-3p and miR-133a-3p are potential mediators of EAT arrhythmogenicity.

Identification of Genetic Variants Associated with Severe Myocardial Bridging through Whole-Exome Sequencing.

Myocardial bridging (MB) is a congenital coronary artery anomaly and an important cause of angina. The genetic basis of MB is currently unknown. This study used a whole-exome sequencing technique and analyzed genotypic differences. Eight coronary angiography-confirmed cases of severe MB and eight age- and sex-matched control patients were investigated. In total, 139 rare variants that are potentially pathogenic for severe MB were identified in 132 genes. Genes with multiple rare variants or co-predicted by ClinVar and CADD/REVEL for severe MB were collected, from which heart-specific genes were selected under the guidance of tissue expression levels. Functional annotation indicated significant genetic associations with abnormal skeletal muscle mass, cardiomyopathies, and transmembrane ion channels. Candidate genes were reviewed regarding the functions and locations of each individual gene product. Among the gene candidates for severe MB, rare variants in DMD, SGCA, and TTN were determined to be the most crucial. The results suggest that altered anchoring proteins on the cell membrane and intracellular sarcomere unit of cardiomyocytes play a role in the development of the missed trajectory of coronary vessels. Additional studies are required to support the diagnostic application of cardiac sarcoglycan and dystroglycan complexes in patients with severe MB.

Beta-adrenoreactivity of erythrocyte membranes and expression of beta1-adrenergic receptors of cardiomyocytes in patients with heart failure with different left ventricular ejection fraction

Introduction. More and more studies demonstrate the importance of determining the beta-adrenergic reactivity of erythrocyte membranes (beta-ARM) for predicting the severity of cardiovascular diseases, including chronic heart failure. But the presence of general patterns between the change in beta-ARM and the expression of beta1adrenergic receptors on the membranes of cardiomyocytes, depending on the left ventricular ejection fraction (LVEF) remains questionable.Aim: To evaluate beta-ARM and expression of beta1-adrenergic receptors of cardiomyocytes in groups of patients with heart failure with preserved (≥ 50%) and reduced (< 50%) LV EF.Materials and methods. The study included 100 patients with chronic heart failure of ischemic origin. Groups of 79 patients with LVEF ≥ 50% and 21 patients with LVEF < 50% were formed. The levels of epinephrine and norepinephrine in blood plasma and the expression of beta1-adrenergic receptors in myocardial samples were assessed using enzyme immunoassay. Beta-ARM was determined in whole blood by assessing the inhibition of hemolysis of erythrocytes placed in a hypoosmotic medium in the presence of a selective beta-blocker.Results. In the group with LV EF < 50%, beta-ARM was increased (p = 0.037), amounting to 31.9% (23.2; 40.1), compared with the group with LVEF ≥ 50%, where beta-ARM was 21.4% (15.0; 31.7). At the same time, in patients with LV EF < 50%, the expression of beta1-AR on cardiomyocyte membranes was significantly lower than in the other group (p = 0.037): 2.7 (2.1; 3.0) ng/mg and 3.1 (2.4; 3.9) ng/mg total protein, respectively. However, plasma levels of epinephrine and noradrenaline were comparable in the study groups.Conclusion. Patients with heart failure with LVEF < 50% were characterized by increased beta-ARM and reduced expression of beta1-AR on cardiomyocyte membranes compared with the group with LVEF ≥ 50% with comparable levels of circulating catecholamines.

REMODELING OF EXTRACELLULARLY RECORDED ACTION POTENTIALS OF RAT HEART SUBEPICARDIAL CARDIOMYOCYTES AFTER ISCHEMIA REPERFUSION INJURY

A number of systemic heart diseases leading to the development of heart failure (aortic stenosis, hypertension, diabetic cardiomyopathy, reperfusion injury etc.) are accompanied by a pronounced reorganization of the T-system of cardiomyocytes, both in humans and animals. However, structural-functional changes within this membrane compartment of cardiomyocytes following ischemia-reperfusion (IR) have not been thoroughly studied. The aim of the work was to study the remodeling of the T-system in the subepicardial cardiomyocytes of the left ventricle of the rat heart after IR injury using confocal microscopy and extracellular recording methods. The study was carried out after 24 hours, two weeks, and four weeks following IR. A remodeling of action potentials, recorded extracellularly in the cardiomyocyte membrane patches devoid of t-tubule entrances (type 1 eAP), was observed. Starting from 24 hours up to 4 weeks after IR, there was an increase in the duration of their decline time (T90) and the formation of eAP after-hyperpolarization phase, reaching maximum values by the fourth week after IR. A decrease in the second peak’s amplitude of eAPs, measured from cardiomyocyte surface locations with t-tubule openings, was also noticed four weeks after IR. In this investigation, no observable changes in the structural organization of the T-system were found. These data suggest that functional modifications of the epicardial cardiomyocyte T-system after IR injury may precede its structural modifications.