Neonatal Cardiomyocytes Research Articles

Skeletal myocyte-specific knockout of Ptpn1 and Ptpn2 enhances inflammatory response in muscle and increases mortality in polymicrobial sepsis

Abstract Background Critically ill patients often suffer from heart and skeletal muscle atrophy and failure (intensive care unit acquired weakness; ICUAW). Inflammation and sepsis are major risk factors for ICUAW. Critically ill patients with sepsis frequently develop insulin resistance that decreases protein synthesis and increases protein degradation in muscle eventually leading to ICUAW. The link between inflammation and insulin resistance is not well understood, but protein tyrosine phosphatases (PTP), that inhibit insulin signaling via insulin receptor (INSR) dephosphorylation as well as inflammation-associated pathways, are implicated. Hypothesis: We hypothesized that PTP1B and TC-PTP mediate inflammation-induced insulin resistance and muscle atrophy in heart and skeletal muscle. Methods and Results qRT-PCR analyses showed that the proinflammatory cytokines tumor necrosis factor (TNF), interleukin (IL)-1β, and IL-6 caused a ~2-fold increase in Ptpn1/PTP1B and Ptpn2/TC-PTP mRNA expression in C2C12 myocytes as well as in neonatal rat ventricular cardiomyocytes. Inhibition of the IL-6/GP130 pathway by siRNA and inhibitors in myocytes in vitro and skeletal muscle specific gp130 knockout mice in vivo attenuated IL-6-induced Ptpn1/PTP1B and Ptpn2/TC-PTP mRNA expression. CLP-induced polymicrobial sepsis was associated with a significant increase in gene expression and protein content of pro-inflammatory cytokines (Tnf, Il1b, Ifng) and leukocyte markers (CD68, CD11c, F4/80) in muscle of skeletal myocyte specific double knockout (Ptpn1+Ptpn2loxP/loxP, MCK cre, dKO) compared to wildtype (Ptpn1+Ptpn2loxP/loxP) mice in sepsis. This also resulted in a lower survival rate of dKO mice in sepsis (42% WT vs. 23% dKO). Immunohistological analyses revealed centralized nuclei, macrophage/monocyte infiltration and enhanced regeneration (Myh3, Pax7, Ki67) in tibialis anterior muscle of septic dKO mice. A strong activation of the NLRP3-inflammasome, as indicated by cleavage of caspase 1, GSDMD, GSDME and caspase 8, indicative for a pronounced inflammation and activated cell death pathways was observed in muscle of septic dKO mice. Conclusion The proinflammatory cytokine IL-6 increases the expression of Ptpn1 and Ptpn2 and enhances overall PTP activity. Selective inhibition of the IL-6R/GP130 signaling pathway by JAK2- and STAT3-inhibition as well as Gp130 deletion in myocytes in vivo attenuates those effects. Muscle-specific deletion of Ptpn1 and Ptpn2 in skeletal muscle leads to increased mortality, enhanced pro-inflammatory response and induced inflammatory, programmed cell death pathways in sepsis. Our data show that Ptpn1 and Ptpn2 play a key anti-inflammatory role in skeletal muscle.

The STRIPAK associated kinase MST4 is a novel mediator in heart failure pathogenesis

Abstract Heart failure still poses substantial challenges in understanding its underlying molecular pathways. We recently found the mammalian STE20-like kinase 4 (MST4) significantly upregulated in human failing hearts (5.7-fold in DCM and 3.8-fold ICM) as well as in heart failure animal models (i.e. 2.5-fold in Calsarcin-1-KO, 2.5-fold in MLP-KO). MST4 is a member of striatin interacting phosphatases and kinases complexes (STRIPAK), an emerging group of multiprotein complexes responsible for integrating cellular signalling pathways. We provide the first evidence of functional STRIPAK complexes containing MST4 by co-immunoprecipitation and interactomics experiments in cardiomyocytes. Moreover, we found MST4 to interact with several intercalated disc proteins like beta-Catenin or Desmin. Adenoviral overexpression of MST4 in neonatal rat ventricular cardiomyocytes (NRVCM) results in a significant increase in cell size (+13%). This was not accompanied by the activation of fetal genes such as NPPA, NPPB or RCAN1.4, but rather by an increase in phosphorylation of Protein kinase B (PKB/Akt, 3.3-fold). Akt is usually associated with physiological hypertrophy, indicating that MST4 may be involved in protective pathways in cardiomyocytes. In support of this notion, MST4 overexpression reduced apoptotic activity (cleavage of Caspase 3 -69%, Caspase 7 -80%, Parp1 -27%) and improved both contractility (fractional shortening cell +23%, fractional shortening sarcomere +30%, time to max. contraction cell -10%, time to max. contraction sarcomere -12%) and relaxation parameters (time to max. relaxation velocity cell -12%) in isolated adult rat ventricular cardiomyocytes. In order to identify cardiac targets of MST4, we performed a comprehensive analysis of the phosphoproteome of NRVCM overexpressing MST4 compared with control-virus +/- pharmacological inhibitor at two time points. We identified more than 70,000 peptides including more than 20,000 phosphopeptides in more than 4,000 protein groups. Using linear modelling with a reduced data set containing regulated features selected by thresholds for p-value and fold change, we observed a consistent MST4 effect and a number of enriched gene ontology terms. Here, we found several potential MST4 targets and effects on protein expression levels associated with intercalated disc proteins. In human heart samples, MST4 was also localized to the intercalated disc region by immunostaining. Interesting proteins in that context that might help explain some of the observed cardioprotective MST4 effects are Connexin 43, Desmin, Ryanodine receptor, Phospholemman, Junctophilin2, Myozap or Frizzled 1. Taken together, our data imply that MST4 upregulation in human heart failure serves an adaptive role which may offer translational opportunities.Graphical Abstract

Downregulation of CPEB3 exacerbates cardiac injury by inhibiting cardiomyocyte adaptive metabolic reprogramming after myocardial infarction

Abstract Background Adaptive metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis in hypoxia plays a protective role in cardiomyocyte survival by reducing reactive oxygen species (ROS) and increasing ATP. Cytoplasmic polyadenylation element binding protein 3 (CPEB3) influences protein translation by modulating 3' untranslated region (3'UTR) lengths through alternative polyadenylation (APA). However, the role of CPEB3 in cardiomyocyte after myocardial infarction (MI) remains unknown. Purpose This study aims to explore the function of CPEB3 in cardiomyocyte after MI. Methods Three GEO databases of mice MI (GSE110209, GSE114695 and GSE236374) were analyzed to identify CPEB3 dysregulation. Cardiomyocyte-specific CPEB3 knockout mice and CPEB3-knockdown neonatal mouse cardiomyocytes (NMCMs), RNA sequencing, flow cytometry, TUNEL staining, Seahorse, ROS and ATP measurements were used to investigate the impact of CPEB3. APA events analysis, RIP sequencing, CUT-TAG, 3’RACE, polysome profiling and dual luciferase report were performed to elucidate the mechanisms of CPEB3. Recombinant adeno-associated virus carrying cardiac troponin T promoter was used to evaluate the therapeutic efficacy of CPEB3 in mice with MI. Results CPEB3 expression was markedly reduced in the heart tissue after MI and in NMCMs after hypoxia. Cardiomyocyte-specific CPEB3 deletion aggravated cardiomyocyte apoptosis, enlarged infarct size, and exacerbated cardiac injury after MI. RNA sequencing revealed that CPEB3 deficiency predominantly inhibited glycolysis-related pathway, with a notable decrease in pyruvate dehydrogenase kinase 1 (PDK1) expression. In CPEB3-knockdown NMCMs, adaptive metabolic reprogramming from OXPHOS to glycolysis and ATP level were decreased in hypoxia, whereas ROS production was significantly enhanced, all of which resulted in apoptosis and were regulated by PDK1. Besides, PDK1 overexpression counteracted the effects of CPEB3 knockdown on metabolic pattern, ROS, ATP and apoptosis. Mechanistically, CPEB3 deficiency led to a shortened 3’UTR of the transcription factor forkhead box O3 (FOXO3), and a decline of FOXO3 protein level. CPEB3 protein was confirmed to directly bind to FOXO3 mRNA, and the translation efficiency of FOXO3 was decreased with CPEB3 knockdown. Furthermore, FOXO3 was found to activate the transcription of PDK1, and FOXO3 overexpression alleviated the decline of PDK1 and attenuated CPEB3 knockdown-induced apoptosis in hypoxia. Finally, cardiomyocyte-specific CPEB3 overexpression reduced cardiomyocyte apoptosis, suppressed myocardial fibrosis, and improved cardiac function by upregulating FOXO3 and PDK1 levels after MI. Conclusion CPEB3 could promote FOXO3 protein expression via APA of FOXO3 3’UTR, activate transcription of PDK1 and ultimately protect cardiomyocyte from apoptosis by restoring metabolic balance in hypoxia. CPEB3 may serve as a promising therapeutic target for cardiomyocyte apoptosis after MI.Schematic diagram

Mammalian Diaphanous-related formin 1 (mDia1) inducing diabetic cardiomyopathy by regulating metabolic reprogramming

Abstract Background Mammalian Diaphanous-related formin 1 (mDia1) is one of the formin family proteins and acts as a rho-effector molecule which is involved in actin organization. Previous studies showed that mDia1 aggravated compensatory cardiac hypertrophy in response to pressure overload. However, the role of mDia1 in diabetic cardiomyopathy and its mechanisms remain elusive. Objective To clarify the mechanisms of mDia1 in DCM through mediating metabolic reprogramming. Methods 5-week-old of mDia1 ko mice were used to induce DCM by feeding high-fat diet (HFD) for 16 weeks. The blood glucose and body weight of the mice were measured regularly. Echocardiography was used to evaluate cardiac function. HE and WGA staining were used to evaluate hypertrophy of cardiomyocytes. Transmission electron microscopy and mitotracker staining were used to observe the morphology of mitochondrial in heart. Proteomics and metabolomics were used to explore the metabolites in hearts of mice. Primary neonatal cardiomyocytes were isolated and stimulated with 35 mM glucose and 250 μM palmitate in vitro. Adenovirus expressing shRNA of mDia1 was used to knockdown mDia1 in cardiomyocytes. Mitochondria were isolated from primary cardiomyocytes to verify the translocation of mDia1. Co-Immunoprecipitation was used to confirm the interaction between mDia1 with succinate dehydrogenase subunits A (SDHA). Results mDia1 had no effects to blood glucose and body weight after 16-week HFD feeding. Loss of mDia1 ameliorated diastolic dysfunction in DCM mice with shorter isovolumic relaxation time (IVRT) and increased E/A ratio in left ventricular. In addition, cardiac hypertrophy was significantly improved in mDia1 ko mice. mDia1 deficiency tended to maintain the normal mitochondrial structure. Results of proteomics showed that mDia1 was involved in mitochondrial function, ATP synthesis and metabolic pathways. Metabolomics showed that HFD induced accumulation of α-ketoglutaric acid in wild type mice compared with mDia1 ko mice. However, mDia1 ko significantly increased fumaric acid but did not affect succinyl-CoA and succinic acid compared with wild type mice. Furthermore，we found that mDia1 translocated into mitochondrial with high glucose and palmitate stimuli. Results of CoIP showed mDia1 interacted with SDHA both in cardiomyocytes and HEK293 cells. Conclusions mDia1 deteriorated DCM through translocating into mitochondrial and interacting with SDHA, a process dependent on metabolic pathways.Figure of Mechanism

ALDH2 mediates the effects of sodium-glucose cotransporter 2 inhibitors (SGLT2i) on improving cardiac remodeling

BackgroundSodium-glucose cotransporter-2 inhibitors (SGLT2i) are now recommended for patients with heart failure, but the mechanisms that underlie the protective role of SGLT2i in cardiac remodeling remain unclear. Aldehyde dehydrogenase 2 (ALDH2) effectively prevents cardiac remodeling. Here, the key role of ALDH2 in the efficacy of SGLT2i on cardiac remodeling was studied.MethodsAnalysis of multiple transcriptomic datasets and two-sample Mendelian randomization were performed to find out the differentially expressed genes between pathological cardiac hypertrophy models (patients) and controls. A pathological cardiac hypertrophy mouse model was established via transverse aortic constriction (TAC) or isoproterenol (ISO). Cardiomyocyte-specific ALDH2 knockout mice (ALDH2CMKO) and littermate control mice (ALDH2flox/flox) were generated to determine the critical role of ALDH2 in the preventive effects of dapagliflozin (DAPA) on cardiac remodeling. RNA sequencing, gene knockdown or overexpression, bisulfite sequencing PCR, and luciferase reporter assays were performed to explore the underlying molecular mechanisms involved.ResultsOnly ALDH2 was differentially expressed when the differentially expressed genes obtained via Mendelian analysis and the differentially expressed genes obtained from the multiple transcriptome datasets were combined. Mendelian analysis revealed that ALDH2 was negatively related to the severity of myocardial hypertrophy in patients. DAPA alleviated cardiac remodeling in mouse hearts subjected to TAC or ISO. ALDH2 expression was reduced, whereas ALDH2 expression was restored by DAPA in hypertrophic hearts. Cardiomyocyte specific ALDH2 knockout abolished the protective role of DAPA in preventing cardiac remodeling. ALDH2 expression and activity were increased in DAPA-treated neonatal rat primary cardiomyocytes (NRCMs), H9C2 cells and AC16 cells. Moreover, DAPA upregulated ALDH2 in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes. Sodium/proton exchanger 1 (NHE1) inhibition contributed to the regulation of ALDH2 by DAPA. DAPA suppressed the production of reactive oxygen species (ROS), downregulated DNA methyltransferase 1 (DNMT1) and subsequently reduced the ALDH2 promoter methylation level. Further studies revealed that DAPA enhanced the binding of nuclear transcription factor Y, subunit A (NFYA) to the promoter region of ALDH2, which was due to the decreased promoter methylation level of ALDH2.ConclusionsThe upregulation of ALDH2 plays a critical role in the protection of DAPA against cardiac remodeling. DAPA enhances the binding of NFYA to the ALDH2 promoter by reducing the ALDH2 promoter methylation level through NHE1/ROS/DNMT1 pathway.Graphical abstract

Pharmacological Preconditioning with Fenofibrate in Cardiomyocyte Cultures of Neonatal Rats Subjected to Hypoxia/Reoxygenation, High Glucose, and Their Combination

Pharmacological preconditioning is an alternative to protect the heart against the consequences of damage from ischemia/reperfusion (I/R). It is based on the administration of specific drugs that imitate the effect of ischemic preconditioning (IPC). Peroxisomal proliferator-activated receptors (PPARs) can prevent apoptosis in pathologies such as I/R and heart failure. Therefore, our objective was to determine if the stimulation of PPARα with fenofibrate (feno) decreases the apoptotic process induced by hypoxia/reoxygenation (HR), high glucose (HG), and HR/HG. For that purpose, cardiomyocyte cultures were divided into the following groups: Group 1—control (Ctrl); Group 2—HR; Group 3—HR + 10 μM feno; Group 4—HG, (25 mM glucose); Group 5—HG + feno; Group 6—HR/HG, and Group 7—HR/HG + feno. Our results indicate that cell viability decreases in neonatal cardiomyocytes undergoing HR, HG, and their combination, while feno improved cell viability. Feno treatment decreased apoptosis compared with HG-, HR-, or HG/HR-vehicle-treated. Nuclear- and mitochondrial-apoptosis markers increased in neonatal cardiomyocytes from HR, HG, and HR/HG; while the cytotoxicity decreased in cells treated with feno. In addition, the expression of Bax, Bad, and caspase 9 decreased due to feno, while 14-3-3ɛ and Bcl2 were increased. Inner mitochondrial cytochrome C increased with feno in every condition, as well as mitochondrial activity. Feno treatment prevented injury in the ultrastructure and in the mitochondrial membranes. Thus, our results suggest that feno decreases apoptosis in neonatal cardiomyocytes, improving the ultrastructure of mitochondria in the pathological conditions studied.

Stochastic and alternating pacing paradigms to assess the stability of cardiac conduction

Reentry, the most common cause of severe arrhythmias, is initiated by slow conduction and conduction block. Hence, evaluating conduction velocity and conduction block is of primary importance. However, the assessment of cardiac conduction safety in experimental and clinical settings remains elusive. To identify markers of conduction instability that can be determined experimentally, we developed an approach based on new pacing paradigms. Conduction across a cardiac tissue expansion was assessed in computer simulations and in experiments using cultures of neonatal murine cardiomyocytes on microelectrode arrays. Simulated and in vitro tissues were paced at a progressively increasing rate, with stochastic or alternating variations of cycle length, until conduction block occurred. Increasing pacing rate led to conduction block near the expansion. When stochastic or alternating variations were introduced into the pacing protocol, the standard deviation and the amplitude of alternating variations of local conduction times emerged as markers of unstable conduction prone to block. In both simulations and experiments, conduction delays were prolonged at the expansion but increased only slightly during the pacing protocol. In contrast, these markers of instability increased several-fold, early before block occurrence. The first and second moments of these two metrics provided an estimation of the site of block and the accuracy of this estimation. Therefore, when beat-to-beat variations of pacing cycle length are introduced into a pacing protocol, the local variability of conduction permits to predict sites of block. Our pacing paradigms may have translational applications in clinical cardiac electrophysiology, particularly in identifying ablation targets during mapping procedures.

Mettl1 knockdown alleviates cardiac I/R injury in mice by inactivating the Mettl1-CYLD-P53 positive feedback loop.

The N7-methylguanosine (m7G) methyltransferase Mettl1 has been recently implicated in cardiac repair and fibrosis. In this study we investigated the role of Mettl1 in mouse cardiomyocytes injury and the underlying mechanisms. Cardiac ischemia/reperfusion (I/R) I/R model was established in mice by ligation of the left anterior descending coronary artery (LAD) for 45 min followed by reperfusion for 24 h. We showed the mRNA and protein levels of Mettl1 were significantly upregulated in mouse I/R hearts and H2O2-treated neonatal mouse cardiomyocytes (NMCMs). Mettl1 knockdown markedly ameliorated cardiac I/R injury, evidenced by decreased infarct size, apoptosis, and improved cardiac function. Overexpression of Mettl1 triggered cardiomyocytes apoptosis in vivo and in vitro. By performing RNA sequencing combined with m7G methylated RNA sequencing in Mettl1-overexpressing mouse hearts, we revealed that Mettl1 catalyzed m7G modification of the deubiquitinase cylindromatosis (CYLD) mRNA to increase the expression of CYLD, which enhanced the stability of P53 via abrogating its ubiquitination degradation. Vice versa, P53 served as a transcriptional factor to positively regulate Mettl1 expression during I/R injury. Knockdown of CYLD mitigated cardiomyocytes apoptosis induced by Mettl1 overexpression or oxidative stress. From the available drug-targets databases and literature, we identified 4 small molecule inhibitors of m7G modification. Sinefungin, one of the Mettl1 inhibitors exerted profound protection against cardiac I/R injury in vivo and in vitro. Collectively, this study has identified Mettl1 as a key regulator of cardiomyocyte apoptosis, and targeting the Mettl1-CYLD-P53 positive feedback circuit may represent a novel therapeutic avenue for alleviating cardiac I/R injury.

Macrophages enhance sodium channel expression in cardiomyocytes.

Cardiac macrophages facilitate electrical conduction through the atrioventricular-node (AV) in mice. A possible role for cardiomyocyte-macrophage coupling on the effect of antiarrhythmic therapy has not been investigated yet. Holter monitoring was conducted in LysMCrexCsf1rLsL-DTR mice (MMDTR) under baseline conditions and after an elctrophysiological stress test by flecainide. In vivo effects were recapitulated in vitro by patch-clamp experiments. The underlying mechanism was characterized by expression and localization analysis of connexin43 (Cx43) and voltage-gated-sodium-channel-5 (Nav1.5). ECG monitoring in MMDTR mice did not show any significant conduction abnormalities but a significantly attenuated flecainide-induced extension of RR- and PP-intervals. Patch-clamp analysis revealed that the application of flecainide to neonatal rat ventricular cardiomyocytes (CMs) changed their resting-membrane-potential (RMP) to more negative potentials and decreased action-potential-duration (APD50). Coupling of macrophages to CMs significantly enhances the effects of flecainide, with a further reduction of the RMP and APD50, mediated by an upregulation of Cx43 and Nav1.5 surface expression. Macrophage depletion in mice does not correlate with cardiac electric conduction delay. Cardiac macrophages amplify the effects of flecainide on electrophysiological properties of cardiomyocytes in vivo and in vitro. Mechanistically, formation of macrophage-cardiomyocyte cell-cell-contacts via Cx43 facilitates the recruitment of Nav1.5 to the cell membrane increasing flecainide effects.

Colchicine Prevents Cardiac Rupture in Mice with Myocardial Infarction by Inhibiting P53-Dependent Apoptosis.

Cardiac rupture is a fatal complication following myocardial infarction (MI) and there are currently no effective pharmacological strategies for preventing this condition. In this study, we investigated the effect of colchicine on post-infarct cardiac rupture in mice and its underlying mechanisms.We induced MI in mice by permanently ligating the left anterior descending artery. Oral colchicine or vehicle was administered at a dose of 0.1 mg/kg/day from day 1 to day 7 after MI. Cultured neonatal cardiomyocytes and fibroblasts were exposed to normoxia or anoxia and treated with colchicine.Colchicine significantly improved the survival rate (colchicine, n = 46: 82.6% versus vehicle, n = 42: 61.9%, P < 0.05) at 1 week after MI. Histological analysis revealed colchicine significantly reduced the infarct size and the number of macrophages around the infarct area. Colchicine decreased apoptosis in the myocardium of the border zone and cultured cardiomyocytes and fibroblasts as assessed by TUNEL assay. Colchicine also attenuated the activation of p53 and decreased the expression of cleaved-caspase 3 and bax, as assessed by Western blotting.Colchicine prevents cardiac rupture via inhibition of apoptosis, which is attributable to the downregulation of p53 activity. Our findings suggest that colchicine may be a prospective preventive medicine for cardiac rupture, however, large clinical trials are required.

SGLT2 inhibitors protect against diabetic cardiomyopathy and atrial fibrillation through a CaMKII independent mechanism.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as an important mediator of the increasingly evident cardioprotective benefits exerted by sodium-glucose transport protein 2 channel inhibitors (SGLT2i). However, the exact nature of the relationship between CaMKII and SGLT2i remains unclear. Here, we find that empagliflozin but not dapagliflozin attenuated susceptibility to atrial fibrillation (AF) in a type 2 diabetic (T2D) mouse model. However, both empagliflozin and dapagliflozin protected from diabetic cardiomyopathy in T2D mice. We then used real-time microscopy of neonatal rat ventricular cardiomyocytes (NRVMs) with the CaMKII biosensor - CaMKAR to demonstrate that direct inhibition of CaMKII is not essential for the effects of SGLT2i in these cells. Therefore, we conclude that the benefits of SGLT2i in heart disease likely occur through indirect modulation of CaMKII activity, or possibly through an alternative pathway altogether.

Leiomodin 2 neonatal dilated cardiomyopathy mutation results in altered actin gene signatures and cardiomyocyte dysfunction

Neonatal dilated cardiomyopathy (DCM) is a poorly understood muscular disease of the heart. Several homozygous biallelic variants in LMOD2, the gene encoding the actin-binding protein Leiomodin 2, have been identified to result in severe DCM. Collectively, LMOD2-related cardiomyopathies present with cardiac dilation and decreased heart contractility, often resulting in neonatal death. Thus, it is evident that Lmod2 is essential to normal human cardiac muscle function. This study aimed to understand the underlying pathophysiology and signaling pathways related to the first reported LMOD2 variant (c.1193 G > A, p.Trp398*). Using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and a mouse model harboring the homologous mutation to the patient, we discovered dysregulated actin-thin filament lengths, altered contractility and calcium handling properties, as well as alterations in the serum response factor (SRF)-dependent signaling pathway. These findings reveal that LMOD2 may be regulating SRF activity in an actin-dependent manner and provide a potential new strategy for the development of biologically active molecules to target LMOD2-related cardiomyopathies.

IDI2-AS1 influences the development of acute myocardial infarction by regulating NR4A2 through microRNA-33b-5p

To explore the effect and correlation of long non-coding RNA (lncRNA) IDI2-AS1/microRNA-33b-5p (miR-33b-5p)/nuclear receptor-associated protein NR4A2 competitive endogenous RNA (ceRNA) regulatory network on acute myocardial infarction (AMI), and to verify whether IDI2-AS1 regulates NR4A2 through miR-33b-5p to affect the occurrence and development of myocardial infarction. The miRNA and mRNA expression chips related to myocardial infarction were obtained from gene expression omnibus (GEO), and the differential expression was analyzed. The upstream regulatory mechanism of NR4A2 was predicted using TargetScan database. Thirty-two male C57/BL6 mice were divided into Sham group, AMI model group, miR-33b-5p mimic group [miR-33b-5p mimic lentivirus (5×107 TU) was injected locally into the heart tissue during ligation] and miR-33b-5p inhibitor group [miR-33b-5p inhibitor lentivirus (5×107 TU) was injected locally into the heart tissue during ligation] according to random number table method, with 8 mice per group. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were asseessed by echocardiography, left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were calculated. After the last weighing, the anesthetized mice were sacrificed and the heart tissues were taken. Masson staining of the heart tissues was observed under light microscope, myocardial collagen volume fraction (CVF) and infarct size were calculated. Cardiomyocytes of SPF grade SD rats were collected. They were divided into normal control group (control group), ischemia-hypoxia model group, miR-33b-5p mimic transfection group (miR-33b-5p mimic transfection group before ischemia and hypoxia treatment) and miR-33b-5p inhibitor transfection group (miR-33b-5p inhibitor transfection group before ischemia and hypoxia treatment). The activity of caspase-3/7 in cardiomyocytes was measured. The levels of interleukins (IL-1β, IL-6) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The levels of malondialdehyde (MDA), superoxide dismutase (SOD), creatine kinase (CK), MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) were detected by colorimetry. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of apoptosis-related proteins Bax and Bcl-2, cytochrome C (Cyt C) and IDI2-AS1/miR-33b-5p/NR4A2 regulatory axis genes. The myocardial infarction microarray analysis showed that NR4A2 expression was significantly up-regulated in myocardial infarction, with predicted upstream regulatory mechanisms indicating its possible influence through the IDI2-AS1/miR-33b-5p/NR4A2 regulatory axis. Echocardiographic detection showed that compared with AMI model group and miR-33b-5p inhibitor group, LVEF and LVFS in the heart tissue of mice in miR-33b-5p mimic group were significantly increased, while the levels of LVEDD, LVESD, CK, CK-MB and LDH were significantly decreased, with statistical significance. Light microscope showed myocardial fibrosis and myocardial infarction in AMI model group and miR-33b-5p inhibitor group. In the miR-33b-5p mimic group, the degree of myocardial fibrosis was decreased and the myocardial infarction size was significantly reduced. Compared with AMI model group and miR-33b-5p inhibitor group, the levels of MDA, IL-1β, IL-6, TNF-α and the expressions of Bax and Cyt C in the heart tissue of mice in miR-33b-5p mimic group were significantly decreased, while the levels of SOD and Bcl-2 expression were significantly increased, and the differences were statistically significant. The expressions of IDI2-AS1 and NR4A2 in the heart tissue of mice in miR-33b-5p mimic group were significantly lower than those in AMI model group and miR-33b-5p inhibitor group [IDI2-AS1 (2-ΔΔCt): 1.96±0.08 vs. 2.73±0.08, 3.10±0.05, NR4A2 (2-ΔΔCt): 2.36±0.07 vs. 3.16±0.08, 3.80±0.08, all P < 0.01]. The expression of miR-33b-5p was significantly higher than that of AMI model group and miR-33b-5p inhibitor group (2-ΔΔCt: 0.88±0.07 vs. 0.57±0.07, 0.23±0.01, both P < 0.01). The cell experiment results showed that the caspase-3/7 activity of rat neonatal cardiomyocytes in the miR-33b-5p mimic transfection group was significantly lower than that in the ischemia-hypoxia model group and the miR-33b-5p inhibitor transfection group, suggesting that miR-33b-5p can significantly reduce the apoptosis level of the ischemia-hypoxia model. The levels of peroxidation and inflammation indexes, important genes of apoptosis pathway and the expression of IDI2-AS1/miR-33b-5p/NR4A2 regulatory axis of rat neonatal cardiomyocytes in all groups were consistent with the above. IDI2-AS1 can regulate NR4A2 through miR-33b-5p, thus affecting the occurrence and development of AMI.

Altered O-glycosylation of β1-adrenergic receptor N-terminal single-nucleotide variants modulates receptor processing and functional activity.

N-terminal nonsynonymous single-nucleotide polymorphisms (SNPs) of G protein-coupled receptors (GPCRs) are common and often affect receptor post-translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human β1-adrenergic receptor (β1AR) is O-glycosylated in the N-terminal extracellular domain by polypeptide GalNAc transferase-2 that co-regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O-glycosylation assays, analysis of native receptor N-terminal O-glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O-glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O-glycosites that enhanced or inhibited cleavage at the adjacent sites (P52↓L53 and R31↓L32), respectively, or abolished the major site at R31↓L32 (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full-length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol-mediated signaling in an enhanced bystander bioluminescence energy transfer β-arrestin2 recruitment assay in a coordinated manner with the common C-terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a β1AR gain-of-function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.

The Kv4 potassium channel modulator NS5806 attenuates cardiac hypertrophy in vivo and in vitro

The compound NS5806 is a Kv4 channel modulator. This study investigated the chronic effects of NS5806 on cardiac hypertrophy induced by transverse aortic constriction (TAC) in mice in vivo and on neonatal rat ventricular cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) in vitro. Four weeks after TAC, NS5806 was administered by gavage for 4 weeks. Echocardiograms revealed pronounced left ventricular (LV) hypertrophy in TAC-treated mice compared with sham mice. NS5806 attenuated LV hypertrophy, as manifested by the restoration of LV wall thickness and weight and the reversal of contractile dysfunction in TAC-treated mice. NS5806 also blunted the TAC-induced increases in the expression of cardiac hypertrophic and fibrotic genes, including ANP, BNP and TGF-β. Electrophysiological recordings revealed a significant prolongation of action potential duration and QT intervals, accompanied by an increase in susceptibility to ventricular arrhythmias in mice with cardiac hypertrophy. However, NS5806 restored these alterations in electrical parameters and thus reduced the incidence of mouse sudden death. Furthermore, NS5806 abrogated the downregulation of the Kv4 protein in the hypertrophic myocardium but did not influence the reduction in Kv4 mRNA expression. In addition, NS5806 suppressed in vitro cardiomyocyte hypertrophy. The results provide novel insight for further ion channel modulator development as a potential treatment option for cardiac hypertrophy.

Fibronectin type III domain containing 4 alleviates myocardial ischemia/reperfusion injury via the Nrf2-dependent antioxidant pathway

Fibronectin type III domain containing 4 (FNDC4) is highly homologous with FNDC5, which possesses various cardiometabolic protective functions. Emerging evidence suggests a noteworthy involvement of FNDC4 in fat metabolism and inflammatory processes. This study aimed to characterize the role of FNDC4 in myocardial ischemia/reperfusion (MI/R) injury and decrypt its underlying mechanisms. MI/R models of mice were established to investigate the alteration of FNDC4 in plasma and myocardium. We observed that plasma FNDC4 in MI/R-injury mice and patients experiencing acute myocardial infarction were both significantly reduced as opposed to their respective controls. Likewise, FNDC4 expression of myocardium decreased markedly in MI/R mice compared to the sham-operated group. Mice of FNDC4 knockout and myocardial overexpression were further introduced to elucidate the role of FNDC4 in MI/R injury by detecting cardiomyocyte apoptosis, myocardial infarct size, and cardiac function. Ablation of FNDC4 exacerbated cardiac dysfunction, increased myocardial infarction area and cardiomyocyte apoptosis when matched with wild-type mice post-MI/R. In contrast, FNDC4 overexpression through intramyocardial injection of rAAV9-Fndc4 significantly ameliorated cardiac function, reduced myocardial infarction area and cardiomyocyte apoptosis compared to sham group. Additionally, hypoxia-reoxygenation (H/R) was used to induce cardiomyocyte apoptosis, and to further elucidate the direct effects of FNDC4 on cardiomyocytes in vitro, and the results demonstrated that neonatal rat ventricular cardiomyocytes overexpressing FNDC4 showed less H/R-induced apoptosis, as evidenced by cleaved caspase 3 expression, TUNEL staining and flow cytometry. By performing RNA-seq analysis followed by cause-effect analysis, ERK1/2-Nrf2 pathway-mediated antioxidative effects were responsible for the protective roles of FNDC4 on cardiomyocytes. In summary, FNDC4 exerts cardioprotection against MI/R injury predominantly through mitigating oxidative stress responses and reducing cardiomyocyte apoptosis. These insights solidify the proposition of FNDC4 as a potential therapeutic aim for tackling MI/R damage.

Advancing Myocardial Infarction Treatment: Harnessing Multi-Layered Recellularized Cardiac Patches with Fetal Myocardial Scaffolds and Acellular Amniotic Membrane.

Myocardial infarction (MI) is a leading cause of irreversible functional cardiac tissue loss, requiring novel regenerative strategies. This study assessed the potential therapeutic efficacy of recellularized cardiac patches, incorporating fetal myocardial scaffolds with rat fetal cardiomyocytes and acellular human amniotic membrane, in adult Wistar rat models of MI. Decellularized myocardial tissue was obtained from 14 to 16 week-old human fetuses that had been aborted. Chemical detergents (0.1% EDTA and 0.2% sodium dodecyl sulfate) were used to prepare the fetal extracellular matrix (ECM), which was characterized for bio-scaffold microstructure and biocompatibility via scanning electron microscopy (SEM) and MTT assay, respectively. Neonatal cardiomyocytes were extracted from the ventricles of one-day-old Wistar rats' littermates and characterized through immunostaining against Connexin-43 and α-smooth muscle actin. The isolated cells were seeded onto decellularized tissues and covered with decellularized amniotic membrane. Sixteen healthy adult Wistar rats were systematically allocated to control and MI groups. MI was induced via arterial ligation. Fourteen days post-operation, the MI group was received the engineered patches. Following a two-week post-implantation period, the animals were euthanized, and the hearts were harvested for the graft evaluation. Histological analysis, DAPI staining, and ultra-structural examination corroborated the successful depletion of cellular elements, while maintaining the integrity of the fetal ECM and architecture. Subsequent histological and immunohistochemichal (IHC) evaluations confirmed effective cardiomyocyte seeding on the scaffolds. The application of these engineered patches in MI models resulted in increased angiogenesis, reduced fibrosis, and restricted scar tissue formation, with the implanted cardiomyocytes remaining viable at graft sites, indicating prospective in vivo cell viability. This study suggests that multi-layered recellularized cardiac patches are a promising surgical intervention for myocardial infarction, showcasing significant potential by promoting angiogenesis, mitigating fibrosis, and minimizing scar tissue formation in MI models. These features are pivotal for enhancing the therapeutic outcomes in MI patients, focusing on the restoration of the myocardial structure and function post-infarction.

PiR112710 attenuates diabetic cardiomyopathy through inhibiting Txnip/NLRP3-mediated pyroptosis in db/db mice

PIWI-interacting RNAs (piRNAs) are involved in the regulation of hypertrophic cardiomyopathy, heart failure and myocardial methylation. However, their functions and the underlying molecular mechanisms in diabetic cardiomyopathy (DCM) have yet to be fully elucidated. In the present study, a pyroptosis-associated piRNA (piR112710) was identified that ameliorates cardiac remodeling through targeting the activation of inflammasomes and mitochondrial dysfunction that are mediated via the thioredoxin-interacting protein (Txnip)/NLRP3 signaling axis. Subsequently, the cardioprotective effects of piR112710 on both the myocardium from db/db mice and cardiomyocytes from neonatal mice that were incubated with a high concentration of glucose combined with palmitate were examined. piR112710 was found to significantly improve cardiac dysfunction in db/db mice, characterized by improved echocardiography, lower levels of fibrosis, attenuated expression levels of inflammatory factors and pyroptosis-associated proteins (namely, Txnip, ASC, NLRP3, caspase-1 and GSDMD-N), and enhanced myocardial mitochondrial respiratory functions. In cultured neonatal mice cardiomyocytes, piR112710 deficiency and high glucose along with palmitate treatment led to significantly upregulated expression levels of pyroptosis associated proteins and collagens, oxidative stress, mitochondrial dysfunction and increased levels of inflammatory factors. Supplementation with piR112710, however, led to a reversal of the aforementioned changes induced by high glucose and palmitate. Mechanistically, the cardioprotective effect of piR112710 appears to be dependent upon effective elimination of reactive oxygen species and inactivation of the Txnip/NLRP3 signaling axis. Taken together, the findings of the present study have revealed that the piRNA-mediated inhibitory mechanism involving the Txnip/NLRP3 axis may participate in the regulation of pyroptosis, which protects against DCM both in vivo and in vitro. piR112710 may therefore be a potential therapeutic target for the reduction of myocardial injury caused by cardiomyocyte pyroptosis in DCM.

Abstract We039: Adducin Promotes Cardiomyocyte Sarcomere Disassembly

Introduction: Recent interest in understanding the cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. Despite advancements, the intricacies of cardiomyocyte mitosis, especially the breakdown of sarcomeres to enable daughter cardiomyocytes' division, remain largely unexplored. Hypothesis: This study investigates the role of Adducin in the disassembly of sarcomeres during cardiomyocyte mitosis, hypothesizing that Adducin serves as a crucial regulator in this process. Goals: Our objective is to elucidate the function of Adducin in sarcomere disassembly during the mitosis of mammalian cardiomyocytes and understand how this process is regulated. Methods: We examined the expression patterns of α/γ-Adducins, particularly in neonatal cardiomyocytes undergoing mitosis. Subsequently, we experimented with the overexpression of various α-Adducin splices and phosphorus in vitro and in vivo, with a keen interest in the phosphorylation effects of α-Adducin on sarcomere disassembly. Results: Here, we identify Adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-Adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phosphoforms of α-Adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-Adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Additionally, co-overexpressing this α-Adducin variant with γ-Adducin in adult mice stabilizes the Adducin complex and maintains sarcomere disassembly, facilitated through interactions with α-Actinin. Conclusion: The study highlights Adducin, particularly α-Adducin phosphorylated at Thr445/Thr480, as a pivotal regulator of sarcomere disassembly during neonatal cardiomyocyte mitosis. This mechanism is essential for coordinating the cytoskeletal changes necessary for the mitotic process in cardiomyocytes, offering insights into potential therapeutic targets for cardiomyopathy.

Abstract We034: Hjurp Promotes Cardiomyocyte Proliferation and Heart Regeneration by Mediating CenpA Assembly

Background: Loss of cardiomyocytes without replenishment is one of the main cellular mechanisms leading to heart failure. Promoting cardiomyocyte proliferation and heart regeneration has become a therapeutic strategy to rescue heart failure. Therefore, we need to understand the differences between neonatal cardiomyocytes and adult cardiomyocytes, summarize the mechanism of cardiomyocyte exit from the cell cycle, and find potential intervention targets. Method: Combined analysis of bulk RNA-seq, ATAC-seq, and histone CUT&amp;Tag of 1-day-old mouse and 7-day-old mouse cardiomyocytes to screen for Hjurp . In vitro , siRNA was used to knock down Hjurp in neonatal rat cardiomyocytes, and adenovirus was used to overexpress Hjurp . RT-qPCR and immunofluorescence were used to detect cardiomyocyte proliferation and cardiomyocyte number. In vivo , adenovirus was injected into the mouse heart in situ to knock down Hjurp . Immunofluorescence was used to detect cardiomyocyte proliferation. Masson staining was used to detect the proportion of myocardial fibrosis and echocardiography was used to detect cardiac function to reflect cardiac regeneration and repair. Results: HJURP was identified by RNA-seq, ATAC-seq, and histone CUT&amp;Tag as a key regulator of cardiomyocyte proliferation during neonatal heart regeneration in mice. The expression levels of Hjurp and Cenp-A decrease with the weakening of cardiomyocyte proliferation ability. In vitro , Hjurp overexpression promoted whereas Hjurp knockdown attenuated cardiomyocyte proliferation. In vivo , cardiomyocyte proliferation and heart regeneration were suspended in neonatal mice after cardiac injury when Hjurp was knockdown in cardiomyocytes. Conclusion: Our study confirms that HJURP is necessary for cardiomyocyte proliferation and cardiac regeneration after cardiac injury. As an assembly element of centromeres during the cell cycle, overexpression of HJURP is sufficient to promote cardiomyocyte proliferation, which may be accomplished by promoting CENP-A assembly. Thus, HJURP might serve as a potential novel target in heart regeneration after myocardial infarction.