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Purpose of Review Formation of the heart requires the coordinated addition of multiple progenitor sources which have undergone different pathways of specification and differentiation. In this review, I aim to put into context how recent studies defining cardiac progenitor heterogeneity build on our understanding of early heart development and also discuss the questions raised by this new insight. Recent Findings With the development of sequencing technologies and imaging approaches, it has been possible to define, at high temporal resolution, the molecular profile and anatomical location of cardiac progenitors at the single-cell level, during the formation of the mammalian heart. Summary Given the recent progress in our understanding of early heart development and technical advances in high-resolution time-lapse imaging and lineage analysis, we are now in a position of great potential, allowing us to resolve heart formation at previously impossible levels of detail. Understanding how this essential organ forms not only addresses questions of fundamental biological significance but also provides a blueprint for strategies to both treat and model heart disease.

Cardiac dysfunction and remodeling are serious complications of sepsis and are the main causes of death in sepsis. RCAN1 is a feedback regulator of cardiac hypertrophy. Here, we aim to investigate the role of RCAN1 in septic cardiomyopathy.Mice were randomly divided into control-WT, control-RCAN1Our data show that RCAN1 was downregulated in septic mouse heart and LPS-induced cardiomyocytes. RCAN1Our finding demonstrated that RCAN1 deficiency aggravated mitochondrial injury and septic cardiomyopathy through activating CaMKII. RCAN1 serves as a novel therapeutic target for treatment of sepsis-related cardiac remodeling and dysfunction.

Acute myocardial infarction (AMI) leads to anoxia and ischemia of cardiomyocytes, followed by apoptosis. This study investigated the protective effect of ginsenoside Rg1 (Rg1) on myocardial ischemia injury in rats with AMI. Rats were randomly divided into five groups: group A (blank control group), group B (hypoxia/reoxygenation group), group C (hypoxia/reoxygenation + 10 mg/L Rg1), group D (hypoxia/reoxygenation + 20 mg/L Rg1) and group E (hypoxia/reoxygenation + 40 mg/L Rg1). The survival rate, apoptosis rate, expression of cyclin-dependent kinase 4 (CDK4), fibroblast growth factor 9 (FGF9), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), microvessel density and myocardial infarction area of rats in each group were compared. The expressions of CDK4 and FGF9, the contents of SOD and GSH-Px in groups C, D and E injected with Rg1 were significantly promoted compared to group B without Rg1 injection (P lt; 0.05). The survival rate of myocardial cells was significantly increased while the apoptosis rate was significantly decreased in group C, D, E compared to group B (P lt; 0.05). On the 3rd, 7th and 10th day following Rg1 treatment, the infarct area of E group was significantly decreased in three groups C, D, E, and the microvessel density of infarct area was significantly increased compared with group B (P lt; 0.05). So, Rg1 can improve the survival rate of myocardial cells, reduce the apoptosis rate and the area of myocardial infarction, and increase the microvessel density of infarct area, thus playing a protective role in ischemic myocardial cells of AMI rats.

Several computational studies have been undertaken to explore the Ca2+-induced Ca2+ release (CICR) events in cardiac myocytes and along with experimental studies it has given us invaluable insight into the mechanism of CICR from spark/blink initiation to termination and regulation, and their interplay under normal and pathological conditions. The computational modelling of this mechanism has mainly been investigated using coupled differential equations (DEs). However, there is a lack of computational investigation into (1) how the different formulation of coupled DEs capture the Ca2+ movement in the cytosol and sarcoplasmic reticulum (SR), (2) the buffer and dye inclusion in both compartments, and (3) the effect of buffer and dye properties on the calcium behaviour. This work is set out to explore (1) the effect of different coupled formulation of DEs on spark/blink occurrence, (2) the inclusion of improved sarcoplasmic buffering properties, and (3) the effects of cytosolic and sarcoplasmic dye and buffer properties on Ca2+ movement. The simulation results show large discrepancies between different formulations of the governing equations. Additionally, extension of the model to include sarcoplasmic buffering properties show normalised fluorescent dye profiles to be in good agreement with experimental and amongst its one- and two-dimensional representations.

Autophagy is known as a conserved mechanism in order to preserve cell survival under various stress conditions via maintaining cellular homeostasis. Although autophagy is active in the heart at baseline and plays a critical role in cell survival, inappropriate activation of autophagy following ischemia/reperfusion (I/R) injury leads to cell death.The distinct functions of autophagy in myocardial I/R injury condition have been examined in numerous studies, however, contradicting results have been achieved in this field. These studies have documented that autophagy acts as a double-edged sword in myocardial I/R injury. Clarifying the exact role of autophagy in determining the health or death of cardiomyocytes under I/R injury is very helpful to achieve better cardioprotection in prospective clinical studies. Thus, autophagy may be an interesting target for the treatment or prevention of myocardial I/R injury. But before considering this matter, it is necessary to address the gaps in our knowledge about the complex role of autophagy in myocardial I/R injury.In this review, by providing updated data about the role of autophagy in the heart during ischemia and reperfusion, we tried to provide more insights in this context and encourage scientists to pay special attention towards manipulating autophagy as an intriguing and powerful approach in cardioprotection.

To examine the effect of Shenmai Injection (SMJ) on ferroptosis during myocardial ischemia reperfusion (I/R) injury in rats and the underlying mechanism.A total of 120 SPF-grade adult male SD rats, weighing 220-250 g were randomly divided into different groups according to a random number table. Myocardial I/R model was established by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion. SMJ was injected intraperitoneally at the onset of 120 min of reperfusion, and erastin (an agonist of ferroptosis), ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) and ML385 (an inhibitor of nuclear factor erythroid-2 related factor 2 (Nrf2)) were administered intraperitoneally separately 30 min before myocardial ischemia as different pretreatments. Cardiac function before ischemia, after ischemia and after reperfusion was analysed. Pathological changes in the myocardium and the ultrastructure of cardiomyocytes were observed, and the myocardial infarction area was measured. Additionally, the concentration of FeCompared with the sham group, I/R significantly injured heart tissues, as evidenced by the disordered, ruptured and oedematous myocardial fibres; the increases in infarct size, serum CK-MB, cTnI and MDA levels, and myocardial FeFerroptosis is involved in myocardial I/R injury. Pretreatment with SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis, thereby providing a strategy for the prevention and treatment of ischemic heart diseases.

Tax1 banding protein 1 (Tax1bp1) was originally identified as an NF-κB regulatory protein that participated in inflammatory, antiviral and innate immune processes. Tax1bp1 also functions as an autophagy receptor that plays a role in autophagy. Our previous study shows that Tax1bp1 protects against cardiomyopathy in STZ-induced diabetic mice. In this study we investigated the role of Tax1bp1 in heart failure. Pressure overload-induced heart failure model was established in mice by aortic banding (AB) surgery, and angiotensin II (Ang II)-induced heart failure model was established by infusion of Ang II through osmotic minipump for 4 weeks. We showed that the expression levels of Tax1bp1 in the heart were markedly increased 2 and 4 weeks after AB surgery. Knockdown of Tax1bp1 in mouse hearts significantly ameliorated both AB- and Ang II infusion-induced heart failure parameters. On the contrary, AB-induced heart failure was aggravated in cardiac-specific Tax1bp1 transgenic mice. Similar results were observed in neonatal rat cardiomyocytes (NRCMs) under Ang II insult. We demonstrated that the pro-heart failure effect of Tax1bp1 resulted from its interaction with the E3 ligase ITCH to promote the transcription factor P73 ubiquitination and degradation, causing enhanced BCL2 interacting protein 3 (BNIP3)-mediated cardiomyocyte apoptosis. Knockdown ITCH or BNIP3 in NRCMs significantly reduced Ang II-induced apoptosis in vitro. Similarly, BNIP3 knockdown attenuated heart failure in cardiac-specific Tax1bp1 transgenic mice. In the left ventricles of heart failure patients, Tax1bp1 expression level was significantly increased; Tax1bp1 gene expression was negatively correlated with left ventricular ejection fraction in heart failure patients. Collectively, the Tax1bp1 increase in heart failure enhances ITCH-P73-BNIP3-mediated cardiomyocyte apoptosis and induced cardiac injury. Tax1bp1 may serve as a potent therapeutic target for the treatment of heart failure.• Cardiac Tax1bp1 transgene mice were more vulnerable to cardiac dysfunction under stress.• Cardiac Tax1bp1 transgene mice were more vulnerable to cardiac dysfunction under stress.• Knockout of Tax1bp1 in mouse hearts ameliorated heart failure induced by pressure overload.• Tax1bp1 interacts with the E3 ligase Itch to promote P73 ubiquitination and degradation, causing enhanced BNIP3-mediated apoptosis.• Tax1bp1 may become a target of new therapeutic methods for treating heart failure.

The adult zebrafish heart has a high capacity for regeneration following injury. However, the composition of the regenerative niche has remained largely elusive. Here, we dissected the diversity of activated cell states in the regenerating zebrafish heart based on single-cell transcriptomics and spatiotemporal analysis. We observed the emergence of several transient cell states with fibroblast characteristics following injury, and we outlined the proregenerative function of collagen-12-expressing fibroblasts. To understand the cascade of events leading to heart regeneration, we determined the origin of these cell states by high-throughput lineage tracing. We found that activated fibroblasts were derived from two separate sources: the epicardium and the endocardium. Mechanistically, we determined Wnt signalling as a regulator of the endocardial fibroblast response. In summary, our work identifies specialized activated fibroblast cell states that contribute to heart regeneration, thereby opening up possible approaches to modulating the regenerative capacity of the vertebrate heart.

Background Studies have shown that histone H3 methylation is involved in regulating the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs). KDM5B can specifically reduce the level of histone 3 lysine 4 trimethylation (H3K4me3), thereby activating the expression of related genes and participating in biological processes such as cell differentiation, embryonic development and tumor formation. Whether KDM5B is involved in the regulation of BMSCs differentiation into cardiomyocytes through the above manner has not been reported. Objective To investigate the effect of KDM5B on the induction and differentiation of swine BMSCs into myocardial cells in vitro. Methods Swine bone marrow BMSCs were isolated and cultured, and the overexpression, interference expression and blank vector of KMD5B were constructed and transfected by lentivirus. BMSCs was induced to differentiate into cardiomyocytes by 5-azacytidine (5-AZA) in vitro, and the differentiation efficiency was compared by immunofluorescence, RT-PCR, Western Blot and whole-cell patch clamp detection. Result Compared with the control group, the expression levels of histone H3K4me3 and pluripotency gene Nanog in KDM5B overexpression group were significantly decreased, while the expression level of key myocardial gene HCN4 and myocardial marker gene α-Actin and cTNT were significantly increased, and the Na+ current density on the surface of differentiated myocardial cell membrane was significantly increased. Meanwhile, the corresponding results of the KDM5B silent expression group were just opposite. Conclusions It indicated that enhanced KDM5B expression could promote the differentiation of BMSCs into cardiomyocytes and improve the differentiation efficiency by controlling H3K4 methylation levels.

Restoring the blood perfusion of ischemic heart tissues is the main treatment for myocardial ischemia. However, the accompanying myocardial ischemia reperfusion injury (IRI) would aggravate myocardial damage. Previous studies have confirmed that aryl hydrocarbon receptor (AhR) is closely correlated to kidney and intestinal IRI. The present study aimed to explore the relationship between AhR and myocardial IRI.An oxygen glucose deprivation/reoxygenation (OGD/R) model of H9c2 cells and an ischemia/reperfusion (I/R) model of Sprague-Dawley rat myocardium were established. OGD/R cells and myocardial IRI rats were treated with different concentrations of the AhR antagonist CH-223191 or agonist 6-formylindolo[3,2-b] carbazole (FICZ). Under the conditions of normoxia and hypoxia/reoxygenation, the activity of cardiomyocytes, lactate dehydrogenase (LDH) and cell reactive oxygen species (ROS) were detected. In rats, myocardial pathological damage and markers of myocardial injury were detected.According to the results of the cell viability, LDH and ROS tests in vitro, both CH-223191 and FICZ showed no myocardial protection under OGD/R conditions. However, the histological staining and analysis of myocardial injury marker LDH in vitro revealed that CH-223191 could significantly reduce the myocardial IRI.AhR exhibited a different effect on myocardial IRI in vitro and in vivo. In vivo, CH-223191 could significantly alleviate the myocardial IRI, suggesting that inhibition of AhR may play a role in myocardial protection, and AhR may serve as a potential treatment target for myocardial IRI.

Mitophagy, a mitochondria-specific form of autophagy, removes dysfunctional mitochondria and is hence an essential process contributing to mitochondrial quality control. PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are critical molecules involved in stress-induced mitophagy, but the intracellular signaling mechanisms by which this pathway is regulated are unclear. We tested the hypothesis that signaling through RhoA, a small GTPase, induces mitophagy via modulation of the PINK1/Parkin pathway as a protective mechanism against ischemic stress. We demonstrate that expression of constitutively active RhoA as well as sphingosine-1-phosphate induced activation of endogenous RhoA in cardiomyocytes result in an accumulation of PINK1 at mitochondria. This is accompanied by translocation of Parkin to mitochondria and ubiquitination of mitochondrial proteins leading to recognition of mitochondria by autophagosomes and their lysosomal degradation. Expression of RhoA in cardiomyocytes confers protection against ischemia, and this cardioprotection is attenuated by siRNA-mediated PINK1 knockdown. In vivo myocardial infarction elicits increases in mitochondrial PINK1, Parkin, and ubiquitinated mitochondrial proteins. AAV9-mediated RhoA expression potentiates these responses and a concurrent decrease in infarct size is observed. Interestingly, induction of mitochondrial PINK1 accumulation in response to RhoA signaling is neither mediated through its transcriptional upregulation nor dependent on depolarization of the mitochondrial membrane, the canonical mechanism for PINK1 accumulation. Instead, our results reveal that RhoA signaling inhibits PINK1 cleavage, thereby stabilizing PINK1 protein at mitochondria. We further show that active RhoA localizes at mitochondria and interacts with PINK1, and that the mitochondrial localization of RhoA is regulated by its downstream effector protein kinase D. These findings demonstrate that RhoA activation engages a unique mechanism to regulate PINK1 accumulation, induce mitophagy and protect against ischemic stress, and implicates regulation of RhoA signaling as a potential strategy to enhance mitophagy and confer protection under stress conditions.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), has been confirmed to recruit enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) to regulate cardiomyocyte apoptosis in diabetic cardiomyopathy. However, whether the similar regulatory axis exists in sepsis-induced myocardial dysfunction (SIMD) has not been clearly established. The current study sought to define the mechanism governing MALAT1-mediated EZH2 in SIMD. MALAT1 was significantly upregulated in lipopolysaccharide-induced cardiomyocytes. Depletion of MALAT1 by caudal vein injection of small interfering RNA targeting MALAT1 alleviated myocardial injury in SIMD rats, restored cardiac function, reduced oxidative stress production and fibrosis, and inhibited inflammatory factors and apoptosis in myocardial tissues. Moreover, MALAT1 bound to EZH2 and promoted EZH2 activity in the nucleus of cardiomyocytes. EZH2 repressed ubiquitin-specific peptidase 22 (USP22) expression through H3K27me3 modification. EZH2 elevation aggravated the cardiac injury in SIMD rats, while USP22 upregulation inhibited the effect of EZH2, which reduced the cardiac injury in SIMD rats. Taken together, MALAT1 decreased USP22 expression by interacting with EZH2, thereby worsening SIMD, highlighting an attractive therapeutic strategy for SIMD.

Cardiovascular diseases (CVDs) are globally known to be important causes of mortality and disabilities. Common treatment strategies for CVDs, such as pharmacological therapeutics impose serious challenges due to the failure of treatments for myocardial necrosis. By contrast, stem cells (SCs) based therapies are seen to be promising approaches to CVDs treatment. In such approaches, cardiomyocytes are differentiated from SCs. To fulfill SCs complete potential, the method should be appointed to generate cardiomyocytes with more mature structure and well-functioning operations. For heart repairing applications, a greatly scalable and medical-grade cardiomyocyte generation must be used. Nonetheless, there are some challenges such as immune rejection, arrhythmogenesis, tumorigenesis, and graft cell death potential. Herein, we discuss the types of potential SCs, and commonly used methods including embryoid bodies related techniques, co-culture, mechanical stimulation, and electrical stimulation and their applications, advantages and limitations in this field. An estimated 17.9 million people died from CVDs in 2019, representing 32 % of all global deaths. Of these deaths, 85 % were due to heart attack and stroke.

Identification of early biomarkers of heart injury and drug-induced cardiotoxicity is important to eliminate harmful drug candidates early in preclinical development and to prevent severe drug effects. The main objective of this study was to investigate the expression of microRNAs (miRNAs) in human-induced pluripotent stem cell cardiomyocytes (hiPSC-CM) in response to a broad range of cardiotoxic drugs. Next generation sequencing was applied to hiPSC-CM treated for 72 h with 40 drugs falling into the categories of functional (i.e., ion channel blockers), structural (changes in cardiomyocytes structure), and general (causing both functional and structural) cardiotoxicants as well as non-cardiotoxic drugs. The largest changes in miRNAs expression were observed after treatments with structural or general cardiotoxicants. The number of deregulated miRNAs was the highest for idarubicin, mitoxantrone, and bortezomib treatments. RT-qPCR validation confirmed upregulation of several miRNAs across multiple treatments at therapeutically relevant concentrations: hsa-miR-187-3p, hsa-miR-146b-5p, hsa-miR-182-5p (anthracyclines); hsa-miR-365a-5p, hsa-miR-185-3p, hsa-miR-184, hsa-miR-182-5p (kinase inhibitors); hsa-miR-182-5p, hsa-miR-126-3p and hsa-miR-96-5p (common some anthracyclines, kinase inhibitors and bortezomib). Further investigations showed that an upregulation of hsa-miR-187-3p and hsa-miR-182-5p could serve as a potential biomarker of structural cardiotoxicity and/or an additional endpoint to characterize cardiac injury in vitro.

Doxorubicin (Dox), an effective therapy in different types of cancer, is known to exhibit cardiotoxic effects. Despite previous studies indicating the benefits of dapagliflozin (DAPA) in patients experiencing heart failure, it remains uncertain whether DAPA exerts a protective effect on Dox-induced cardiac dysfunction. Signal transducer and activator of transcription 3 (STAT3) participates in various mechanisms of cardioprotection. Herein, we aimed to investigate the effects of DAPA on Dox-induced cardiotoxicity and the role of STAT3. Sprague-Dawley rats were pretreated with oral DAPA for 6 weeks followed by Dox for 4 weeks. Sequential echocardiography was applied to assess cardiac function. For in vitro analysis, cardiomyocytes were treated with 10 μM DAPA and subsequently exposed to 1 μM Dox. The expression of reactive oxygen species- and apoptosis-related proteins was measured. Using STAT3 siRNA, we further examined the effects of STAT3 effect on DAPA-associated protection against Dox-induced apoptosis. In rats treated with Dox, DAPA significantly reduced cardiac fibrosis and improved cardiac function and hemodynamics. Additionally, DAPA effectively inhibited Dox-induced apoptosis and reactive oxygen species (ROS) in cardiomyocytes. Mechanistically, we showed that DAPA decreased cardiac expression of Bax and cleaved caspase 3 but increased Bcl-2 expression. DAPA also significantly rescued Dox-suppressed STAT3 expression. Conversely, knocking down STAT3 in cardiomyocytes reversed the DAPA-related protective effects on Dox-induced cell apoptosis and ROS. Collectively, our findings indicate that DAPA could be useful for preventing Dox-induced cardiotoxicity by restoring STAT3.

This study aims to explore the mechanism underlying miR-142-3p regulating myocardial injury induced by coronary microembolization (CME) through ATXN1L. miR-142-3p overexpression or ATXN1L knockout adenovirus vectors were injected into rats before CME treatment. Cardiac functions were examined by echocardiography, and pathologies of myocardial tissues were assessed. Then, serum cTnI and IL-1β contents and concentrations of IL-1β and IL-18 in cell supernatant were measured. Immunofluorescence determined the localization of histone deacetylase 3 (HDAC3). The interaction between miR-142-3p and ATXN1L as well as the binding between HDAC3 and histone 3 (H3) was identified. The binding of ATXN1L and HDAC3 to NOL3 promoter was verified using ChIP. The levels of ATXN1L, NOL3, and miR-142-3p as well as apoptosis- and pyroptosis-related proteins and acetyl-histone 3 (ac-H3) were evaluated. CME treatment impaired the cardiac functions in rats and increased cTnI content. CME rats showed microinfarction foci in myocardial tissues. After CME treatment, miR-142-3p and NOL3 were modestly expressed while ATXN1L content was elevated, in addition to increases in apoptosis and pyroptosis. miR-142-3p overexpression or ATXN1L knockout alleviated CME-induced myocardial injury, cardiomyocyte apoptosis, and pyroptosis in myocardial tissues. miR-142-3p regulated ATXN1L expression in a targeted manner. In the cellular context, miR-142-3p overexpression attenuated apoptosis and pyroptosis in cardiomyocytes, which was partly counteracted by ATXN1L overexpression. ATXN1L functioned on cardiomyocytes by promoting deacetylation of H3 through HDAC3 and thus inhibited NOL3 expression. Inhibition of HDAC3 or overexpression of NOL3 ameliorated the promotive effects of ATXN1L on cardiomyocyte apoptosis and pyroptosis. In vivo and in vitro evidence in this study supported that miR-142-3p could attenuate CME-induced myocardial injury via ATXN1L/HDAC3/NOL3. HIGHLIGHTS: CME model witnessed aberrant expression of miR-142-3p, ATXN1L, and NOL3; miR-142-3p negatively regulated ATXN1L; miR-142-3p mediated CME-induced myocardial injury through ATXN1L; ATXN1L promoted deacetylation of H3 through HDAC3 and thus inhibited NOL3 expression; ATXN1L acted on cardiomyocyte apoptosis and pyroptosis through HDAC3/NOL3 axis.

Exosomes-related microRNAs (miRNAs) have been considered to be the significant biomarkers contributing to the development of atrial fibrillation (AF). We observed the implicit mechanism of exosomes-miR-148a derived from bone marrow mesenchymal stem cells (BMSCs) in AF. The AF cell and mice models were established firstly. QRT-PCR and Western blot analysis were applied to detect the expression of miR-148a, SPARC-associated modular calcium-binding protein 2 (SMOC2), Bcl-2, Bax, and caspase-3. BMSCs were separated from healthy mice and exosomes were obtained from BMSCs. BMSCs were transfected with mimics and inhibitor, and HL-1 cells were treated with mimics and pcDNA3.1. MTT assay were used to detect cell viability of cells. Flow cytometric analysis and TUNEL analysis were used for detecting cell apoptosis of cells. In our study, exosomes derived from BMSCs inhibited the development of AF, and miR-148a acted a vital role in this segment. SMOC2 was a target gene of miR-148a and promoted apoptosis of HL-1 cells. Additionally, miR-148a mimics decreased cellular apoptosis, eliminated SMOC2 expression, and elevated Bcl-2 expression in AF-treated cells. Collectively, miR-148a overexpressed in BMSC-exosomes restrained cardiomyocytes apoptosis by inhibiting SMOC2.

The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity. To enable the application of CasRx in vivo, we established a Credependent CasRx knock-in mouse. Using these mice, we specifically knocked down the expression of Meis1 and Hoxb13 in cardiomyocytes, which induced cardiac regeneration after myocardial infarction. We also knocked down the lncRNA Mhrt in cardiomyocytes with the CasRx knock-in mice, causing hypertrophic cardiomyopathy. In summary, we generated a Credependent CasRx knock-in mouse that can efficiently knock down coding gene and lncRNA expression in specific somatic cells. This in vivo CRISPR-CasRx system is promising for gene function research and disease modeling.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have been reported to exhibit immature embryonic or fetal cardiomyocyte-like phenotypes. To enhance the maturation of hESC-CMs, we identified a natural steroidal alkaloid, tomatidine, as a new substance that stimulates the maturation of hESC-CMs. Treatment of human embryonic stem cells with tomatidine during cardiomyocyte differentiation stimulated the expression of several cardiomyocyte-specific markers and increased the density of T-tubules. Furthermore, tomatidine treatment augmented the number and size of mitochondria and enhanced the formation of mitochondrial lamellar cristae. Tomatidine treatment stimulated mitochondrial functions, including mitochondrial membrane potential, oxidative phosphorylation, and ATP production, in hESC-CMs. Tomatidine-treated hESC-CMs were more sensitive to doxorubicin-induced cardiotoxicity than the control cells. In conclusion, the present study suggests that tomatidine promotes the differentiation of stem cells to adult cardiomyocytes by accelerating mitochondrial biogenesis and maturation and that tomatidine-treated mature hESC-CMs can be used for cardiotoxicity screening and cardiac disease modeling.

Background Gout is usually found in patients with atrial fibrillation (AF). K+ efflux is a common trigger of NLRP3 inflammasome activation which is involved in the pathogenesis of AF. We investigated the role of the K+ channel Kv1.5 in monosodium urate crystal (MSU)-induced activation of the NLRP3 inflammasome and electrical remodeling in mouse and human macrophages J774.1 and THP-1, and mouse atrial myocytes HL-1. Methods and Results Macrophages, primed with lipopolysaccharide (LPS), were stimulated by MSU. HL-1 cells were incubated with the conditioned medium (CM) from MSU-stimulated macrophages. Western blot, ELISA and patch clamp were used. MSU induced caspase-1 expression in LPS-primed J774.1 cells and IL-1β secretion, suggesting NLRP3 inflammasome activation. A selective Kv1.5 inhibitor, diphenyl phosphine oxide-1 (DPO-1), and siRNAs against Kv1.5 suppressed the levels of caspase-1 and IL-1β. MSU reduced intracellular K+ concentration which was prevented by DPO-1 and siRNAs against Kv1.5. MSU increased expression of Hsp70, and Kv1.5 on the plasma membrane. siRNAs against Hsp70 were suppressed but heat shock increased the expression of Hsp70, caspase-1, IL-1β, and Kv1.5 in MSU-stimulated J774.1 cells. The CM from MSU-stimulated macrophages enhanced the expression of caspase-1, IL-1β and Kv1.5 with increased Kv1.5-mediated currents that shortened action potential duration in HL-1 cells. These responses were abolished by DPO-1 and a siRNA against Kv1.5. Conclusions Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome in macrophages. MSUrelated activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are via macrophages. Kv1.5 may have therapeutic value for diseases related to gout-induced activation of the NLRP3 inflammsome, including AF.

Cardiomyocyte injury is a typical feature in cardiovascular diseases. Changes in cardiomyocytes strongly affect the progression of cardiovascular diseases. This work aimed to investigate the biological function and potential mechanism of action of miR-150-5p in cardiomyocytes.A myocardial ischemia (MI) injury rat model was constructed to detect miR-150-5p and tetratricopeptide repeat domain 5 (TTC5) expression during heart ischemia injury. Primary cardiomyocytes were isolated for in vitro study. CCK-8 assays were used to detect cardiomyocyte viability. Western blots were used to detect TTC5 and P53 expression. qPCR was utilized to measure RNA expression of miR-150-5p and TTC5. The TUNEL assay was used to determine cell apoptosis. ELISA was used to determine cytokine (TNF-α, IL-1β, IL-6, and IL-8) levels in heart tissues and cell culture supernatants. A dual-luciferase reporter assay was carried out to verify the binding ability between miR-150-5p and TTC5. Oxygen-glucose deprivation (OGD) treatment significantly inhibited cell viability. Ultrasound-targeted microbubble destruction (UTMD)-mediated uptake of miR-150-5p inverted these results. Additionally, UTMD-mediated uptake of miR-150-5p retarded the effects of OGD treatment on cell apoptosis. Besides, UTMD-mediated uptake of miR-150-5p counteracted the effects of OGD treatment on the inflammatory response by regulating cytokine (TNF-α, IL-1β, IL-6, and IL-8) levels. For the mechanism of the protective effect on the heart, we predicted and confirmed that miR-150-5p bound to TTC5 and inhibited TTC5 expression.UTMD-mediated uptake of miR-150-5p attenuated OGD-induced primary cardiomyocyte injury by inhibiting TTC5 expression. This discovery contributes toward further understanding the progression of primary cardiomyocyte injury.

Myocardial ischemia-reperfusion injury (MI/RI), a complication of myocardial injury, is associated with high rates of mortality and disability. We aimed to explore the effect of nicorandil™ against MI/RI and investigated the underlying molecular mechanisms. In this in vitro study, hypoxia/reoxygenation (H/R) processing of H9c2 cells significantly suppressed the expressions of IL33 and ST2, reduced cell viability, increased production of reactive oxygen species, downregulated protein expression of Bcl-2, upregulated protein expressions of Bax, cleaved caspase3, and cleaved PARP, increased intracellular calcium overload, and induced cell apoptosis. Nicorandil processing reduced H/R-induced H9c2 cell damage. Nicorandil processing ameliorated the H/R-induced inhibition of the IL33 and ST2 expression in H9c2 cells. 5-Hydroxydecanoate blocked the effects of nicorandil on H9c2 cell viability, ROS production, and apoptosis and inhibited both IL33 and ST2. Similarly, the protective effect of nicorandil was restrained after inhibition of the IL33/ST2 pathway. Our findings suggest that the protective effect of nicorandil against H/R-induced H9c2 cell apoptosis was mediated through IL33/ST2 signaling pathway.

A breakthrough in oncology research was the discovery of doxorubicin (Dox) in the 1960’s. Unlike other chemotherapy drugs, Dox was determined to have a greater therapeutic index. Since its discovery, Dox has, in part, contributed to the 5–10-year survival increase in cancer patient outcomes. Unfortunately, despite its efficacy, both in adult and pediatric cancers, the clinical significance of Dox is tainted by its adverse side effects, which usually manifest as cardiotoxicity. The issue stems from Dox’s lack of specificity which prevents it from accurately distinguishing between cancer cells and healthy cell lines, like cardiomyocytes. In addition, the high binding affinity of Dox to topoisomerases, which are abundantly found in cancer and cardiac cells in different isoforms, potentiates DNA damage. In both cell lines, Dox induces cytotoxicity by stimulating the production of pro-oxidants whilst inhibiting antioxidant enzymatic activity. Given that the cardiac muscle has an inherently low antioxidant capacity makes it susceptible to oxidative damage thereby, allowing the accumulation of Dox within the myocardium. Subsequently, Dox drives the activation of cell death pathways, such as ferroptosis, necroptosis and apoptosis by triggering numerous cellular responses that have been implicated in diseases. To date, the exact mechanism by which Dox induces the cardiotoxicity remains an aspect of much interest in cardio-oncology research. Hence, the current review summarizes the proposed mechanisms that are associated with the onset and progression of DIC.

Diabetes mellitus (DM)-induced cardiac morbidities have been the leading cause of death among diabetic patients. Recently, sodium-glucose cotransporter-2 (SGLT-2) inhibitors including empagliflozin (EMPA), which have been approved for the treatment of DM, have gained attention for their cardioprotective effect. The mechanism by which SGLT-2 inhibitors exert their cardioprotective effect remains unclear. Recent studies have suggested that EMPA exerts its cardioprotective effect by inhibiting the Na+/H+ exchanger (NHE), a group of membrane proteins that regulate intracellular pH and cell volume. Increased activity and expression of NHE isoform 1 (NHE1), the predominant isoform expressed in the heart, leads to cardiac hypertrophy. p90 ribosomal s6 kinase (p90 RSK) has been demonstrated to stimulate NHE1 activity. In our study, H9c2 cardiomyoblasts were treated with angiotensin II (ANG) to activate NHE1 and generate a hypertrophic model. We aimed to understand whether EMPA reverses the ANG-induced hypertrophic response and to elucidate the molecular pathway contributing to the cardioprotective effect of EMPA. Our study demonstrated that ANG-induced hypertrophy of H9c2 cardiomyoblasts is accompanied with increased SGLT-1 and NHE1 protein expression, an effect which is prevented in the presence of EMPA. EMPA reduces ANG-induced hypertrophy through the inhibition of SGLT-1 and NHE1 expression.Other Information Published in: Molecular and Cellular Biochemistry License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1007/s11010-022-04411-6

The renin-angiotensin system (RAS) plays a crucial role and coordinates multiple body functions through its hormonal mechanism. The RAS is supported in its function by numerous peptides such as angiotensin II (Ang II), Ang IV, Ang III, angiotensin (1-7) and (1-9). The system formed by ACE2/Ang(1-7)/MASr is a regulatory pathway within the RAS system and its functions are different from those of the ACE/Ang II/AT-1r system. Recently, it has been discovered that a key role of the RAS and the ACE2/Ang(1-7)/MASr system is in inflammatory processes such as cardiac hypertrophy and heart failure. Studies are ongoing to better understand and comprehend the function of the RAS in inflammation. Recent evidence associates AT-1r antagonists with a cardioprotective, anti-inflammatory, and anti-hypertrophic role. In this in vitro study, we demonstrate the protective role of treatment (50 and 200 μM) of an AT-1r antagonist, irbesartan, on hypoxia and inflammation-induced damage in cardiomyocytes.

Purpose of Review Exciting pre-clinical data presents pluripotent stem cell-derived cardiomyocytes (PSC-CM) as a novel therapeutic prospect following myocardial infarction, and worldwide clinical trials are imminent. However, despite notable advances, several challenges remain. Here, we review PSC-CM pre-clinical studies, identifying key translational hurdles. We further discuss cell production and characterization strategies, identifying markers that may help generate cells which overcome these barriers. Recent Findings PSC-CMs can robustly repopulate infarcted myocardium with functional, force generating cardiomyocytes. However, current differentiation protocols produce immature and heterogenous cardiomyocytes, creating related issues such as arrhythmogenicity, immunogenicity and poor engraftment. Recent efforts have enhanced our understanding of cardiovascular developmental biology. This knowledge may help implement novel differentiation or gene editing strategies that could overcome these limitations. Summary PSC-CMs are an exciting therapeutic prospect. Despite substantial recent advances, limitations of the technology remain. However, with our continued and increasing biological understanding, these issues are addressable, with several worldwide clinical trials anticipated in the coming years.

BACKGROUND: During cardiogenesis, cardiac cells receive various stimuli, such as biomechanical and chemical cues, from the surrounding microenvironment, and these signals induce the maturation of heart cells. Mechanical force, especially tensile force in the heart, is one of the key stimuli that induce cardiomyocyte (CM) maturation through mechanotransduction, a process through which physical cues are transformed into biological responses. However, the effects and mechanisms of tensile force on cell maturation are poorly studied. METHODS: In this study, we developed a cyclic stretch system that mimics the mechanical environment of the heart by loading tensile force to human-induced pluripotent stem cell (hiPSC)-derived CMs. hiPSC-CMs cultured with the cyclic stretch system analyzed morphological change, immunofluorescent staining, expression of maturation markers in mRNA, and beating properties compared to static cultures. RESULTS: hiPSC-CMs cultured with the cyclic stretch system showed increased cell alignment, sarcomere length and expression of maturation markers in mRNA, such as TNNI3, MYL2 and TTN, compared to static cultures. Especially, the expression of genes related to nuclear mechanotransduction, such as Yap1, Lamin A/C, plectin, and desmin, was increased in the cyclically stretched hiPSC-CMs. Furthermore, the volume of the nucleus was increased by as much as 120% in the cyclic stretch group. CONCLUSION: These results revealed that nuclear mechanotransduction induced by tensile force is involved in CM maturation. Together, these findings provide novel evidence suggesting that nuclear mechanotransduction induced by tensile force is involved in the regulation of cardiac maturation.

Myocardial infarction is one of the most lethal diseases in cardiovascular diseases. In the present work, we aimed to elucidate the molecular and functional association long non-coding RNA (lncRNA) X-inactive specific transcript (XIST), microRNA (miR)-191-5p, and TNF receptor-associated factor 3 (TRAF3). Human cardiomyocyte primary cells (HCMs) were stimulated by hypoxia to establish a model of myocardial injury in vitro. The relative expressions of XIST, miR-191-5p, and TRAF3 were measured using quantitative real-time polymerase chain reaction (qRT-PCR) assay. The capabilities of proliferation and apoptosis were determined using cell counting kit (CCK-8) and flow cytometry assays, respectively. The molecular interactions were verified using dual luciferase assay. The protein contents of TRAF3, Bcl-2, and Bax were calculated using western blot assay. XIST was significantly increased, but miR-191-5p was reduced in hypoxia-treated HCMs compared to that in control group. Either downregulated XIST or enforced miR-191-5p markedly enhanced cell viability and restrained cell apoptotic rate in hypoxia-treated HCMs. Mechanistically, XIST directly interacted with miR-191-5p to competitive releasing TRAF3 expression. Importantly, overexpression of TRAF3 dramatically diminished the protective effects of XIST knockdown on hypoxia-triggered HCMs injury. Collectively, our data elucidated a novel "lncRNA XIST/miR-191-5p/TRAF3" molecular network in vitro, indicating that the reduced lncRNA XIST-protected HCMs from hypoxia-induced cell injury by regulating miR-191-5p/TRAF3 signaling, which might provide some convincing evidences for further understanding the influences of "lncRNA-miRNA-mRNA" network in the development of MI.

Despite direct reprogramming of human cardiac fibroblasts into induced cardiomyocytes (iCM) holds great potential for heart regeneration, the mechanisms are poorly understood. Whether other human somatic cells could be reprogrammed into cardiomyocytes is also unknown. Here, we report human urine cells (hUCs) could be converted into CM-like cells from different donors and the related chromatin accessibility dynamics (CAD) by assay for transposase accessible chromatin(ATAC)-seq. hUCs transduced by MEF2C, TBX5, MESP1 and MYOCD but without GATA4 expressed multiple cardiac specific genes, exhibited Ca

Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) is an enveloped single-stranded RNA virus that can lead to respiratory symptoms and damage many organs such as heart, kidney, intestine, brain and liver. It has not been clearly documented whether myocardial injury is caused by direct infection of cardiomyocytes, lung injury, or other unknown mechanisms. The gene expression profile of GSE150392 was obtained from the Gene Expression Omnibus (GEO) database. The processing of high-throughput sequencing data and the screening of differentially expressed genes (DEGs) were implemented by R software. The R software was employed to analyze the Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The protein-protein interaction (PPI) network of the DEGs was constructed by the STRING website. The Cytoscape software was applied for the visualization of PPI network and the identification of hub genes. The statistical analysis was performed by the GraphPad Prism software to verify the hub genes. A total of 516 up-regulated genes and 191 down-regulated genes were screened out. The top 1 enrichment items of GO in biological process (BP), Cellular Component (CC), and Molecular Function (MF) were type I interferon signaling pathway, sarcomere, and receptor ligand activity, respectively. The top 10 enrichment pathways, including TNF signaling pathway, were identified by KEGG enrichment analysis. A PPI network was established, consisting of 613 nodes and 3,993 edges. The 12 hub genes were confirmed as statistically significant, which was verified by GSE151879 dataset. In conclusion, the hub genes of human iPSC-cardiomyocytes infected with SARS-CoV-2 were identified through bioinformatics analysis, which may be used as biomarkers for further research.

Impaired autophagic flux induces aging-related ischemia vulnerability, which is the hallmark pathology in cardiac aging. Our previous work has confirmed that the accumulation of charged multivesicular body protein 2B (CHMP2B), a subunit of the ESCRT-III complex, in the heart can impair autophagy flux. However, whether CHMP2B accumulation contributes to aging-related intolerance to ischemia/reperfusion (I/R) injury and the regulatory mechanism for CHMP2B in aged heart remain elusive. The cardiac CHMP2B level was significantly higher in aged human myocardium than that in young myocardium. Increased CHMP2B were shown to inhibit autophagic flux leading to the deterioration of MI/R injury in aged mice hearts. Interestingly, a negative correlation was observed between SIRT6 and CHMP2B expression in human heart samples. Specific activation of SIRT6 suppressed CHMP2B accumulation and ameliorated autophagy flux in aged hearts. Using myocardial-specific SIRT6 heterozygous knockout mice and recovery experiments confirmed that SIRT6 regulated myocardial CHMP2B levels. Finally, activation of SIRT6 decreased acetylation of FoxO1 to promote its transcriptional function on Atrogin-1, a muscle-specific ubiquitin ligase, which subsequently enhanced the degradation of CHMP2B by Atrogin-1. This is a novel mechanism for SIRT6 against aging-related myocardial ischemia vulnerability, particularly by preventing excessive accumulation of autophagy key factors.

To estimate the cardioprotective mechanism of cordycepin on myocardial ischemia/reperfusion (I/R) injury. The left anterior descending artery of mice was ligated transiently to establish the myocardial I/R model. TTC/Evans Blue staining and TUNEL assay were performed to quantify the infarct size and apoptosis index. The cardiac function was evaluated by echocardiography. Neonatal rat ventricular cardiomyocytes (NRVCs) underwent hypoxia and reoxygenation (H/R). MTS and LDH were detected to measured cell viability and necrosis respectively. The results suggested that cordycepin could markedly decrease apoptosis, reduce infarct size, and improve cardiac function in mice subjected to I/R injury, alongside with enhanced autophagy. In NRVCs, cordycepin treatment obviously reduced ROS production. In addition, cordycepin partly promoted autophagy in the context of H/R injury by regulating AMPK/mTOR pathway. Our data demonstrated that cordycepin exerts cardio-protective effect and promotes cardiac functional recovery following myocardial I/R by enhancing autophagy via AMPK-mTOR signaling pathway.

Acute myocardial infarction (AMI) is a severe and even fatal cardiovascular disease. The effect of transcription factors on AMI is intensively explored. Our experiment attempts to probe the role of Sox9 in cardiomyocyte apoptosis after AMI. AMI cell model was established in AC16 cells by hypoxia treatment. Cell viability and apoptosis were assessed. Then, the levels of BAX, Bcl-2, Sox9, miR-223-3p, and MEF2C were detected. The binding relation between Sox9 and miR-223-3p and between miR-223-3p and MEF2C was verified. The expression of miR-223-3p was upregulated using the miR-223-3p mimic, and collaborative experiments were conducted as si-Sox9 or si-MEF2C was transfected into cells to inhibit the expression of Sox9 or MEF2C. Sox9 was highly expressed in cardiomyocyte apoptosis after hypoxia, while Sox9 silencing protected hypoxia-treated cardiomyocytes from apoptosis by enhancing cell viability, quenching apoptosis, and reducing activity of caspase-3 and caspase-9. Essentially, Sox9 bound to the miR-223-3p promoter region to upregulate its expression. miR-223-3p targeted MEF2C transcription. miR-223-3p overexpression and MEF2C silencing could counteract the suppressive role of Sox9 silencing in hypoxia-treated cardiomyocyte apoptosis. Sox9 exacerbated hypoxia-induced cardiomyocyte apoptosis by promoting miR-223-3p expression and inhibiting MEF2C transcription.

In this work, we investigated the in vitro and in vivo functions of bisphosphonate of zoledronate (Zd) in hypoxia/reoxygenation (H/R) injured human embryonic stem cell-derived cardiomyocytes (hES-CMs). In the in vitro setting, the effects of Zd on hES-CM survival and differentiation were examined. We found that low and medium concentrations (2 µm) of Zd did not induce cell death of hES-CMs. 0.5 µm Zd protected H/R-induced hES-CM apoptosis but did not affect key differentiation proteins, including hcTnl, PECM-1 Cnx43 and Pan-Cadherin. In addition, Zd-induced TrkA/B phosphorylation and promoted VEGF to counter the apoptotic effect of H/R injury. In the in vivo animal model of myocardial infarction, Zd treatment promoted the survival of hES-CMs by inducing PECAM1 and hcTnl. Thus, we concluded that Zd protected H/R-induced hES-CM apoptosis in vitro and promoted hES-CM survival in vivo. These data may facilitate the development of human embryonic stem cells into clinical applications for patients with ischemic heart disease.

Latest clinical research shows that trimetazidine therapy during the perioperative period relieves endothelial dysfunction in patients with unstable angina induced by percutaneous coronary intervention. In this study we investigated the effects of TMZ on myocardial angiogenesis in pressure overload-induced cardiac hypertrophy mice. Cardiac hypertrophy was induced in mice by transverse aortic constriction (TAC) surgery. TAC mice were administered trimetazidine (2.8 mg/100 µL, i.g.) for 28 consecutive days. We showed that trimetazidine administration significantly increased blood vessel density in the left ventricular myocardium and abrogated cardiac dysfunction in TAC mice. Co-administration of a specific HSF1 inhibitor KRIBB11 (1.25 mg/100 µL, i.h.) abrogated the angiogenesis-promoting effects of trimetazidine in TAC mice. Using luciferase reporter and electrophoretic mobility shift assays we demonstrated that the transcription factor HSF1 bound to the promoter region of VEGF-A, and the transcriptional activity of HSF1 was enhanced upon trimetazidine treatment. In molecular docking analysis we found that trimetazidine directly bound to Akt via a hydrogen bond with Asp292 and a pi–pi bond with Trp80. In norepinephrine-treated HUVECs, we showed that trimetazidine significantly increased the phosphorylation of Akt and the synergistic nuclear translocation of Akt and HSF1, as well as the binding of Akt and HSF1 in the nucleus. These results suggest that trimetazidine enhances myocardial angiogenesis through a direct interaction with Akt and promotion of nuclear translocation of HSF1, and that trimetazidine may be used for the treatment of myocardial angiogenic disorders in hypertensive patients.

Obesity is an important independent risk factor for cardiovascular diseases, remaining an important health concern worldwide. Evidence shows that saturated fatty acid-induced inflammation in cardiomyocytes contributes to obesity-related cardiomyopathy. Dapagliflozin (Dapa), a selective SGLT2 inhibitor, exerts a favorable preventive activity in heart failure. In this study, we investigated the protective effect of Dapa against cardiomyopathy caused by high fat diet-induced obesity in vitro and in vivo. Cultured rat cardiomyocyte H9c2 cells were pretreated with Dapa (1, 2.5 μM) for 1.5 h, followed by treatment with palmitic acid (PA, 200 μM) for 24 h. We showed that Dapa pretreatment concentration-dependently attenuated PA-induced cell hypertrophy, fibrosis and apoptosis. Transcriptome analysis revealed that inhibition of PA-activated MAPK/AP-1 pathway contributed to the protective effect of Dapa in H9c2 cells, and this was confirmed by anti-p-cJUN fluorescence staining assay. Using surface plasmon resonance analysis we found the direct binding of Dapa with NHE1. Gain and loss of function experiments further demonstrated the role of NHE1 in the protection of Dapa. In vivo experiments were conducted in mice fed a high fat diet for 5 months. The mice were administered Dapa (1 mg·kg

Doxorubicin (DOX) is a potent chemotherapeutic agent; however, it causes severe heart injury via apoptosis induction in many patients. DOX-induced cardiotoxicity is attenuated by activated autophagy in the heart. We previously found that programmed cell death 1 (Pdcd1), an immune checkpoint receptor, inhibits DOX-induced cardiomyocyte apoptosis. In this study, we investigated whether autophagy contributes to the protective role of Pdcd1 against DOX-induced cardiomyocyte apoptosis. We also examined the role of Pdcd1 in DOX-induced apoptosis in cancer cells. Rat cardiomyocyte cell line H9c2 and human cancer cell lines K562 and MCF-7 were transfected with Pdcd1-encoding plasmid DNA to establish Pdcd1-overexpressing cells. Apoptosis and autophagy were determined using a luciferase assay. In H9c2 cells, DOX-induced apoptosis and viability reduction occurred through caspase activation. In particular, Pdcd1 overexpression activated the autophagy pathway through the inhibition of the mammalian target of rapamycin, a major negative regulator of autophagy. Moreover, it prevented DOX-induced cardiomyocyte apoptosis; a similar cardioprotection was observed when normal H9c2 cells (without Pdcd1 overexpression) were treated with rapamycin, an autophagy inducer, before the DOX treatment. Conversely, in cancer cells, Pdcd1 overexpression increased both basal and DOX-induced apoptosis. The role of Pdcd1 in DOX-induced apoptosis in cardiomyocytes and cancer cells was opposing. Pdcd1 signaling prevented DOX-induced apoptosis in cardiomyocytes, through autophagy induction; it enhanced DOX-induced apoptosis in cancer cells. Therefore, Pdcd1 could be a critical molecule for more effective and safer DOX chemotherapy.

DSH-20, the active ingredient of Salvia miltiorrhiza flower extract, is used to treat cardiovascular diseases. However, its mechanism of action remains unclear. Herein, we investigated the intervention of DSH-20 in HDSH-20 reduced H

Purpose of Review We review therapeutic approaches aimed at restoring function of the failing heart by targeting mitochondrial reactive oxygen species (ROS), ion handling, and substrate utilization for adenosine triphosphate (ATP) production. Recent Findings Mitochondria-targeted therapies have been tested in animal models of and humans with heart failure (HF). Cardiac benefits of sodium/glucose cotransporter 2 inhibitors might be partly explained by their effects on ion handling and metabolism of cardiac myocytes. Summary The large energy requirements of the heart are met by oxidative phosphorylation in mitochondria, which is tightly regulated by the turnover of ATP that fuels cardiac contraction and relaxation. In heart failure (HF), this mechano-energetic coupling is disrupted, leading to bioenergetic mismatch and production of ROS that drive the progression of cardiac dysfunction. Furthermore, HF is accompanied by changes in substrate uptake and oxidation that are considered detrimental for mitochondrial oxidative metabolism and negatively affect cardiac efficiency. Mitochondria lie at the crossroads of metabolic and energetic dysfunction in HF and represent ideal therapeutic targets.

Esophageal Cancer-Related Gene 4 (Ecrg4) expressed in cardiomyocytes and the cardiac conduction system is downregulated during cardiac ischemia and atrial fibrillation. To explore whether Ecrg4 plays any role in doxorubicin (DOX)-induced cardiotoxicity. Rats and neonatal rat cardiomyocytes (NRCMs) were employed to study the effect of DOX on Ecrg4 transcription. Bioinformatics combined with promoter analysis were used to map the rat Ecrg4 promoter. ChIP assay was used to evaluate the binding of Sp1 to the Ecrg4 promoter. Transient transfection was used to study the effect of Sp1 on the expression of endogenous Ecrg4. DOX decreased endogenous Ecrg4 gene expression in the heart and cultured NRCMs. In silico analysis showed that the 5'UTR immediately upstream of the start codon ATG, harbors a putative promoter that is GC-rich, and contains CpG islands, multiple overlapping Sp1sites. Transcription is initiated mainly on the 'C' at - 15. Serial 5'-deletion combined with dual-luciferase assays showed that the rat Ecrg4 core promoter resides at - 1/- 800. Sp1 transactivated Ecrg4 gene, which was almost abolished by DOX. Furthermore, ChIP assay showed that Sp1 specifically bound to the Ecrg4 promoter was interrupted by DOX. Finally, DOX suppressed Sp1 protein expression, and restoration of Sp1 increased Ecrg4 expression that was resistant to DOX-induced Ecrg4 downregulation. Importantly, cardiomyocyte-specific loss of Ecrg4 significantly enriched the differentially expressed proteins in the signaling pathways commonly involved in DOX-induced cardiotoxicity. Our results indicate that Sp1 mediates DOX-induced suppression of Ecrg4, which may contribute indirectly to its cardiotoxicity.

Molecular mechanisms underlying myocardial ischemia/reperfusion (MI/R) injury and effective strategies to treat MI/R injury are both in shortage. Although pyroptosis of cardiomyocytes and the protective role of cardiac fibroblasts (CFs) have been well recognized as targets to reduce MI/R injury and sudden cardiac death (SCD), the connection has not yet been established. Here, we showed that CFs protected cardiomyocytes against MI/R-induced injury through suppression of pyroptosis. A novel molecular mechanism underpinning this effect was further identified. Under hypoxia/reoxygenation condition, CFs were found to secrete exosomes, which contain increased level of microRNA-133a (miR-133a). These exosomes then delivered miR-133a into cardiomyocytes to target ELAVL1 and repressed cardiomyocyte pyroptosis. Based on this finding, we successfully developed a new strategy that used exosomes derived from CFs with overexpressed miR-133a to enhance the therapeutic outcomes for the MI/R injury. Overall, our results provide a novel molecular basis for understanding and treating MI/R injury, and our study also provides novel insight for the postmortem diagnosis of MI/R injury induced SCD by using exosome biomarker in forensic.

Acute myocardial infarction (AMI) is an ischemic heart disease with high mortality. AMI-induced hypoxia will trigger serious myocardial injury, such as cardiomyocyte apoptosis. miRNAs have been reported to be involved in the development of AMI. Our previous study revealed that hypoxia regulates the miRNAome of rat cardiomyoblast cells (H9c2), including many known "hypoxamiRs." This study aimed to investigate the potential function of miR-361-3p in the hypoxic response of cardiomyocytes. H9c2 cells were cultured in hypoxic condition and rat AMI model was established by ligating the coronary artery. Cell apoptosis and miR-361-3p expression were measured in hypoxia-exposed H9c2 cell and myocardium of AMI rat. Gain- and loss-of-function analyses in vitro were performed to assess the effect of miR-361-3p in hypoxia-induced cardiomyocyte injury. Hypoxia induced notable changes in cell morphology, triggered cell apoptosis, increased cell membrane damage, and meanwhile decreased miR-361-3p expression in a time-dependent manner. AMI induced cell apoptosis in rat myocardium accompanied by downregulation of miR-361-3p. miR-361-3p overexpression markedly reduced hypoxia-induced cardiomyocyte injury; however, its downregulation had an opposite effect. Functionally, miR-361-3p mitigated hypoxia injury by inhibiting apoptosis via targeting apoptosis initiators caspase-2/-8/-9. This study revealed that miR-361-3p has a cardioprotective effect on hypoxia-induced cardiomyocyte injury, suggesting it may be a novel therapeutic target for hypoxia-related cardiac diseases.

The nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is associated with doxorubicin (DOX)-induced cardiac injury. It has been reported that microRNA-24-3p (miR-24-3p) may regulate the Keapl by mRNA degradation, whereas Keapl can suppress the activation of Nrf2. However, the role of miR-24-3p in DOX-related cardiotoxicity remains unclear.The mice receiving DOX were used as cardiac injury model. In this study, an adenoassociated virus 9 system was used to deliver miR-24-3p or miR-scramble to mice hearts. The echocardiographic and hemodynamic analyses were used to evaluate the effects of miR-24-3p on cardiac function under DOX stimulation. ELISA and RT-PCR were used to detect protein or mRNA expressions associated with cardiac injury, inflammation response, apoptosis and oxidative stress. Western Blot were used for quantitative analysis of the roles of miR-24-3p in regulating Nrf2 expression. H9C2 cells used to verify the role of miR-24-3p in vitro.We found that miR-24-3p mRNA was significantly decreased in DOX-treated mice and cardiomyocytes. Overexpression of miR-24-3p blocked cardiac injury caused by DOX injection, as reflected by the reduction in the levels of cardiac troponin I, creatinine kinase isoenzyme MB and the N-terminal pro brain natriuretic peptide. Furthermore, miR-24-3p reduced oxidative stress and cell loss without affecting the inflammation response. As expected, we found that Nrf2 was upregulated by miR-24-3p supplementation, and that the protective efforts of miR-24-3p supplementation were abolished when Nrf2 was silenced.The results from this study suggest that miR-24-3p protects cardiomyocytes against DOX-induced heart injury via activation of the Nrf2 pathway. miR-24-3p supplementation may be a novel strategy to counteract the cardiac side effects of DOX treatment.

Increasing exploration of rare-earth elements (REEs) has resulted in a high REEs' exposure risk. Owing to their persistence and accumulation of REEs in the environment, their adverse effects have caused widespread concern. However, limited toxicological data are available for the adverse effects of yttrium (Y) and its underlying mechanisms of action. In the present study, H9c2 cardiomyocytes were used in vitro model to investigate the cardiotoxicity of yttrium chloride (YCl

This study investigated how miR-136-5p partially affected cardiomyocyte pyroptosis in rats with coronary microembolization (CME). The cardiac function and structure of rats with CME were evaluated using echocardiography, hematoxylin and eosin staining, Masson staining, and troponin I level. Pyroptosis was induced by lipopolysaccharide (LPS) in isolated rat cardiomyocytes and evaluated by the expression of caspase-1, NOD-like receptor family pyrin domain-containing 3, interleukin-1β, and gasdermin D-N. After cell transfection, the expression of Ataxin-1 like (ATXN1L), pyrin domain-containing 1 (PYDC1), and pyroptosis-related proteins was assessed. Dual-luciferase reporter and immunoprecipitation assays were used to verify the relationships among miR-136-5p, ATXN1L, and capicua (CIC). MiR-136-5p was under-expressed, whereas ATXN1L was overexpressed in rats with CME and in LPS-treated primary cardiomyocytes. MiR-136-5p targeted ATXN1L, and ATXN1L bound to CIC to suppress PYDC1 expression. MiR-136-5p overexpression suppressed pyroptosis by inhibiting the binding of ATXN1L with CIC and promoting PYDC1 expression, which was reversed by simultaneous elevation of ATXN1L. In conclusion, miR-136-5p suppressed pyroptosis by upregulating PYDC1 via ATXN1L/CIC axis, thereby attenuating cardiac damage caused by CME.

Stem cell therapy is developing as a valuable therapeutic trend for heart diseases. Most recent studies are aimed to find the most appropriate types of stem cells for the treatment of myocardial infarction (MI). The animal models have shown that bone marrow-derived mesenchymal stem cells (BMSCs) are a possible, safe, and efficient type of stem cell used in MI. The previous study demonstrated that 5-Azacytidine (5-Aza) could promote cardiac differentiation in stem cells.This study used 5-Aza to induce cardiomyocyte differentiation in BMSCs both in static and microfluidic cell culture systems. For this purpose, we investigated the differentiation by using real-time PCR and Immunocytochemistry (ICC) Analysis.Our results showed that 5-Aza could cause to express cardiac markers in BMSCs as indicated by real-time PCR and immunocytochemistry (ICC). However, BMSCs are exposed to both 5-Aza and shear stress, and their synergistic effects could significantly induce cardiac gene expressions in BMSCs. This level of gene expression was observed neither in 5-Aza nor in shear stress groups only.These results demonstrate that when BMSCs expose to 5-Aza as well as mechanical cues such as shear stress, the cardiac gene expression can be increased dramatically.

This study aimed to investigate the relationship between miR-21 and lipopolysaccharide (LPS)-induced myocardial injury and its molecular and regulatory mechanisms.We constructed LPS-mediated myocardial injury model using C57BL/6J mice and H9c2 cells. In-vivo, in-vitro, RIP and dual-luciferase reporter assays were used to determine the effect of miR-21 on myocardial injury.In-vivo and in-vitro results showed that the expression of miR-21 was increased in LPS-treated H9c2 cells and myocardial tissues of mice, and the pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) and NF-κB pathway were activated in LPS-treated H9c2 cells. Besides, the B-cell lymphoma-2 (Bcl-2) and cyclin-dependent kinase 6 (CDK6) expression levels decreased, while Bax and cleaved caspase 9 levels increased in LPS-treated H9c2 cells. Inhibition of miR-21 could suppress LPS-induced apoptosis, inflammatory reactions and NF-κB activation to attenuate LPS-induced myocardial injury in H9c2 cells, and effectively improve survival of mice with sepsis. Most importantly, Bcl-2 and CDK6 were found to be the direct target of miR-21 using dual-luciferase reporter and RNA immunoprecipitation assays. Further gain-of-function assay demonstrated that Bcl-2 or CDK6 over-expression promoted the protective effects of miR-21 inhibitor on LPS-mediated myocardial cells.Our findings revealed that the down-regulation or antagonism of miR-21 protects myocardial cells against LPS-induced apoptosis and inflammation through up-regulating Bcl-2 and CDK6 expression, which provided a new insight for prevention and treatment of myocardial injury.

Increasing investigations have focused on long non-coding RNAs (lncRNAs) in various human diseases, including acute myocardial infarction (AMI). Although lncRNA HOTTIP has been identified to play an important role in coronary artery diseases, its role and specific mechanism in AMI remain unclear. To investigate the potential role of HOTTIP in MI, HOTTIP expression in hypoxia-treated cardiomyocytes and myocardial tissues of MI mice was evaluated. The potential targets of HOTTIP and miR-92a-2 were predicted using Starbase and Targetscan. To further determine the cardio-protective effects of HOTTIP in vivo, si-HOTTIP and miR-92a-2 mimics were individually or co-injected into mice through intramyocardial injection. Moreover, their roles were further confirmed in rescue experiments. HOTTIP was significantly upregulated in ischemic myocardium of MI mice and hypoxia-induced cardiomyocytes. Moreover, HOTTIP knockdown markedly promoted cardiomyocyte growth and inhibited cardiomyocyte apoptosis in vitro. Luciferase reporter assay showed that HOTTIP could directly sponge miR-92a-2 to negatively regulate miR-92a-2 expression. In addition, c-Met was identified as a direct target of miR-92a-2, and their correlation was confirmed by luciferase reporter assay. MiR-92a-2 overexpression significantly enhanced the protective effect of HOTTIP knockdown against AMI through partially inhibiting c-Met expression. Our results demonstrated that HOTTIP downregulation attenuated AMI progression via the targeting miR-92a-2/c-Met axis and suggested that HOTTIP might be a potential therapeutic target for AMI.

Human pluripotent stem cells (hPSCs) have great potential for disease modeling, drug discovery, and regenerative medicine as they can differentiate into many different functional cell types via directed differentiation. However, the application of disease modeling is limited due to a time-consuming and labor-intensive process of introducing known pathogenic mutations into hPSCs. Base editing is a newly developed technology that enables the facile introduction of point mutations into specific loci within the genome of living cells without unwanted genome injured. We describe an optimized stepwise protocol to introduce disease-specific mutations of long QT syndrome (LQTs) into hPSCs. We highlight technical issues, especially those associated with introducing a point mutation to obtain isogenic hPSCs without inserting any resistance cassette and reproducible cardiomyocyte differentiation. Based on the protocol, we succeeded in getting hPSCs carrying LQTs pathogenic mutation with excellent efficiency (31.7% of heterozygous clones, 9.1% of homozygous clones) in less than 20 days. In addition, we also provide protocols to analyze electrophysiological of hPSC-derived cardiomyocytes using multi-electrode arrays. This protocol is also applicable to introduce other disease-specific mutations into hPSCs. Graphical abstract

This study investigated the optimum transport condition for heart tissue to recover single-cell cardiomyocytes for future in-vitro or in-vivo studies. The heart tissues were obtained from removing excessive myocardium discharged during the repair surgery of an excessive right atrial hypertrophy due to a congenital disease. The transportation temperature studied was the most used temperature (4 °C) or the conventional condition, compared to a physiological temperature(37 °C). The heart tissues were transported from the operating theatre to the lab maintained less than 30 min consistently. Single-cell isolation was enzymatically and mechanically performed using collagenase-V (160 U/mg) and proteinase-XXIV (7-14 U/mg) following the previously described protocol. The impact of temperature differences was observed by the density of cells harvested per mg tissue, cell viability, and the senescence signals, identified by the p21, p53 and caspase-9 mRNA expressions. Results the heart tissue transported at 37 °C yielded significantly higher viable cell density (p 0.01) yielded viable cells significantly higher density (p 0.01) than the 4 °C; 2,335 ± 849 cells per mg tissue, and 732 ± 425 cells per mg tissue, respectively. The percentage of viable cells in both groups showed no difference. Although the 37 °C group expressed the apoptosis genes such as p21, p53 and caspase9 by 2.5-, 5.41-, 5-fold respectively (p 0.05). Nonetheless, the Nk×2.5 and MHC genes were expressed 1,7- and 3.56-fold higher than the 4 °C. and the c-Kit+ expression was 17.56-fold, however, statistically insignificant. Conclusion When needed for single-cell isolation, a heart tissue transported at 37 °C yielded higher cell density per mg tissue compared to at 4 °C, while other indicators of gene expressions for apoptosis, cardiac structural proteins, cardiac progenitor cells showed no difference. Further investigations of the isolated cells at different temperature conditions towards their proliferation and differentiation capacities in a 3-D scaffold would be essential.