Cardiac-specific deletion of heat shock protein 60 induces mitochondrial stress and disrupts heart development in mice.

Congenital heart diseases are the most common birth defects around the world. Emerging evidence suggests that mitochondrial homeostasis is required for normal heart development. In mitochondria, a series of molecular chaperones including heat shock protein 60 (HSP60) are engaged in assisting the import and folding of mitochondrial proteins. However, it remains largely obscure whether and how these mitochondrial chaperones regulate cardiac development. Here, we generated a cardiac-specific Hspd1 deletion mouse model by αMHC-Cre and investigated the role of HSP60 in cardiac development. We observed that deletion of HSP60 in embryonic cardiomyocytes resulted in abnormal heart development and embryonic lethality, characterized by reduced cardiac cell proliferation and thinner ventricular walls, highlighting an essential role of cardiac HSP60 in embryonic heart development and survival. Our results also demonstrated that HSP60 deficiency caused significant downregulation of mitochondrial ETC subunits and induced mitochondrial stress. Analysis of gene expression revealed that P21 that negatively regulates cell proliferation is significantly upregulated in HSP60 knockout hearts. Moreover, HSP60 deficiency induced activation of eIF2α-ATF4 pathway, further indicating the underlying mitochondrial stress in cardiomyocytes after HSP60 deletion. Taken together, our study demonstrated that regular function of mitochondrial chaperones is pivotal for maintaining normal mitochondrial homeostasis and embryonic heart development.

Epigenetic determinants and non-myocardial signaling pathways contributing to heart growth and regeneration.

Congenital heart disease is the most common birth defect worldwide. Defective cardiac myogenesis is either a major presentation or associated with many types of congenital heart disease. Non-myocardial tissues, including endocardium and epicardium, function as a supporting hub for myocardial growth and maturation during heart development. Recent research findings suggest an emerging role of epigenetics in nonmyocytes supporting myocardial development. Understanding how growth signaling pathways in non-myocardial tissues are regulated by epigenetic factors will likely identify new disease mechanisms for congenital heart diseases and shed lights for novel therapeutic strategies for heart regeneration.

RICH1 is a novel key suppressor of isoproterenol­ or angiotensin II­induced cardiomyocyte hypertrophy.

Cardiac hypertrophy is one of the key processes in the development of heart failure. Notably, small GTPases and GTPase­activating proteins (GAPs) serve essential roles in cardiac hypertrophy. RhoGAP interacting with CIP4 homologs protein 1 (RICH1) is a RhoGAP that can regulate Cdc42/Rac1 and F­actin dynamics. RICH1 is involved in cell proliferation and adhesion; however, to the best of our knowledge, its role in cardiac hypertrophy remains unknown. In the present study, the role of RICH1 in cardiomyocyte hypertrophy was assessed. Cell viability was analyzed using the Cell Counting Kit­8 assay and cells surface area (CSA) was determined by cell fluorescence staining. Reverse transcription­quantitative PCR and western blotting were used to assess the mRNA expression levels of hypertrophic marker genes, such as Nppa, Nppb and Myh7, and the protein expression levels of RICH1, respectively. RICH1 was shown to be downregulated in isoproterenol (ISO)­ or angiotensin II (Ang II)­treated H9c2 cells. Notably, overexpression of RICH1 attenuated the upregulation of hypertrophy­related markers, such as Nppa, Nppb and Myh7, and the enlargement of CSA induced by ISO and Ang II. By contrast, the knockdown of RICH1 exacerbated these effects. These findings suggested that RICH1 may be a novel suppressor of ISO­ or Ang II­induced cardiomyocyte hypertrophy. The results of the present study will be beneficial to further studies assessing the role of RICH1 and its downstream molecules in inhibiting cardiac hypertrophy.

The molecular mechanism of MiR-26a-5p regulates autophagy and activates NLRP3 inflammasome to mediate cardiomyocyte hypertrophy.

OBJECTIVE: Many studies have found that miR-26a-5p plays an essential role in the progression of pathological cardiac hypertrophy, however, there is still no evidence on whether miR-26a-5p is related to the activation of autophagy and NLRP3 inflammasome. And the mechanism of miR-26a-5p and NLRP3 inflammasome aggravating pathological cardiac hypertrophy remain unclear. METHODS: Cardiomyocytes were treated with 200µM PE to induce cardiac hypertrophy and intervened with 10mM NLRP3 inhibitor INF39. In addition, we also used the MiR-26a-5p mimic and inhibitor to transfect PE-induced cardiac hypertrophy. RT-qPCR and western blotting were used to detect the expressions of miR-26a-5p, NLRP3, ASC and Caspase-1 in each group, and we used α-SMA immunofluorescence to detect the change of cardiomyocyte area. The expression levels of autophagy proteins LC3, beclin-1 and p62 were detected by western blotting. Finally, we induced the SD rat cardiac hypertrophy model through aortic constriction (TAC) surgery. In the experimental group, rats were intervened with MiR-26a-5p mimic, MiR-26a-5p inhibitor, autophagy inhibitor 3-MA, and autophagy activator Rapamycin. RESULTS: In cell experiments, we observed that the expression of miR-26a-5p was associated with cardiomyocyte hypertrophy and increased surface area. Furthermore, miR-26a-5p facilitated autophagy and activated the NLRP3 inflammasome pathway, which caused changes in the expression of genes and proteins including LC3, beclin-1, p62, ACS, NLRP3, and Caspase-1. We discovered similar outcomes in the TAC rat model, where miR-26a-5p expression corresponded with cardiomyocyte enlargement and fibrosis in the cardiac interstitial and perivascular regions. In conclusion, miR-26a-5p has the potential to regulate autophagy and activate the NLRP3 inflammasome, contributing to the development of cardiomyocyte hypertrophy. CONCLUSION: Our study found a relationship between the expression of miR-26a-5p and cardiomyocyte hypertrophy. The mechanism behind this relationship appears to involve the activation of the NLRP3 inflammasome pathway, which is caused by miR-26a-5p promoting autophagy. Targeting the expression of miR-26a-5p, as well as inhibiting the activation of autophagy and the NLRP3 inflammasome pathway, could offer additional treatments for pathological cardiac hypertrophy.

MicroRNA-34a-Dependent Attenuation of Angiogenesis in Right Ventricular Failure.

BACKGROUND: The right ventricle (RV) is at risk in patients with complex congenital heart disease involving right-sided obstructive lesions. We have shown that capillary rarefaction occurs early in the pressure-loaded RV. Here we test the hypothesis that microRNA (miR)-34a, which is induced in RV hypertrophy and RV failure (RVF), blocks the hypoxia-inducible factor-1α-vascular endothelial growth factor (VEGF) axis, leading to the attenuated angiogenic response and increased susceptibility to RV failure. METHODS AND RESULTS: Mice underwent pulmonary artery banding to induce RV hypertrophy and RVF. Capillary rarefaction occurred immediately. Although hypoxia-inducible factor-1α expression increased (0.12±0.01 versus 0.22±0.03, P=0.05), VEGF expression decreased (0.61±0.03 versus 0.22±0.05, P=0.01). miR-34a expression was most upregulated in fibroblasts (4-fold), but also in cardiomyocytes and endothelial cells (2-fold). Overexpression of miR-34a in endothelial cells increased cell senescence (10±3% versus 22±2%, P<0.05) by suppressing sirtulin 1 expression, and decreased tube formation by 50% via suppression of hypoxia-inducible factor-1α, VEGF A, VEGF B, and VEGF receptor 2. miR-34a was induced by stretch, transforming growth factor-ß1, adrenergic stimulation, and hypoxia in cardiac fibroblasts and cardiomyocytes. In mice with RVF, locked nucleic acid-antimiR-34a improved RV shortening fraction and survival half-time and restored capillarity and VEGF expression. In children with congenital heart disease-related RVF, RV capillarity was decreased and miR-34a increased 5-fold. CONCLUSIONS: In summary, miR-34a from fibroblasts, cardiomyocytes, and endothelial cells mediates capillary rarefaction by suppressing the hypoxia-inducible factor-1α-VEGF axis in RV hypertrophy/RVF, raising the potential for anti-miR-34a therapeutics in patients with at-risk RVs.

Generation and characterization of a human induced pluripotent stem cell line heterozygous for a NOTCH1 mutation (NCHi014-A).

NOTCH1 signaling is crucial for cardiovascular development. Numerous studies have identified heterozygous NOTCH1 loss of function and missense variants associated with a spectrum of congenital heart diseases (CHD). We generated induced pluripotent stem cells (iPSC) from a healthy individual to develop a model for NOTCH1+/- iPSC to study the molecular pathogenesis of CHD. NOTCH1+/-iPSC (NCHi014-A) have normal morphology and karyotype, are identical to the parental cell line, express pluripotency markers and have the ability to differentiate to the three germ layers. NOTCH1+/- iPSC can be used as a tool to study the cellular and molecular mechanisms underlying NOTCH1-associated human CHD.

Kcnh2 deletion is associated with rat embryonic development defects via destruction of KCNH2­integrin ß1 complex.

The Kv11.1 potassium channel encoded by the Kcnh2 gene is crucial in conducting the rapid delayed rectifier K+ current in cardiomyocytes. Homozygous mutation in Kcnh2 is embryonically lethal in humans and mice. However, the molecular signaling pathway of intrauterine fetal loss is unclear. The present study generated a Kcnh2 knockout rat based on edited rat embryonic stem cells (rESCs). Kcnh2 knockout was embryonic lethal on day 11.5 of development due to a heart configuration defect. Experiments with human embryonic heart single cells (6.5­7 weeks post­conception) suggested that potassium voltage­gated channel subfamily H member 2 (KCNH2) plays a crucial role in the development of compact cardiomyocytes. By contrast, apoptosis was found to be triggered in the homozygous embryos, which could be attributed to the failure of KCNH2 to form a complex with integrin ß1 that was essential for preventing the process of apoptosis via inhibition of forkhead box O3A. Destruction of the KCNH2/integrin ß1 complex reduced the phosphorylation level of AKT and deactivated the glycogen synthase kinase 3 ß (GSK­3ß)/ß­catenin pathway, which caused early developmental abnormalities in rats. The present work reveals a basic mechanism by which KCNH2 maintains intact embryonic heart development.

Changes in glutamic oxaloacetic transaminase 2 during rat physiological and pathological cardiomyocyte hypertrophy.

BACKGROUND: Physiological and pathological cardiomyocyte hypertrophy are important pathophysiological processes of adult congenital heart disease-associated ventricular hypertrophy. Glutamic oxaloacetic transaminase (GOT) is a vital marker of myocardial injury. This study aimed to investigate the changes in GOT levels during physiological and pathological cardiomyocyte hypertrophy in rats. METHODS: RNA-seq analysis and colorimetric methods were used to evaluate the changes in GOT mRNA and activity, respectively. GOT2 protein expression was detected by western blotting and immunofluorescence. Hematoxylin-eosin and wheat germ agglutinin methods were used to observe changes in rat cardiomyocyte morphology. RESULTS: In juvenile rat hearts, GOT mRNA expression and activity, and GOT2 protein level increased with age-related physiological cardiomyocyte hypertrophy; however, GOT2 protein level was reduced in hypoxia-induced pathological cardiomyocyte hypertrophy. CONCLUSIONS: GOT2 may regulate physiological and pathological myocardial hypertrophy in rats. We speculated that the low GOT2 level contributed to the rapid occurrence of pathological cardiomyocyte hypertrophy, causing strong plasticity of right ventricular cardiomyocytes in the early postnatal period and heart failure in adulthood.

Gene editing and cardiac disease modelling for the interpretation of genetic variants of uncertain significance in congenital heart disease.

BACKGROUND: Genomic sequencing in congenital heart disease (CHD) patients often discovers novel genetic variants, which are classified as variants of uncertain significance (VUS). Functional analysis of each VUS is required in specialised laboratories, to determine whether the VUS is disease causative or not, leading to lengthy diagnostic delays. We investigated stem cell cardiac disease modelling and transcriptomics for the purpose of genetic variant classification using a GATA4 (p.Arg283Cys) VUS in a patient with CHD. METHODS: We performed high efficiency CRISPR gene editing with homology directed repair in induced pluripotent stem cells (iPSCs), followed by rapid clonal selection with amplicon sequencing. Genetic variant and healthy matched control cells were compared using cardiomyocyte disease modelling and transcriptomics. RESULTS: Genetic variant and healthy cardiomyocytes similarly expressed Troponin T (cTNNT), and GATA4. Transcriptomics analysis of cardiomyocyte differentiation identified changes consistent with the patient's clinical human phenotype ontology terms. Further, transcriptomics revealed changes in calcium signalling, and cardiomyocyte adrenergic signalling in the variant cells. Functional testing demonstrated, altered action potentials in GATA4 genetic variant cardiomyocytes were consistent with patient cardiac abnormalities. CONCLUSIONS: This work provides in vivo functional studies supportive of a damaging effect on the gene or gene product. Furthermore, we demonstrate the utility of iPSCs, CRISPR gene editing and cardiac disease modelling for genetic variant interpretation. The method can readily be applied to other genetic variants in GATA4 or other genes in cardiac disease, providing a centralised assessment pathway for patient genetic variant interpretation.

Skeletal defects and bone metabolism in Noonan, Costello and cardio-facio-cutaneous syndromes.

Noonan, Costello and Cardio-facio-cutaneous syndromes belong to a group of disorders named RASopathies due to their common pathogenetic origin that lies on the Ras/MAPK signaling pathway. Genetics has eased, at least in part, the distinction of these entities as they are presented with overlapping clinical features which, sometimes, become more pronounced with age. Distinctive face, cardiac and skeletal defects are among the primary abnormalities seen in these patients. Skeletal dysmorphisms range from mild to severe and may include anterior chest wall anomalies, scoliosis, kyphosis, short stature, hand anomalies, muscle weakness, osteopenia or/and osteoporosis. Patients usually have increased serum concentrations of bone resorption markers, while markers of bone formation are within normal range. The causative molecular defects encompass the members of the Ras/MAPK/ERK pathway and the adjacent cascades, important for the maintenance of normal bone homeostasis. It has been suggested that modulation of the expression of specific molecules involved in the processes of bone remodeling may affect the osteogenic fate decision, potentially, bringing out new pharmaceutical targets. Currently, the laboratory imprint of bone metabolism on the clinical picture of the affected individuals is not clear, maybe due to the rarity of these syndromes, the small number of the recruited patients and the methods used for the description of their clinical and biochemical profiles.

Transpulmonary Expression of Exosomal microRNAs in Idiopathic and Congenital Heart Disease-Related Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary artery hypertension (PAH) is a fatal disease characterized by a complex pathogenesis. Exosomes containing microRNAs (miRs) have emerged as a novel biomarker. Transpulmonary exosomal miRs offer valuable insights into pulmonary circulation microenvironments. Hereby, we aimed to explore the potentials of transpulmonary exosomal miRs as differentiating factors between idiopathic PAH and congenital heart disease (CHD)-related PAH. METHODS AND RESULTS: During right heart catheterization, we collected exosomes at pulmonary arteries in 25 patients diagnosed with idiopathic PAH and 20 patients with CHD-related PAH. Next-generation sequencing identified several candidate exosomal miRs. Using quantitative polymerase chain reaction, we validated the expressions of these miRs and revealed significantly elevated expressions of miR-21, miR-139-5p, miR-155-5p, let-7f-5p, miR-328-3p, miR-330-3p, and miR-103a-3p in patients with CHD-related PAH, in contrast to patients with idiopathic PAH. Among these miRs, miR-21 exhibited the highest expression in patients with CHD-related PAH. These findings were further corroborated in an external cohort comprising 10 patients with idiopathic PAH and 8 patients with CHD-related PAH. Using an in vitro flow model simulating the shear stress experienced by pulmonary endothelial cells, we observed a significant upregulation of miR-21. Suppressing miR-21 rescued the shear stress-induced downregulation of the RAS/phosphatidylinositol 3-kinase/protein kinase B pathway, leading to a mitigation of apoptosis. CONCLUSIONS: Our study identified a pronounced expression of transpulmonary exosomal miR-21, particularly in patients with CHD-related PAH, through next-generation sequencing analysis. Further investigation is warranted to elucidate the regulatory mechanisms involving miR-21 in the pathophysiology of PAH.

LOC102549726/miR-760-3p network is involved in the progression of ISO-induced pathological cardiomyocyte hypertrophy via endoplasmic reticulum stress.

Pathological cardiac hypertrophy (CH) is featured by myocyte enlargement and cardiac malfunction. Multiple signaling pathways have been implicated in diverse pathological and physiological processes in CH. However, the function of LOC102549726/miR-760-3p network in CH remains unclear. Here, we characterize the functional role of LOC102549726/miR-760-3p network in CH and delineate the underlying mechanism. The expression of LncRNA LOC102549726 and hypertrophic markers was significantly increased compared to the control, while the level of miR-760-3p was decreased. Next, we examined ER stress response in a hypertrophic cardiomyocyte model. The expression of ER stress markers was greatly enhanced after incubation with ISO. The hypertrophic reaction, ER stress response, and increased potassium and calcium ion channels were alleviated by genetic downregulation of LOC102549726. It has been demonstrated that LOC102549726 functions as a competitive endogenous RNA (ceRNA) of miR-760-3p. Overexpression of miR-760-3p decreased cell surface area and substantially mitigated ER stress response; protein levels of potassium and calcium channels were also significantly up-regulated compared to the NC control. In contrast, miR-760-3p inhibition increased cell size, aggravated CH and ER stress responses, and reduced ion channels. Collectively, in this study we demonstrated that the LOC102549726/miR-760-3p network was a crucial regulator of CH development. Ion channels mediate the ER stress response and may be a downstream sensor of the LOC102549726/miR-760-3p network. Therefore, these findings advance our understanding of pathological CH and provide new insights into therapeutic targets for cardiac remodeling.

Increased Ca2+ Transient Underlies RyR2-Related Left Ventricular Noncompaction.

BACKGROUND: A loss-of-function cardiac ryanodine receptor (RyR2) mutation, I4855M+/-, has recently been linked to a new cardiac disorder termed RyR2 Ca2+ release deficiency syndrome (CRDS) as well as left ventricular noncompaction (LVNC). The mechanism by which RyR2 loss-of-function causes CRDS has been extensively studied, but the mechanism underlying RyR2 loss-of-function-associated LVNC is unknown. Here, we determined the impact of a CRDS-LVNC-associated RyR2-I4855M+/- loss-of-function mutation on cardiac structure and function. METHODS: We generated a mouse model expressing the CRDS-LVNC-associated RyR2-I4855M+/- mutation. Histological analysis, echocardiography, ECG recording, and intact heart Ca2+ imaging were performed to characterize the structural and functional consequences of the RyR2-I4855M+/- mutation. RESULTS: As in humans, RyR2-I4855M+/- mice displayed LVNC characterized by cardiac hypertrabeculation and noncompaction. RyR2-I4855M+/- mice were highly susceptible to electrical stimulation-induced ventricular arrhythmias but protected from stress-induced ventricular arrhythmias. Unexpectedly, the RyR2-I4855M+/- mutation increased the peak Ca2+ transient but did not alter the L-type Ca2+ current, suggesting an increase in Ca2+-induced Ca2+ release gain. The RyR2-I4855M+/- mutation abolished sarcoplasmic reticulum store overload-induced Ca2+ release or Ca2+ leak, elevated sarcoplasmic reticulum Ca2+ load, prolonged Ca2+ transient decay, and elevated end-diastolic Ca2+ level upon rapid pacing. Immunoblotting revealed increased level of phosphorylated CaMKII (Ca2+-calmodulin dependent protein kinases II) but unchanged levels of CaMKII, calcineurin, and other Ca2+ handling proteins in the RyR2-I4855M+/- mutant compared with wild type. CONCLUSIONS: The RyR2-I4855M+/- mutant mice represent the first RyR2-associated LVNC animal model that recapitulates the CRDS-LVNC overlapping phenotype in humans. The RyR2-I4855M+/- mutation increases the peak Ca2+ transient by increasing the Ca2+-induced Ca2+ release gain and the end-diastolic Ca2+ level by prolonging Ca2+ transient decay. Our data suggest that the increased peak-systolic and end-diastolic Ca2+ levels may underlie RyR2-associated LVNC.

CFAP45, a heterotaxy and congenital heart disease gene, affects cilia stability.

Congenital heart disease (CHD) is the most common and lethal birth defect, affecting 1.3 million individuals worldwide. During early embryogenesis, errors in Left-Right (LR) patterning called Heterotaxy (Htx) can lead to severe CHD. Many of the genetic underpinnings of Htx/CHD remain unknown. In analyzing a family with Htx/CHD using whole-exome sequencing, we identified a homozygous recessive missense mutation in CFAP45 in two affected siblings. CFAP45 belongs to the coiled-coil domain-containing protein family, and its role in development is emerging. When we depleted Cfap45 in frog embryos, we detected abnormalities in cardiac looping and global markers of LR patterning, recapitulating the patient's heterotaxy phenotype. In vertebrates, laterality is broken at the Left-Right Organizer (LRO) by motile monocilia that generate leftward fluid flow. When we analyzed the LRO in embryos depleted of Cfap45, we discovered "bulges" within the cilia of these monociliated cells. In addition, epidermal multiciliated cells lost cilia with Cfap45 depletion. Via live confocal imaging, we found that Cfap45 localizes in a punctate but static position within the ciliary axoneme, and depletion leads to loss of cilia stability and eventual detachment from the cell's apical surface. This work demonstrates that in Xenopus, Cfap45 is required to sustain cilia stability in multiciliated and monociliated cells, providing a plausible mechanism for its role in heterotaxy and congenital heart disease.

The cardiorespiratory optimal point as a discriminator of lesion severity in adults with congenital heart disease.

BACKGROUND: Peak oxygen consumption (VO2peak), which depends on maximal exertion and is reduced in adults with congenital heart disease (ACHD), is associated with lesion severity. The lowest ventilatory equivalent for oxygen (the minimum value of VE/VO2) reflects the cardiorespiratory optimal point (COP) as best possible respiration-circulatory interaction and may discriminate between lesion types without the need for maximal exertion. However, data on COP in ACHD is scarce. METHODS: We retrospectively analyzed stable ACHD with moderate (N.=13) and severe lesions (N.=17) reporting to our outpatient clinic undergoing cardiopulmonary exercise testing. The primary outcome of the study was the difference of COP between moderate and severe lesions. Secondary outcomes were between group differences of the submaximal variable exercise oxygen uptake efficiency slope (OUES) and peak O2 pulse (O2pulsemax) as a surrogate for peripheral oxygen extraction and stroke volume increase during exercise. RESULTS: The group of severe lesions displayed higher COP (29.5±7.0 vs. 25.2±6.2, P=0.028) as well as lower O2pulsemax (13.3±8.4 vs. 14.9±3.4 mL/beat/kg 102, P=0.038). VO2peak (17.4±6.5 vs. 20.8±8.5 mL/kg/min, P=0.286) and OUES (1.5±0.7 vs. 1.8±0.9, P=0.613) showed a trend towards lower values in severe lesions. COP was a better between group discriminator than O2pulsemax (area under the curve 73.8% vs. 72.4%). CONCLUSIONS: As a submaximal variable, COP discriminated between moderate and severe lesions and may prove beneficial in a highly vulnerable population that is often unable to undergo exertional testing.

Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced cardiac anomalies through reconciliation of autophagy and ferroptosis.

Lipopolysaccharide (LPS) from Gram-negative bacteria is a major contributor to cardiovascular failure, but the signaling mechanisms underlying its stress response are not fully understood. This study aimed to investigate the effect of the antioxidant enzyme catalase on LPS-induced cardiac abnormalities and the mechanisms involved, with particular focus on the interplay between autophagy, ferroptosis, and apoptosis. Cardiac-specific catalase (CAT) overexpression and wild-type (WT) mice were stimulated with LPS (6 mg/kg, intravenous injection), and cardiac morphology and function were evaluated. Oxidative stress, ferroptosis, apoptosis, and mitochondrial status were monitored, and survival curves were plotted based on the results of LPS stimulation. The results showed that, compared with WT mice, mice overexpressing catalase had a higher survival rate under LPS stimulation. Ultrasound echocardiography, cardiomyocyte characteristics, and Masson's trichrome staining showed that LPS inhibited cardiac function and caused cardiac fibrosis, while catalase alleviated these adverse effects. LPS increased apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), increased O2·- production, induced inflammation (TNF-α), autophagy, iron toxicity, and carbonyl damage, and significantly damaged mitochondria (mitochondrial membrane potential, mitochondrial proteins, and ultrastructure). These effects were significantly alleviated by catalase. Interestingly, the antioxidant N-acetylcysteine, autophagy inhibitor 3-methyladenine, and ferroptosis inhibitor lipostatin-1 all eliminated the LPS-induced contraction dysfunction and ferroptosis (using lipid peroxidation). Induction of ferroptosis could eliminate the cardioprotective effect of NAC. In conclusion, catalase rescues LPS-induced cardiac dysfunction by regulating oxidative stress, autophagy, ferroptosis, apoptosis, and mitochondrial damage in cardiomyocytes.

Deletion of PDK1 Caused Cardiac Malmorphogenesis and Heart Defects Due to Profound Protein Phosphorylation Changes Mediated by SHP2.

Phosphoinositide-dependent protein kinase-1 (PDK1), a master kinase and involved in multiple signaling transduction, participates in regulating embryonic cardiac development and postnatal cardiac remodeling. Germline PDK1 knockout mice displayed no heart development; in this article, we deleted PDK1 in heart tissue with different cre to characterize the temporospatial features and find the relevance with congenital heart disease(CHD), furthermore to investigate the underlying mechanism. Knocking out PDK1 with Nkx2.5-cre, the heart showed prominent pulmonic stenosis. Ablated PDK1 with Mef2cSHF-cre, the second heart field (SHF) exhibited severe hypoplasia. And deleted PDK1 with αMHC-cre, the mice displayed dilated heart disease, protein analysis indicated PI3K and ERK were activated; meanwhile, PDK1-AKT-GSK3, and S6K-S6 were disrupted; phosphorylation level of Akt473, S6k421/424, and Gsk3α21 enhanced; however, Akt308, S6k389, and Gsk3ß9 decreased. In mechanism investigation, we found SHP2 membrane localization and phosphorylation level of SHP2542 elevated, which suggested SHP2 likely mediated the disruption.

Effects of ketamine-induced H3K9 hypoacetylation during pregnancy on cardiogenesis of mouse offspring.

BACKGROUND: Prenatal exposure to adverse factors can cause congenital heart defects. Ketamine, a widely used anesthetic drug, produces several adverse reactions such as tachycardia, hypertension, and laryngospasm, especially in pediatric patients. This study aimed to detect the effects of ketamine exposure during pregnancy on the cardiogenesis of mouse offspring and the potential mechanisms. METHODS: In this study, ketamine at an addictive dose (5 mg/kg) was administered to mice during early gestation to explore the epigenetic mechanism of its causing cardiac dysplasia. The cardiac morphology of the mouse offspring was observed through hematoxylin-eosin staining and transmission electron microscopy. The heart function of one-month-old neonates was detected by echocardiography. The expression of cardiomyogenesis-related genes was detected by western blot and RT-qPCR. The acetylation level of histone H3K9 at the Mlc2 promoter and its deacetylase level and activity were detected by CHIP-qPCR, RT-qPCR, and ELISA, respectively. RESULTS: Our data revealed that ketamine exposure during pregnancy could cause cardiac enlargement, myocardial sarcomere disorganization, and decreased cardiac contractile function in mouse offspring. Moreover, ketamine reduced the expression of Myh6, Myh7, Mlc2, Mef2c, and cTnI. The histone H3K9 acetylation level at the Mlc2 promoter was down-regulated by increasing the histone deacetylase activity and HDAC3 level upon ketamine administration. CONCLUSIONS: Our work indicates that H3K9 acetylation is a vital player in cardiac dysplasia in offspring caused by prenatal ketamine exposure and HDAC3 is a key regulatory factor.

Loss of GLTSCR1 causes congenital heart defects by regulating NPPA transcription.

Precise and specific spatiotemporal domains of gene expression regulation are critical for embryonic development. Recent studies have identified GLTSCR1 as a gene transcriptional elongation regulator in cancer research. However, the function of GLTSCR1, especially in embryonic development, remains poorly understood. Here, we found that GLTSCR1 was essential for cardiac development because Gltscr1 knockout (Gltscr1-/-) led to embryonic lethality in mice with severe congenital heart defects (CHDs). Ventricular septal defect and double outflow right ventricular were also observed in neural crest cells with conditional deletion of Gltscr1, which were associated with neonatal lethality in mice. Mechanistically, GLTSCR1 deletion promoted NPPA expression by coordinating the CHD risk G allele of rs56153133 in the NPPA enhancer and releasing the transcription factor ZNF740-binding site on the NPPA promoter. These findings demonstrated that GLTSCR1 acts as a candidate CHD-related gene.

Establishing a human embryonic stem cell line (SKLRMe005-A) from a blastocyst with congenital heart disease (CHD).

GATA binding protein 6 (GATA6) is an important transcription factor of cardiovascular endothelial cells, has the potential to regulate the process of cardiac development. Consequently, its abnormal expression is related to congenital heart disease.Human GATA6 gene clones were on chromosome 18 q11.1- q 11.2, consists of 7 exons and 6 intron.Now, a human embryonic stem cell line with GATA6 c.620_647del (p.S208Afs*77) mutation was generated. Interestingly, the ESC line displayed a normal karyotype, pluripotency and morphology of stem cells.This cell line has the ability to undergo differentiation into three germ layers in vivo.