Cardiomyocyte Overexpression Research Articles

Downregulation of HSPA12A protects heart against sepsis through suppressing mTOR-mediated inflammatory response in cardiomyocytes

ObjectiveOver-activated immune response in hearts is the main pathological feature of septic cardiomyopathy, a fatal complication of sepsis with high mortality. Autophagy is capable to limit immune response by removing inflammatory mediators. Heat shock protein A12A (HSPA12A) encodes an atypical member of HSP70 family. This study aimed to investigate the role of HSPA12A in septic cardiomyopathy. MethodsSepsis was induced by cecal ligation and puncture (CLP) for 6 h in mice in vivo or by LPS treatment for 24 h in primary cardiomyocytes in vitro. HSPA12A knockout (Hspa12a−/−) mice were generated by cre-loxp system. Echocardiography was performed to assess cardiac function. TUNEL and propidium iodide (PI) staining was used to indicate cardiomyocyte death. Inflammation-related factors were examined by qPCR and immunoblotting. Autophagy was evaluated by levels of LC3-II and p62. ResultsSepsis decreased HSPA12A expression in hearts and cardiomyocytes, while HSPA12A knockout in mice attenuated sepsis-induced cardiomyocyte death and cardiac dysfunction. Sepsis-induced activation of TLR4/MyD88/NF-κB-mediated inflammation was inhibited in hearts by HSPA12A knockout whereas was enhanced by HSPA12A overexpression in cardiomyocytes. Moreover, HSPA12A overexpression activated mTOR and inhibited autophagy in cardiomyocytes, while inhibition of mTOR by rapamycin diminished the HSPA12A-induced autophagy inhibition, inflammation activation, and cardiomyocyte death in septic cardiomyocytes. ConclusionDownregulation of HSPA12A inhibited mTOR to activated autophagy, thereby suppressed inflammatory response, and ultimately attenuated septic cardiomyopathy. Our findings identified HSPA12A as a driver for septic cardiomyopathy development, and strategies that inhibit HSPA12A in cardiomyocytes might be a potential therapeutic intervention.

Nrf2/NRF1 signaling activation and crosstalk amplify mitochondrial biogenesis in the treatment of triptolide-induced cardiotoxicity using calycosin

Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates both oxidative stress and mitochondrial biogenesis. Our previous study reported the cardioprotection of calycosin against triptolide toxicity through promoting mitochondrial biogenesis by activating nuclear respiratory factor 1 (NRF1), a coregulatory effect contributed by Nrf2 was not fully elucidated. This work aimed at investigating the involvement of Nrf2 in mitochondrial protection and elucidating Nrf2/NRF1 signaling crosstalk on amplifying the detoxification of calycosin. Results indicated that calycosin inhibited cardiomyocytes apoptosis and F-actin depolymerization following triptolide exposure. Cardiac contraction was improved by calycosin through increasing both fractional shortening (FS%) and ejection fraction (EF%). This enhanced contractile capacity of heart was benefited from mitochondrial protection reflected by ultrastructure improvement, augment in mitochondrial mass and ATP production. NRF1 overexpression in cardiomyocytes increased mitochondrial mass and DNA copy number, whereas NRF1 knockdown mitigated calycosin-mediated enhancement in mitochondrial mass. For nuclear Nrf2, it was upregulated by calycosin in a way of disrupting Nrf2-Keap1 (Kelch-like ECH associated protein 1) interaction, followed by inhibiting ubiquitination and degradation. The involvement of Nrf2 in mitochondrial protection was validated by the results that both Nrf2 knockdown and Nrf2 inhibitor blocked the calycosin effects on mitochondrial biogenesis and respiration. In the case of calycosin treatment, its effect on NRF1 and Nrf2 upregulations were respectively blocked by PGCα/Nrf2 and NRF1 knockdown, indicative of the mutual regulation between Nrf2 and NRF1. Accordingly, calycosin activated Nrf2/NRF1 and the signaling crosstalk, leading to mitochondrial biogenesis amplification, which would become a novel mechanism of calycosin against triptolide-induced cardiotoxicity.

HDAC7 promotes cardiomyocyte proliferation by suppressing Myocyte Enhancer Factor 2.

Postnatal mammalian cardiomyocytes (CMs) rapidly lose proliferative capacity and exit the cell cycle and undergo further differentiation and maturation. Cell cycle activation has been a major strategy to stimulate postnatal CM proliferation, albeit achieving modest effects. One impediment is that postnatal CMs may need to undergo dedifferentiation before proliferation, if not simultaneously. Here, we report that overexpression of Hdac7 in neonatal mouse CMs results in significant CM dedifferentiation and proliferation. Mechanistically, we show that HDAC7-mediated CM proliferation is contingent on dedifferentiation, which is accomplished through suppressing MEF2. Hdac7 overexpression in CM shifts the chromatin state from binding MEF2, which favors the differentiation transcriptional program to AP-1, which favors the proliferative transcriptional program. Further, we found that HDAC7 interacts with minichromosome maintenance complex (MCM) components to initiate cell cycle progression. Our findings reveal that HDAC7 promotes CM proliferation by its dual action on CM dedifferentiation and proliferation, uncovering a potential new strategy for heart regeneration/repair.

Key subdomains of mesencephalic astrocyte-derived neurotrophic factor attenuate myocardial ischemia/reperfusion injury by JAK1/STAT1/NF-κB signaling pathway

BackgroundMyocardial ischemia/reperfusion (I/R) injury is a common pathological process in clinical practice. Developing effective therapeutic strategies to reduce or prevent this injury is crucial. The article aimed to investigate the role and mechanism of mesencephalic astrocyte-derived neurotrophic factor (MANF) and its key subdomains in modulating myocardial I/R-induced cardiomyocyte apoptosis.MethodsMANF stable knockout cell line and MANF mutant overexpression plasmids were constructed. The effects of MANF and mutants on apoptosis and endoplasmic reticulum (ER) stress related proteins were evaluated in hypoxia/reoxygenation-induced HL-1 cardiomyocytes by western blot, immunofluorescence, Tunel and flow cytometry. Echocardiography, ELISA, TTC and Masson were used to observe the effects of recombinant MANF protein (rMANF) on cardiac function in myocardial I/R mice.ResultsThis study observed increased expression of MANF in both myocardial infarction patients and I/R mice. MANF overexpression in cardiomyocytes decreased ER stress-induced apoptosis, while MANF knockout exacerbated it. rMANF improved cardiac function in I/R mice by reducing injury and inflammation. This study specifically demonstrates that mutations in the α-helix of MANF were more effective in reducing ER stress and cardiomyocyte apoptosis. Mechanistically, MANF and the α-helix mutant attenuated I/R injury by inhibiting the JAK1/STAT1/NF-κB signaling pathway in addition to reducing ER stress-induced apoptosis.ConclusionThese findings highlight MANF and its subdomains as critical regulators of myocardial I/R injury, offering promising therapeutic targets with significant clinical implications for I/R-related diseases.

GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia

Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.

Cardiomyocyte-specific RXFP1 overexpression protects against pressure overload-induced cardiac dysfunction independently of relaxin

Heart failure (HF) prevalence is rising due to reduced early mortality and demographic change. Relaxin (RLN) mediates protective effects in the cardiovascular system through Relaxin-receptor 1 (RXFP1). Cardiac overexpression of RXFP1 with additional RLN supplementation attenuated HF in the pressure-overload transverse aortic constriction (TAC) model. Here, we hypothesized that robust transgenic RXFP1 overexpression in cardiomyocytes (CM) protects from TAC-induced HF even in the absence of RLN.Hence, transgenic mice with a CM-specific overexpression of human RXFP1 (hRXFP1tg) were generated. Receptor functionality was demonstrated by in vivo hemodynamics, where the administration of RLN induced positive inotropy strictly in hRXFP1tg. An increase in phospholamban-phosphorylation at serine 16 was identified as a molecular correlate. hRXFP1tg were protected from TAC without additional RLN administration, presenting not only less decline in systolic left ventricular (LV) function but also abrogated LV dilation and pulmonary congestion compared to WT mice. Molecularly, transgenic hearts exhibited not only a significantly attenuated fetal and fibrotic gene activation but also demonstrated less fibrotic tissue and CM hypertrophy in histological sections. These protective effects were evident in both sexes. Similar cardioprotective effects of hRXFP1tg were detectable in a RLN-knockout model, suggesting an alternative mechanism of receptor activation through intrinsic activity, alternative endogenous ligands or crosstalk with other receptors.In summary, CM-specific RXFP1 overexpression provides protection against TAC even in the absence of endogenous RLN. This suggests RXFP1 overexpression as a potential therapeutic approach for HF, offering baseline protection with optional RLN supplementation for specific activation.

Proline metabolic reprogramming modulates cardiac remodeling induced by pressure overload in the heart.

Metabolic reprogramming is critical in the onset of pressure overload-induced cardiac remodeling. Our study reveals that proline dehydrogenase (PRODH), the key enzyme in proline metabolism, reprograms cardiomyocyte metabolism to protect against cardiac remodeling. We induced cardiac remodeling using transverse aortic constriction (TAC) in both cardiac-specific PRODH knockout and overexpression mice. Our results indicate that PRODH expression is suppressed after TAC. Cardiac-specific PRODH knockout mice exhibited worsened cardiac dysfunction, while mice with PRODH overexpression demonstrated a protective effect. In addition, we simulated cardiomyocyte hypertrophy in vitro using neonatal rat ventricular myocytes treated with phenylephrine. Through RNA sequencing, metabolomics, and metabolic flux analysis, we elucidated that PRODH overexpression in cardiomyocytes redirects proline catabolism to replenish tricarboxylic acid cycle intermediates, enhance energy production, and restore glutathione redox balance. Our findings suggest PRODH as a modulator of cardiac bioenergetics and redox homeostasis during cardiac remodeling induced by pressure overload. This highlights the potential of PRODH as a therapeutic target for cardiac remodeling.

Unveiling G-protein coupled receptor kinase-5 inhibitors for chronic degenerative diseases: Multilayered prioritization employing explainable machine learning-driven multi-class QSAR, ligand-based pharmacophore and free energy-inspired molecular simulation

GRK5 holds a pivotal role in cellular signaling pathways, with its overexpression in cardiomyocytes, neuronal cells, and tumor cells strongly associated with various chronic degenerative diseases, which highlights the urgent need for potential inhibitors. In this study, multiclass classification-based QSAR models were developed using diverse machine learning algorithms. These models were built from curated compounds with experimentally derived GRK5 inhibitory activity. Additionally, a pharmacophore model was constructed using active compounds from the dataset. Among the models, the SVM-based approach proved most effective and was initially used to screen DrugBank compounds within the applicability domain. Compounds showing significant GRK5 inhibitory potential underwent evaluation for key pharmacophoric features. Prospective compounds were subjected to molecular docking to assess binding affinity towards GRK5's key active site amino acid residues. Stability at the binding site was analyzed through 200 ns molecular dynamics simulations. MM-GBSA analysis quantified individual free energy components contributing to the total binding energy with respect to binding site residues. Metadynamics analysis, including PCA, FEL, and PDF, provided crucial insights into conformational changes of both apo and holo forms of GRK5 at defined energy states. The study identifies DB02844 (S-Adenosyl-1,8-Diamino-3-Thiooctane) and DB13155 (Esculin) as promising GRK5 inhibitors, warranting further in vitro and in vivo validation studies.

Rap1GAP exacerbates myocardial infarction by regulating the AMPK/SIRT1/NF-κB signaling pathway

Rap1 GTPase-activating protein (Rap1GAP) is an important tumor suppressor. The purpose of this study was to investigate the role of Rap1GAP in myocardial infarction (MI) and its potential mechanism. Left anterior descending coronary artery ligation was performed on cardiac-specific Rap1GAP conditional knockout (Rap1GAP-CKO) mice and control mice with MI. Seven days after MI, Rap1GAP expression in the hearts of control mice peaked, the expression of proapoptotic markers (Bax and cleaved caspase-3) increased, the expression of antiapoptotic factors (Bcl-2) decreased, and the expression of the inflammatory factors IL-6 and TNF-α increased; thus, apoptosis occurred, inflammation, infarct size, and left ventricular dysfunction increased, while the heart changes caused by MI were alleviated in Rap1GAP-CKO mice. Mouse heart tissue was obtained for transcriptome sequencing, and gene set enrichment analysis (GSEA) was used to analyze Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We found that Rap1GAP was associated with the AMPK and NF-κB signaling pathways and that Rap1GAP inhibited AMPK/SIRT1 and activated the NF-κB signaling pathway in model animals. Similar results were observed in primary rat myocardial cells subjected to oxygen–glucose deprivation (OGD) to induce ischemia and hypoxia. Activating AMPK with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reversed the damage caused by Rap1GAP overexpression in cardiomyocytes. In addition, the coimmunoprecipitation results showed that exogenous Rap1GAP interacted with AMPK. Rap1GAP was verified to regulate the AMPK SIRT1/NF-κB signaling pathway and exacerbate the damage to myocardial cells caused by ischemia and hypoxia. In conclusion, our results suggest that Rap1GAP promotes MI by modulating the AMPK/SIRT1/NF-κB signaling pathway and that Rap1GAP may be a therapeutic target for MI treatment in the future.

Hypoxia-induced stabilization of HIF2A promotes cardiomyocyte proliferation by attenuating DNA damage.

Gradual exposure to a chronic hypoxic environment leads to cardiomyocyte proliferation and improved cardiac function in mouse models through a reduction in oxidative DNA damage. However, the upstream transcriptional events that link chronic hypoxia to DNA damage have remained obscure. We sought to determine whether hypoxia signaling mediated by the hypoxia-inducible factor 1 or 2 (HIF1A or HIF2A) underlies the proliferation phenotype that is induced by chronic hypoxia. We used genetic loss-of-function models using cardiomyocyte-specific HIF1A and HIF2A gene deletions in chronic hypoxia. We additionally characterized a cardiomyocyte-specific HIF2A overexpression mouse model in normoxia during aging and upon injury. We performed transcriptional profiling with RNA-sequencing on cardiac tissue, from which we verified candidates at the protein level. We find that HIF2A - rather than HIF1A - mediates hypoxia-induced cardiomyocyte proliferation. Ectopic, oxygen-insensitive HIF2A expression in cardiomyocytes reveals the cell-autonomous role of HIF2A in cardiomyocyte proliferation. HIF2A overexpression in cardiomyocytes elicits cardiac regeneration and improvement in systolic function after myocardial infarction in adult mice. RNA-sequencing reveals that ectopic HIF2A expression attenuates DNA damage pathways, which was confirmed with immunoblot and immunofluorescence. Our study provides mechanistic insights about a new approach to induce cardiomyocyte renewal and mitigate cardiac injury in the adult mammalian heart. In light of evidence that DNA damage accrues in cardiomyocytes with aging, these findings may help to usher in a new therapeutic approach to overcome such age-related changes and achieve regeneration.

Cardiomyocyte-specific beta3-adrenergic receptor expression reduces infarct size inhibiting the autophagic flux in mice

Abstract Background Since the discovery of β3-adrenergic receptor (β3AR), the third class of β-adrenergic receptors, expression in in both cardiomyocytes and endothelial cells of the cardiovascular system, it has come to the focus due to it´s possible implication in cardiovascular diseases. Although it´s expression is low compared to β1 and β2 subtypes (1), its been demonstrated that β3AR agonists have a cardioprotective effect in ischemia/reperfusion (IR) injury though inhibition of mitochondrial pore opening (2). Purpose In this project we determined the cellular origin of the β3AR cardioprotection and its role on cellular autophagic machinery. Methods Mice with overexpression of the β3AR in cardiomyocytes or endothelial cells and their control littermates were subjected to left coronary artery occlusion for 45 min followed reperfusion after 24h and 7days. Left ventricular function was assessed by echocardiography at day 7. Then, heart samples were collected at baseline and 24h and 7 days after IRI. Mitochondrial number were analyzed by transmission electron microscopy. Proteins related to autophagy signaling pathways were measured by western blot and RT-qPCR. Results Cardiomyocyte-specific β3AR overexpression protects the heart upon IR injury, reduced cardiac fibrosis which improved cardiac function and maintained heart mass. Studying the effects at baseline to understand the molecular mechanisms of cardioprotection, we found that β3AR overexpression in cardiomyocytes showed a reduction in autophagy markers such as LC3B and Parkin, meaning there is an alteration in the autophagic flux. Conclusions Cardiomyocyte-specific overexpression of β3-adrenergic receptor reduces infarct size and protects the heart affecting the autophagy machinery. Our results shed light on the role of the β3AR in the mitochondrial quality control system, offering evidence of its potential use as a target to decrease IR injury in patients with acute myocardial infarction.

The STING-IRF3 Signaling Pathway, Mediated by Endoplasmic Reticulum Stress, Contributes to Impaired Myocardial Autophagic Flux After Ischemia/Reperfusion.

This study aimed to determine whether endoplasmic reticulum (ER) stress is involved in impaired autophagy after myocardial ischemia/reperfusion (M-I/R) and elucidate the underlying mechanisms. The expression levels of stimulator of interferon gene (STING) and interferon regulatory transcription factor 3 (IRF3) phosphorylation increased in M-I/R heart tissues and hypoxia-treated/reoxygenation-treated H9c2 cells. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) significantly suppressed the stimulation of STING-IRF3 transcription and alleviated cardiac dysfunction caused by M-I/R injury. In addition, 4-PBA reversed ischemia-induced/reperfusion-induced autophagic flux dysfunction, as demonstrated by a decrease in p 62 and LC3 levels. Similarly, the protective effect of STING deficiency on myocardial cell damage was achieved by the recovery of autophagic flux. Conversely, the protective effect of 4-PBA against hypoxia/reoxygenation injury in cardiomyocytes was offset by STING overexpression, wherein the activated STING-IRF3 pathway promoted the expression of Rubicon (a negatively-regulated autophagic molecule) by binding to the Rubicon promoter. Rubicon ablation effectively counteracts the adverse effects of STING overexpression in cardiomyocytes. The data showed that STING-IRF3 signaling of ER stress receptors is particularly important in the progression of physiological M-I/R caused by the inhibition of autophagic flow in vivo and in vitro.

ESCRT-III Component CHMP4C Attenuates Cardiac Hypertrophy by Targeting the Endo-Lysosomal Degradation of EGFR.

Cardiac hypertrophy and subsequent heart failure impose a considerable burden on public health worldwide. Impaired protein degradation, especially endo-lysosome-mediated degradation of membrane proteins, is associated with cardiac hypertrophy progression. CHMP4C (charged multivesicular body protein 4C), a critical constituent of multivesicular bodies, is involved in cellular trafficking and signaling. However, the specific role of CHMP4C in the progression of cardiac hypertrophy remains largely unknown. Mouse models with CHMP4C knockout or cardiadc-specific overexpression were subjected to transverse aortic constriction surgery for 4 weeks. Cardiac morphology and function were assessed through histological staining and echocardiography. Confocal imaging and coimmunoprecipitation assays were performed to identify the direct target of CHMP4C. An EGFR (epidermal growth factor receptor) inhibitor was administrated to determine whether effects of CHMP4C on cardiac hypertrophy were EGFR dependent. CHMP4C was significantly upregulated in both pressure-overloaded mice and spontaneously hypertensive rats. Compared with wild-type mice, CHMP4C deficiency exacerbated transverse aortic constriction-induced cardiac hypertrophy, whereas CHMP4C overexpression in cardiomyocytes attenuated cardiac dysfunction. Mechanistically, the effect of CHMP4C on cardiac hypertrophy relied on the EGFR signaling pathway. Fluorescent staining and coimmunoprecipitation assays confirmed that CHMP4C interacts directly with EGFR and promotes lysosome-mediated degradation of activated EGFR, thus attenuating cardiac hypertrophy. Notably, an EGFR inhibitor canertinib counteracted the exacerbation of cardiac hypertrophy induced by CHMP4C knockdown in vitro and in vivo. CHMP4C represses cardiac hypertrophy by modulating lysosomal degradation of EGFR and is a potential therapeutic candidate for cardiac hypertrophy.

Current insights into the role of miRNA-125 in cardiovascular disease: potential biological markers and therapeutic targets

Recently, miRNAs are being used as diagnostic markers for various pathological conditions. This review analyzed the main studies devoted to the role of miRNA-125 in the development of cardiovascular diseases. Members of the miRNA-125 family are involved in cell differentiation, proliferation, and apoptosis by targeting mRNAs associated with these cellular processes. This miRNA can enhance or inhibit pathological processes such as oncogenesis, muscle abnormalities, neurological disorders, and others. Members of the miRNA-125 family also influence the development and function of immune cells and are involved in immunological defense. Research shows that the miRNA-125 family is associated with cardiac development. They also play an important role in pathophysiological conditions of the cardiovascular system. However, the same miRNA-125 family members play different roles in different pathological processes. For example, miRNA-125b overexpression in cardiomyocytes can inhibit their apoptosis and inflammatory response. However, miRNA-125b is also a regulator of cardiac fibrosis; its overexpression in cardiac fibroblasts can enhance their proliferation. Therefore, in pathological conditions, miRNA-125b excess aggravates myocardial fibrosis and remodeling, destroys the original morphological structure of the heart, disrupts neovascularization processes, and aggravates apoptosis of cardiomyocytes in the damaged area. Thus, to avoid adverse reactions, the optimal dose and timing of therapeutic intervention using members of the miRNA-125 family, their inhibitors, and mimetics must be carefully determined. An expanded and accurate understanding of miRNA-125 functions in gene regulatory networks associated with cardiovascular pathology will enable the development of novel and innovative therapeutic strategies.

UGCG modulates heart hypertrophy through B4GalT5-mediated mitochondrial oxidative stress and the ERK signaling pathway

Mechanical pressure overload and other stimuli often contribute to heart hypertrophy, a significant factor in the induction of heart failure. The UDP-glucose ceramide glycosyltransferase (UGCG) enzyme plays a crucial role in the metabolism of sphingolipids through the production of glucosylceramide. However, its role in heart hypertrophy remains unknown. In this study, UGCG was induced in response to pressure overload in vivo and phenylephrine stimulation in vitro. Additionally, UGCG downregulation ameliorated cardiomyocyte hypertrophy, improved cardiomyocyte mitochondrial oxidative stress, and reduced the ERK signaling pathway. Conversely, UGCG overexpression in cardiomyocytes promoted heart hypertrophy development, aggravated mitochondrial oxidative stress, and stimulated ERK signaling. Furthermore, the interaction between beta-1,4-galactosyltransferase 5 (B4GalT5), which catalyses the synthesis of lactosylceramide, and UGCG was identified, which also functions as a synergistic molecule of UGCG. Notably, limiting the expression of B4GalT5 impaired the capacity of UGCG to promote myocardial hypertrophy, suggesting that B4GalT5 acts as an intermediary for UGCG. Overall, this study highlights the potential of UGCG as a modulator of heart hypertrophy, rendering it a potential target for combating heart hypertrophy.

Abstract P1084: Cardiomyocyte-specific Overexpression Of Ccnd2 Via Modified Mrnafor Remuscularization In Hearts With Myocardial Infarction

Introduction: The adult mammalian heart has limited regenerative capacity due to postnatal cardiomyocyte (CM) cell cyclearrest. With the success of modified mRNA (modRNA) vaccine against SARS-CoV-2, modRNA technology offers efficient, transient, safe, nonimmunogenic, and controlled mRNA delivery without any risk of genomic integration. Using our previously reported cardiomyocyte specific modRNA translation system (CM SMRTs), we investigated the myocardial regenerative potential and therapeutic outcome of transient and specific cardiomyocyte overexpression of cyclin D2 (CCND2) in a pig model of myocardial infarction (MI). Methods: Modified mRNA was synthesized by in vitro transcription. AMI was induced by occlusion of the left anterior descending coronary artery for 60 minutes with reperfusion. Pigs were randomly assigned to vehicle group (1ml sucrose-citrate buffer, n=6), nGFP-CM SMRTs group (7.5mg CM-specific nuclear GFP modRNA, n=7)and CCND2-CM SMRTs group (7.5mg CM-specific CCND2 modRNA, n=8). CM cell cycle activity was measured 3 days after injection by immunostaining for proliferation marker Ki67, M-phase marker phospho-histone H3 (PH3) and cytokinetic marker aurora B kinase (AuB). Cardiac function was evaluated by echo and cardiac MRI (cMRI) on day 28; infarct size was determined via late gadolinium enhancement cMRI; arrhythmia incidence was assessed via implanted loop recorders. Results: The short-term in vivo study demonstrated a highly specific overexpression of targeted genes in CMs. CCND2-CM SMRTs significantly promoted CM cell cycle activation that was evidenced by significant increase of cell cycle markers compared with nGFP-CM SMRTs group: Ki67 (4.21±0.34% vs. 0.81±0.05%), PH3 (0.51±0.06% vs. 0.13±0.03%), and Symmetric AuB (0.035±0.007/mm 2 vs. 0.000±0.000/mm 2 ), 3 days after AMI and modRNA treatment. The long-term follow-up of the animals also showed improved heart function (EF: 50.34±1.94% vs. 38.60±1.18%) and decreased scar size (8.80±1.07% vs. 22.77±2.77%). The frequency of arrhythmic events in animals between all groups were similar. Conclusion: The results in large animal model of AMI demonstrate remuscularization and therapeutic potential of cardiomyocyte-specific overexpression of CCND2 via modRNA.

Tripartite motif‑containing 14 may aggravate cardiac hypertrophy via the AKT signalling pathway in neonatal rat cardiomyocytes and transgenic mice.

Tripartite motif‑containing 14 (TRIM14) is an E3 ubiquitin ligase that primarily participates in the natural immune response and in tumour development via ubiquitination. However, the role of TRIM14 in cardiac hypertrophy is not currently clear. The present study examined the role of TRIM14 in cardiac hypertrophy and its potential molecular mechanism. TRIM14 was overexpressed in neonatal rat cardiomyocytes using adenovirus and cardiomyocyte hypertrophy was induced using phenylephrine (PE). Cardiomyocyte hypertrophy was assessed by measuring cardiomyocyte surface area and markers of hypertrophy. In addition, TRIM14‑transgenic (TRIM14‑TG) mice were created and cardiac hypertrophy was induced using transverse aortic constriction (TAC). Cardiac function, heart weight‑to‑body weight ratio (HW/BW), cardiomyocyte cross‑sectional area, cardiac fibrosis and hypertrophic markers were further examined. The expression of AKT signalling pathway‑related proteins was detected. TRIM14 overexpression in cardiomyocytes promoted PE‑induced increases in cardiomyocyte surface area and hypertrophic markers. TRIM14‑TG mice developed worse cardiac function, greater HW/BW, cross‑sectional area and cardiac fibrosis, and higher levels of hypertrophic markers in response to TAC. TRIM14 overexpression also increased the phosphorylation levels of AKT, GSK‑3β, mTOR and p70S6K invivo and invitro. To the best our knowledge, the present study was the first to reveal that overexpression of TRIM14 aggravated cardiac hypertrophy invivo and invitro, which may be related to activation of the AKT signalling pathway.

USF2 activates RhoB/ROCK pathway by transcriptional inhibition of miR-206 to promote pyroptosis in septic cardiomyocytes.

Septic cardiomyopathy (SCM) is one of the most serious complications of sepsis. The present study investigated the role and mechanism of upstream stimulatory factor 2 (USF2) in SCM. Serum samples were extracted from SCM patients and healthy individuals. A murine model of sepsis was induced by caecal ligation and puncture (CLP) surgery. Myocardial injury was examined by echocardiography and HE staining. ELISA assay evaluated myocardial markers (CK-MB, cTnI) and inflammatory cytokines (TNF-α, IL-1β, IL-18). Primary mouse cardiomyocytes were treated with lipopolysaccharide (LPS) to simulate sepsis in vitro. RT-qPCR and Western blot were used for analyzing gene and protein levels. CCK-8 assay assessed cell viability. NLRP3 was detected by immunofluorescence. ChIP, RIP and dual luciferase reporter assays were conducted to validate the molecular associations. USF2 was increased in serum from SCM patients, septic mice and primary cardiomyocytes. USF2 silencing improved the survival of septic mice and attenuated sepsis-induced myocardial pyroptosis and inflammation in vitro and in vivo. Mechanistically, USF2 could directly bind to the promoter of miR-206 to transcriptionally inhibit its expression. Moreover, RhoB was confirmed as a target of miR-206 and could promote ROCK activation and NLRP3 inflammasome formation. Moreover, overexpression of RhoB remarkably reversed the protection against LPS-induced inflammation and pyroptosis mediated by USF2 deletion or miR-206 overexpression in cardiomyocytes. The above findings elucidated that USF2 knockdown exerted a cardioprotective effect on sepsis by decreasing pyroptosis and inflammation via miR-206/RhoB/ROCK pathway, suggesting that USF2 may be a novel drug target in SCM.

The inhibitory effect of vitamin D on myocardial homocysteine levels involves activation of Nrf2-mediated methionine synthase

BackgroundHomocysteine (Hcy) is a synthetic amino acid containing sulfhydryl group, which is an intermediate product of the deep metabolic pathway of methionine and cysteine. The abnormal increase in fasting plasma total Hcy concentration caused by various factors is called hyperhomocysteine (HHcy). HHcy is closely relevant to the occurrence and progression of diverse cardiovascular and cerebrovascular diseases, such as coronary heart disease, hypertension and diabetes, etc. Vitamin D/vitamin D receptor (VDR) pathway is pointed out that prevent cardiovascular disease by reducing serum homocysteine levels. Our research is designed to explore the potential mechanism of vitamin D in the prevention and treatment of HHcy. Methods and resultsThe Hcy and 25(OH)D3 levels in mouse myocardial tissue, serum or myocardial cells were detected using ELISA kits. The expression levels of VDR, Nrf2 and methionine synthase (MTR) were observed using Western blotting, immunohistochemistry and real time polymerase chain reaction (PCR). General information of the mice, including diet, water intake and body weight, was recorded. Vitamin D up-regulated the mRNA and protein expression of Nrf2 and MTR in mouse myocardial tissue and cells. CHIP assay determined that the combination of Nrf2 binding to the S1 site of the MTR promoter in cardiomyocytes using traditional PCR and real time PCR. Dual Luciferase Assay was applied to detect the transcriptional control of Nrf2 on MTR. The up-regulation effect of Nrf2 on MTR was verified by Nrf2 knockout and overexpression in cardiomyocytes. The role of Nrf2 in vitamin D inhibition of Hcy was revealed using Nrf2-knockdown HL-1 cells and Nrf2 heterozygous mice. Western blotting, real time PCR, IHC staining and ELISA showed that Nrf2 deficiency could restrain the increase in MTR expression and the decrease in Hcy level induced by vitamin D. The transcriptional activities of Nrf2/MTR were activated by vitamin D/VDR with a decrease in Hcy. ConclusionVitamin D/VDR upregulates MTR in an Nrf2-dependent manner, thereby reducing the risk of HHcy.

Alzheimer's disease-related presenilin 1 M146L mutation disrupts SERCA2a regulation and increases propensity of Ca waves in ventricular cardiomyocytes.

Presenilin 1 (PS1) is a transmembrane protein expressed in the endoplasmic reticulum (ER) of different cell types. It has been shown that Ca mishandling due to PS1 mutations contributes to the Alzheimer's disease (AD)-related neurodegeneration. PS1 is also expressed in the heart. Several clinical studies revealed that PS1 mutations are associated with cardiomyopathies. We previously showed that PS1 interacts with the cardiac ER Ca ATPase (SERCA2a) and regulates its function. Here, we studied the role of AD-related PS1M146L mutation in regulation of intracellular Ca homeostasis. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1M146L revealed that the mutation does not disrupt the interaction between these two proteins. Measurements of SERCA2a-mediated Ca transport showed that at low ER Ca loads ([Ca]ER≤0.15 mM), both PS1WT and PS1M146L enhance ER Ca uptake by a similar level (∼40%) compared to control (no PS1 expression). At high ER Ca loads ([Ca]ER≥0.35 mM), however, PS1WT reduced ER Ca uptake and ER Ca load, whereas PS1M146L was less effective in regulating SERCA2a function. We used in vivo gene delivery in mouse cardiac wall to study the effect of PS1WT and PS1M146L overexpression on Ca regulation in ventricular myocytes. We found that both PS1WT and PS1M146L localized predominantly at the sarcoplasmic reticulum. Analysis of cytosolic Ca dynamics revealed that PS1WT overexpression in cardiomyocytes increases diastolic Ca and decreases Ca transient amplitudes. In contrast, PS1M146L expression increased propensity of spontaneous Ca waves. These results illustrate that the lack of SERCA2a regulation by PS1M146L at high ER Ca loads can lead to ER Ca overload and Ca waves. These defects in Ca regulation might contribute to the onset of cardiomyopathies in patients with PS1 mutations.