Hearts Of Knockout Mice Research Articles

Reduced Sox7 contributes to abnormal valve development resulting in congenital aortic stenosis

Abstract Background Abnormal valve development is the most common congenital heart malformation. During valve remodeling, abnormal changes in valve cell proliferation, apoptosis, and extracellular matrix synthesis affect valve development, resulting in malformations. The molecular mechanisms underlying pathophysiology of aortic stenos is remain unclear. Purpose We would like to explore how Sox7 is involved in aortic valve development. Methods and Results We proved Sox7 was mainly expressed in endocardial and mesenchymal cells of aortic valves by immunofluorescence staining determined by at different stages of mice (E10.5～8 weeks) and human embryos and RNA-seq of E9.5 mouse embryonic hearts. We constructed a conditional Sox7 floxed allele in endothelial cells using an endocardial specific Cre. Echocardiography data showed that Nfat-KO mice had increased blood velocity, pressure gradient across aortic valves, left ventricular mass and dilated aortic roots. Then, HE and Sirius red staining to examine histological changes in aortic valves of Nfat-KO mice were performed, and we observed a remarkable increase in the size and increased staining of fibrotic cells in aortic valves of KO mice. Next, we collected embryonic hearts of Sox7 CNC-specific knockout mice (WNT1-KO) and controls at E16.5. According to HE staining results and echocardiography analysis, there was no difference in aortic valves between WNT1-KO mice and controls. Meanwhile, we collected Nfat-KO mice and control embryos at E11.5 days. HE staining results showed that EndMT process was not impaired. Interestingly, leaflets and cusps of Nfat-KO mice showed significantly augmented proliferation levels, decreased apoptosis levels, and calcium deposition in fibrin layer. A significant decrease in MMP9 levels were observed in both mRNA and protein levels. Co-transfection of Sox7-expressing vector and MMP9 luciferase in MEEC suggested that Sox7 significantly activated MMP9. We used adenovirus to overexpress Sox7 in valvular interstitial cells. Compared with that in control group, the Sox7-overexpression group increased protein level of MMP9 and cleaved caspase-3, and increased Tunel-positive rate and lower Ki-67 positive rate. Finally, we detected expression of Sox7 protein in human fetal aortic valves derived from patients with aortic stenosis using immunofluorescence. The results demonstrated that the number of Sox7 positive mesenchymal cells in human fetal aortic valves of patients was significantly lower than that in control group. Conclusion We demonstrated that targeted inactivation of Sox7 in the endocardium of mice affects valve development and leads to aortic stenosis. Mechanistically, no influence of Sox7 on the EndMT process of the OFT cushion. Moreover, CNC-derived Sox7 was not involved in aortic valve development. Interestingly, we found that Sox7 regulated ECM remodeling through matrix MMP9 and Sox7 had an impact on apoptosis, proliferation, and calcification.

Tegaserod Stimulates 5-HT4 Serotonin Receptors in the Isolated Human Atrium.

Tegaserod (1-{[(5-methoxy-1H-indol-3-yl)methyliden]amino}-3-pentylguanidine) is a potent agonist at human recombinant 5-HT4 serotonin receptors. Consequently, tegaserod is utilized in the treatment of bowel diseases. The objective of this study was to test the hypothesis that tegaserod stimulates human cardiac atrial 5-HT4-receptors via cyclic adenosine monophosphate (cAMP)-dependent pathways. Tegaserod exerted positive inotropic effects (PIEs) and positive chronotropic effects (PCEs) in isolated left and right atrial preparations, respectively, from mice with cardiac-specific overexpression of the human 5-HT4 serotonin receptor (5-HT4-TG) in a concentration- and time-dependent manner. However, no effect was observed in the hearts of littermates of wild-type mice (WT). Western blot analysis revealed that the expression of 5-HT4 receptors was significantly higher in 5-HT4-TG mice compared to WT mice. The specificity of the signal for the 5-HT4 receptor was confirmed by the absence of the signal in the hearts of 5-HT4 receptor knockout mice. Furthermore, tegaserod increased the force of contraction (at concentrations as low as 10 nM), reduced the time of tension relaxation, and increased the rate of tension development in isolated electrically stimulated (at a rate of 60 beats per minute) human right atrial preparations (HAPs, obtained during open-heart surgery) when administered alone. The potency and efficacy of tegaserod to raise the force of contraction were enhanced in the presence of cilostamide, a phosphodiesterase III inhibitor. The positive inotropic effect of tegaserod in HAPs was found to be attenuated by the 5-HT4 serotonin receptor antagonist GR 125487 (0.1 µM). The efficacy of tegaserod (10 µM) in raising the force of contraction in HAPs was less pronounced than that of serotonin (10 µM) or isoprenaline (1 µM). Tegaserod shifted the concentration-response curve of the force of contraction to serotonin to the right in HAPs, indicating that it is a partial agonist at 5-HT4 serotonin receptors in this model. We propose that the mechanism of action of tegaserod in HAPs involves cAMP-dependent phosphorylation of cardiac regulatory proteins.

Abstract Tu095: Cycloastragenol Alleviates Cardiac Fibrosis through Inhibiting Pyroptosis Activated by NLRP3 Inflammasome

Cardiac fibrosis is cardiac interstitial remodeling characterized by an enhanced extracellular matrix deposition. Acute sympathetic stress causes cardiac cells to immediately undergo pyroptosis, which induces cardiac inflammation and damage, and ultimately results in cardiac fibrosis. Our previous study has demonstrated the inhibitory effects of cycloastragenol (CAG), an active form of astragaloside IV isolated from Astragalus membranaceus, on isoproterenol (ISO)-induced cardiac fibrosis through blocking the NLRP3 signaling pathway. It is yet unknown how CAG affects pyroptosis of cardiac cells and whether its ability to prevent cardiac fibrosis coincides with this process. In this study, 5 mg/kg ISO was subcutaneously injected into wild-type (WT), NLRP3 knockout (KO), and GSDMD KO mice for 7 days to induce cardiac fibrosis. CAG (125 mg/kg/d) was administrated to the mice intragastrically at the start of the ISO injection. Primary cardiomyocytes and cardiac fibroblasts were isolated from neonatal rat hearts and treated with 10 μM ISO either with or without CAG. The results show that CAG alleviates cardiac fibrosis in WT mice, while lost this efficacy in NLRP3 KO mice. GSDMD KO mice do not develop ISO-induced cardiac fibrosis, indicating the key role of pyroptosis in cardiac fibrosis. Pyroptosis mainly occurs in mouse hearts during the initial stages of ISO induction (1 and 24 hours after ISO injection), and CAG significantly reduces pyroptosis of heart cells in WT mice, while CAG is unable to prevent pyroptosis in NLRP3 KO mouse hearts. The TGF-/Smads signaling pathway is also inhibited by CAG in WT mice but not in NLRP3 KO mice. Additionally, CAG prevents the in vitro pyroptosis of cardiomyocytes induced by ISO directly and cardiac fibroblasts caused by LPS+ATP. These results suggest that the principal mechanism of the anti-cardiac fibrosis effect of CAG mainly depends on its inhibition of pyroptosis mediated by NLRP3 inflammasome.

Vaccine Therapy for Heart Failure Targeting the Inflammatory Cytokine Igfbp7.

The heart comprises many types of cells such as cardiomyocytes, endothelial cells (ECs), fibroblasts, smooth muscle cells, pericytes, and blood cells. Every cell type responds to various stressors (eg, hemodynamic overload and ischemia) and changes its properties and interrelationships among cells. To date, heart failure research has focused mainly on cardiomyocytes; however, other types of cells and their cell-to-cell interactions might also be important in the pathogenesis of heart failure. Pressure overload was imposed on mice by transverse aortic constriction and the vascular structure of the heart was examined using a tissue transparency technique. Functional and molecular analyses including single-cell RNA sequencing were performed on the hearts of wild-type mice and EC-specific gene knockout mice. Metabolites in heart tissue were measured by capillary electrophoresis-time of flight-mass spectrometry system. The vaccine was prepared by conjugating the synthesized epitope peptides with keyhole limpet hemocyanin and administered to mice with aluminum hydroxide as an adjuvant. Tissue samples from heart failure patients were used for single-nucleus RNA sequencing to examine gene expression in ECs and perform pathway analysis in cardiomyocytes. Pressure overload induced the development of intricately entwined blood vessels in murine hearts, leading to the accumulation of replication stress and DNA damage in cardiac ECs. Inhibition of cell proliferation by a cyclin-dependent kinase inhibitor reduced DNA damage in ECs and ameliorated transverse aortic constriction-induced cardiac dysfunction. Single-cell RNA sequencing analysis revealed upregulation of Igfbp7 (insulin-like growth factor-binding protein 7) expression in the senescent ECs and downregulation of insulin signaling and oxidative phosphorylation in cardiomyocytes of murine and human failing hearts. Overexpression of Igfbp7 in the murine heart using AAV9 (adeno-associated virus serotype 9) exacerbated cardiac dysfunction, while EC-specific deletion of Igfbp7 and the vaccine targeting Igfbp7 ameliorated cardiac dysfunction with increased oxidative phosphorylation in cardiomyocytes under pressure overload. Igfbp7 produced by senescent ECs causes cardiac dysfunction and vaccine therapy targeting Igfbp7 may be useful to prevent the development of heart failure.

In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation

BackgroundInsulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent.MethodsInsulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements.ResultsWe quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided.ConclusionWe present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.Graphical

Loss of Cavin-2 destabilizes phosphatase and tensin homologue and enhances Akt signalling pathway in cardiomyocytes.

Specific cavins and caveolins, known as caveola-related proteins, have been implicated in cardiac hypertrophy and myocardial injury. Cavin-2 forms complexes with other caveola-related proteins, but the role of Cavin-2 in cardiomyocytes (CMs) is poorly understood. Here, we investigated an unknown function of Cavin-2 in CMs. Under cardiac stress-free conditions, systemic Cavin-2 knockout (KO) induced mild and significant CM hypertrophy. Cavin-2 KO suppressed phosphatase and tensin homologue (PTEN) associated with Akt signalling, whereas there was no difference in Akt activity between the hearts of the wild-type and the Cavin-2 KO mice under cardiac stress-free conditions. However, after swim training, CM hypertrophy was more facilitated with enhanced phosphoinositide 3-kinase (PI3K)-Akt activity in the hearts of Cavin-2 KO mice. Cavin-2 knockdown neonatal rat CMs (NRCMs) using adenovirus expressing Cavin-2 short hairpin RNA were hypertrophied and resistant to hypoxia and H2O2-induced apoptosis. Cavin-2 knockdown increased Akt phosphorylation in NRCMs, and an Akt inhibitor inhibited Cavin-2 knockdown-induced anti-apoptotic responses in a dose-dependent manner. Cavin-2 knockdown increased phosphatidylinositol-3,4,5-triphosphate production and attenuated PTEN at the membrane fraction of NRCMs. Immunostaining and immunoprecipitation showed that Cavin-2 was associated with PTEN at the plasma membrane of NRCMs. A protein stability assay showed that Cavin-2 knockdown promoted PTEN destabilization in NRCMs. In an Angiotensin II (2-week continuous infusion)-induced pathological cardiac hypertrophy model, CM hypertrophy and CM apoptosis were suppressed in CM-specific Cavin-2 conditional KO (Cavin-2 cKO) mice. Because Cavin-2 cKO mouse hearts showed increased Akt activity but not decreased extracellular signal-regulated kinase activity, suppression of pathological hypertrophy by Cavin-2 loss may be due to increased survival of healthy CMs. Cavin-2 plays a negative regulator in the PI3K-Akt signalling in CMs through interaction with PTEN. Loss of Cavin-2 enhances Akt activity by promoting PTEN destabilization, which promotes physiological CM hypertrophy and may enhance Akt-mediated cardioprotective effects against pathological CM hypertrophy.

Cardiac-Specific Suppression of Valosin-Containing Protein Induces Progressive Heart Failure and Premature Mortality Correlating with Temporal Dysregulations in mTOR Complex 2 and Protein Phosphatase 1.

Valosin-containing protein (VCP), an ATPase-associated protein, is emerging as a crucial regulator in cardiac pathologies. However, the pivotal role of VCP in the heart under physiological conditions remains undetermined. In this study, we tested a hypothesis that sufficient VCP expression is required for cardiac development and physiological cardiac function. Thus, we generated a cardiac-specific VCP knockout (KO) mouse model and assessed the consequences of VCP suppression on the heart through physiological and molecular studies at baseline. Our results reveal that homozygous KO mice are embryonically lethal, whereas heterozygous KO mice with a reduction in VCP by ~40% in the heart are viable at birth but progressively develop heart failure and succumb to mortality at the age of 10 to 12 months. The suppression of VCP induced a selective activation of the mammalian target of rapamycin complex 1 (mTORC1) but not mTORC2 at the early age of 12 weeks. The prolonged suppression of VCP increased the expression (by ~2 folds) and nuclear translocation (by >4 folds) of protein phosphatase 1 (PP1), a key mediator of protein dephosphorylation, accompanied by a remarked reduction (~80%) in AKTSer473 phosphorylation in VCP KO mouse hearts at a later age but not the early stage. These temporal molecular alterations were highly associated with the progressive decline in cardiac function. Overall, our findings shed light on the essential role of VCP in the heart under physiological conditions, providing new insights into molecular mechanisms in the development of heart failure.

Deletion of Interleukin-1β Converting Enzyme Alters Mouse Cardiac Structure and Function.

Interleukin-1β converting enzyme (ICE, caspase-1) is a thiol protease that cleaves the pro-inflammatory cytokine precursors of IL-1β and IL-18 into active forms. Given the association between caspase-1 and cardiovascular pathology, we analyzed the hearts of ICE knockout (ICE KO) mice to test the hypothesis that caspase-1 plays a significant role in cardiac morphology and function. We characterized the histological and functional changes in the hearts of ICE KO mice compared to the Wild type. The cardiomyocytes from the neonatal ICE KO mice showed an impaired response to oxidative stress. Subsequently, the hearts from the ICE KO mice were hypertrophied, with a significant increase in the left ventricular and septal wall thickness and a greater LV mass/body weight ratio. The ICE KO mice hearts exhibited irregular myofibril arrangements and disruption of the cristae in the mitochondrial structure. Proapoptotic proteins that were significantly increased in the hearts of ICE KO versus the Wild type included pErk, pJNK, p53, Fas, Bax, and caspase 3. Further, the antiapoptotic proteins Bag-1 and Bcl-2 are activated in ICE KO hearts. Functionally, there was an increase in the left ventricular epicardial diameter and volume in ICE KO. In conclusion, our findings support the important role of caspase-1 in maintaining cardiac health; specifically, a significant decrease in caspase-1 is detrimental to the cardiovascular system.

Emerging contaminants at trace levels of pesticides perturbs biomolecules in different organs in mice: Role of peroxisome proliferator-activated receptor-alpha

Objectives: Information is lacking on the consequences of chronic exposure to emerging contaminants at environmentally relevant (trace concentrations) on biomolecules. Environmental exposure to these chemical mixtures happens at trace concentrations and at multiple molecular interactions. The consequences of trace concentrations of multiple pesticides (MPs) on the regulation of selected biomolecules nitric oxide (NO), thiols, superoxide dismutase (SOD), and glutathione S-transferase (GST) in the tissues from wild type (WT) and genetically deficient- peroxisome proliferator-activated receptor-alpha (PPARα) knockout (Null) mice were investigated. Material and Methods: Mice were exposed to trace concentrations of MPs: Atrazine, dieldrin, endrin, endosulfan, and anthracene (1–100 ng/L) in drinking water for 6 weeks. Organs were collected and homogenized; NO, protein and non-protein thiol levels, as well as SOD and GST activities were determined. Results: Differential and organ selective effects of the treatments were observed in the WT and PPARα knockout. Increased NO levels were observed in the organs from WT with limited increase in the kidney (Null). SOD activity was decreased in the organs from the WT and was increased in the PPARα knockout when compared to the control. Thiol level was significantly increased in the heart and spleen in the WT and in the heart of the PPARα knockout mice when compared to the control. Non-protein thiol concentration was reduced in the heart and kidney (WT) and reduced in the liver of the PPARα knockout when compared to the control. GST activity was significantly decreased in the liver and spleen (WT) and was significantly elevated in all organs in the PPARα knockout mice when compared to the WT. Conclusion: The low concentrations of MPs may have caused selective dysregulation of biomolecules in different organs of the body. These effects observed may be influenced by genetic status such as in PPARα deficiency. These results present a scenario that implicates nanoconcentrations of series of organic contaminants that can cause cellular and molecular dysregulations of biomolecules precipitating toxicity and pathology that can be a threat to human health. Further, investigation into the molecular mechanism(s) and signaling pathway(s) implicated in these dysregulations is warranted.

Ubiquitin specific protease 38 aggravates pathological cardiac remodeling by stabilizing phospho-TBK1.

Chronic pressure overload can cause pathological cardiac remodeling and eventually heart failure. The ubiquitin specific protease (USP) family proteins play a prominent role in regulating substrate protein degradation and cardiac structural and functional homeostasis. Although USP38 is expressed in the heart, uncertainty exists regarding the function of USP38 in pathological cardiac remodeling. We constructed and generated cardiac specific USP38 knockout mice and cardiac specific USP38 overexpression mice to assess the role of USP38 in pathological cardiac remodeling. Furthermore, we used co-immunoprecipitation (Co-IP) assays and western blot analysis to identify the molecular interaction events. Here, we reported that the expression of USP38 is significantly elevated under a hypertrophic condition in vivo and in vitro. USP38 deletion significantly mitigates cardiomyocyte enlargement in vitro and hypertrophic effect induced by pressure overload, while overexpression of USP38 markedly aggravates cardiac hypertrophy and remodeling. Mechanistically, USP38 interacts with TANK-binding kinase 1 (TBK1) and removes K48-linked polyubiquitination of TBK1, stabilizing p-TBK1 and promoting the activation of its downstream mediators. Overexpression of TBK1 in the heart of cardiac specific USP38 knockout mice partially counteracts the benefit of USP38 deletion on pathological cardiac remodeling. The TBK1 inhibitor Amlexanox significantly alleviates pressure overload induced-cardiac hypertrophy and myocardial fibrosis in mice with USP38 overexpression. Our results demonstrate that USP38 serves as a positive regulator of pathological cardiac remodeling and suggest that targeting the USP38-TBK1 axis is a promising treatment strategy for hypertrophic heart failure.

Cardiomyocyte-specific disruption of the circadian BMAL1–REV-ERBα/β regulatory network impacts distinct miRNA species in the murine heart

Circadian disruption increases cardiovascular disease (CVD) risk, through poorly understood mechanisms. Given that small RNA species are critical modulators of cardiac physiology/pathology, we sought to determine the extent to which cardiomyocyte circadian clock (CCC) disruption impacts cardiac small RNA species. Accordingly, we collected hearts from cardiomyocyte-specific Bmal1 knockout (CBK; a model of CCC disruption) and littermate control (CON) mice at multiple times of the day, followed by small RNA-seq. The data reveal 47 differentially expressed miRNAs species in CBK hearts. Subsequent bioinformatic analyses predict that differentially expressed miRNA species in CBK hearts influence processes such as circadian rhythmicity, cellular signaling, and metabolism. Of the induced miRNAs in CBK hearts, 7 are predicted to be targeted by the transcriptional repressors REV-ERBα/β (integral circadian clock components that are directly regulated by BMAL1). Similar to CBK hearts, cardiomyocyte-specific Rev-erbα/β double knockout (CM-RevDKO) mouse hearts exhibit increased let-7c-1-3p, miR-23b-5p, miR-139-3p, miR-5123, and miR-7068-3p levels. Importantly, 19 putative targets of these 5 miRNAs are commonly repressed in CBK and CM-RevDKO heart (of which 16 are targeted by let-7c-1-3p). These observations suggest that disruption of the circadian BMAL1–REV-ERBα/β regulatory network in the heart induces distinct miRNAs, whose mRNA targets impact critical cellular functions.

Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

This study found that the level of neuroepithelial cell-transforming gene 1 protein (NET1) was significantly increased in a mouse cardiac fibrosis model. Moreover, the expression level of NET1 was increased in cardiac fibrosis induced by TGF-β1, suggesting that NET1 was involved in the pathological process of cardiac fibrosis. Overexpression of NET1 promoted β-catenin expression in the nucleus and significantly increased the proliferation and migration of cardiac fibroblasts. NET1 may form a complex with β-catenin through GSK3β. Knockdown of β-catenin alleviated the effects of NET1 overexpression on collagen production and cell migration. In the heart of NET1 knockout mice, NET1 knockout can reduce the expression of β-catenin, α-SMA, and collagen content induced by MI. In conclusion, NET1 may regulate the activation of Wnt/β-catenin and TGF/Smads signaling pathway, promote collagen synthesis in fibroblasts, and participate in cardiac fibrosis. Thus, NET1 may be a potential therapeutic target in cardiac fibrosis.

Role of selenoprotein M knockdown in the melatonin antagonism of nickel-induced apoptosis and endoplasmic reticulum stress in mouse heart.

The aim of this study was to investigate the role of selenoprotein M (SelM) in endoplasmic reticulum stress and apoptosis in nickel-exposed mouse hearts and to explore the detoxifying effects of melatonin. At 21 d after intraperitoneal injection of nickel chloride (NiCl2) and/or melatonin into male wild-type (WT) and SelM knockout (KO) C57BL/6J mice, NiCl2 was found to induce changes in the microstructure and ultrastructure of the hearts of both WT and SelM KO mice, which were caused by oxidative stress, endoplasmic reticulum stress, and apoptosis, as evidenced by decreases in malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) activity. Changes in the messenger RNA (mRNA) and protein expression of genes related to endoplasmic reticulum stress (activating transcription factor 4 (ATF4), inositol-requiring protein 1 (IRE1), c-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP)) and apoptosis (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Caspase-9, and Caspase-12) were also observed. Notably, the observed damage was worse in SelM KO mice. Furthermore, melatonin alleviated the heart injury caused by NiCl2 in WT mice but could not exert a good protective effect in the heart of SelM KO mice. Overall, the findings suggested that the antioxidant capacity of SelM, as well as its modulation of endoplasmic reticulum stress and apoptosis, plays important roles in nickel-induced heart injury.

Btg1 and Btg2 regulate neonatal cardiomyocyte cell cycle arrest

Rodent cardiomyocytes undergo mitotic arrest in the first postnatal week. Here, we investigate the role of transcriptional co-regulator Btg2 (B-cell translocation gene 2) and functionally-similar homolog Btg1 in postnatal cardiomyocyte cell cycling and maturation. Btg1 and Btg2 (Btg1/2) are expressed in neonatal C57BL/6 mouse left ventricles coincident with cardiomyocyte cell cycle arrest. Btg1/2 constitutive double knockout (DKO) mouse hearts exhibit increased pHH3+ mitotic cardiomyocytes compared to Wildtype at postnatal day (P)7, but not at P30. Similarly, neonatal AAV9-mediated Btg1/2 double knockdown (DKD) mouse hearts exhibit increased EdU+ mitotic cardiomyocytes compared to Scramble AAV9-shRNA controls at P7, but not at P14. In neonatal rat ventricular myocyte (NRVM) cultures, siRNA-mediated Btg1/2 single and double knockdown cohorts showed increased EdU+ cardiomyocytes compared to Scramble siRNA controls, without increase in binucleation or nuclear DNA content. RNAseq analyses of Btg1/2-depleted NRVMs support a role for Btg1/2 in inhibiting cell proliferation, and in modulating reactive oxygen species response pathways, implicated in neonatal cardiomyocyte cell cycle arrest. Together, these data identify Btg1 and Btg2 as novel contributing factors in mammalian cardiomyocyte cell cycle arrest after birth.

Quantification and Proteomic Characterization of β-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid β-oxidation, especially β-hydroxybutyrate, are fatty energy–supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-hydroxybutyrate–mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.

Abstract 9506: Calpain Activation in Early Reperfusion Triggers Pyroptosis Resulting in Myocardial Ischemia/Reperfusion Injury

Sterile inflammation contributes to reperfusion injury. During myocardial ischemia/reperfusion (I/R), inflammasomes proteolytically activate caspase-1 (cas-1) which then kills cardiomyocytes by liberating N-terminal fragments of gasdermin D which form large pores in the sarcolemma (pyroptosis). We and others find hearts from cas-1 knockout (KO) mice have less myocardial infarction (MI) after I/R than those of wild type (WT) hearts. Pharmacologic cas-1 blockade when administered prior to ischemia or at the onset of reperfusion provides similar protection against MI but protection is lost when blockade is started 10min after reperfusion revealing that cas-1 does its killing in the first 10min of reperfusion. We wondered what could trigger cas-1 activation at reperfusion. Ca ++ floods myocytes at reperfusion making Ca ++ a suspect. We tested if adenyl cyclase isoform 10 (AC10), which is stimulated by Ca ++ and in one study promoted I/R injury in cardiomyocytes, could be responsible. However, there were no differences in infarct size among AC10 -/- KO, AC10 -/+ heterozygous, and WT buffer-perfused mouse hearts after 50min global ischemia/2h reperfusion. Ca ++ also activates calpain which releases membrane-bound inactive procas-1 which is required for infammasome assembly. Adding calpain antagonist calpeptin (CPT) to the perfusate of isolated AC10 -/+ hearts reduced MI from 90% of risk zone to 55% (p&lt;0.001). Cas-1 KO mouse hearts were similarly protected after I/R (40% infarction) and treating KO hearts with CPT resulted in no added protection suggesting both cas-1 and calpain are steps in the same mechanism of cell killing. This result was amplified by lack of added protection when mice received combined CPT and emricasan (EMRI), a pan-cas blocker. In contrast, EMRI plus preconditioning does give additive protection. Hence calpain activity in the initial minutes of reperfusion is also involved in injurious pyroptosis and may be a viable target in treatment of acute MI.

The novel ageing-induced long non-coding RNA MIRIAL controls endothelial cell and mitochondrial function

Abstract Vascular ageing is a key risk factor for cardiovascular diseases and is characterised by a continuous decline in endothelial cell function. Despite progress in recent years, the molecular mechanisms for this deterioration remain incompletely understood. Long non-coding RNAs (lncRNAs) are a heterogeneous class of RNAs that have been shown to regulate gene expression and protein function, however, little is known about their role in the ageing-associated dysregulation of endothelial cell (EC) function. In this study, we aimed to identify and functionally characterise a novel ageing-regulated lncRNA in ECs. Using RNA sequencing data of cardiac ECs derived from 12 weeks young and 20 months old mice, we identified Mirial as an ageing-induced lncRNA (1.32-fold, p=0.00005). Mirial is conserved between mice and humans and has no obvious coding potential. GapmeR-mediated silencing of MIRIAL in human umbilical vein ECs (HUVECs) decreased cell proliferation by 50%, migration by 24% (p=0.045) and basal angiogenic sprouting by 53% (p=0.0029), without affecting apoptosis or senescence. Additionally, silencing of MIRIAL increases mitochondrial mass (1.8-fold, p&amp;lt;0.01) and spare respiratory capacity (1.95-fold). Preliminary data from the hearts of Mirial knockout mice confirm the elevated mitochondrial mass after Mirial ablation (1.26-fold, p=0.05). In HUVECS, MIRIAL is mainly associated with the chromatin (80%), suggesting a role in the regulation of gene expression. Pathway analysis showed an overrepresentation of p53 target genes that were upregulated upon MIRIAL knockdown, which was validated using qRT-PCR (1.8–5.2-fold increases). Interestingly, this effect is fully dependent on the presence of p53. Moreover, p53 and phospho-p53 (Ser15) were both increased (1.8-fold, p=0.01 and 2.9-fold, p=0.02, respectively) after MIRIAL silencing. Pulldown of MIRIAL identified DDX5 and MRPL41 as direct p53 interactors and RNA immunoprecipitation revealed that MIRIAL physically interacts with p53 (3.75-fold enrichment, p&amp;lt;0.01). Gene set enrichment analysis of RNA sequencing data revealed that 10% of deregulated genes after MIRIAL knockdown have a binding site for Forkhead Box O (FoxO) transcription factors. In particular, FoxO1 is known as one of the key players in endothelial proliferation and regulation of angiogenesis as well as in mitochondrial biogenesis. Taken together, MIRIAL is an ageing-induced lncRNA in endothelial cells acting as a key regulator of metabolic and cellular function. MIRIAL promotes cell proliferation, migration and basal angiogenic sprouting while decreasing mitochondrial function. We hypothesise that MIRIAL influences these cellular functions by affecting the p53 pathway and mitochondrial respiration through FoxO signalling. The results from the present study suggest that modulation of cellular MIRIAL expression may be a promising strategy to prevent or even reverse ageing-induced functional decline of ECs, both in vitro and in vivo. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft - Collaborative Research Centre (SFB) 834 - Project B9Deutsche Forschungsgemeinschaft - Collaborative Research Centre/Transregio (TRR) 267 - Project B4

Genetic Deletion of Galectin-3 Exacerbates Age-Related Myocardial Hypertrophy and Fibrosis in Mice.

Aging is accompanied by progressive and adverse cardiac remodeling characterized by myocardial hypertrophy, fibrosis, and dysfunction. We previously reported that galectin-3 (Gal-3) is a critical regulator of inflammation and fibrosis associated with hypertensive heart disease and myocardial infarction. Nevertheless, the role and mechanism of Gal-3 in age-related cardiac remodeling have not been previously investigated. We hypothesized that Gal-3 plays a critical role in cardiac aging and that its deficiency exacerbates the underlying mechanisms of myocardial hypertrophy and fibrosis. Male C57BL/6 (control) (n=24) and Gal-3 knockout (KO) (n=29) mice were studied at 24 months of age to evaluate the role of Gal-3 in cardiac aging. We assessed 1) survival rate; 2) systolic blood pressure (SBP) by plethysmography; 3) myocardial hypertrophy, apoptosis, and fibrosis by quantification of histological and immunohistochemical analysis; 4) cardiac expression of angiotensin (Ang) II, Ang (1-7) by Radioimmunoassay; 5) transforming growth factor-β (TGF-β), sirtuin (SIRT) 1, SIRT 7 and metalloproteinase 9 (MMP-9) by RT-qPCR and 6) ventricular remodeling and function by echocardiography. We found that aged Gal-3 KO mice had a lower survival rate and exhibited exacerbated myocardial hypertrophy and fibrosis without changes in SBP. Similarly, myocardial apoptosis and MMP-9 mRNA expression was significantly increased in the hearts of Gal-3 KO mice compared to controls. Additionally, cardiac Ang II and TGF-β expression were higher in aged Gal-3 KO mice while SIRT1 and SIRT7 expression were reduced. Our findings strongly suggest that Gal-3 is involved in age-related cardiac remodeling by regulating critical mechanisms associated with the development of pathological hypertrophy. The gene deletion of Gal-3 reduced the lifespan and markedly increased age-dependent mechanisms of myocardial hypertrophy, apoptosis, and fibrosis, including Ang-II, TGF-β, and MMP-9. At the same time, there was diminished cardiac-specific expression of SIRT1 and SIRT7, which are extensively implicated in delaying age-dependent cardiomyopathies.

Abstract P3075: Hippo Pathway In Cardiac Fibroblast Regulates Macrophage Recruitment Following Injury

Background: The evolutionary conserved Hippo signaling pathway (HSP) is an inhibitor of tissue regeneration, including the Lats1/2 kinases that phosphorylate and inactivate YAP and TAZ. Emerging data indicate that HSP regulates innate immunity, inflammation, and autoimmune diseases. However, the role of HSP in post-MI inflammation is poorly understood. Methods: Single-cell RNA-sequencing (scRNA-Seq) and spatial transcriptomics were utilized in cardiac fibroblast (CF)-specific-Lats1/2 knockout (Lats CKO) mouse heart. Flow cytometry, immunofluorescence (IF), and echocardiogram (Echo) were used to describe the phenotype in ventricle of Lats CKO mouse heart with/out Ccr2 knockout. Results: We integrated our scRNA-Seq data in Lats CKO mouse heart with three published data sets of MI mouse heart. A CF subcluster with elevated YAP activity and expressing chemokines Ccl2 and Ccl7 (Ccl2/Ccl7+ CFs) and macrophages (Macs) expressing Ccr2, the receptor for Ccl2/Ccl7 (Ccr2+ Macs), were identified, which suggests a signaling transduction from Ccl2/Ccl7+ CFs to Ccr2+ Macs in post-MI hearts. To figure out the function and mechanism of this specific cell communication, we employed a Lats CKO mice model which mimics the post-MI heart and exhibits high YAP activity and conformed the cell communication between Ccl2/Ccl7+ CFs and Ccr2+ Macs by spatial transcriptomics. Furthermore, Ccr2 loss of function in Lats CKO mice suppressed Mac expansion and CF proliferation. Consistently, Ccr2 knockout reversed the phenotype presenting smaller left ventricle chamber in Lats CKO mouse heart because of CF proliferation. Conclusion: YAP target genes Ccl2 and Ccl7 in CFs are responsible for Ccr2+ Macs recruitment and the recruited Ccr2+ Macs in turn promote CF proliferation post-MI.

Abstract P3040: Mitochondrial Membrane Protein Sigmar1 Regulates Mitochondrial Ultrastructure And Respiratory Functions In The Heart

Background: Sigma-1 receptor (Sigmar1) is multifunctional, ubiquitously expressed chaperone protein in mammalian cell systems. Earlier biochemical and immuno-electron microscopic analysis revealed Sigmar1’s cellular localization depends on cell and tissue systems, explaining its involvement in regulating diverse cellular and molecular functions. However, Sigmar1’s localization at organelle levels, membrane tropism, and direct molecular functions in cardiomyocytes remain elusive. Objective: We conducted systematic biochemical, microscopic, and functional assays to define Sigmar1’s subcellular localization, membrane topology, and functions in the heart. Methods and Results: Immuno-labeled quantum dots (QDs) in transmission electron microscopy revealed anti-Sigmar1 labeled QDs on the mitochondrial membranes, lysosomes, and at the sarcoplasmic reticulum-mitochondria interface. Subcellular fractionation of heart lysates also confirmed Sigmar1’s distribution in purified mitochondrial and lysosomal fractions. We further confirmed Sigmar1’s mitochondrial localization by Sigmar1’s colocalization with Mito-Tracker Red labeled isolated cardiac mitochondria. Biochemical experiments constituting alkaline extraction, detergent solubilization, and dose-dependent Proteinase K digestion on isolated heart mitochondria demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1’s structural requirement for mitochondrial localization by expressing Flag-tagged Sigmar1 fragments demonstrated N-terminal required for mitochondrial localization. High-resolution mitochondrial respirometry studies showed that genetic loss of Sigmar1 in primary cardiomyocytes reduces mitochondrial respiration. Sigmar1 knock-out mice hearts also showed the accumulation of abnormal, irregular-shaped giant mitochondria and defects in mitochondrial respiration. Conclusions: Overall, our studies demonstrated that Sigmar1 is an integral mitochondrial membrane protein and is essential in maintaining mitochondrial ultrastructure and respiratory functions in the heart.