Mouse Myocardium Research Articles

Extracellular flux analysis in intact cardiac tissue slices-A novel tool to investigate cardiac substrate metabolism in mouse myocardium.

Cardiac pathologies are accompanied by alterations in substrate metabolism, and extracellular flux analysis is a standard tool to investigate metabolic disturbances, especially in immortalized cell lines. However, preparations of primary cells, such as adult cardiomyocytes require enzymatic dissociation and cultivation affecting metabolism. Therefore, we developed a flux analyzer-based method for the assessment of substrate metabolism in intact vibratome-sliced mouse heart tissue. Oxygen consumption rates were determined using a Seahorse XFe24-analyzer and "islet capture plates." We demonstrate that tissue slices are suitable for extracellular flux analysis and metabolize both free fatty acids (FFA) and glucose/glutamine. Functional integrity of tissue slices was proven by optical mapping-based assessment of action potentials. In a proof-of-principle approach, the sensitivity of the method was tested by analyzing substrate metabolism in the remote myocardium after myocardial infarction (I/R). Here, I/R increased uncoupled OCR compared with sham animals indicating a stimulated metabolic capacity. This increase was caused by a higher glucose/glutamine metabolism, whereas FFA oxidation was unchanged. In conclusion, we describe a novel method to analyze cardiac substrate metabolism in intact cardiac tissue slices by extracellular flux analysis. The proof-of-principle experiment demonstrated that this approach has a sensitivity allowing the investigation of pathophysiologically relevant disturbances in cardiac substrate metabolism.

Expression profiles and bioinformatic analysis of microRNAs in myocardium of diabetic cardiomyopathy mice.

MicroRNAs (miRNAs) can regulate expression of target genes at post transcriptional level, and mediate the pathophysiological process of many diseases. The study will illuminate the miRNA expression profiles of diabetic cardiomyopathy (DCM), seeking probable biomarkers of DCM at early stage and determining a target for the treatment of DCM. Db/db mice were used as an animal model of type 2 diabetes mellitus. At 22 weeks of age, cardiac function was evaluated by echocardiography, and the structural changes in myocardium were evaluated by HE staining and TEM. The miRNA expression profiles were detected using miRNA sequencing and differentially expressed miRNAs were validated by real-time PCR. Bioinformatic analysis was used to analyze target genes of these miRNAs and relevant pathways in DCM. The results showed that 40 miRNAs were differentially expressed, including 28 upregulated miRNAs and 12 downregulated miRNAs. GO and KEGG pathway analysis showed that the target genes of up-regulated miRNAs were involved in 66 pathways, including Wnt, p53 and calcium signaling pathways, as well as FOXO and apoptosis signaling pathways, etc. The target genes of down-regulated miRNAs were involved in 68 pathways, including mitophagy, Ras and MAPK signaling pathways, etc. Moreover, some differentially expressed miRNAs were found in myocardium of DCM for the first time, such as miR-7225-5p, miR-696, miR-3470a, miR-3470b, miR-6240, miR-6538, miR-5128, miR-1195, miR-203-3p and miR-330-5p. It is hoped that a few novel molecular pathways or targets of treatment for DCM would be found through understanding the expression features of miRNAs in diabetic myocardium.

A combination of Sophoraflavescens alkaloids and Panaxquinquefolium saponins attenuates coxsackievirus B3‑induced acute myocarditis in mice via NF‑κB signaling.

Timely treatment of viral myocarditis (VMC), a form of cardiac inflammation caused by viral infections, can reduce the occurrence of dilated cardiomyopathy and sudden death. Our previous study demonstrated the anti-inflammatory and anti-fibrotic effects of KX, a combination of Sophora flavescens alkaloids and Panax quinquefolium saponins, on an autoimmune myocarditis model in vivo. The present study explored the effects of KX on coxsackievirus B3 (CVB3)-induced acute VMC in mice. Mice were randomly divided into four groups: Control, VMC, KX-high (275 mg/kg) and KX-low (138 mg/kg). Mice in the VMC, KX-high and KX-low groups received injections of CVB3 to establish the VMC model, and those in the KX-high and KX-low groups also received KX by gavage (10 ml/kg) 2 h after virus injection until euthanasia was performed on day 7 or 21. Mice in the control group received an equal KX volume of purified water. The levels of lactate dehydrogenase (LDH), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTn-I), IL-1β, IL-6, TNF-α and high-sensitive C-reactive protein (hs-CRP) in mouse serum was measured using ELISA. Myocardial tissue structure and degree of injury were observed using hematoxylin and eosin staining. Western blotting and reverse transcription-quantitative PCR were performed to detect the expression levels of NF-κB pathway-related mRNA and protein in myocardial tissue. The results showed that the inflammation and myocardial damage levels of the mice in the VMC group were higher at 7 days than those at 21 days. At both 7 and 21 days, KX decreased the serum CK-MB, LDH, cTn-I, IL-6, TNF-α and hs-CRP levels, and inhibited NF-κB pathway-related mRNA and protein expression in the myocardium of mice. These findings indicated that KX may reduce the inflammatory response and attenuate the pathological damage in the acute and subacute phases of CVB3-induced VMC through the NF-κB pathway.

Exertional heart stroke causes long-term obesity and cardiac hypertrophy in mice

Exertional heat stroke (EHS) is a life-threatening illness that can lead to short- and long-term adverse health outcomes. Several human epidemiology studies have shown that heat stroke exposure is highly associated with the development of cardiovascular disease later in life. However, whether EHS causes heart disease or individuals with predispositions to heart disease are more susceptible to EHS is unknown. Previously, our laboratory demonstrated metabolic abnormalities in the myocardium of mice, two weeks after EHS, characterized by lipid accumulation. In this study, we hypothesized that EHS exposure in mice leads to long-term susceptibilities to cardiovascular disease that is accelerated by co-exposure to a Western diet. METHODS: Sixty-four male (n=32) and female (n=32) C57BL/6 mice were exposed to either EHS (forced wheel running in 34.5°C for males and 37.5°C for females) or sham exercise controls (EXC, forced wheel running in ~22.5°C). Fourteen days later, mice were placed on either a Western diet (WD) or a standard diet (SD) and then followed for 9 additional weeks. At week 12, post interventions (EHS or EXC), animals were euthanized and samples collected for analysis. RESULTS: Male mice exposed to EHS with either WD ( P = 0.0001) or SD ( P = 0.0001) gained more body mass over the 9-week diet period compared to diet matched EXC controls. Also, regardless of diet, male mice exposed to EHS consumed more food compared to matched EXC (WD: P < 0.005 and SD: P < 0.04). However, only female mice exposed to EHS with WD gained more body mass compared to female EXC mice with WD ( P < 0.04). At the end of the study, and regardless of diet, male mice exposed to EHS showed enlarged hearts in terms of absolute mass (WD: P=0.0241, SD: P=0.0069) and relative mass/tibia length (WD: P=0.013, SD P=0.03). On the other hand, EHS female mice exposed to WD showed enlarged heart mass compared to EHS on SD ( P=0.0005). CONCLUSIONS: These results demonstrate that EHS exposure in mice leads to a long-term metabolic disorder characterized by greatly accelerated weight gain, greater appetite and cardiac hypertrophy. Effects were more evident and consistent in males. Such a response is typical of early stages of metabolic syndrome and would likely contribute to eventual cardiac disease. Therefore, the data is consistent with EHS exposure being a risk factor for long term heart disease. U.S. Army Grant BA180078 and from King Saud University, Saudi Arabia This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Trim65 attenuates isoproterenol-induced cardiac hypertrophy by promoting autophagy and ameliorating mitochondrial dysfunction via the Jak1/Stat1 signaling pathway

Pathological cardiac hypertrophy is a major cause of heart failure, and there is no effective approach for its prevention or treatment. The Trim family is a recently identified family of E3 ubiquitin ligases that regulate cardiac hypertrophy. Trim65, which is a member of the Trim family, previous studies have not determined whether Trim65 affects cardiac hypertrophy. In this study, the effects of Trim65 on isoproterenol (ISO)-induced cardiac hypertrophy and the underlying mechanisms were investigated. In contrast to C57BL/6 mice, Trim65-knockout (Trim65-KO) mice developed more severe myocardial hypertrophy, fibrosis and cardiac dysfunction after being intraperitoneally injected with ISO for 2 weeks. Transmission electron microscopy (TEM) revealed that the autophagic flux was inhibited, mitochondria were swollen, and mitochondrial cristae were lost or decreased in the myocardium of Trim65-KO mice. In vitro studies demonstrated that overexpression of Trim65 inhibited ISO-induced cardiomyocyte hypertrophy by increasing mitochondrial density and membrane potential, and the Stat1 inhibitor fludarabine attenuated the effect of Trim65 knockdown on ISO-induced cardiomyocyte hypertrophy by reducing Reactive oxygen species (ROS) production and increasing the mitochondrial density and membrane potential. Our findings provide the first link between Trim65 and mitochondria, and we found for the first time that Trim65 inhibits mitochondria-dependent apoptosis and autophagy via the Jak1/Stat1 signalling pathway, ultimately attenuating ISO-induced cardiac hypertrophy; this effect of Trim65 might be mediated via the regulation of Jak1 ubiquitination. Taking these findings together, we suggest that genes that are related to mitochondria-dependent apoptosis and that are associated with Trim65 could be promising therapeutic targets for cardiac hypertrophy.

Altered acylcarnitine metabolism and inflexible mitochondrial fuel utilization characterize the loss of neonatal myocardial regeneration capacity

Myocardial regeneration capacity declines during the first week after birth, and this decline is linked to adaptation to oxidative metabolism. Utilizing this regenerative window, we characterized the metabolic changes in myocardial injury in 1-day-old regeneration-competent and 7-day-old regeneration-compromised mice. The mice were either sham-operated or received left anterior descending coronary artery ligation to induce myocardial infarction (MI) and acute ischemic heart failure. Myocardial samples were collected 21 days after operations for metabolomic, transcriptomic and proteomic analyses. Phenotypic characterizations were carried out using echocardiography, histology and mitochondrial structural and functional assessments. In both groups, MI induced an early decline in cardiac function that persisted in the regeneration-compromised mice over time. By integrating the findings from metabolomic, transcriptomic and proteomic examinations, we linked regeneration failure to the accumulation of long-chain acylcarnitines and insufficient metabolic capacity for fatty acid beta-oxidation. Decreased expression of the redox-sensitive mitochondrial Slc25a20 carnitine-acylcarnitine translocase together with a decreased reduced:oxidized glutathione ratio in the myocardium in the regeneration-compromised mice pointed to a defect in the redox-sensitive acylcarnitine transport to the mitochondrial matrix. Rather than a forced shift from the preferred adult myocardial oxidative fuel source, our results suggest the facilitation of mitochondrial fatty acid transport and improvement of the beta-oxidation pathway as a means to overcome the metabolic barrier for repair and regeneration in adult mammals after MI and heart failure.

Thioredoxin Reductase 2 Synergizes with Cytochrome c, Somatic to Alleviate Doxorubicin-Induced Oxidative Stress in Cardiomyocytes and Mouse Myocardium.

Doxorubicin (DOX) may cause multiple side effects, which include cardiotoxicity. Hence, to ascertain the impact of thioredoxin reductase 2 (TXNRD2) and cytochrome c, somatic (CYCS) on DOX-induced oxidative stress (OS) in cardiomyocytes and mouse myocardium, this study was implemented. DOX was utilized to treat cardiomyocytes and mice, and TXNRD2 and CYCS expression in cell supernatant and mouse myocardial tissues was detected. TXNRD2 and/or CYCS were overexpressed in DOX-induced cardiomyocytes and mice. In cardiomyocytes, cell viability and the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione (GSH) were measured. In mice, pathologic changes of the heart, ejection fraction (EF), fractional shortening (FS), and heart weight (HW) /tibial length (TL) ratio, and the contents of lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB), and cardiac troponin I (cTnI) were analyzed. To assess the binding between TXNRD2 and CYCS, coimmunoprecipitation and glutathione S-transferase pull-down assays were performed. TXNRD2 and CYCS were downregulated in DOX-treated cardiomyocytes and mice. Mechanistically, TXNRD2 interacted with CYCS. Overexpression of TXNRD2 or CYCS augmented viability and SOD, CAT, and GSH levels but reduced ROS and MDA contents in DOX-induced cardiomyocytes, which was further facilitated by simultaneous overexpression of TXNRD2 or CYCS. Moreover, TXNRD2 or CYCS upregulation improved the pathologic changes in myocardial tissues, along with increases in EF, FS, and HW/TL ratio of the heart and SOD, CAT, and GSH levels and decreases in LDH, CK-MB, cTnI, ROS, and MDA levels. TXNRD2 coordinated with CYCS to alleviate DOX-induced OS in cardiomyocytes and mouse myocardium.

Disrupting methyl-CpG-binding protein 2 expression induces the transformation of induced pluripotent stem cell cardiomyocytes into pacemaker-like cells by insulin gene enhancer binding protein 1.

The experiment will explore whether interfering with the expression of methyl-CpG-binding protein 2 (MecP2) can enhance the ability of insulin gene enhancer binding protein 1 (ISL1) to induce iPSC-CMs to differentiate into pacemaker-like cells. Differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) can be induced via the regulation of the Wnt signaling pathway. Real-time quantitative PCR (qPCR), western blotting, immunofluorescence staining, and patch-clamp technique were used to analyze the ability of ISL1 to induce the transformation of iPSC-CMs into pacemaker-like cells. Calcium spark, patch-clamp technique, and real-time qPCR were used to verify whether disrupting the expression of MeCP2 enhanced this ability of ISL1 to induce the differentiation of iPSC-CMs into pacemaker cells. Transplant pacemaker-like cardiomyocytes into the myocardium of mice to observe whether the pacemaker cells can survive in the tissue for a long time. RT-qPCR and patch-clamp analyses showed that overexpression of ISL1 induced the successful differentiation of iPSC-CMs into pacemaker cells. ISL1-overexpressing pacemaker-like cells possessed typical characteristics of pacemaker morphology, including action potential and If inward current. Chromatin immunoprecipitation results showed that MeCP2 bound to the promoter region of HCN4. Following disruption of MeCP2 expression, the gene expression of sinoatrial node-specific transcription factors, If inward current, and cardiac rhythm changes in iPSC-CMs resembled those of sinoatrial node pacemaker cells. Therefore, ISL1 induced the differentiation of iPSC-CMs into pacemaker-like cells, and knockdown of MeCP2 increased this effect. Frozen section results showed that surviving pacemaker-like cells could still be observed in myocardial tissue after 45 days. Experiments have found that interfering with the expression of MeCP2 can increase the ability of ISL1 to induce iPSC-CM cells to differentiate into pacemaker-like cells. And the pacemaker-like cells obtained in this experiment can survive in myocardial tissue for a long time.

The alkaloids of Corydalis hendersonii Hemsl. contribute to the cardioprotective effect against ischemic injury in mice by attenuating cardiomyocyte apoptosis via p38 MAPK signaling pathway

BackgroundThere is a characteristic Tibetan folk medicine in China named Corydalis hendersonii Hemsl. (CH) has been used for treatment of cardiovascular related diseases, called “plethora” in Tibetan medicine. Previous studies demonstrated that ethanol extract of CH showed anti-acute myocardial infarction (AMI) effect through inhibiting fibrosis and inflammation. Rich alkaloids fraction (RAF) is isolated from CH, but whether RAF possessing an equivalent effect with the CH ethanol extract and by which mechanism it protects against AMI has not yet reported. The paper aimed to study the potential role of RAF on myocardial injured mice and its underlying mechanism.Materials and methodsLiquid chromatography mass spectrometry-ion trap-time of flight (LCMS-IT-TOF) was used to analyze the chemical profile and isolate pure compounds. The ligation of left anterior descending (LAD) of coronary artery in mice was used to evaluate the in vivo anti-AMI effect, by dividing into eight groups: Sham, Model, Fosinopril (10 mg/kg, i.g.), total extract (TE, 400 mg/kg, i.g.), poor alkaloids fraction (PAF, 300 mg/kg, i.g.), and RAF (25, 50, and 100 mg/kg, respectively, i.g.) groups. Echocardiography was used to evaluate mice heart function through the index of left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameter (LVEDd), fractional shortening (FS) and ejection fraction (EF). We detected the lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in the serum and the plasma level of angiotensin II (AngII). The apoptosis of mice myocardial tissue was verified by TUNEL assay. The expression of p38 mitogen-activated protein kinases (p38 MAPK), Bcl-2 and Bcl-2-associated X protein (Bax) were detected through immunofluorescence staining, qRT-PCR and western blot in mice heart tissue and H9c2 cells.ResultsEchocardiography data indicated that the values of LVEDd and LVEDs were reduced and the values of FS and EF were improved by TE and RAF significantly. RAF also decreased the levels of LDH, CK-MB and AngII and significantly inhibited inflammatory cells in the marginal zone of myocardial infarction. The TUNEL assay results showed that RAF significantly attenuated cell apoptosis. Immunofluorescence and qRT-PCR assay showed that RAF inhibited p38 MAPK, Bax, and Bcl-2 proteins in mice myocardium. Western blot results validated that the expressions of key proteins were inhibited by RAF. Also, the apoptotic cells and apoptosis-related proteins were dramatically reduced by RAF in vivo and in vitro. Besides, RAF and PAF were analyzed by LCMS-IT-TOF to identify the main compounds and to demonstrate the difference between them. The results showed that a total of 14 alkaloids were identified, which indicated that the isoquinoline alkaloids were the main ingredients in RAF may contributing to the cardioprotective effect in mice.ConclusionsRAF improves cardiac function by inhibiting apoptosis via p38 MAPK signaling pathway, and RAF contributes to the effect against myocardial ischemic injury of TE in mice, which provides a substantial reference for the clinical application against ischemia heart disease and quality control of CH.Graphical

Sema3A alleviates viral myocarditis by modulating SIRT1 to regulate cardiomyocyte mitophagy.

Viral myocarditis (VMC) is a common myocardial inflammatory disease characterized by inflammatory cell infiltration and cardiomyocyte necrosis. Sema3A was reported to reduce cardiac inflammation and improve cardiac function after myocardial infarction, but its role in VMC remains to be explored. Here, a VMC mouse model was established by infection with CVB3, and Sema3A was overexpressed in vivo by intraventricular injection of an adenovirus-mediated Sema3A expression vector (Ad-Sema3A). We found that Sema3A overexpression attenuated CVB3-induced cardiac dysfunction and tissue inflammation. And Sema3A also reduced macrophage accumulation and NLRP3 inflammasome activation in the myocardium of VMC mice. In vitro, LPS was used to stimulate primary splenic macrophages to mimic the macrophage activation state in vivo. Activated macrophages were co-cultured with primary mouse cardiomyocytes to evaluate macrophage infiltration-induced cardiomyocyte damage. Ectopic expression of Sema3A in cardiomyocytes effectively protected cardiomyocytes from activated macrophage-induced inflammation, apoptosis, and ROS accumulation. Mechanistically, cardiomyocyte-expressed Sema3A mitigated macrophage infiltration-caused cardiomyocyte dysfunction by promoting cardiomyocyte mitophagy and hindering NLRP3 inflammasome activation. Furthermore, NAM (a SIRT1 inhibitor) reversed the protective effect of Sema3A against activated macrophage-induced cardiomyocyte dysfunction by suppressing cardiomyocyte mitophagy. In conclusion, Sema3A promoted cardiomyocyte mitophagy and suppressed inflammasome activation by regulating SIRT1, thereby attenuating macrophage infiltration-induced cardiomyocyte injury in VMC.

Identification of iron metabolism-related genes in the circulation and myocardium of patients with sepsis via applied bioinformatics analysis.

Early diagnosis of septic cardiomyopathy is essential to reduce the mortality rate of sepsis. Previous studies indicated that iron metabolism plays a vital role in sepsis-induced cardiomyopathy. Here, we aimed to identify shared iron metabolism-related genes (IMRGs) in the myocardium and blood monocytes of patients with sepsis and to determine their prognostic signature. First, an applied bioinformatics-based analysis was conducted to identify shared IMRGs differentially expressed in the myocardium and peripheral blood monocytes of patients with sepsis. Second, Cytoscape was used to construct a protein-protein interaction network, and immune infiltration of the septic myocardium was assessed using single-sample gene set enrichment analysis. In addition, a prognostic prediction model for IMRGs was established by Cox regression analysis. Finally, the expression of key mRNAs in the myocardium of mice with sepsis was verified using quantitative polymerase chain reaction analysis. We screened common differentially expressed genes in septic myocardium and blood monocytes and identified 14 that were related to iron metabolism. We found that HBB, SLC25A37, SLC11A1, and HMOX1 strongly correlated with monocytes and neutrophils, whereas HMOX1 and SLC11A1 strongly correlated with macrophages. We then established a prognostic model (HIF1A and SLC25A37) using the common differentially expressed IMRGs. The prognostic model we established was expected to better aid in diagnosing septic cardiomyopathy. Moreover, we verified these genes using datasets and experiments and found a significant difference between the sepsis and control groups. Common differential expression of IMRGs was identified in blood monocytes and myocardium between sepsis and control groups, among which HIF1A and SLC25A37 might predict prognosis in septic cardiomyopathy. The study may help us deeply understand the molecular mechanisms of iron metabolism and aid in the diagnosis and treatment of septic cardiomyopathy.

The Combined Treatment of Curcumin with Verapamil Ameliorates the Cardiovascular Pathology in a Williams–Beuren Syndrome Mouse Model

Williams-Beuren syndrome (WBS) is a rare disorder caused by a recurrent microdeletion with hallmarks of cardiovascular manifestations, mainly supra-valvular aortic stenosis (SVAS). Unfortunately, there is currently no efficient treatment. We investigated the effect of chronic oral treatment with curcumin and verapamil on the cardiovascular phenotype of a murine model of WBS harbouring a similar deletion, CD (complete deletion) mice. We analysed systolic blood pressure in vivo and the histopathology of the ascending aorta and the left ventricular myocardium to determine the effects of treatments and their underlying mechanism. Molecular analysis showed significantly upregulated xanthine oxidoreductase (XOR) expression in the aorta and left ventricular myocardium of CD mice. This overexpression is concomitant with increased levels of nitrated proteins as a result of byproduct-mediated oxidative stress damage, indicating that XOR-generated oxidative stress impacts the pathophysiology of cardiovascular manifestations in WBS. Only the combined therapy of curcumin and verapamil resulted in a significant improvement of cardiovascular parameters via activation of the nuclear factor erythroid 2 (NRF2) and reduction of XOR and nitrated protein levels. Our data suggested that the inhibition of XOR and oxidative stress damage could help prevent the severe cardiovascular injuries of this disorder.

Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis

BackgroundIncreased acid sphingomyelinase (ASMase) activity is associated with insulin resistance and cardiac dysfunction. However, the effects of ASMase on diabetic cardiomyopathy (DCM) and the molecular mechanism(s) underlying remain to be elucidated. We here investigated whether ASMase caused DCM through NADPH oxidase 4-mediated apoptosis.Methods and resultsWe used pharmacological and genetic approaches coupled with study of murine and cell line samples to reveal the mechanisms initiated by ASMase in diabetic hearts. The protein expression and activity of ASMase were upregulated, meanwhile ceramide accumulation was increased in the myocardium of HFD mice. Inhibition of ASMase with imipramine (20 mg Kg−1 d−1) or siRNA reduced cardiomyocyte apoptosis, fibrosis, and mitigated cardiac hypertrophy and cardiac dysfunction in HFD mice. The similar effects were observed in cardiomyocytes treated with high glucose (HG, 30 mmol L−1) + palmitic acid (PA, 100 μmol L−1) or C16 ceramide (CER, 20 μmol L−1). Interestingly, the cardioprotective effect of ASMase inhibition was not accompanied by reduced ceramide accumulation, indicating a ceramide-independent manner. The mechanism may involve activated NADPH oxidase 4 (NOX4), increased ROS generation and triggered apoptosis. Suppression of NOX4 with apocynin prevented HG + PA and CER incubation induced Nppb and Myh7 pro-hypertrophic gene expression, ROS production and apoptosis in H9c2 cells. Furthermore, cardiomyocyte-specific ASMase knockout (ASMaseMyh6KO) restored HFD-induced cardiac dysfunction, remodeling, and apoptosis, whereas NOX4 protein expression was downregulated.ConclusionsThese results demonstrated that HFD-mediated activation of cardiomyocyte ASMase could increase NOX4 expression, which may stimulate oxidative stress, apoptosis, and then cause metabolic cardiomyopathy.

Lymphatic insufficiency leads to distinct myocardial infarct content assessed by magnetic resonance TRAFFn, T1ρ and T2 relaxation times

The role of cardiac lymphatics in the pathogenesis of myocardial infarction (MI) is unclear. Lymphatic system regulates cardiac physiological processes such as edema and tissue fluid balance, which affect MI pathogenesis. Recently, MI and fibrosis have been assessed using endogenous contrast in magnetic resonance imaging (MRI) based on the relaxation along a fictitious field with rank n (RAFFn). We extended the RAFFn applications to evaluate the effects of lymphatic insufficiency on MI with comparison to longitudinal rotating frame (T1ρ) and T2 relaxation times. MI was induced in transgenic (TG) mice expressing soluble decoy VEGF receptor 3 that reduces lymphatic vessel formation and their wild-type (WT) control littermates for comparison. The RAFFn relaxation times with rank 2 (TRAFF2), and rank 4 (TRAFF4), T1ρ and T2 were acquired at time points 0, 3, 7, 21 and 42 days after the MI at 9.4 T. Infarct sizes were determined based on TRAFF2, TRAFF4, T1ρ and T2 relaxation time maps. The area of differences (AOD) was calculated based on the MI areas determined on T2 and TRAFF2, TRAFF4 or T1ρ relaxation time maps. Hematoxylin–eosin and Sirius red stained histology sections were prepared to confirm MI locations and sizes. MI was detected as increased TRAFF2, TRAFF4, T1ρ and T2 relaxation times. Infarct sizes were similar on all relaxation time maps during the experimental period. Significantly larger AOD values were found together with increased AOD values in the TG group compared to the WT group. Histology confirmed these findings. The lymphatic deficiency was found to increase cardiac edema in MI. The combination of TRAFF2 (or TRAFF4) and T2 characterizes MI and edema in the myocardium in both lymphatic insufficiency and normal mice without any contrast agents.

Filamin C is Essential for mammalian myocardial integrity.

FLNC, encoding filamin C, is one of the most mutated genes in dilated and hypertrophic cardiomyopathy. However, the precise role of filamin C in mammalian heart remains unclear. In this study, we demonstrated Flnc global (FlncgKO) and cardiomyocyte-specific knockout (FlnccKO) mice died in utero from severely ruptured ventricular myocardium, indicating filamin C is required to maintain the structural integrity of myocardium in the mammalian heart. Contrary to the common belief that filamin C acts as an integrin inactivator, we observed attenuated activation of β1 integrin specifically in the myocardium of FlncgKO mice. Although deleting β1 integrin from cardiomyocytes did not recapitulate the heart rupture phenotype in Flnc knockout mice, deleting both β1 integrin and filamin C from cardiomyocytes resulted in much more severe heart ruptures than deleting filamin C alone. Our results demonstrated that filamin C works in concert with β1 integrin to maintain the structural integrity of myocardium during mammalian heart development.

Impact of Prenatal Alcohol Exposure on the Development and Myocardium of Adult Mice: Morphometric Changes, Transcriptional Modulation of Genes Related to Cardiac Dysfunction, and Antioxidant Cardioprotection.

The impact of prenatal alcohol exposure (PAE) varies considerably between individuals, leading to morphological and genetic changes. However, minor changes usually go undetected in PAE children. We investigated PAE's effects on gene transcription of genes related to cardiac dysfunction signaling in mouse myocardium and morphological changes. C57Bl/6 mice were subjected to a 10% PAE protocol. In postnatal days 2 and 60 (PN2 and PN60), morphometric measurements in the offspring were performed. Ventricular samples of the heart were collected in PN60 from male offspring for quantification of mRNA expression of 47 genes of nine myocardial signal transduction pathways related to cardiovascular dysfunction. Animals from the PAE group presented low birth weight than the Control group, but the differences were abolished in adult mice. In contrast, the mice's size was similar in PN2; however, PAE mice were oversized at PN60 compared with the Control group. Cardiac and ventricular indexes were increased in PAE mice. PAE modulated the mRNA expression of 43 genes, especially increasing the expressions of genes essential for maladaptive tissue remodeling. PAE animals presented increased antioxidant enzyme activities in the myocardium. In summary, PAE animals presented morphometric changes, transcription of cardiac dysfunction-related genes, and increased antioxidant protection in the myocardium.

MicroRNA-155-5p in serum derived-exosomes promotes ischaemia-reperfusion injury by reducing CypD ubiquitination by NEDD4.

Recovery of blood flow is a therapeutic approach for myocardial infarction but paradoxically induces injury to the myocardium. Exosomes (exos) are pivotal mediators for intercellular communication that can be released by different cells and are involved in cardiovascular diseases. This study aimed to explore the possible effects and mechanisms of miR-155-5p loaded by serum-derived exos in myocardial infarction reperfusion injury (MIRI). Exos were isolated from mouse serum after induction of ischaemia reperfusion (I/R) and injected into I/R-treated mice to assess cardiac function, infarction size, and cardiomyocyte apoptosis. Primary cardiomyocytes were transfected with miR-155-5p inhibitor before treatment with oxygen-glucose deprivation and re-oxygenation (OGD/R) and exos derived from the serum of I/R-treated mice (I/R-Exos), in which Bcl-2, Bax, and cleaved-caspase-3 levels were detected. The interactions among miR-155-5p, NEDD4, and CypD were evaluated. miR-155-5p level was evidently increased in I/R-Exos than in exos from the serum of sham-operated mice (P<0.05). In comparison with the I/R group, the I/R-Exos+I/R group had increased infarct size, elevated miR-155-5p expression, and boosted apoptotic rate in mouse myocardium (P<0.05). In mice treated with I/R-Exos and I/R, miR-155-5p inhibition reduced cardiac infarct size and apoptosis (P<0.05). NEDD4 was a target gene of miR-155-5p and promoted CypD ubiquitination. Cardiomyocyte apoptosis was markedly increased in the miR-155-5p inhibitor+shNEDD4+OGD/R group versus the miR-155-5p inhibitor+OGD/R group (P<0.05), but decreased in the miR-155-5p inhibitor+shNEDD4+shCypD+OGD/R group than in the miR-155-5p inhibitor+shNEDD4+OGD/R group (P<0.05). miR-155-5p in I/R-Exos may facilitate MIRI by inhibiting CypD ubiquitination via targeting NEDD4.

β-arrestin2 Mediates the Arginine Vasopressin-Induced Expression of IL-1β in Murine Hearts.

Circulating levels of arginine vasopressin (AVP) are elevated during cardiac stress and this could be a factor in cardiac inflammation and fibrosis. Herein, we studied the effects of AVP on interleukin-1β (IL-1β) production and the role(s) of β-arrestin2-dependent signaling in murine heart. The levels of IL-1β mRNA and protein in adult rat cardiofibroblasts (ARCFs) was measured using quantitative PCR and ELISA, respectively. The activity of β-arrestin2 was manipulated using either pharmacologic inhibitors or through recombinant β-arrestin2 over-expression. These experiments were conducted to determine the roles of β-arrestin2 in the regulation of AVP-induced IL-1β and NLRP3 inflammasome production. The phosphorylation and activation of NF-κB induced by AVP was measured by immunoblotting. β-arrestin2 knockout (KO) mice were used to investigate whether β-arrestin2 mediated the AVP-induced production of IL-1β and NLRP3, as well as the phosphorylation of the NF-κB p65 subunitin mouse myocardium. Prism GraphPad software(version 8.0), was used for all statistical analyses. AVP induced the expression of IL-1β in a time-dependent manner in ARCFs but not in cultured adult rat cardiomyocytes (ARCMs). The inhibition of NF-κB with pyrrolidinedithiocarbamic acid (PDTC) prevented the AVP-induced phosphorylation of NF-κB and production of IL-1β and NLRP3 in ARCFs. The deletion of β-arrestin2 blocked the phosphorylation of p65 and the expression of NLRP3 and IL-1β induced by AVP in both mouse hearts and in ARCFs. AVP promotes IL-1β expression through β-arrestin2-mediated NF-κB signaling in murine heart.

Cardiac Piezo1 Exacerbates Lethal Ventricular Arrhythmogenesis by Linking Mechanical Stress with Ca2+ Handling After Myocardial Infarction.

Ventricular arrhythmogenesis is a key cause of sudden cardiac death following myocardial infarction (MI). Accumulating data show that ischemia, sympathetic activation, and inflammation contribute to arrhythmogenesis. However, the role and mechanisms of abnormal mechanical stress in ventricular arrhythmia following MI remain undefined. We aimed to examine the impact of increased mechanical stress and identify the role of the key sensor Piezo1 in ventricular arrhythmogenesis in MI. Concomitant with increased ventricular pressure, Piezo1, as a newly recognized mechano-sensitive cation channel, was the most up-regulated mechanosensor in the myocardium of patients with advanced heart failure. Piezo1 was mainly located at the intercalated discs and T-tubules of cardiomyocytes, which are responsible for intracellular calcium homeostasis and intercellular communication. Cardiomyocyte-conditional Piezo1 knockout mice (Piezo1Cko) exhibited preserved cardiac function after MI. Piezo1Cko mice also displayed a dramatically decreased mortality in response to the programmed electrical stimulation after MI with a markedly reduced incidence of ventricular tachycardia. In contrast, activation of Piezo1 in mouse myocardium increased the electrical instability as indicated by prolonged QT interval and sagging ST segment. Mechanistically, Piezo1 impaired intracellular calcium cycling dynamics by mediating the intracellular Ca2+ overload and increasing the activation of Ca2+-modulated signaling, CaMKII, and calpain, which led to the enhancement of phosphorylation of RyR2 and further increment of Ca2+ leaking, finally provoking cardiac arrhythmias. Furthermore, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Piezo1 activation remarkably triggered cellular arrhythmogenic remodeling by significantly shortening the duration of the action potential, inducing early afterdepolarization, and enhancing triggered activity.This study uncovered a proarrhythmic role of Piezo1 during cardiac remodeling, which is achieved by regulating Ca2+ handling, implying a promising therapeutic target in sudden cardiac death and heart failure.

Novel Approach for Assessing Postinfarct Myocardial Injury and Inflammation Using Hybrid Somatostatin Receptor Positron Emission Tomography/Magnetic Resonance Imaging.

Novel Approach for Assessing Postinfarct Myocardial Injury and Inflammation Using Hybrid Somatostatin Receptor Positron Emission Tomography/Magnetic Resonance Imaging.