Mouse Cardiomyocytes Research Articles

Abstract 12168: Pi3kγ is a Key Modulator of Cardiac Metabolism Rewiring Occurring in Doxorubicin-Treated Mice

Introduction: Doxorubicin (DOX) is a potent chemotherapeutic drug, whose use is hampered by the onset of long-term cardiotoxicity and lack of effective therapeutic approaches. Recently, metabolic perturbations have emerged as an early predictor factor of cardiac dysfunction, but how DOX affects cardiac metabolism is still unknown. We have previously demonstrated that phosphoinositide 3-kinase γ (PI3Kγ) is a major driver of DOX-evoked cardiotoxicity through mitophagy inhibition and accumulation of dysfunctional mitochondria. Hypothesis: Here we sought to explore the role of PI3Kγ in modulating cardiac metabolism upon DOX administration. Methods: Metabolomic analysis, glycolysis and oxidative phosphorylation were measured in whole hearts from wild-type (WT) and knock-in mice expressing a kinase-inactive PI3Kγ (kinase-dead; KD) treated with DOX (4 mg/kg on day 0, 7 and 14). Similar analyses were performed in WT and KD hearts infected with AAV9-shATG7 to explore the role of autophagy. Moreover, glucose uptake and GLUT-4 expression on the plasma membrane were measured in WT and KD neonatal mouse cardiomyocytes (NMCs) exposed to 1μM DOX. Results: Metabolomic analysis revealed an increased level of succinate and malic acid in DOX-treated hearts, suggesting defects in the TCA cycle. In agreement, DOX promotes the accumulation of metabolites normally catabolised in the TCA cycle, such as aminoacids and pyruvate, indicating a severe mitochondria dysfunction. Accordingly, fatty acid oxidation and electron transport chain activity were impaired in DOX-treated hearts at day 3 and exacerbated at day 42 after DOX. At the same time, glycolysis was upregulated. These changes were prevented in KD animals. Interestingly, upon autophagy inhibition, KD mice lost their cardioprotection at the mitochondrial level, but they still failed to upregulate glycolysis. This suggests a new autophagy-independent mechanism through which PI3Kγ regulates glucose utilisation. In agreement with the pharmacological and genetic inhibition of PI3Kγ abrogates glucose uptake in NMCs and normalises the plasma membrane exposure of GLUT-4. Conclusions: Overall, these results promote PI3Kγ as a master regulator of metabolic rewiring occurring in DOX-treated cardiomyocytes.

Abstract 10586: CREG1-FBXO27-LAMP2 Axis Promotes Autophagy Function of Cardiomyocytes and Alleviates Diabetic Cardiomyopathy

Introduction: Diabetic cardiomyopathy is a chronic complication of diabetes in the absence of coronary artery disease, arterial hypertension and valvular disease. Autophagy plays an important role in the development of diabetic cardiomyopathy. Cellular repressor of E1A-stimulated genes 1 (CREG1) is an important myocardial protective factor. The aim of this study was to investigate the effects and mechanisms of CREG1 in diabetic cardiomyopathy. Methods: Male C57BL/6J mice, CREG1 transgenic mice, and CREG1 cardiac-specific knockout mice were used to establish a type 2 diabetic model. Small animal ultrasound, Masson’s staining, and western blotting were used to evaluate cardiac function, myocardial fibrosis and autophagy. Neonatal mouse cardiomyocytes (NMCMs) were isolated and cultured. NMCMs were infected with CREG1- overexpressing adenovirus or small interfering RNA, followed by stimulation with palmitic acid (PA), real-time PCR, western blotting and immunofluorescence staining were used. Results: CREG1 protein expression was decreased in the myocardium of diabetic cardiomyopathy. CREG1 deficiency in the heart aggravated cardiac dysfunction, cardiac hypertrophy, and fibrosis in mice with diabetic cardiomyopathy, accompanied by aggravated autophagy dysfunction. CREG1 overexpression improved cardiac function and ameliorated cardiac hypertrophy and fibrosis in diabetic cardiomyopathy, by improving autophagy. CREG1 protein expression was decreased in PA-stimulated NMCMs. CREG1 knockdown aggravated cardiomyocyte hypertrophy and inhibited autophagy, which were reversed by resveratrol treatment. Conversely, CREG1 overexpression inhibited cardiomyocyte hypertrophy and improved autophagy, which were reversed by chloroquine or bafilomycin A1 treatment. Overexpression of LAMP2 reversed the effect of CREG1 knockdown on the PA-induced inhibition of cardiomyocyte autophagy. CREG1 inhibited the LAMP2 protein degradation by inhibiting the expression of F-box protein 27 (FBXO27). Conclusions: CREG1-FBXO27-LAMP2 axis alleviated diabetic cardiomyopathy by promoting cardiomyocyte autophagy. Our findings might help clarify new roles of CREG1 in the development of diabetic cardiomyopathy.

Pathophysiological Role of Variants of the Promoter Region of CITED2 Gene in Sporadic Tetralogy of Fallot Patients with Cellular Function Verification.

Tetralogy of Fallot (TOF) is a common congenital heart malformation. Genetic variants in the CITED2 coding region are known to be significantly associated with cardiac malformation, but the role of variants in the CITED2 promoter region in the development of TOF remains unclear. In this study, we investigated CITED2 promoter variants in the DNA of 605 subjects, including 312 TOF patients and 293 unrelated healthy controls, by Sanger sequencing. We identified nine CITED2 gene promoter variants (including one novel heterozygous variant). Six were found only in patients with TOF and none in the control group. The transcriptional activity of the CITED2 gene promoter in mouse cardiomyocyte (HL-1) cells was significantly altered by the six variants (p < 0.05). The results of the electrophoretic mobility change assay and JASPAR database analysis showed that these variants generated or destroyed a series of possible transcription factor binding sites, resulting in changes in the CITED2 protein expression. We conclude that CITED2 promoter variants in TOF patients affect transcriptional activity and may be involved in the occurrence and progression of TOF. These findings may provide new insights into molecular pathogenesis and potential therapeutic insights in patients with TOF.

Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption

Kinetic analysis of intracellular calcium (Ca2+) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca2+ kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca2+ kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca2+ amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly.

Network pharmacology-based and experimental identification of the effects of Renshen Yangrong decoction on myocardial infarction.

Objective: Myocardial infarction (MI) is one of the leading causes of death worldwide. Currently, the drugs used to treat MI have various side effects. Emerging evidence supports the protective effects of Renshen Yangrong Decoction (RSYRD) in cardiovascular diseases (CVDs) treatments, with few side effect reports. However, the role of RSYRD in MI remains unclear. In this study, network pharmacological analysis was combined with experiments in vivo and in vitro to validate the effects of RSYRD in the treatment during the early stage of MI. Methods: Firstly, network pharmacology analysis was performed to search for the potential targets and signaling pathways of RSYRD in the early stage of MI. Then, the protein-protein interaction (PPI) network was constructed to identify the core genes of RSYRD that may play a key role in MI. At last, the treatment effectiveness of RSYRD on MI was verified via experiments in vitro and in vivo. Results: RSYRD contained fifty-six bioactive components. Eighty-eight intersections between RSYRD and MI targets and thirteen core genes were screened. KEGG and GO functional enrichment analyses predicted that RSYRD might play a therapeutic role in MI through oxidative stress, apoptosis, and immune-inflammatory signaling pathways. In vivo and in vitro experiment results revealed that significant apoptosis occurred in myocardial tissue in the early stage of MI. Moreover, the levels of reactive oxide species (ROS), TNF-α, and IL-6 increased markedly. After RSYRD administration, they significantly decreased. At the mechanistic level, RSYRD could reduce ROS production to alleviate cell apoptosis. Conclusion: RSYRD could reduce neonatal mouse cardiomyocytes (NMCMs) apoptosis by lowering ROS production induced by hypoxia and improve the cardiac function of mice 3days post-MI. RSYRD could also reduce the levels of TNF-α and IL-6 in the serum of mice.

SV40 large T antigen-induced immortalization reprograms mouse cardiomyocyte progenitors with mesenchymal stem cell characteristics and osteogenic potential

SV40 large T antigen-induced immortalization reprograms mouse cardiomyocyte progenitors with mesenchymal stem cell characteristics and osteogenic potential

Titin truncating variant cardiomyopathy and related sarcomere insufficiency causes high energy demand resulting in mitochondrial dysfunction, autophagosome formation, and apoptosis

Abstract Background/Objectives Titin truncating variants (TTNtv) are the most prevalent genetic cause of dilated cardiomyopathy (DCM), resulting in upregulation of cardiac transcripts of oxidative phosphorylation (1,2). However, the underlying molecular mechanism(s) and cellular consequences of these findings remain unknown. Methods and results To gain insight into the metabolic changes and cellular consequences of a TTNtv, metabolic, mitochondrial, and survival pathways were studied in human TTNtv DCM hearts and isolated cardiomyocytes of TTNtv mice. TTNtv resulted in a significant increase of cardiac transcripts of glycolysis, citric acid cycle, mitochondrial fission, autophagy, and apoptosis when comparing RNAseq in 24 TTNtv and 27 mutation-negative DCM cardiac biopsies. Furthermore, a decrease in the area of myofibrils in human TTNtv hearts (TTNtv vs. mutation-negative DCM: 46%, and 62%, P=0.001), and an increase of mitochondrial (49% and 31%, P=0,001) and autophagosome areas (4% and 2%, P=0.002) was observed using transmission electron microscopy (TEM). Similar patterns of cardiomyocyte disorganization and stress could be seen in TTNtv hearts of mice even without a phenotype. Additionally, observed swollen mitochondria by TEM and decreased quantity of OXPHOS proteins by immunoblotting in murine TTNtv hearts indicate mitochondrial stress. Mitochondrial oxygen consumption at baseline and the maximum respiration in TTNtv cardiomyocytes of mice increased by a factor of 1.8 and 1.5 respectively (both P≤0.05), compared to WT. Furthermore, palmitate oxidation in TTNtv cardiomyocytes increased by 1.3 fold (P=0.005) compared to WT mice, suggestive of increased energy demand in TTNtv. Conclusion Myofibrillar insufficiency in human TTNtv DCM augments the cardiac oxygen and energy consumption, leading to pronounced morphological and functional mitochondrial decompensation. Swelling, damage and fission of mitochondria is further characterized by autophagosome formation and increased apoptosis pathways in TTNtv hearts. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Double-Dose consortium by Dutch Cardiovascular Alliance (DCVA)

PCSK9 modulates the Wnt/beta-catenin signaling pathway in myocardial ischemia/reperfusion injury

Abstract Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein that affects cholesterol homeostasis. Recent research has found that PCSK9 has various effects on the heart that are unrelated to LDL cholesterol regulation. The Wnt/β-catenin signaling pathway plays a crucial role during heart development, and it is re-activated in response to cardiac injury. Low-density lipoprotein receptor-related proteins 5 (LRP5) act as co-receptors of Wnt ligands and are indispensable for Wnt/β-catenin signal transduction. However, it is not fully elucidated whether other members of the LDLR-superfamily may be targets of PCSK9. Purpose This study aimed to determine if LRP5 is a PCSK9 target, study the association between PCSK9 and Wnt/β-catenin signaling, and elucidate its effect on myocardial infarction in patients with ischemic cardiomyopathy. Methods The expression of Lrp5, phospho-β-catenin, total β-catenin was evaluated by western blot analysis, and the effects of overexpressed PCSK9 were tested under normoxia, hypoxia, or hypoxia/re-oxygenation (H/R) in mouse cardiomyocytes (HL-1). The transcriptional activity of β-catenin was assessed using the TOP-Flash/FOP-Flash luciferase reporter assay. In addition, the impact on various downstream targets of the Wnt/β-catenin signaling pathway was assessed using a quantitative real-time polymerase chain reaction. To examine whether PCSK9 regulates injury of cardiomyocytes in vivo, we subjected transgenic mice with cardiac-specific overexpression of PCSK9 (PCSK9 TG) and wild-type (WT) mice to either sham surgery or ischemia/reperfusion (I/R) surgery. Results Under hypoxic conditions, the Wnt/β-catenin signaling pathway-related genes were downregulated in HL-1 cells, as evidenced by lower Lrp5 and active phospho-β-catenin expression levels (0.5-fold, n=3, p&amp;lt;0.01). After H/R, the Wnt/β-catenin-related genes were recovered (1.5-fold, p&amp;lt;0.01) in the control group but not in the PCSK9 overexpressed group. In the luciferase reporter assay results, PCSK9 overexpression inhibited the recovery of β-catenin transcriptional activity after H/R, in contrast to the control group. Furthermore, mRNA levels of Axin2, Cyclin D1, which are the Wnt/β-catenin signaling downstream pathway targets, were down-regulated under hypoxia and recovered after H/R but did not recover in PCSK9 overexpressed cells. In the mouse I/R model, the overall protein levels of the Wnt/β-catenin signaling-related genes were down-regulated in PCSK9 TG mice compared to WT mice after I/R injury. Conclusions These results indicated that the regulation of PCSK9 is closely associated with the Wnt/β-catenin signaling pathway which may play a crucial role in damaged cardiomyocytes. It suggests that the regulation of PCSK9 could be a therapeutic target in patients with ischemic cardiomyopathy. Funding Acknowledgement Type of funding sources: None.

Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes: potential implication in Doxorubicin-induced cardiotoxicity

Abstract Background The treatment with doxorubicin, a powerful chemotherapeutic agent, has been shown to be associated with an increased risk of lethal heart failure. Although various types of cell death pathway such as apoptosis and ferroptosis have been shown to be involved in the development of doxorubicin-induced cardiotoxicity, DIC, the involvement of necroptosis, a novel programmed necrosis induced by translocation of activated mixed lineage kinase domain-like protein, MLKL, to plasma membrane, remains unclear. Purpose The aim of this study was to determine whether necroptosis is involved in the development of DIC. Methods and results DIC was induced in C57BL/6J mice by intraperitoneal injection of doxorubicin at a dose of 10 mg/kg 3 times for a week. Eight days after the commencement of injection, echocardiographic analyses showed that left ventricular ejection fraction assessed by echocardiography was significantly lower in the doxorubicin-treated mice than in the vehicle-treated mice (44.0±13.7 vs. 70.5±3.7%), indicating the development of DIC. Immunoblot analysis showed that MLKL protein level was higher by 1.6 fold in the doxorubicin-treated mice than in the vehicle-treated mice. Interestingly, immunohistochemical analysis showed that signals of phospho-Ser345-MLKL, an activated form of MLKL, was found in the nuclei in addition to cytosol and intercalated discs of cardiomyocytes in the doxorubicin-treated mice. To get novel insight into significance of nuclear MLKL accumulation, a leucine-rich nuclear export signal (NES) spanning amino acids 280–284 of rat MLKL was identified by site-directed mutation analyses, and H9c2 cells, cultured rat cardiomyoblasts, were transfected with expression constructs for nucleus-directed MLKL (FLAG-mtNES-MLKL) or its wild type (FLAG-WT-MLKL). Percentage of FLAG-positive cells stained with Zombie Red, a fluorescent dye that is non-permeant to live cells, was higher in FLAG-mtNES-MLKL-transfected cells than in FLAG-WT-MLKL-transfected cells (80.0±3.5% vs. 6.3±1.3%, p&amp;lt;0.05), whereas percentage of cells immunostained with cleaved caspase-3 to FLAG-positive cells was similar in the two groups. The effect of the MLKL mutant on necroptosis was attenuated by treatment with GppNHp, an inhibitor of Ran-mediated nuclear protein import. Conclusion Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes, which may contribute to progression of DIC. Funding Acknowledgement Type of funding sources: None.

Oxidative activation of CaMKII mediates arrhythmias and contractile dysfunction in an obstructive sleep apnea mouse model

Abstract Background/Introduction Atrial arrhythmias frequently occur in patients with obstructive sleep apnea (OSA), but the underlying mechanisms remain insufficiently understood. We recently demonstrated that CaMKII-dependent pro-arrhythmic activity is increased in patients with OSA, but the mechanisms of CaMKII activation remain unknown. Interestingly, OSA can lead to increased reactive oxygen species production, which may facilitate CaMKII activation by oxidation at methionine residues 281/282. Purpose We tested if oxidation of CaMKII is involved in the development of arrhythmias and contractile dysfunction in vitro and in vivo in a mouse model of obstructive sleep apnea by tongue enlargement. Methods Experiments were performed using 41 wild-type (WT) and 25 genetically modified mice lacking oxidative CaMKII activation due to mutation of methionine 281/282 to valine (MMVV mice). Polytetrafluorethylene (PTFE, 100 μl) was injected into the tongue of 23 WT and 12 MMVV mice to induce OSA by sustained tongue enlargement as previously established. Whole body plethysmography was performed to confirm success of intervention. Echocardiography was performed at baseline and after 8 weeks to assess left-ventricular function. After 8 weeks, isolated atrial and ventricular cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 AM (5 μM, 15 min) and analyzed with epifluorescence microscopy under regular electrical field stimulation (1 Hz). Results PTFE injection resulted in an increased frequency of inspiratory flow limitations (IFLs/h) from 27.4±5.95 to 59.6±6.22 in PTFE mice (p&amp;lt;0.001, fig. 1A). Interestingly, in WT PTFE mice, left ventricular ejection fraction (LVEF, in %) was reduced at 8 weeks post procedure from 57.0±1.36 to 51.5±1.85 (p=0.001, fig. 1B). Importantly, mice lacking oxidative CaMKII activation were protected from such decline in contractile function, and LVEF was higher at 8 weeks vs. WT PTFE (p&amp;lt;0.001, fig. 1B). Congruent with deterioration of contractility in vivo, the Ca transient amplitude after 30s pause (normalized to steady-state before pause) was decreased in ventricular cardiomyocytes of WT PTFE mice indicating increased SR Ca leak (p for interaction genotype x PTFE = 0.014, fig. 1C). In contrast to WT, the post-pause ratio was increased in MMVV PTFE mice (p=0.018, fig. 1C). In addition to contractile function, cellular arrhythmic events were analyzed as non-stimulated pro-arrhythmic events (NSEs) during steady-state electrical field stimulation at 1 Hz (fig. 1D, red arrows). NSE frequency was increased in atrial cardiomyocytes from WT PTFE mice with 0.050±0.005 compared to 0.018±0.003 s–1 in WT CTRL (p&amp;lt;0.001, fig. 1D). Intriguingly, MMVV mice were protected from NSEs after PTFE treatment (0.020±0.004, p&amp;lt;0.001 vs. WT PTFE, Fig. 1D). Conclusion In a mouse model of obstructive sleep apnea, contractile dysfunction and pro-arrhythmic activity are modulated by oxidative CaMKII activation, which may have therapeutic implications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft; Medical Faculty at University of Regensburg

Kir2.1 dysfunction at the sarcolemma and the sarcoplasmic reticulum causes arrhythmias in a mouse model of Andersen–Tawil syndrome type 1

Andersen–Tawil syndrome type 1 (ATS1) is associated with life-threatening arrhythmias of unknown mechanism. In this study, we generated and characterized a mouse model of ATS1 carrying the trafficking-deficient mutant Kir2.1Δ314-315 channel. The mutant mouse recapitulates the electrophysiological phenotype of ATS1, with QT prolongation exacerbated by flecainide or isoproterenol, drug-induced QRS prolongation, increased vulnerability to reentrant arrhythmias and multifocal discharges resembling catecholaminergic polymorphic ventricular tachycardia (CPVT). Kir2.1Δ314-315 cardiomyocytes display significantly reduced inward rectifier K+ and Na+ currents, depolarized resting membrane potential and prolonged action potentials. We show that, in wild-type mouse cardiomyocytes and skeletal muscle cells, Kir2.1 channels localize to sarcoplasmic reticulum (SR) microdomains, contributing to intracellular Ca2+ homeostasis. Kir2.1Δ314-315 cardiomyocytes exhibit defective SR Kir2.1 localization and function, as intact and permeabilized Kir2.1Δ314-315 cardiomyocytes display abnormal spontaneous Ca2+ release events. Overall, defective Kir2.1 channel function at the sarcolemma and the SR explain the life-threatening arrhythmias in ATS1 and its overlap with CPVT.

SARS-CoV-2 Nsp6 damages Drosophila heart and mouse cardiomyocytes through MGA/MAX complex-mediated increased glycolysis

SARS-CoV-2 infection causes COVID-19, a severe acute respiratory disease associated with cardiovascular complications including long-term outcomes. The presence of virus in cardiac tissue of patients with COVID-19 suggests this is a direct, rather than secondary, effect of infection. Here, by expressing individual SARS-CoV-2 proteins in the Drosophila heart, we demonstrate interaction of virus Nsp6 with host proteins of the MGA/MAX complex (MGA, PCGF6 and TFDP1). Complementing transcriptomic data from the fly heart reveal that this interaction blocks the antagonistic MGA/MAX complex, which shifts the balance towards MYC/MAX and activates glycolysis—with similar findings in mouse cardiomyocytes. Further, the Nsp6-induced glycolysis disrupts cardiac mitochondrial function, known to increase reactive oxygen species (ROS) in heart failure; this could explain COVID-19-associated cardiac pathology. Inhibiting the glycolysis pathway by 2-deoxy-D-glucose (2DG) treatment attenuates the Nsp6-induced cardiac phenotype in flies and mice. These findings point to glycolysis as a potential pharmacological target for treating COVID-19-associated heart failure.

Diesel-derived PM2.5 induces impairment of cardiac movement followed by mitochondria dysfunction in cardiomyocytes.

Particulate matter (PM) in polluted air can be exposed to the human body through inhalation, ingestion, and skin contact, accumulating in various organs throughout the body. Organ accumulation of PM is a growing health concern, particularly in the cardiovascular system. PM emissions are formed in the air by solid particles, liquid droplets, and fuel – particularly diesel – combustion. PM2.5 (size < 2.5 μm particle) is a major risk factor for approximately 200,000 premature deaths annually caused by air pollution. This study assessed the deleterious effects of diesel-derived PM2.5 exposure in HL-1 mouse cardiomyocyte cell lines. The PM2.5-induced biological changes, including ultrastructure, intracellular reactive oxygen species (ROS) generation, viability, and intracellular ATP levels, were analyzed. Moreover, we analyzed changes in transcriptomics using RNA sequencing and metabolomics using gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in PM2.5-treated HL-1 cells. Ultrastructural analysis using transmission electron microscopy revealed disruption of mitochondrial cristae structures in a PM2.5 dose-dependent manner. The elevation of ROS levels and reduction in cell viability and ATP levels were similarly observed in a PM2.5 dose-dependently. In addition, 6,005 genes were differentially expressed (fold change cut-off ± 4) from a total of 45,777 identified genes, and 20 amino acids (AAs) were differentially expressed (fold change cut-off ± 1.2) from a total of 28 identified AAs profiles. Using bioinformatic analysis with ingenuity pathway analysis (IPA) software, we found that the changes in the transcriptome and metabolome are highly related to changes in biological functions, including homeostasis of Ca2+, depolarization of mitochondria, the function of mitochondria, synthesis of ATP, and cardiomyopathy. Moreover, an integrated single omics network was constructed by combining the transcriptome and the metabolome. In silico prediction analysis with IPA predicted that upregulation of mitochondria depolarization, ROS generation, cardiomyopathy, suppression of Ca2+ homeostasis, mitochondrial function, and ATP synthesis occurred in PM2.5-treated HL-1 cells. In particular, the cardiac movement of HL-1 was significantly reduced after PM2.5 treatment. In conclusion, our results assessed the harmful effects of PM2.5 on mitochondrial function and analyzed the biological changes related to cardiac movement, which is potentially associated with cardiovascular diseases.

Silencing of Long Noncoding RNA TUG1 Ameliorates Atherosclerosis-Induced Myocardial Injury by Upregulating microRNA-30b-3p and Downregulating Brd4.

Long noncoding RNAs and microRNAs (miRNAs) are emerging biomarkers involved in human diseases, and we focused on the roles of long noncoding RNA taurine upregulated gene 1 (TUG1) and miR-30b-3p in the related mechanisms of atherosclerosis-induced myocardial injury. ApoE-deficient mice were fed with high-fat diet to establish atherosclerotic models and then were subjected to either TUG1 downregulation or miR-30b-3p upregulation treatment. The serum myocardial enzymes, inflammatory biomarkers, pathological changes, intramyocardial macrophage infiltration, and apoptosis of cardiomyocytes in atherosclerotic mice were determined. The expression of TUG1, miR-30b-3p, and bromodomain protein 4 (Brd4) in atherosclerotic models was evaluated. Moreover, the correlations of TUG1, miR-30b-3p, and Brd4 were verified. TUG1 and Brd4 were increased while miR-30b-3p was decreased in atherosclerotic mice. The silenced TUG1 or elevated miR-30b-3p attenuated atherosclerosis-induced myocardial injury mainly by reducing serum myocardial enzyme content and inflammatory response, improving pathological changes, and preventing macrophage infiltration and cardiomyocyte apoptosis in atherosclerotic mice. Mechanistically, TUG1 could competitively bind with miR-30b-3p to prevent the degradation of its target gene Brd4. This study reveals that the silencing of TUG1 ameliorates atherosclerosis-induced myocardial injury by upregulating miR-30b-3p and downregulating Brd4, which may provide novel targets for atherosclerosis treatment.

Deficiency of a novel lncRNA-HRAT protects against myocardial ischemia reperfusion injury by targeting miR-370-3p/RNF41 pathway.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) contribute to myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms by which lncRNAs modulate myocardial I/R injury have not been thoroughly examined and require further investigation. A novel lncRNA named lncRNA-hypoxia/reoxygenation (H/R)-associated transcript (lncRNA-HRAT) was identified by RNA sequencing analysis. The expression of lncRNA-HRAT exhibited a significant increase in the I/R mice hearts and cardiomyocytes treated with H/R. LncRNA-HRAT overexpression facilitates H/R-induced cardiomyocyte apoptosis. Furthermore, cardiomyocyte-specific deficiency of lncRNA-HRAT in vivo after I/R decreased creatine kinase (CK) release in the serum, reduced myocardial infarct area, and improved cardiac dysfunction. Molecular mechanistic investigations revealed that lncRNA-HRAT serves as a competing endogenous RNA (ceRNA) of miR-370-3p, thus upregulating the expression of ring finger protein 41 (RNF41), thereby aggravating apoptosis in cardiomyocytes induced by H/R. This study revealed that the lncRNA-HRAT/miR-370-3p/RNF41 pathway regulates cardiomyocyte apoptosis and myocardial injury. These findings suggest that targeted inhibition of lncRNA-HRAT may offer a novel therapeutic method to prevent myocardial I/R injury.

Retraction: 'The protease inhibitor UCF-101 ameliorates streptozotocin-induced mouse cardiomyocyte contractile dysfunction in vitro: role of AMP-activated protein kinase'.

Retraction: [Qun Li, The protease inhibitor UCF-101 ameliorates streptozotocin-induced mouse cardiomyocyte contractile dysfunction in vitro: role of AMP-activated protein kinase, Experimental Physiology 2009, 94(9), pp. 984-994. (https://doi.org/10.1113/expphysiol.2009.049189)]. The above article, published online on 14 August 2009 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the corresponding author, the journal Editor-in-Chief Professor Mike Tipton, The Physiological Society, and John Wiley & Sons Ltd. The retraction has been agreed following an institutional investigation by the University of Wyoming. This investigation cited data irregularities, image reuse, and manipulation in a number of figures, specifically: Re-use of α-tubulin blot of Figure 7A in 7C, as well as reuse of α-tubulin blot of Figure 7B in 7D. Re-sizing and re-use of α-tubulin bands of Figure 3A in Figure 5A. α-tubulin reported within two distinct sets of AMPK and pAMPK data in Figure 5A. These concerns were put to the corresponding author, who confirmed that loading controls had been reused inappropriately throughout the manuscript, albeit accidentally. The corresponding author resupplied Western blot images and data, but these could not be verified. The extent of image reuse and inappropriate use of controls significantly undermined confidence in the paper's data and subsequent conclusions. This retraction includes the corrigendum published online on 04 February 2016 (https://doi.org/10.1113/EP085709). Corresponding author JR agreed to this retraction. QL and LKH could not be reached. REFERENCES Li, Q., Hueckstaedt, L. K., & Ren, J. (2009). The protease inhibitor UCF-101 ameliorates streptozotocin-induced mouse cardiomyocyte contractile dysfunction in vitro: role of AMP-activated protein kinase. Experimental Physiology, 94(9), 984-994. https://doi.org/10.1113/expphysiol.2009.049189 (2016). Corrigendum. Experimental Physiology, 101(3), 451-451. https://doi.org/10.1113/EP085709.

The protective effects of Clerodendranthus spicatus (Thunb.) C. Y. Wu extract on oxidative stress induced by 2,2'-azo (2-methylpropamidine) dihydrochloride in HL-1 mouse cardiomyocytes.

To investigate the protective effects of Clerodendranthus spicatus (Thunb.) C. Y. Wu extract (CSTE) on oxidative stress injury in HL-1 mouse cardiomyocytes induced by 2,2'-azo (2-methylpropamidine) dihydrochloride (AAPH, 1 mmol/L), HL-1 cells were co-cultured with different concentrations (10–100 μg/mL) of the CSTE for 24 h. A cell damage model was established by continuously culturing the cells in Dulbecco's Modified Eagle Medium plus AAPH for 4 h. Cell survival rates were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, and by measuring intracellular malondialdehyde (MDA) content. MDA and total reactive oxygen species (ROS) levels were determined by thiobarbituric acid colorimetry and the 2',7'-dihydrodichlorofluorescent sodium yellow diacetate probe, respectively. Apoptosis was measured by flow cytometry. The intracellular catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione s-transferase (GST), γ-glutamylcysteine synthetase (γ-GCS), and glutathione (GSH) contents were determined by colorimetric methods. CSTE content was determined by high performance liquid chromatography. The CSTE pretreatment improved survival rates in damaged HL-1 cells, reduced total intracellular ROS and MDA levels, and reduced apoptosis. The CSTE also increased the activities of the antioxidant enzymes (CAT, SOD, GSH-Px, and GST), as well as the γ-GCS and GSH levels in damaged cells. Real-time fluorescence quantitative polymerase chain reaction analysis indicated that the CSTE upregulated CAT, SOD1, and GSH-Px mRNA expression levels. Additionally, the CSTE reduced MDA and ROS levels in HL-1 cells by improving the endogenous antioxidant system; thus, alleviating the oxidative stress damage caused by AAPH. Our compositional analyses revealed that the CSTE contained caffeic acid, isoquercetin, rosmarinic acid, luteolin, and baicalin. The CSTE demonstrates antioxidant and protective effects in myocardial cells.

Cardiomyocyte-Specific Long Noncoding RNA Regulates Alternative Splicing of the Triadin Gene in the Heart.

Abnormalities in Ca2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive. We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as, a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart. We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA. These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing.

MLKL-mediated necroptosis is a target for cardiac protection in mouse models of type-1 diabetes

BackgroundCardiomyocyte death contributes to cardiac pathology of diabetes. Studies have shown that the RIPK3/MLKL necroptosis signaling is activated in diabetic hearts. Deletion of RIPK3 was reported to attenuate myocardial injury and heart dysfunction in streptozocin (STZ)-induced diabetic mice, suggesting a potential role of necroptosis in diabetic cardiomyopathy. This study characterized cardiomyocyte necroptosis in diabetic hearts and investigated whether MLKL-mediated necroptosis is a target for cardiac protection in diabetes.MethodsType 1 diabetes was induced in RIPK3 knockout, MLKL knockout and wild-type mice. Akita Type-1 diabetic mice were injected with shRNA for MLKL. Myocardial function was assessed by echocardiography. Immuno-histological analyses determined cardiomyocyte death and fibrosis in the heart. Cultured adult mouse cardiomyocytes were incubated with high glucose in the presence of various drugs. Cell death and phosphorylation of RIPK3 and MLKL were analysed.ResultsWe showed that the levels of phosphorylated RIPK3 and MLKL were higher in high glucose-stimulated cardiomyocytes and hearts of STZ-induced type-1 diabetic mice, akita mice and type-1 diabetic monkeys when compared to non-diabetic controls. Inhibition of RIPK3 by its pharmacological inhibitor or gene deletion, or MLKL deletion prevented high glucose-induced MLKL phosphorylation and attenuated necroptosis in cardiomyocytes. In STZ-induced type-1 diabetic mice, cardiomyocyte necroptosis was present along with elevated cardiac troponin I in serum and MLKL oligomerization, and co-localized with phosphorylated MLKL. Deletion of RIPK3 or MLKL prevented MLKL phosphorylation and cardiac necroptosis, attenuated serum cardiac troponin I levels, reduced myocardial collagen deposition and improved myocardial function in STZ-injected mice. Additionally, shRNA-mediated down-regulation of MLKL reduced cardiomyocyte necroptosis in akita mice. Interestingly, incubation with anti-diabetic drugs (empagliflozin and metformin) prevented phosphorylation of RIPK3 and MLKL, and reduced cell death in high glucose-induced cardiomyocytes.ConclusionsWe have provided evidence that cardiomyocyte necroptosis is present in diabetic hearts and that MLKL-mediated cardiomyocyte necroptosis contributes to diabetic cardiomyopathy. These findings highlight MLKL-mediated necroptosis as a target for cardiac protection in diabetes.

HMOX1 silencing prevents doxorubicin-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis by downregulating CTGF.

Doxorubicin is a type of effective antitumor drug but can contribute to cardiomyocyte injuries. We aimed to dissect the mechanism of the HMOX1/CTGF axis in DOX-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis. Bioinformatics analysis was conducted to retrieve differentially expressed genes in a DOX-induced mouse model. Mouse cardiomyocytes, HL-1 cells, were induced with l µM DOX, after which gain- or loss-of-function assays were applied. CCK-8, fluorescent probe assay, flow cytometry, and corresponding kits were employed to detect cell viability, ROS levels, mitochondrial membrane potential and cell apoptosis, and GSH and Fe2+ contents, respectively. qRT-PCR or Western blot assay was adopted to test HMOX1, CTGF, BCL-2, Caspase3, Cleaved-Caspase3, and GPX4 expression. Bioinformatics analysis showed that HMOX1 and CTGF were highly expressed in DOX-induced mice and correlated with each other. Also, HMOX1 and CTGF expression was high in HL-1 cells after DOX treatment, along with an obvious decrease in cell viability and GSH and GPX4 expression, an increase in ROS levels, apoptosis, and Fe2+ contents, and mitochondrial membrane potential dysfunction or loss. HMOX1 or CTGF silencing diminished cell apoptosis, Cleaved-Caspase3 expression, Fe2+ contents, and ROS levels, enhanced cell viability and the expression of GSH, GPX4, and BCL-2, and recovered mitochondrial membrane potential in DOX-induced HL-1 cells. Nevertheless, the effects of HMOX1 silencing on the viability, apoptosis, ferroptosis, and mitochondrial dysfunction of DOX-induced HL-1 cells were counteracted by CTGF overexpression. In conclusion, HMOX1 silencing decreased CTGF expression to alleviate DOX-induced injury, mitochondrial dysfunction, and ferroptosis of mouse cardiomyocytes.