Primary Neonatal Rat Research Articles

Cyclovirobuxine D Ameliorates Experimental Diabetic Cardiomyopathy by Inhibiting Cardiomyocyte Pyroptosis via NLRP3 in vivo and in vitro.

Diabetic cardiomyopathy (DCM) is one of the common complications of diabetic patients, which can induce myocardial hypertrophy, cardiac fibrosis, and heart failure. Growing evidence has shown that the occurrence and development of DCM are accompanied by pyroptosis which is an NLRP3-mediated intense inflammatory cell death. Cyclovirobuxine D (CVB-D) has been shown to significantly ameliorate DCM and anti-inflammatory effects associated with cardiomyopathy, but it is unclear whether it has an effect on cardiomyocyte pyroptosis accompanying DCM. Therefore, the purpose of the present study was to explore the ameliorating effect of CVB-D on cardiomyocyte pyroptosis associated with DCM and its molecular regulation mechanism. Type 2 diabetes in C57BL/6 mice was reproduced by the high-fat and high-glucose diet (HFD) combined with low-dose streptozotocin (STZ). The characteristics of DCM were evaluated by cardiac ultrasonography, serum detection, and histopathological staining. The results suggested that CVB-D could significantly alleviate the cardiac pathology of DCM. Then, we explored the mechanism of CVB-D on primary neonatal rat cardiomyocyte (PNRCM) injury with high glucose (HG) in vitro to simulate the physiological environment of DCM. Preincubation with CVB-D could significantly increase cell viability, attenuate cytopathological changes and inhibit the expression levels of pyroptosis-related proteins. Further research found that the myocardial improvement effect of CVB-D was related to its inhibition of NLRP3 expression. In conclusion, our data suggest that CVB-D can ameliorate DCM by inhibiting cardiomyocyte pyroptosis via NLRP3, providing a novel molecular target for CVB-D clinical application.

ANGPTL8 is a negative regulator in pathological cardiac hypertrophy

Pathological cardiac hypertrophy is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the role of angiopoietin-like protein 8 (ANGPTL8) in pathological cardiac hypertrophy. We found that serum ANGPTL8 levels were significantly increased in hypertensive patients with cardiac hypertrophy and in mice with cardiac hypertrophy induced by Ang II or TAC. Furthermore, the secretion of ANGPTL8 from the liver was increased during hypertrophic processes, which were triggered by Ang II. In the Ang II- and transverse aortic constriction (TAC)-induced mouse cardiac hypertrophy model, ANGPTL8 deficiency remarkably accelerated cardiac hypertrophy and fibrosis with deteriorating cardiac dysfunction. Accordingly, both recombinant human full-length ANGPTL8 (rANGPTL8) protein and ANGPTL8 overexpression significantly mitigated Ang II-induced cell enlargement in primary neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. Mechanistically, the antihypertrophic effects of ANGPTL8 depended on inhibiting Akt and GSK-3β activation, and the Akt activator SC-79 abolished the antihypertrophic effects of rANGPTL8 in vitro. Moreover, we demonstrated that ANGPTL8 directly bound to the paired Ig-like receptor PIRB (LILRB3) by RNA-seq and immunoprecipitation-mass screening. Remarkably, the antihypertrophic effects of ANGPTL8 were largely blocked by anti-LILRB3 and siRNA-LILRB3. Our study indicated that ANGPTL8 served as a novel negative regulator of pathological cardiac hypertrophy by binding to LILRB3 (PIRB) and inhibiting Akt/GSK3β activation, suggesting that ANGPTL8 may provide synergistic effects in combination with AT1 blockers and become a therapeutic target for cardiac hypertrophy and heart failure.

Measuring hypertrophy in neonatal rat primary cardiomyocytes and human iPSC-derived cardiomyocytes.

In the heart, left ventricular hypertrophy is initially an adaptive mechanism that increases wall thickness to preserve normal cardiac output and function in the face of coronary artery disease or hypertension. Cardiac hypertrophy develops in response to pressure and volume overload but can also be seen in inherited cardiomyopathies. As the wall thickens, it becomes stiffer impairing the distribution of oxygenated blood to the rest of the body. With complex cellular signalling and transcriptional networks involved in the establishment of the hypertrophic state, several model systems have been developed to better understand the molecular drivers of disease. Immortalized cardiomyocyte cell lines, primary rodent and larger animal models have all helped understand the pathological mechanisms underlying cardiac hypertrophy. Induced pluripotent stem cell-derived cardiomyocytes are also used and have the additional benefit of providing access to human samples with direct disease relevance as when generated from patients suffering from hypertrophic cardiomyopathies. Here, we briefly review in vitro and in vivo model systems that have been used to model hypertrophy and provide detailed methods to isolate primary neonatal rat cardiomyocytes as well as to generate cardiomyocytes from human iPSCs. We also describe how to model hypertrophy in a "dish" using gene expression analysis and immunofluorescence combined with automated high-content imaging.

Glucose Fluctuations Aggravate Myocardial Fibrosis via the Nuclear Factor-κB-Mediated Nucleotide-Binding Oligomerization Domain-Like Receptor Protein 3 Inflammasome Activation.

BackgroundGlucose fluctuations may be associated with myocardial fibrosis. This study aimed to investigate the underlying mechanisms of glucose fluctuation-related myocardial fibrosis.MethodsStreptozotocin (STZ)-injected type 1 diabetic rats were randomized to five groups: the controlled blood glucose (CBG) group, uncontrolled blood glucose (UBG) group, fluctuated blood glucose (FBG) group, FBG rats injected with 0.9% sodium chloride (NaCl) (FBG + NaCl) group, and FBG rats injected with MCC950 (FBG + MCC950) group. Eight weeks later, left ventricular function was evaluated by echocardiography and myocardial fibrosis was observed by Masson trichrome staining. The primary neonatal rat cardiac fibroblasts were cultured with different concentrations of glucose in vitro.ResultsThe left ventricular function was impaired and myocardial fibrosis was aggravated most significantly in the FBG group compared with the CBG and UBG groups. The levels of interleukin (IL)-1β, IL-18, transforming growth factor-β1 (TGF-β1), collagen type 1 (collagen I), nuclear factor (NF)-κB, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome were significantly increased in the FBG group. In vitro, the inhibition of NF-κB and inflammasome reversed these effects. In vivo, NLRP3 inhibition with MCC950 reversed left ventricular systolic dysfunction and myocardial fibrosis induced by glucose fluctuations.ConclusionGlucose fluctuations promote diabetic myocardial fibrosis by the NF-κB-mediated inflammasome activation.

Extracellular vesicles enclosed-miR-421 suppresses air pollution (PM2.5 )-induced cardiac dysfunction via ACE2 signalling.

Air pollution, via ambient PM2.5, is a big threat to public health since it associates with increased hospitalisation, incidence rate and mortality of cardiopulmonary injury. However, the potential mediators of pulmonary injury in PM2.5‐induced cardiovascular disorder are not fully understood. To investigate a potential cross talk between lung and heart upon PM2.5 exposure, intratracheal instillation in vivo, organ culture ex vivo and human bronchial epithelial cells (Beas‐2B) culture in vitro experiments were performed respectively. The exposed supernatants of Beas‐2B were collected to treat primary neonatal rat cardiomyocytes (NRCMs). Upon intratracheal instillation, subacute PM2.5 exposure caused cardiac dysfunction, which was time‐dependent secondary to lung injury in mice, thereby demonstrating a cross‐talk between lungs and heart potentially mediated via small extracellular vesicles (sEV). We isolated sEV from PM2.5‐exposed mice serum and Beas‐2B supernatants to analyse the change of sEV subpopulations in response to PM2.5. Single particle interferometric reflectance imaging sensing analysis (SP‐IRIS) demonstrated that PM2.5 increased CD63/CD81/CD9 positive particles. Our results indicated that respiratory system‐derived sEV containing miR‐421 contributed to cardiac dysfunction post‐PM2.5 exposure. Inhibition of miR‐421 by AAV9‐miR421‐sponge could significantly reverse PM2.5‐induced cardiac dysfunction in mice. We identified that cardiac angiotensin converting enzyme 2 (ACE2) was a downstream target of sEV‐miR421, and induced myocardial cell apoptosis and cardiac dysfunction. In addition, we observed that GW4869 (an inhibitor of sEV release) or diminazene aceturate (DIZE, an activator of ACE2) treatment could attenuate PM2.5‐induced cardiac dysfunction in vivo. Taken together, our results suggest that PM2.5 exposure promotes sEV‐linked miR421 release after lung injury and hereby contributes to PM2.5‐induced cardiac dysfunction via suppressing ACE2.

PPARγ Mediates the Cardioprotective Roles of Danlou Tablet After Acute Myocardial Ischemia-Reperfusion Injury.

Ischemic heart disease is one of the biggest threats to human life in the world. Reperfusion therapy is an effective strategy to reduce infarct size and ischemic injury. However, reperfusion process may cause secondary myocardial injury which is defined as ischemia-reperfusion injury (IRI). Exploring potential therapeutic strategy to attenuate IRI is extremely important. Danlou tablet (Dan), a Chinese herbal compound consisting of ten herbs, has been identified to be protective for the heart. However, the mechanism of Dan-induced cardioprotection after acute reperfusion was unelucidated. In this study, to investigate the role and mechanism of Dan in myocardial IRI, we performed acute IRI modeling in mice and oxygen-glucose deprivation–reperfusion (OGD/R)-induced apoptosis in primary neonatal rat cardiomyocytes (NRCMs). We found that Dan had protective effect against acute IRI in mice, as evidenced by reduced infarct size, TUNEL-positive cardiomyocytes (CMs), and Bax/Bcl2 ratio and cleaved-caspase 3/caspase 3 ratio in vivo. Meanwhile, Dan inhibited OGD/R-induced apoptosis of NRCMs in vitro. Mechanistically, Dan could activate proliferator-activated receptor gamma (PPARγ) in both IRI hearts and OGD/R-stressed NRCMs, while inhibition of PPARγ attenuated the protective effect of Dan against IRI in vivo and OGD/R-induced CM apoptosis in vitro. These data reveal that Dan attenuates acute myocardial IRI and CM apoptosis through activating PPARγ. Our findings may extend the knowledge of Chinese medicine and provide potential strategy for the precise treatment of ischemic heart diseases.

Endoplasmic reticulum stress mediates homocysteine-induced hypertrophy of cardiac cells through activation of cyclic nucleotide phosphodiesterase 1C.

Although the association of elevated homocysteine level with cardiac hypertrophy has been reported, the molecular mechanisms by which homocysteine induces cardiac hypertrophy remain inadequately understood. In this study we aim to uncover the roles of cyclic nucleotide phosphodiesterase 1 (PDE1) and endoplasmic reticulum (ER) stress and their relationship to advance the mechanistic understanding of homocysteine-induced cardiac cell hypertrophy. H9c2 cells and primary neonatal rat cardiomyocytes are exposed to homocysteine with or without ER stress inhibitor TUDCA or PDE1-specific inhibitor Lu AF58027, or transfected with siRNAs targeting PDE1 isoforms prior to homocysteine-exposure. Cell surface area is measured and ultrastructure is examined by transmission electron microscopy. Hypertrophic markers, PDE1 isoforms, and ER stress molecules are detected by q-PCR and western blot analysis. Intracellular cGMP and cAMP are measured by ELISA. The results show that homocysteine causes the enlargement of H9c2 cells, increases the expressions of hypertrophic markers β-MHC and ANP, upregulates PDE1A and PDE1C, promotes the expressions of ER stress molecules, and causes ER dilatation and degranulation. TUDCA and Lu AF58027 downregulate β-MHC and ANP, and alleviate cell enlargement. TUDCA decreases PDE1A and PDE1C levels. Silencing of PDE1C inhibits homocysteine-induced hypertrophy, whereas PDE1A knockdown has minor effect. Both cAMP and cGMP are decreased after homocysteine-exposure, while only cAMP is restored by Lu AF58027 and TUDCA. TUDCA and Lu AF58027 also inhibit cell enlargement, downregulate ANP, β-MHC and PDE1C, and enhance cAMP level in homocysteine-exposed primary cardiomyocytes. ER stress mediates homocysteine-induced hypertrophy of cardiac cells via upregulating PDE1C expression Cyclic nucleotide, especially cAMP, is the downstream mediator of the ER stress-PDE1C signaling axis in homocysteine-induced cell hypertrophy.

Ecklonia stolonifera Okamura Extract Suppresses Myocardial Infarction-Induced Left Ventricular Systolic Dysfunction by Inhibiting p300-HAT Activity.

Ecklonia stolonifera Okamura extract (ESE) has been reported to have various bioactive effects, but its effects on cardiovascular disease have not yet been investigated. First, primary neonatal rat cultured cardiomyocytes were treated with ESE and stimulated with phenylephrine (PE) for 48 h. ESE (1000 µg/mL) significantly suppressed PE-induced cardiomyocyte hypertrophy, hypertrophy-related gene transcription, and the acetylation of histone H3K9. An in vitro p300-HAT assay indicated that ESE directly inhibited p300-HAT activity. Next, one week after myocardial infarction (MI) surgery, rats (left ventricular fractional shortening (LVFS) < 40%) were randomly assigned to three groups: vehicle (saline, n = 9), ESE (0.3 g/kg, n = 10), or ESE (1 g/kg, n = 10). Daily oral administration was carried out for 8 weeks. After treatment, LVFS was significantly higher in the ESE (1 g/kg) group than in the vehicle group. The ESE treatments also significantly suppressed MI-induced increases in myocardial cell diameter, perivascular fibrosis, hypertrophy- and fibrosis-related gene transcription, and the acetylation of histone H3K9. These results suggest that ESE suppressed both hypertrophic responses in cardiomyocytes and the development of heart failure in rats by inhibiting p300-HAT activity. Thus, this dietary extract is a potential novel therapeutic strategy for heart failure in humans.

A20 (TNFAIP3) alleviates viral myocarditis through ADAR1/miR-1a-3p-dependent regulation

ObjectiveTo investigate the effect of A20 and how A20 is regulated in viral myocarditis (VMC).MethodsBABL/C mice, primary neonatal rat cardiomyocytes and H9c2 cells were infected with Coxsackie virus B3 (CVB3) to establish animal and cellular models of VMC. H&E staining revealed the pathologic condition of myocardium. ELISA measured the serum levels of creatine kinase, creatine kinase isoenzyme and cardiac troponin I. The effects of A20, miR-1a-3p and ADAR1 were investigated using gain and loss of function approaches. ELISA measured the levels of IL-6, IL-18 and TNF-α in serum or cell culture supernatant. TUNEL staining and flow cytometry assessed the apoptosis of myocardium and cardiomyocytes, respectively. RNA-binding protein immunoprecipitation and dual-luciferase reporter assays verified the binding between A20 and miR-1a-3p. Co-immunoprecipitation assay verified the binding between ADAR1 and Dicer.ResultsA20 was underexpressed and miR-1a-3p was overexpressed in the myocardium of VMC mice as well as in CVB3-infected cardiomyocytes. Overexpression of A20 suppressed cardiomyocyte inflammation and apoptosis in vivo and in vitro. miR-1a-3p promoted CVB3-induced inflammation and apoptosis in cardiomyocytes by binding to A20. The expression of miR-1a-3p was regulated by ADAR1. ADAR1 promoted the slicing of miR-1a-3p precursor by binding to Dicer.ConclusionA20, regulated by ADAR1/miR-1a-3p, suppresses inflammation and cardiomyocyte apoptosis in VMC.

Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy.

Doxorubicin (DOX)-induced cardiomyopathy constitutes dose-dependent cardiac toxicity, culminating in fatal heart failure progression. Cardiac toxicity limits effective and subsequent use of DOX in chemotherapy regimens in pediatric, adult, and recurrent cancer patients. DOX-induced profound alterations in mitochondrial morphology, dynamics, and defects in mitochondrial energy metabolism in the heart comprise key stressors in DOX-induced cardiotoxicity. Hence, the discovery of novel molecular targets and therapeutics to mitigate DOX-induced mitochondrial dysfunctions are imperative. Herein, we provided two laboratory protocols to monitor DOX-induced alterations in mitochondrial morphology and respiration in isolated primary neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes are extensively used to monitor signaling mechanisms regulating cardiomyopathy in vitro. Therefore, these protocols will help researchers study the effects of novel pharmacological and genetic manipulations against DOX-induced alterations in mitochondrial morphology and energy metabolism in cardiomyocytes.

Calenduloside E protects against myocardial ischemia-reperfusion injury induced calcium overload by enhancing autophagy and inhibiting L-type Ca2+ channels through BAG3.

Calenduloside E (CE) is a saponin isolated from Aralia elata (Miq) Seem, which has anti-cardiovascular disease effects. This study aims to evaluate the anti-myocardial ischemia-reperfusion injury (MIRI) mechanisms of CE and regulation of BAG3 on calcium overload. We adopted siRNA to interfere with BAG3 expression in H9c2 cardiomyocytes and used adenovirus to interfere with BAG3 expression (Ad-BAG3) in primary neonatal rat cardiomyocytes (PNRCMs) to clarify the role of BAG3 in mitigating MIRI by CE. The results showed that CE reduced calcium overload, and Ad-BAG3 had a significant regulatory effect on L-type Ca2+ channels (LTCC) but no effects on other calcium-related proteins. And BAG3 and LTCC were colocalized in myocardial tissue and BAG3 inhibited LTCC expression. Surprisingly, CE had no regulatory effect on LTCC mRNA, but CE promoted LTCC degradation through the autophagy-lysosomal pathway rather than the ubiquitination-protease pathway. Autophagy inhibitor played a negative regulation of cardiomyocyte contraction rhythm and field potential signals. Ad-BAG3 inhibited autophagy by regulating the expression of autophagy-related proteins and autophagy agonist treatment suppressed calcium overload. Therefore, CE promoted autophagy through BAG3, thereby regulating LTCC expression, inhibiting calcium overload, and ultimately reducing MIRI.

Pervasive cytoquakes in the actomyosin cortex across cell types and substrate stiffness.

The actomyosin cytoskeleton enables cells to resist deformation, crawl, change their shape and sense their surroundings. Despite decades of study, how its molecular constituents can assemble together to form a network with the observed mechanics of cells remains poorly understood. Recently, it has been shown that the actomyosin cortex of quiescent cells can undergo frequent, abrupt reconfigurations and displacements, called cytoquakes. Notably, such fluctuations are not predicted by current physical models of actomyosin networks, and their prevalence across cell types and mechanical environments has not previously been studied. Using micropost array detectors, we have performed high-resolution measurements of the dynamic mechanical fluctuations of cells' actomyosin cortex and stress fiber networks. This reveals cortical dynamics dominated by cytoquakes-intermittent events with a fat-tailed distribution of displacements, sometimes spanning microposts separated by 4μm, in all cell types studied. These included 3T3 fibroblasts, where cytoquakes persisted over substrate stiffnesses spanning the tissue-relevant range of 4.3kPa-17kPa, and primary neonatal rat cardiac fibroblasts and myofibroblasts, human embryonic kidney cells and human bone osteosarcoma epithelial (U2OS) cells, where cytoquakes were observed on substrates in the same stiffness range. Overall, these findings suggest that the cortex self-organizes into a marginally stable mechanical state whose physics may contribute to cell mechanical properties, active behavior and mechanosensing.

RRM2 Improves Cardiomyocyte Proliferation after Myocardial Ischemia Reperfusion Injury through the Hippo-YAP Pathway.

Objective Ribonucleotide reductase M2 (RRM2) as an enzyme that catalyzes the deoxyreduction of nucleosides to deoxyribonucleoside triphosphate (dNTP) has been extensively studied, and it plays a crucial role in regulating cell proliferation. However, its role in ischemia-reperfusion injury (I/RI) is still unclear. Methods SD rats were used as the research object to detect the expression of RRM2 in the myocardium by constructing an I/RI model. At the same time, primary SD neonatal rat cardiomyocytes were extracted, and hypoxia/reoxygenation (H/R) treatment simulated the I/RI model. Using transfection technology to overexpress RRM2 in cardiomyocytes, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of RRM2, Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and immunofluorescence staining was used to detect Ki67 and EdU-positive cells. Western blot (WB) technology was used to detect YAP and its phosphorylation expression. Results qRT-PCR results indicated that the expression of RRM2 was inhibited in the model group, and cardiomyocytes overexpressing RRM2 can obviously promote the proliferation of primary cardiomyocytes and improve the damage of cardiac structure and function caused by I/R. At the same time, RRM2 can promote the increase of YAP protein expression and the increase of Cyclin D1 mRNA expression. Conclusion RRM2 expression was downregulated in myocardial tissue with I/R. After overexpression of RRM2, cardiomyocyte proliferation was upregulated and the Hippo-YAP signaling pathway was activated.

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia.

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1β, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).

Amelioratory effect of neoandrographolide on myocardial ischemic-reperfusion injury by its anti-inflammatory and anti-apoptotic activities.

In the present study, we aimed to evaluate the cardioprotective effect of neoandrographolide (Neo) on myocardial ischemia/reperfusion injury (I/R) models and explore its possible mechanism. We randomly and equally divided male mice into sham-operation, I/R, and I/R + Neo groups. H9C2 cell line and primary neonatal rat cardiomyocytes were induced into the simulated I/R's status and used to further validate the Neo's role in vitro. Heart systolic function, indexes of myocardial injury (IMI), infarct size, pathological change, cell apoptosis, inflammatory cytokines, and indexes related to apoptotic and NF-κB signaling pathways were analyzed in vivo or in vitro after the Neo treatment. Compared to the I/R group, Neo significantly suppressed IMI, infarct size, inflammatory cell infiltration, cell apoptosis, inflammatory cytokines, bax, cleaved caspase-3, P-IKBa, and P-NF-κB protein expressions, and the translocation of NF-kB subunit p65 from the cytoplasm to the nucleus in vivo or in vitro. Still, ejected fraction, fractional shortening, and the bcl-2 protein expression were notably increased after the Neo treatment. Neo could be developed into a new drug for treating myocardial I/R by inhibiting myocardial inflammation and apoptosis, which was closely related to suppressing the activation of bax/bcl-2 and NF-κB signaling pathways.

Nobiletin Attenuates Pathological Cardiac Remodeling after Myocardial Infarction via Activating PPARγ and PGC1α.

Materials and Methods C57BL/6 mice were treated with coronary artery ligation to generate an MI model, followed by treatment for 3 weeks with NOB (50 mg/kg/d) or vehicle (50 mg/kg/d), with or without the peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor T0070907 (1 mg/kg/d). Cardiac function (echocardiography, survival rate, Evans blue, and triphenyl tetrazolium chloride staining), fibrosis (Masson's trichrome staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB)), hypertrophy (haematoxylin-eosin staining, wheat germ agglutinin staining, and qRT-PCR), and apoptosis (WB and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining) were evaluated. Hypoxia-induced apoptosis (TUNEL, WB) and phenylephrine- (PE-) induced pathological hypertrophy (immunofluorescence staining, qRT-PCR) models were established in primary neonatal rat ventricular myocytes (NRVMs). The effects of NOB with or without T0070907 were examined for the expression of PPARγ and PPARγ coactivator 1α (PGC1α) by WB in mice and NRVMs. The potential downstream effectors of PPARγ were further analyzed by WB in mice. Results Following MI in mice, NOB intervention enhanced cardiac function across three predominant dimensions of pathological cardiac remodeling, which reflected in decreasing cardiac fibrosis, apoptosis, and hypertrophy decompensation. NOB intervention also alleviated apoptosis and hypertrophy in NRVMs. NOB intervention upregulated PPARγ and PGC1α in vivo and in vitro. Furthermore, the PPARγ inhibitor abolished the protective effects of NOB against pathological cardiac remodeling during the progression from MI to CHF. The potential downstream effectors of PPARγ were nuclear factor erythroid 2-related factor 2 (Nrf-2) and heme oxygenase 1 (HO-1). Conclusions Our findings suggested that NOB alleviates pathological cardiac remodeling after MI via PPARγ and PGC1α upregulation.

De-ubiquitination of p300 by USP12 Critically Enhances METTL3 Expression and Ang II-induced cardiac hypertrophy

Stresses, such as neurohumoral activation, induced pathological cardiac hypertrophy is the main risk factor for heart failure. The ubiquitin-proteasome system (UPS) plays a key role in maintaining protein homeostasis and cardiac function. However, research on the role and mechanism of deubiquitinating enzymes (DUBs) in cardiac hypertrophy is limited. Here, we observe that the deubiquitinating enzyme ubiquitin-specific protease 12(USP12) is upregulated in Ang II-induced hypertrophic hearts and primary neonatal rat cardiomyocytes (NRCMs). Inhibition of USP12 ameliorate Ang II-induced myocardial hypertrophy, while overexpression of USP12 have the opposite effect. USP12 deficiency also significantly attenuate the phenotype of Ang II-induced cardiac hypertrophy in vivo. Moreover, we demonstrate that USP12 aggravate Ang II-induced cardiac hypertrophy by enhancing METTL3, a methyltransferase which catalyze N6-methyladenosine (m6A) modification on messenger RNA and acts as a harmful factor in pathological cardiac hypertrophy. Upregulation of METTL3 reverse the reduction of myocardial hypertrophy induced by USP12 silencing in NRCMs. In contrast, knockdown of METTL3 attenuate the aggravation of myocardial hypertrophy in USP12-overexpressing NRCMs. Furthermore, we discover that USP12 promote the expression of METTL3 via upregulating p300. Mechanistically, USP12 binds and stabilizes p300, thereby activating the transcription of its downstream gene METTL3. Finally, our data show that USP12 is partially dependent on the stabilization of p300 to activate METTL3 expression and promote myocardial hypertrophy. Taken together, our results demonstrate that USP12 acts as a pro-hypertrophic deubiquitinating enzyme via enhancing p300/METTL3 axis, indicating that targeting USP12 could be a potential treatment strategy for pathological cardiac hypertrophy.

MiR-141 attenuates sepsis-induced cardiomyopathy by targeting DAPK1.

To discuss the possible effects of microRNA-141 (miR-141) in sepsis-induced cardiomyopathy (SIC) via targeting death-associated protein kinase 1 (DAPK1). An SIC mouse model was constructed by abdominal injection of lipopolysaccharide (LPS) and divided into control, LPS, LPS + pre-miR-141, and LPS + anti-miR-141 groups. Hemodynamic indicators and heart function indexes of mice were detected. ELISA was used to determine the serum levels of inflammatory cytokines, while TUNEL staining to observe the apoptosis of myocardial cells of mice, as well as qRT-PCR and Western blotting to clarify the expression of miR-141 and DAPK1. Lastly, in vitro experiment was also conducted on the primary neonatal rat ventricular cardiomyocytes (NRVCMs) to validate the results. Mice in the LPS group, as compared to the control group, had lower left ventricular ejection fraction, left ventricular fractional shortening, left ventricular systolic pressure, and ±dp/dt, but a higher left ventricular end-diastolic pressure, while the serum expression of IL-1β, IL-6, TNF-α, and cTn-T was up-regulated evidently with the increased apoptotic index of myocardial tissues. However, miR-141 and Bcl-2/Bax were down-regulated with elevated DAPK1 and cleaved caspase-3. The above changes were ameliorated in mice from the LPS + pre-miR-141 group relative to the LPS group, while those in the LPS + anti-miR-141 group were further deteriorated. In vitro experiment showed that miR-141 overexpression could reduce the apoptosis of LPS-induced NRVCMs and the levels of inflammatory cytokines with the increased cell viability. MiR-141 could decrease inflammatory response and reduce myocardial cell apoptosis by targeting DAPK1, thereby playing the promising protective role in SIC.

Traditional Chinese Medication Qiliqiangxin Attenuates Diabetic Cardiomyopathy via Activating PPARγ.

Background: Diabetic cardiomyopathy is the primary complication associated with diabetes mellitus and also is a major cause of death and disability. Limited pharmacological therapies are available for diabetic cardiomyopathy. Qiliqiangxin (QLQX), a Chinese medication, has been proven to be beneficial for heart failure patients. However, the role and the underlying protective mechanisms of QLQX in diabetic cardiomyopathy remain largely unexplored.Methods: Primary neonatal rat cardiomyocytes (NRCMs) were treated with glucose (HG, 40 mM) to establish the hyperglycemia-induced apoptosis model in vitro. Streptozotocin (STZ, 50 mg/kg/day for 5 consecutive days) was intraperitoneally injected into mice to establish the diabetic cardiomyopathy model in vivo. Various analyses including qRT-PCR, western blot, immunofluorescence [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining] histology (hematoxylin–eosin and Masson's trichrome staining), and cardiac function (echocardiography) were performed in these mice. QLQX (0.5 μg/ml in vitro and 0.5 g/kg/day in vivo) was used in this study.Results: QLQX attenuated hyperglycemia-induced cardiomyocyte apoptosis via activating peroxisome proliferation-activated receptor γ (PPARγ). In vivo, QLQX treatment protected mice against STZ-induced cardiac dysfunction and pathological remodeling.Conclusions: QLQX attenuates diabetic cardiomyopathy via activating PPARγ.

Conductive polypyrrole-encapsulated silk fibroin fibers for cardiac tissue engineering

Polypyrrole (PPy) has been utilized in smart scaffolds to improve the functionality of the engineered cardiac tissue. Compared to the commonly used aqueous coating, here, PPy was blended into silk fibroin (SF) solution to electrospin conductive PPy-encapsulated SF nanofibers. Combinations of various SF concentrations (5%, 7%, and 12%) and different PPy-to-SF ratios (15:85, 30:70, and 40:60) were compared. PPy reduced the fiber diameter (0.431 ± 0.060 μm), better-mimicking the myocardium fibrils. Conductive mats with 7% SF showed the closest mechanical properties (1.437 ± 0.044 MPa) to the native myocardium; meanwhile, a PPy-to-SF ratio of 15:85 exhibited sufficient electrical conductivity for cardiomyocytes (CMs). In vitro studies using three different types of CM demonstrated that the hybrid mats support CM contraction. Primary neonatal rat CMs on the mat with a PPy-to-SF ratio of 15:85 were elongated and orientated anisotropically with locally organized sarcomeric striations. By contrast, human-induced pluripotent stem cell derived-CMs on the mat with a PPy-to-SF ratio of 30:70 exhibited the strongest contractions. Contraction synchrony was further improved by external stimulation. Taken together, these findings indicated the great potential of the PPy-encapsulated SF electrospun mat for cardiac tissue engineering.