Neonatal Mouse Cardiomyocytes Research Articles

Myofibril Assembly in Cultured Mouse Neonatal Cardiomyocytes.

The formation of myofibrils was analyzed in neonatal mouse cardiomyocytes grown in culture and stained with fluorescent antibodies directed against myofibrillar proteins. The cardiomyocyte cultures also were exposed to siRNA probes to test the role of nonmuscle myosin IIB expression in the formation of myofibrils. In culture, new myofibrils formed in the spreading cell margins surrounding contractile myofibrils previously assembled in utero. Observations indicated that assembly of mature myofibrils occurred in three-stages, as previously reported in cultured mouse skeletal muscle. Premyofibrils, characterized by minisarcomeres with nonmuscle myosin IIB and muscle-specific alpha-actinin bound to actin filaments, formed in the first stage; followed by nascent myofibrils, the second stage when muscle myosin II and titin were first detected. In the mature myofibril stage muscle myosin II filaments aligned in periodic A-Bands; late assembling proteins, including myomesin and telethonin, were integrated in the sarcomeres, and nonmuscle IIB was absent from the sarcomeres. Treatment of the cultured neonatal cardiomyocytes with gene-specific siRNAs for nonmuscle myosin IIB, led to a marked decrease in the formation of premyofibrils, and subsequently of mature myofibrils. Anat Rec, 301:2067-2079, 2018. © 2018 Wiley Periodicals, Inc.

Calpain-2 protects against heat stress-induced cardiomyocyte apoptosis and heart dysfunction by blocking p38 mitogen-activated protein kinase activation.

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain-2 and its downstream signal pathway in heat stress-induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat-shock response, as indicated by upregulated heat-shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase-3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain-2 expression and increased phosphorylation of p38, extraceIIuIar signaI-reguIated protein kinase (ERK1/2) and c-Jun N-terminaI kinase (JNK). Calpain-2 overexpression abrogated heat stress-induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress-induced apoptosis in MNCs. In cardiac-specific calpain-2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferasedUTP nick end labelling assays, respectively. M-mode echocardiography also demonstrated that calpain-2 overexpression significantly improved heatstroke-induced decreases in ventricular end-diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain-2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction.

Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice.

BackgroundCardiac remodeling is a pathophysiological process that involves various changes in heart, including cardiac hypertrophy and fibrosis. Cardiac remodeling following pathological stimuli is common trigger leading to cardiac maladaptation and onset of heart failure, and their pathogenesis remains unclear.MethodsHeart specimens of tetralogy of Fallot (TOF) patients, myocardial infarction (MI) and transverse aortic constriction (TAC) mouse models were collected to determine changes of microtubule associated protein 4 (MAP4) phosphorylation. MAP4 (S667A, S737E and S760E) knock in (MAP4 KI) mouse and cultured neonatal mouse cardiomyocytes or fibroblasts were used to investigate changes of cardiac phenotypes and possible mechanisms with a variety of approaches, including functional, histocytological and pathological observations.FindingsElevated cardiac phosphorylation of MAP4 (S737 and S760) was observed in TOF patients, MI and TAC mouse models. In MAP4 KI mice, age-dependent cardiac phenotypes, including cardiac hypertrophy, fibrosis, diastolic and systolic dysfunction were observed. In addition, increased cardiomyocyte apoptosis together with microtubule disassembly and mitochondrial translocation of phosphorylated MAP4 was detected prior to the onset of cardiac remodeling, and p38/MAPK was demonstrated to be the possible signaling pathway that mediated MAP4 (S737 and S760) phosphorylation.InterpretationOur data reveal for the first time that MAP4 drives pathological cardiac remodeling through its phosphorylation. These findings bear the therapeutic potential to ameliorate pathological cardiac remodeling by attenuating MAP4 phosphorylation.FundThis work was supported by the Key Program of National Natural Science Foundation of China (No.81430042) and National Natural Science Foundation of China (No.81671913).

Calcium‑sensing receptors in human peripheral Tlymphocytes and AMI: Cause and effect.

Acute myocardial infarction (AMI) is a disease associated with inflammation. Tlymphocytes are involved by secreting cytokines and inflammatory factors. In our previous study, it was found that the Tlymphocytes exhibited certain functional changes, the onset of which was induced by modulating calcium‑sensing receptor (CaSR) in AMI. In the present study, western blotting was used to verified the expression of Tlymphocyte CaSR and pathway proteins, including phosphorylated extracellular signal‑regulated kinase (P‑ERK)1/2 and phosphorylated c‑Jun N‑terminal kinase (P‑JNK), and used cytometric bead array to detect the secretion of interleukin (IL)‑4, IL‑6, IL‑10 and tumor necrosis factor (TNF)‑α in AMI onset, the results demonstrated that they were all increased. In addition, the expression of Tlymphocyte pathway proteins, including P‑ERK1/2 and P‑JNK, and the secretion of IL‑4, IL‑6, IL‑10 and TNF‑α decreased after Tlymphocytes being transfected by CaSR small interfering RNA. By contrast, the neonatal mouse cardiomyocytes under hypoxia and hypoxia/re‑oxygenation exhibited ultrastructural damage, increased apoptosis, increased production of lactate dehydrogenase (LDH) and malondialdehyde, and reduced superoxide dismutase; these indicators changed extensively when cardiomyocytes were co‑cultured with Tlymphocytes. However, the effects were reversed when the cardiomyocytes were co‑cultured with CaSR‑silenced Tlymphocytes. These results indicated that CaSR may modulate Tlymphocytes to release cytokines through mitogen‑activated protein kinase pathways and affect cardiomyocyte injury. The relationship between AMI and Tlymphocyte CaSR is reciprocal.

Treatment with placental growth factor attenuates myocardial ischemia/reperfusion injury.

Studies have established that oxidative stress plays an important role in the pathology of myocardial ischemia/reperfusion injury (MIRI). Vascular endothelial growth factor receptor 1 (VEGFR1) activation was reported to reduce oxidative stress and apoptosis. In the present study, we tested the hypothesis that the activation of VEGFR1 by placental growth factor (PlGF) could reduce MIRI by regulating oxidative stress. Mouse hearts and neonatal mouse cardiomyocytes were subjected to ischemia/reperfusion (I/R) and oxygen glucose deprivation (OGD), respectively. PlGF pretreatment markedly ameliorated I/R injury, as demonstrated by reduced infarct size and improved cardiac function. The protection was associated with a reduction of cardiomyocyte apoptosis. Similarly, our in vitro study showed that PlGF treatment improved cell viability and reduced cardiomyocyte apoptosis. Also, activation of VEGFR1 by PlGF suppressed intracellular and mitochondrial reactive oxygen species (ROS) generation. However, VEGFR1 neutralizing monoclonal antibody, which preventing PlGF binding, totally blocked this protective effect. In conclusion, activation of VEGFR1 could protect heart from I/R injury by suppression of oxidative stress and apoptosis.

Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of miR-125b.

Autophagy can preserve cell viability under conditions of mild ischemic stress by degrading damaged organelles for ATP production, but under conditions of severe ischemia, it can promote cell death and worsen cardiac performance. Mesenchymal stem cells (MSCs) are cardioprotective when tested in animal models of myocardial infarction, but whether these benefits occur through the regulation of autophagy is unknown. To determine whether transplanted MSCs reduce the rate of autophagic degradation (autophagic flux) in infarcted hearts and if so, to characterize the mechanisms involved. Treatment with transplanted MSCs improved cardiac function and infarct size while reducing apoptosis and measures of autophagic flux (bafilomycin A1-induced LC3-II [microtubule-associated protein 1 light chain 3] accumulation and autophagosome/autolysosome prevalence) in infarcted mouse hearts. In hypoxia and serum deprivation-cultured neonatal mouse cardiomyocytes, autophagic flux and cell death, as well as p53-Bnip3 (B-cell lymphoma 2-interacting protein 3) signaling, declined when the cells were cultured with MSCs or MSC-secreted exosomes (MSC-exo), but the changes associated with MSC-exo were largely abolished by pretreatment with the exosomal inhibitor GW4869. Furthermore, a mimic of the exosomal oligonucleotide miR-125b reduced, whereas an anti-miR-125b oligonucleotide increased, autophagic flux and cell death, via modulating p53-Bnip3 signaling in hypoxia and serum deprivation-cultured neonatal mouse cardiomyocytes. In the in vivo mouse myocardial infarction model, MSC-exo, but not the exosomes obtained from MSCs pretreated with the anti-miR-125b oligonucleotide (MSC-exoanti-miR-125b), recapitulated the same results as the in vitro experiments. Moreover, measurements of infarct size and cardiac function were significantly better in groups that were treated with MSC-exo than the MSC-exoanti-miR-125b group. The beneficial effects offered by MSC transplantation after myocardial infarction are at least partially because of improved autophagic flux through excreted exosome containing mainly miR-125b-5p.

MicroRNA-19a/b-3p protect the heart from hypertension-induced pathological cardiac hypertrophy through PDE5A.

Aim:PDE5A is a leading factor contributing to cGMP signaling and cardiac hypertrophy. However, microRNA-mediated posttranscriptional regulation of PDE5A has not been reported. The aim of this study is to screen the microRNAs that are able to regulate PDE5A and explore the function of the microRNAs in cardiac hypertrophy and remodeling.Methods and Results:Although miR-19a/b-3p (microRNA-19a-3p and microRNA-19b-3p) have been reported to be differentially expressed during cardiac hypertrophy, the direct targets and the functions of this microRNA family for regulation of cardiac hypertrophy have not yet been investigated. The present study identified some direct targets and the underlying functions of miR-19a/b-3p by using bioinformatics tools and gene manipulations within mouse neonatal cardiomyocytes. Transfection of miR-19a/b-3p down-regulated endogenous expressions of PDE5A at both mRNA and protein levels with real-time PCR and western blot. Luciferase reporter assays showed that PDE5A was a direct target of miR-19a/b-3p. In mouse models of cardiac hypertrophy, we found that miR-19a/b-3p was expressed in cardiomyocytes and that its expression was reduced in pressure overload-induced hypertrophic hearts. miR-19a/b-3p transgenic mice prevented the progress of cardiac hypertrophy and cardiac remodeling in response to angiotensin II infusion with echocardiographic assessment and pressure–volume relation analysis.Conclusion:Our study elucidates that PDE5A is a novel direct target of miR-19a/b-3p, and demonstrates that antihypertrophic roles of the miR-19a/b-3p family in Ang II-induced hypertrophy and cardiac remodeling, suggests that endogenous miR-19a/b-3p might have clinical potential to suppress cardiac hypertrophy and heart failure.

Ablation of toll-like receptor 4 attenuates aging-induced myocardial remodeling and contractile dysfunction through NCoRI-HDAC1-mediated regulation of autophagy

Aging is usually accompanied with overt structural and functional changes as well as suppressed autophagy in the heart although the precise regulatory mechanisms are somewhat unknown. Here we evaluated the role of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in cardiac aging and the underlying mechanism with a focus on autophagy. Cardiac geometry and function were monitored in young or old wild-type (WT) and TLR4 knockout (TLR4−/−) mice using echocardiography, IonOptix® edge-detection and fura-2 techniques. Levels of autophagy and mitophagy, nuclear receptor corepressor 1 (NCoR1) and histone deacetylase I (HDAC1) were examined using western blot. Transmission electronic microscopy (TEM) was employed to monitor myocardial ultrastructure. Our results revealed that TLR4 ablation alleviated advanced aging (24 months)-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte cross-sectional area), contractile function and intracellular Ca2+ handling as well as autophagy and mitophagy [Beclin-1, Atg5, LC3B, PTEN-induced putative kinase 1 (PINK1), Parkin and p62]. Aging downregulated levels of NCoR1 and HDAC1 as well as their interaction, the effects were significantly attenuated or negated by TLR4 ablation. Advanced aging disturbed myocardial ultrastructure as evidenced by loss of myofilament alignment and swollen mitochondria, which was obliterated by TLR4 ablation. Moreover, aging suppressed autophagy (GFP-LC3B puncta) in neonatal mouse cardiomyocytes, the effect of which was negated by the TLR4 inhibitor CLI-095. Inhibition of HDCA1 using apicidin cancelled off CLI095-induced beneficial response of GFP-LC3B puncta against aging. Our data collectively indicate a role for TLR4-mediated autophagy in cardiac remodeling and contractile dysfunction in aging through a HDAC1-NCoR1-dependent mechanism.

(‑)‑Epicatechin protects against myocardial ischemia‑induced cardiac injury via activation of the PTEN/PI3K/AKT pathway.

Flavonol (−)-epicatechin (EPI) is primarily contained in green tea (Camellia sinensis) and cocoa beans (Theobroma cacao), and has been demonstrated to be beneficial for the health of the cardiovascular system. However, the effect and the underlying mechanism of EPI on myocardial ischemia induced cardiac injury has not yet been determined. Therefore, the present study aimed to detect whether EPI inhibited myocardial ischemia injury. An in vivo mouse myocardial ischemia model was induced by the ligation of left descending coronary artery for 7 days. EPI (1 mg/kg/day) was administrated 10 days prior to myocardial ischemia operation. The in vitro mouse myocardial ischemia model was induced by cultivating neonatal mouse cardiomyocytes under anoxia condition for 12 h. Cardiomyocytes were treated with EPI (5 µM) for 1 h and then incubated under anoxia conditions. Mouse hearts and cultured cardiomyocytes were used for hematoxylin-eosin, masson, ultrasonography, terminal dUTP nick end-labeling, immunofluorescence, western blotting and MTT assays. Results revealed that myocardial ischemia-induced mouse cardiac injury was significantly inhibited by EPI, as evidenced by decreased myocardial apoptosis, cardiac fibrosis and myocardial hypertrophy and improved cardiac function. In addition, it was confirmed that EPI serves a protective effect against myocardial ischemia via the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which was reversed by the PI3K inhibitor, LY294002. The protective role of EPI in myocardial apoptosis was further confirmed on mouse cardiomyocytes following anoxia treatment in vitro. In conclusion, the data suggested that EPI protects against myocardial ischemia induced cardiac injury through the PTEN/PI3K/AKT signaling pathway in vivo and in vitro, which may provide clinical therapeutic approaches and targets for cardiac ischemia injury.

High-density lipoprotein protects cardiomyocytes against necrosis induced by oxygen and glucose deprivation through SR-B1, PI3K, and AKT1 and 2.

The cardioprotective lipoprotein HDL (high-density lipoprotein) prevents myocardial infarction and cardiomyocyte death due to ischemia/reperfusion injury. The scavenger receptor class B, type 1 (SR-B1) is a high-affinity HDL receptor and has been shown to mediate HDL-dependent lipid transport as well as signaling in a variety of different cell types. The contribution of SR-B1 in cardiomyocytes to the protective effects of HDL on cardiomyocyte survival following ischemia has not yet been studied. Here, we use a model of simulated ischemia (oxygen and glucose deprivation, OGD) to assess the mechanistic involvement of SR-B1, PI3K (phosphatidylinositol-3-kinase), and AKT in HDL-mediated protection of cardiomyocytes from cell death. Neonatal mouse cardiomyocytes and immortalized human ventricular cardiomyocytes, subjected to OGD for 4 h, underwent substantial cell death due to necrosis but not necroptosis or apoptosis. Pretreatment of cells with HDL, but not low-density lipoprotein, protected them against OGD-induced necrosis. HDL-mediated protection was lost in cardiomyocytes from SR-B1−/− mice or when SR-B1 was knocked down in human immortalized ventricular cardiomyocytes. HDL treatment induced the phosphorylation of AKT in cardiomyocytes in an SR-B1-dependent manner. Finally, chemical inhibition of PI3K or AKT or silencing of either AKT1 or AKT2 gene expression abolished HDL-mediated protection against OGD-induced necrosis of cardiomyocytes. These results are the first to identify a role of SR-B1 in mediating the protective effects of HDL against necrosis in cardiomyocytes, and to identify AKT activation downstream of SR-B1 in cardiomyocytes.

Deletion of protein kinase B2 preserves cardiac function by blocking interleukin-6-mediated injury and restores blood pressure during angiotensin II/high-salt-diet-induced hypertension.

Protein kinase B2 (AKT2) is implicated in cardiomyocyte survival during various stress conditions. However, the role of AKT2 in heart function, cardiac hypertrophy and blood pressure (BP) control during hypertension is not fully understood. Therefore, we sought to determine whether the deletion of AKT2 protects cardiac function during angiotensin II/high-salt-diet (AngII/HSD) treatment and find out the signaling pathway. Male C57BL/6J (wild type), AKT2 knockout and interleukin (IL)-6 knockout mice were fed a 4% NaCl diet for 5 weeks. In the last week, mice were split in two groups and infused subcutaneously with either vehicle or AngII (1.5 μg/h per mouse) for 1 week. Then, BP and cardiac function were assessed. Immunohistology of IL-6 and monocyte chemoattractant protein 1 was performed to detect inflammation in the heart. Masson's trichrome staining was performed to evaluate cardiac fibrosis. Heart tissue homogenates and neonatal mice cardiomyocytes were collected to analyze oxidative stress. Compared with wild-type mice, AKT2 knockout mice maintained BP and showed better left ventricle ejection fraction, lower level of fibrosis, reduced oxidative stress, reduced IL-6 expression and less macrophage infiltration, when treated with AngII/HSD. IL-6 knockout mice treated with AngII/HSD also showed alleviated left ventricular function, fibrosis, oxidative stress and macrophage infiltration compared with wild type. AKT2 deficiency prevents the development of AngII/HSD-induced hypertension, cardiac dysfunction and myocardial injury including oxidative stress, fibrosis and inflammation by suppressing IL-6 expression. These data reveal an important role of the AKT2-IL-6 pathway in mediating AngII/HSD-induced hypertension and cardiomyopathy.

Interleukin 13 Promotes Cardiomyocyte Proliferation and Heart Regeneration In Vivo

AimsPreliminary data shows that the cytokine interleukin 13 (IL13) can promote neonatal mouse cardiomyocyte (CM) proliferation in vitro. From this, we hypothesize that IL13 and its downstream signaling pathway(s) play a role in stimulating mammalian heart cell cycle activity in vivo. We aim to test the role of endogenous, and exogenously administered, IL13 in promoting cardiomyocyte cell cycle activity and heart regeneration in vivo.Methods and ResultsTo understand if IL13 could promote CM proliferation in vivo, we collected whole hearts from C57/BL6J and IL13−/− neonatal mice. Heart halves were saved for histology and immunohistochemistry (IHC) and ventricle pieces for RNA extraction and protein lysates. To measure proliferation, we sectioned neonatal hearts for IHC and stained them with Proliferating Cell Nuclear Antigen (PCNA) or Phosphorylated Histone H3 (pH3). To ensure the proliferating cells were CM specific, we dual‐stained PCNA with Myocyte Enhancer Factor 2C (Mef2C) and pH3 with Cardiac Troponin T2 (cTnnt). We found CMs in IL13−/− mice show a decrease in PCNA when compared to BL6 wildtype CMs, indicative of decreased proliferation in the absence of IL13. When analyzed by qRT‐PCR, our IL13−/− mice also showed an increase in hypertrophic markers, specifically Brain Natriuretic peptide (nppb). Nppb is primarily expressed by the heart ventricles upon myocardial stretch and is shown to be upregulated during heart failure. Additionally, we found that by administering recombinant IL13 (rIL13) to the IL13−/− mice, we were able to rescue some of these phenotypes, namely increasing PCNA‐ and pH3‐positive CMs and decreasing nppb expression. To elucidate the downstream signaling pathways of IL13, we treated cultured neonatal rat CMs with rIL13 or no treatment. We preformed western blot and RNAsequencing on these CMs treated with rIL13 and both of these methods indicated a strong initial activation of Erk1/2 and Akt signaling pathways.ConclusionsOur data suggests that endogenous and exogenously administered IL13 can promote CM cell cycle activity. IL13−/− mice show decreased levels of CM proliferation, which was in part rescued when administered rIL13. We also found that rIL13 treatment in CMs activates Erk1/2 and Akt pathways, which have been shown to be pro‐proliferative and pro‐survival in CMs, respectively. In the future, IL13 and its corresponding signaling pathways could potentially be targeted to promote pro‐regenerative therapies in cardiac tissue.Support or Funding InformationAmerican Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome

The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias and sudden death. In the present day, only 30% of BrS cases have known genetic causes. Using whole-exome sequencing in a large pedigree with affected members, we identified a rare variant (p.R211H) in RRAD, the gene encoding Rad GTPase. The aim of this work was to elucidate the mechanisms by which the RRAD p.R211H variant leads to BrS. The study was performed in two cell models, i.e., cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) reprogrammed from the proband carrying the familial mutation and his healthy brother (non-carrier) and neonatal mouse cardiomyocytes (NMC) infected with adenoviruses encoding wildtype human RAD (ad_WT-RAD) or GFP (ad_GFP), and knock-in mouse mo del carrying the equivalent p.R210H Rad mutation (KI mice). iPSC-CMs from the proband displayed a severe decrease of INa and a moderate decrease of ICaL. Overexpression of WT-RAD in NMC triggered similar impact on both currents. The iPSC-CMs carrying the variant exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations. Combined with the electrical phenotype, these cells showed cortical distribution of actin, cell rounding and reduced focal adhesion count. In mice, Rad was predominantly expressed in the right ventricle outflow tract compared to the other cardiac compartments. This pattern of expression was not altered by the variant. KI mice displayed ventricular conduction disorders under ajmaline challenge and histological anomalies, including fibrosis development, in the right ventricular wall. Mutation in the RAD GTPase recapitulate the typical electrophysiological signature of Brugada syndrome coupled with cytoskeleton disturbances in cardiomyocytes derived from the patient. KI mice showed both cardiac electrical and structural disturbances.

Fibroblast growth factor-21 prevents diabetic cardiomyopathy via AMPK-mediated antioxidation and lipid-lowering effects in the heart

Our previous studies showed that both exogenous and endogenous FGF21 inhibited cardiac apoptosis at the early stage of type 1 diabetes. Whether FGF21 induces preventive effect on type 2 diabetes-induced cardiomyopathy was investigated in the present study. High-fat-diet/streptozotocin-induced type 2 diabetes was established in both wild-type (WT) and FGF21-knockout (FGF21-KO) mice followed by treating with FGF21 for 4 months. Diabetic cardiomyopathy (DCM) was diagnosed by significant cardiac dysfunction, remodeling, and cardiac lipid accumulation associated with increased apoptosis, inflammation, and oxidative stress, which was aggravated in FGF21-KO mice. However, the cardiac damage above was prevented by administration of FGF21. Further studies demonstrated that the metabolic regulating effect of FGF21 is not enough, contributing to FGF21-induced significant cardiac protection under diabetic conditions. Therefore, other protective mechanisms must exist. The in vivo cardiac damage was mimicked in primary neonatal or adult mouse cardiomyocytes treated with HG/Pal, which was inhibited by FGF21 treatment. Knockdown of AMPKα1/2, AKT2, or NRF2 with their siRNAs revealed that FGF21 protected cardiomyocytes from HG/Pal partially via upregulating AMPK–AKT2–NRF2-mediated antioxidative pathway. Additionally, knockdown of AMPK suppressed fatty acid β-oxidation via inhibition of ACC–CPT-1 pathway. And, inhibition of fatty acid β-oxidation partially blocked FGF21-induced protection in cardiomyocytes. Further, in vitro and in vivo studies indicated that FGF21-induced cardiac protection against type 2 diabetes was mainly attributed to lipotoxicity rather than glucose toxicity. These results demonstrate that FGF21 functions physiologically and pharmacologically to prevent type 2 diabetic lipotoxicity-induced cardiomyopathy through activation of both AMPK–AKT2–NRF2-mediated antioxidative pathway and AMPK–ACC–CPT-1-mediated lipid-lowering effect in the heart.

High content analysis identifies unique morphological features of reprogrammed cardiomyocytes

Direct reprogramming of fibroblasts into cardiomyocytes is a promising approach for cardiac regeneration but still faces challenges in efficiently generating mature cardiomyocytes. Systematic optimization of reprogramming protocols requires scalable, objective methods to assess cellular phenotype beyond what is captured by transcriptional signatures alone. To address this question, we automatically segmented reprogrammed cardiomyocytes from immunofluorescence images and analyzed cell morphology. We also introduce a method to quantify sarcomere structure using Haralick texture features, called SarcOmere Texture Analysis (SOTA). We show that induced cardiac-like myocytes (iCLMs) are highly variable in expression of cardiomyocyte markers, producing subtypes that are not typically seen in vivo. Compared to neonatal mouse cardiomyocytes, iCLMs have more variable cell size and shape, have less organized sarcomere structure, and demonstrate reduced sarcomere length. Taken together, these results indicate that traditional methods of assessing cardiomyocyte reprogramming by quantifying induction of cardiomyocyte marker proteins may not be sufficient to predict functionality. The automated image analysis methods described in this study may enable more systematic approaches for improving reprogramming techniques above and beyond existing algorithms that rely heavily on transcriptome profiling.

Nuclear Localization Leucine-Rich-Repeat Protein 1 Deficiency Protects Against Cardiac Hypertrophy by Pressure Overload

Background/Aims: Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a cytoplasmic protein, involved in autoimmune diseases, mammalian reproduction, neuronal cell death, and stroke. However, the role of NLRP1 in cardiac hypertrophy remains unclear. We used in vivo and in vitro models to investigate the effects of NLRP1 on cardiac hypertrophy. Methods: We used NLRP1-deficient mice and cultured neonatal rat cardiomyocytes with gain and loss of NLRP1 function. Cardiac hypertrophy was estimated by echocardiographic and hemodynamic measurements, and by pathological and molecular analysis. Results: Eight weeks after aortic banding (AB), NLRP1 deficiency significantly inhibited aortic banding–induced cardiac hypertrophy, inflammation, and fibrosis. Activation of MAPK, NF-κB, and TGF-β/Smad pathways was reduced in NLRP1-knockout (KO) mice compared with that in wild-type (WT) mice. Consistent with these results, in vitro studies, performed using cultured neonatal mouse cardiomyocytes, confirmed that NLRP1 deficiency protects against cardiomyocyte hypertrophy induced by isoproterenol (PE); this protective activity was associated with the arrest of MAPK and NF-κB signaling. Conclusions: Our data illustrates that NLRP1 plays a crucial role in the development of cardiac hypertrophy via positive regulation of the MAPK, NF-κB, and TGF-β/Smad signaling pathways.

Downregulation of Long Non-Coding RNA Kcnq1ot1: An Important Mechanism of Arsenic Trioxide-Induced Long QT Syndrome

Background/Aims: Arsenic trioxide (ATO) is a known anti-acute promyelocytic leukemia (APL) reagent, whose clinical applications are limited by its serious cardiac toxicity and fatal adverse effects, such as sudden cardiac death resulting from long QT syndrome (LQTS). The mechanisms of cardiac arrhythmia due to ATO exposure still need to be elucidated. Long non-coding RNAs (lncRNAs) are emerging as major regulators of various pathophysiological processes. This study aimed to explore the involvement of lncRNAs in ATO-induced LQTS in vivo and in vitro. Methods: For in vivo experiments, mice were administered ATO through the tail vein. For in vitro experiments, ATO was added to the culture medium of primary cultured neonatal mouse cardiomyocytes. To evaluate the effect of lncRNA Kcnq1ot1, siRNA and lentivirus-shRNA were synthesized to knockdown lncRNA Kcnq1ot1. Results: After ATO treatment, the Kcnq1ot1 and Kcnq1 expression levels were down regulated. lncRNA Kcnq1ot1 knockdown prolonged the action potential duration (APD) in vitro and exerted LQTS in vivo. Correspondingly, Kcnq1 expression was decreased after silencing lncRNA Kcnq1ot1. However, the knockdown of Kcnq1 exerted no effect on lncRNA Kcnq1ot1 expression. Conclusions: To our knowledge, this report is the first to demonstrate that lncRNA Kcnq1ot1 downregulation is responsible for QT interval prolongation induced by ATO at least partially by repressing Kcnq1 expression. lncRNA Kcnq1ot1 has important pathophysiological functions in the heart and could become a novel antiarrhythmic target.

Activation of M3 Muscarinic Acetylcholine Receptors Delayed Cardiac Aging by Inhibiting the Caspase-1/IL-1β Signaling Pathway

Background/Aims: Because the prevalence of age-related cardiac impairment increases as the human lifespan increases, it is important to combat the effects of aging. Recently, the cardiac M3 muscarinic acetylcholine receptor (M3-mAChR) has been demonstrated to play important roles in cardiac development and in the pathogenesis of cardiac diseases. However, the role of M3-mAChR in aging remains largely unknown. Therefore, the aim of this study was to investigate the involvement of M3-mAChR in the progression of cardiac aging. Methods: We established a cardiac aging model in mice through subcutaneous injection with D-galactose at a dose of 100 mg/kg/day for 6 weeks. D-galactose was also used to induce aging in primary cultured neonatal mouse cardiomyocytes. The myocardium from mice was stained with hematoxylin and eosin for histological analysis. The protein expression levels of p53 and p21 were determined using western blotting. The mRNA and protein expression levels of M3-mAChR, caspase-1, and interleukin (IL)-1β were determined using real-time PCR, immunohistochemical staining, and western blotting. Results: The expression of M3-mAChR was down-regulated in the myocardium from aged mice and D-galactose-treated mice, while the expression levels of caspase-1 and its downstream molecule IL-1β were significantly increased. The M3-mAChR agonist choline reduced the increase in caspase-1 in cardiomyocytes induced by D-galactose, which was reversed by the M3-mAChR antagonist 4-DAMP. Moreover, 4-DAMP promoted D-galactose-induced cardiomyocyte aging, which was attenuated by a caspase-1 inhibitor. Conclusion: Activation of M3-mAChR delayed cardiac aging by inhibiting the caspase-1/IL-1β signaling pathway.

Abstract 21308: Mesenchymal Stem Cells Driven by Exosomal Mir-125b Protect Myocardial Infarction via P53-Mediated Autophagy Signalling

Background and Objectives: Bone marrow derived mesenchymal stem cells (MSCs) transplantation confers protection against adverse cardiac remodeling mainly through paracrine effects, however, it remains largely unknow whether autophagy is involved in the protection offered by MSCs therapy. In the present study, we used the mice myocardial infarction(MI)model to investigate whether and how MSCs modulates autophagy activity to protect the injured myocardium. Methods and Results: Using a well established methodology of MSCs therapy for treating myocardial infarction, we reproduced the data that MSCs can reduce myocardial apoptosis to improve cardiac function post-MI, which was closely associated with attenuated autophagy flux in cardiac tissue. To further investigate how MSCs modulate autophagic activity, we co-cultured MSCs with neonatal mice cardiomyocytes (NMCMs) to test whether NMCMs co-cultured with MSCs exhibited enhanced tolerance to hypoxia. We showed that NMCMs exposed to hypoxia had increased autophagic flux, co-culture with MSCs resulted in a decrease in autophagic flux which was associated with improved apoptosis. Interestingly, NMCMs exposed to hypoxia exhibited up-regulated levels of p53 and its downward target Bnip3 at both mRNA and protein levels. Importantly, NMCMs co-cultured with MSCs had decreased p53 and bnip3 expression levels, suggesting that MSCs protected hypoxic cardiomyocytes via modulating autophagic flux through suppressing p53/bnip3 pathways. Exosome has been considered as the major paracrine components of MSCs, therefore, we extracted the exosomes from MSCs and it displayed the similar effects with MSCs on the autophagy modulation. We found miR-125b was an important component of exosome, inhibition of miR-125b in exosome abolished the effect of autophagy modulation via p53/Bnip3 signal pathway. Conclusion: MSCs, can exert the paracrine effects via miR-125b containing exosomes to block p53/Bnip3 pathway and decrease the autophagic related cell death, thus conferring protection for the cardiomyocytes.

HDL protects against doxorubicin-induced cardiotoxicity in a scavenger receptor class B type 1-, PI3K-, and Akt-dependent manner.

Doxorubicin is a widely used chemotherapeutic with deleterious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes in vitro against doxorubicin-induced apoptosis. Scavenger receptor class B type 1 (SR-B1), a high-affinity HDL receptor, mediates cytoprotective signaling by HDL through Akt. Here, we assessed whether increased HDL levels protect against doxorubicin-induced cardiotoxicity in vivo and in cardiomyocytes in culture and explored the intracellular signaling mechanisms involved, particularly the role of SR-B1. Transgenic mice with increased HDL levels through overexpression of human apolipoprotein A1 (apoA1Tg/Tg) and wild-type mice (apoA1+/+) with normal HDL levels were treated repeatedly with doxorubicin. After treatment, apoA1+/+ mice displayed cardiac dysfunction, as evidenced by reduced left ventricular end-systolic pressure and +dP/d t, and histological analysis revealed cardiomyocyte atrophy and increased cardiomyocyte apoptosis after doxorubicin treatment. In contrast, apoA1Tg/Tg mice were protected against doxorubicin-induced cardiac dysfunction and cardiomyocyte atrophy and apoptosis. When SR-B1 was knocked out, however, overexpression of apoA1 did not protect against doxorubicin-induced cardiotoxicity. Using primary neonatal mouse cardiomyocytes and human immortalized ventricular cardiomyocytes in combination with genetic knockout, inhibitors, or siRNA-mediated knockdown, we demonstrated that SR-B1 is required for HDL-mediated protection of cardiomyocytes against doxorubicin-induced apoptosis in vitro via a pathway involving phosphatidylinositol 3-kinase and Akt1/2. Our findings provide proof of concept that raising apoA1 to supraphysiological levels can dramatically protect against doxorubicin-induced cardiotoxicity via a pathway that is mediated by SR-B1 and involves Akt1/2 activation in cardiomyocytes. NEW & NOTEWORTHY We have identified an important role for the scavenger receptor class B type 1 in facilitating high-density lipoprotein-mediated protection of cardiomyocytes against stress-induced apoptosis and shown that increasing plasma high-density lipoprotein protects against the deleterious side effects of the chemotherapeutic and cardiotoxic drug doxorubicin.