Primary Neonatal Cardiomyocytes Research Articles

THBru attenuates diabetic cardiomyopathy by inhibiting RAGE-dependent inflammation.

Diabetic cardiomyopathy (DCM) is a complication of diabetes mellitus characterized by heart failure and cardiac remodeling. Previous studies show that tetrahydroberberrubine (THBru) retrogrades cardiac aging by promoting PHB2-mediated mitochondrial autophagy and prevents peritoneal adhesion by suppressing inflammation. In this study we investigated whether THBru exerted protective effect against DCM in db/db mice and potential mechanisms. Eight-week-old male db/db mice were administered THBru (25, 50 mg·kg-1·d-1, i.g.) for 12 weeks. Cardiac function was assessed using echocardiography. We showed that THBru administration significantly improved both cardiac systolic and diastolic function, as well as attenuated cardiac remodeling in db/db mice. In primary neonatal mouse cardiomyocytes (NMCMs), THBru (20, 40 μM) dose-dependently ameliorated high glucose (HG)-induced cell damage, hypertrophy, inflammatory cytokines release, and reactive oxygen species (ROS) production. Using Autodock, surface plasmon resonance (SPR) and DARTS analyses, we revealed that THBru bound to the domain of the receptor for advanced glycosylation end products (RAGE), subsequently leading to inactivation of the PI3K/AKT/NF-κB pathway. Importantly, overexpression of RAGE in NMCMs reversed HG-induced inactivation of the PI3K/AKT/NF-κB pathway and subsequently counteracted the beneficial effects mediated by THBru. We conclude that THBru acts as an inhibitor of RAGE, leading to inactivation of the PI3K/AKT/NF-κB pathway. This action effectively alleviates the inflammatory responses and oxidative stress in cardiomyocytes, ultimately leading to ameliorated DCM.

Cardiomyocyte maturation alters molecular stress response capacities and determines cell survival upon mitochondrial dysfunction

Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart.

FOXO1 reduces STAT3 activation and causes impaired mitochondrial quality control in diabetic cardiomyopathy.

To investigate the role of FOXO1 in STAT3 activation and mitochondrial quality control in the diabetic heart. Type 1 diabetes mellitus (T1DM) was induced in rats by a single intraperitoneal injection of 60 mg · kg-1 streptozotocin (STZ), while type 2 diabetes mellitus (T2DM) was induced in rats with a high-fat diet through intraperitoneal injection of 35 mg · kg-1 STZ. Primary neonatal mouse cardiomyocytes and H9c2 cells were exposed to low glucose (5.5 mM) or high glucose (HG; 30 mM) with or without treatment with the FOXO1 inhibitor AS1842856 (1 μM) for 24 hours. In addition, the diabetic db/db mice (aged 8 weeks) and sex- and age-matched non-diabetic db/+ mice were treated with vehicle or AS1842856 by oral gavage for 15 days at a dose of 5 mg · kg-1 · d-1 . Rats with T1DM or T2DM had excessive cardiac FOXO1 activation, accompanied by decreased STAT3 activation. Immunofluorescence and immunoprecipitation analysis showed colocalization and association of FOXO1 and STAT3 under basal conditions in isolated cardiomyocytes. Selective inhibition of FOXO1 activation by AS1842856 or FOXO1 siRNA transfection improved STAT3 activation, mitophagy and mitochondrial fusion, and decreased mitochondrial fission in isolated cardiomyocytes exposed to HG. Transfection with STAT3 siRNA further reduced mitophagy, mitochondrial fusion and increased mitochondrial fission in HG-treated cardiomyocytes. AS1842856 alleviated cardiac dysfunction, pathological damage and improved STAT3 activation, mitophagy and mitochondrial dynamics in diabetic db/db mice. Additionally, AS1842856 improved mitochondrial function indicated by increased mitochondrial membrane potential and adenosine triphosphate production and decreased mitochondrial reactive oxygen species production in isolated cardiomyocytes exposed to HG. Excessive FOXO1 activation during diabetes reduces STAT3 activation, with subsequent impairment of mitochondrial quality, ultimately promoting the development of diabetic cardiomyopathy.

Mitophagy-dependent cardioprotection of resistance training on heart failure.

Resistance exercise is an indispensable mode of exercise rehabilitation for heart failure. Here we elucidate the cardiac effects of resistance training alone or combined with different aerobic trainings on heart failure and explore the critical regulation of mitophagy. The chronic heart failure model was constructed by transverse aortic constriction surgery, followed by 8 wk of resistance training (RT), moderate-intensity continuous training combined with resistance training (MRT), and high-intensity interval training combined with resistance training (HRT), and subsequently analyzed the changes of maximum load, cardiac structure and function, and myocardial mitophagic activity. The role and signaling of mitophagy in exercise protection of heart failure were investigated by knockdown of Hif1α and Parkin genes in primary neonatal cardiomyocytes. RT and especially MRT improved maximum load (P < 0.0001), myocardial morphology and fibrosis (P < 0.0001), reduced left ventricular diameter and enhanced left ventricular systolic function (P < 0.01), and enhanced myocardial mitophagic activity and HIF1α expression (P < 0.05) in heart failure mice. However, HRT had no obvious protective effect on ventricular diameter and function or mitophagy. The abilities of exercise stimulation to regulate reactive oxygen species, adenosine triphosphate, and brain natriuretic peptide were impaired after knockdown of Hif1α and Parkin genes inhibited mitophagy in failing cardiomyocytes (P < 0.05). Different exercise modalities provide discrepant cardiovascular effects on heart failure, and MRT exhibits optimal protection. The HIF1α-Parkin-mitophagy pathway is involved in the protection and regulation of exercise on heart failure.NEW & NOTEWORTHY Impaired myocardial mitophagy is implicated in the pathogenesis of heart failure. Resistance training alone or combined with different aerobic trainings provide discrepant cardiovascular effects on heart failure, and the cardioprotective function depends on HIF1α-Parkin-mitophagy pathway.

Abstract P3161: Engineering Mesenchymal Stem Cell-derived Extracellular Vesicles To Enhance Their Therapeutic Efficacy In Cardiovascular Disease

Background: Extracellular vesicles derived from cardiosphere-derived cells (CDC-EVs) have demonstrated the ability to regulate cardiomyocyte apoptosis and induce cardiomyocyte proliferation in pre-clinical models of acute MI. While CDC-EVs exhibit cardioprotective effects, they require invasive intramyocardial injections to achieve therapeutic efficacy, detracting from their translatability. Mesenchymal stem cells (MSC) also generate cardioprotective EVs (MSC-EVs), which have demonstrated therapeutic efficacy in models of cardiac disease following intravenous administration. However, MSC-EVs lack several of the cardioprotective miRs expressed by CDC-EVs, including miR146a, which has been shown to modulate cardiomyocyte apoptosis. The objective of our current study was to determine the effect of enriching MSC-EVs with cardioprotective CDC-EV miRNA on cardiomyocyte apoptosis. Methods: MSCs were transfected with miR-146a-5p mimics using Trans-IT TKO in serum free growth media. EVs were isolated using a combination of ultrafiltration and size exclusion chromatography (SEC) and characterized by size, morphology and expression of surface markers. RT-PCR was performed on EV samples to quantify miR-146a expression. Primary neonatal mouse cardiomyocytes were treated with MSC-EVs, miR-146a loaded MSC-EVs, CDC-EVs or matched volume vehicle control. Cardiomyocyte apoptosis was assessed by a TUNEL assay. Results: Treatment with miR-146a loaded MSC EVs reduced cardiomyocyte apoptosis compared with cardiomyocytes treated with unmodified MSC exosomes (14±1% vs. 31±3%) (mean ± SEM, p=0.001, one-way ANOVA). miR-146a loaded MSC-EVs and CDC-EVs demonstrated a similar level of reduction in cardiomyocyte apoptosis (14±1% vs. 15±1%). There was no difference in the level of apoptosis between cardiomyocytes treated with unmodified MSC exosomes and cardiomyocytes treated with vehicle control (31±3% vs. 27±1%) (mean ± SEM, p=0.366, one-way ANOVA). Conclusion: Engineering MSC-EVs to express miR-146a enhances their ability to reduce cardiomyocyte apoptosis in vitro . This highlights the potential of cargo engineering as a means to improve the therapeutic efficacy of EVs for cardiovascular disease.

Resveratrol prevents Ang II-induced cardiac hypertrophy by inhibition of NF-κB signaling

BackgroundPathological cardiac hypertrophy is a hallmark of various cardiovascular diseases (CVD) including chronic heart failure (HF) and an important target for the treatment of these diseases. Aberrant activation of Angiotensin II (Ang II)/AT1R signaling pathway is one of the main triggers of cardiac hypertrophy, which further gives rise to excessive inflammation that is mediated by the key transcription factor NF-κB. Resveratrol (REV) is a natural polyphenol with multiple anti-inflammatory and anti-oxidative effects, however the ability of REV in preventing Ang II-induced cardiac hypertrophy in combination with NF-κB signaling activation remains unclear. MethodsMurine models of cardiac hypertrophy was conducted via implantation of Ang II osmotic pumps. Primary neonatal rat cardiomyocyte and heart tissues were examined to determine the effect and underlying mechanism of REV in preventing Ang II-induced cardiac hypertrophy. ResultsAdministrations of REV significantly prevented Ang II-induced cardiac hypertrophy, as well as robustly attenuated Ang II-induced cardiac fibrosis, and cardiac dysfunction. Furthermore, REV not only directly prevented Ang II/AT1R signal transductions, but also prevented Ang II-induced expressions of pro-inflammatory cytokines and activation of NF-κB signaling pathway. ConclusionsOur study provides important new mechanistic insight into the cardioprotective effects of REV in preventing Ang II-induced cardiac hypertrophy via inhibiting adverse NF-κB signaling activation. Our findings further suggest the therapeutic potential of REV as a promising drug for the treatment of cardiac hypertrophy and heart failure.

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human iPSC-derived cardiomyocytes presented a renewed opportunity. Here, we conducted a comparative study, assessing cellular signaling through cardiac G protein-coupled receptors (GPCRs) in rat neonatal cardiomyocytes (RNCMs) and human induced pluripotent stem cell-derived cardiomyocytes. Genetically encoded biosensors were used to explore GPCR-mediated nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) activities in both cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human cardiomyocytes. In line with this, bulk RNA-seq revealed key differences in the expression patterns of GPCRs, G proteins and downstream effector expression levels. Our study demonstrates that human stem cell-derived models of the cardiomyocyte offer distinct advantages for understanding cellular signaling in the heart.

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress, mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway.

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1S616 , mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.

Trastuzumab potentiates doxorubicin-induced cardiotoxicity via activating the NLRP3 inflammasome in vivo and in vitro

Trastuzumab (Tra), the first humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2), is commonly used alongside doxorubicin (Dox) as a combination therapy in HER2-positive breast cancer. Unfortunately, this leads to a more severe cardiotoxicity than Dox alone. NLRP3 inflammasome is known to be involved in Dox-induced cardiotoxicity and multiple cardiovascular diseases. However, whether the NLRP3 inflammasome contributes to the synergistic cardiotoxicity of Tra has not been elucidated. In this study, primary neonatal rat cardiomyocyte (PNRC), H9c2 cells and mice were treated with Dox (15 mg/kg in mice or 1 μM in cardiomyocyte) or Tra (15.75 mg/kg in mice or 1 μM in cardiomyocyte), or Dox combined Tra as cardiotoxicity models to investigate this question. Our results demonstrated that Tra significantly potentiated Dox-induced cardiomyocyte apoptosis and cardiac dysfunction. These were accompanied by the increased expressions of NLRP3 inflammasome components (NLRP3, ASC and cleaved caspase-1), the secretion of IL-β and the pronounced production of ROS. Inhibiting the activation of NLRP3 inflammasome by NLRP3 silencing significantly reduced cell apoptosis and ROS production in Dox combined Tra-treated PNRC. Compared with the wild type mice, the systolic dysfunction, myocardial hypertrophy, cardiomyocyte apoptosis and oxidative stress induced by Dox combined Tra were alleviated in NLRP3 gene knockout mice. Our data revealed that the co-activation of NLRP3 inflammasome by Tra promoted the inflammation, oxidative stress and cardiomyocytes apoptosis in Dox combined Tra-induced cardiotoxicity model both in vivo and in vitro. Our results suggest that NLRP3 inhibition is a promising cardioprotective strategy in Dox/Tra combination therapy.

CRISPR/Cas9 Knock-Out in Primary Neonatal and Adult Cardiomyocytes Reveals Distinct cAMP Dynamics Regulation by Various PDE2A and PDE3A Isoforms.

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.

Ischemia/reperfusion-induced MiD51 upregulation recruits Drp1 to mitochondria and contributes to myocardial injury

The translocation of Drp1 from the cytosol to mitochondria leads to Drp1 activation and mitochondrial fission in myocardial ischemia/reperfusion (MI/R). However, the molecular mechanism underlying mitochondrial Drp1 translocation remains poorly understood. Mitochondrial Drp1 recruitment relies on 4 binding partners including MiD49, MiD51, Mff and Fis1. This study was to elucidate which one facilitate mitochondrial Drp1 translocation and its role in MI/R injury. MI/R was induced by ligating the left anterior descending coronary artery for 30 min and subsequent reperfusion for 3 h. Primary neonatal cardiomyocytes were subjected to hypoxia for 2 h and reoxygenation for 4 h. SiRNA or Adeno-associated virus (AAV) expressing shRNA was used to knock down the key binding partner in vitro or in vivo respectively. The expression of MiD51 rather than other binding partners (MiD49, Mff or Fis1) was increased after MI/R. MiD51 knockdown inhibited hypoxia/reoxygenation (H/R) or ischemia/reperfusion (I/R)-induced mitochondrial Drp1 translocation. SiRNA-induced knockdown of MiD51 suppressed mitochondrial oxidative stress, improved mitochondrial function and alleviate cellular injury in H/R cardiomyocytes. AAV-mediated knockdown of MiD51 reduced myocardial injury and improved cardiac function in the I/R hearts, while mitochondrial Drp1 translocation and cardiac function were not affected by MiD51 knockdown in the hearts without I/R. MiD51 is identified as the binding partner that promotes mitochondrial Drp1 translocation and contributes to MI/R injury. Inhibition of MiD51 may be a potential therapeutic target to alleviate MI/R injury.

BNP protects against diabetic cardiomyopathy by promoting Opa1-mediated mitochondrial fusion via activating the PKG-STAT3 pathway

Brain natriuretic peptide (BNP) belongs to the family of natriuretic peptides, which are responsible for a wide range of actions. Diabetic cardiomyopathy (DCM) is often associated with increased BNP levels. This present research intends to explore the role of BNP in the development of DCM and the underlying mechanisms. Diabetes was induced in mice using streptozotocin (STZ). Primary neonatal cardiomyocytes were treated with high glucose. It was found that the levels of plasma BNP started to increase at 8 weeks after diabetes, which preceded the development of DCM. Addition of exogenous BNP promoted Opa1-mediated mitochondrial fusion, inhibited mitochondrial oxidative stress, preserved mitochondrial respiratory capacity and prevented the development of DCM, while knockdown of endogenous BNP exacerbated mitochondrial dysfunction and accelerated DCM. Opa1 knockdown attenuated the aforementioned protective action of BNP both in vivo and in vitro. BNP-induced mitochondrial fusion requires the activation of STAT3, which facilitated Opa1 transcription by binding to its promoter regions. PKG, a crucial signaling biomolecule in the BNP signaling pathway, interacted with STAT3 and induced its activation. Knockdown of NPRA (the receptor of BNP) or PKG blunted the promoting effect of BNP on STAT3 phosphorylation and Opa1-mediated mitochondrial fusion. The results of this study demonstrate for the first time that there is a rise in BNP during the early stages of DCM as a compensatory protection mechanism. BNP is a novel mitochondrial fusion activator in protecting against hyperglycemia-induced mitochondrial oxidative injury and DCM through the activation of NPRA-PKG-STAT3-Opa1 signaling pathway.

CRISPR/Cas9 knockout in primary neonatal and adult cardiomyocytes reveals distinct cAMP dynamics regulation by various PDE2A and PDE3A isoforms

CRISPR/Cas9 knockout in primary neonatal and adult cardiomyocytes reveals distinct cAMP dynamics regulation by various PDE2A and PDE3A isoforms

CRISPR/Cas9 knockout in primary neonatal and adult cardiomyocytes reveals distinct cAMP dynamics regulation by various PDE2A and PDE3A isoforms

CRISPR/Cas9 knockout in primary neonatal and adult cardiomyocytes reveals distinct cAMP dynamics regulation by various PDE2A and PDE3A isoforms

Exogenous spermidine alleviates diabetic cardiomyopathy via suppressing reactive oxygen species, endoplasmic reticulum stress, and Pannexin-1-mediated ferroptosis.

Diabetic cardiomyopathy (DCM) is a serious complication and death cause of diabetes mellitus (DM). Recent cardiology studies suggest that spermidine (SPD) has cardioprotective effects. Here, we verified the hypothesis of SPD's protective effects on DCM. Therefore, db/db mice and primary neonatal mouse cardiomyocytes were used to observe the effects of SPD. Immunoblotting showed that ornithine decarboxylase (ODC) and SPD/spermine N1-acetyltransferase (SSAT) were downregulated and upregulated in the myocardium of db/db mice, respectively. We found that diabetic mice showed cardiac dysfunction in 12 weeks. Conversely, exogenous SPD could improve cardiac functions and reduce the deposition of collagens, myocardial damage, reactive oxygen species (ROS) levels, and endoplasmic reticulum stress (ERS) in diabetic mouse hearts. Our results also demonstrated that cardiomyocytes displayed ferroptosis and then activated Pannexin-1 expression, which resulted in the increase of the extracellular adenosine triphosphate (ATP). Subsequently, increased ATP as a paracrine molecule combined to purinergic receptor P2X7 to activate ERK1/2 signaling pathway in cardiomyocytes and activated NCOA4-mediated ferroptinophagy to promote lipid peroxidation and ferroptosis. Interestingly, SPD could reverse these molecular processes. Our findings indicate an important new mechanism for DCM and suggest that SPD has potential applicability to protect against deterioration of cardiac function with DCM.

Dual Specificity Phosphatase 3 (DUSP3) Knockdown Alleviates Acute Myocardial Infarction Damage via Inhibiting Apoptosis and Inflammation.

Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response and is associated with ischemia/reperfusion (I/R). However, the precise function of DUSP3 in acute myocardial infarction (AMI) remains to be established. In this study, the AMI model in vivo was established in mice by permanent left anterior descending coronary artery (LAD) occlusion, and primary neonatal mouse cardiomyocytes were treated with hypoxia for 12 hours to mimic AMI in vitro. Sh-DUSP3 and AAV9-sh-DUSP3 were used to knock down the DUSP3 expression. LVEF%, LVFS%, SOD1, and HO-1 level, and TTC staining were used to test the cardiac function. Flow cytometric analysis, Western blot, and TUNEL staining were used to investigate the effect of DUSP3 knockdown on apoptosis. Moreover, we detect inflammatory factors expression and oxidative stress by ELISA. Besides, we investigate DUSP3 expression by RT-qPCR. Our findings determined the role of DUSP3 in the progression of AMI. And demonstrated that DUSP3 knockdown alleviated oxidative stress, inflammation, and apoptosis. In addition, our results indicated that DUSP3 knockdown could regulate the expression of p-NF-κB, ICAM1, and VCAM1. Our results demonstrated that the knockdown of DUSP3 could effectively alleviate AMI symptoms and be mediated through the NF-κB signaling pathway.

PS-B06-8: EXPLORING THE ROLE OF NECROPTOSIS IN HYPERTENSION

Objective: Altered immune cell activation plays a key role in promoting hypertension. A major question remains as to the immune system becomes activated during hypertension. A possibility is necroptosis, which a newly described form of cell death alerts the immune system to dying cells, causing inflammation. Using mice genetically unable to undergo necroptosis, we aimed to determine whether necroptosis contributes to inflammation and fibrosis or alters blood pressure during hypertension. To determine whether factors present during hypertension impact necroptosis, we examined whether Angiotensin II sensitizes human and murine cells to necroptosis. Design and Methods: Monocytes were sorted from peripheral blood mononuclear cells from 3 normotensive and 3 untreated hypertensive participants diagnosed with 24-h blood pressure (BP) monitoring. Necroptosis was induced in human monocytes using hTNFα (10ug/ml), SMAC mimetic (500 nM), zVAD (20uM), and in primary neonatal murine cardiomyocytes and fibroblasts from 3 litters of C57BL/6J mice using mTNFα (10ug/ml), SMAC mimetic (100 nM), zVAD (10uM), in the presence or absence of angiotensin II (Ang-II, 5 ug/ml). Viability of human cells was measured using propidium iodide by flow cytometry, and in murine cells using CellTitre-Glo Luminescent Cell Viability Assay. Hypertension was induced in male Mlkl-/- mice, which cannot undergo necroptosis, and wild-type (WT) littermate controls by implanting minipumps containing Ang-II (0.5 mg/kg/day for 28 days; n = 6/genotype). Blood pressure (BP) was measured weekly by tail-cuff. Results: Monocytes from hypertensive participants were not more susceptible to necroptosis than normotensive participants (% viability ± SD; 48% ± 0.11 vs 51% ± 0.04, P = 0.79). This was not altered by Ang-II treatment in normotensive (51% ± 0.04 vs 56% ± 0.11 with Ang-II, P = 0.98) or hypertensive participants (48% ± 0.11 vs 56% ± 0.18 with Ang-II, P = 0.92). Moreover, Ang-II did not alter the viability of primary murine cardiomyocytes in vitro (42% ± 23 vs 40.1 ± 17.8 with Ang-II, P = 0.79). Primary mouse cardiac fibroblasts were unable to undergo necroptosis. In vivo, there was no difference in BP of Ang-II-treated Mlkl-/- and WT control mice at 4 weeks (P = 0.87). Conclusions: Our findings showed that, compared to normotensive participants, monocytes from hypertensive participants were not more susceptible to necroptosis. Furthermore, Ang-II did not directly act on the necroptosis pathway in both mouse cardiac fibroblasts or human monocytes. Changes in BP were not observed between WT and MLKL knockout mice upon Ang-II treatment, suggesting necroptosis does not impact BP regulation. Further analyses are being performed to determine whether inflammation or tissue fibrosis are altered in the absence of necroptosis.

722 NATURAL ACTIVATORS OF AUTOPHAGY PROTECT THE HEART FROM DOX-INDUCED CARDIOMYOPATHY

Abstract Introduction Heart failure is a frequent death cause for oncological patients who received treatment with doxorubicin (DOX). DOX administration inhibits autophagy in cardiomyocytes and causes myocardial damage. Natural activators of autophagy (NAA) such as trehalose, a natural disaccharide, and spermidine, a putrescine-derived polyamine initially isolated from semen, were previously found to be promising candidates to recover the autophagic flux and treat cardiovascular diseases in mice. Hypothesis The administration of natural activators of autophagy protects the heart from DOX-induced myocardial injury. Methods C57BL/6J WT mice were fed ad libitum with trehalose or spermidine in drinking water and received 3 weekly injections of DOX, reaching a final cumulative dose of 15 mg/kg. Echocardiographic, histological and biochemical analyses were performed 6 weeks after the first administration of DOX. Also, in vitro experimentation was performed on neonatal rat primary cardiomyocytes. Results Mice treated with DOX had reduced systolic function (FS: 44±1.05% vs. 34.1±2.33%, n=6-8), but trehalose administration preserved left ventricular fractional shortening (FS: 34.1 [Office1]±2.33% vs. 45.1±0.75%, n=6-8). Autophagy-deficient Beclin1+/- mice had reduced FS and were not protected by trehalose administration (35.4±2.48% vs. 45.1±0.75%, n=7-8). Mice treated with DOX developed fibrosis (0.1±0.08% vs. 8.6±3.5%, n=5), but this effect was reduced in trehalose-fed mice that received DOX (8.6±3.5% vs. 2±0.68%, n=4-5). DOX administration increased the levels of damaged-mitochondria disposal (2.6±0.74 vs. 9.4±1.7 mitophagic bodies per TEM field, n=13) and trehalose administration further boosted this effect (9.4±1.7 vs. 24.1±1.45, n=13). The mRFP-eGFP-Lc3-dots assay was used for evaluating the autophagic flux in neonatal primary cardiomyocytes and a reduction of autophagy was observed in cells treated with DOX (51.9±4.57 vs. 29.9±3.52 red dots per cell, n=34-51), while trehalose recovered the autophagic flux (29.9±3.52 vs. 121.5±8.02 red dots per cell, n=33-34). Mitochondrial biogenesis reporter ‘MitoTimer’ mice that received DOX showed increased levels of biogenesis (0.8±0.38 vs. 3.3±0.9, n=5-6), while mice receiving DOX and trehalose did not (3.3±0.9 vs. 0.3±0.12, n=5-6). Similarly, spermidine preserved systolic function (FS: 29.4±2.03 vs. 39±3.22, n=5-6) and increased myocardial autophagy. Conclusion NAA-mediated mitophagy induction protects the heart from developing DOX-induced cardiomyopathy by disposing of damaged mitochondria. [Office1]Le virgole diventano punti

Role of Scl39a13/ZIP13 in cardiovascular homeostasis.

Zinc plays a critical role in many physiological processes, and disruption of zinc homeostasis induces various disorders, such as growth retardation, osteopenia, immune deficiency, and inflammation. However, how the imbalance in zinc homeostasis leads to heart disease is not yet fully understood. Cardiovascular diseases are a major cause of death worldwide, and the development of novel therapeutic targets to treat it is urgently needed. We report that a zinc transporter, ZIP13, regulates cardiovascular homeostasis. We found that the expression level of Zip13 mRNA was diminished in both primary neonatal cardiomyocytes and mouse heart tissues treated with the cardiotoxic agent doxycycline. Primary neonatal cardiomyocytes from Zip13 gene-knockout (KO) mice exhibited abnormal irregular arrhythmic beating. RNA-seq analysis identified 606 differentially expressed genes in Zip13-KO mouse-derived primary neonatal cardiomyocytes and Gene ontology (GO) analysis revealed that both inflammation- and cell adhesion-related genes were significantly enriched. In addition, telemetry echocardiography analysis suggested that arrhythmias were likely to occur in Zip13-KO mice, in which elevated levels of the cardiac fibrosis marker Col1a1, vascular inflammation-related gene eNOS, and Golgi-related molecule GM130 were observed. These results indicate the physiological importance of ZIP13—it maintains cardiovascular homeostasis by resolving inflammation and stress response. Our findings suggest that optimizing ZIP13 expression and/or function may improve cardiovascular disease management.

Tetrahydroberberrubine retards heart aging in mice by promoting PHB2-mediated mitophagy.

Heart aging is characterized by left ventricular hypertrophy and diastolic dysfunction, which in turn induces a variety of cardiovascular diseases. There is still no therapeutic drug to ameliorate cardiac abnormities in heart aging. In this study we investigated the protective effects of berberine (BBR) and its derivative tetrahydroberberrubine (THBru) against heart aging process. Heart aging was induced in mice by injection of D-galactose (D-gal, 120 mg · kg-1 · d-1, sc.) for 12 weeks. Meanwhile the mice were orally treated with berberine (50 mg · kg-1 · d-1) or THBru (25, 50 mg · kg-1 · d-1) for 12 weeks. We showed that BBR and THBru treatment significantly mitigated diastolic dysfunction and cardiac remodeling in D-gal-induced aging mice. Furthermore, treatment with BBR (40 μM) and THBru (20, 40 μM) inhibited D-gal-induced senescence in primary neonatal mouse cardiomyocytes in vitro. Overall, THBru exhibited higher efficacy than BBR at the same dose. We found that the levels of mitophagy were significantly decreased during the aging process in vivo and in vitro, THBru and BBR promoted mitophagy with different potencies. We demonstrated that the mitophagy-inducing effects of THBru resulted from increased mRNA stability of prohibitin 2 (PHB2), a pivotal factor during mitophagy, thereby upregulating PHB2 protein expression. Knockdown of PHB2 effectively reversed the antisenescence effects of THBru in D-gal-treated cardiomyocytes. On the contrary, overexpression of PHB2 promoted mitophagy and retarded cardiomyocyte senescence, as THBru did. In conclusion, this study identifies THBru as a potent antiaging medicine that induces PHB2-mediated mitophagy and suggests its clinical application prospects.