Neonatal Cardiomyocytes Research Articles

Role of exchange protein directly activated by cAMP in cardiac aging

The aging of the heart is characterized by the presence of fibrosis, hypertrophy, inflammation, apoptosis, and senescence. It has been shown that an increase of cAMP in the aging heart aggravates age-related pathologies, but the players involved in this process have not been determined yet. In this sense, cAMP exerts its function through different effectors among which the Exchange protein directly activated by cAMP 1 (EPAC1) is involved in cardiac remodeling leading to heart failure making this protein a potential target to modulate cardiac aging. This project aims to determine the potential role of EPAC1 in cardiac aging and its possible mechanism of action. In rat neonatal cardiomyocytes we used EPAC's specific activator 8-CPT and inhibitor CE3F4 and evaluated Beta-galactosidase activity and DNA damage to determine senescence activation. For in vivo studies we used 6 month-old and 24 month-old WT or KO EPAC1 mice. To analyse diastolic dysfunction, we performed cardiac echography before and after an acute injection of Isoproterenol. We also evaluated cardiomyocyte cross-sectional areas by immunostaining and the presence of inflammation and senescence markers by qPCR. Finally, we used human samples of young, old patients and HFpEF patients and measured the levels of expression of EPAC1 by qPCR. We found that activating EPAC1 induced both beta-galactosidase activation and DNA damage and that both processes could be reversed by CE3F4. We also found that Doxorubicin-induced senescence was decreased by blocking EPAC1 activation. In vivo, we showed that EPAC1 protein expression was increased in old mice. In addition, we found that after Isoproterenol stress, isovolumic relaxation time (IVRT) was unchanged in old EPAC1 KO mice compared to WT. Immunostaining analysis demonstrated that cardiomyocyte hypertrophy was decreased in Old KO mice compared to WT. Interestingly, we found that senescence and inflammation markers were increased in the WT but not in the EPAC1 KO mice. Finally, mRNA expression of EPAC1 was upregulated in HFpEF patients compared to young subjects. Our findings show that EPAC1 promotes the detrimental process of cardiac aging by increasing senescence, inflammation, and cellular hypertrophy highlighting its potential as a therapeutic target for cardiovascular diseases related to aging.

Ucp2 regulates neonatal cardiomyocytes proliferation through modulation of their mitochondrial metabolism

A major block to mammalian cardiac regeneration is the limited capacity of adult cardiomyocytes (CMs) to proliferate in response to injury. CMs proliferation is linked to oxidative metabolism: at birth, neonatal CMs endure a metabolic switch, with high reliance on fatty acid oxidation. This leads to increased mtROS production that has been shown to mediate their cell-cycle arrest. Thus, studying mitochondrial activity in the neonate is critical to understand constrains on CMs proliferation, regardless of their age. In this context, we focus of Ucp2, a mitochondrial carrier that plays a key role in regulation of cell fuel preferences and mtROS production. Our goal is to investigate whether Ucp2 can regulate neonatal CMs proliferation through modulation of their oxidative metabolism. Using inducible and complete Ucp2 KOs mice models together with RNAseq, flow cytometry and Seahorse analysis, we have addressed the role of Ucp2 in regulation of neonatal CMs proliferation, mtROS levels, mitochondrial biogenesis and metabolic activity. Being expressed at low levels during development, Ucp2 is upregulated from P1 to P7. Its expression then decreases to become barely detectable in the adult, thus correlating with CMs proliferative period. We show that neonatal Ucp2-/- CMs display a reduced mitochondrial respiration (OCR) that is not compensated by glycolysis. This low OCR is accompanied by a strong decrease in mitochondrial mass, together with alterations of ETC complexes expression, and to an increased reliance on glutaminolysis. In addition to decreased ATP/ADP ratio, we show that this impaired metabolic activity leads to reduction of CMs proliferation, a phenotype that is mediated by p38MAPK signaling pathway. We are currently assessing the consequences of loss of Ucp2 on neonatal heart metabolites levels and mitochondrial network. We hypothesize that Ucp2, through the export of metabolites from mitochondria to cytosol, takes part in mitochondrial retrograde signaling leading to regulation of CMs maturation and proliferation.

The whole transcriptome analysis and the circRNA-lncRNA network construction in arsenic trioxide-treated mice myocardium

Background/AimsArsenic trioxide (ATO) is an effective anti-cancer drug. Nonetheless, it possesses cardiotoxic effects which limit its clinical application. The present study aims to elucidate the molecular basis of ATO-induced cardiotoxicity through using whole transcriptome analysis. MethodsThe whole transcriptome in ATO-treated mice myocardium was analyzed using RNA sequencing technique. These results were confirmed by real-time PCR. The lncRNA-mRNA and circRNA-mRNA co-expression networks were constructed. Finally, a circRNA-lncRNA co-regulated competing endogenous RNA (ceRNA) network was constructed. GO and KEGG pathway analyses were performed. The expression levels of Txnip and Spp1 in ATO-treated neonatal mouse cardiomyocytes were validated by real-time PCR. ResultsA total of 113 mRNAs, 159 lncRNAs, 35 miRNAs, and 94 circRNAs were differentially expressed in ATO-treated mice myocardium. A lncRNA-circRNA co-regulation network was constructed. Function annotation revealed that aberrantly expressed genes may be enriched in the ‘Wnt signaling pathway’, ‘Hippo signaling pathway’, ‘Notch signaling pathway’, etc. Finally, the expression levels of Txnip and Spp1 were validated in ATO-treated cardiomyocytes, which was in accordance with the RNA-sequencing results. ConclusionATO altered coding and noncoding RNA profiles in myocardium of mice. The ATO-related lncRNA-circRNA co-regulation network was constructed. Genes in the co-regulation network are likely to play important roles in the cardiotoxicity of ATO. This study provides new insights into the prevention and treatment of ATO-induced cardiotoxicity.

Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56–2.2 × 106 cells/heart) and viability (~70–100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

Targeting Adiponectin Receptor 1 Phosphorylation Against Ischemic Heart Failure.

Despite significantly reduced acute myocardial infarction (MI) mortality in recent years, ischemic heart failure continues to escalate. Therapeutic interventions effectively reversing pathological remodeling are an urgent unmet medical need. We recently demonstrated that AdipoR1 (APN [adiponectin] receptor 1) phosphorylation by GRK2 (G-protein-coupled receptor kinase 2) contributes to maladaptive remodeling in the ischemic heart. The current study clarified the underlying mechanisms leading to AdipoR1 phosphorylative desensitization and investigated whether blocking AdipoR1 phosphorylation may restore its protective signaling, reversing post-MI remodeling. Specific sites and underlying molecular mechanisms responsible for AdipoR1 phosphorylative desensitization were investigated in vitro (neonatal and adult cardiomyocytes). The effects of AdipoR1 phosphorylation inhibition upon APN post-MI remodeling and heart failure progression were investigated in vivo. Among 4 previously identified sites sensitive to GRK2 phosphorylation, alanine substitution of Ser205 (AdipoR1S205A), but not other 3 sites, rescued GRK2-suppressed AdipoR1 functions, restoring APN-induced cell salvage kinase activation and reducing oxidative cell death. The molecular investigation followed by functional determination demonstrated that AdipoR1 phosphorylation promoted clathrin-dependent (not caveolae) endocytosis and lysosomal-mediated (not proteasome) degradation, reducing AdipoR1 protein level and suppressing AdipoR1-mediated cytoprotective action. GRK2-induced AdipoR1 endocytosis and degradation were blocked by AdipoR1S205A overexpression. Moreover, AdipoR1S205E (pseudophosphorylation) phenocopied GRK2 effects, promoted AdipoR1 endocytosis and degradation, and inhibited AdipoR1 biological function. Most importantly, AdipoR1 function was preserved during heart failure development in AdipoR1-KO (AdipoR1 knockout) mice reexpressing hAdipoR1S205A. APN administration in the failing heart reversed post-MI remodeling and improved cardiac function. However, reexpressing hAdipoR1WT in AdipoR1-KO mice failed to restore APN cardioprotection. Ser205 is responsible for AdipoR1 phosphorylative desensitization in the failing heart. Blockade of AdipoR1 phosphorylation followed by pharmacological APN administration is a novel therapy effective in reversing post-MI remodeling and mitigating heart failure progression.

Green Manufacturing of Flexible Sensors with a Giant Gauge Factor: Bridging Effect of CNT and Electric Field Enhancement at the Percolation Threshold.

Toxic organic solvents are commonly used to disperse nanomaterials in the manufacturing of flexible conductive composites (e.g., graphene-PDMS). The dry-blended method avoids toxic organic solvent usage but leads to poor performance. Here, we proposed an innovative manufacturing method by adapting the traditional dry-blended method, including two key steps: minor CNT bridging and high-frequency electric field enhancement at the percolation threshold of graphene-PDMS. Significant improvement was achieved in the electrical conductivity (1528 times), the giant gauge factor (>8767.54), and the piezoresistive strain range (30 times) over the traditional dry-blended method. Further applications in measurements of culturing rat neonatal cardiomyocytes and mouse hearts proved that the proposed method has great potential for the manufacturing of nontoxic flexible sensors.

Stimulation of Cardiomyocyte Proliferation Is Dependent on Species and Level of Maturation.

The heart is one of the least regenerative organs. This is in large part due to the inability of adult mammalian cardiomyocytes to proliferate and divide. In recent years, a number of small molecules and molecular targets have been identified to stimulate cardiomyocyte proliferation, including p38 inhibition, YAP-Tead activation, fibroblast growth factor 1 and Neuregulin 1. Despite these exciting initial findings, a therapeutic approach to enhance cardiomyocyte proliferation in vivo is still lacking. We hypothesized that a more comprehensive in vitro validation using live-cell imaging and assessment of the proliferative effects on various cardiomyocyte sources might identify the most potent proliferative stimuli. Here, we used previously published stimuli to determine their proliferative effect on cardiomyocytes from different species and isolated from different developmental timepoints. Although all stimuli enhanced DNA synthesis and Histone H3 phosphorylation in neonatal rat ventricular cardiomyocytes to similar degrees, these effects varied substantially in mouse cardiomyocytes and human iPSC-derived cardiomyocytes. Our results highlight p21 inhibition and Yap-Tead activation as potent proliferative strategies to induce cultured cardiomyocyte cell cycle activity across mouse, rat and human cardiomyocytes.

Identification of electrical rotational activity in noisy cardiac tissue recordings using a deep neural network

Abstract Funding Acknowledgements Type of funding sources: None. Background Deep learning is increasingly used in modern biomedical research and applications due to the substantial availability of large clinical datasets. These approaches are invaluable in tasks involving noisy imaging data, such as tumour segmentation in histological images. In cardiology, a deep learning approach could be helpful in real-time tracking of the sources of arrhythmia, i.e. electrical rotational activity in the heart. However, the existing optical or electrophysiological recordings that could be used for training such a model are recorded under highly variable conditions and are not always annotated, thereby requiring data augmentation. Purpose To use deep neural networks trained on synthetic data to obtain concise (low dimensional) representations of noisy optical mapping recordings of cardiac arrhythmias and rapidly locate spiral wave centres. Methods To overcome the lack of experimental training data, a digital twin of a neonatal rat ventricular cardiomyocyte monolayer was used to create a large synthetic training dataset of noiseless spiral wave recordings. Spiral wave centres were detected and labelled by making use of classical algorithms which are proven to work well on noiseless data. After labelling the centres, noise was added to the spiral wave recordings to simulate realistic experimental measurements. Subsequently, these data were fed into three different deep learning architectures: 1) a variational auto-encoder (VAE) to denoise optical mapping recordings of cardiac arrhythmias in an unsupervised manner, 2) a convolutional neural network (CNN) to detect the spiral centres, and 3) a combination of both to denoise the recording and detect centres simultaneously. Results After training on synthetic datasets, each architecture could accurately predict what it was designed for (noiseless wave fronts, spiral centres including chirality, or both) on both synthetic and experimental data. These spiral centre detection results were compared with 5 classical methods of denoising and spiral centre detection for accuracy and speed. Our method was as accurate as the best performing yet slow classical algorithm, which can only detect centres after observing a full rotation cycle (~300ms). At the same time, it was as fast as the fastest classical method, needing only 30ms after enabling the algorithm to detect spiral wave centres. This allows quasi-real-time tracking of arrhythmic sources. Conclusion This study reveals that modern deep learning strategies in combination with synthetic simulation datasets can be used on experimental measurements to aid in the development of new technologies, here applied to the detection of spiral wave centres in optical mapping recordings of cardiac arrhythmias. Given the combination of speed and accuracy at which these algorithms produce results, further exploration and refinement may improve the identification of targets for catheter ablation, thereby potentially improving the outcome.

Trapped re-entry: a dormant source of arrhythmia

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This study was supported by the European Research Council (Starting grant 716509) to D.A. Pijnappels. Background Diseased atria are characterized by functional and structural heterogeneities (e.g. dense fibrotic regions), which add to abnormal impulse generation and propagation, like ectopy and block. These heterogeneities are thought to play a role in the origin of complex fractionated atrial electrograms (CFAEs) under sinus rhythm (SR) in patients with atrial fibrillation (AF), but also in the onset and perpetuation (e.g. re-entry) of this disorder. The underlying mechanisms, however, remain incompletely understood. Objective To test the hypothesis that dense local fibrotic regions can create an electrically isolated conduction pathway in which re-entry can be established via ectopy and block to become "trapped" (giving rise to CFAEs under SR), only to be "released" under dynamic tissue changes at a connecting isthmus (causing acute onset of arrhythmia). Methods The geometry of such a pathway, under which re-entry could be trapped and released, was explored in vitro using optogenetics by creating conduction blocks of any shape by means of light-gated depolarizing ion channels (CatCh) and patterned illumination. Insight from these studies was used for complementary investigation in a digital twin of the human atria to assess clinical relevance. Results Optical mapping studies, in monolayers of CatCh-activated neonatal rat atrial cardiomyocytes, revealed that re-entry can be established and trapped by creating an electrically isolated pathway with a bulk-connecting isthmus causing source-sink mismatch. A tachyarrhythmia was shown to exist locally with SR prevailing in the bulk of the monolayer. Next, conditions were found under which re-entry could escape this pathway, thereby converting a local dormant arrhythmic source into an active driver with global impact. Escape could be established by overcoming the source-sink mismatch through widening of the isthmus or a reduction of the gap junctional coupling. In a digital twin of the human atria, it was revealed that the conditions for "trapped re-entry" and its release can be realized as well. Unipolar pseudo-electrograms derived from these complementary computational 3D studies showed CFAEs at the site of "trapped re-entry" in coexistence with normal electrograms of SR in the bulk of the atria. Upon release of the re-entry, acute arrhythmia onset occurred, affecting the complete atria as evidenced by wave front and electrogram visualization. Conclusion This study reveals that "trapped re-entry", a previously undesignated phenomenon, can explain the observation of two designated ones: CFAEs under SR and acute arrhythmia onset. Further exploration of the concept of "trapped re-entry" may not only expand our understanding of AF initiation and perpetuation, but also termination, including ablation strategies by site-directed targeting.

Reshaping the Preterm Heart: Shifting Cardiac Renin-Angiotensin System Towards Cardioprotection in Rats Exposed to Neonatal High-Oxygen Stress.

Background:Approximately 10% of infants are born preterm. Preterm birth leads to short and long-term changes in cardiac shape and function. By using a rat model of neonatal high-oxygen (80%O2) exposure, mimicking the premature hyperoxic transition to the extrauterine environment, we revealed a major role of the renin-angiotensin system peptide Angio II (angiotensin II) and its receptor AT1 (angiotensin receptor type 1) on neonatal O2-induced cardiomyopathy. Here, we tested whether treatment with either orally active compounds of the peptides Angio-(1–7) or alamandine included in cyclodextrin could prevent postnatal cardiac remodeling and the programming of cardiomyopathy induced by neonatal high-O2 exposure.Methods:Sprague-Dawley pups were exposed to room air or 80% O2 from postnatal day 3 (P3) to P10. Neonatal rats were treated orally from P3 to P10 and assessed at P10 and P28. Left ventricular (LV) shapes were characterized by tridimensional computational atlases of ultrasound images in addition to histomorphometry.Results:At P10, high O2-exposed rats presented a smaller, globular and hypertrophied LV shape versus controls. Treatment with cyclodextrin–Angio-(1–7) significantly improved LV function in the O2-exposed neonatal rats and slightly changed LV shape. Cyclodextrin-alamandine and cyclodextrin–Angio-(1–7) treatments similarly reduced hypertrophy at P10 as well as LV remodeling and dysfunction at P28. Both treatments upregulated cardiac angiotensin-converting enzyme 2 in O2-exposed rats at P10 and P28.Conclusions:Our findings demonstrate LV remodeling changes induced by O2-stress and the potential benefits of treatments targeting the cardioprotective renin-angiotensin system axis, supporting the neonatal period as an important window for interventions aiming at preventing cardiomyopathy in people born preterm.

The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro

Remdesivir is a prodrug of a nucleoside analog and the first antiviral therapeutic approved for coronavirus disease. Recent cardiac safety concerns and reports on remdesivir-related acute kidney injury call for a better characterization of remdesivir toxicity and understanding of the underlying mechanisms. Here, we performed an in vitro toxicity assessment of remdesivir around clinically relevant concentrations (Cmax 9 µM) using H9c2 rat cardiomyoblasts, neonatal mouse cardiomyocytes (NMCM), rat NRK-52E and human RPTEC/TERT1 cells as cell models for the assessment of cardiotoxicity or nephrotoxicity, respectively. Due to the known potential of nucleoside analogs for the induction of mitochondrial toxicity, we assessed mitochondrial function in response to remdesivir treatment, early proteomic changes in NMCM and RPTEC/TERT1 cells and the contractile function of NMCM. Short-term treatments (24 h) of H9c2 and NRK-52E cells with remdesivir adversely affected cell viability by inhibition of proliferation as determined by significantly decreased 3H-thymidine uptake. Mitochondrial toxicity of remdesivir (1.6–3.1 µM) in cardiac cells was evident by a significant decrease in oxygen consumption, a collapse of mitochondrial membrane potential and an increase in lactate secretion after a 24–48-h treatment. This was supported by early proteomic changes of respiratory chain proteins and intermediate filaments that are typically involved in mitochondrial reorganization. Functionally, an impedance-based analysis showed that remdesivir (6.25 µM) affected the beat rate and contractility of NMCM. In conclusion, we identified adverse effects of remdesivir in cardiac and kidney cells at clinically relevant concentrations, suggesting a careful evaluation of therapeutic use in patients at risk for cardiovascular or kidney disease.

Fasudil Ameliorates Osteoporosis Following Myocardial Infarction by Regulating Cardiac Calcitonin Secretion.

We hypothesis that Rho kinase inhibitor fasudil ameliorates osteoporosis following myocardial infarction (MI) by regulating cardiac calcitonin secretion. A mice model of MI and cultured neonatal cardiomyocytes exposed to hypoxia and serum deprivation (H/SD), and fibroblasts exposed to TGF-β were used, respectively. Cardiac function in vivo was assessed with echocardiography. Osteoporosis in vivo was assessed with X-ray and micro-CT. In vivo and in vitro studies used histological and immunohistochemical techniques, along with western blots. In mice post-MI, fasudil ameliorates the microstructure and bone metabolism of the lumbar, improved cardiac function, and attenuated myocardial fibrosis. In vitro, fasudil or αCGRP could effectively inhibit the proliferation of primary fibroblasts treated with TGF-β. Moreover, fasudil ameliorates the cardiac calcitonin secretion induced by MI in vivo or by H/SD in vitro. Our findings suggest that fasudil improved MI-induced osteoporosis by promoting cardiac secreting calcitonin.

Higenamine Attenuates Doxorubicin-Induced Cardiac Remodeling and Myocyte Apoptosis by Suppressing AMPK Activation

Background: As an effective antitumor drug, doxorubicin (DOX) is primarily used to treat solid tumors and hematologic malignancies. However, increasing evidence has emerged indicating its cardiotoxicity, and few solutions have been proposed to counter this side effect. Higenamine (HG) is a natural compound widely found in many Chinese herbs and also serves as a component in many healthcare products. Several studies have demonstrated its cardioprotective effect in different models, but little is known about the underlying influences of HG against myocardial damage from DOX-induced chronic cardiotoxicity. Methods and Results: C57BL/6 mice and neonatal rat ventricular cardiomyocytes (NRVMs) were used to evaluate the cardioprotective effect of HG against DOX-induced myocardial damage. In mice, DOX (intraperitoneally injected 5 mg/kg every 3 days for 4 weeks) significantly increased cardiomyocyte apoptosis, cardiac atrophy, and cardiac dysfunction, which were significantly attenuated by HG (intragastrically administered with 10 mg/kg every day for 4 weeks). In NRVMs, DOX (3 μM for 24 h) significantly increased cell apoptosis and the level of reactive oxygen species while reducing the level of superoxide dismutase and mitochondrial membrane potential. Remarkably, HG can reverse these pathological changes caused by DOX. Interestingly, the protective effect of HG on DOX-induced cardiotoxicity was independent of the activation of the beta-2 adrenergic receptor (β2-AR), known for mediating the effect of HG on antagonizing ischemia/reperfusion-induced cardiac apoptosis. Furthermore, HG attenuated the abnormal activation of phosphorylated adenosine-activated protein kinase (AMPK). Consistently, AMPK agonists (AICAR) can eliminate these pharmacological actions of HG. Conclusion: Collectively, our results suggested that HG alleviated DOX-induced chronic myocardial injury by suppressing AMPK activation and ROS production.

Role of Cardiomyocyte-Derived Exosomal MicroRNA-146a-5p in Macrophage Polarization and Activation.

Myocardial infarction arises from an excessive or prolonged inflammatory response, leading to ventricular remodeling or impaired cardiac function. Macrophages exhibit different polarization types associated with inflammation both at steady state and after myocardial infarction. Exosomal miR-146a-5p has been identified as an important molecule in the cardiovascular field in recent years. However, the effect of cardiomyocyte-derived exosomal miR-146a-5p on macrophages has not yet been elucidated. Initially, we found that exosomes with low expression of miR-146a-5p derived from myocardial infarction tissues modulated macrophage polarization. To determine whether cardiomyocyte-derived exosomal miR-146a-5p mediated macrophage polarization, we treated macrophages with exosomes rich in miR-146a-5p collected from neonatal mouse cardiomyocytes. The effects of exosomal miR-146a-5p on macrophage polarization were measured using RT-qPCR, transwell assays, and western blotting. The results showed that the increased expression of miR-146a-5p promoted M1 macrophage polarization, inhibited M2 macrophage polarization, and increased the expression of VEGFA. However, the decreased expression of exosomalmiR-146a-5p showed the opposite trends. Interestingly, in contrast to treatment with the solitary miR-146a-5p mimic, exosomal miR-146a-5p derived from neonatal mouse cardiomyocytes reduced TNFα and iNOS expression. In addition, when macrophages were activated by the miR-146a-5p mimic or exosomal miR-146a-5p, the expression of TNF receptor-associated factor 6 (TRAF6), a target gene of miR-146a-5p, was reduced significantly. Taken together, these findings indicate that exosomal miR-146a-5p derived from cardiomyocytes could stimulate M1 macrophage polarization to induce an inflammatory reaction, while targeting TRAF6, exerting an anti-inflammatory effect. Exosomal miR-146a-5p plays important roles in macrophages, illuminating a novel potential therapeutic target in myocardial infarction.

CXCL10 is a novel anti-angiogenic factor downstream of p53 in cardiomyocytes.

Tumor suppressor protein p53 plays crucial roles in the onset of heart failure. p53 activation results in cardiac dysfunction, at least partially by suppressing angiogenesis. Though p53 has been reported to reduce VEGF production by inhibiting hypoxia‐inducible factor, the anti‐angiogenic property of p53 remains to be fully elucidated in cardiomyocytes. To explore the molecular signals downstream of p53 that regulate vascular function, especially under normoxic conditions, DNA microarray was performed using p53‐overexpressing rat neonatal cardiomyocytes. Among genes induced by more than 2‐fold, we focused on CXCL10, an anti‐angiogenic chemokine. Real‐time PCR revealed that p53 upregulated the CXCL10 expression as well as p21, a well‐known downstream target of p53. Since p53 is known to be activated by doxorubicin (Doxo), we examined the effects of Doxo on the expression of CXCL10 and found that Doxo enhanced the CXCL10 expression, accompanied by p53 induction. Importantly, Doxo‐induced CXCL10 was abrogated by siRNA knockdown of p53, indicating that p53 activation is necessary for Doxo‐induced CXCL10. Next, we examined the effect of hypoxic condition on p53‐mediated induction of CXCL10. Interestingly, CXCL10 was induced by hypoxia and its induction was potentiated by the overexpression of p53. Finally, the conditioned media from cultured cardiomyocytes expressing p53 decreased the tube formation of endothelial cells compared with control, analyzed by angiogenesis assay. However, the inhibition of CXCR3, the receptor of CXCL10, restored the tube formation. These data indicate that CXCL10 is a novel anti‐angiogenic factor downstream of p53 in cardiomyocytes and could contribute to the suppression of vascular function by p53.

Tumor Necrosis Factor-α-Induced Protein 8-Like 2 Ameliorates Cardiac Hypertrophy by Targeting TLR4 in Macrophages.

Background Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2), a novel immunoregulatory protein, has been reported to regulate inflammation and apoptosis. The role of TIPE2 in cardiovascular disease, especially cardiac hypertrophy, has not been elucidated. Thus, the aim of the present study was to explore the role of TIPE2 in cardiac hypertrophy. Methods Mice were subjected to aortic banding (AB) to induce an adverse hypertrophic model. To overexpress TIPE2, mice were injected with a lentiviral vector expressing TIPE2. Echocardiographic and hemodynamic analyses were used to evaluate cardiac function. Neonatal rat cardiomyocytes (NRCMs) and mouse peritoneal macrophages (MPMs) were isolated and stimulated with angiotensin II. NRCMs and MPM were also cocultured and stimulated with angiotensin II. Cells were transfected with Lenti-TIPE2 to overexpress TIPE2. Results TIPE2 expression levels were downregulated in hypertrophic mouse hearts and in macrophages in heart tissue. TIPE2 overexpression attenuated pressure overload-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction. Moreover, we found that TIPE2 overexpression in neonatal cardiomyocytes did not relieve the angiotensin II-induced hypertrophic response in vitro. Furthermore, TIPE2 overexpression downregulated TLR4 and NF-κB signaling in macrophages but not in cardiomyocytes, which led to diminished inflammation in macrophages and consequently reduced the activation of hypertrophic Akt signaling in cardiomyocytes. TLR4 inhibition by TAK-242 did not enhance the antihypertrophic effect of TIPE2 overexpression. Conclusions The present study indicated that TIPE2 represses macrophage activation by targeting TLR4, subsequently inhibiting cardiac hypertrophy.

LRP5 regulates cardiomyocyte proliferation and neonatal heart regeneration by the AKT/P21 pathway.

The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low‐density lipoprotein receptor‐related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac‐specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin‐dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.

Alleviation of doxorubicin-induced cardiomyocyte death through miR-147-y-mediated mitophagy

Doxorubicin (DOX) is a commonly used antitumor drug. However, it may cause severe cardiotoxicity, apoptosis being a major change. A recent report indicates that miR-147 expression is decreased in the myocardium of a myocardial infarction model, suggesting a potential role of this miRNA in DOX-induced cardiomyocyte toxicity. In this study, freshly isolated neonatal pig cardiomyocytes were used; following transfection of a miR-147-y mimic, the cell death induced by DOX was alleviated, represented by augmented mitophagy [indicated by a decrease in P62, and increases in LC3, PINK1, parkin mRNA, LC3Ⅱ/Ⅰ, beclin-1, PINK1, and parkin including p-parkin (Ser65) protein expression], prohibited cell apoptosis as determined by TUNEL staining, and the suppression of caspase-3 transcription and cleaved caspase-3 translation. In cells transfected with an miR-147-y inhibitor, DOX-induced mitophagy was decreased, while apoptosis was increased. Additionally, RAPTOR gene silencing in cardiomyocytes exposed to DOX increased the rate of mitophagy and decreased that of apoptosis as compared with the treatment with DOX alone. Moreover, RAPTOR overexpression downregulated the rate of mitophagy and increased that of apoptosis in cells exposed to DOX. RAPTOR was confirmed as the target gene of miR-147-y based on the results of luciferase reporter gene assays and the opposite effects of the miR-147-y mimic and miR-147-y inhibitor on RAPTOR expression. In summary, our study suggests that miR-147-y mediates DOX-induced cardiomyocyte mitophagy while suppresses apoptosis by targeting RAPTOR, thus playing a protective role in DOX-induced cardiomyocyte damage.

Inhibition of BKCa channels protects neonatal hearts against myocardial ischemia and reperfusion injury

BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channel’s activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes, which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL-positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.

Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated “striated muscle contraction” as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.