Cultures Of Neonatal Rat Cardiomyocytes Research Articles

Fluorescence Signal Kinetics of a New Ca2+ Probebound to Cardiac Ryanodine Receptors Point to Micro-Domain Detection of Ca2+

To probe the micro-domains of Ca2+-signaling, we constructed a probe, calstabi-cam, targeted to cardiac ryanodine receptors (RyR2) by FKBP12.6 (calstabin 2) incorporating calmodulin and EYFP as Ca2+-sensor. 48-72h after infection of cultured rat neonatal cardiomyocytes (rNCM), global measurements of calstabi-cam fluorescence were accompanied by simultaneous recordings of Ca2+ channel (ICa) and Na+/Ca2+-exchanger (INCX) and compared to measurements of fluo-4 Ca-transients. rNCMs continued spontaneous beating after dialysis with Cs+-based internal solutions (0.1-0.2mM EGTA at −50mV) as they generated calstabi-cam fluorescence signals that coincided with INCX oscillations, like those measured by fluo-4 that were blocked by 10µM ryanodine. Activation of ICa (∼5pA/pF) triggered slowly relaxing Ca-transients of calstabi-cam fluorescence (ΔF/F0 = −0.11; n=14). Similarly, short caffeine pulses also activated long lasting calstabi-cam signals (-ΔF/F0=0.15±0.012; n=28) accompanied by INCX (1.57±0.13pA/pF; n=28). Calstabi-cam signals activated by rapid Na+ withdrawal was significantly delayed (∼230ms) compared to those triggered by ICa (∼13ms) or caffeine (∼35ms). Similar results were found in feline adult cardiac myocytes where 1µM Isoproterenol increased ICa significantly without enhancing the associated calstabi-cam transients. These results suggest that the new probe responds more effectively to Ca2+ that is released into dyadic clefts by RyR2 than by sarcolemmal Ca2+ influx via Na/Ca exchanger. The extremely slow decay kinetics of Calstabi-cam signal is consistent with the notion that [Ca2+] in the dyadic cleft may rise to much higher values than those of cytosol.

Carbon monoxide releasing molecule-2 attenuated ischemia/reperfusion-induced apoptosis in cardiomyocytes via a mitochondrial pathway

Carbon monoxide (CO) is an endogenous gaseous transmitter that exerts multi-protection in ischemia/reperfusion (I/R) injury, but few experimental studies regarding CO on myocardial I/R-induced apoptosis, as well as its underlying mechanism have been conducted. The present study was designed to investigate whether CO released from CO-releasing molecule-2 (CORM-2) is capable of ameliorating myocardial I/R-induced apoptosis via a mitochondrial apoptotic pathway. Primary cultures of neonatal rat cardiomyocytes were randomly distributed into four groups: Control, I/R (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion), CORM-2 and inactive CORM-2 (iCORM-2) groups (20 µM CORM-2 and 20 µM iCORM-2 were administered at the beginning of reperfusion following ischemia, respectively). Flow cytometric analysis showed that CORM-2 treatment significantly decreased apoptosis of cardiomyocytes triggered by simulated I/R. CORM-2 partially recovered mitochondrial respiration and ultrastructure alteration, and lowered caspase-3 expression and the release of cytochrome c. Furthermore, CORM-2 partly reduced BAK/BAX expression in mitochondria, as well as the BAX level in the cytoplasm. Cardioprotection is lost when CORM-2 is replaced by iCORM-2. CORM-2 treatment, at the time of reperfusion, was concluded to attenuate myocardial I/R-induced apoptosis. The protection mechanisms may be targeted to the mitochondria and involved in the inhibition of the BAK/BAX‑mediated intrinsic pathway.

Angiotensin II and the ERK pathway mediate the induction of leptin by mechanical cyclic stretch in cultured rat neonatal cardiomyocytes

Mechanical cyclic stretch of cardiomyocytes causes cardiac hypertrophy through cardiac-restricted gene expression. Leptin induces cardiomyocyte hypertrophy in response to myocardial stress. In the present study, we evaluated the expression of leptin under cyclic stretch and its role in regulating genetic transcription in cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to cyclic stretch, and the expression levels of leptin, ROS (reactive oxygen species) and AngII (angiotensin II) were evaluated. Signal transduction inhibitors were used to identify the pathway of leptin expression. EMSAs were used to identify the binding of leptin/STAT3 (signal transducer and activator of transcription 3) and luciferase assays were used to identify the transcription of leptin in cardiomyocytes. The study also used an invivo model of AV (aortocaval) shunt in rats to investigate leptin, ROS and AngII expression. Leptin and leptin receptor levels increased after cyclic stretch with the earlier expression of AngII and ROS. Leptin expression was suppressed by AngII receptor blockers, an ROS scavenger [NAC (N-acetylcysteine)], an ERK (extracellular-signal-regulated kinase) pathway inhibitor (PD98059) and ERK siRNA. Binding of leptin/STAT3 was identified by EMSAs, and luciferase assays confirmed the transcription of leptin in neonatal cardiomyocytes after cyclic stretch. Increased MHC (myosin heavy chain) expression and [3H]-proline incorporation in cardiomyocytes was detected after cyclic stretch, which were inhibited by leptin siRNA and NAC. The invivo model of AV shunt also demonstrated increased levels of plasma and myocardial leptin, ROS and AngII expression after cyclic stretch. Mechanical cyclic stretch in cardiomyocytes increased leptin expression mediated by the induction of AngII, ROS and the ERK pathway to cause cardiomyocyte hypertrophy. Myocardial hypertrophy can be identified by increased transcriptional activity and an enhanced hypertrophic phenotype of cardiomyocytes.

Protease Activated Receptor-2 Contributes to Heart Failure

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.

COX-2 is involved in ET-1-induced hypertrophy of neonatal rat cardiomyocytes: Role of NFATc3

Endothelin-1 (ET-1) is a critical molecule that involved in heart failure. It has been proved that ET-1 stimulation results in cardiac hypertrophy both in vitro and in vivo, but the mechanisms underlying remain largely unknown. In this study, we reported that cyclooxygenase-2 (COX-2) might be an important mediator of hypertrophic responses to ET-1 stimulation. In the cultured rat neonatal cardiomyocytes, ET-1 significantly upregulated the expression and activity of COX-2, which was accompanied by increase in cell surface area and BNP mRNA level. In contrast, ET-1-dependent cardiomyocyte hypertrophy was abolished by COX-2 selective inhibitors, NS-398 and celecoxib, or by COX-2 RNA interference, but the inhibitory effects could be diminished by pretreatment with PGE2. Furthermore, cyclosporin A (CsA) and knockdown of nuclear factor of activated T-cells c3 (NFATc3) inhibited the expression of COX-2 induced by ET-1, and NFATc3 could also bound to the -GGAAA- sequence in the promoter region of rat COX-2 gene, indicating that calcineurin/NFATc3 signaling participated in the transcriptional regulation of COX-2 following ET-1 treatment. These findings provided further insight into the roles of ET-1 in cardiac hypertrophy and suggested a potential therapeutic strategy against cardiac hypertrophy by inhibiting COX-2.

Abstract 18111: Engineered Cell Therapy With Embryonic Stem Cell-Derived Cardiomyocytes Encapsulated in Injectable Nanomatrix Gel Enhanced Engraftment and Promoted Cardiac Repair in Experimental Myocardial Infarction

Background: A significant barrier to the therapeutic use of stem cells is poor cell engraftment in vivo. To overcome this hurdle, studies have attempted to engineer stem cells using biomaterials. Particularly, injectable self-assembling nanomatrix gel has emerged as a highly promising biomaterial by providing a natural extracellular matrix-like microenvironment. Here, we evaluate the therapeutic potential of cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) encapsulated with injectable nanomatrix gel consisting of peptide amphiphiles incorporating cell adhesive ligand RGDS (PA-RGDS) on experimental myocardial infarction (MI). Methods and results: First, we examined the survival of CMs on RGDS. We cultured rat neonatal CMs on PA-RGDS for 7 days and found that higher than 90% of the CMs remained alive with well-defined CM structures as determined by live/dead and immuno stainings. Next, to test if PA-RGDS can increase the retention of CMs in the heart, we intramyocardially injected DiI (fluorescent probe)-labeled CMs into the uninjured hearts with or without PA-RGDS. Flow cytometry analysis with enzymatically digested heart tissues harvested at day 7 showed a ~3 fold higher rate of mESC-CM engraftment in the mice receiving CMs with PA-RGDS compared to those receiving only mESC-CMs (11.3% ± 0.4 vs 4.4% ± 0.2). Finally, we investigated the therapeutic effects of mESC-CMs with PA-RGDS on the MI in comparison with PBS control, CM alone, and PA-RGDS alone. Weekly echocardiography demonstrated that three groups (CM, PA-RGDS and CM+PA-RGDS) showed higher ejection fractions than the PBS group at week 2. However, in the CM or PA-RGDS alone groups, cardiac function was dramatically reduced between 3-4 weeks. In contrast, the CM+PA-RGDS group maintained cardiac function for up to 12 weeks. Confocal microscopic examination showed that injected CMs in PA-RGDS engrafted robustly in myocardium, formed clusters, integrated into host myocardium, and expressed representative CM proteins such as cTNT and MHC6/7. Conclusions: This study demonstrated that implantation of ESC-derived CMs encapsulated with PA-RGDS improves cell retention and cardiac function post-MI. This engineered cell therapy can develop into a novel option for treating MI.

Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-β in cardiac myocytes

The expression of MURC (muscle-restricted coiled-coil protein), a hypertrophy-regulated gene, increases during pressure overload. Hypoxia can cause myocardial hypertrophy; however, how hypoxia affects the regulation of MURC in cardiomyocytes undergoing hypertrophy is still unknown. The aim of the present study was to test the hypothesis that hypoxia induces MURC expression in cardiomyocytes during hypertrophy. The expression of MURC was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia and in an in vivo model of AMI (acute myocardial infarction) to induce myocardial hypoxia in adult rats. MURC protein and mRNA expression were significantly enhanced by hypoxia. MURC proteins induced by hypoxia were significantly blocked after the addition of PD98059 or ERK (extracellular-signal-regulated kinase) siRNA 30 min before hypoxia. Gel-shift assay showed increased DNA-binding activity of SRF (serum response factor) after hypoxia. PD98059, ERK siRNA and an anti-TGF-β (transforming growth factor-β) antibody abolished the SRF-binding activity enhanced by hypoxia or exogenous administration of TGF-β. A luciferase promoter assay demonstrated increased transcriptional activity of SRF in cardiomyocytes by hypoxia. Increased βMHC (β-myosin heavy chain) and BNP (B-type natriuretic peptide) protein expression and increased protein synthesis was identified after hypoxia with the presence of MURC in hypertrophic cardiomyocytes. MURC siRNA inhibited the hypertrophic marker protein expression and protein synthesis induced by hypoxia. AMI in adult rats also demonstrated increased MURC protein expression in the left ventricular myocardium. In conclusion, hypoxia in cultured rat neonatal cardiomyocytes increased MURC expression via the induction of TGF-β, SRF and the ERK pathway. These findings suggest that MURC plays a role in hypoxia-induced hypertrophy in cardiomyocytes.

Proteome Modulation in H9c2 Cardiac Cells by microRNAs miR-378 and miR-378

MicroRNAs are a novel class of powerful endogenous regulators of gene expression. MiR-378 and miR-378* are localized in the first intron of the Ppargc1b gene that codes the transcriptional co-activator PGC-1β. The latter regulates energy expenditure as well as mitochondrial biogenesis. The miR-378:miR-378* hairpin is highly expressed in cardiac cells. To better assess their role in cardiomyocytes, we identified miR-378 and miR-378* targets via a proteomic screen. We established H9c2 cellular models of overexpression of miR-378 and miR-378* and identified a total of 86 down-regulated proteins in the presence of either one of these miRs. Functional annotation clustering showed that miR-378 and miR-378* regulate related pathways in cardiomyocytes, including energy metabolism, notably glycolysis, cytoskeleton, notably actin filaments and muscle contraction. Using bioinformatics algorithms we found that 20 proteins were predicted as direct targets of the miRs. We validated eight of these targets by quantitative RT-PCR and luciferase reporter assay. We found that miR-378 targets lactate dehydrogenase A and impacts on cell proliferation and survival whereas miR-378* targets cytoskeleton proteins actin and vimentin. Proteins involved in endoplasmic reticulum stress response such as chaperone and/or calcium buffering proteins GRP78, PPIA (cyclophilin A), calumenin, and GMMPA involved in glycosylation are repressed by these miRs. Our results show that the miR-378/378* hairpin establishes a connection among energy metabolism, cytoskeleton remodeling, and endoplasmic reticulum function through post-transcriptional regulation of key proteins involved in theses pathways.

Biokinetically-Based In Vitro Cardiotoxicity of Residual Oil Fly Ash: Hazard Identification and Mechanisms of Injury

Epidemiological studies have associated air pollution particulate matter (PM) exposure with adverse cardiovascular effects. Identification of causal PM sources is critically needed to support regulatory decisions to protect public health. This research examines the in vitro cardiotoxicity of bioavailable constituents of residual oil fly ash (ROFA) employing in vivo, biokinetically-based, concentrations determined from their pulmonary deposition. Pulmonary deposition of ROFA led to a rapid increase in plasma vanadium (V) levels that were prolonged in hypertensive animals without systemic inflammation. ROFA cardiotoxicity was evaluated using neonatal rat cardiomyocyte (RCM) cultures exposed to particle-free leachates of ROFA (ROFA-L) at levels present in exposed rat plasma. Cardiotoxicity was observed at low levels (3.13μg/mL) of ROFA-L 24h post-exposure. Dimethylthiourea (28mM) inhibited ROFA-L-induced cytotoxicity at high (25-12.5μg/mL) doses, suggesting that oxidative stress is responsible at high ROFA-L doses. Cardiotoxicity could not be reproduced using a V+Ni+Fe mixture or a ROFA-L depleted of these metals, suggesting that ROFA-L cardiotoxicity requires the full complement of bioavailable constituents. Susceptibility of RCMs to ROFA-L-induced cytotoxicity was increased following tyrosine phosphorylation inhibition, suggesting that phosphotyrosine signaling pathways play a critical role in regulating ROFA-L-induced cardiotoxicity. These data demonstrate that bioavailable constituents of ROFA are capable of direct adverse cardiac effects.

Globular Adiponectin Attenuates Myocardial Ischemia/Reperfusion Injury by Upregulating Endoplasmic Reticulum Ca2+-ATPase Activity and Inhibiting Endoplasmic Reticulum Stress

The aim of this study was to explore the mechanisms underlying the effects of globular adiponectin (gAd) on myocardial ischemia/reperfusion (I/R) injury. An in vivo myocardial I/R model and an in vitro neonatal rat cardiomyocyte hypoxia/reoxygenation (H/R) model simulating I/R injury in vivo were adopted to investigate whether and how the cardioprotective effects of gAd are mediated by the inhibition of endoplasmic reticulum (ER) stress. gAd (1 μg/g, intravenously) attenuated the myocardial infarct size, myocardial enzyme activity, and apoptosis in rats with I/R, and similar protection was observed in primary cultures of neonatal rat cardiomyocytes. The protective effects of gAd were associated with the suppression of ER stress, as evidenced by reversing the upregulation of 78-kDa glucose-regulated protein, C/EBP homologous protein, and caspase-12 that were induced by H/R and thapsigargin. In addition, gAd conferred resistance to ER stress and cardiomyocyte injury by modulating ER Ca²⁺-ATPase (SERCA) activity. Moreover, gAd further increased H/R-enhanced Akt phosphorylation. The protective effects of gAd on ER stress and SERCA activity were abolished by preincubation of rat neonatal cardiomyocytes with the PI3K inhibitor LY294002. Consistent with this finding, I/R-induced ER stress and SERCA dysfunction were also significantly ameliorated by gAd. These effects involved PI3K/Akt signaling pathway. The protective effects of gAd during I/R are mediated, at least in part, by modulating SERCA activity and consequently suppressing ER stress via the activation of PI3K/Akt signaling.

Homocysteine Restricts Copper Availability Leading to Suppression of Cytochrome C Oxidase Activity in Phenylephrine-Treated Cardiomyocytes

Cardiomyocyte hypertrophy induced by phenylephrine (PE) is accompanied by suppression of cytochrome c oxidase (CCO) activity, and copper (Cu) supplementation restores CCO activity and reverses the hypertrophy. The present study was aimed to understand the mechanism of PE-induced decrease in CCO activity. Primary cultures of neonatal rat cardiomyocytes were treated with PE at a final concentration of l00 µM in cultures for 72 h to induce cell hypertrophy. The CCO activity was determined by enzymatic assay and changes in CCO subunit COX-IV as well as copper chaperones for CCO (COX17, SCO2, and COX11) were determined by Western blotting. PE treatment increased both intracellular and extracellular homocysteine concentrations and decreased intracellular Cu concentrations. Studies in vitro found that homocysteine and Cu form complexes. Inhibition of the intracellular homocysteine synthesis in the PE-treated cardiomyocytes prevented the increase in the extracellular homocysteine concentration, retained the intracellular Cu concentration, and preserved the CCO activity. PE treatment decreased protein concentrations of the COX-IV, and the Cu chaperones COX17, COX11, and SCO2. These PE effects were prevented by either inhibition of the intracellular homocysteine synthesis or Cu supplementation. Therefore, PE-induced elevation of homocysteine restricts Cu availability through its interaction with Cu and suppression of Cu chaperones, leading to the decrease in CCO enzyme activity.

Tbx20 promotes cardiomyocyte proliferation and persistence of fetal characteristics in adult mouse hearts

While differentiated cardiomyocytes proliferate prior to birth, adult cardiomyocytes in mammals exhibit relatively little proliferative activity. The T-box transcription factor Tbx20 is necessary and sufficient to promote prenatal cardiomyocyte proliferation, and Tbx20 also is required for adult cardiac homeostasis. The ability of Tbx20 to promote post-natal and adult cardiomyocyte proliferation was examined in mice with cardiomyocyte-specific Tbx20 gain-of-function beginning in the fetal period. In adult hearts, increased Tbx20 expression promotes cardiomyocyte proliferation and results in increased numbers of small, cycling, mononucleated cardiomyocytes, marked by persistent expression of fetal contractile protein genes. In adult cardiomyocytes in vivo and in neonatal rat cardiomyocytes in culture, Tbx20 promotes the activation of BMP2/pSmad1/5/8 and PI3K/AKT/GSK3β/β-catenin signaling pathways concomitant with increased cell proliferation. Inhibition of PI3K/AKT/GSK3β/β-catenin signaling reduces, but does not eliminate, Tbx20-mediated increases in cell proliferation, providing evidence for parallel regulatory pathways downstream of BMP/Smad1/5/8 signaling in promoting cardiomyocyte proliferation after birth. Thus, Tbx20 overexpression beginning in the fetal period activates multiple cardiac proliferative pathways after birth and maintains adult cardiomyocytes in an immature state in vivo.

Modulation of Staurosporine-Activated Volume-Sensitive Outwardly Rectifying Cl- Channel by PI3K/Akt in Cardiomyocytes

Chloride (Cl⁻) channels participate in the regulation of cardiac function in response to stress although the underlying regulatory mechanism remains poorly understood. This study was designed to examine the impact of the pro-apoptotic stimulus staurosporine (STS) on the volume-sensitive outwardly rectifying Cl⁻ current (I(Cl,Vol)) in cardiomyocytes and possible regulatory mechanism involved with a focus on phosphatidylinositol-3 kinase (PI3K)/Akt. Primary cultured rat neonatal cardiomyocytes were subjected to hypotonic and isotonic environment in the presence or absence of STS prior to whole-cell voltage-clamp evaluation of Cl⁻ current. Whole-cell recordings revealed that STS activated an outwardly rectifying Cl⁻ current with phenotypic properties reminiscent of I(Cl,Vol). These currents were outwardly rectifying with a time-dependent inactivation at positive potentials and were sensitive to 4,4'-diisothiocya-natostilbene- 2,2'- disulfonicacid (DIDS), a non-selective Cl⁻ channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl)oxybutyric acid (DCPIB), a selective VSOR Cl⁻ channel blocker. DIDS and DCPIB inhibited I(Cl,Vol) by 92.6% ± 7.3% and 78.4% ± 5.5%, respectively. Our data further revealed that the PI3K inhibitor LY294002 facilitated the current with the peak amplitude of 19.54 ± 2.70 pA/pF. To the contrary, insulin partially inhibited the current amplitude with the peak current amplitude of 15.4 ± 2.13 pA/pF. Taken together, our data depicted staurosporine is capable of activating I(Cl,Vol) channel in cardiomyocytes via possibly a PI3K/Akt-dependent mechanism.

Engraftment Patterns of Human Adult Mesenchymal Stem Cells Expose Electrotonic and Paracrine Proarrhythmic Mechanisms in Myocardial Cell Cultures

After intramyocardial injection, mesenchymal stem cells (MSCs) may engraft and influence host myocardium. However, engraftment rate and pattern of distribution are difficult to control in vivo, hampering assessment of potential adverse effects. In this study, the role of the engraftment patterns of MSCs on arrhythmicity in controllable in vitro models is investigated. Cocultures of 4×10(5) neonatal rat cardiomyocytes and 7% or 28% adult human MSCs (hMSCs) in diffuse or clustered distribution patterns were prepared. Electrophysiological effects were studied by optical mapping and patch-clamping. In diffuse cocultures, hMSCs dose-dependently decreased neonatal rat cardiomyocyte excitability, slowed conduction, and prolonged action potential duration until 90% repolarization (APD90). Triggered activity (14% versus 0% in controls) and increased inducibility of re-entry (53% versus 6% in controls) were observed in 28% hMSC cocultures. MSC clusters increased APD90, slowed conduction locally, and increased re-entry inducibility (23%), without increasing triggered activity. Pharmacological heterocellular electric uncoupling increased excitability and conduction velocity to 133% in 28% hMSC cocultures, but did not alter APD90. Transwell experiments showed that hMSCs dose-dependently increased APD90, APD dispersion, inducibility of re-entry and affected specific ion channel protein levels, whereas excitability was unaltered. Incubation with hMSC-derived exosomes did not increase APD in neonatal rat cardiomyocyte cultures. Adult hMSCs affect arrhythmicity of neonatal rat cardiomyocyte cultures by heterocellular coupling leading to depolarization-induced conduction slowing and by direct release of paracrine factors that negatively affect repolarization rate. The extent of these detrimental effects depends on the number and distribution pattern of hMSCs. These results suggest that caution should be urged against potential adverse effects of myocardial hMSC engraftment.

The Next Regeneration: Identifying Cardiac Regeneration–Inducing MicroRNAs Using Functional Screening

As medical and interventional therapies improve, more patients are surviving myocardial infarction. As a consequence, the prevalence of patients surviving with compromised cardiac function is increasing, leading to significant morbidity and mortality. Unlike most organs in the body, it has long been accepted that the myocardium cannot regenerate in the adult because cardiomyocyte proliferation rapidly ceases after birth. Although this prevailing view has gradually given way to one that cardiomyocytes do have a modest capacity for regeneration, the factors that regulate these cells from entering the cell cycle have remained elusive. Eulalio et al hypothesized that microRNAs (miRNAs) serve as molecular regulators that maintain cardiomyocytes in quiescence and that manipulation of these regulators could improve cardiac regeneration and thus function. The investigators used an unbiased functional screening approach for miRNAs able to induce reentry of cardiomyocytes into the cell cycle. Cultures of neonatal rat cardiomyocytes were transfected with a whole-genome miRNA library of 875 mimics and stained for proliferative markers Ki67 and 5-ethynyl-29-deoxyuridine (EdU) and cardiomyocyte marker α-actinin, followed by high-throughput microscopy. miRNAs that increased neonatal rat cardiomyocyte proliferation 2-fold were selected and then also tested in the mouse. The investigators next selected miRNAs to test in postnatal cardiomyocytes isolated from 7-day-old …

Sweet Chestnut (Castanea sativaMill.) Bark Extract: Cardiovascular Activity and Myocyte Protection against Oxidative Damage

This work was aimed at evaluating the cardioprotective effects of Castanea sativa Mill. (CSM) bark extract characterized in its phenolic composition by HPLC-DAD-MS analysis. The study was performed using primary cultures of neonatal rat cardiomyocytes to investigate the antioxidant and cytoprotective effects of CSM bark extract and isolated guinea pig left and right atria, left papillary muscle, and aorta to evaluate its direct effect on cholinergic and adrenergic response. In cultured cardiomyocytes the CSM bark extract reduced intracellular reactive oxygen species formation and improved cell viability following oxidative stress in dose-dependent manner. Moreover, the extract decreased the contraction induced by noradrenaline (1 μM) in guinea pig aortic strips and induced transient negative chronotropic and positive inotropic effects without involvement of cholinergic or adrenergic receptors in the guinea pig atria. Our results indicate that CSM bark extract exhibits antioxidant activity and might induce cardioprotective effect.

Resveratrol Attenuates Ischemia/Reperfusion Injury in Neonatal Cardiomyocytes and Its Underlying Mechanism

This study was designed to investigate whether Resveratrol (Res) could be a prophylactic factor in the prevention of I/R injury and to shed light on its underlying mechanism. Primary culture of neonatal rat cardiomyocytes were randomly distributed into three groups: the normal group (cultured cardiomyocytes were in normal conditions), the I/R group (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion), and the Res+I/R group (100 µmol/L Res was administered before cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion). To test the extent of cardiomyocyte injury, several indices were detected including cell viability, LDH activity, Na+-K+-ATPase and Ca2+-ATPase activity. To test apoptotic cell death, caspase-3 activity and the expression of Bcl-2/Bax were detected. To explore the underlying mechanism, several inhibitors, intracellular calcium, SOD activity and MDA content were used to identify some key molecules involved. Res increased cell viability, Na+-K+-ATPase and Ca2+-ATPase activity, Bcl-2 expression, and SOD level. While LDH activity, capase-3 activity, Bax expression, intracellular calcium and MDA content were decreased by Res. And the effect of Res was blocked completely by either L-NAME (an eNOS inhibitor) or MB (a cGMP inhibitor), and partly by either DS (a PKC inhibitor) or Glybenclamide (a KATP inhibitor). Our results suggest that Res attenuates I/R injury in cardiomyocytes by preventing cell apoptosis, decreasing LDH release and increasing ATPase activity. NO, cGMP, PKC and KATP may play an important role in the protective role of Res. Moreover, Res enhances the capacity of anti-oxygen free radical and alleviates intracellular calcium overload in cardiomyocytes.

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Introduction: Previous results suggest maternal gestational under nutrition selectively impairs right ventricular mitochondrial response to acute hypoxia. Hypothesis: After an acute ischemic event, ventricular cardiomyocyte viability in intrauterine growth restricted (IUGR) rat pups is lower than controls (CTR) while mitochondrial reactive oxygen species (ROS) production is increased. RV cardiomyocytes have less viability, and increased ROS, post ischemia compared to left ventricular (LV), and the IUGR RV group with the least. Methods: Pregnant Sprague Dawley rats were subjected to caloric restriction in the 3rd trimester- an established caloric restrictive diet to model IUGR (NR). NR and CTR neonatal hearts were harvested from age 2-3 day pups. RV and LV cardiomyocytes were cultured separately. On day 5 of culture, cells underwent 6 hours of oxygen glucose deprivation (OGD), and cell viability was assessed using an MTT Cell Proliferation Assay 18 hour later. Mitochondrial ROS production was measured by total superoxide production using confocal microscopy. Results: Baseline cell viability in CTR RV was 47% (p?0.01-19) of CTR LV.and NR RV was 30% NR LV (p?0.01-23). Total ROS in CTR RV was 118% of CTR LV (p?0.34) and NR RV was 140% NR LV (p?0.001). A significant increase in ROS production was seen in NR compared to CTR. Total ROS in NR LV was 174% CTR LV (p?0.01) and NR RV was 170% CTR RV (p?0.0001).No difference in cell viability was found following OGD (each group compared to its baseline), CTR LV=56%, CTR RV=61%, NR LV=53% and NR RV=56% (p?0.17). All cells for confocal microscopy died post OGD. Conclusions: Cell viability, using primary neonatal rat cardiomyocyte cultures, is significantly less in RV cardiomyocytes compared to LV. Cell viability was further reduced in cardiomyocytes isolated from NR pups, least in NR RV. OGD, simulating ischemia, decreased cell viability in both NR and CTR. ROS production is greater in mitochondria from cardiomyocytes isolated from NR pups. ROS production was greater NR RV compared to NR LV. Less viability and increased ROS production within the NR group may indicate a defect in bioenergetics as a result of intrauterine nutritional restriction.

Keeping the beat. Focus on “Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro”

despite 20–30 years of extensive research into the cellular and molecular mechanisms underlying the development of cardiovascular disease (CVD), CVD remains the leading cause of death (25% all-cause mortality) in men and women. Nevertheless, advances in CVD therapeutics, coupled with improved

Phytochemical profile and nutraceutical value of old and modern common wheat cultivars.

Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention as they have strong antioxidant properties and can protect against many degenerative diseases. Aim of this study was to profile grain phenolic extracts of one modern and five old common wheat (Triticum aestivum L.) varieties and to evaluate their potential antiproliferative or cytoprotective effect in different cell culture systems.Wheat extracts were characterized in terms of antioxidant activity and phenolic composition (HPLC/ESI-TOF-MS profile, polyphenol and flavonoid contents). Results showed that antioxidant activity (FRAP and DPPH) is mostly influenced by flavonoid (both bound and free) content and by the ratio flavonoids/polyphenols. Using a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes, the potential antiproliferative or cytoprotective effects of different wheat genotypes were evaluated in terms of intracellular reactive oxygen species levels and cell viability. All tested wheat phenolic extracts exerted dose-dependent cytoprotective and antiproliferative effects on cardiomyocytes and HL60 cells, respectively. Due to the peculiar phenolic pattern of each wheat variety, a significant genotype effect was highlighted. On the whole, the most relevant scavenging effect was found for the old variety Verna. No significant differences in terms of anti-proliferative activities among wheat genotypes was observed. Results reported in this study evidenced a correspondence between the in vitro antioxidant activity and potential healthy properties of different extracts. This suggests that an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases.