Primary Cardiomyocyte Cultures Research Articles

Exogenous H2S contributes to recovery of ischemic post-conditioning-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2-STAT3 pathways in the aging cardiomyocytes.

BackgroundHydrogen sulfide (H2S), a third member of gasotransmitter family along with nitric oxide and carbon monoxide, generated from mainly catalyzed by cystathionine-lyase, possesses important functions in the cardiovascular system. Ischemic post-conditioning (PC) strongly protects against the hypoxia/reoxygenation (H/R)-induced injury and apoptosis of cardiomyocytes. However, PC protection is ineffective in the aging cardiomyocytes. Whether H2S restores PC-induced cardioprotection by decrease of reactive oxygen species (ROS) level in the aging cardiomyocytes is unknown.MethodsThe aging cardiomyocytes were induced by treatment of primary cultures of neonatal cardiomyocytes using d-galactose and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. ROS level was analyzed using spectrofluorimeter. Related protein expressions were detected through Western blot.ResultsTreatment of NaHS (a H2S donor) protected against H/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The supplementation of NaHS also decreased the activity of LDH and CK, MDA contents, ROS levels and the phosphorylation of IκBα, NF-κB, JNK2 and STAT3, and increased cell viability, the expression of Bcl-2, the activity of SOD, CAT and GSH-PX. PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the addition of NaHS. The beneficial role of NaHS was similar to the supply of N-acetyl-cysteine (NAC, an inhibitor of ROS), Ammonium pyrrolidinedithiocarbamate (PDTC, an inhibitor of NF-κB) and AG 490 (an inhibitor of JNK2), respectively, during PC.ConclusionOur results suggest that exogenous H2S contributes to recovery of PC-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2/STAT3 pathways in the aging cardiomyocytes.

0015 : Epac signalling in doxorubicin-induced cardiotoxicity: a novel implication in death pathways

The main mechanisms underlying doxorubicin (Dox)-induced cardiotoxicity involve classically reactive oxygen species generation, DNA intercalation and topoisomerase II (TopII) inhibition which lead ultimately to cardiomyocyte death. However, new signalling pathways are now emerging. β-adrenergic signalling with Epac (exchange protein directly activated by cAMP) implication could be worth investigating as Epac activates small G proteins (Rac1, Rho) known to be involved in dox-induced cardiotoxicity. Therefore, we have investigated the Epac role in Dox cardiotoxicity in both in vivo (C57Bl63/ Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro model (primary culture of neonatal rat cardiomyocytes (NRVM), 24h, Dox 1μM). In vivo, long-term follow up showed the development of dilated cardiomyopathy (DCM) 15 weeks post treatment associated with Ca2+ homeostasis dysfunction in Dox-treated mice. We also observed a time-dependent modulation of Epac1, Epac2, Rho A and Rac1 expression with alterated, compensated and final decompensated phase at, 2, 6 and 15 weeks respectively. In vitro, Dox treatments led to a global alteration of Epac signalling: Dox treatment led to a reduced Epac 1 / 2 expressions while Epac1 activity is increased. Dox also decreased Rap1 and Rac1 expression and activities. Moreover, the inhibition of Epac1 (ESI09, CE3F4, shEpac1), but not Epac2, prevented Doxinduced DNA-TopIIβ cleavable complex/DNA damage in part by TopIIβ downregulation. We also reported that Dox induces cell death in cardiomyocytes through activation of the mitochondrial caspase dependent apoptotic pathway which as again prevented by specific EPAC1 inhibition. These results were confirmed in vivo as Dox-induced cardiotoxicity was prevented in Epac1 KO as evidenced by lack of DCM, alteration of cardiac function and calcium homeostasis (15 weeks). In conclusion, Epac could be a valuable therapeutic target to prevent Anthacyclines-induced cardiomyopathy. The author hereby declares no conflict of interest

Regression of pressure overload‐induced cardiac hypertrophy by trientine‐induced copper redistribution to the heart in rats

Sustained pressure overload leads to pathological cardiac hypertrophy, and copper efflux from the heart with an increase in plasma copper concentrations. Dietary copper supplementation replenishes copper content and reverses the pathogenesis in mice with cardiac hypertrophy. We recently observed that copper‐trientine complex increased copper uptake by primary cultures of neonatal rat cardiomyocytes. The present study was undertaken to test the hypothesis that trientine can redistribute copper from plasma to the copper‐depleted heart tissue. Adult male Sprague‐Dawley rats were subjected to ascending aortic constriction (AAC). Sixteen weeks after the operation, cardiac hypertrophy was observed by echocardiography and trientine at a dose of 8 mg/kg, twice a day, was administered orally for 6 weeks. At the end of the treatment, copper concentrations were significantly increased in the heart and significantly reduced in the plasma. Along with copper redistribution, cardiac hypertrophy was reversed almost back to the normal as judged by IVSD and LVPWD, and cardiac function was improved as indicated by EF and FS. In addition, the level of cardiac fibrosis was reduced as reflected by the decrease in the content of collagens type I and III and the reduced cross linking of the collagens. This study demonstrates that trientine, although traditionally considered as a copper chelator, is capable of redistributing copper from plasma to the heart leading to regression of pathological cardiac hypertrophy and improved cardiac function.Support or Funding InformationNational Science Foundation of China (81230004 and 81300109).

Urotensin II induction of neonatal cardiomyocyte hypertrophy involves the CaMKII/PLN/SERCA 2a signaling pathway

Although studies have shown that Urotensin II (UII) can induce cardiomyocyte hypertrophy and UII-induced cardiomyocyte hypertrophy model has been widely used for hypertrophy research, but its precise mechanism remains unknown. Recent researches have demonstrated that UII-induced cardiomyocyte hypertrophy has a relationship with the changes of intracellular Ca2+ concentration. Therefore, the aim of this study was to investigate the mechanisms of cardiomyocyte hypertrophy induced by UII and to explore whether the calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulating of phospholamban (PLN) Thr17–phosphorylation signaling pathway contributed to UII-induced cardiomyocyte hypertrophy. Primary cultures of neonatal rat cardiomyocytes were stimulated for 48h with UII. Cell size, protein/DNA contents and intracellular Ca2+ were determined. Phosphorylated and total forms of CaMKII, PLN and the total amount of serco/endo-plasmic reticulum ATPases (SERCA 2a) were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the CaMKII inhibitor, KN-93. These results showed that UII increased cell size, protein/DNA ratio and intracellular Ca2+, consistent with a hypertrophic response. Furthermore, the phosphorylation of CaMKII and its downstream target PLN (Thr17), SERCA 2a levels were up-regulated by UII treatment. Conversely, treatment with KN-93 reversed all those effects of UII. Taken together, the results suggest that UII can induce cardiomyocyte hypertrophy through CaMKII-mediated up-regulating of PLN Thr17–phosphorylation signaling pathway.

Sirt3 protects mitochondrial DNA damage and blocks the development of doxorubicin-induced cardiomyopathy in mice

Doxorubicin (Doxo) is a chemotherapeutic drug widely used to treat variety of cancers. One of the most serious side effects of Doxo is its dose-dependent and delayed toxicity to the heart. Doxo is known to induce cardiac mitochondrial damage. Recently, the mitochondrial sirtuin SIRT3 has been shown to protect mitochondria from oxidative stress. Here we show that overexpression of SIRT3 protects the heart from toxicity of Doxo by preventing the drug-induced mitochondrial DNA (mtDNA) damage. Doxo treatment caused depletion of Sirt3 levels both in primary cultures of cardiomyocytes and in mouse hearts, which led to massive acetylation of mitochondrial proteins. Doxo-induced toxicity to cardiomyocytes was associated with increased reactive oxygen species (ROS) production, mitochondrial fragmentation, and cell death. Overexpression of SIRT3 helped to attenuate Doxo-induced ROS levels and cardiomyocyte death. Sirt3 knockout (Sirt3.KO) mice could not endure the full dose of Doxo treatment, developed exacerbated cardiac hypertrophy, and died during the course of treatment, whereas Sirt3 transgenic (Sirt3.tg) mice were protected against Doxo-induced cardiotoxicity. Along with Sirt3, we also observed a concomitant decrease in levels of oxoguanine-DNA glycosylase-1 (OGG1), a major DNA glycosylase that hydrolyzes oxidized-guanine (8-oxo-dG) to guanine. Depletion of OGG1 levels was associated with increased mtDNA damage. Sirt3.KO mice and Doxo-treated mice showed increased 8-oxo-dG adducts in DNA and corresponding increase in mtDNA damage, whereas, 8-oxo-dG adducts and mtDNA damage were markedly reduced in Sirt3 overexpressing transgenic mice hearts. These results thus demonstrated that Sirt3 activation protects the heart from Doxo-induced cardiotoxicity by maintaining OGG1 levels and protecting mitochondria from DNA damage.

CaV1.2 Interaction with AT1R Reduces Receptor Internalization

The cardiac L type calcium channel is a multi-subunit complex that includes CaV1.2 as the pore-forming subunit that co-assembles with the auxiliary CaVa2d1 subunit and with the CaVb subunit. The main role of these auxiliary subunits is to modify the amount of channels inserted in the plasma membrane. Similarly, trafficking of these channels is controlled by direct interaction with other proteins such as some G-protein coupled receptors (GPCR). Angiotensin II (AngII) type I receptor (AT1R) is a GPCR well known to be involved in the intracellular calcium regulation at the heart, modulating cardiomyocytes contractibility. In this work we show, by co-immunoprecipitation, that AT1R forms a macro-complex with CaV1.2 in primary culture of newborn rat cardiomyocytes and in an heterologous system. Standard biochemical approaches were used to demonstrate that these proteins colocalize at the plasma membrane and to determine the region of CaV1.2 responsible for this interaction. The functional impact of this complex was studied in terms of L-type currents inhibition and in terms of internalization of AT1R. Although inhibition of endogenous L-type current in cardiomyocytes was found to be dependent on the CaVb2 transcriptional start site (TSS) expressed, the presence of the macro-complex was found to be independent on the variant expressed. The effect of this interaction over AT1R internalization was studied in cells stimulated with AngII at different times. As expected, in cell expressing only the AT1R and stimulated with AngII, the receptor is readily internalized after 15 min, however, in cells expressing also CaV1.2, AT1R internalization (and CaV1.2) is observed only after 30 minutes of AngII stimulation. Overall, our results demonstrate that AT1R interaction with CaV1.2 increases the residence time at the plasma membrane of the receptor and induce L-type channel internalization after prolonged AngII exposure.

Differential Gene Expression Patterns in Chicken Cardiomyocytes during Hydrogen Peroxide-Induced Apoptosis.

Hydrogen peroxide (H2O2) is both an exogenous and endogenous cytotoxic agent that can reliably induce apoptosis in numerous cell types for studies on apoptosis signaling pathways. However, little is known of these apoptotic processes in myocardial cells of chicken, a species prone to progressive heart failure. Sequencing of mRNA transcripts (RNA-Seq) allows for the identification of differentially expressed genes under various physiological and pathological conditions to elucidate the molecular pathways involved, including cellular responses to exogenous and endogenous toxins. We used RNA-seq to examine genes differentially expressed during H2O2-induced apoptosis in primary cultures of embryonic chicken cardiomyocytes. Following control or H2O2 treatment, RNA was extracted and sequencing performed to identify novel transcripts up- or downregulated in the H2O2 treatment group and construct protein−protein interaction networks. Of the 19,268 known and 2,160 novel transcripts identified in both control and H2O2 treatment groups, 4,650 showed significant differential expression. Among them, 55.63% were upregulated and 44.37% downregulated. Initiation of apoptosis by H2O2 was associated with upregulation of caspase-8, caspase-9, and caspase-3, and downregulation of anti-apoptotic genes API5 and TRIA1. Many other differentially expressed genes were associated with metabolic pathways (including ‘Fatty acid metabolism’, ‘Alanine, aspartate, and glutamate metabolism’, and ‘Biosynthesis of unsaturated fatty acids’) and cell signaling pathways (including ‘PPAR signaling pathway’, ‘Adipocytokine signaling pathway’, ‘TGF-beta signaling pathway’, ‘MAPK signaling pathway’, and ‘p53 signaling pathway’). In chicken cardiomyocytes, H2O2 alters the expression of numerous genes linked to cell signaling and metabolism as well as genes directly associated with apoptosis. In particular, H2O2 also affects the biosynthesis and processing of proteins and unsaturated fatty acids. These results highlight the value of RNA-seq for revealing unexpected molecular contributors to oxidative stress responses, thereby identifying novel potential therapeutic targets.

Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures.

Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients.

Impact of miR-208 and its Target Gene Nemo-Like Kinase on the Protective Effect of Ginsenoside Rb1 in Hypoxia/Ischemia Injuried Cardiomyocytes

Background/Aims: Ginsenoside Rb1 (GS-Rb1) is one of the most important active pharmacological extracts of the Traditional Chinese Medicine ginseng, with extensive evidence of its cardioprotective properties. Mir-208 has been shown to act as a biomarker of acute myocardial infarction in vivo studies including man. However the impact of miR-208 on the protective effect of GS-Rb1 in hypoxia/ischemia injured cardiomyocytes remains unclear. The current study aims to investigate the target gene of miR-208 and the impact on the protective effect of GS-Rb1 in hypoxia/ischemia (H/I) injuried cardiomyocytes. Materials and Methods: Primary cultures of neonatal rat cardiomyocytes (NRCMs) was subjected to the H/I conditions with or without GS-Rb1. Cell viability was calculated by MTT assay and confirmed by flow cytometry analysis. Mir-208 was then detected by qRT-PCR. Luciferase reporter assay was carried out to detect the target gene of Mir-208. Then the NRCMs were transfected with miR-208 mimics and inhibitors to evaluate the impact on cardioprotective properties of Rb1. Results: The miR-208 expression level was clearly upregulated in the H/I treated NRCMs accompanied by the percentage of the apoptotic cells which could be reversed by GS-Rb1 pretreatment. The nemo-like kinase (NLK) mRNA and protein expression levels were decreased in H/I group measured by RT-PCR and western blotting. Luciferase activity assay was then carried out to identify that NLK may be a direct target of mir-208. MTT assay showed that miR-208 inhibitor slightly decreased the protective effect of Rb1 on the H/I impaired NRCMs. However, results showed no statistical difference. Conclusions: These findings proved that NLK was a direct target of mir-208 and miR-208 act indirectly during Rb1 protecting H/I impaired NRCMs and further researches were needed to explore the relationship that microRNAs and other signal pathways in the protective effect of GS-Rb1 on the hypoxia/ischemia injuries in cardiomyocytes.

Curcumin Protects Neonatal Rat Cardiomyocytes against High Glucose-Induced Apoptosis via PI3K/Akt Signalling Pathway.

The function of curcumin on NADPH oxidase-related ROS production and cardiac apoptosis, together with the modulation of protein signalling pathways, was investigated in cardiomyocytes. Primary cultures of neonatal rat cardiomyocytes were exposed to 30 mmol/L high glucose with or without curcumin. Cell viability, apoptosis, superoxide formation, the expression of NADPH oxidase subunits, and potential regulatory molecules, Akt and GSK-3β, were assessed in cardiomyocytes. Cardiomyocytes exposure to high glucose led to an increase in both cell apoptosis and intracellular ROS levels, which were strongly prevented by curcumin treatment (10 μM). In addition, treatment with curcumin remarkably suppressed the increased activity of Rac1, as well as the enhanced expression of gp91phox and p47phox induced by high glucose. Lipid peroxidation and SOD were reversed in the presence of curcumin. Furthermore, curcumin treatment markedly inhibited the reduced Bcl-2/Bax ratio elicited by high glucose exposure. Moreover, curcumin significantly increased Akt and GSK-3β phosphorylation in cardiomyocytes treated with high glucose. In addition, LY294002 blocked the effects of curcumin on cardiomyocytes exposure to high glucose. In conclusion, these results demonstrated that curcumin attenuated high glucose-induced cardiomyocyte apoptosis by inhibiting NADPH-mediated oxidative stress and this protective effect is most likely mediated by PI3K/Akt-related signalling pathway.

The association of depressed angiogenic factors with reduced capillary density in the Rhesus monkey model of myocardial ischemia.

Depressed capillary density is associated with myocardial ischemic infarction, in which hypoxia-inducible factor 1α (HIF-1α) is increased. The present study was undertaken to examine changes in the angiogenic factors whose expression is regulated by HIF-1 and their relation to the depressed capillary density in the Rhesus monkey model of myocardial ischemic infarction. Male Rhesus monkeys 2-3 years old were subjected to myocardial ischemia by permanent ligation of left anterior descending (LAD) artery leading to the development of myocardial infarction. Eight weeks after LAD ligation, copper concentrations, myocardial histological changes and capillary density were examined, along with Western blot and immunohistochemical analysis of angiogenic factors and detection of HIF-1 activity. Capillary density was significantly decreased but the concentrations of HIF-1α and HIF-1β were significantly increased in the infarct area. However, the levels of mRNA and protein for VEGF and VEGFR1 were significantly decreased. Other HIF-1 regulated angiogenic factors, including Tie-2, Ang-1 and FGF-1, were also significantly depressed, but vascular destabilizing factor Ang-2 was significantly increased. Copper concentrations were depressed in the infarct area. Copper-independent HIF-1 activity was increased shown by the elevated mRNA level of IGF-2, a HIF-1 target gene. Removal of copper by a copper chelator, tetraethylenepentamine, from primary cultures of neonatal rat cardiomyocytes also suppressed the expression of HIF-1 regulated VEGF and BNIP3, but not IGF-2. The data suggest that under ischemic conditions, copper loss suppressed the expression of critical angiogenic genes regulated by HIF-1, but did not affect copper-independent HIF-1 activation of gene expression. This copper-dependent dysregulation of angiogenic gene expression would contribute to the pathogenesis of myocardial ischemic infarction.

Rapid effects of aldosterone in primary cultures of cardiomyocytes – do they suggest the existence of a membrane-bound receptor?

Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca2+. Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.

Zinc-induced metallothionein overexpression prevents doxorubicin toxicity in cardiomyocytes by regulating the peroxiredoxins

1. Cardiotoxicity is an important factor that limits the clinical use of doxorubicin (Dox). Metallothionein (MT) can antagonize the Dox-induced cardiotoxicity. Using a proteomics approach we have detected that major peroxiredoxins (Prxs) may be involved in this process. In the present study, we further investigate the mechanisms of the MT effects against Dox-induced cytotoxicity and the interactions between MT and Prxs.2. We have established a primary cardiomyocyte culture system from MT-I/II null (MT−/−) and corresponding wild type (MT+/+) neonatal mice, and pretreated the MT+/+ cardiomyocytes with ZnCl2 to establish the MT overexpression cardiomyocyte model.3. Based on the results, in MT+/+ cardiomyocytes, ZnCl2 pretreatment significantly increased the cardiomyocytes MT levels and inhibited the cardiotoxicity of Dox; it can resist LDH leakage, cardiomyocyte apoptosis, DNA damage, ROS accumulation and inhibit the decrease in activity of antioxidant enzymes induced by Dox. Moreover, ZnCl2 enhanced the expression of Prx-2, -3, -5 and -6, it can inhibit the expression of Prxs decrease in MT+/+ cardiomyocytes induced by Dox, but had no effect in MT−/− cardiomyocytes.4. Therefore, the present study suggests that ZnCl2 can protect the cardiomyocytes from the Dox-induced oxidative injury and can inhibit the changes in Prxs expression through induced MT overexpression.

Matrix Metalloproteinases in Primary Culture of Cardiomyocytes.

The highly organized contractile apparatus of cardiomyocytes in heart tissue allows for their continuous contractility, whereas extracellular matrix components are synthesized and spatially organized by fibroblasts and endothelial cells. However, reorganization of the cardiomyocyte contractile apparatus occurs upon their 2D cultivation, which is accompanied by transient loss of their contractility and acquired capability of extracellular matrix synthesis (Bildyug, N. B., and Pinaev, G. P. (2013) Tsitologiya, 55, 713-724). In this study, matrix metalloproteinases were investigated at different times of cardiomyocyte 2D cultivation and 3D cultivation in collagen gels. It was found that cardiomyocytes in 2D culture synthesize matrix metalloproteinases MMP-2 and MMP-9, wherein their amount varies with the cultivation time. The peak MMP-9 amount is at early cultivation time, when the reorganization of cardiomyocyte contractile apparatus occurs, and the MMP-2 peak precedes the recovery of the initial organization of their contractile apparatus. Upon cardiomyocyte cultivation in 3D collagen gels, in which case their contractile apparatus does not rearrange, a steady small amount of MMP-2 and MMP-9 is observed. These data indicate that the cardiomyocyte contractile apparatus reorganization in culture is associated with synthesis and spatial organization of their own extracellular matrix.

Expression of calcineurin, calpastatin and heat shock proteins during ischemia and reperfusion

ObjectiveCalcineurin (CaN) interacts with calpains (Calpn) and causes cellular damage eventually leading to cell death. Calpastatin (Calp) is a specific Calpn inhibitor, along with CaN stimulation has been implicated in reduced cell death and self-repair. Molecular chaperones, heat shock proteins (Hsp70 and Hsp90) acts as regulators in Calpn signaling. This study aims to elucidate the role of CaN, Calp and Hsps during induced ischemia and reperfusion in primary cardiomyocyte cultures (murine). Methods and resultsProtein expression was analyzed concurrently with viability using flow cytometry (FACS) in ischemia- and reperfusion-induced murine cardiomyocyte cultures. The expression of Hsp70 and Hsp90, both being molecular chaperones, increased during ischemia with a concurrent increase in death of cells expressing these proteins. The relative expression of Hsp70 and Hsp90 during ischemia with respect to CaN was enhanced in comparison to Calp. Reperfusion slightly decreased the number of cells expressing these chaperones. There was no increase in death of cells co-expressing Hsp70 and Hsp90 along with CaN and Calp. CaN expression peaked during ischemia and subsequent reperfusion reduced its expression and cell death. Calp expression increased both during ischemia and subsequent reperfusion but cell death decreased during reperfusion. ConclusionThe present study adds to the existing knowledge that Hsp70, Hsp90, CaN and Calp interact with each other and play significant role in cardio protection.

SIRT2 regulates microtubule stabilization in diabetic cardiomyopathy

Stable microtubules (MTs) is involved the mechanism of diabetic cardiomyopathy (DCM), which is induced by acetylation of α-tubulin. The present study investigated whether SIRT2, a deacetylase, regulates MT stability through α-tubulin deacetylation in DCM and whether the receptor of advanced glycation end products (AGEs) signaling pathway is involved in this effect. Type 1 diabetic mellitus (T1DM) rats model was established by a single intraperitoneal injection of streptozotocin (STZ, 65mg/kg), and neonatal rat cardiomyocytes were also cultured. Heart function was detected by Doppler. MT stability was elevated by β-tubulin expression density. The protein expression of SIRT2, acetylated α-tubulin and AGEs receptor were detected by immunohistochemistry or Western blots. The interaction of SIRT2 and acetylated α-tubulin was detected by Co-immunoprecipitation. In an animal model of T1DM, Western blots and immunohistochemistry revealed downregulation of SIRT2 but upregulation of the acetylated α-tubulin protein. These effects were reduced by treatment of aminoguanidine, an inhibitor of AGEs production. HDAC6 expression did not regulated in heart. In primary cultures of neonatal rat cardiomyocytes, the AGEs treatment impaired the SIRT2/acetylated α-tubulin signaling pathway, and SIRT2-overexpression reversed the function of AGEs on cardiomyocytes. In addition, gene silencing of AGEs receptor alleviated the impairment effect of AGEs on cardiomyocytes. In conclusion, these data demonstrate that AGEs/AGEs receptor promote MT stabilization via the suppression of the SIRT2/acetylated α-tubulin signaling pathway in DCM development.

Perfluorooctanoic acid-induced toxicity in primary cultures of chicken embryo cardiomyocytes.

Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that induces developmental cardiotoxicity. It is detectable in late stage chicken embryos and hatchling chickens. To investigate mechanism(s) of cardiotoxicity, primary cultures of cardiomyocytes were prepared from 10-day-old chicken embryos that were (A) pre-exposed to vehicle or 2 mg of PFOA/kg of egg weight in ovo or (B) incubated with PFOA in vitro at concentrations ranging from 0 to 100 µg/mL in medium for 1 or 36 h. When viability was assessed, survival of cardiomyocytes prepared from pre-exposed embryos did not differ from vehicle controls, even under conditions of serum starvation designed to challenge the cells. However, 1 h of exposure to 100 µg/mL of PFOA in vitro and 36 h of exposure to 75 and 100 µg/mL PFOA in vitro decreased viability. When contractility was evaluated, cardiomyocytes cultured from pre-exposed embryos exhibited decreases in time to maximum departure velocity and cell length at peak contraction, whereas cardiomyocytes exposed in vitro exhibited a reduction in the 50% relaxation time at a concentration of 1 µg/mL relative to vehicle controls. Morphological assessment revealed decreased cardiomyocytes axial length following in ovo PFOA exposure and 24 h in vitro PFOA 50 µg/mL exposure. Reactive oxygen species (ROS) generation, which was evaluated only in cardiomyocytes exposed to PFOA in vitro, was significantly elevated following incubation with 50 µg/mL of PFOA for 1 h. These data indicate that while in vitro exposure to relatively high concentrations of PFOA can induce cytotoxicity and ROS, developmental cardiotoxicity observed in ovo is not likely mediated via PFOA-induced overt cytotoxicity, but likely by altering early cardiac morphologic and function processes. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1580-1590, 2016.

NF‐қB Activation is Required for Thyroid Hormone Induced Cardiomyocyte Hypertrophy

The nuclear factor NF‐қB promotes inflammatory response and mediates cardiac growth by different hypertrophic stimuli. We have previous results obtained by microarray experiments indicating that thyroid hormone (triiodotyronine, T3) up‐regulates pro‐inflammatory genes. Considering that, we investigated the influence of T3 on NF‐қB signaling and the possible participation of this nuclear factor in cardiomyocyte hypertrophy induced by T3. Primary cultures of neonatal rat ventricular cardiomyocytes were treated with T3 for 10, 30 minutes and 24 hours and the NF‐қB signaling was analyzed by western blot. Also, cardiomyocytes were treated with an inhibitor of NF‐қB (JSH‐23) followed by treatment with T3 for evaluation of mRNA expression of atrial natriuretic factor (ANF) and cell surface area analysis. Data were presented as mean ± SD and were analyzed using one‐way ANOVA and Tukey multiple comparisons test. Values of p<0.05 were considered statistically significant. T3 rapidly increased the nuclear protein expression of NF‐қB without alterations on IKK and IқB expression. The inhibition of NF‐қB reduced both ANF mRNA levels and the surface area of cardiomyocytes compared to T3 treated cells. These results confirm that NF‐қB is required for development of cardiomyocyte hypertrophy by T3. The possible interactions of NF‐қB with genes associated with cardiac growth are being investigated. Financial support: Fapesp (2013/22480‐4 and 2011/23352‐4).

Estrogen-Related Receptor α (ERRα) is required for adaptive increases in PGC-1 isoform expression during electrically stimulated contraction of adult cardiomyocytes in sustained hypoxic conditions

Background and objectivesIn adult myocardium, Estrogen-Related Receptor α (ERRα) programs energetic capacity of cardiomyocytes by regulating expression of target genes required for mitochondrial biogenesis, fatty acid metabolism and oxidative phosphorylation. Transcriptional activation by ERRα is dependent on the α or β isoform of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1). This study utilized a model of continuously contracting adult cardiomyocytes to determine the effects of sustained oxygen reduction (hypoxia) on ERRα target gene expression. Methods and resultsAdult feline cardiomyocytes in primary culture were electrically stimulated to contract at 1Hz in either normoxia (21% O2) or hypoxia (0.5% O2). Compared to normoxia, hypoxia increased PGC-1α mRNA and PGC-1β mRNA levels by 16-fold and 14-fold after 24h. ERRα mRNA levels were increased 3-fold by hypoxia over the same time period. Treatment of cardiomyocytes with XCT-790, an ERRα inverse agonist, caused knockdown of ERRα protein expression. The increases in PGC-1 mRNA levels in response to hypoxia were blocked by XCT-790 treatment, which indicates that expression of PGC-1 isoforms is dependent on ERRα activity. The products of two ERRα target genes required for energy metabolism, Cox6c mRNA and Fabp3 mRNA, increased by 4.5-fold and 3.5 fold after 24h of hypoxia as compared to normoxic controls. These increases were blocked by XCT-790 treatment of hypoxic cardiomyocytes with a concomitant decrease in ERRα expression. ConclusionsERRα activity is required to increase expression of PGC-1 isoforms and downstream target genes as part of the adaptive response of contracting adult cardiomyocytes to sustained hypoxia.

Effects of 5-fluorouracil on morphology, cell cycle, proliferation, apoptosis, autophagy and ROS production in endothelial cells and cardiomyocytes.

Antimetabolites are a class of effective anticancer drugs interfering in essential biochemical processes. 5-Fluorouracil (5-FU) and its prodrug Capecitabine are widely used in the treatment of several solid tumors (gastro-intestinal, gynecological, head and neck, breast carcinomas). Therapy with fluoropyrimidines is associated with a wide range of adverse effects, including diarrhea, dehydration, abdominal pain, nausea, stomatitis, and hand-foot syndrome. Among the 5-FU side effects, increasing attention is given to cardiovascular toxicities induced at different levels and intensities. Since the mechanisms related to 5-FU-induced cardiotoxicity are still unclear, we examined the effects of 5-FU on primary cell cultures of human cardiomyocytes and endothelial cells, which represent two key components of the cardiovascular system. We analyzed at the cellular and molecular level 5-FU effects on cell proliferation, cell cycle, survival and induction of apoptosis, in an experimental cardioncology approach. We observed autophagic features at the ultrastructural and molecular levels, in particular in 5-FU exposed cardiomyocytes. Reactive oxygen species (ROS) elevation characterized the endothelial response. These responses were prevented by a ROS scavenger. We found induction of a senescent phenotype on both cell types treated with 5-FU. In vivo, in a xenograft model of colon cancer, we showed that 5-FU treatment induced ultrastructural changes in the endothelium of various organs. Taken together, our data suggest that 5-FU can affect, both at the cellular and molecular levels, two key cell types of the cardiovascular system, potentially explaining some manifestations of 5-FU-induced cardiovascular toxicity.