Primary Cultured Rat Cardiomyocytes Research Articles

Abstract 17696: Perm1 Stabilizes Nrf2 via Keap1 Oxidation to Protect the Heart Against Reperfusion Injury

Introduction: Reperfusion after ischemia (IR) produces reactive oxygen species (ROS) that promote myocardial injury. Nrf2 is a transcription factor that promotes transcription of antioxidant enzymes. Keap1 is a negative regulator of Nrf2. When oxidated by ROS, Keap1 releases Nrf2, leading to Nrf2 activation. Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) is a scaffold or adaptor protein that maintains cellular metabolism. However, whether and how Perm1 protects the heart against IR injury via Nrf2 remains unknown. Methods: We used systemic Perm1 knockout (KO) mice and adeno-associated virus (AAV)-induced cardiac-specific Perm1 overexpression to investigate the pathological role of Perm1 in IR injury. Results: IR-induced myocardial infarction was greater in Perm1KO mice than in control mice. Consistently, infarction was smaller in mice with Perm1 overexpression (Infarction size/area at risk (AAR): AAV-GFP as a control 0.41 and AAV-Perm1 0.28*; p<0.05). Myocardial oxidative stress, evaluated via increases in dityrosine, 4HNE and GSSG/GSH, after IR was greater in Perm1KO but less in Perm1 overexpression mice. Expression of IR-induced Nrf2 target genes, such as catalase, superoxide dismutase 2, heme oxygenase 1, NADH quinone dehydrogenase 1 and thioredoxin 1, was inhibited in Perm1KO but promoted in overexpression mice. The detrimental effects observed in Perm1KO mice were normalized by ML334, an inhibitor of Keap1-Nrf2 interaction (Infarction size/AAR: Wild 0.42; Perm1KO 0.58; Wild with ML334 0.37 and Perm1KO with ML334 0.42*; p<0.05 vs. Perm1KO), suggesting that Perm1 protects the heart via Nrf2. In primary cultured rat cardiomyocytes, Perm1 was bound to Keap1 and promoted its oxidation, thereby stabilizing and activating Nrf2. Perm1-induced Nrf2 activation was verified with reporter gene assays using a consensus binding sequence of Nrf2, termed antioxidant response element. In an in vitro system using recombinant proteins, Perm1 directly bound to Keap1. Conclusions: These results suggest that Perm1 is a novel regulator of cellular redox homeostasis through Nrf2 activation via Keap1 oxidation that protects the heart against IR injury.

Abstract MP241: Roflumilast-mediated Phosphodiesterase 4d Inhibition Reverses Diabetes-associated Cardiac Dysfunction And Remodeling: Effects Beyond Glucose Lowering

Introduction: Patients with type 2 diabetes (T2DM) have a substantial risk of developing cardiovascular disease. Our recent studies reveal that hyperinsulinemia attenuates cardiac contractility by inducing expression of phosphodiesterase 4D (PDE4D) that increases cAMP degradation. Furthermore, there is growing evidence that PDE4 dysregulation is of pathophysiological importance in metabolic disorders. Hypothesis: We propose that inhibition of PDE4D might ameliorate diabetes-associated cardiac dysfunction, in addition to lowering glucose. Methods: Male C57BL/6J mice fed with high-fat diet (HFD) were treated with PDE4 inhibitor roflumilast (currently used to treat chronic obstructive pulmonary disease, COPD). Myocardial structure, contractile function and remodeling were evaluated. For mechanistic studies, specific silencing of cardiac PDE4D and overexpression of miR-1 were administrated in mice undergoing high-fat feeding. These studies were complemented by in vitro analysis in primary cultured rat cardiomyocytes and cardiac fibroblasts. Results: Mice on HFD developed greater body weight compared to normal chow diet-fed mice, manifesting hyperglycemia, hyperinsulinemia, cardiac contractile dysfunction and remodeling, and cardiac PDE4D5 upregulation by 24 weeks. The expression of PDE4D5 was also elevated in human hearts with diabetes. In comparison with vehicle-treated HFD controls, PDE4 inhibitor roflumilast (1mg/kg/day) can prevent and even reverse hyperglycemia and cardiac dysfunction, accompanied by the decrease of cardiac PDE4D expression. In addition, cardiac miR-1 level was reduced in HFD mice, which was restored by PDE4 inhibitor roflumilast treatment. Either cardiac specific PDE4D5 knockdown or miR-1 overexpression significantly reversed cardiac dysfunction in HFD-mice, despite persistence of hyperglycemia and glucose intolerance. Gain- and loss-of-function studies of PDE4D in cardiomyocytes implicated that suppression of PDE4D protected cardiac hypertrophy via SERCA2a-mediated miR-1 restoration. Moreover, inhibition of PDE4D prevented insulin-activated TGF-β1 signaling which promotes miR-1 reduction in cardiac fibroblasts and subsequent fibrosis. Conclusions: These studies elucidate a novel mechanism by which PDE4D contributes to HFD-induced heart failure through reducing miR-1 expression in both cardiomyocytes and cardiac fibroblasts and suggest a therapeutic potential of PDE4 inhibitor roflumilast in preventing or treating cardiac dysfunction associated with diabetes.

Effects of docosahexaenoic acid or arachidonic acid supplementation on gene expression and contractile force of rat cardiomyocytes in primary culture

While fatty acids play essential roles in the physiology of the myocardium, conventional culture media contain little lipid. We previously revealed that rat neonatal myocardium mainly contains docosahexaenoic (DHA), linoleic (LA), and arachidonic (AA) acids as polyunsaturated fatty acids (PUFAs), and these contents in cultured cardiomyocytes derived from fetal rats were markedly lower than those in the neonatal myocardium. In this study, we first assessed the effects of supplementation of DHA, LA, or AA on the fatty acid contents and the percentage change of contractile area in primarily cultured rat cardiomyocytes. Based on this assessment, we then evaluated the effects of DHA or AA supplementation on mRNA expression and further directly measured the contractile force of cardiomyocytes with the supplementations. This study revealed that percentage change of contractile area was maximized under 20 μM DHA or 50 μM AA supplementation while LA supplementation did not affect this contraction index, and that a widespread upregulation tendency of the mRNA expression related to differentiation, maturity, fatty acid metabolism, and cell adhesion was seen in the cultured cardiomyocytes with supplementation of DHA or AA. In particular, upregulation of the gene expression of cellular adhesion molecules connexin43 and N-cadherin were remarkable, whereas the effects on differentiation and maturation were less pronounced. Correspondingly, the increase of the percentage change of the contractile area of cardiomyocyte clusters in culture dishes with the supplementations was significant, whereas the enhancement of the contractile force was modest. These results suggest that supplementation of DHA or AA to the fetal cardiomyocyte culture may play effective roles in preventing the de-differentiation of the cardiomyocytes in culture and that the enhancement of the contractile performance may be mainly attributed to the improvement of intercellular connection.

Role of IgE-FcεR1 in Pathological Cardiac Remodeling and Dysfunction.

Immunoglobulin E (IgE) belongs to a class of immunoglobulins involved in immune response to specific allergens. However, the roles of IgE and IgE receptor (FcεR1) in pathological cardiac remodeling and heart failure are unknown. Serum IgE levels and cardiac FcεR1 expression were assessed in diseased hearts from human and mouse. The role of FcεR1 signaling in pathological cardiac remodeling was explored in vivo by FcεR1 genetic depletion, anti-IgE antibodies, and bone marrow transplantation. The roles of the IgE-FcεR1 pathway were further evaluated in vitro in primary cultured rat cardiomyocytes and cardiac fibroblasts (CFs). RNA sequencing and bioinformatic analyses were used to identify biochemical changes and signaling pathways that are regulated by IgE/FcεR1. Serum IgE levels were significantly elevated in patients with heart failure as well as in 2 mouse cardiac disease models induced by chronic pressure overload via transverse aortic constriction and chronic angiotensin II infusion. Interestingly, FcεR1 expression levels were also significantly upregulated in failing hearts from human and mouse. Blockade of the IgE-FcεR1 pathway by FcεR1 knockout alleviated transverse aortic constriction- or angiotensin II-induced pathological cardiac remodeling or dysfunction. Anti-IgE antibodies (including the clinical drug omalizumab) also significantly alleviated angiotensin II-induced cardiac remodeling. Bone marrow transplantation experiments indicated that IgE-induced cardiac remodeling was mediated through non-bone marrow-derived cells. FcεR1 was found to be expressed in both cardiomyocytes and CFs. In cultured rat cardiomyocytes, IgE-induced cardiomyocyte hypertrophy and hypertrophic marker expression were abolished by depleting FcεR1. In cultured rat CFs, IgE-induced CF activation and matrix protein production were also blocked by FcεR1 deficiency. RNA sequencing and signaling pathway analyses revealed that transforming growth factor-β may be a critical mediator, and blocking transforming growth factor-β indeed alleviated IgE-induced cardiomyocyte hypertrophy and cardiac fibroblast activation in vitro. Our findings suggest that IgE induction plays a causative role in pathological cardiac remodeling, at least partially via the activation of IgE-FcεR1 signaling in cardiomyocytes and CFs. Therapeutic strategies targeting the IgE-FcεR1 axis may be effective for managing IgE-mediated cardiac remodeling.

An α2-adrenoceptor agonist: Dexmedetomidine induces protective cardiomyocyte hypertrophy through mitochondrial-AMPK pathway

Aims: Dexmedetomidine (Dex) as a highly selective α2-adrenoceptor agonist, was widely used anesthetic in perioperative settings, whether Dex induces cardiac hypertrophy during perioperative administration is unknown.Methods: The effects of Dex on cardiac hypertrophy were explored using the transverse aortic constriction model and neonatal rat cardiomyocytes.Results: We reported that Dex induces cardiomyocyte hypertrophy with activated ERK, AKT, PKC and inactivated AMPK in both wild-type mice and primary cultured rat cardiomyocytes. Additionally, pre-administration of Dex protects against transverse aortic constriction induced-heart failure in mice. We found that Dex up-regulates the activation of ERK, AKT, and PKC via suppression of AMPK activation in rat cardiomyocytes. However, suppression of mitochondrial coupling efficiency and membrane potential by FCCP blocks Dex induced AMPK inactivation as well as ERK, AKT, and PKC activation. All of these effects are blocked by the α2-adrenoceptor antagonist atipamezole.Conclusion: The present study demonstrates Dex preconditioning induces cardiac hypertrophy that protects against heart failure through mitochondria-AMPK pathway in perioperative settings.

Protein acetylation, a new way of regulating glucose metabolism in diabetic heart

At rest, the heart favors fatty acids to produce its energy switching to glucose in response to insulin at postprandial state. Diabetic heart features metabolic adaptation defaults and is no longer able to switch, producing its energy exclusively through fatty acid oxidation. This metabolic inflexibility is a major factor of cardiac dysfunction. Observations made in high-fat diet mouse model lead to an increase in cardiac protein acetylation. We hypothesized that modulation of acetylation impacts the ability of the heart to uptake glucose with a special focus on tubulin. Microtubules playing a key role in GLUT4 translocation to the plasma membrane, we evaluated the impact of tubulin acetylation on cardiac glucose uptake. The model is primary cultured rat cardiomyocytes. Acetylation levels and signaling pathways are evaluated by western blotting. The impact of long term incubation (20 h) with different pharmacological reducers of acetylation (garcinol 10 μM and anacardic acid 25 μM) on basal and insulin (3 nM, 30 min)-stimulated glucose uptake are measured following the detritiation rate of 2-3H-glucose. Tubacin, the inhibitor of tubulin deacetylase and an adenovirus overexpressing a non-acetylable form of tubulin are used to modulate tubulin acetylation. Insulin induces a 6-fold increase in glucose transport. Pharmacologically inhibiting global acetylation increases basal glucose transport similarly to insulin stimulation. By contrast, increasing specifically tubulin acetylation using tubacin inhibits 40% of basal and insulin-induced glucose transport. Accordingly, the overexpression of a dominant negative form and non-acetylable form of tubulin decreases endogenous tubulin acetylation while increasing basal glucose transport. Finally, targeting tubulin acetylation partially restores fatty acid-mediated inhibition of glucose transport. There is a clear correlation between tubulin acetylation and glucose uptake level.

Reduced mitochondrial response sensitivity is involved in the anti‑apoptotic effect of dexmedetomidine pretreatment in cardiomyocytes.

Dexmedetomidine is a commonly used α2-adreno-ceptor agonist, which affects various organs, including providing beneficial effects on the heart. However, the mechanism underlying the cardiac benefit remains to be fully elucidated. In the present study, it was demonstrated that dexmedetomidine pretreatment on primary cultured rat cardiomyocytes protected against reactive oxygen species (ROS)‑induced apoptosis. In terms of the potential mechanism, it was demonstrated that dexmedetomidine inhibited mitochondrial biogenesis and mitochondrial respiratory complexes, but with increased coupling efficiency. However, dexmedetomidine upregulated mitochondrial membrane potential (Δψm) and resisted against the loss of Δψm induced by carbonilcyanide p‑triflouromethoxyphenylhydrazone. Due to the importance of mitochondria affecting ROS, the present study investigated the dexmedetomidine‑suppressed mitochondrial response to H2O2 stimulation, which was explained by suppressed ROS levels and the suppression of the increased oxygen consumption rate. Results demonstrated for the first time, to the best of our knowledge, a novel protective mechanism for dexmedetomidine on cardiomyocytes through the attenuated response of mitochondria towards H2O2, which had a protective effect against ROS‑induced apoptosis.

Effects of docosahexaenoic or arachidonic acid on contractile function in rat cardiomyocytes in primary culture

Effects of docosahexaenoic or arachidonic acid on contractile function in rat cardiomyocytes in primary culture

Effect of Three Statins on Glucose Uptake of Cardiomyocytes and its Mechanism.

BackgroundThe aim of this study was to investigate the effects of different statins on glucose uptake and to confirm its mechanism in primary cultured rat cardiomyocytes after administration of atorvastatin, pravastatin, and rosuvastatin.Material/MethodsPrimary cultured rat cardiomyocytes were randomly assigned to 5 groups: normal control group (OB), insulin group (S1), statin 1-μM (S2), 5-μM (S3), and 10-μM (S4) groups for 3 different statins. The 2-[3H]-DG uptake of each group was determined and the mRNA and protein expression levels of glucose transporter type 4 (GLUT4), insulin receptor substrate (IRs), and RhoA were assessed.ResultsAfter treatment with different concentrations of statins and insulin, the 2-[3H]-DG uptake showed a significant negative correlation with the concentration of atorvastatin (P<0.05), and no significant correlation with pravastatin and rosuvastatin. The mRNA and protein expression levels of GLUT4 and IRs-1 in primary cultured cardiomyocytes were both significantly reduced by atorvastatin treatment (P<0.05). Pravastatin and rosuvastatin showed no significant effects on GLUT4 and IRs-1 expression. The mRNA and protein expression levels of RhoA both showed no significant difference when treated with the 3 statins.ConclusionsThese results confirm that atorvastatin can inhibit insulin-induced glucose uptake in primary cultured rat cardiomyocytes by regulating the PI3K/Akt insulin signal transduction pathway.

Effect of icariin on hypoxia/reoxygenation injury in neonatal rat cardiomyocytes

To investigate the effect of icariin on myocardial hypoxia reoxygenation injury and the possible mechanism. Neonatal Sprague-Dawley rat cardiomyocytes in primary culture were treated with different concentrations of icariin for 24 h prior to hypoxia/reoxygenation injury. Cardiomyocyte apoptosis was evaluated with Tunel staining. The expression levels of apoptosis proteins were detected by Western blotting. The nuclear translocation of p65 was evaluated by immunofluorescence. The p65 signaling pathway was also detected by Western blotting. Myocardial apoptosis rate significantly increased after hypoxia/reoxygenation (control: 1.5% ± 0.1%; 23.4% ± 1.3%, P<0.05). While icariin significantly reduced cardiomyocyte apoptosis induced by hypoxia/reoxygenation (1 µmol/L icariin: 7.2% ± 0.9%; 10 µmol/L icariin: 3.9% ± 0.8%, both P<0.05). Western blot showed that the expression levels of pro-apoptotic protein, Bax, increased significantly (control: 0.19 ± 0.05; 0.41 ± 0.03, P<0.05), while the expression of anti-apoptotic protein, B-Cell CLL/Lymphoma 2 (BCL-2), was significantly reduced (control: 0.15 ± 0.02; 0.03 ± 0.01, P<0.05) after hypoxia/reoxygenation. Notably, icariin reduced the expression of Bax (1 µmol/L icariin: 0.29 ± 0.01; 10 µmol/L icariin: 0.33 ± 0.03, both P<0.05) and increased expression of BCL-2 (1 µmol/L icariin: 0.10 ± 0.03; 10 µmol/L icariin: 0.11 ± 0.02, both P<0.05). Immunofluorescence showed that NFκB-p65 nuclear translocation in cardiomyocytes was increased after hypoxia/reoxygenation (control: 3.6% ± 0.5%; 89.5% ± 4.8%, P<0.05), while icariin reduced the nuclear translocation of p65 (1 µmol/L icariin: 32.6% ± 2.3%; 10 µmol/L icariin: 10.6% ± 1.0%, both P<0.05). Moreover, icariin reduced the activation of p65 and phosphorylation of IKBα induced by hypoxia/reoxygenation in cardiomyocytes. Icariin can protect cardiomyocytes against hypoxia reoxygenation injury, which may be via blocking p65 signaling pathway.

20-Hydroxyeicosatetraenoic Acid Is a Key Mediator of Angiotensin II-induced Apoptosis in Cardiac Myocytes.

Cardiomyocyte apoptosis is involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both Angiotensin II (Ang II) and 20-hydroxyeicosatetraenoic acid (20-HETE) induce apoptosis in cardiomyocytes. Here, we examined the relationship between 20-HETE and Ang II in cardiomyocyte apoptosis. Apoptosis was examined using flow cytometry in primary cultured rat cardiomyocytes treated with control, Ang II, and Ang II plus HET0016 (a 20-HETE formation inhibitor). The results demonstrated that the treatment of cardiomyocytes with Ang II or 20-HETE significantly increased the percentage of apoptotic cells and that Ang II-induced apoptosis was markedly attenuated by HET0016 or losartan (an AT1 receptor antagonist). In apoptotic mechanism experiments, Ang II or 20-HETE treatment significantly reduced mitochondrial membrane potential, indicating that a mitochondria-dependent mechanism is involved. Ang II-induced alteration in mitochondrial membrane potential was significantly attenuated by HET0016. Treatment of cardiomyocytes with Ang II also increased superoxide production, and this effect of Ang II was attenuated by HET0016. Treatment of cardiomyocytes with Ang II significantly increased CYP4A1 expression and 20-HETE production, as measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes by a mitochondrial superoxide-dependent pathway.

α-Enolase plays a catalytically independent role in doxorubicin-induced cardiomyocyte apoptosis and mitochondrial dysfunction

Backgroundα-Enolase is a glycolytic enzyme with “second jobs” beyond its catalytic activity. However, its possible contribution to cardiac dysfunction remains to be determined. The present study aimed to investigate the role of α-enolase in doxorubicin (Dox)-induced cardiomyopathy as well as the underlying mechanisms. Experimental approachesThe expression of α-enolase was detected in rat hearts and primary cultured rat cardiomyocytes with or without Dox administration. An adenovirus carrying short-hairpin interfering RNA targeting α-enolase was constructed and transduced specifically into the heart by intramyocardial injection. Heart function, cell apoptosis and mitochondrial function were measured following Dox administration. In addition, by using gain- and loss-of-function approaches to regulate α-enolase expression in primary cultured rat cardiomyocytes, we investigated the role of endogenous, wide type and catalytically inactive mutant α-enolase in cardiomyocyte apoptosis and ATP generation. Furthermore, the involvement of α-enolase in AMPK phosphorylation was also studied. Key resultsThe mRNA and protein expression of cardiac α-enolase was significantly upregulated by Dox. Genetic silencing of α-enolase in rat hearts and cultured cardiomyocytes attenuated Dox-induced apoptosis and mitochondrial dysfunction. In contrast, overexpression of wide-type or catalytically inactive α-enolase in cardiomyocytes mimicked the detrimental role of Dox in inducing apoptosis and ATP reduction. AMPK dephosphorylation was further demonstrated to be involved in the proapoptotic and ATP-depriving effects of α-enolase. ConclusionOur findings provided the evidence that α-enolase has a catalytically independent role in inducing cardiomyocyte apoptosis and mitochondrial dysfunction, which could be at least partially contributed to the inhibition of AMPK phosphorylation.

Cardiomyocyte cytokinesis score: a potential method for cardiomyocyte proliferation.

One of the most important indicators of myocardial regeneration is cardiomyocyte proliferation. However, it is difficult to distinguish cardiomyocytes in the regenerating stage from binucleated or multinucleated myocytes by conventional morphometric techniques. As cell cycle progression (CCP) scores have been successfully applied to the evaluation of the proliferation of cancer cells, we sought to establish a multi-gene score to evaluate cardiomyocyte proliferation in this study. Given the disturbances of nuclear division without cell division that occurs in cardiomyocytes, ten cytokinesis-correlated genes (Anln, Aurkb, Cenpa, Kif4, Kif23, Prc1, RhoA, Spin1, TACC2, and CDC42) were chosen to establish the cardiomyocyte cytokinesis score (CC-Score). The expression levels of these genes in H9C2 rat cardiomyoblast cells, the proliferation of which were stimulated or inhibited, were detected using qRT-PCR. To confirm the feasibility of the CC-Score system, four conventional methods for evaluating cardiomyocyte proliferation, including the MTT assay, BrdU assay, immunofluorescence, and flow cytometry analysis, were used in each group. The results of the CC-Score in the assessment of the proliferation of H9C2 cells were consistent with those of four commonly used proliferative assay methods. We conclude that the CC-Score can be used to assess the proliferation status of H9C2 cells, and suggest that the CC-Score may be a potential method for the assessment of cardiomyocyte proliferation in myocardial regeneration. However, validation studies utilizing primary cultured rat cardiomyocytes and heart tissue are warranted.

Activation of an apoptotic signal transduction pathway involved in the upregulation of calpain and apoptosis-inducing factor in aldosterone-induced primary cultured cardiomyocytes

In this study, aldosterone (ALD)-induced apoptosis of cardiomyocyte was evaluated based on the previous studies, and the roles of calpain signaling were clarified. Primary cultured rat cardiomyocytes were injured by ALD (0.01-10 μM) for varying time periods. Then, the effects of ethylene glycol tetraacetic acid (EGTA) (0.5 mM), calpeptin (2.5 μM), and spironoclactone (10 μM) were evaluated on cardiomyocytes activated by ALD. Cardiomyocytes that were injured by ALD were assayed by the MTT and LDH leakage ratio. Apoptosis was evaluated by a TUNEL assay, annexin V/PI staining, and caspase-3 activity. The expression of cleavage of Bid (tBid), calpain and apoptosis-inducing factor (AIF) was evaluated by western blot analysis. ALD increased calpain expression and caspase-3 activity and promoted Bid cleavage. It also induced the release of AIF from mitochondria into the cytosol. The upregulation of calpain, tBid and caspase-3 activity were further inhibited by treatment with EGTA in the presence of ALD. Additionally, AIF levels in the cytosol decreased due to EGTA but not due to calpeptin. This was also accompanied by a significant decrease in apoptosis. Furthermore, treatment with spironoclactone not only attenuated the pro-apoptotic effect of ALD but reversed the ALD-induced increase of calpain and AIF levels.

Induction of MicroRNA-24 by HIF-1 Protects Against Ischemic Injury in Rat Cardiomyocytes

MicroRNAs are emerging as important regulators of cardiac function. This study investigated the role of microRNA-24 (miR-24) in ischemic cardiomyocytes, based on the observation that miR-24 expression was significantly enhanced in the ischemic myocardium of rats. Using primary cultured rat cardiomyocytes, cell injury was induced by ischemic conditions, and the cells were evaluated for changes in lactate dehydrogenase (LDH) release, cell viability, apoptosis and necrosis. The results showed that miR-24 was increased in myocytes exposed to ischemia. When miR-24 was further overexpressed in ischemic myocytes using the mimic RNA sequence, LDH release was reduced, cell viability was enhanced, and apoptosis and necrosis rates were both decreased. By contrast, a deficiency in miR-24 resulted in the largest LDH release, lowest cell viability and highest apoptosis and necrosis rates in normal and ischemic myocytes, with significant changes compared to that of non-transfected myocytes. Additionally, the mRNA and protein levels of the pro-apoptotic gene, BCL2L11, were down-regulated by miR-24 overexpression and up-regulated by miR-24 deficiency. The luciferase reporter assay confirmed BCL2L11 to be a target of miR-24. Overall, this study showed a protective role for miR-24 against myocardial ischemia by inhibiting BCL2L11, and may represent a potential novel treatment for ischemic heart disease.

Expression of Glucocorticoid-Induced Leucine Zipper (GILZ) in Cardiomyocytes

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressive effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the glucocorticoid receptor (GR), a cytosolic protein that translocates to the nuclei and functions to alter transcription upon ligand binding. Among a long list of genes activated by GCs is the glucocorticoid-induced leucine zipper (GILZ). GC-induced GILZ expression has been well established in lymphocytes and mediates GC-induced apoptosis. Unlike lymphocytes, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression in cardiomyocytes in vivo and in vitro. Expression of GILZ in mouse hearts as a result of GC administration was confirmed by Western blot analyses. GCs induced dose- and time-dependent elevation of GILZ expression in primary cultured rat cardiomyocytes, with dexamethasone (Dex) as low as 0.1 μM being effective. Time course analysis indicated that GILZ protein levels increased at 8 h and peaked at 48 h after exposure to 1 μM Dex. H9c2(2-1) cell line showed a similar response of GILZ induction by Dex as primary cultured rat cardiomyocytes, providing a convenient model for studying the biological significance of GILZ expression. With corticosterone (CT), an endogenous form of corticosteroids in rodents, 0.1-2.5 μM was found to induce GILZ in H9c2(2-1) cells. Time course analysis with 1 μM CT indicated induction of GILZ at 6 h with peak expression at 18 h. Inhibition of the GR by mifepristone led to blunting of GILZ induction by GCs. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs, pointing to H9c2(2-1) cells as a valid model for studying the biological function of GILZ in cardiomyocytes.

EFFECTS OF WEDELOLACTONE ON THE PROTEIN EXPRESSION OF APOPTOSIS-ASSOCIATED BCL-2, BAX AND PARP (89KD) IN THE PRIMARY CULTURED RAT CARDIOMYOCYTES SUBJECTED TO ANOXIA/REOXYGENATION INJURY

ObjectivesTo study the effects of Wedelolactone (Wed) on the protein expression of apoptosis-associated Bcl-2, Bax and PARP (89KD) in the primary cultured rat cardiomyocytes subjected to anoxia/reoxygenation injury.MethodsThe primary cultured...

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

Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro

Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.

Role of ERK1/2 activation in microtubule stabilization and glucose transport in cardiomyocytes

We previously demonstrated that microtubule disruption impairs stimulation of glucose uptake in cardiomyocytes and that 9-cis retinoic acid (9cRA) treatment preserved both microtubule integrity and stimulated glucose transport. Herein we investigated whether 1) activation of the extracellular signal-regulated kinases (ERK1/2) is responsible for microtubule destabilization and 2) ERK1/2 inactivation may explain the positive effects of 9cRA on glucose uptake and microtubule stabilization. Adult rat cardiomyocytes in primary culture showed increased basal ERK1/2 phosphorylation. Cardiomyocytes exposed to inhibitors of the ERK1/2 kinase mitogen/extracellular signal-regulated kinase (MEK) 1/2 had preserved microtubular scaffold, including microtubule-organizing centers (MTOC), together with increased insulin and metabolic stress-stimulated glucose transport as well as signaling, thus replicating the effects of 9cRA treatment. Although 9cRA treatment did not significantly reduce global ERK1/2 activation, it markedly reduced perinuclear-activated ERK1/2 at the location of MTOC. 9cRA also triggered relocation of the ERK1/2 phosphatase mitogen-activated protein kinase phosphatase-3 from the cytosol to the nucleus. These results indicate that, in cardiomyocytes, microtubule destabilization, leading to impaired stimulation of glucose transport, is mediated by ERK1/2 activation, impacting on the MTOC. 9cRA acid restores stimulated glucose transport indirectly through compartmentalized inactivation of ERK1/2.