Primary Neonatal Rat Cardiomyocytes Research Articles

Abstract 390: Dysregulation of ATF2 in Dilated Cardiomyopathy Caused by FBXO32 Mutation

Background: Dilated cardiomyopathy (DCM) is a common form of cardiomyopathy causing systolic dysfunction and heart failure. Rare variants in more than 30 genes mostly encoding sarcomeric proteins and proteins of the extracellular matrix have been implicated in familial DCM to date. We recently identified a novel mutation (Gly243Arg) in FBXO32 causing familial DCM through abnormal SKP1/CUL/F-BOX (SCF) complex formation and defects in proteins regulating the autophagy/lysosome machinery (Al-Yacoub, Genome Biology, 2016). Objective: To explore in more details the mechanisms by which the defective SCF FBXO32 complex leads to the development of DCM. Methodology: Using a PCR-based microarray, we screened for mRNAs significantly dysregulated in the heart of the patient carrying the FBXO32 mutation compared to control and idiopathic human hearts. Subsequently, we validated dysregulation of a candidate gene using immunoblot analysis and tested the effect of the mutant or wild-type FBXO32 on the novel candidate identified in primary neonatal rat cardiomyocytes. Results: We found a robust up-regulation in mRNA expression of the Activating transcription Factor 2 (ATF2), a member of the leucine zipper family of DNA binding proteins, which plays a critical role in cardiac development. ATF2 protein level was also strongly increased in the heart with the FBXO32 mutation compared to control hearts and to hearts of idiopathic origin. Expression of the mutant FBXO32 protein in primary cardiomyocytes enhanced ATF2 protein expression compared to cells expressing the wild-type FBXO32 protein. Since FBXO32 is member of the SCF complex and has ubiquitin ligase activity, experiments are now investigating whether FBXO32 directly regulates ATF2 protein stability and the role of ATF2 in autophagy flux regulation in dilated cardiomyopathy. Conclusion: Our results indicate that abnormal SCF activity due to the FBXO32 mutation stabilizes the AFT2 transcription factor and suggest a new mechanism by aberrant SCF activity causes DCM in human.

Activation of volume-sensitive Cl- channel mediates autophagy-related cell death in myocardial ischaemia/reperfusion injury.

Excessive reactive oxygen species (ROS) plays an important role in myocardial ischemia/reperfusion (I/R) injury, which triggers not only myocardial cellular apoptosis but also autophagy-related cell death, in which volume-sensitive outwardly rectifying (VSOR) Cl− channel-activated by ROS contributes to cell apoptotic volume decrease, playing an incipient incident of cellular apoptosis. However, whether VSOR Cl− channel concurrently participates in autophagy-related cell death regulation remains unclear. To illuminate the issue, studies underwent in myocardial vitro and vivo I/R model. Rats were performed to ischemia 30 minutes and subsequent reperfusion 24-96 hours, ROS scavenger (NAC), VSOR Cl− channel blocker (DCPIB) and autophagy inhibitor (3MA) were administered respectively. Results showed that oxidative stress, LC3-II stain and inflammation in myocardial tissue were markedly increased, lysosome associated membrane protein-2 (LAMP2) were significantly reduced with I/R group as compared with sham group, reperfusion significantly led to damage in myocardial tissue and heart function, whereas the disorder could be rescued through these agents. Moreover, primary neonatal rat cardiomyocytes hypoxia/reoxygenation model were administered, results showed that VSOR Cl− channel-activated by reoxygenation could cause both cell volume decrease and intracellular acidification, which further increased LC3 and depleted of LAMP2, resulting in autophagy-related cell death. Interestingly, VSOR Cl− channel-blocked by DCPIB could stably maintain the cell volume, intracellular pH, abundant LAMP2 and autophagic intensity regardless of ROS intension derived from reoxygenation injury or adding H2O2. These results first demonstrate that VSOR Cl− channel-activated is a pivotal event to trigger autophagy-related death, which reveals a novel therapeutic target to decrease myocardial I/R injury.

A functional variant in the coding region of CAMTA2 is associated with left ventricular hypertrophy by affecting the activation of Nkx2.5-dependent transcription.

The calmodulin-binding transcription activator 2 (CAMTA2) promotes transcription of genes involved in cardiac hypertrophy through its interaction with Nkx2.5 and is an indispensable transcription coactivator for cardiac hypertrophy. We hypothesized that variants in the coding region of CAMTA2 would affect its function and confer a risk of cardiac hypertrophy. The effects of the variant rs238234 on the activity of the atrial natriuretic factor promoter and on the cardiomyocytes hypertrophy were assessed in the H9C2 cell line and primary neonatal rat cardiomyocytes, respectively. Furthermore, the association of this variant with left ventricular hypertrophy (LVH) was tested in hypertensive patients with and without hypertrophy (N = 325 and 697), and this analysis was replicated in an independent population of 987 hypertensive patients without hypertrophy and 463 hypertensive patients with hypertrophy. We found that the G allele of rs238234 activated the atrial natriuretic factor promoter more strongly than the C allele. The cell size of cardiomyocytes was larger in the presence of the Ad-CAMTA2 G allele, and the G allele was associated with significantly increased susceptibility to LVH in hypertensive [odds ratio (OR), 1.29; P = 0.009]. In the discovery cohort, after adjusting for age and sex, the GG genotype was significantly associated with increased LVH risk (OR, 1.75; P = 0.015). There was little attenuation of the ORs (1.62; P < 0.05) when adjusting for BMI, heart rate, blood pressure, smoking, and drinking and further adjusting all covariates including lipid levels and other major risk factors. However, the GC genotype did not show any association with LVH using three regressive models. Replication in the second study yielded similar results. Our results provide evidence that the rs238234 GG genotype in the coding region of CAMTA2 may increase the risk of LVH by affecting the activation of Nkx2.5-dependent transcription.

Ischemic postconditioning protects the heart against ischemia-reperfusion injury via neuronal nitric oxide synthase in the sarcoplasmic reticulum and mitochondria.

As a result of its spatial confinement in cardiomyocytes, neuronal nitric oxide synthase (nNOS) is thought to regulate mitochondrial and sarcoplasmic reticulum (SR) function by maintaining nitroso-redox balance and Ca2+ cycling. Thus, we hypothesize that ischemic postconditioning (IPostC) protects hearts against ischemic/reperfusion (I/R) injury through an nNOS-mediated pathway. Isolated mouse hearts were subjected to I/R injury in a Langendorff apparatus, H9C2 cells and primary neonatal rat cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) in vitro. IPostC, compared with I/R, decreased infarct size and improved cardiac function, and the selective nNOS inhibitors abolished these effects. IPostC recovered nNOS activity and arginase expression. IPostC also increased AMP kinase (AMPK) phosphorylation and alleviated oxidative stress, and nNOS and AMPK inhibition abolished these effects. IPostC increased nitrotyrosine production in the cytosol but decreased it in mitochondria. Enhanced phospholamban (PLB) phosphorylation, normalized SR function and decreased Ca2+ overload were observed following the recovery of nNOS activity, and nNOS inhibition abolished these effects. Similar effects of IPostC were demonstrated in cardiomyocytes in vitro. IPostC decreased oxidative stress partially by regulating uncoupled nNOS and the nNOS/AMPK/peroxisome proliferator-activated receptor gamma coactivator 1 alpha/superoxide dismutase axis, and improved SR function through increasing SR Ca2+ load. These results suggest that IPostC protected hearts against I/R injury via an nNOS-mediated pathway.

SIRT6 suppresses isoproterenol-induced cardiac hypertrophy through activation of autophagy

Reduction in autophagy has been reported to contribute to the pathogenesis of cardiac hypertrophy. However, the molecular pathways leading to impaired autophagy at the presence of hypertrophic stimuli remain to be elucidated. The present study aimed to investigate the role of sirtuin 6 (SIRT6), a sirtuin family member, in regulating cardiomyocyte autophagy, and its implication in prevention of cardiac hypertrophy. Primary neonatal rat cardiomyocytes (NRCMs) or Sprague-Dawley (SD) rats were submitted to isoproterenol (ISO) treatment, and then the hypertrophic responses and changes in autophagy activity were measured. The influence of SIRT6 on autophagy was observed in cultured NRCMs with gain- and loss-of-function approaches to regulate SIRT6 expression, and further confirmed invivo by intramyocardial delivery of an adenovirus vector encoding SIRT6 cDNA. In addition, the involvement of SIRT6-mediated autophagy in attenuation of cardiomyocyte hypertrophy induced by ISO was determined basing on genetic or pharmaceutical disruption of autophagy, and the underlying mechanism was preliminarily explored. ISO-caused cardiac hypertrophy accompanying with a significant decrease in autophagy activity. SIRT6 overexpression enhanced autophagy in NRCMs and in rat hearts, whereas knockdown of SIRT6 by RNA interference led to suppression of cardiomyocyte autophagy. Furthermore, the protective effect of SIRT6 against ISO-stimulated hypertrophy was associated with induction of autophagy. SIRT6 promoted nuclear retention of forkhead box O3 transcription factor possibly via attenuating Aktsignaling, which was responsible for autophagy activation. Our findings revealedthat SIRT6 positively regulates autophagy in cardiomyocytes, which may help to ameliorate ISO-induced cardiac hypertrophy.

β-Catenin overexpression causes an increase in inflammatory cytokines and NF-κB activation in cardiomyocytes.

β-Catenin has been implicated in various developmental and physiological processes. Defective Wnt signaling can result in different cardiac and vascular abnormalities and is activated under pathological conditions such as inflammation and obesity. In this study, roles of β-catenin in inflammation in cardiomyocytes were investigated. 10 samples from hearts of patients with acute infarction and 10 from normal ones were collected in order to access roles of β-catenin in cardiomyocytes. H9c2 cardiomyoblasts and primary neonatal rat cardiomyocytes were transfected with porcine cytomegalovirus (pCMV)-β-catenin plasmid in order to overexpress β-catenin. Protein level of β-catenin protein was increased in human acute infarction tissues compared to ones from normal patients. The transcription factor had increased nuclear localization in cardiomyocytes of the Wistar rats with cardiac hypertension. Furthermore, expression of fibrosis protein markers increased. Protein expression of β-catenin was increased in human acute infarction inflammatory heart tissues and in hearts of inflammatory obesity rats. After pCMV-β-catenin plasmid was transfected in a dose-dependent manner, inflammation protein markers, TNF-α and IL-8, were upregulated in hypertensive neonatal rat cardiomyocytes and H9c2 cardiomyoblasts. In addition, overexpression of β-catenin induced activation and nuclear localization of NF-κB. Therefore, β-catenin is a potential molecular target for treatment of inflammation and fibrosis in cardiomyocytes.

Kallikrein-related peptidase 8 is expressed in myocardium and induces cardiac hypertrophy.

The tissue kallikrein-related peptidase family (KLK) is a group of trypsin- and chymotrypsin-like serine proteases that share a similar homology to parent tissue kallikrein (KLK1). KLK1 is identified in heart and has anti-hypertrophic effects. However, whether other KLK family members play a role in regulating cardiac function remains unknown. In the present study, we demonstrated for the first time that KLK8 was expressed in myocardium. KLK8 expression was upregulated in left ventricle of cardiac hypertrophy models. Both intra-cardiac adenovirus-mediated and transgenic-mediated KLK8 overexpression led to cardiac hypertrophy in vivo. In primary neonatal rat cardiomyocytes, KLK8 knockdown inhibited phenylephrine (PE)-induced cardiomyocyte hypertrophy, whereas KLK8 overexpression promoted cardiomyocyte hypertrophy via a serine protease activity-dependent but kinin receptor-independent pathway. KLK8 overexpression increased epidermal growth factor (EGF) production, which was blocked by the inhibitors of serine protease. EGF receptor (EGFR) antagonist and EGFR knockdown reversed the hypertrophy induced by KLK8 overexpression. KLK8-induced cardiomyocyte hypertrophy was also significantly decreased by blocking the protease-activated receptor 1 (PAR1) or PAR2 pathway. Our data suggest that KLK8 may promote cardiomyocyte hypertrophy through EGF signaling- and PARs-dependent but a kinin receptor-independent pathway. It is implied that different KLK family members can subtly regulate cardiac function and remodeling.

Liensinine- and Neferine-Induced Cardiotoxicity in Primary Neonatal Rat Cardiomyocytes and Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Due to drug-induced potential congestive heart failure and irreversible dilated cardiomyopathies, preclinical evaluation of cardiac dysfunction is important to assess the safety of traditional or novel treatments. The embryos of Nelumbo nucifera Gaertner seeds are a homology of traditional Chinese medicine and food. In this study, we applied the real time cellular analysis (RTCA) Cardio system, which can real-time monitor the contractility of cardiomyocytes (CMs), to evaluate drug safety in rat neonatal CMs and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). This study showed detailed biomechanical CM contractility in vitro, and provided insights into the cardiac dysfunctions associated with liensinine and neferine treatment. These effects exhibited dose and time-dependent recovery. Neferine showed stronger blocking effect in rat neonatal CMs than liensinine. In addition, the effects of liensinine and neferine were further evaluated on hiPS-CMs. Our study also indicated that both liensinine and neferine can cause disruption of calcium homeostasis. For the first time, we demonstrated the potential cardiac side effects of liensinine or neferine. While the same inhibition was observed on hiPS-CMs, more importantly, this study introduced an efficient and effective approach to evaluate the cardiotoxicity of the existing and novel drug candidates.

Astragaloside IV protects cardiomyocytes from anoxia/reoxygenation injury by upregulating the expression of Hes1 protein.

Astragaloside IV (ASI), a traditional Chinese medicine, is a main active ingredient of Astragalus membranaceus. Many clinical studies have found that ASI protects cardiomyocytes in cardiovascular diseases, but the underlying mechanisms remain obscure. The aim of this study was to investigate the molecular mechanisms responsible for the protective effects of ASI in cardiomyocytes from anoxia/reoxygenation (A/R) injury. According to the previous studies, we hypothesized that the cardioprotective effects of ASI against A/R injury might be associated with Notch1/Hes1 signaling pathway. In this study, neonatal rat primary cardiomyocytes were preconditioned with ASI prior to A/R injury. Our results showed that ASI effectively increased the cell viability, decreased the content of MDA, decreased the activities of CPK and LDH, increased the activities of GSH-Px and SOD, and reduced the reactive oxygen species (ROS) generation and the loss of mitochondrial membrane potential (Δψm). ASI inhibited the mitochondrial permeability transition pore (mPTP) opening and activation of caspase-3, and finally decreased the cell apoptosis in cardiomyocytes. Furthermore, ASI upregulated Hes1 protein expression. However, pretreatment with DAPT, a Notch1 inhibitor, effectively attenuated the cardioprotective effects of ASI against A/R injury, except MDA, SOD, GSH-Px, and the ROS generation. Taken together, we demonstrated that ASI could protect against A/R injury via the Notch1/Hes1 signaling pathway.

Hypoxic preconditioning protects cardiomyocytes against hypoxia/reoxygenation-induced cell apoptosis via sphingosine kinase 2 and FAK/AKT pathway

Previous studies have demonstrated that hypoxic preconditioning (HPC) alleviates hypoxia/reoxygenation (H/R) injury. However, the impact and mechanism involved were not fully understood. This study aimed to evaluate the effect of HPC on H/R injury in cardiomyocytes and investigate the molecular mechanisms involved. In our study, primary neonatal rat cardiomyocytes were isolated and characterized by immunofluorescence staining. We established H/R models in vitro to mimic ischemia/reperfusion (I/R) injury in vivo. Primary cardiomyocytes were exposed to HPC and then subjected to H/R. SphK2 expression was determined by quantitative real-time PCR and Western blotting. Cell apoptosis was measured by Hoechst staining. H9c2 cells were transfected with SphK2 siRNA or pcDNA3.1-SphK2 plasmid. The transfection efficiency was evaluated 48h post-transfection. After H/R, cell apoptosis rate was determined by Annexin V–FITC/PI and caspase-3/-9 activity was measured. The activation of FAK/AKT pathway was evaluated by Western blotting. Our results showed that HPC significantly increased SphK2 expression in primary cardiomyocytes under normal or H/R condition and protected against H/R-induced cell apoptosis, whereas SphK2 inhibitor K145 abolished the cardioprotective effect of HPC. HPC markedly reduced the cell apoptosis rate of H9c2, decreased the activities of caspase-3 and -9 and increased p-FAK and p-AKT levels, which were reversed by SphK2 knockdown. Additionally, SphK2 overexpression exerted a similar effect with HPC on cell apoptosis and FAK/AKT. Inhibition of H9c2 cell apoptosis induced by HPC and SphK2 overexpression was abolished by PI3K/AKT inhibitor LY294002. These results indicate that HPC may protect cardiomyocytes against H/R injury via SphK2 and the downstream FAK/AKT signaling pathway. Our findings provided important evidences for the protective role of HPC in ameliorating myocardial H/R injury.

Role of endogenous TNF-α in cardiomyocyte apoptosis induced by bacteria lipoprotein and the protective effect of IL-10

Cardiomyocyte apoptosis is thought to play an important role in sepsis-induced cardiodepression. Previous studies mainly focused on the role of exogenous TNF-α in sepsis-induced cardiac damage, however, the role of endogenous TNF-α is rarely known. Therefore, we hypothesized that endogenous TNF-α also contributed to sepsis-induced cardiomyocyte apoptosis. Primary neonatal rat cardiomyocytes were time- and dose-dependently stimulated with BLP and TNF-α. In separate experiments, cells were treated with TNF-α antagonist and IL-10, respectively, to determine effects of endogenous TNF-α and exogenous IL-10 on BLP-induced cardiomyocyte apoptosis. After treatment, apoptosis was evaluated by nuclear condensation, membrane permeability change, caspase-3 activation, and pro- to anti-apoptotic protein (bax to bcl-2) expression. Treatment of cardiomyocytes with BLP and TNF-α both significantly induced caspase-3 activation in a time- and dose-dependent manner and caused apparent nuclear condensation and increased membrane permeability. TNF-α antagonist pretreatment attenuated BLP-induced caspase-3 activation, and downregulated bax/bcl-2 ratio. In addition, administration of IL-10 inhibited TNF-α production and suppressed cardiomyocyte apoptosis induced by BLP. Our data suggest that endogenous TNF-α play an important role in BLP-induced cardiomyocyte apoptosis and IL-10 protect cardiomyocytes from BLP-induced apoptosis, an effect partially through inhibition of endogenous TNF-α production.

Kaempferol protects cardiomyocytes against anoxia/reoxygenation injury via mitochondrial pathway mediated by SIRT1

Mitochondria-mediated apoptosis is a critical mechanism of anoxia/ reoxygenation (A/R)-induced injury in cardiomyocytes. Kaempferol (Kae) is a natural polyphenol and a type of flavonoid, which has been demonstrated to protect myocardium against ischemia/reperfusion (I/R) injury. However, the mechanism is still not fully elucidated. We hypothesize that Kae may improve the mitochondrial function during I/R injury via a potential signal pathway. In this study, an in vitro I/R model was replicated on neonatal rat primary cardiomyocytes by A/R treatment. Cell viability was monitored by the 3-(4,5-dimethylthiazol- 2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay. The levels of intracellular reactive oxygen species, mitochondrial membrane potential (Δψm) and apoptosis were determined by flow cytometry. Protein expression was detected by Western Blotting. mPTP opening and the activity of caspase-3 were measured by colorimetric method. The results showed that Kae effectively enhanced the cell viability and decreased the LDH release in cardiomyocytes subjected to A/R injury. Kae reduced the A/R-induced reactive oxygen species generation, the loss of Δψm, and the release of cytochrome c from mitochondria into cytosol. Kae inhibited the A/R-stimulated mPTP opening and activation of caspase-3, and ultimate decrease in cardiomyocytes apoptosis. Furthermore, we found Kae up-regulated Human Silent Information Regulator Type 1 (SIRT1) expression, indicating SIRT1 signal pathway likely involved the cardioprotection of Kae. Sirtinol, a SIRT1 inhibitor, abolished the protective effect of Kae in cardiomyocytes subjected to A/R. Additionally, Kae significantly increased the expression of Bcl-2. Thus, we firstly demonstrate that Kae protects cardiomyocytes against A/R injury through mitochondrial pathway mediated by SIRT1.

Interleukin-27, a novel cytokine induced by ischemia-reperfusion injury in rat hearts, mediates cardioprotective effects via the gp130/STAT3 pathway.

Patients with coronary artery disease show high serum levels of interleukin (IL)-27, a novel member of the IL-6 family. However, the function of IL-27 in hearts suffering ischemia/reperfusion (IR) injury is unclear. Here, we showed increased expression of mRNA for the IL-27 subunits, EBI3 and p28, in rat hearts after 40 min of coronary ligation and release for 7 days. This increase was associated with a peak in the release of the cardiac enzyme, creatine kinase-MB, on day 2 post-release. Moreover, levels of IL-27 receptor subunit gp130 mRNA, but not those of subunit WSX-1 mRNA, decreased in post-ischemic hearts. These results suggest that increased IL-27 production may compensate for receptor downregulation during myocardial recovery. Lactate dehydrogenase release and crystal violet staining revealed that IL-27 or IL-6 significantly attenuated severe hypoxia (SH, 2 % O2)-induced cell damage in H9c2 cardiomyoblasts and primary rat neonatal cardiomyocytes. Incubating cardiomyocytes with IL-27 or IL-6 resulted in time-dependent activation of signal transducers and activators of transcription 3 (STAT3). Interestingly, IL-27-induced STAT3 activation was attenuated by pre-treatment with a gp130-neutralizing antibody. Blocking gp130 also reduced the cytoprotective effects of IL-27 or IL-6. Moreover, IL-27-mediated protection against SH was blocked by stattic, a small-molecule inhibitor of STAT3. IL-27 markedly improved post-ischemic recovery and reduced tissue damage in isolated perfused hearts when administered 5 min before reperfusion. These results indicate that IL-27 protects the myocardium against IR injury and facilitates the recovery of damaged cardiomyocytes via the gp130/STAT3 pathway.

Cardiomyocyte lipotoxicity is mediated by Il-6 and causes down-regulation of PPARs

Here we sought to evaluate the effect of palmitate on cytokine and PPAR activity/expression. We investigated the effect of BSA conjugated palmitate and oleate on PPAR activity, PPAR-α and δ expression, as well as the expression of cytokines and key factors responsible for β-oxidation by qRT-PCR and western blotting in primary rat neonatal cardiomyocytes (NCMs). Furthermore we evaluated the effect of anti-inflammatory actions of AICAR and PPAR agonists on cytokine expression and cell death in palmitate treated NCMs. We found that palmitate caused down regulation of PPARs and increased cytokine expression and cell death, all of which was significantly attenuated by the co-administration of either AICAR or PPAR agonists. This work supports the pro-inflammatory actions of intracellular lipid and provides further insight into the pathological mechanism of cardiac lipotoxicity as occurs in diabetic hearts.

Fluid driving system for a micropump by differentiating iPS cells into cardiomyocytes on a tent-like structure

A number of recent studies have exploited the sizes and functional properties of microdevices and cellular mechanical components to construct bio-microactuators. We previously developed bio-micropumps powered by cardiomyocytes that utilizes glucose in the medium as chemical energy. To fabricate the pump, however, primary neonatal rat cardiomyocytes are indispensable. The operation of harvesting primary cells is inconvenient and ethically not adequate due to the need for animals sacrifice. In contrast, induced pluripotent stem (iPS) cells are obtained from subcutaneous tissue. Their most significant properties are that they proliferate indefinitely and can be differentiated into many kinds of cells, including cardiomyocytes, and also have no ethical issue differently from ES cells. By exploiting these properties of iPS cells, the above issues will be addressed. Based on this concept, we constructed a system for driving fluids as a principal component of a micropump by differentiating iPS cells into spontaneously beating cardiomyocytes. Cellular contractile force was transmitted to fluid in a microchannel by a tent-like thin membrane. The microchip was irradiated with O2 plasma and coated with gelatin to attach the cells. Embryoid bodies (EBs) of mouse-derived iPS cells were seeded on the microchip and incubated at 37°C without Leukemia Inhibitory Factor (LIF) to differentiate them into cardiomyocytes. About 2 weeks later, EB beating and periodical oscillation of fluid in a microchannel connected to a diaphragm chamber was observed. The theoretical flow rate assuming the use of ideal check valves (Q) was 6.9nL/min. Our device presents a reasonable alternative to normal cardiomyocytes for preliminary investigations requiring bio-actuating pumps.

Distribution of formins in cardiac muscle: FHOD1 is a component of intercalated discs and costameres

The formin homology domain-containing protein1 (FHOD1) suppresses actin polymerization by inhibiting nucleation, but bundles actin filaments and caps filament barbed ends. Two polyclonal antibodies against FHOD1 were generated against (i) its N-terminal sequence (residues 1–339) and (ii) a peptide corresponding the sequence from position 358–371, which is unique for FHOD1 and does not occur in its close relative FHOD3. After affinity purification both antibodies specifically stain purified full length FHOD1 and a band of similar molecular mass in homogenates of cardiac muscle. The antibody against the N-terminus of FHOD1 was used for immunostaining cells of established lines, primary neonatal (NRC) and adult (ARC) rat cardiomyocytes and demonstrated the presence of FHOD1 in HeLa and fibroblastic cells along stress fibers and within presumed lamellipodia and actin arcs. In NRCs and ARCs we observed a prominent staining of presumed intercalated discs (ICD). Immunostaining of sections of hearts with both anti-FHOD1 antibodies confirmed the presence of FHOD1 in ICDs and double immunostaining demonstrated its colocalisation with cadherin, plakoglobin and a probably slightly shifted localization to connexin43. Similarly, immunostaining of isolated mouse or pig ICDs corroborated the presence of FHOD1 and its colocalisation with the mentioned cell junctional components. Anti-FHOD1 immunoblots of isolated ICDs demonstrated the presence of an immunoreactive band comigrating with purified FHOD1. Conversely, an anti-peptide antibody specific for FHOD3 with no cross-reactivity against FHOD1 immunostained on sections of cardiac muscle and ARCs the myofibrils in a cross-striated pattern but not the ICDs. In addition, the anti-peptide-FHOD1 antibody stained the lateral sarcolemma of ARCs in a banded pattern. Double immunostaining with anti-cadherin and -integrin-ß1 indicated the additional localization of FHOD1 in costameres. Immunostaining of cardiac muscle sections or ARCs with antibodies against mDia3-FH2-domain showed colocalisation with cadherin along the lateral border of cardiomyocytes suggesting also its presence in costameres.

Sodium hydrosulfide attenuates hyperhomocysteinemia rat myocardial injury through cardiac mitochondrial protection.

Hydrogen sulfide (H2S) plays an important role during rat myocardial injury. However, little is known about the role of H2S in hyperhomocysteinemia (HHcy)-induced cardiac dysfunction as well as the underlying mechanisms. In this study, we investigated whether sodium hydrosulfide (NaHS, a H2S donor) influences methionine-induced HHcy rat myocardial injury in intact rat hearts and primary neonatal rat cardiomyocytes. HHcy rats were induced by methionine (2.0g/kg) and the daily administration of 80μmol/L NaHS in the HHcy+NaHS treatment group. At the end of 4, 8, and 12weeks, the ultrastructural alterations and functions of the hearts were observed using transmission electron microscopy and echocardiography system. The percentage of apoptotic cardiomyocytes, the mitochondrial membrane potential, and the production of reactive oxygen species (ROS) were measured. The expressions of cystathionine-γ-lyase (CSE), Bax and Bcl-2, caspase-3, phospho-endothelial nitric oxide synthase and the mitochondrial NOX4 and cytochrome c were analyzed by Western blotting. The results showed the cardiac dysfunction, the ultrastructural changes, and the apoptotic rate increase in the HHcy rat hearts. In the primary neonatal rat cardiomyocytes of HHcy group, ROS production was increased markedly, whereas the expression of CSE was decreased. However, treatment with NaHS significantly improved the HHcy rat hearts function, the ultrastructural changes, and decreased the levels of ROS in the primary neonatal rat cardiomyocytes administrated with HHcy group. Furthermore, NaHS down-regulated the expression of mitochondrial NOX4 and caspase-3 and Bax and inhibited the release of cytochrome c from mitochondria. In conclusion, H2S is involved in the attenuation of HHcy myocardial injury through the protection of cardiac mitochondria.

Live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope's autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

β-adrenergic receptor-dependent alterations in murine cardiac transcript expression are differentially regulated by gefitinib in vivo.

β-adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to promote cardioprotection in a mouse model of heart failure and we recently showed that this mechanism leads to enhanced cell survival in part via regulation of apoptotic transcript expression in isolated primary rat neonatal cardiomyocytes. Thus, we hypothesized that this process could regulate cardiac transcript expression in vivo. To comprehensively assess cardiac transcript alterations in response to acute βAR-dependent EGFR transactivation, we performed whole transcriptome analysis of hearts from C57BL/6 mice given i.p. injections of the βAR agonist isoproterenol in the presence or absence of the EGFR antagonist gefitinib for 1 hour. Total cardiac RNA from each treatment group underwent transcriptome analysis, revealing a substantial number of transcripts regulated by each treatment. Gefitinib alone significantly altered the expression of 405 transcripts, while isoproterenol either alone or in conjunction with gefitinib significantly altered 493 and 698 distinct transcripts, respectively. Further statistical analysis was performed, confirming 473 transcripts whose regulation by isoproterenol were significantly altered by gefitinib (isoproterenol-induced up/downregulation antagonized/promoted by gefinitib), including several known to be involved in the regulation of numerous processes including cell death and survival. Thus, βAR-dependent regulation of cardiac transcript expression in vivo can be modulated by the EGFR antagonist gefitinib.