Neonatal Rat Ventricular Cells Research Articles

MicroRNA-128 inhibition attenuates myocardial ischemia/reperfusion injury-induced cardiomyocyte apoptosis by the targeted activation of peroxisome proliferator-activated receptor gamma.

The aim of the present study was to investigate the effects of microRNA (miR)-128 inhibition on the targeted activation of peroxisome proliferator-activated receptor gamma (PPARG) and on cardiomyocyte apoptosis induced by myocardial ischemia/reperfusion (I/R) injury. In vitro, the expression of PPARG was detected by reverse transcription-quantitative polymerase chain reaction and western blotting in neonatal rat ventricular myocytes (NRVMs) and HEK293 cells transfected with the mimics or inhibitors of miR-128 or control RNA. Luciferase reporter assays were used to identify whether PPARG is a direct target of miR-128. In vivo, miR-128 was knocked down via ear vein injection of antagomir-128 in a rabbit myocardial I/R injury model. Western blotting investigated the activation of Akt [phosphorylated (p)-Akt] and the expression of total-Akt, PPARG and myeloid leukemia cell differentiation protein-1 (Mcl-1) in the myocardium. Cardiomyocyte apoptosis was examined with transmission electron microscropy and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. PPARG mRNA and protein were downregulated in NRVMs transfected with miR-128 mimics, but upregulated by antagomir-128 compared with control. This indicates that PPARG is a direct miR-128 target. Activation of Akt (p-Akt), Mcl-1 and PPARG expression in the myocardium were increased by miR-128 inhibition. Furthermore, miR-128 antagomirs significantly reduced apoptosis in hearts subjected to I/R injury, which was blocked by the PPARG inhibitor GW9662. In conclusion, miR-128 inhibition attenuated I/R injury-induced cardiomyocyte apoptosis by the targeted activation of PPARG signaling.

Hsp90aa1: a novel target gene of miR-1 in cardiac ischemia/reperfusion injury.

The role of microRNA-1 (miR-1) in ischemia/reperfusion (I/R)-induced injury is not well illustrated. The present study aimed to investigate the expression and potential target of miR-1 in the myocardium of a rat model of I/R. The apoptosis of cardiomyocytes in the ischemic rat myocardium increased on day 1, then attenuated on day 3 and day 7 post-I/R. Heat shot protein 90 (Hsp90) aa1 mRNA expression was decreased post-I/R, and Hsp90aa1 protein level was decreased on day1 post-I/R, but was reversed on day 3 and day 7 post-I/R. MiR-1 was downregulated post-I/R, and repression of miR-1 in cultured neonatal rat ventricular cells (NRVCs) led to an increase of Bcl-2 and decreases of Bax and active caspase-3. Dual luciferase reporter assays revealed that miR-1 interacted with the 310–315 nt site at the 3′UTR of Hsp90aa1, and miR-1 was verified to inhibit Hsp90aa1 expression at the posttranscriptional level. Over-expression of Hsp90aa1 could attenuate oxygen-glucose deprivation (OGD)-induced apoptosis of NRVCs. Additionally, miR-1 mimic, in parallel to Hsp90aa1 siRNA, could enhance OGD-induced apoptosis of NRVCs. Taken together, our results reveal that Hsp90aa1 is a novel target of miR-1, and repression of miR-1 may contribute to the recovery of Hsp90aa1 during myocardial I/R.

MiR-223-3p as a Novel MicroRNA Regulator of Expression of Voltage-Gated K+ Channel Kv4.2 in Acute Myocardial Infarction

Background/Aims: Acute myocardial infarction (AMI) is a devastating cardiovascular disease with a high rate of morbidity and mortality, partly due to enhanced arrhythmogenicity. MicroRNAs (miRNAs) have been shown to participate in the regulation of cardiac ion channels and the associated arrhythmias. The purpose of this study was to test our hypothesis that miR-223-3p contributes to the electrical disorders in AMI via modulating KCND2, the gene encoding voltage-gated channel Kv4.2 that carries transient outward K⁺ current I_to. Methods: AMI model was established in male Sprague-Dawley (SD) rats by left anterior descending artery (LAD) ligation. Evans blue and TTC staining was used to measure infarct area. I_to was recorded in isolated ventricular cardiomyocytes or cultured neonatal rat ventricular cells (NRVCs) by whole-cell patch-clamp techniques. Western blot analysis was employed to detect the protein level of Kv4.2 and real-time RT-PCR to determine the transcript level of miR-223-3p. Luciferase assay was used to examine the interaction between miR-223-3p and KCND2 in cultured NRVCs. Results: Expression of miR-223-3p was remarkably upregulated in AMI relative to sham control rats. On the contrary, the protein level of Kv4.2 and I_to density were significantly decreased in AMI. Consistently, transfection of miR-223-3p mimic markedly reduced Kv4.2 protein level and Ito current in cultured NRVCs. Co-transfection of AMO-223-3p (an antisense inhibitor of miR-223-3p) reversed the repressive effect of miR-223-3p. Luciferase assay showed that miR-223-3p, but not the negative control, substantially suppressed the luciferase activity, confirming the direct binding of miR-223-3p to the seed site within the KCND2 sequence. Finally, direct intramuscular injection of AMO-223-3p into the ischemic myocardium to knockdown endogenous miR-223-3p decreased the propensity of ischemic arrhythmias. Conclusions: Upregulation of miR-223-3p in AMI repressed the expression of KCND2/Kv4.2 resulting in reduction of I_to density that can cause APD prolongation and promote arrhythmias in AMI, and therefore knockdown of endogenous miR-223-3p might be considered a new approach for antiarrhythmic therapy of ischemic arrhythmias.

Apolipoprotein-J prevents angiotensin II-induced apoptosis in neonatal rat ventricular cells.

BackgroundUp-regulation of angiotensin II (AngII) occurs in cardiac diseases, such as congestive heart failure, cardiac hypertrophy, myocardial ischemia and atrial fibrillation, which represent major health problems. Evidence from in vivo studies suggests that the level of Apolipoprotein-J (ApoJ) is also elevated but plays a protective role in cardiovascular disease. This study aimed to evaluate the protective effects of ApoJ against cytotoxicity of AngII in neonatal rat ventricular cells (NRVCs).Methods and resultsIn culture, NRVCs were damaged by exposure to AngII, and ApoJ overexpression using an adenovirus vector significantly reduced the AngII-induced cell injury. ApoJ also prevented AngII from augmenting Nox2/gp91phox expression. The reactive oxygen species (ROS) scavenger, Mn(III)TBAP, showed similar results of attenuating AngII-induced cell damage. Furthermore, ApoJ overexpression increased phosphorylation of Akt, and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 diminished the antioxidant effects of ApoJ, and prevented the protective effect of ApoJ against the cytotoxicity of AngII. Moreover, upregulation of nuclear factor κB (NF-κB) p65 expression and phosphorylation of p38 mitogen-activated protein kinase (MAPK) mediated by AngII in cultured NRVCs were significantly inhibited by overexpression of ApoJ. The p38 MAPK inhibitor SB203580 and the NF-κB inhibitor PDTC protected NRVCs from injury caused by AngII.ConclusionsApoJ serves as a cytoprotective protein in NRVCs against cytotoxicity of AngII through the PI3K-Akt-ROS and MAPK/ NF-κB pathways.

Engineered heart slices for electrophysiological and contractile studies

A major consideration in the design of engineered cardiac tissues for the faithful representation of physiological behavior is the recapitulation of the complex topography and biochemistry of native tissue. In this study we present engineered heart slices (EHS), which consist of neonatal rat ventricular cells (NRVCs) seeded onto thin slices of decellularized cardiac tissue that retain important aspects of native extracellular matrix (ECM). To form EHS, rat or pig ventricular tissue was sectioned into 300 μm-thick, 5 to 16 mm-diameter disks, which were subsequently decellularized using detergents, spread on coverslips, and seeded with NRVCs. The organized fiber structure of the ECM remained after decellularization and promoted cell elongation and alignment, resulting in an anisotropic, functional tissue that could be electrically paced. Contraction decreased at higher pacing rates, and optical mapping revealed electrical conduction that was anisotropic with a ratio of approximately 2.0, rate-dependent shortening of the action potential and slowing of conduction, and slowing of conduction by the sodium channel blocker lidocaine. Reentrant arrhythmias could also be pace-induced and terminated. EHS constitute an attractive in vitro cardiac tissue in which cardiac cells are cultured on thin slices of decellularized cardiac ECM that provide important biochemical, structural, and mechanical cues absent in traditional cell cultures.

Bone marrow mesenchymal stem cell transplantation retards the natural senescence of rat hearts.

Bone marrow mesenchymal stem cells (BMSCs) have been shown to offer a wide variety of cellular functions including the protective effects on damaged hearts. Here we investigated the antiaging properties of BMSCs and the underlying mechanism in a cellular model of cardiomyocyte senescence and a rat model of aging hearts. Neonatal rat ventricular cells (NRVCs) and BMSCs were cocultured in the same dish with a semipermeable membrane to separate the two populations. Monocultured NRVCs displayed the senescence-associated phenotypes, characterized by an increase in the number of β-galactosidase-positive cells and decreases in the degradation and disappearance of cellular organelles in a time-dependent manner. The levels of reactive oxygen species and malondialdehyde were elevated, whereas the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase were decreased, along with upregulation of p53, p21(Cip1/Waf1), and p16(INK4a) in the aging cardiomyocytes. These deleterious alterations were abrogated in aging NRVCs cocultured with BMSCs. Qualitatively, the same senescent phenotypes were consistently observed in aging rat hearts. Notably, BMSC transplantation significantly prevented these detrimental alterations and improved the impaired cardiac function in the aging rats. In summary, BMSCs possess strong antisenescence action on the aging NRVCs and hearts and can improve cardiac function after transplantation in aging rats. The present study, therefore, provides an alternative approach for the treatment of heart failure in the elderly population.

Aggravation of cardiac myofibroblast arrhythmogeneicity by mechanical stress.

Myofibroblasts (MFBs) as appearing in the myocardium during fibrotic remodelling induce slow conduction following heterocellular gap junctional coupling with cardiomyocytes (CMCs) in bioengineered tissue preparations kept under isometric conditions. In this study, we investigated the hypothesis that strain as developed during diastolic filling of the heart chambers may modulate MFB-dependent slow conduction. Effects of defined levels of strain on single-cell electrophysiology (patch clamp) and impulse conduction in patterned growth cell strands (optical mapping) were investigated in neonatal rat ventricular cell cultures (Wistar) grown on flexible substrates. While 10.5% strain only minimally affected conduction times in control CMC strands (+3.2%, n.s.), it caused a significant slowing of conduction in the fibrosis model consisting of CMC strands coated with MFBs (conduction times +26.3%). Increased sensitivity to strain of the fibrosis model was due to activation of mechanosensitive channels (MSCs) in both CMCs and MFBs that aggravated the MFB-dependent baseline depolarization of CMCs. As found in non-strained preparations, baseline depolarization of CMCs was partly due to the presence of constitutively active MSCs in coupled MFBs. Constitutive activity of MSCs was not dependent on the contractile state of MFBs, because neither stimulation (thrombin) nor suppression (blebbistatin) thereof significantly affected conduction velocities in the non-strained fibrosis model. The findings demonstrate that both constitutive and strain-induced activity of MSCs in MFBs significantly enhance their depolarizing effect on electrotonically coupled CMCs. Ensuing aggravation of slow conduction may contribute to the precipitation of strain-related arrhythmias in fibrotically remodelled hearts.

Let-7e replacement yields potent anti-arrhythmic efficacy via targeting beta 1-adrenergic receptor in rat heart.

Beta-adrenoceptor (β-AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let-7e in the up-regulation of β1-AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β1-AR expression was significantly up-regulated and let-7a, c, d, e and i were markedly down-regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let-7e suppressed β1-AR expression at the protein level, without affecting β1-AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let-7e by let-7e antisense inhibitor (AMO-let-7e) enhanced β1-AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let-7e (len-pre-let-7e) significantly inhibited β1-AR expression in rats, whereas len-AMO-let-7e up-regulated β1-AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β1-AR by endogenous let-7e. Len-negative control (len-NC) did not produce significant influence on β1-AR expression. Len-pre-let-7e also profoundly reduced the up-regulation of β1-AR induced by AMI and this effect was abolished by len-AMO-let-7e. Importantly, len-pre-let-7e application significantly reduced arrhythmia incidence after AMI in rats and its anti-arrhythmic effect was cancelled by len-AMO-let-7e. Notably, anti-arrhythmic efficacy of len-pre-let-7e was similar to propranolol, a non-selective β-AR blocker and metoprolol, a selective β1-AR blocker. Down-regulation of let-7e contributes to the adverse increase in β1-AR expression in AMI and let-7e supplement may be a new therapeutic approach for preventing adverse β1-AR up-regulation and treating AMI-induced arrhythmia.

MicroRNA-328 as a regulator of cardiac hypertrophy

Cardiac hypertrophy is a primary predictor of progressive heart disease that often results in heart failure. Growing evidence has demonstrated that microRNAs (miRNAs) play a critical role in regulating cardiac hypertrophy. This study was designed to evaluate the effect of miR-328 on cardiac hypertrophy and the potential molecular mechanisms. We found that transgenic overexpression of miR-328 in the heart induced cardiac hypertrophy in mice, which was accompanied by reduced SERCA2a level increased intracellular calcium concentration and calcineurin protein level, and enhanced NFATc3 nuclear translocation. However, normalization of miR-328 level by its antisense chemically modified with locked nucleic acid (LNA-antimiR-328) reversed the changes. Forced expression of miR-328 resulted in cardiomyocyte hypertrophy in cultured neonatal rat ventricular cells, which was accompanied by downregulation of SERCA2a expression and activation of the calcineurin/NFATc3 signaling pathway. These changes were abolished by LNA-antimiR-328. We validated the SERCA2a as a direct target for miR-328. MiR-328 expression was upregulated in cardiomyocyte treated with isoproterenol (ISO) to induce hypertrophy; while knockdown of miR-328 attenuated the hypertrophic responses. The level of miR-328 was significantly elevated in a mouse model of hypertrophy by thoracic aortic banding (TAC). Consistently, SERCA2a was downregulated, whereas calcineurin were upregulated, and NFATc3 nuclear translocation was enhanced. In contrast, hypertrophy in these mice was significantly alleviated when treated with miR-328 antisense. MiR-328 promotes cardiac hypertrophy by targeting SERCA2a. Our study therefore uncovered a novel molecular mechanism for cardiac hypertrophy and indicated miR-328 as a potential therapeutic target for this cardiac condition.

Downregulation of the miR-30 family microRNAs contributes to endoplasmic reticulum stress in cardiac muscle and vascular smooth muscle cells

BackgroundEndoplasmic reticulum (ER) stress is a common subcellular response to stresses and central to ER stress is increased expression of glucose-regulated protein 78 (GRP78). However, the mechanisms for GRP78 upregulation remained poorly understood. Our study goal was to shed light on this issue. MethodsH2O2 was used to create cellular models of ER stress in neonatal rat ventricular cells (NRVCs) and rat aorta vascular smooth muscle cells (RAVSMCs). Molecular Biology techniques were used to quantify protein and mRNA levels. Luciferase reporter gene assay was employed to investigate miRNA targeting. MTT assay and ELISA were used to detect cell death. ResultsMiRNAs belonging to the miR-30 family including miR-30a, b, c, d and e were all downregulated in ER stress induced by H2O2 in cardiovascular cells NRVCs and RAVSMCs, along with the upregulation of GRP78, cleaved ATF6, CHOP, and cleaved caspase-12. GRP78 was confirmed to be a target gene for miR-30. Artificial knockdown of miR-30 by antimiR-30 triggered the phenotypic ER stress with significant GRP78/ATF6/CHOP/caspase-12 upregulations and cell death, while miR-30 replacement mitigated ER stress. Knockdown of CHOP by siRNA regulated all members of the miR-30 family whereas sequestration of C/EBP transcription factor by its decoy downregulated miR-30 miRNAs. ConclusionsCollectively, downregulation of the miR-30 family miRNAs contributes to the ER stress and the associated upregulation of GRP78 in the cardiovascular system. The participation of miR-30 creates a positive feedback loop in the ER stress signaling pathway. MiR-30 replacement may be a viable approach for alleviating disorders associated with ER stress.

Acute slowing of cardiac conduction in response to myofibroblast coupling to cardiomyocytes through N-cadherin

The electrophysiological consequences of cardiomyocyte and myofibroblast interactions remain unclear, and the contribution of mechanical coupling between these two cell types is still poorly understood. In this study, we examined the time course and mechanisms by which addition of myofibroblasts activated by transforming growth factor-beta (TGF-β) influence the conduction velocity (CV) of neonatal rat ventricular cell monolayers. We observed that myofibroblasts affected CV within 30min of contact and that these effects were temporally correlated with membrane deformation of cardiomyocytes by the myofibroblasts. Expression of dominant negative RhoA in the myofibroblasts impaired both myofibroblast contraction and myofibroblast-induced slowing of cardiac conduction, whereas overexpression of constitutive RhoA had little effect. To determine the importance of mechanical coupling between these cell types, we examined the expression of the two primary cadherins in the heart (N- and OB-cadherin) at cell–cell contacts formed between myofibroblasts and cardiomyocytes. Although OB-cadherin was frequently found at myofibroblast–myofibroblast contacts, very little expression was observed at myofibroblast–cardiomyocyte contacts. The myofibroblast-induced slowing of cardiac conduction was not prevented by silencing of OB-cadherin in the myofibroblasts, and could be reversed by inhibitors of mechanosensitive channels (gadolinium or streptomycin) and cellular contraction (blebbistatin). In contrast, N-cadherin expression was commonly observed at myofibroblast–cardiomyocyte contacts, and silencing of N-cadherin in myofibroblasts prevented the myofibroblast-dependent slowing of cardiac conduction. We propose that myofibroblasts can impair the electrophysiological function of cardiac tissue through the application of contractile force to the cardiomyocyte membrane via N-cadherin junctions.

MiR-17-5p Impairs Trafficking of H-ERG K+ Channel Protein by Targeting Multiple ER Stress-Related Chaperones during Chronic Oxidative Stress

BackgroundTo investigate if microRNAs (miRNAs) play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. MethodsWe treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Luciferase reporter gene assay was used to study miRNA and target interactions. Whole-cell patch-clamp techniques were employed to record h-ERG K+ current.ResultsH-ERG trafficking was impaired by H2O2 after 48 h treatment, accompanied by reciprocal changes of expression between miR-17-5p seed miRNAs and several chaperones (Hsp70, Hsc70, CANX, and Golga2), with the former upregulated and the latter downregulated. We established these chaperones as targets for miR-17-5p. Application miR-17-5p inhibitor rescued H2O2-induced impairment of h-ERG trafficking. Upregulation of endogenous by H2O2 or forced miR-17-5p expression either reduced h-ERG current. Sequestration of AP1 by its decoy molecule eliminated the upregulation of miR-17-5p, and ameliorated impairment of h-ERG trafficking.ConclusionsCollectively, deregulation of the miR-17-5p seed family miRNAs can cause severe impairment of h-ERG trafficking through targeting multiple ER stress-related chaperones, and activation of AP1 likely accounts for the deleterious upregulation of these miRNAs, in the setting of prolonged duration of oxidative stress. These findings revealed the role of miRNAs in h-ERG trafficking, which may contribute to the cardiac electrical disturbances associated with oxidative stress.

Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro

A major limitation to cardiac tissue engineering and regenerative medicine strategies is the lack of proliferation of postnatal cardiomyocytes. The extracellular matrix (ECM) is altered during heart development, and studies suggest that it plays an important role in regulating myocyte proliferation. Here, the effects of fetal, neonatal and adult cardiac ECM on the expansion of neonatal rat ventricular cells in vitro are studied. At 24h, overall cell attachment was lowest on fetal ECM; however, ∼80% of the cells were cardiomyocytes, while many non-myocytes attached to older ECM and poly-l-lysine controls. After 5days, the cardiomyocyte population remained highest on fetal ECM, with a 4-fold increase in number. Significantly more cardiomyocytes stained positively for the mitotic marker phospho-histone H3 on fetal ECM compared with other substrates at 5days, suggesting that proliferation may be a major mechanism of cardiomyocyte expansion on young ECM. Further study of the beneficial properties of early developmental aged cardiac ECM could advance the design of novel biomaterials aimed at promoting cardiac regeneration.

Early growth response 1 is an early signal inducing Cav3.2 T-type calcium channels during cardiac hypertrophy

The Cav3.2 T-channel plays a pivotal role in inducing calcineurin/nuclear factor of activated T cell (NFAT) signalling during cardiac hypertrophy. Because calcineurin/NFAT signalling is induced early after pressure overload, we hypothesized that Cav3.2 is induced by an early signal. Our aim is to investigate when and how Cav3.2 is induced during cardiac hypertrophy. The evolutionary conserved promoter Cav3.2-3500 from mouse genome was validated to express the reporter gene as endogenous Cav3.2 in cell lines and transgenic (Tg; Cav3.2-3500-Luc) mice. The early induction of luciferase in Tg mice and Cav3.2 mRNA in wild-type mice after transverse aortic banding (TAB) surgery supported our hypothesis that Cav3.2 is induced early during cardiac hypertrophy. The TAB-responding element [-81 to -41 bp upstream of the transcription start site (TSS) of mouse Cav3.2] was identified by in vivo gene transfer by injecting reporter constructs into the left ventricle followed by TAB surgery. Electrophoresis mobility shift assay and chromatin immunoprecipitation assays revealed that Egr1 bound to the TAB-responding element of Cav3.2. Egr1 level was increased with increased Cav3.2 mRNA level at 3 days after TAB. To demonstrate that Egr1 indeed regulates Cav3.2 expression after hypertrophic stimulation, knockdown of Egr1 with short hairpin RNA prevented the phenylephrine-induced up-regulation of Cav3.2 expression and cellular hypertrophy in neonatal rat ventricular myocytes (NRVMs) and H9c2 cells. Furthermore, overexpression of Cav3.2 in Egr1-knockdown cells restored the phenylephrine-induced hypertrophy. Cav3.2 is induced early by Egr1 during cardiac hypertrophy and Cav3.2 is an important mediator of Egr1 in regulating cardiac hypertrophy.

Extracellular Matrix of Mechanically Stretched Cardiac Fibroblasts Improves Viability and Metabolic Activity of Ventricular Cells

Background: In heart, the extracellular matrix (ECM), produced by cardiac fibroblasts, is a potent regulator of heart,s function and growth, and provides a supportive scaffold for heart cells in vitro and in vivo. Cardiac fibroblasts are subjected to mechanical loading all the time in vivo. Therefore, the influences of mechanical loading on formation and bioactivity of cardiac fibroblasts, ECM should be investigated.Methods: Rat cardiac fibroblasts were cultured on silicone elastic membranes and stimulated with mechanical cyclic stretch. After removing the cells, the ECMs coated on the membranes were prepared, some ECMs were treated with heparinase II (GAG-lyase), then the collagen, glycosaminoglycan (GAG) and ECM proteins were assayed. Isolated neonatal rat ventricular cells were seeded on ECM-coated membranes, the viability and lactate dehydrogenase (LDH) activity of the cells after 1-7 days of culture was assayed. In addition, the ATPase activity and related protein level, glucose consumption ratio and lactic acid production ratio of the ventricular cells were analyzed by spectrophotometric methods and Western blot.Results: The cyclic stretch increased collagen and GAG levels of the ECMs, and elevated protein levels of collagen I and fibronectin. Compared with the ECMs produced by unstretched cardiac fibroblasts, the ECMs of mechanically stretched fibroblasts improved viability and LDH activity, elevated the Na+/K+-ATPase activity, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and SERCA 2a protein level, glucose consumption ratio and lactic acid production ratio of ventricular cells seeded on them. The treatment with heparinase II reduced GAG levels of these ECMs, and lowered these metabolism-related indices of ventricular cells cultured on the ECMs.Conclusions: Mechanical stretch promotes ECM formation of cardiac fibroblasts in vitro, the ECM of mechanically stretched cardiac fibroblasts improves metabolic activity of ventricular cells cultured in vitro, and the GAG of the ECMs is involved in regulating metabolic activity of ventricular cells.

Cardiac Fibroblast-Derived Extracellular Matrix Produced <I>In Vitro</I> Stimulates Growth and Metabolism of Cultured Ventricular Cells

Cardiac fibroblasts (CFs) produce extracellular matrix (ECM) which is a potent regulator of heart cell function and growth, and provides a supportive microenvironment for heart cells. Therefore, CF-derived ECM produced in vitro is very suitable for heart-cell culturing and cardiac tissue engineering. The aim of this study was to investigate the effect of CF-derived ECM produced in vitro on the growth and metabolism of cultured ventricular cells. CF-derived ECM-coated cell culture dishes were prepared by culturing rat CFs and then decellularizing the cultures. Isolated neonatal rat ventricular cells were seeded on ECM-coated, collagen I-coated or uncoated dishes, and the growth of cells after 1-5 days of culture was assayed with MTT reagent. In addition, cellular metabolic activity was analyzed by spectrophotometric methods and protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) by Western blotting. The relative growth of ventricular cells was better on ECM-coated than on uncoated or collagen I-coated dishes. Furthermore, the glucose consumption ratio, lactic acid production ratio, Na(+)/K(+)-ATPase activity, SERCA activity and protein levels of SERCA2a were all higher in cells on the ECM-coated dishes. In conclusion, cardiac fi broblast-derived ECM produced in vitro stimulates the growth and metabolism of cultured ventricular cells. This study indicates that the bioactivity of the ECM supports heart cell growth in vitro, and this might be useful for cardiac tissue engineering.

AAV-Mediated Knock-Down of HRC Exacerbates Transverse Aorta Constriction-Induced Heart Failure

BackgroundHistidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca2+ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca2+ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca2+ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively.Methodology/Principal FindingsAAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca2+ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca2+ load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.Conclusions/SignificanceIncreased Ca2+ leak and cytosolic Ca2+ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca2+ cycling.

The ability of phosphodiesterase-5 inhibitors sildenafil and ordonafil to reverse L-NAME induced cardiac hypertrophy in the rabbit: possible role of calcineurin and p38

Phosphodiesterase 5 inhibitors (PDE-5Is) can suppress and (or) reverse pressure overload induced myocardial hypertrophy. This study investigated the suppressive effect of 2 PDE-5Is (sildenafil and ordonafil) on N-nitro-l-arginine methyl ester (L-NAME)-induced cardiac hypertrophy in rabbit heart, and examined their possible mechanism of action. L-NAME increased left ventricular thickness to 6.1± 0.18 mm from 4.6 ± 0.13 mm (p < 0.05), which regressed after treatment with either sildenafil or ordonafil to 5.1 ± 0.1 mm and 4.8 ± 0.2 mm, respectively (p < 0.05). Phenylephrine increased neonatal rat ventricular myocyte cell surface area to 131% ± 3% of the control value, which was associated with significant increment in ERK1/2 to 143% ± 5% of the control value (p < 0.05). Ordonafil and sildenafil decreased cell surface area to 95% ± 3% and 90% ± 1% of the control value, respectively. Both drugs decreased ERK1/2 to 88% ± 4% of the control value. Calcineurin activity was significantly decreased after 1 h of treatment with 0.1 mg·L(-1) ordonafil (1.15 ± 0.05, p < 0.05). For sildenafil (0.1 mg·L(-1)), calcineurin activity significantly decreased only after 24 h of incubation (22%). Also p38 activation was attenuated by ordonafil and sildenafil (0.1 mg·L(-1)). It is suggested that both drugs have the ability to reverse L-NAME-induced cardiac hypertrophy and suppress phenylphrine-induced myocyte hypertrophy, and that these effects may be mediated through the attenuation of calcineurin and its downstream signaling pathways (p38) in neonatal rat ventricular myocytes.

Abstract 134: Electrical Stimulation Improves Calcium Handling in Engineered Heart Tissue

Purpose: Despite considerable advances in cardiac tissue engineering, morphology and functionality of engineered heart tissue (EHT) needs to be further improved, likely by making use of more “physiologic” engineering paradigms. We hypothesized that electrical stimulation may improve calcium handling in EHTs through enhanced maturation of the excitation coupling apparatus. Methods: EHTs were prepared from neonatal rat ventricular heart cells and after one week, transferred to dynamic stretchers. EHTs were cultured with (2 or 4 Hz) or without (non-stimulated ‘NS’) electrical stimulation from day 7 to 13 (Ionoptix C-Pacer: 2 ms square biphasic pulse, 4 Volts). At day 13, functional (twitch tension at 2 Hz field stimulation) were performed, and EHTs were processed for Western Blot analysis. In addition, EHT-derived single cardiomyocytes loaded with fura-2 (1 µM) were analyzed using an Ionoptix setup to assess alterations in calcium handling. Results: Culture under electrical enhanced twitch tension of EHTs (2 Hz vs. NS: 0.83 ± 0.05 vs. 0.62 ± 0.03, p&lt;0.0001, n=8/group), lowered excitation thresholds, and increased upstroke velocity. Assessment of sarcoplasmic reticulum (SR) functionality by post-rest potentiation revealed improved calcium storage capacity in EHTs (NS vs. 2 Hz vs. 4 Hz: 10.2 ± 1.4 vs. 11.7 ± 1.4 vs. 17.8 ± 1.3 % at 30 s rest; p&lt;0.05, n=7/7/5). Ryanodine (1 µM, to block SR-calcium release) reduced EHT twitch tension to 88.3 ± 2.1 % [NS] vs. 87.2 ± 2.4 % [2 Hz] vs. 75.1 ± 4.5 % [4 Hz] (n=8/group, p&lt;0.05) baseline force, indicating greater reliance on the SR for force generation, particularly in the 4 Hz stimulated group. Ryanodine receptor and SERCA protein expression increased with 4 Hz stimulation. In isolated cardiomyocytes, 4 Hz stimulated cells displayed faster upstroke velocity and relaxation times, approaching that of cardiomyocytes isolated from day 13 postnatal rats. Conclusions: Electrical stimulation of dynamically loaded EHTs contributes to increased functionality and appears to improve excitation-contraction coupling through enhanced Calcium storage and release capacity. This combined biomimetic stimulation method may overcome the sub-physiologic SR functionality of classical cell culture models.

Engraftment of human embryonic stem cell derived cardiomyocytes improves conduction in an arrhythmogenic in vitro model

In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-β treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275 ± 36 ms and conduction velocity (CV) of 10.6 ± 4.2 cm/s (n = 3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4 ± 0.9 cm/s, n = 35 vs. 30.1 ± 3.2 cm/s, n = 20 in the longitudinal direction and 4.3 ± 0.3 cm/s vs. 9.3 ± 0.9 cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6 ± 1.3 cm/s and 3.1 ± 0.5 cm/s, n = 11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.