Transfection Of Cardiomyocytes Research Articles

Vps4a Regulates Autophagic Flux to Prevent Hypertrophic Cardiomyopathy.

Autophagy has stabilizing functions for cardiomyocytes. Recent studies indicate that an impairment in the autophagy pathway can seriously affect morphology and function, potentially leading to heart failure. However, the role and the underlying mechanism of the endosomal sorting complex required for transport (ESCRT) family protein, in particular the AAA-ATPase vacuolar protein sorting 4a (Vps4a), in regulating myocardial autophagy remains unclear. In the present study, cardiomyocyte-specific Vps4a knockout mice were generated by crossing Vps4aflox/flox (Vps4afl/fl) with Myh6-cre transgenic mice. As a result, we observed a partially dilated left ventricular (LV) chamber, a significant increase in heart weight to body weight ratio (HW/BW), and heart weight to tibial length ratio (HW/TL), hypertrophic cardiomyopathy and early lethality starting at 3 months of age. Hematoxylin-eosin (HE), immunofluorescence assay (IFA), and Western blot (WB) revealed autophagosome accumulation in cardiomyocytes. A transcriptome-based analysis and autophagic flux tracking by AAV-RFP-GFP-LC3 showed that the autophagic flux was blocked in Vps4a knockout cardiomyocytes. In addition, we provided in vitro evidence demonstrating that Vps4a and LC3 were partially co-localized in cardiomyocytes, and the knockdown of Vps4a led to the accumulation of autophagosomes in cardiomyocytes. Similarly, the transfection of cardiomyocytes with adenovirus (Adv) mCherry-GFP-LC3 further indicated that the autophagic flux was blocked in cells with deficient levels of Vps4a. Finally, an electron microscope (EM) showed that the compromised sealing of autophagosome blocked the autophagic flux in Vps4a-depleted cardiomyocytes. These findings revealed that Vps4a contributed to the sealing of autophagosomes in cardiomyocytes. Therefore, we demonstrated that Vps4a deletion could block the autophagic flux, leading to the accumulation of degradation substances and compromised cardiac function. Overall, this study provides insights into a new theoretical basis for which autophagy may represent a therapeutic target for cardiovascular diseases.

SiRNA Delivery against Myocardial Ischemia Reperfusion Injury Mediated by Reversibly Camouflaged Biomimetic Nanocomplexes.

siRNA-mediated management of myocardial ischemia reperfusion (IR) injury is greatly hampered by the inefficient myocardial enrichment and cardiomyocyte transfection. Herein, nanocomplexes (NCs) reversibly camouflaged with a platelet-macrophage hybrid membrane (HM) are developed to efficiently deliver Sav1 siRNA (siSav1) into cardiomyocytes, suppressing the Hippo pathway and inducing cardiomyocyte regeneration. The biomimetic BSPC@HM NCs consist of a cationic nanocore assembled from a membrane-penetrating helical polypeptide (P-Ben) and siSav1, a charge-reversal intermediate layer of poly(l-lysine)-cis-aconitic acid (PC), and an outer shell of HM. Due to HM-mediated inflammation homing and microthrombus targeting, intravenously injected BSPC@HM NCs can efficiently accumulate in the IR-injured myocardium, where the acidic inflammatory microenvironment triggers charge reversal of PC to shed off both HM and PC layers and allow the penetration of the exposed P-Ben/siSav1 NCs into cardiomyocytes. In rats and pigs, BSPC@HM NCs remarkably downregulates Sav1 in IR-injured myocardium, promotes myocardium regeneration, suppresses myocardial apoptosis, and recovers cardiac functions. This study reports a bioinspired strategy to overcome the multiple systemic barriers against myocardial siRNA delivery, and holds profound potential for gene therapy against cardiac injuries.

Exosomal miR‑152‑5p/ARHGAP6/ROCK axis regulates apoptosis and fibrosis in cardiomyocytes.

Acute myocardial infarction (AMI) is a fatal cardiovascular disease with a high mortality rate. The discovery of effective biomarkers is crucial for the diagnosis and treatment of AMI. In the present study, miRNA sequencing and reverse transcription-quantitative polymerase chain reaction techniques revealed that the expression of exosome derived miR-152-5p was significantly downregulated in patients with AMI compared with healthy controls. A series of functional validation experiments were then performed using H9c2 cardiomyocytes. Following transfection of the cardiomyocytes using an miR-152-5p inhibitor, immunofluorescence staining of a-smooth muscle actin revealed a marked increase in fibrosis. Western blotting revealed that the expression levels of the apoptotic protein Bax, TNF-α and collagen-associated proteins were significantly increased, whereas those of the apoptosis-inhibiting factor Bcl-2 and vascular endothelial growth factor A were significantly decreased. Furthermore, the binding of Rho GTPase-activating protein 6 (ARHGAP6) to miR-152-5p was predicted using an online database and verified using a dual-luciferase reporter gene assay. The transfection of cardiomyocytes with miR-152-5p mimics was found to inhibit the activation of ARHGAP6 and Rho-associated coiled-coil containing kinase 2 (ROCK2). These results suggest that miR-152-5p targets ARHGAP6 through the ROCK signaling pathway to inhibit AMI, which implies that miR-152-5p may be a diagnostic indicator and potential target for treatment of myocardial infarction.

PGAM5-Mediated PHB2 Dephosphorylation Contributes to Diabetic Cardiomyopathy by Disrupting Mitochondrial Quality Surveillance.

Disruption of the mitochondrial quality surveillance (MQS) system contributes to mitochondrial dysfunction in diabetic cardiomyopathy (DCM). In this study, we observed that cardiac expression of phosphoglycerate mutase 5 (PGAM5), a mitochondrial Ser/Thr protein phosphatase, is upregulated in mice with streptozotocin-induced DCM. Notably, DCM-related cardiac structural and functional deficits were negated in cardiomyocyte-specific Pgam5 knockout (Pgam5CKO ) mice. Hyperglycemic stress impaired adenosine triphosphate production, reduced respiratory activity, and prolonged mitochondrial permeability transition pore opening in acutely isolated neonatal cardiomyocytes from control Pgam5f/f mice, and these effects were markedly prevented in cardiomyocytes from Pgam5CKO mice. Likewise, three main MQS-governed processes-namely, mitochondrial fission/fusion cycling, mitophagy, and biogenesis-were disrupted by hyperglycemia in Pgam5f/f , but not in Pgam5CKO , cardiomyocytes. On the basis of bioinformatics prediction of interaction between PGAM5 and prohibitin 2 (PHB2), an inner mitochondrial membrane-associated scaffolding protein, co-immunoprecipitation, and immunoblot assays demonstrated that PGAM5 dephosphorylates PHB2 on Ser91. Transfection of cardiomyocytes with phosphodefective or phosphomimetic Ser91 mutants of PHB2 confirmed a critical role for PGAM5-mediated dephosphorylation of PHB2 in mitochondrial dysfunction associated with hyperglycemic stress. Furthermore, knockin mice expressing phosphomimetic PHB2S91D were resistant to diabetes-induced cardiac dysfunction. Our findings highlight the PGAM-PHB2 axis as a novel and critical regulator of mitochondrial dysfunction in DCM.

Telmisartan cardioprotects from the ischaemic/hypoxic damage through a miR-1-dependent pathway.

The aim of this study was to investigate whether telmisartan protects the heart from the ischaemia/reperfusion damage through a local microRNA‐1 modulation. Studies on the myocardial ischaemia/reperfusion injury in vivo and on the cardiomyocyte hypoxia/reoxygenation damage in vitro were done. In vivo, male Sprague‐Dawley rats administered for 3 weeks with telmisartan 12 mg/kg/d by gastric gavage underwent ischaemia/reperfusion of the left descending coronary artery. In these rats, infarct size measurement, ELISA, immunohistochemistry (IHC) and reverse transcriptase real‐time polymerase chain reaction showed that expressions of connexin 43, potassium voltage‐gated channel subfamily Q member 1 and the protein Bcl‐2 were significantly increased by telmisartan in the reperfused myocardium, paralleled by microRNA‐1 down‐regulation. In vitro, the transfection of cardiomyocytes with microRNA‐1 reduced the expressions of connexin 43, potassium voltage‐gated channel subfamily Q member 1 and Bcl‐2 in the cells. Telmisartan (50 µmol/L) 60 minutes before hypoxia/reoxygenation, while not affecting the levels of miR‐1 in transfected cells in normoxic condition, almost abolished the increment of miR‐1 induced by the hypoxia/reoxygenation to transfected cells. All together, telmisartan cardioprotected against the myocardial damage through the microRNA‐1 modulation, and consequent modifications of its downstream target connexin 43, potassium voltage‐gated channel subfamily Q member 1 and Bcl‐2.

Progress in Gene Therapy for Chronic Heart Failure.

Chronic heart failure (CHF) is still the leading cause of morbidity and mortality worldwide, and carries with it large economic and social burdens. Although steady and substantial progress has been made in reducing mortality from heart failure using conventional treatments, novel pharmacologic and surgical interventions have not been effective in extending five year survival rates. Therefore, it is necessary to explore new therapies. Gene therapy was introduced in 1970s with the development of recombinant DNA technology. Due to recent progress in the understanding of myocardial metabolism and application of vector based gene transfer strategies in animal models and initial clinical trials, gene therapy possibly affords an ideal treatment alternative for CHF. In last 2 decades, much research has been done on gene therapy, using various genes, signal transduction passages and delivery methods to treat advanced heart failure. Current research in ischemic heart disease (IHD) mainly focuses on stimulating angiogenesis, modifying the coronary vascular environment, and improving the vascular endothelial function with localized gene coated catheters and stents. Compared with standard ischemic heart disease treatment, the main goal of gene therapy for CHF is to inhibit apoptosis, reduce the undesirable remodeling and increase contractility through the most efficient cardiomyocyte transfection [Katz 2012a]. In this paper, we review various gene transfer technologies in ischemic heart disease and heart failure models, and discuss the advantages and disadvantages of these strategies in vector-mediated cardiac gene delivery, with the main focus on the high efficiency approach of a molecular cardiac surgery delivery system.

Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1.

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism.Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 μg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection of HCMs with a hsa-miR-128-3p mimic eliminated the protective effects of TXL.Conclusions: The miR-128-3p/p70s6k1 signaling pathway is involved in protection by TXL against HCM apoptosis during H/R. Overexpression of p70s6k1 is, therefore, a potential new strategy for alleviating myocardial reperfusion injury.

Rac1 GTPase regulates 11β hydroxysteroid dehydrogenase type 2 and fibrotic remodeling

The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression (r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression (r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling.

Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway.

Underexpression of CACNA1C Caused by Overexpression of microRNA-29a Underlies the Pathogenesis of Atrial Fibrillation.

BackgroundThe objective of this study was to investigate the molecular mechanism of atrial fibrillation (AF), as well as the negative regulatory relationship between miR-29a-3p and CACNA1C.Material/MethodsWe searched the online miRNA database (www.mirdb.org) and identified the miR-29a-3p binding sequence within the 3′-UTR of the target gene, and then conducted luciferase assay to verify it. The cells were transfected with miR-29a-3p and ICa,L was determined in those cells.ResultsWe validated CACNA1C to be the direct target gene of miR-29a-3p. We also established the negative regulatory relationship between miR-29a-3p and CACNA1C via studying the relative luciferase activity. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of CACNA1C among different groups of cells treated with scramble control, 30nM miR-29a-3p mimics, and 60nM miR-29a-3p mimics, indicating a negative regulatory relationship between miR-29a-3p and CACNA1C. We next analyzed whether miR-29a-3p transfection in cardiomyocytes produced the effects on the ICa,L induced by electrical remodeling, and found a tonic inhibition of IBa by endogenous miR-29a-3p in atrial myocytes.ConclusionsWe validated the negative regulation between miR-29a-3p and CACNA1C, and found that miR-29a-3p might a potential therapeutic target in the treatment of AF.

Hydrogen Sulfide Protects Cardiomyocytes against Apoptosis in Ischemia/Reperfusion through MiR-1-Regulated Histone Deacetylase 4 Pathway

Background/Aims: Hydrogen sulfide (H<Sub>2</Sub>S) is a powerful inhibitor of cardiomyocytes apoptosis following ischemia/reperfusion (IR) injury, but the underlying mechanism remains unclear. Our previous study showed that microRNA-1 (miR-1) was upregulated by 2.21 fold in the IR group compared with that in the H<Sub>2</Sub>S preconditioned group. MiR-206 affected the process of cardiomyocytes hypertrophy by regulating histone deacetylase 4 (HDAC4). HDAC4 is also known to play an anti-apoptotic role in tumor cells, but its role in the myocardium has not been reported. The aim of this study was to test whether H<Sub>2</Sub>S could inhibit apoptosis of cardiomyocytes through HDAC4 regulation by miR-1 in IR. Methods: Cardiomyocytes of neonatal rats were subjected to hypoxia/reoxygenation (HR) injury with or without H<Sub>2</Sub>S pretreatment to simulate IR injury Cardiomyocytes were transfected with miR-1 mimic or HDAC4 siRNA to evaluate whether the miR-1-HDAC4 signaling pathway was involved in the protective effect of H<Sub>2</Sub>S. Results: HR increased cell apoptosis and caspase-3 cleavage, upregulated miR-1, and downregulated HDAC4. H<Sub>2</Sub>S preconditioning attenuated the apoptosis of cardiomyocytes, caspase-3 cleavage and LDH release, and enhanced cell viability In addition, H<Sub>2</Sub>S downregulated miR-1, and preserved HDAC4 expression. HDAC4 protein was down-regulated by miR-1 mimic. Transfection of cardiomyocytes with miR-1 mimic partially reduced the protective effect of H<Sub>2</Sub>S. Meanwhile, transfection of cardiomyocytes with siRNA to HDAC4 partially abrogated the protective effect of H<Sub>2</Sub>S. Conclusions: The miR-1-HDAC4 signaling pathway is involved in the protective effect of H<Sub>2</Sub>S against the apoptosis of cardiomyocytes during the IR injury process.

Na+/H+ exchanger isoform 1-induced osteopontin expression facilitates cardiomyocyte hypertrophy.

Enhanced expression and activity of the Na+/H+ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.

Abstract 359: Negative Regulation Of The Fgf-16 Gene Promoter By Doxorubicin Is Associated With A Decrease In Nkx2.5 Binding

Doxorubicin (DOX) is a widely used and effective anti-cancer drug, but it is also cardiotoxic, which can lead to heart failure, and so strategies are needed to protect the heart. Fibroblast growth factor 16 (FGF-16) is preferentially expressed and released from cardiomyocytes after birth. Evidence suggests that FGF-16 decreases the risk of heart damage and limits the negative effects of heart remodeling (hypertrophy and fibrosis) after injury in vivo. Exogenous addition of FGF-16 also increased resistance to the loss of contractility in an isolated heart model of acute DOX-induced injury. Thus, how endogenous FGF-16 production and by extension function is affected by DOX treatment is of interest. The FGF-16 gene is highly conserved between human and murine species. Alignment of sequences indicates a conserved Nkx2.5 binding site in the proximal promoter region that is associated with a previously characterized TATA box. Nkx2.5 is an important factor in vertebrate heart development and congenital disease. Furthermore, Nkx2.5 RNA levels are decreased with DOX treatment. Thus, the possibility that DOX negatively affects FGF-16, perhaps through an effect on Nkx2.5 levels or binding, was investigated. Neonatal rat cardiomyocytes were treated with DOX, and FGF-16 RNA levels decreased 75% within 6 hours as assessed by qPCR. Inhibition of transcription with actinomycinD had no effect on the DOX-induced decrease in FGF-16 RNA levels. Further support for an effect of DOX on FGF-16 transcription was obtained by transfection of cardiomyocytes with a hybrid 747 bp mouse FGF-16 promoter/luciferase reporter gene that was treated with DOX; a significant decrease in luciferase activity was observed. Electrophoretic mobility shift and chromatin immunoprecipitation assays suggest reduced Nkx2.5 protein-DNA interaction with this site after DOX treatment in vitro and in situ, respectively. These data indicate that DOX decreases FGF-16 RNA expression and this correlates with a decrease in Nkx2.5 levels and association with the proximal promoter region. A direct effect of Nkx2.5 on FGF-16 promoter activity awaits further testing. Thus, a decrease or loss of FGF-16 synthesis might contribute to the DOX-induced damage and/or response to injury.

MicroRNA-150 inhibits expression of adiponectin receptor 2 and is a potential therapeutic target in patients with chronic heart failure

Adiponectin is an anti-inflammatory adipocytokine believed to be involved in the pathogenesis of chronic heart failure (CHF). We aimed to characterize the expression of adiponectin and its receptors in CHF and to assess the impact of microRNAs on the cardiac adiponectin system. Expression of adiponectin and adiponectin receptors (ADIPOR1 and ADIPOR2) was studied by qPCR and immunohistochemistry in myocardial tissues of patients with end-stage CHF and control subjects. MicroRNA binding was evaluated by cloning of an ADIPOR2 3´-untranslated-region reporter construct and subsequent transfection experiments. Effects of miRNA transfection were analyzed in cardiomyocyte cell cultures by qPCR and Western blotting. Gene silencing of ADIPOR2 was performed by siRNA transfection, and the effects of hypoxia/serum starvation were analyzed by flow cytometry. Although CHF patients displayed elevated plasma adiponectin levels, myocardial adiponectin expression generally was very low. In CHF, cardiac ADIPOR1 expression increased by >4-fold, whereas the increase in ADIPOR2 was less than 2-fold. Reporter gene assays on constructs containing the ADIPOR2-3'-untranslated region suggest that microRNA-150 specifically repressed ADIPOR2 expression. Transfection of cardiomyocytes with premiR-150 precursor molecules resulted in 60% down-regulation of ADIPOR2 mRNA and a significant reduction of ADIPOR2 protein expression. MicroRNA-150 was substantially expressed in both normal and CHF myocardium, with a 1.7-fold higher expression in CHF. Finally, knock-down experiments elucidated a stress-protective role of ADIPOR2 in cardiomyocytes. MicroRNA-150 counteracts ADIPOR2 up-regulation in CHF and thus may contribute to adiponectin resistance. Targeting microRNA-150 may be a future strategy to restore cardioprotective adiponectin effects.

GW24-e2121 Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signalling pathway

ObjectivesTo examine the inhibitory effects of adenosine monophosphate-activated protein kinase (AMPK) activation on cardiomyocyte hypertrophy in vitro and to investigate the underlying molecular mechanisms.MethodsCultured neonatal rat cardiomyocytes were treated with...

P42/p44-MAPK and PI3K are sufficient for IL-6 family cytokines/gp130 to signal to hypertrophy and survival in cardiomyocytes in the absence of JAK/STAT activation

The effect of differential signalling by IL-6 and leukaemia inhibitory factor (LIF) which signal by gp130 homodimerisation or LIFRβ/gp130 heterodimerisation on survival and hypertrophy was studied in neonatal rat cardiomyocytes. Both LIF and IL-6 [in the absence of soluble IL-6 receptor (sIL-6Rα)] activated Erk1/2, JNK1/2, p38-MAPK and PI3K signalling peaking at 20min and induced cytoprotection against simulated ischemia-reperfusion injury which was blocked by the MEK1/2 inhibitor PD98059 but not the p38-MAPK inhibitor SB203580. In the absence of sIL-6R, IL-6 did not induce STAT1/3 phosphorylation, whereas IL-6/sIL-6R and LIF induced STAT1 and STAT3 phosphorylation. Furthermore, IL-6/sIL-6R induced phosphorylation of STAT1 Tyr701 and STAT3 Tyr705 were enhanced by SB203580. IL-6 and pheneylephrine (PE), but not LIF, induced cardiomyocyte iNOS expression and nitric oxide (NO) production. IL-6, LIF and PE induced cardiomyocyte hypertrophy, but with phenotypic differences in ANF and SERCA2 expression and myofilament organisation with IL-6 more resembling PE than LIF. Transfection of cardiomyocytes with full length or truncated chimaeric gp130 cytoplasmic domain/Erythropoietin receptor (EpoR) extracellular domain fusion constructs showed that the membrane proximal Box 1 and Box 2 containing region of gp130 was necessary and sufficient for MAPK and PI3K activation; hypertrophy; SERCA2 expression and iNOS/NO induction in the absence of JAK/STAT activation. In conclusion, IL-6 can signal in cardiomyocytes independent of sIL-6R and STAT1/3 and furthermore, that Erk1/2 and PI3K activation by IL-6 are both necessary and sufficient for induced cardioprotection. In addition, p38-MAPK may act as a negative feedback regulator of JAK/STAT activation in cardiomyocytes.

Targeted Gene Delivery to Ischemic Myocardium by Homing Peptide-Guided Polymeric Carrier

Myocardial ischemia needs an alternative treatment such as gene therapy for the direct protection of cardiomyocytes against necrosis or apoptosis and to prevent the development of myocardial fibrosis and cardiac dysfunction. Despite the utility of gene therapy, its therapeutic use is limited due to inadequate transfection in cardiomyocytes and difficulty in directing to ischemic myocardium. Here, we present a polymeric gene carrier that is capable of targeting ischemic myocardium, resulting in high localization within the ischemic zone of the left ventricle (LV) of an ischemia/reperfusion (I/R) rat model upon systemic administration. Cystamine bisacrylamide-diamino hexane (CD) polymer was modified with the ischemic myocardium-targeted peptide (IMTP) and D-9-arginine (9R) for dual effects of the homing to ischemic myocardium and enhanced transfection efficiency with minimized polymer use. Conjugation of IMTP and 9R to CD led to an increase in transfection under hypoxia and significantly reduced the amount of polymer required for high transfection. Finally, we confirmed targeting of IMTP-CD-9R/DNA polyplex to ischemic myocardium and enhanced gene expression in LV of the I/R rat after tail vein injection. This study provides a clue that gene therapy for the treatment of myocardial ischemia can be achieved by using homing peptide-guided gene delivery systems.

Cross-talk between inflammation and angiotensin II: Studies based on direct transfection of cardiomyocytes with AT1R and AT2R cDNA

Ischemic myocardium exhibits inflammation, local angiotensin II (Ang II) generation and up-regulation of LOX-1, a lectin-like ox-LDL receptor. To define the inter-active roles of Ang II and inflammation in furthering tissue injury, cultured HL-1 cardiomyocytes were treated with Ang II. Ang II treatment up-regulated the expression of Ang II type 1 (AT1R) and type 2 (AT2R) receptors as well as LOX-1. Ang II also activated p44/42MAPK, p38MAPK, c-Jun and NF-κB, and increased the expression of inflammation-related genes (interleukins-6, interleukins-10, tumor necrosis factor-α, intercellular adhesion molecule-1). To study how inflammation per se might affect expression of Ang II receptors and LOX-1, cultured, cardiomyocytes were treated with lipopolysaccharide (LPS). Like Ang II, LPS increased the expression of AT1R, AT2R and LOX-1. LPS also activated mitogen-acticated protein kinase (MAPKs), c-Jun and NF-κB, and pro-inflammatory genes. The selective inhibitors of MAPKs, c-Jun and NF-κB each blocked the transcription of LOX-1 and pro-inflammatory genes in response to Ang II as well as LPS. These observations suggested a positive feedback between Ang II and inflammation. To delineate the role of AT1R and AT2R in LOX-1 expression, another set of cardiomyocytes were transfected with AT1R or AT2R cDNA. Forced over-expression of AT1R resulted in activation of MAPKs, c-Jun and NF-κB, up-regulation of inflammatory genes and LOX-1; on the other hand forced AT2R over-expression induced up-regulation of pro-apoptotic signals (pro-IL-1β and IL-1β), and decreased LOX-1 expression. These studies show that both Ang II and inflammation mediator LPS up-regulate AT1R, AT2R and LOX-1 expression. Up-regulation of AT1R promotes inflammation and LOX-1 expression, whereas up-regulation of AT2R promotes apoptosis signals and decreases LOX-1 expression.

Electric pulses augment reporter gene expression in the beating heart

Gene therapy of the heart has been attempted in a number of clinical trials with the injection of naked DNA, although quantitative information on myocellular transfection rates is not available. The present study aimed to quantify the efficacy of electropulsing protocols that differ in pulse duration and number to stimulate transfection of cardiomyocytes and to determine the impact on myocardial integrity. Reporter plasmid for constitutive expression of green fluorescent protein (GFP) was injected into the left ventricle of beating hearts of adult, male Lewis rats. Four electrotransfer protocols consisting of repeated long pulses (8 × 20 ms), trains of short pulses (eight trains of either 60 or 80 × 100 µs) or their combination were compared with control procedures concerning the degree of GFP expression and the effect on infiltration, fibrosis and apoptosis. All tested protocols produced GFP expression at the site of plasmid injection. Continuous pulses were most effective and increased the number of GFP-positive cardiomyocytes by more than 300-fold compared to plasmid injection alone (p < 0.05). Concomitantly, the incidence of macrophage infiltration, fibrosis and cell death was increased. Trains of short pulses reduced macrophage infiltration and fibrosis by four- and two-fold, respectively, although they were 20-fold less efficient in stimulating cardiomyocyte transfection. GFP expression co-related to delivered electric energy, infiltration and fibrosis, although not apoptosis. The data imply that electropulsing of the myocardium promotes the overexpression of exogenous protein in mature cardiomyocytes in relation to an injury component. Fractionation of pulses is indicated as a option for sophisticated gene therapeutic approaches to the heart.

Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signalling pathway

AimTo examine the inhibitory effects of adenosine monophosphate-activated protein kinase (AMPK) activation on cardiomyocytes hypertrophy and explore the underlying molecular mechanisms.MethodsCultured neonatal rat cardiomyocytes were treated with specific AMPK activator...