Neonatal Rat Ventricular Cardiomyocytes Research Articles

Discovery of a first-in-class inhibitor of sulfide:quinone oxidoreductase that protects against adverse cardiac remodelling and heart failure.

Hydrogen sulfide (H2S) is a potent signalling molecule that activates diverse cardioprotective pathways by post-translational modification (persulfidation) of cysteine residues in upstream protein targets. Heart failure patients with reduced ejection fraction (HFrEF) exhibit low levels of H2S. Sulfide:quinone oxidoreductase (SQOR) catalyses the first irreversible step in the metabolism of H2S and plays a key role in regulating H2S-mediated signalling. Here, the aim of this study was to discover a first-in-class inhibitor of human SQOR and evaluate its cardioprotective effect in an animal model of HFrEF. We identified a potent inhibitor of human SQOR (STI1, IC50 = 29 nM) by high-throughput screening of a small-molecule library, followed by focused medicinal chemistry optimization and structure-based design. STI1 is a competitive inhibitor that binds with high selectivity to the coenzyme Q-binding pocket in SQOR. STI1 exhibited very low cytotoxicity and attenuated the hypertrophic response of neonatal rat ventricular cardiomyocytes and H9c2 cells induced by neurohormonal stressors. A mouse HFrEF model was produced by transverse aortic constriction (TAC). Treatment of TAC mice with STI1 mitigated the development of cardiomegaly, pulmonary congestion, dilatation of the left ventricle, and cardiac fibrosis and decreased the pressure gradient across the aortic constriction. Moreover, STI1 dramatically improved survival, preserved cardiac function, and prevented the progression to HFrEF by impeding the transition from compensated to decompensated left ventricle hypertrophy. We demonstrate that the coenzyme Q-binding pocket in human SQOR is a druggable target and establish proof of concept for the potential of SQOR inhibitors to provide a novel therapeutic approach for the treatment of HFrEF.

Dual role of miR-1 in the development and function of sinoatrial cells

miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC).We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1.miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating If currents; miR1OE SAN-like cells instead have only fast-activating If with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and If alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1.In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.

STIM2 protein regulates Orai1-mediated store-operated Ca2+ entry in cardiomyocytes

Stromal interaction molecules, STIM1 and STIM2, are ER Ca 2+ sensors that initiate store-operated Ca 2+ entry (SOCE). It has been recently identified three STIM2 splice variants (STIM2.1, 2.2 and 2.3), STIM2.2 being SOCE activator whereas STIM2.1 will be SOCE inhibitor. The role of STIM1-mediated SOCE has been highlighted in cardiac pathobiology but the one of STIM 2 .1 and STIM2.2 is unknown. To assess whether and how STIM2.1 and STIM2.2 regulate SOCE in neonatal rat ventricular cardiomyocytes (NRVMs). To analyze the function of STIM2.1 and STIM2.2 variants, the NRVMs were transfected with STIM2.2 or STIM2.1 in combination or not with STIM1 or dn-Orai1 106A mutant plasmids or with a mixture of 4 STIM2 siRNAs. At rest, both native STIM2 and exogenous STIM2.2 proteins present a reticular organization. Live cell confocal imaging in STIM2.2 overexpressing cells showed that STIM2.2 is aggregated and translocated towards the sarcolemma following the SR Ca 2+ store depletion. With Ca 2+ imaging, we found that STIM2.2 overexpression significantly increases SOCE, similarly to STIM1 overexpression, but the STIM1 and STIM2.2 co-expression has no additional effect. Importantly, the STIM2.2-enhanced SOCE is suppressed by functional Orai1 inhibition, either by co-expressing STIM2.2 with the dn-Orai1 106A mutant or with the selective Orai1 inhibitor, JPIII. In addition, the JPIII-sensitive Isoc current is also higher in STIM2.2 overexpressing cells. Nonetheless, silencing of endogenous STIM2 also increases SOCE. By co-immunoprecipitation, we found that STIM2, STIM1 and Orai1 interact together suggesting that all these proteins form a complex and constitute functional SOCE machinery. We demonstrated that, in overexpressing system, STIM2.2 functions together with STIM1 as a positive regulator of Orai1-mediated SOCE and that, in endogenous system, the STIM2.1 variant may be the predominant variant that negatively regulates SOCE in NRVMs.

GPR55 regulates the responsiveness to, but does not dimerise with, α1A-adrenoceptors

Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α1A-AR) in the cardiovascular system and the potential to influence function/signalling activities. GPR55 and α1A-AR mediated changes in both cardiac and vascular function was assessed in male wild-type (WT) and GPR55 homozygous knockout (GPR55-/-) mice by pressure volume loop analysis and isolated vessel myography, respectively. Dimerization of GPR55 with the α1A-AR was examined in transfected Chinese hamster ovary-K1 (CHO-K1) cells via Bioluminescence Resonance Energy Transfer (BRET). GPR55 and α1A-AR mediated signalling (extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation) was investigated in neonatal rat ventricular cardiomyocytes using AlphaScreen proximity assays. GPR55-/- mice exhibited both enhanced pressor and inotropic responses to A61603 (α1A-AR agonist), while in isolated vessels, A61603 induced vasoconstriction was attenuated by a GPR55-dependent mechanism. Conversely, GPR55-mediated vasorelaxation was not altered by pharmacological blockade of α1A-ARs with tamsulosin. While cellular studies demonstrated that GPR55 and α1A-AR failed to dimerize, pharmacological blockade of GPR55 altered α1A-AR mediated signalling and reduced ERK1/2 phosphorylation. Taken together, this study provides evidence that GPR55 and α1A-AR do not dimerize to form heteromers, but do interact at the signalling level to modulate the function of α1A-AR in the cardiovascular system.

MiR-96 promotes myocardial infarction-induced apoptosis by targeting XIAP

Acute myocardial infarction (AMI) has becoming a common leading cause of sudden death worldwide. MiR-96 has been identified that can target anti-apoptotic related genes in various human diseases. However, its role in AMI remains unclear. In this study, we found that miR-96 was significantly upregulated in the ischemic heart of MI mice (mice with myocardial infarction) and also in the H2O2-treated neonatal rat ventricular cardiomyocytes (CMs). In response H2O2, miR-96 inhibitor could significantly promote cell viability and reduce cell apoptosis of CMs, and inhibit the expression of Cleaved caspase-3 and Bax, while promote Bcl-2 expression. In addition, downregulation of miR-96 remarkedly reduced the infarct size and the percentages of apoptotic cells in the heart tissues of MI mice, and then protected against the damaged cardiac function. Moreover, we identified that XIAP (X-linked inhibitor of apoptosis) acted as a direct target gene of miR-96, meanwhile si-XIAP could obviously reverse miR-96 inhibitor induced protective effect in H2O2-treated CMs Taken together, our study demonstrated that miR-96 promoted AMI progression by directly targeting XIAP, and inhibiting the anti-apoptotic function of XIAP (Graphical abstract), which provided a novel therapeutic target for AMI treatment.

Pinoresinol diglucoside (PDG) attenuates cardiac hypertrophy via AKT/mTOR/NF-κB signaling in pressure overload-induced rats

Ethnopharmacological relevancePinoresinol diglucoside (PDG), the active compound extracted from Eucommia ulmoides, Styrax sp. and Forsythia suspensa, plays the roles in regulating hypertension, inflammation and oxidative stress. AimsConsidering that hypertension and inflammation has been proved to contribute to cardiac remodeling, we tested the effects of PDG on cardiac hypertrophy (CM). MethodsMale Sprague Dawley (SD) rats were used to construct hypertrophic rats by partial abdominal aortic constriction (AAC)-surgery. PDG solution (2 mg/ml) was used to treat AAC-induced rats by intraperitoneal injection at low dose (L-PDG, 2.5 mg/kg per day), medium dose (M-PDG, 5 mg/kg per day), and high dose (H-PDG, 7.5 mg/kg per day) for 3 weeks post AAC-surgery. CM was evaluated by the ratio of left ventricular weight to body weight ratio (LVW/BW), left ventricular wall thickness by H&E staining, and collagen content deposit by Masson's staining. Further, isoproterenol (ISO) and phenylephrine (PE) were used to produce cellular models of CM in neonatal rat ventricular cardiomyocytes (NRVMs). PDG pre-treated NRVMs 2 h at low dose (L-PDG, 2.5 μg/ml), medium dose (M-PDG, 5 μg/ml), and high dose (H-PDG, 7.5 μg/ml) for 24 h with or without PE- and ISO-stimulation. CM was evaluated by the expressions of hypertrophic biomarkers. Next, the hypertrophic biomarkers and pro-inflammatory cytokines were measured using quantitative real-time PCR (qRT-PCR), the expressions of protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/transcription factor nuclear factor-kappa B (NF-kB) signaling pathway were determined by Western blotting. ResultsPDG treatment prevented cardiac histomorphology damages, decreased upregulations of hypertrophic biomarkers, and prevented fibrosis and inflammation after pressure overload resulting from AAC-surgery. Consistently, PDG remarkably inhibited the changes of cardiomyocyte hypertrophic biomarkers and inflammatory responses in cellular models of CM. Interestingly, PDG administration inhibited the activation of AKT/mTOR/NF-kB signaling pathway both in vivo and in vitro. ConclusionsPDG prevents AAC-induced CM in vivo, PE- and ISO-induced CM in vitro. The AKT/mTOR/NF-kB signaling pathway could be the potential therapeutic target involved in the protection of PDG. These findings provide novel evidence that PDG might be a promising therapeutic strategy for CM.

Different roles of BAG3 in cardiac physiological hypertrophy and pathological remodeling

Heart failure is one of the leading causes of death worldwide. A stimulated heart undergoes either adaptive physiological hypertrophy, which can maintain a normal heart function, or maladaptive pathological remodeling, which can deteriorate heart function. These 2 kinds of remodeling often co-occur at the early stages of many heart diseases and have important effects on cardiac function. The Bcl2-associated athanogene 3 (BAG3) protein is highly expressed in the heart and has many functions. However, it is unknown how BAG3 is regulated and what its function is during physiological hypertrophy and pathological remodeling. We generated tamoxifen-induced, heart-specific heterozygous and homozygous BAG3 knockout mouse models (BAG3 protein level decreased by approximately 40% and 80% in the hearts after tamoxifen administration). BAG3 knockout models were subjected to swimming training or phenylephrine (PE) infusion to induce cardiac physiological hypertrophy and pathological remodeling. Neonatal rat ventricular cardiomyocytes (NRVCs) were used to study BAG3 functions and mechanisms in vitro. We found that BAG3 was upregulated in physiological hypertrophy and in pathological remodeling both in vivo and in vitro. Heterozygous or homozygous knockout BAG3 in mouse hearts and knockdown of BAG3 in the NRVCs blunted physiological hypertrophy and aggravated pathological remodeling, while overexpression of BAG3 promoted physiological hypertrophy and inhibited pathological remodeling in NRVCs. Mechanistically, BAG3 overexpression in NRVCs promoted physiological hypertrophy by activating the protein kinase B (AKT)/mammalian (or mechanistic) target of rapamycin (mTOR) pathway. BAG3 knockdown in NRVCs aggravated pathological remodeling through activation of the calcineurin/nuclear factor of activated T cells 2 (NFATc2) pathway. Because BAG3 has a dual role in cardiac remodeling, heart-specific regulation of BAG3 may be an effective therapeutic strategy to protect against deterioration of heart function and heart failure caused by many heart diseases.

Kir6.1 improves cardiac dysfunction in diabetic cardiomyopathy via the AKT-FoxO1 signalling pathway.

Previous studies have shown that the expression of inwardly rectifying potassium channel 6.1 (Kir6.1) in heart mitochondria is significantly reduced in type 1 diabetes. However, whether its expression and function are changed and what role it plays in type 2 diabetic cardiomyopathy (DCM) have not been reported. This study investigated the role and mechanism of Kir6.1 in DCM. We found that the cardiac function and the Kir6.1 expression in DCM mice were decreased. We generated mice overexpressing or lacking Kir6.1 gene specifically in the heart. Kir6.1 overexpression improved cardiac dysfunction in DCM. Cardiac‐specific Kir6.1 knockout aggravated cardiac dysfunction. Kir6.1 regulated the phosphorylation of AKT and Foxo1 in DCM. We further found that Kir6.1 overexpression also improved cardiomyocyte dysfunction and up‐regulated the phosphorylation of AKT and FoxO1 in neonatal rat ventricular cardiomyocytes with insulin resistance. Furthermore, FoxO1 activation down‐regulated the expression of Kir6.1 and decreased the mitochondrial membrane potential (ΔΨm) in cardiomyocytes. FoxO1 inactivation up‐regulated the expression of Kir6.1 and increased the ΔΨm in cardiomyocytes. Chromatin immunoprecipitation assay demonstrated that the Kir6.1 promoter region contains a functional FoxO1‐binding site. In conclusion, Kir6.1 improves cardiac dysfunction in DCM, probably through the AKT‐FoxO1 signalling pathway.

Proarrhythmic Electrical Remodeling by Noncardiomyocytes at Interfaces With Cardiomyocytes Under Oxidative Stress.

Life-threatening ventricular arrhythmias, typically arising from interfaces between fibrosis and surviving cardiomyocytes, are feared sequelae of structurally remodeled hearts under oxidative stress. Incomplete understanding of the proarrhythmic electrical remodeling by fibrosis limits the development of novel antiarrhythmic strategies. To define the mechanistic determinants of the proarrhythmia in electrical crosstalk between cardiomyocytes and noncardiomyocytes, we developed a novel in vitro model of interface between neonatal rat ventricular cardiomyocytes (NRVMs) and controls [NRVMs or connexin43 (Cx43)-deficient HeLa cells] vs. Cx43+ noncardiomyocytes [aged rat ventricular myofibroblasts (ARVFs) or HeLaCx43 cells]. We performed high-speed voltage-sensitive optical imaging at baseline and following acute H2O2 exposure. In NRVM-NRVM and NRVM-HeLa controls, no arrhythmias occurred under either experimental condition. In the NRVM-ARVF and NRVM-HeLaCx43 groups, Cx43+ noncardiomyocytes enabled passive decremental propagation of electrical impulses and impaired NRVM activation and repolarization, thereby slowing conduction and prolonging action potential duration. Following H2O2 exposure, arrhythmia triggers, automaticity, and non-reentrant and reentrant arrhythmias emerged. This study reveals that myofibroblasts (which generate cardiac fibrosis) and other noncardiomyocytes can induce not only structural remodeling but also electrical remodeling and that electrical remodeling by noncardiomyocytes can be particularly arrhythmogenic in the presence of an oxidative burst. Synergistic electrical remodeling between H2O2 and noncardiomyocytes may account for the clinical arrhythmogenicity of myofibroblasts at fibrotic interfaces with cardiomyocytes in ischemic/non-ischemic cardiomyopathies. Understanding the enhanced arrhythmogenicity of synergistic electrical remodeling by H2O2 and noncardiomyocytes may guide novel safe-by-design antiarrhythmic strategies for next-generation iatrogenic interfaces between surviving native cardiomyocytes and exogenous stem cells or engineered tissues in cardiac regenerative therapies.

Klotho attenuated Doxorubicin-induced cardiomyopathy by alleviating Dynamin-related protein 1 - mediated mitochondrial dysfunction

Doxorubicin (Dox)-induced cardiotoxicity could lead to dilated cardiomyopathy and heart failure. Our previous study reported the protective effects of Klotho against hyperglycemia-induced cardiomyopathy. We investigated whether Klotho alleviated Dox-induced cardiotoxicity. Neonatal rat ventricular cardiomyocytes and H9c2 cells were incubated with 5 μM Dox for 24 h with or without Klotho (0.1 μg/mL). Dox-induced cardiotoxicity model was approached in C57BL/6 mice. Cardiac function and serum enzyme activity, apoptosis and mitochondrial dysfunction were measured. We found that pretreatment with Klotho significantly reduced Dox-induced apoptosis in cardiomyocytes. In Dox-treated mice, Klotho also suppressed cardiac cell death and improved cardiac function. Moreover, the expression of Dynamin-related protein 1 (Drp1) was increased after Dox-treatment both in vitro and in vivo, which was related to apoptosis in cardiomyocytes. In vitro experiments, Drp1 ser 616 phosphorylation post-Dox stimulation could be significantly attenuated by Klotho or Drp1 specific inhibitor Mdivi-1. Overexpression of Drp1 in cardiomyocytes increased Dox-induced heart injury which could also be attenuated by Klotho. This study demonstrated that Klotho alleviated Dox-induced cardiotoxicity by reducing apoptosis and mitochondrial fission through down-regulating Drp1 expression. Our findings highlighted new targets for the therapy of Dox-induced cardiomyopathy.

Mitochondrial Fission and Mitophagy Coordinately Restrict High Glucose Toxicity in Cardiomyocytes.

Hyperglycemia-induced mitochondrial dysfunction plays a key role in the pathogenesis of diabetic cardiomyopathy. Injured mitochondrial segments are separated by mitochondrial fission and eliminated by autophagic sequestration and subsequent degradation in the lysosome, a process termed mitophagy. However, it remains poorly understood how high glucose affects the activities of, and the relationship between, mitochondrial fission and mitophagy in cardiomyocytes. In this study, we determined the functional roles of mitochondrial fission and mitophagy in hyperglycemia-induced cardiomyocyte injury. High glucose (30 mM, HG) reduced mitochondrial connectivity and particle size and increased mitochondrial number in neonatal rat ventricular cardiomyocytes, suggesting an enhanced mitochondrial fragmentation. SiRNA knockdown of the pro-fission factor dynamin-related protein 1 (DRP1) restored mitochondrial size but did not affect HG toxicity, and Mdivi-1, a DRP1 inhibitor, even increased HG-induced cardiomyocyte injury, as shown by superoxide production, mitochondrial membrane potential and cell death. However, DRP1 overexpression triggered mitochondrial fragmentation and mitigated HG-induced cardiomyocyte injury, suggesting that the increased mitochondrial fission is beneficial, rather than detrimental, to cardiomyocytes cultured under HG conditions. This is in contrast to the prevailing hypothesis that mitochondrial fragmentation mediates or contributes to HG cardiotoxicity. Meanwhile, HG reduced mitophagy flux as determined by the difference in the levels of mitochondria-associated LC3-II or the numbers of mitophagy foci indicated by the novel dual fluorescent reporter mt-Rosella in the absence and presence of the lysosomal inhibitors. The ability of HG to induce mitochondrial fragmentation and inhibit mitophagy was reproduced in adult mouse cardiomyocytes. Overexpression of Parkin, a positive regulator of mitophagy, or treatment with CCCP, a mitochondrial uncoupler, induced mitophagy and attenuated HG-induced cardiomyocyte death, while Parkin knockdown had opposite effects, suggesting an essential role of mitophagy in cardiomyocyte survival under HG conditions. Strikingly, Parkin overexpression increased mitochondrial fragmentation, while DRP1 overexpression accelerated mitophagy flux, demonstrating a reciprocal activation loop that controls mitochondrial fission and mitophagy. Thus, strategies that promote the mutual positive interaction between mitochondrial fission and mitophagy while simultaneously maintain their levels within the physiological range would be expected to improve mitochondrial health, alleviating hyperglycemic cardiotoxicity.

Abstract 12947: Development of Molecular Targeted Therapy Against Right Ventricular Failure: Involvement in A Network of Immunocompetent Cells

Backgrounds: Right ventricular (RV) failure plays a critical role in right heart failure and left heart failure. However, there is no specific therapy developed for RV failure. To elucidate a novel therapeutic target against RV failure, we focus on differentially expressed genes in RV to develop novel therapeutics for RV failure. Methods: Microarray analysis using several parts of adult murine heart was conducted and differentially expressed genes (DEGs) were applied to pathway analysis by Ingenuity Pathway Analysis R . Molecular mechanism was examined by using neonatal rat ventricular cardiomyocyte (NRVM) in vitro . To understand the function of target molecule in vivo , we induced RV failure by pulmonary artery constriction (PAC) in mice and inhibition experiments were performed using these RV failure model mice. Results: In microarray analysis for RV, left ventricle and ventricular septum, 995 genes were extracted as DEGs in RV. Pathway analysis revealed that alternative complement pathway-related genes were significantly up-regulated in RV. Moreover, complement factor D (Cfd) and C3a was a potential upstream factor attributable to unique feature of RV. Administration of C3a recombinant protein to NRVM phosphorylated several MAP kinases. Moreover, in C3KO PAC mice, RV dysfunction was significantly suppressed, and histological study suggested that RV fibrosis was significantly suppressed by comparing to wild type PAC mice. Furthermore, administration of C3a receptor antagonist to wild type PAC mice dramatically improved RV dysfunction and reduced RV fibrosis. Additionally, in vivo electrophysiological study revealed that the inducibility of ventricular arrhythmia was increased in wild type PAC mice, but ventricular arrhythmia was significantly attenuated in C3KO PAC mice. Conclusion: We revealed that complement C3a was highly produced in RV, and genetic or chemical blockade of C3a ameliorates RV dysfunction and RV fibrosis in RV failure model mice. C3a was to be a potent bioactive protein for immunocompetent cells that played an important role in modulating RV function. Accordingly, we demonstrated that the blockade of C3a had a potential role for novel therapeutic target of RV failure.

Abstract 13914: Ischemic Preconditioning Induces MG53 Secretion From the Heart Through H 2 O 2 /PKC-delta Signaling

Introduction: Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion (I/R) injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (or TRIM72), not only plays an essential role in IPC-mediated cardioprotection, but also as a myokine/cardiokine, can be secreted from the heart and skeletal muscle in response to metabolic stress in addition to its intracellular actions. Hypothesis: We hypothesized that IPC-mediated cardioprotection is causally related to MG53 secretion and figured out the underlying mechanism. Methods and Results: Using proteomic analysis in conjunction with genetic and pharmacological approaches, we examined MG53 secretion in response to IPC and explored the underlying mechanism using rodents in in vivo , isolated perfused hearts, and cultured neonatal rat ventricular cardiomyocytes. IPC profoundly increased perfusate MG53 levels in mouse hearts by 5.50 ± 0.32 and 4.26 ± 0.40 folds from baseline over 0-60 and 60-120 min of reperfusion, respectively. Mechanistically, IPC-induced MG53 secretion is dependent on H 2 O 2 -evoked, Src-mediated phosphorylation of PKC-δ-Y311. Functionally, systemic delivery of recombinant human MG53 proteins (rhMG53) to mimic elevated circulating MG53 not only restored IPC function in MG53-deficient mice, but also protected rodent hearts from I/R injury even in the absence of IPC. Treatment of rhMG53 overtly decreased the infarct size (IF/AAR) induced by I/R compared to the BSA-treated control group (11.9 ± 1.8% vs 27.3 ± 2.0%, P <0.01), and reduced the mortality from 44.7% to 5.3% in rats. Moreover, H 2 O 2 augmented MG53 secretion, and MG53 knockdown exacerbated H 2 O 2 -induced cell injury in human embryonic stem cell-derived cardiomyocytes. Conclusions: In conclusion, IPC and oxidative stress can trigger MG53 secretion from the heart via an H 2 O 2 -PKC-δ-dependent mechanism, and secreted MG53 acts as an essential factor conveying IPC-induced cardioprotection against ischemia/reperfusion injury. Recombinant MG53 proteins can be developed into a novel treatment for various diseases of human heart in which the endogenous MG53 is low.

The study on optogenetic tachyarrhythmia termination under pathological conditions from the single cell to the whole heart

Abstract Background Ventricular tachyarrhytmias (VTs) are common among patients suffering from cardiac remodeling and cause significant morbidity and mortality. Current research and treatment options for such VTs are suboptimal, hence new strategies are urgently needed. Optogenetics offers efficacious means to control cardiac rhythm, including shock-free VT termination. However, this has not been demonstrated in diseased hearts in vivo, while clinical translation would not only require such demonstration, but also an in-depth understanding of cellular responses. Purpose To assess the optogenetic response at the cardiac cell, tissue, and whole heart level in terms of rhtyhm control under pathological conditions by an integrative experimental platform including in vitro and in vivo models of cardiac disease. Methods Remodeling was induced in neonatal rat ventricular cardiomyocytes (NRVMs) by phenylephrine (PE) exposure. Pathological conditions leading to ventricular remodeling were mimicked by transverse aortic constriction (TAC) surgery in adult rats. The light-activated ion channel ReaChR was ectopically expressed in NRVMs and in hearts of TAC and sham animals by viral vector-based gene delivery. Results Electrical and structural remodeling was evidenced by elongated action potential durations (p<0.05) and increased cell capacitance (p<0.05) in PE-treated, but not in control cells (CTL). Light-induced ionic currents in ReaChR-expressing PE-treated and CTL NRVMs displayed comparable kinetic properties and current densities (p>0.05). Illumination (1 s) caused a sudden shift in membrane potential leading to a plateau at −7.3 mV for PE-treated and −18.9 mV for CTL cells (p>0.05). Hearts explanted from TAC animals showed increased average heart weight to body weight ratio, ventricular fibrosis and expression of hypertrophy markers (ANP, aSkMA, p<0.05), while tissue preparations showed significant APD increase compared to sham. In vivo gene delivery resulted in expression of the ReaChR-citrine transgene in ∼80% of isolated ventricular myocytes (VMs). Photocurrent densities were not different (p>0.05) in VMs from TAC and sham animals, which currents led to comparable shifts in membrane potential (65.3 mV for TAC and 63.9 mV for CTL). In line with this, illumination caused marked depolarization in tissue preparations (from −77.6 to −16.4 mV) in TAC animals as assessed by conventional sharp electrode measurements. Importantly, as anticipated, electrically-induced VT episodes could be terminated in open chest experiments in TAC animals (n=6; 76.3% of cases) by epicardial illumination in vivo. Conclusions Key operational parameters of the optogenetic response remained unaffected in models of cardiac disease, which allowed efficacious optogenetic VT termination in the diseased rat heart exhibiting structural and electrical remodeling. These findings corroborate the translational potential of shock-free therapy of cardiac arrhythmia by optogenetics. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This work was supported by personal funding from the Netherlands Organization for Scientific Research (NWO, Vidi grant 1714336 to D.A.P.). D.A.P. is also a recipient of the European Research Council (ERC), Starting grant (716509). Additional support was provided by the Netherlands Heart Institute (ICIN grant 230.148-04 to A.A.F.d.V.).

Citri Reticulatae Pericarpium protects against isoproterenol-induced chronic heart failure via activation of PPARγ.

BackgroundAccumulated clinical trials and animal studies showed that Qiliqiangxin (QLQX), a traditional Chinese medicine formula containing extracts of 11 herbs, exerts beneficial effects on chronic heart failure (HF). Citri Reticulatae Pericarpium (CRP), one herbal medicine in QLQX, has been widely used in treatment against digestive, respiratory and cardiovascular diseases (CVDs) in China. However, the cardiac protective effects and mechanisms of CRP are still unclear.MethodsThe effects of CRP were investigated in isoproterenol (ISO)-induced chronic HF mice model and neonatal rat ventricular cardiomyocytes (NRVMs) treated with ISO. Echocardiography was used to determine cardiac function. Hematoxylin-eosin (HE) staining and α-actinin immunofluorescent staining were used to measure cardiomyocyte size. Cardiac fibrosis was evaluated by Masson’s trichrome staining. The expression of atrial natriuretic polypeptide (ANP) and brain natriuretic polypeptide (BNP) were determined by quantitative real time PCR (qRT-PCR). Western blot was applied to examine the expression of peroxisome proliferator-activated receptor gamma (PPARγ), PPARγ coactivator-1α (PGC-1α), fibrosis-related and apoptosis-related proteins.ResultsWe found that CRP could significantly attenuate ISO-induced cardiac dysfunction, inhibit cardiac pathological hypertrophy and alleviate myocardial fibrosis and apoptosis. Mechanistically, the downregulation of PPARγ and PGC-1α in ISO-injected mice hearts and ISO-treated NRVMs could be reversed by CRP treatment. The beneficial effects of CRP against ISO-induced HF were abolished by PPARγ inhibitor (T0070907), suggesting that CRP-mediated PPARγ upregulation was essential for the preventive effect of CRP on ISO-induced cardiac dysfunction.ConclusionsIn conclusion, our study demonstrated that CRP attenuates ISO-induced cardiac remodeling via PPARγ activation, which represents a new application for CRP in the prevention of chronic HF.

Boron improves cardiac contractility and fibrotic remodeling following myocardial infarction injury

Myocardial fibrosis is a major determinant of clinical outcomes in heart failure (HF) patients. It is characterized by the emergence of myofibroblasts and early activation of pro-fibrotic signaling pathways before adverse ventricular remodeling and progression of HF. Boron has been reported in recent years to augment the innate immune system and cell proliferation, which play an important role in the repair and regeneration of the injured tissue. Currently, the effect of boron on cardiac contractility and remodeling is unknown. In this study, we investigated, for the first time, the effect of boron supplementation on cardiac function, myocardial fibrosis, apoptosis and regeneration in a rat model myocardial infarction (MI)-induced HF. MI was induced in animals and borax, a sodium salt of boron, was administered for 7 days, p.o., 21 days post-injury at a dose level of 4 mg/kg body weight. Transthoracic echocardiographic analysis showed a significant improvement in systolic and diastolic functions with boron treatment compared to saline control. In addition, boron administration showed a marked reduction in myocardial fibrosis and apoptosis in the injured hearts, highlighting a protective effect of boron in the ischemic heart. Interestingly, we observed a tenfold increase of nuclei in thin myocardial sections stained positive for the cell cycle marker Ki67 in the MI boron-treated rats compared to saline, indicative of increased cardiomyocyte cell cycle activity in MI hearts, highlighting its potential role in regeneration post-injury. We similarly observed increased Ki67 and BrdU staining in cultured fresh neonatal rat ventricular cardiomyocytes. Collectively, the results show that boron positively impacted MI-induced HF and attenuated cardiac fibrosis and apoptosis, two prominent features of HF. Importantly, boron has the potential to induce cardiomyocyte cell cycle entry and potentially cardiac tissue regeneration after injury. Boron might be beneficial as a supplement in MI and may be a good candidate substance for anti-fibrosis approach.

Optimization of heart-targeting agents for therapeutic delivery in cardiomyocytes

Cardiovascular diseases represent the major cause of death in western industrialized countries. In particular, 50% of heart failure patients die within 5-years. Although usual treatments exist like β-blockers, they impact the quality of life due to side effects and off-targets. Thus, targeting specifically cardiomyocytes for drug delivery would certainly represent an improvement over classical ways of administration. Interestingly, two peptides were previously described to exhibit a preferential cardiac tropism (i.e. CTP: APWHLSSQYSRT and PCM: WLSEAGPVVTVRALRGTGSW). However, CTP and PCM peptides efficacy in terms of cell penetration remains low and to our knowledge no optimization has been reported. The aim of this work was to optimize original CTP and PCM peptides and to characterize their respective uptake efficacy and cytotoxicity in cardiomyocytes. Over 80 different CTP- and PCM-derived peptides coupled with 6-carboxyfluoresceine (6-CF) were developed. All constructs were assessed for cellular uptake in neonatal rat ventricular cardiomyocytes (NRVC). Cellular uptake efficacy and corresponding viability were quantified by flow cytometry as percentage of 6-CF-positive cells. High content imaging was performed to localize 6-CF-peptides in NRVC. Two of the derived peptides, 6-CF-CTP1 and 6-CF-PCM2, exhibited, respectively, a 6.5- and 5.5-fold increase over original 6-CF-CTP and 6-CF-PCM sequences in live cellular uptake. None of the original or derived 6-CF-CTP and 6-CF-PCM-peptides exhibited cytotoxicity from 1 to 50 μM. 6-CF peptides are distributed in NRVC cytoplasm. We also performed 4 °C incubations which did not prevent the NRVC uptake, excluding an energy-driven process. This study displays potential for optimization of the CTP or PCM peptide sequences to increase their cellular uptake efficacy without cytotoxicity. It raises the potential use of heart targeting peptides for cardiac drug delivery.

A PKA/AKAP signaling complex is essential for regulation of ventricular cardiomyocytes Cx43 gap junction communication

In cardiomyocytes, β-adrenergic receptor (β-AR) stimulation activates protein kinase A (PKA), which increases electrical conduction velocity. Connexin-43 (Cx43) form gap junction (GJ) channels at the intercalated discs of cardiomyocytes and ensure electrical impulse propagation. This is essential for coordinated cardiac contractions. In myocardial infarction (MI), Cx43 is altered that affects GJ communication, electrical conduction leading to fatal arrhythmias. Regardless the cause, the β-AR system is activated after a MI. Considering the key role of Cx43, we investigated the functional regulation of GJ by PKA-anchored through A-Kinase Anchoring Protein (AKAP) in cardiac physiology and pathophysiology (MI). Neonatal rat ventricular cardiomyocytes were cultured with peptides that inhibit PKA or displace PKA anchoring from AKAP in basal condition or after β-AR stimulation. Cx43 phosphorylation level and GJ communication were assessed by immunoblot and GAP-Fluorescence Loss In Photobleaching experiments. A Cx43-PKA-AKAP signaling complex in cardiomyocytes and heart was investigated by immunofluorescence, immunoblot or immunoprecipitation. Proximity ligation assays were performed in cells and tissue in physiological conditions and after MI. Our experiments point to a PKA-scaffold protein required for PKA-mediated regulation of Cx43 phosphorylation and GJ communication. We show that the AKAP named ezrin forms a molecular complex with PKA and Cx43 in rat heart at the cellular and tissue levels. While PKA and ezrin expression levels remain constant, Cx43 showed a significant reduction after a MI. Interestingly, we noticed a decreased in Cx43 vicinity with ezrin at the ID of ventricular cardiomyocytes. These data suggest for a role of PKA-ezrin-Cx43 complex in the regulation of GJ communication in cardiac physiology. A deregulation of this complex could explain partly the altered electrical propagation observed after a MI.

Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment.

BackgroundCardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling.Methods and ResultsMyofibroblast activation was modulated in vitro by combining stiffness cues with TGFβ1 (transforming growth factor β 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFβ1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGFβ1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy.ConclusionsCulturing CFs on stiff substrates, treating with TGFβ1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGFβ1 caused myocyte hypertrophy, most likely through insulin growth factor 1.

MicroRNA-183 as a Novel Regulator Protects Against Cardiomyocytes Hypertrophy via Targeting TIAM1.

MicroRNAs serve as important regulators of the pathogenesis of cardiac hypertrophy. Among them, miR-183 is well documented as a novel tumor suppressor in previous studies, whereas it exhibits a downregulated expression in cardiac hypertrophy recently. The present study was aimed to examine the effect of miR-183 on cardiomyocytes hypertrophy. Angiotensin II (Ang II) was used for establishment of cardiac hypertrophy model in vitro. Neonatal rat ventricular cardiomyocytes transfected with miR-183 mimic or negative control were further utilized for the phenotype analysis. Moreover, the bioinformatics analysis and luciferase reporter assays were used for exploring the potential target of miR-183 in cardiomyocytes. We observed a significant decreased expression of miR-183 in hypertrophic cardiomyocytes. Overexpression of miR-183 significantly attenuated the cardiomyocytes size morphologically and prohypertrophic genes expression. Moreover, we demonstrated that TIAM1 was a direct target gene of miR-183 verified by bioinformatics analysis and luciferase reporter assays, which showed a decreased mRNA and protein expression in the cardiomyocytes transfected with miR-183 upon Ang II stimulation. Additionally, the downregulated TIAM1 expression was required for the attenuated effect of miR-183 on cardiomyocytes hypertrophy. Taken together, these evidences indicated that miR-183 acted as a cardioprotective regulator for the development of cardiomyocytes hypertrophy via directly regulation of TIAM1.