ISO-induced Cardiac Hypertrophy Research Articles

Mechanistic insights to the cardioprotective effect of blueberry nutraceutical extract in isoprenaline-induced cardiac hypertrophy

BackgroundLowbush blueberry extract (Vaccinium angustifolium) is abundant with polyphenols (such as chlorogenic acid) with high antioxidant profile. It has received great interest due to its protective role in many disorders such as heart diseases and neurological disorders. HypothesisWe hypothesized that blueberry leaf extract might have a protective effect against cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis. MethodBlueberry leaf nutraceutical extract was administered orally to male albino rats at three different doses (25, 50 and 100 mg/kg/day of the extract, equivalent to 3.4, 6.8 and 13.6 mg of chlorogenic acid, respectively) once daily for 28 consecutive days against a dose of isoprenaline (ISO) (5 mg/kg) for 14 days. ResultsThe results indicated that isoprenaline induced significant myocardial damage, characterized by conduction abnormalities, increased heart-to-body weight ratio, increased serum CKMB, AST, c-TnI and LDH. Pretreatment with blueberry extract at a dose of 50 mg/kg/day (equivalent to 6.8 mg chlorogenic acid) protected against ISO-induced ECG changes, leakage of cardiac enzymes and histopathological changes. Also, ISO caused significant glutathione depletion, lipid peroxidation and reduction in activities of antioxidant catalase enzyme. These effects were prevented by pretreatment with blueberry extract. Additionally, ISO elicited inflammatory effects by increasing the expression of NF-κB, COX-2, TNF-α and IL-6 while pretreatment with blueberry extract significantly inhibited these inflammatory responses. Furthermore, ISO induced fibrosis by increasing the level of TGF-β while pretreatment with blueberry extract significantly reduced it. ConclusionThese findings indicate that blueberry leaf extract possessed a potent protective effect against ISO-induced cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis.

1H NMR-based dynamic metabolomics delineates the therapeutic effects of Baoyuan decoction on isoproterenol-induced cardiac hypertrophy

Cardiac hypertrophy (CH) is a major risk factor for many serious heart diseases. Sustained CH is catastrophic, resulting in cardiac dysfunction that eventually leads to heart failure (HF). Baoyuan decoction (BYD) is a famous traditional Chinese medicine (TCM) formula for supplementing and reinforcing Qi, clinically used for the treatment of cardiovascular diseases (CVDs). However, the therapeutic effects of BYD on CH remain unidentified. We herein investigated the effect of BYD on isoproterenol (ISO)-induced CH in rats and the underlying mechanisms by comprehensive pharmacodynamics and 1H NMR-based dynamic metabolomics analysis of the plasma and urine samples. Results showed that BYD treatment markedly attenuated ISO-induced CH as evidenced by decreasing the left ventricular wall thickness, pathological cardiomyocyte hypertrophy, myocardial collagen fiber deposition and apoptosis, and plasma natriuretic peptide levels. Multivariate trajectory analysis revealed that the BYD treatment could restore the CH-disturbed plasma and urinary metabolite profiles towards the normal metabolic status featuring with a time-dependent tendency. Moreover, the key metabolic alterations in CH rats at different BYD-treated time stages involved energy metabolism, oxidative stress responses, amino acid metabolism, and gut microbiota metabolism. Of particularly, the significant roles of BYD for treating CH lie in the improvement of cardiac energy generation and antioxidant capacity. Our investigation provides a holistic view of BYD for therapeutic intervention of CH through monitoring of the dynamic metabolic changes and the results indicate that BYD may be applied as a potential agent for treating CH.

Abstract 010: Dual Loss of RGS2 and 5 Exacerbates the Progression of Cardiac Hypertrophy in Mice Chronically Treated With Isoproterenol

This study determined the effect of chronic isoproterenol (ISO) treatment on cardiac structure and function in male mice without or with dual loss of regulator of G protein signaling (RGS) 2 and 5. RGS2 and 5 act as GTPase-activating proteins (GAPs) that preferentially terminate signaling via G q/11 - and G i/o class G proteins, by accelerating GTP hydrolysis. Deletion of either RGS2 or 5 increases susceptibility to cardiovascular disease. However, the effects of dual absence of the two RGS proteins on normal physiology and disease are unknown. Using mice concurrently lacking RGS2 and 5 ( Rgs2/5 dbKO) and wild type (WT) cohort, we determined how the dual absence of both RGS proteins affects cardiac response to chronic β-adrenergic receptor stimulation. WT and Rgs2/5 dbKO mice were infused with saline or 30 μg/g/day of ISO for 3 or 14 days. At baseline, Rgs2/5 dbKO mice showed cardiac hypertrophy (WT: 9.06 ± 0.34 vs. dbKO: 10.16 ± 0.32 mg/mm tibia length; p <0.05) and left ventricular chamber dilation by echocardiography, without tissue fibrosis. ISO infusion for 3 days caused and augmented cardiac hypertrophy in WT (SAL: 9.65 ± 0.38 vs. ISO: 11.68 ± 0.45 mg/mm tibia length; p <0.05) and Rgs2/5 dbKO (SAL: 9.97 ± 0.43 vs. ISO: 12.57 ± 0.69 mg/mm tibia length; p <0.01) mice, respectively, as well as interstitial fibrosis and increased expression of hypertrophic and heart failure gene markers, including Nppa , Serca , Mybpc3 and Tnni3 . Sub-chronic ISO infusion also caused a greater decrease in percent fractional shortening by day 3, in Rgs2/5 dbKO relative to WT mice (WT: -4.22 ± 4.15 vs. dbKO: -7.69 ± 3.86 %; p <0.05). In WT mice, cardiac hypertrophy and left ventricular dilation were lower after 14-day compared to 3-day ISO infusion, but similar to saline-treated control levels (ΔSAL to 3d-ISO: 21.04 ± 0.82 vs. ΔSAL to 14d-ISO: 15.05 ± 0.66 %; p <0.05). This was accompanied by a robust increase in Rgs5 but not Rgs2 mRNA expression in WT mice. In contrast, Rgs2/5 dbKO mice continued to show abnormal cardiac structure and function after 14-day ISO infusion. Together, these data suggest that increased expression of RGS5 compensates for the lack of change in RGS2 expression and/or function and protects against transition from ISO-induced cardiac hypertrophy to heart failure.

Integrated Dissection of Cysteine Oxidative Post-translational Modification Proteome During Cardiac Hypertrophy.

Cysteine oxidative modification of cellular proteins is crucial for many aspects of cardiac hypertrophy development. However, integrated dissection of multiple types of cysteine oxidative post-translational modifications (O-PTM) of proteomes in cardiac hypertrophy is currently missing. Here we developed a novel discovery platform that encompasses a customized biotin switch-based quantitative proteomics pipeline and an advanced analytic workflow to comprehensively profile the landscape of cysteine O-PTM in an ISO-induced cardiac hypertrophy mouse model. Specifically, we identified a total of 1655 proteins containing 3324 oxidized cysteine sites by at least one of the following three modifications: reversible cysteine O-PTM, cysteine sulfinylation (CysSO2H), and cysteine sulfonylation (CysSO3H). Analyzing the hypertrophy signatures that are reproducibly discovered from this computational workflow unveiled four biological processes with increased cysteine O-PTM. Among them, protein phosphorylation, creatine metabolism, and response to elevated Ca2+ pathways exhibited an elevation of cysteine O-PTM in early stages, whereas glucose metabolism enzymes were increasingly modified in later stages, illustrating a temporal regulatory map in cardiac hypertrophy. Our cysteine O-PTM platform depicts a dynamic and integrated landscape of the cysteine oxidative proteome, through the extracted molecular signatures, and provides critical mechanistic insights in cardiac hypertrophy. Data are available via ProteomeXchange with identifier PXD010336.

JMJD3 inhibition protects against isoproterenol-induced cardiac hypertrophy by suppressing β-MHC expression.

Jumonji domain-containing protein D3 (JMJD3), a histone 3 lysine 27 (H3K27) demethylase, has been extensively studied for their participation in development, cellular physiology and a variety of diseases. However, its potential roles in cardiovascular system remain unknown. In this study, we found that JMJD3 played a pivotal role in the process of cardiac hypertrophy. JMJD3 expression was elevated by isoproterenol (ISO) stimuli both in vitro and in vivo. Overexpression of wild-type JMJD3, but not the demethylase-defective mutant, promoted cardiomyocyte hypertrophy, as implied by increased cardiomyocyte surface area and the expression of hypertrophy marker genes. In contrary, JMJD3 silencing or its inhibitor GSK-J4 suppressed ISO-induced cardiac hypertrophy. Mechanistically, JMJD3 was recruited to demethylate H3K27me3 at the promoter of β-MHC to promote its expression and cardiac hypertrophy. Thus, our results reveal that JMJD3 may be a key epigenetic regulator of β-MHC expression in cardiomyocytes and a potential therapeutic target for cardiac hypertrophy.

Exogenous Hydrogen Sulfide Supplement Attenuates Isoproterenol-Induced Myocardial Hypertrophy in a Sirtuin 3-Dependent Manner.

Hydrogen sulfide (H2S) is a gasotransmitter with a variety of cardiovascular protective effects. Sirtuin 3 (SIRT3) is closely related to mitochondrial function and oxidative stress. We found that NaHS increased SIRT3 expression in the preventive effect on isoproterenol- (ISO-) induced myocardial hypertrophy. We further investigated whether exogenous H2S supplement improved ISO-induced myocardial hypertrophy in a SIRT3-dependent manner. 10-week-old male 129S1/SvImJ (WT) mice and SIRT3 knockout (KO) mice were intraperitoneally injected with NaHS (50 μmol/kg/d) for two weeks and then intraperitoneally injected with ISO (60 mg/kg/d) for another two weeks. In WT mice, NaHS significantly reduced the cardiac index of ISO-induced mice, decreased the cross-sectional area of cardiomyocytes, and inhibited the expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA. The activity of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) in the myocardium was increased, but the level of malondialdehyde (MDA) was decreased. The fluorescence intensity of dihydroethidium staining for superoxide anion was attenuated. Optic atrophy 1 (OPA1) expression was upregulated, while dynamin-related protein 1 (DRP1) expression was downregulated. ERK, but not P38 and JNK, phosphorylation was downregulated. However, all above protective effects were unavailable in ISO-induced SIRT3 KO mice. Our present study suggested that exogenous H2S supplement inhibited ISO-induced cardiac hypertrophy depending on SIRT3, which might be associated with antioxidant stress.

Specific α7 nicotinic acetylcholine receptor agonist ameliorates isoproterenol-induced cardiac remodelling in mice through TGF-β1/Smad3 pathway.

It is well-accepted that inflammation plays an important role in the development of cardiac remodelling and that therapeutic approaches targeting inflammation can inhibit cardiac remodelling. Although a large amount of evidence indicates that activation of α7 nicotinic acetylcholine receptor (α7nAChR) causes an anti-inflammatory effect, the role of α7nAChR in cardiac remodelling and the underlying mechanism have not been established. To investigate the effect of the specific α7nAChR agonist, PNU282987, on cardiac remodelling induced by isoproterenol (ISO 60mg/kg per day) in mice, the cardiomyocyte cross-sectional area (CSA) and collagen volume fraction were evaluated by hematoxylin and eosin (HE) and Masson staining, respectively. Cardiac function and ventricular wall thickness were measured by echocardiography. The protein expressions of collagen I, matrix metalloproteinase 9 (MMP-9), transforming growth factor β1 (TGF-β1), and Smad3 were analyzed by Western blot. ISO-induced cardiac hypertrophy, characterized by an increase in the heart weight/body weight ratio, CSA and ventricular wall thickness. Moreover, cardiac fibrosis indices, such as collagen volume fraction, MMP-9 and collagen I protein expression, were also increased by ISO. PNU282987 not only attenuated cardiac hypertrophy but also decreased the cardiac fibrosis induced by ISO. Furthermore, PNU282987 suppressed TGF-β1 protein expression and the phosphorylation of Smad3 induced by ISO. In conclusion, PNU282987 ameliorated the cardiac remodelling induced by ISO, which may be related to the TGF-β1/Smad3 pathway. These data imply that the α7nAChR may represent a novel therapeutic target for cardiac remodelling in many cardiovascular diseases.

E2/ER β inhibit ISO-induced cardiac cellular hypertrophy by suppressing Ca2+-calcineurin signaling.

Cardiovascular incidences are markedly higher in men than in pre-menstrual women. However, this advantage in women declines with aging and therefore can be correlated with the sex hormone 17β-Estradiol (E2) which is reported to protect heart cells by acting though estrogen receptors (ERs). In this study we have determined the effect of E2/ERβ against ISO induced cellular hypertrophy in H9c2 cardiomyoblast cells. The results confirm that ISO induced cardiac-hypertrophy by elevating the levels of hypertrophy associated proteins, ANP and BNP and further by upregulating p-CaMKII, calcineurin, p-GATA4 and NFATc3 which was correlated with a significant enlargement of the H9c2 cardiomyoblast. However, overexpression of ERβ and/or administration of E2 inhibited ISO-induced hypertrophy in H9c2 cells. In addition, E2/ERβ also inhibited ISO-induced NFATc3 translocation, and reduced the protein level of downstream marker, BNP. Furthermore, by testing with the calcineurin inhibitor (CsA), it was confirmed that calcineurin acted as a key mediator for the anti-hypertrophic effect of E2/ERβ. In cells treated with calcium blocker (BATPA), the inhibitory effect of E2/ERβ on ISO-induced Ca2+ influx and hypertrophic effects were totally blocked suggesting that E2/ERβ inhibited calcineurin activity to activate I-1 protein and suppress PP1, then induce PLB protein phosphorylation and activation, resulting in Ca2+ reuptake into sarcoplasmic reticulum through SR Ca2+ cycling modification. In conclusion, E2/ERβ suppresses the Ca2+ influx and calcineurin activity induced by ISO to enhance the PLB protein activity and SR Ca2+ cycling.

Abstract 157: Alterations in Signaling Pathways During Regression of Pathological Cardiac Hypertrophy

Introduction: Cardiac hypertrophy is initially, a compensatory mechanism to maintain cardiac output when there is an increased load on the heart. However, if cardiac hypertrophy persists for an extended time, there can be maladaptive changes to the myocardium. Even when the underlying cause of hypertrophy is treated, regression is often minimal or absent. Clinical cases of cardiac regression do exist, including patients receiving bariatric surgery or a left ventricular assist device. While many of the mechanisms leading to cardiac hypertrophy are well understood, little is known about the mechanisms of reversal of hypertrophy and why it is sometimes irreversible. We hypothesized that a reversal of isoproterenol (Iso) induced cardiac hypertrophy in the mouse will be observed within 7 days following the removal of the stimulus and we will be able to identify alterations in signaling pathways. Methods: We induced pathological cardiac hypertrophy with Iso for 7 days, at which peak hypertrophy is achieved. To identify if/when regression occurs, the Iso treatment was stopped and the mice were monitored for 7 days. Heart weights were measured at peak hypertrophy, post-drug days 1, 2, 3 & 7, along with vehicle treated mice (8/group). We used left ventricle tissue for protein analysis and protein degradation activity assays. Results: Regression from cardiac hypertrophy occurs by post-drug day 7 (p=0.016) in the Iso mouse model. p-Akt is increased with Iso treatment and returns to vehicle control levels by post-drug day 7. There is a decrease in p-mTOR and an increase in LC3-II levels at post-drug day 7, indicating a possible role of autophagy in cardiac regression. In addition, there was a decrease in cell size when neonatal rat ventricular myocytes were treated with the Akt inhibitor, Wortmannin, following phenylephrine induced hypertrophy. Conclusion: Regression of Iso-induced cardiac hypertrophy occurs in the mouse after 7 days following the removal of the stimulus. The Akt pathway is activated with Iso treatment and when this pathway is inactivated during regression, autophagy is activated, which may be an important mechanism to degrade proteins and lead to a decrease in cardiac hypertrophy. Finally, when the Akt pathway is inhibited in vitro , hypertrophic cells regress.

P 201 - Role of redox-potential decreasing in the chronic isoproterenol-induced cardiac hypertrophy and its pharmacological correction

This study investigated the efficacy of preventive role of NAD-containing cardiotrophic drug Adenocine® in chronic isopreterenol (ISO)-induced cardiac hypertrophy (CH) on tissue antioxidant status. On the 14 days of ISO-induced CH (10 mg/kg daily ) all male chinchilla rabbits were randomly assigned into 3 groups: control - infusion of 0,9% NaCl; main I - receive 10 mg/kg nebivolol per os in combination with lisinopril (10 mg/kg) infusion (traditional treatment (TT)); and main II - receive intravenously 15 mg/kg of Adenocin dissolved in water for injection during 7 days. The increases of heart weight by 18,6% and heart-to-body weight ratio by 35,5% observed in CH were significantly suppressed in Adenocin-treated group by 15,8 and 31,5% and only by 6,4 and 9,7% respectively after TT. ISO-induced CH is associated with loss of NAD from 6,4±0,3 to 5,1±0,4mMol/mg. TT didn’t cause the restoration NAD/NADH and NAD content in LV myocardium and decrease level of BNP. Adenocin treatment restored the normal level of NAD+/NADH, increased the activity of CuZn–SOD, Mn–SOD,superoxide anion production, catalase activities and decreased the content of MDA while the TT did not. Conclusion The results described the benficial cardioprotective effect of Adenocin on the the activities of both enzymatic and non-enzymatic antioxidants LV myocardium of ISO-induced CHF.

KLF15 protects against isoproterenol-induced cardiac hypertrophy via regulation of cell death and inhibition of Akt/mTOR signaling

Increasing evidence indicate that the Krüppel-like factor KLF15, a member of Cys2/His2 zinc-finger DNA-binding proteins, attenuates cardiac hypertrophy. However, the role of KLF15 in cardiovascular system is largely unknown and the exact molecular mechanism of its protective function is not fully elucidated. In the present study, we established a mouse model of cardiac hypertrophy and found that KLF15 expression was down-regulated in hypertrophic hearts. To evaluate the roles of KLF15 in cardiac hypertrophy, we generated transgenic mice overexpressing KLF15 of KLF15 knockdown mice and subsequently induced cardiac hypertrophy. The results indicated that KLF15 overexpression protects mice from ISO-induced cardiac hypertrophy, with reduced ratios of heart weight (HW)/body weight (BW) and cross-sectional area. We also observed that KLF15 overexpression attenuated cardiac fibrosis, inhibited apoptosis and induced autophagy in cardiomyocytes compared with KLF15 knockdown mice. More importantly, we found that the KLF15 overexpression inhibited the Akt/mTOR signaling pathway. Taken together, our findings imply that KLF15 possesses potential anti-hypertrophic and anti-fibrotic functions, possibly via regulation of cell death pathways and the inhibition of Akt/mTOR axis. KLF15 may constitute an efficient candidate drug for the treatment of heart failure and other cardiovascular diseases.

The role of cytochrome P450 1B1 and its associated mid-chain hydroxyeicosatetraenoic acid metabolites in the development of cardiac hypertrophy induced by isoproterenol.

Numerous experimental studies have demonstrated the role of cytochrome P450 1B1 (CYP1B1) and its associated mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) metabolite in the pathogenesis of cardiac hypertrophy. However, the ability of isoproterenol (ISO) to induce cardiac hypertrophy through mid-chain HETEs has not been investigated yet. Therefore, we hypothesized that ISO induces cardiac hypertrophy through the induction of CYP1B1 and its associated mid-chain HETE metabolites. To test our hypothesis, Sprague-Dawley rats were treated with ISO (5mg/kg i.p.) for 12 and 72h whereas, human ventricular cardiomyocytes RL-14 cells were exposed to 100μM ISO in the presence and absence of 0.5μM tetramethoxystilbene (TMS) a selective CYP1B1 inhibitor, or 25 nM CYP1B1-siRNA. Moreover, RL-14 cells were transiently transfected with the CRISPR-CYP1B1 plasmid. Thereafter, real-time PCR, western blot analysis, and liquid chromatography-electrospray ionization mass spectroscopy were used to determine the level of gene expression, protein expression, and mid-chain HETEs, respectively. Our results showed that ISO induced CYP1B1 protein expression and the level of cardiac mid-chain HETEs in vivo at pre-hypertrophic and hypertrophic stage. In vitro, inhibition of CYP1B1 using TMS or CYP1B1-siRNA significantly attenuates ISO-induced hypertrophy. Furthermore, overexpression of CYP1B1 significantly induced cellular hypertrophy and mid-chain HETEs metabolite. Mechanistically, the protective effect of TMS against cardiac hypertrophy was mediated through the modulation of superoxide anion, mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that CYP1B1 and its associated mid-chain HETE metabolites are directly involved in the ISO-induced cardiac hypertrophy.

Proteomic analysis of the protective effects of aqueous bark extract of Terminalia arjuna (Roxb.) on isoproterenol-induced cardiac hypertrophy in rats

Ethnopharmacological relevanceAqueous bark extract of Terminalia arjuna (TA) has been in use as an ethnomedicine for cardiovascular ailments in the Indian subcontinent for centuries. Studies using hemodynamic, ROS scavenging and anti-inflammatory parameters in animal models have shown its anti-atherogenic, hypotensive, inotropic, anti-inflammatory effects. However, details analysis on its effects on established molecular and cell biological markers are a prerequisite for its wider acceptance to the medical community. Aims of the studyTo test the efficacy of TA extract in ameliorating cardiac hypertrophy induced by ISO in rats. MethodsCardiac hypertrophy was induced by ISO (5mg/kg/day s.c. for 14 days) in rats and a standardized aqueous extract of TA stem bark was orally administered by gavage. Total RNA and protein were isolated from control, ISO, ISO plus TA and TA treated rat hearts and analyzed for the transcripts for the markers of hypertrophy, signaling kinases, transcription factors and total protein profile. ResultsTA extract reversed the induction of fetal genes like β-myosin heavy chain, skeletal α-actin and brain natriuretic peptide in hypertrophic rat hearts. While ISO slightly increased the level of phospho-ERK, TA repressed it to about one third of the base line level. Survival kinase Akt, ER stress marker Grp78 and epigenetic regulator HDAC5 were augmented by ISO and TA restored them by various extents. ISO administration moderately increased the transcription factor NFκB binding activity, while coadministration of TA further increased it. AP-1 binding activity was largely unchanged by ISO treatment but it was upregulated when administered along with TA. MEF2D binding activity was increased by ISO and TA restored it to the baseline level. Global proteomic analysis revealed that TA treatment restored a subset of proteins up- and down-regulated in the hypertrophied hearts. Amongst those restored by TA were purinergic receptor X, myosin light chain 3, tropomyosin, and kininogen; suggesting a nodal role of TA in modulating cardiac function. ConclusionsThis study for the first time reveals that TA partially or completely restores the marker mRNAs, signaling kinases, transcription factors and total protein profile in rat heart, thereby demonstrating its efficacy in preventing ISO-induced cardiac hypertrophy.

Hypertrophic and Inflammatory Markers in Isoproterenol-Induced CardiacHypertrophy and its Pharmacological Correction

Backgrounds: Short-term sustained beta-adrenergic stimulation is a hallmark of sympathetic hyperactivity and a common future in cardiovascular disease. Activation of endogenous cell signaling pathways that negatively regulate cardiac hypertrophy including the decline of NAD and the reduction of the intracellular NAD+/NADH ratio. The hypothesis that prevention of NAD depletion in myocardium may be critical in the treatment of cardiac hypertrophy and inflammatory responses was tested. Methods: On the 7 days of isoproterenol (ISO)-induced cardiac hypertrophy (CH) all animals were randomly assigned into 3 groups: control-without therapy (infusion of 0,9% NaCl); main I-receive 10 mg/kg nebivolol per os in combination with lisinopril infusion; and main II-receive infusion of 15 mg/kg of Nadcin® dissolved in water for injection. All animals were euthanized throughout 7 days after beginning of treatment. Results and conclusion: The increases of heart weight by 18,6% and heart-to-body weight ratio by 35,5% observed in ISO-induced CH were significantly suppressed in lisinopril-nebivolol therapy and monotherapy with NAD+- containing drug, Nadcin®. Therapy with Nadcin® restored the normal level of NAD+/NADH ratio and norepinephrine, in myocardium and cerebral arteries, decrease level of nuclear factor kappa B (NF-kB) and tumor necrosis factor (TNF)- alpha in myocardium, cerebral arteries and blood and as a result blocking the myocardial hypertrophy progression. Treatment with Nadcin® on the ISO-induced model of cardiac injury in rabbits occurs significant anti-hypertrophic and anti-inflammatory effects and occurs preventive action on the redox-potential, NAD+/NADH decreasing and NAD+ depletion of myocardium and redox-potential decreasing in cerebral arteries. Thus, therapeutic strategy for NAD repletion have shown high therapeutic potential for cardiac, vascular and neurologic diseases.

The poly(ADP-ribosyl)ation of FoxO3 mediated by PARP1 participates in isoproterenol-induced cardiac hypertrophy

The Forkhead box-containing protein, O subfamily 3 (FoxO3) transcription factor negatively regulates myocardial hypertrophy, and its transcriptional activity is finely conditioned by diverse posttranslational modifications, such as phosphorylation, acetylation, ubiquitination, methylation and glycosylation. Here, we introduce a novel modification of the FoxO3 protein in cardiomyocytes: poly(ADP-ribosyl)ation (PARylation) mediated by poly(ADP-ribose) polymerase-1 (PARP1). This process catalyzes the NAD+-dependent synthesis of polymers of ADP-ribose (PAR) and their subsequent attachment to target proteins by PARPs. Primary-cultured neonatal rat cardiomyocytes were incubated with isoproterenol (ISO) to induce hypertrophy, or were infected with recombinant adenovirus vectors harboring PARP1 cDNA (Ad-PARP1). Sprague-Dawley (SD) rats were treated with ISO to induce cardiac hypertrophy, or were injected with Ad-PARP1 into the anterior and posterior left ventricular walls. Cardiomyocyte surface area, the mRNA expression of hypertrophic biomarkers, echocardiography, morphometry of the hearts were measured. The PARP1 activity was tested by cellular PAR levels. Interactions of PARP1 and FoxO3 were investigated by co-immunoprecipitation and immunofluorescence technique. PARylation of FoxO3 mediated by PARP1 facilitated its phosphorylation at the T32, S252 and S314 sites, triggered its nucleus export and suppressed its transcriptional activity and target genes expression, ultimately inducing cardiac hypertrophy. Additionally, PARP1 silencing or specific inhibition by 3-Aminobenzamide (3AB) and veliparib (ABT-888) alleviated the inhibition of FoxO3 activity by ISO, thus suppressing ISO-induced cardiac hypertrophy. Our data provide the first evidence that PARP1 exacerbates cardiac hypertrophy by PARylation of FoxO3.

A Periodontal pathogen Porphyromonas gingivalis deteriorates Isoproterenol-Induced myocardial remodeling in mice.

Heart failure is a serious disease induced by several conditions, including hypertrophic cardiomyopathy. Although many reports suggest that there is an association between periodontal disease and cardiovascular disease, the mechanisms have yet to be elucidated. The purpose of this study was to clarify the relationship between periodontal disease and heart disease, especially in cardiac hypertrophy. We used C57BL/6J mice and implanted two types of subcutaneous chambers. First, we subcutaneously implanted a coil-shaped chamber into the back of a mouse. Porphyromonas gingivalis (P.g.), a major periodontal pathogen, was injected into the chamber. Then, an osmotic pump was implanted to infuse isoproterenol. Four weeks after the ISO infusion, we performed echocardiography and harvested the heart and blood. We measured the serum level of anti-P.g.-IgG using ELISA. The mRNA levels of several factors were measured using PCR. We found stronger cardiomyocyte hypertrophy in the ISO(+)/P.g.(+) mice compared with the ISO(+)/P.g.(-) mice. The total square of randomly selected cardiomyocytes was 23% larger in the ISO(+)/P.g.(+) mice than in the ISO(+)/P.g.(-) mice. We detected a higher level of mRNA expression in Toll-like receptor 2 and NADPH oxidase 4 in the ISO(+)/P.g.(-) mice compared with the control group. We revealed that a periodontal pathogen affected ISO-induced cardiac hypertrophy via oxidative stress.

SIRT6 suppresses isoproterenol-induced cardiac hypertrophy through activation of autophagy

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

Abstract 14809: Murc/cavin-4 Regulates Cardiac Function via β-adrenergic Receptor Signaling

Background: Muscle-restricted coiled-coil protein (MURC), also referred to as Cavin-4, is a member of the cavin family, which regulates caveolae function together with caveolins. In cardiomyocytes, caveolin-3, a critical and muscle-specific component of caveolae, regulates caveolar structure and function and forms a complex with MURC/Cavin-4. Caveolin-3 overexpression induces cardiac protection, and caveolin-3 deficiency causes progressive cardiomyopathy. The caveolin-3 scaffolding domain peptide inhibits type 5 adenylyl cyclase (AC5), which is an enzyme to synthesize cAMP and an effector of β-adrenergic receptor (AR) signaling. Mutations in MURC/Cavin-4 have been identified in patients with dilated cardiomyopathy. However, molecular mechanisms of MURC as a caveolar component in cardiac function remain unknown. Methods and results: To assess the role of MURC/Cavin-4 in cardiac function, wild-type (WT) and MURC/Cavin-4-knockout (MURC-KO) mice were subjected to a model of pressure overload induced by transverse aortic constriction (TAC). After TAC, WT mice showed cardiac hypertrophy and heart failure, whereas MURC-KO mice were resistant to pressure overload-induced cardiac hypertrophy and heart failure accompanied by fibrosis. We then focused on β-AR signaling, because β1-AR signaling is one of the essential parts in heart failure. MURC/Cavin-4 was co-localized with β1-AR in cardiomyocytes. Isoproterenol (ISO) treatment induced cardiac hypertrophy with fibrosis in WT mice, while ISO-induced cardiac hypertrophy and fibrosis was markedly attenuated in MURC-KO mice. In cardiomyocytes, MURC/Cavin-4 knockdown suppressed ISO-induced cAMP production, while MURC/Cavin-4 overexpression and caveolin-3 knockdown stimulated cAMP production, indicating that caveolin-3 and MURC/Cavin-4 have opposite effects on cAMP production. MURC/Cavin-4 bound to caveolin-3 and interferes with the association between caveolin-3 and AC5. Moreover, MURC-knockdown suppressed ISO-induced PKA activity and phospholamban phosphorylation. Conclusion: Our findings suggest that MURC/Cavin-4 regulates cardiac function via β-AR signaling.

Orphan Nuclear Receptor Nur77 Inhibits Cardiac Hypertrophic Response to Beta-Adrenergic Stimulation.

The orphan nuclear receptor Nur77 plays critical roles in cardiovascular diseases, and its expression is markedly induced in the heart after beta-adrenergic receptor (β-AR) activation. However, the functional significance of Nur77 in β-AR signaling in the heart remains unclear. By using Northern blot, Western blot, and immunofluorescent staining assays, we showed that Nur77 expression was markedly upregulated in cardiomyocytes in response to multiple hypertrophic stimuli, including isoproterenol (ISO), phenylephrine (PE), and endothelin-1 (ET-1). In a time- and dose-dependent manner, ISO increases Nur77 expression in the nuclei of cardiomyocytes. Overexpression of Nur77 markedly inhibited ISO-induced cardiac hypertrophy by inducing nuclear translocation of Nur77 in cardiomyocytes. Furthermore, cardiac overexpression of Nur77 by intramyocardial injection of Ad-Nur77 substantially inhibited cardiac hypertrophy and ameliorated cardiac dysfunction after chronic infusion of ISO in mice. Mechanistically, we demonstrated that Nur77 functionally interacts with NFATc3 and GATA4 and inhibits their transcriptional activities, which are critical for the development of cardiac hypertrophy. These results demonstrate for the first time that Nur77 is a novel negative regulator for the β-AR-induced cardiac hypertrophy through inhibiting the NFATc3 and GATA4 transcriptional pathways. Targeting Nur77 may represent a potentially novel therapeutic strategy for preventing cardiac hypertrophy and heart failure.

Simvastatin prevents isoproterenol-induced cardiac hypertrophy through modulation of the JAK/STAT pathway.

Simvastatin (SIM) is a lipid-soluble inhibitor of hydroxy-3-methylglutaryl coenzyme A reductase with multiple reported therapeutic benefits. The present study was designed to investigate the effect of pretreatment with SIM on isoproterenol (ISO)-induced cardiac hypertrophy in rats. Twenty-four male albino Wistar rats weighing 180–200 g were divided into four groups. Groups I and III received normal saline while groups II and IV received SIM (10 mg/kg body weight) for 30 days per gavage. In the last 7 days, rats of groups III and IV were administered ISO (5 mg/kg) intraperitoneally to induce cardiac hypertrophy. Administration of ISO induced an increase in heart-to-body weight (HW/BW) ratio, an increase in serum interleukin-6, and elevated systolic and diastolic blood pressure. Serum levels of lipids, cardiovascular risk indices, and cardiac troponin I and creatine phosphokinase-MB showed significant increase in ISO-induced hypertrophic rats. Histopathological examination of heart tissue revealed focal areas of subendocardium degeneration, mononuclear cellular infiltrations, fibrous tissue deposition, and increased thickness of the myocardium of left ventricle. In addition, ISO-administered rats exhibited significant upregulation of cardiac Janus kinase, phosphorylated signal transducer and activator of transcription, and nuclear factor-kappa B. Pretreatment with SIM significantly prevented ISO-induced cardiac hypertrophy, alleviated the altered biochemical parameters, and improved the heart architecture. In conclusion, our study provides evidence that SIM prevented the development of cardiac hypertrophy via modulation of the Janus kinase/signal transducer and activator of transcription-signaling pathway in the heart of ISO-administered animals.