Isoproterenol Treatment Research Articles

Application of Zebrafish Model in the Suppression of Drug-Induced Cardiac Hypertrophy by Traditional Indian Medicine Yogendra Ras.

Zebrafish is an elegant vertebrate employed to model the pathological etiologies of human maladies such as cardiac diseases. Persistent physiological stresses can induce abnormalities in heart functions such as cardiac hypertrophy (CH), which can lead to morbidity and mortality. In the present study, using zebrafish as a study model, efficacy of the traditional Indian Ayurveda medicine “Yogendra Ras” (YDR) was validated in ameliorating drug-induced cardiac hypertrophy. YDR was prepared using traditionally described methods and composed of nano- and micron-sized metal particles. Elemental composition analysis of YDR showed the presence of mainly Au, Sn, and Hg. Cardiac hypertrophy was induced in the zebrafish following a pretreatment with erythromycin (ERY), and the onset and reconciliation of disease by YDR were determined using a treadmill electrocardiogram, heart anatomy analysis, C-reactive protein release, and platelet aggregation time-analysis. YDR treatment of CH-induced zebrafish showed comparable results with the Standard-of-care drug, verapamil, tested in parallel. Under in-vitro conditions, treatment of isoproterenol (ISP)-stimulated murine cardiomyocytes (H9C2) with YDR resulted in the suppression of drug-stimulated biomarkers of oxidative stress: COX-2, NOX-2, NOX-4, ANF, troponin-I, -T, and cardiolipin. Taken together, zebrafish showed a strong disposition as a model for studying the efficacy of Ayurvedic medicines towards drug-induced cardiopathies. YDR provided strong evidence for its capability in modulating drug-induced CH through the restoration of redox homeostasis and exhibited potential as a viable complementary therapy.

Cardiac Overexpression of PDE4B Blunts β-Adrenergic Response and Maladaptive Remodeling in Heart Failure.

The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac β-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca2+ handling and PDE4B is decreased in pressure overload hypertrophy, suggesting that increasing PDE4B in the heart is beneficial in heart failure. We measured PDE4B expression in human cardiac tissues and developed 2 transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B and an adeno-associated virus serotype 9 encoding PDE4B. Myocardial structure and function were evaluated by echocardiography, ECG, and in Langendorff-perfused hearts. Also, cAMP and PKA (cAMP dependent protein kinase) activity were monitored by Förster resonance energy transfer, L-type Ca2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca2+ transients with an Ionoptix system. Heart failure was induced by 2 weeks infusion of isoproterenol or transverse aortic constriction. Cardiac remodeling was evaluated by serial echocardiography, morphometric analysis, and histology. PDE4B protein was decreased in human failing hearts. The first PDE4B-transgenic mouse line (TG15) had a ≈15-fold increase in cardiac cAMP-PDE activity and a ≈30% decrease in cAMP content and fractional shortening associated with a mild cardiac hypertrophy that resorbed with age. Basal ex vivo myocardial function was unchanged, but β-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca2+ current, Ca2+ transients, and cell contraction were blunted. Endurance capacity and life expectancy were normal. Moreover, these mice were protected from systolic dysfunction, hypertrophy, lung congestion, and fibrosis induced by chronic isoproterenol treatment. In the second PDE4B-transgenic mouse line (TG50), markedly higher PDE4B overexpression, resulting in a ≈50-fold increase in cardiac cAMP-PDE activity caused a ≈50% decrease in fractional shortening, hypertrophy, dilatation, and premature death. In contrast, mice injected with adeno-associated virus serotype 9 encoding PDE4B (1012 viral particles/mouse) had a ≈50% increase in cardiac cAMP-PDE activity, which did not modify basal cardiac function but efficiently prevented systolic dysfunction, apoptosis, and fibrosis, while attenuating hypertrophy induced by chronic isoproterenol infusion. Similarly, adeno-associated virus serotype 9 encoding PDE4B slowed contractile deterioration, attenuated hypertrophy and lung congestion, and prevented apoptosis and fibrotic remodeling in transverse aortic constriction. Our results indicate that a moderate increase in PDE4B is cardioprotective and suggest that cardiac gene therapy with PDE4B might constitute a new promising approach to treat heart failure.

Understanding Sarcospan Protein Function by Exposure of Mice to Beta‐Adrenergic Stress Conditions

IntroductionSarcospan (SSPN) is a core component of the dystrophin‐glycoprotein complex (DCG) located at the sarcolemma of muscle fibers. SSPN has been shown to play a major role in membrane stabilization of the heart and other striated muscles, however many of its additional functions remain unknown. Previous studies have shown that SSPN deletion diminishes cardiac β‐adrenergic responsiveness, however it elicits increases in cardiac mass (JAHA, 2015 Dec 23;4(12). Blunted β‐adrenergic responsiveness in SSPN‐null hearts appears to be part of a complex phenotypic presentation that occurs downstream of alterations in sympathetic drive, and a propensity toward metabolic syndrome and insulin resistance.MethodsHere, we document the whole‐body effects of ß‐adrenergic stimulation in three‐month‐old WT and SSPN‐null male and female mice after treatment with an isoproterenol (ISO) dosing regimen (8 mg/kg of body weight/day for 5 days) with tissues collected 2 weeks after treatment and examined for alterations in histology, gene and protein expression. After the treatment regimen, morphometric measurements were recorded and cardiac function assessed by echocardiography.ResultsAfter ISO treatment significant differences were found in sympathetic and cholinergic receptor expression in SSPN‐null cardiac and white adipose tissue (WAT) that were distinct from WT. The response of SSPN‐null mice to ISO treatment displayed distinct sex‐differences. After ISO treatment, WT and SSPN‐null male mice exhibited similar levels of WAT lipolysis, evidenced by loss of body and reduced size of epidydymal fat pads. However, female SSPN‐null mice were protected from ISO treatment induced loss in body weight. Furthermore, WT females had reduced spleen weights after ISO treatment, indicative of normal immune cell migration to other tissues, whereas spleen weights were unchanged in SSPN‐null females after ISO treatment suggesting suppressed immune responsiveness.DiscussionThe goal of this study is to better understand how the cardiac function of SSPN‐null mice appears largely unaltered by β‐adrenergic stimulation, whereas other tissues display overall normal responsiveness to ISO treatment. Since lipolysis is induced through activation of β‐adrenergic receptors, we will examine associated markers in untreated and β‐adrenergic‐stimulated WT and SSPN‐null mice of both sexes.Support or Funding InformationResearch Support: Florida State University, FSU CRC FYAP Award, and AHA 16SDG29120002.

Machine Learning‐supported Characterization of the Oxidative Stress‐Sensitive Posttranslational Modifications in ISO‐induced Murine Cardiac Hypertrophy

IntroductionOxidative stress disrupts cellular redox homeostasis and chemically alters proteins at the proteome scale in the form of oxidative sensitive post‐translational modifications (O‐PTMs). In stressed heart, these O‐PTMs often lead to protein dysfunction and proteotoxicity, which have been implicated as pathological drivers for various cardiovascular diseases. However, little is known about the dynamics of myocardial O‐PTMs during disease progression, or the O‐PTM molecular signatures pertinent to pathophysiological phenotypes of the heart.MethodsWe characterized a large‐scale longitudinal proteomics dataset previously collected from mice under isoproterenol (ISO)‐induced cardiac hypertrophy. Six genetic mouse strains exhibiting distinct susceptibilities to ISO challenge were employed to recapitulate genetic diversity in humans. Left ventricle tissue samples were analyzed at 7 time points following ISO treatment (0, 1, 3, 5, 7, 10, 14 days); cardiac function was measured concomitantly via heart weight/body weight ratios and ejection fraction. Molecular features (e.g., modification site & occupancy) of 7 prevalent types of O‐PTMs were extracted using the Integrated Proteomics Pipeline. Stringent filtering criteria were applied to identify O‐PTMs of high confidence in both Control and ISO‐treatment samples, as well as to enable data‐imputation to recover missing data. A customized analytic pipeline empowered by Machine Learning (ML) approaches was employed to pinpoint a panel of O‐PTMs as molecular signatures. Subsequently, we constructed a multi‐layer Bayesian network to unearth pivotal relationships among molecular signatures and phenotypic features of heart.ResultsWe conducted the first large‐scale O‐PTM analysis in murine cardiac remodeling, which elucidates the impact of ISO‐induced oxidative stress on 7 irreversible O‐PTM subtypes. After data‐imputation, a completed data matrix containing temporal occupancy values of 2,877 O‐PTMs maximally defined the O‐PTM landscapes in healthy and stressed mouse hearts. The ML‐supported feature selection and validation extracted 9 O‐PTMs that are highly indicative of pathophysiological conditions. The Bayesian network modeling unveiled hierarchical relationships among O‐PTM signatures and cardiac function, offering mechanistic insights underlying oxidative stress and cardiac dysfunction. These O‐PTM signatures are enriched in biological pathways supporting energy production, ROS regulation, Ca2+signaling, and ECM remodeling, which are all benchmark processes of cardiac remodeling.ConclusionProteomics captures in remarkable detail the molecular interplays underlying cardiovascular pathophysiology. ML methods support in‐depth analyses to tease out key molecular features unique to disease phenotypes, providing a novel strategy to identify potential diagnostic and therapeutic targets. Application of these cutting‐edge computational approaches on O‐PTM investigations will enhance our capability to substantiate precision medicine.Support or Funding InformationNIH R01HL146739 to D. Wang & D. Ng; NIH R35HL135772 to P. Ping.

Cardiac AKAP12 Signalosome Overexpression Exacerbates Isoproterenol Induced Heart Failure

BackgroundHeart failure (HF), defined as an impaired ability of the heart to efficiently pump blood, is responsible for 1 out of 8 deaths per year in the U.S.A. and is the major cause of death globally. Physiologically, stimulation of β‐adrenergic receptors (β‐AR) by catecholamines, epinephrine (EP) and norepinephrine (NE), increases cardiac contractility. However, in HF, chronic β‐AR stimulation leads to reduced cardiac function due in part to the desensitization of these receptors. Previous in vivo studies reported by our group showed enhanced cardiac function after Isoproterenol (ISO) induced HF in male mice lacking functional cardiac AKAP12 compared to wild‐type (WT) mice. In this study, we aim to investigate the impact of cardiac AKAP12 overexpression (oxAKAP12‐Tg) on HF progression in both male and female mice and understand the underlying mechanisms involved. Our central hypothesis is that cardiac AKAP12 overexpression is involved in heart failure progression by influencing signaling downstream β‐AR.MethodsWT and oxAKAP12‐Tg mice (8–10) weeks old males and females were categorized into three groups; subcutaneous ISO osmotic pump group (ISO; 60 mg/kg/day X 14‐days), vehicle group (ascorbic acid 0.0002% X 14‐days), and sham group (no subcutaneous pump). Echocardiography and ECG were obtained from all groups 2‐days before subcutaneous insertion of the osmotic pumps as well as 7‐days and 14‐days after the osmotic pump insertion, followed by tissue harvest on the 14‐day. Adult mouse ventricular homogenates were used for protein expression levels analysis. Furthermore, CRISPR‐cas9 technology was used to overexpress AKAP12 into AC16 cells, a human cardiomyocyte cell line, to assess gene expression levels upon overnight treatment with 100 nmol of ISO.ResultsCardiac overexpression of AKAP12 in both males and females reduced left ventricular ejection fraction (EF) and fractional shortening (FS). Both males and females treated with ISO and overexpressing AKAP12 had a 22.7±2% reduction in EF and 14.5±2.5% reduction in FS after 14‐days of chronic ISO treatment when compared to control groups. However, the pattern of this decline in cardiac function was different between male mice and female mice; where male mice showed a slow decline in cardiac function over the two week period of chronic ISO treatment while female mice showed a steep decline within the first week of chronic ISO treatment that plateaued during the second week of treatment. Protein expression levels of β‐arrestin1 and β‐arrestin2 were significantly lower in cardiac homogenates of ISO treated oxAKAP12‐Tg when compared to ISO treated WT male and female mice, respectively. No significant differences were observed between control groups. This observation was further supported by QPCR gene expression studies; where we observed a 40% reduction of β‐arrestin‐1 gene expression in ISO treated AC16 overexpressing AKAP12 when compared to the ISO treated WT AC16 cells.ConclusionsCardiac overexpression of AKAP12 negatively influences systolic function in both male and female mice, potentially through affecting the β‐arrestin signaling pathway. Thus, AKAP12 might be involved in biased signaling downstream β‐AR which could serve as a potential target for designing drugs to ameliorate HF.

Evidence for significance of serine 1487 in β-adrenergic regulation of Cav1.2 channel protein function in genetically engineered mice

Stress responses are mediated through activation of the β-adrenergic signaling pathway. Chronic sympathetic stimulation can result in the development of hypertrophy, arrhythmia and sudden cardiac death but the molecular mechanisms are still not understood. We identified a single serine, following phosphorylation by cAMP-dependent protein kinase A, responsible for the increase in function of the human cardiac Cav1.2 channel protein in an artificial cell system. To confirm whether the serine is involved in in vivo channel stimulation by β-adrenergic signaling, we created CRISPR/Cas9 mutant mouse lines with glutamate and alanine-substitution of the serine1487 on Cav1.2 protein to mimic the phosphorylated and the non-phosphorylated protein. The average litter size (6.7 ± 0.7; N = 16 and 6.1 ± 0.4; N = 24 respectively) and survival rate (48.6% vs. 42.5%) were similar in the two lines, but homozygous mutation of Cav1.2 S1487E caused 100% mortality with a median survival of ~1 day. The relative size of the hearts of homozygous S1487A mice were smaller than S1487E (heart weight/tibial length: 7.0 ± 0.1; N = 3 vs 7.8 ± 0.3; N = 3). Administration of intraperitoneal injection of 20 mg/kg isoproterenol increased the heart rate up to 30% in both glutamate and alanine-substituted heterozygous mutants in a similar manner to the negatively genotyped littermates, but did not alter the heart rate of the homozygous S1487A mutants. Chronic isoproterenol treatment (20 mg/kg isoproterenol daily injection for 2 weeks) induced hypertrophy in the wild type and heterozygous S1497A mutant mice, but there were no significant changes in the posterior wall thickness of the heart of homozygous S1487A mutants.

Improved workflow for mass spectrometry–based metabolomics analysis of the heart

MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS-based metabolomics workflow that uses insoluble protein-derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.

Interleukin-9 Aggravates Isoproterenol-Induced Heart Failure by Activating Signal Transducer and Activator of Transcription 3 Signalling

BackgroundPrevious studies have demonstrated that inflammation is closely related to the occurrence and development of heart failure (HF). As an inflammation-related cytokine, interleukin (IL)-9 has been reported to be involved in the development of cardiovascular diseases. However, the role of IL-9 in HF in response to isoproterenol (ISO) stimulation has barely been explored. Thus, this study aimed to investigate whether IL-9 participates in HF and the possible associated mechanisms. MethodsChronic ISO infusion was used to establish an HF model, and the IL-9 levels in mice and isolated cardiomyocytes were measured. In addition, ISO-treated mice received an injection of recombinant mouse IL-9 (rIL-9) or an antimouse IL-9 neutralizing monoclonal antibody (mAb) to investigate the effects of IL-9 on cardiac function, hypertrophy, and fibrosis. ResultsIL-9 levels were significantly increased in mice and isolated cardiomyocytes after ISO treatment. Treatment with rIL-9 resulted in aggravated cardiac dysfunction and amplified cardiac hypertrophy and fibrosis, whereas treatment with the anti-IL-9 neutralizing mAb ameliorated cardiac dysfunction and reduced cardiac hypertrophy and fibrosis in ISO-treated mice. In addition, ISO infusion–induced cardiac inflammation and cardiomyocyte apoptosis was aggravated by rIL-9 but prevented by the anti-IL-9 mAb. IL-9 did not activate signal transducer and activator of transcription (STAT)1 or STAT5 but induced STAT3 phosphorylation in ISO-induced HF. Moreover, S31-201, a specific STAT3 inhibitor, nearly abolished rIL-9-induced increases in cardiac dysfunction, hypertrophy, and fibrosis in response to ISO stimulation. ConclusionsIL-9 aggravated cardiac dysfunction and amplified cardiac hypertrophy and fibrosis in the ISO-induced HF model by activating STAT3 signalling. These data indicate that blocking IL-9 may be an attractive pharmacotherapeutic strategy for the treatment of cardiac hypertrophy and fibrosis induced by chronic β-adrenergic receptor activation to limit the progression of HF.

Diazoxide Modulates Cardiac Hypertrophy by Targeting H2O2 Generation and Mitochondrial Superoxide Dismutase Activity.

Cardiac hypertrophy involves marked wall thickening or chamber enlargement. If sustained, this condition will lead to dysfunctional mitochondria and oxidative stress. Mitochondria have ATP-sensitive K+ channels (mitoKATP) in the inner membrane that modulate the redox status of the cell. We investigated the in vivo effects of mitoKATP opening on oxidative stress in isoproterenol- induced cardiac hypertrophy. Cardiac hypertrophy was induced in Swiss mice treated intraperitoneally with isoproterenol (ISO - 30 mg/kg/day) for 8 days. From day 4, diazoxide (DZX - 5 mg/kg/day) was used in order to open mitoKATP (a clinically relevant therapy scheme) and 5-hydroxydecanoate (5HD - 5 mg/kg/day) or glibenclamide (GLI - 3 mg/kg/day) were used as mitoKATP blockers. Isoproterenol-treated mice had elevated heart weight/tibia length ratios (HW/TL). Additionally, hypertrophic hearts had elevated levels of carbonylated proteins and Thiobarbituric Acid Reactive Substances (TBARS), markers of protein and lipid oxidation. In contrast, mitoKATP opening with DZX avoided ISO effects on gross hypertrophic markers (HW/TL), carbonylated proteins and TBARS, in a manner reversed by 5HD and GLI. Moreover, DZX improved mitochondrial superoxide dismutase activity. This effect was also blocked by 5HD and GLI. Additionally, ex vivo treatment of isoproterenol- induced hypertrophic cardiac tissue with DZX decreased H2O2 production in a manner sensitive to 5HD, indicating that this drug also acutely avoids oxidative stress. Our results suggest that diazoxide blocks oxidative stress and reverses cardiac hypertrophy. This pharmacological intervention could be a potential therapeutic strategy to prevent oxidative stress associated with cardiac hypertrophy.

Aliskiren attenuates cardiac dysfunction by modulation of the mTOR and apoptosis pathways.

Aliskiren (ALS) is well known for its antihypertensive properties. However, the potential underlying the molecular mechanism and the anti-hypertrophic effect of ALS have not yet been fully elucidated. The aim of the present study was to investigate the role of ALS in mammalian target of rapamycin (mTOR) and apoptosis signaling using in vivo and in vitro models of cardiac hypertrophy. A rat model of cardiac hypertrophy was induced by isoproterenol treatment (5 mg·kg-1·day-1) for 4 weeks, with or without ALS treatment at 20 mg·kg-1·day-1. The expression of hypertrophic, fibrotic, and apoptotic markers was determined by RT-qPCR. The protein expression of apoptotic markers mTOR and p-mTOR was assessed by western blot analysis. The proliferation of H9C2 cells was monitored using the MTS assay. Cell apoptosis was analyzed using flow cytometry. In vivo, isoproterenol-treated rats exhibited worse cardiac function, whereas ALS treatment reversed these dysfunctions, which were associated with changes in p-mTOR, Bcl-2, Bax, and cleaved caspase-3 expression, as well as the number of apoptotic cells. In vitro, H9C2 cardiomyocyte viability was significantly inhibited and cardiac hypertrophy was induced by Ang II administration, but ALS reversed Ang II-induced H9C2 cardiomyocyte hypertrophy and death. Furthermore, Ang II triggered the activation of the mTOR and apoptosis pathways in hypertrophic cardiomyocytes that were inhibited by ALS treatment. These results indicated that ALS alleviated cardiac hypertrophy through inhibition of the mTOR and apoptosis pathways in cardiomyocytes.

The effects of resveratrol treatment on caveolin-3 expression and Na+/K+ ATPase activity in rats with isoproterenol-induced myocardial injury.

OBJECTIVE:The present study aims to investigate the therapeutic effects of resveratrol (RES) on isoproterenol (ISO) induced myocardial injury rat model.METHODS:Catecholamine-induced heart damage was induced by ISO treatment for 30 days. The rats were divided into four groups as follows: the control group received saline, the ISO group received 5.0 mg/kg ISO, the RES group received 10 mg/kg RES, and the ISO-RES group received 10 mg/kg RES and 5 mg/kg ISO treatments for 30 days. Following echocardiographic measurements and body weight recorded, the rats were decapitated. Plasma and cardiac tissue samples obtained by decapitation were analyzed using biochemical, histopathological, molecular and immunohistochemical methods.RESULTS:In the ISO group, Na+/K+ ATPase activity and ATP content, GSH, and caveolin-3 levels were low. LDH, CK and lysosomal enzyme activities, MDA level, and MPO activity were found to be high. It was determined that GSH and MDA levels and MPO, Na+/K+ ATPase activity, ATP content caveolin-3 levels changes that arose from ISO treatment were suppressed by RES treatment.CONCLUSION:RES treatment has ameliorated all the functional and biochemical parameters. The results obtained in this study suggest that RES is a promising supplement against catecholamine exposure as it improves antioxidant defense mechanisms in the heart. In the light of above-mentioned data, RES can be assumed as a promising agent in ameliorating the oxidative injury of the myocardium.

Cytoplasm lipids can be modulated through hormone-sensitive lipase and are related to mitochondrial function in porcine IVM oocytes.

Intracellular lipids provide energy for oocyte maturation and development. Triglycerides are the main components of cytoplasm lipid droplets, and hydrolysis of triglycerides requires several lipase-mediated steps. The aim of this study was to determine the effects of the β-adrenoceptor agonist isoproterenol (ISO) and the hormone-sensitive lipase (HSL) inhibitor CAY10499 on the IVM of porcine oocytes. ISO (5mg L-1) and CAY10499 (20mg L-1) had positive and negative effects respectively on invitro oocyte maturation and subsequent embryo development. The rates of polar body extrusion, cleavage and blastocyst formation were significantly higher in the ISO-treated group than the control and CAY10499-treated groups. ISO treatment also upregulated intracellular cAMP levels in comparison with the control group, while CAY10499 significantly increased the triglyceride content of matured oocytes when compared with other groups, consistent with the observed decrease in LIPE (HSL) mRNA levels. Furthermore, the inhibitory effects of CAY10499 included decreases in mitochondrial membrane potential and mitochondrial temperature. These results indicate that ISO has a positive effect on the IVM of porcine oocytes, and that intracellular lipid metabolism can be modulated by CAY10499 through inhibition of HSL and is closely related to mitochondrial function.

Transcriptomic Validation of the Protective Effects of Aqueous Bark Extract of Terminalia arjuna (Roxb.) on Isoproterenol-Induced Cardiac Hypertrophy in Rats.

Aqueous extract of the bark of Terminalia arjuna (TA) is used by a large population in the Indian subcontinent for treating various cardiovascular conditions. Animal experiments have shown its anti-atherogenic, anti-hypertensive, and anti-inflammatory effects. It has several bioactive ingredients with hemodynamic, ROS scavenging, and anti-inflammatory properties. Earlier we have done limited proteomic and transcriptomic analysis to show its efficacy in ameliorating cardiac hypertrophy induced by isoproterenol (ISO) in rats. In the present study we have used high-throughput sequencing of the mRNA from control and treated rat heart to further establish its efficacy. ISO (5 mg/kg/day s.c.) was administered in male adult rats for 14 days to induce cardiac hypertrophy. Standardized aqueous extract TA bark extract was administered orally. Total RNA were isolated from control, ISO, ISO + TA, and TA treated rat hearts and subjected to high throughput sequence analysis. The modulations of the transcript levels were then subjected to bio-informatics analyses using established software. Treatment with ISO downregulated 1,129 genes and upregulated 204 others. Pre-treatment with the TA bark extracts markedly restored that expression pattern with only 97 genes upregulated and 85 genes downregulated. The TA alone group had only 88 upregulated and 26 downregulated genes. The overall profile of expression in ISO + TA and TA alone groups closely matched with the control group. The genes that were modulated included those involved in metabolism, activation of receptors and cell signaling, and cardiovascular and other diseases. Networks associated with those genes included those involved in angiogenesis, extracellular matrix organization, integrin binding, inflammation, drug metabolism, redox metabolism, oxidative phosphorylation, and organization of myofibril. Overlaying of the networks in ISO and ISO_TA group showed that those activated in ISO group were mostly absent in ISO_TA and TA group, suggesting a global effect of the TA extracts. This study for the first time reveals that TA partially or completely restores the gene regulatory network perturbed by ISO treatment in rat heart; signifying its efficacy in checking ISO-induced cardiac hypertrophy.

Kindlin-2 suppresses transcription factor GATA4 through interaction with SUV39H1 to attenuate hypertrophy

Kindlin-2 plays an important role in the regulation of cardiac structure and function. Depletion of Kindlin-2 contributes to cardiac hypertrophy and progressive heart failure, however, the precise mechanisms involved in this process remain unclear. GATA4 is a critical transcription factor in regulating cardiogenesis. We found that Kindlin-2 suppresses the expression of GATA4 through binding to its promoter and prevents cardiomyocytes from hypertrophy induced by isoproterenol (ISO) treatment. Mechanistically, Kindlin-2 interacts with histone methyltransferase SUV39H1 and recruits it to GATA4 promoter leading to the occupancy of histone H3K9 di- and tri-methylation. Furthermore, to confirm the function of Kindlin-2 in vivo, we generated mice with targeted deletion of cardiac Kindlin-2. We found that 6-month-old Kindlin-2 cKO mice have developed hypertrophic cardiomyopathy and that this pathological process can be accelerated by ISO-treatment. GATA4 expression was markedly activated in cardiac tissues of Kindlin-2 cKO mice compared to wild-type animals. Collectively, our data revealed that Kindlin-2 suppresses GATA4 expression by triggering histone H3K9 methylation in part and protects heart from pathological hypertrophy.

Regulation of beta adrenoceptor-mediated myocardial contraction and calcium dynamics by the G protein-coupled estrogen receptor 1

The G protein-coupled estrogen receptor 1 (GPER) produces cardioprotective effects. However, the underlying mechanisms are not well understood. We aimed to investigate the role of GPER in β adrenoceptor-mediated cardiac contraction and myocardial signaling. In anesthetized animals, intrajugular administration of isoproterenol produces a rapid and sustained rise in left ventricular pressure (LVP) and increases ectopic contractions. Administration of the GPER agonist G-1 during the plateau phase of isoproterenol-induced LVP increase rapidly restores LVP to baseline levels and reduces the frequency of ectopic contractions. In freshly isolated cardiomyocytes, isoproterenol potentiates electrically induced peak currents of L-type Ca2+ channels (LTCC) and increases the potential sensitivity of their inactivation. Coadministration of G-1 prevents isoproterenol-induced potentiation of peak LTCC currents and makes channels more sensitive to being inactivated compared to isoproterenol alone. Isoproterenol treatment of cardiomyocytes without electrical stimulation triggers slow-rising Ca2+ signals that are inhibited by the β1AR antagonist metoprolol but not by β2AR antagonist ICI-118551. G-1 pretreatment dose-dependently suppresses isoproterenol-induced total Ca2+ signals and the amplitude and frequency of the intrinsic Ca2+ oscillatory deflections. Pretreatment with the GPER antagonist G-36 produces opposite effects, dose-dependently increasing these signals. ISO promotes robust phosphorylation of Cav1.2 channels at Ser1928. G-1 pretreatment inhibits isoproterenol-stimulated phosphorylation of Cav1.2 at Ser1928, while G-36 pretreatment enhances this signal. Our data indicate that GPER functions as an intrinsic component of β1AR signaling to moderate myocardial Ca2+ dynamics and contraction.

A heart-on-a-chip platform for online monitoring of contractile behavior via digital image processing and piezoelectric sensing technique

Heart-on-a-chip devices have recently emerged as a viable and promising model for drug screening applications, owing to its capability of capturing important biological and physiological parameters of cardiac tissue. However, most heart-on-a-chips are not developed for online and continuous monitoring of contractile behavior, which are the main functional characteristics of cardiac tissue. In this study, we designed and investigated on a heart-on-a-chip platform that provides online monitoring of contractile behavior of a 3D cardiac tissue construct. The contractile behavior include contraction force, frequency, and synchronization. They can be evaluated by an image processing system and a piezoelectric sensing system simultaneously. Based on the deformation of a micro-pillar array embedded within the 3D cardiac tissue upon subjected to cardiac contraction, the image processing system provides in situ multi-site detection of the contractile behavior. At the same time, the piezoelectric sensing system measures the contractile behavior of the entire cardiac tissue construct. A 3D cardiac tissue construct was successfully fabricated. Then the heart-on-a-chip platform was validated by applying various motion patterns on the micro-pillars, which mimicked the contraction patterns of the 3D cardiac tissue. The drug reactivity of the 3D cardiac tissue construct after a treatment of isoproterenol and doxorubicin was evaluated by measuring the contractile behavior via the image processing and the piezoelectric sensing systems. The results from the drug reactivity provided by both these measurement systems were consistent with previous reports, demonstrating the reliability of the heart-on-a-chip platform and its potential for use in cardio-related drug screening applications.

1179A novel exonic variant in TRIM55 gene predisposes to heart failure

Abstract Background/Introduction Heart failure is a potentially lethal cardiac disorder. The genetic variants as well as the molecular signaling pathways accounting for the majority of the heritability of sporadic heart failure are unknown. Here, we screened for novel genetic variants associated with heart failure in the Finnish population. A previously uncharacterized variant in TRIM55 was selected for further validation. Purpose To identify and characterize heart failure-associated genetic variants. Methods Heart failure-associated genetic variants were screened from the FINRISK survey study data by the National Institute of Health and Welfare of Finland (genotype data available for approx. 28,000 individuals, 1,485 individuals with heart failure). A probably damaging heart failure-associated exonic variant in TRIM55 gene (rs138811034), encoding an E140K variant, was functionally characterized in HL-1 mouse cardiomyocytes using CRISPR/Cas9 technology to introduce the alteration in the endogenous locus. Variant cell lines were analyzed with RNA sequencing. The effect of the variant on cellular viability was tested with CTG assays. Hypertrophic response after isoproterenol treatment was studied by quantitating cell surface area with immunofluorescence microscopy. Western analyses of p21 expression and flow cytometry-based cell cycle analyses were carried out to assess effects on cell cycle progression. In zebrafish embryos in vivo, cardiac contractility was videomicroscopically measured after transient CRISPR-mediated knockdown of the trim55a gene. Results HL-1 cells harboring Trim55 E140K/− genotype demonstrated significantly reduced viability as compared to the WT/WT cell (P=0.006), similarly to the cell line with double knockout (−/−) of the gene. E140K/−, −/− and WT/− cell lines demonstrated a significant hypertrophic response to isoproterenol stimulation (P=0.007, P=0.007 and P=0.018, respectively), while the WT/WT cells did not. In the E140K/− and −/− cell lines, p21 protein expression was increased and there were significantly fewer cells in the mitotic G2/M phase of the cell cycle, as compared to the WT/WT cells (P=0.031 and P=0.024, respectively). In the E140K/− and −/− cell lines, RNA sequencing revealed systemic alterations in the genes related to cardiac contractility and stress. In zebrafish embryos with deleted trim55a gene, ejection fraction was reduced as compared to control sgRNA-injected embryos (30.8% ± 6.0% vs 43.9%± 2.2% (95% CIs), respectively, P=0.019). Conclusions TRIM55 E140K variant was statistically associated with heart failure in the Finnish population. In HL-1 cardiomyocytes in vitro, the variant reduced cellular viability and cell cycle progression and promoted hypertrophy. In zebrafish embryos, loss of trim55a caused a significant reduction in cardiac contractility. These findings are consistent with a novel role for TRIM55 polymorphism in predisposing to heart failure. Acknowledgement/Funding Finnish Cultural Foundation (Varsinais-Suomi Regional Fund), Inkeri ja Mauri Vänskän säätiö, Academy of Finland [310507], Sigrid Jusélius Foundation

Isoproterenol Disrupts Intestinal Barriers Activating Gut-Liver-Axis: Effects on Intestinal Mucus and Vascular Barrier as Entry Sites

Background: The gut-liver-axis presents the pathophysiological hallmark for multiple liver diseases and has been proposed to be modulated during stress and shock. Access to the gut-liver-axis needs crossing of the mucus and gut-vascular barrier. The role of β-adrenoreceptor-activation for both barriers has not been defined and is characterized here. Methods: Splanchnic β-adrenergic stimulation was achieved by chronic intraperitoneal application of isoproterenol via alzet-pump in vivo. The intestinal permeability and gut-vascular barrier function was assessed in ileal loop experiments. The extravasation of predefined sizes of fluorescence isothiocyanate (FITC)-dextran molecules in ileal microcirculation was evaluated by intravital confocal laser endomicroscopy in vivo. Mucus parameters thickness, goblet cell count and mucin-expression were assessed by stereomicroscopy, immunostaining and RNA-sequencing respectively. Ileal lamina propria (LP) as well as mesenteric lymph node mononuclear cells was assessed by FACS. Results: Healthy mice lack translocation of 4 kDa-FITC-dextran from the small intestine to the liver, whereas isoproterenol-treated mice demonstrate pathological translocation (PBT). Mucus layer is reduced in thickness with loss of goblet-cells and mucin-2-staining and -expression in isoproterenol-treated animals under standardized gnotobiotic conditions. Isoproterenol disrupts the gut vascular barrier displaying Ileal extravasation of large-sized 70- and 150 kDa-FITC-dextran. This pathological endothelial permeability and accessibility induced by isoproterenol associates with an augmented expression of plasmalemmal-vesicle-associated-protein-1 in intestinal vessel. Ileal LP after isoproterenol treatment contains more CD11c+-dendritic cells (DC) with increased appearance of CCR7+ DC in mesenteric lymph nodes. Conclusions: Isoproterenol impairs the intestinal muco-epithelial and endothelial-vascular barrier promoting PBT to the liver. This barrier dysfunction on multiple levels potentially can contribute to liver injury induced by catecholamines during states of increased β-adrenergic drive.

Protective effect of histone methyltransferase NSD3 on ISO-induced cardiac hypertrophy.

Nuclear receptor-binding SET domain 3 (NSD3) is a lysine methyltransferase that plays important roles in multiple biological activities; however; its potential roles in the cardiovascular system remain unknown. In this study, we found that NSD3 expression is reduced by isoproterenol (ISO) stimuli both invitro and invivo. Overexpression of NSD3 attenuates ISO-induced cardiomyocyte hypertrophy. Mechanistically, ISO treatment decreases H3K27me2/3 modifications on the atrial natriuretic factor (ANF) promoter by suppressing NSD3 and inhibits the association between NSD3 and bromodomain-containing protein 4 (BRD4), thus suppressing the BRD4-mediated H3K27ac modifications, which ultimately promote ANF transcription and cardiomyocyte hypertrophy. In conclusion, NSD3 decreases ANF expression and, thereby, attenuates ISO-induced cardiomyocyte hypertrophy.

Losartan and isoproterenol promote alterations in the local renin-angiotensin system of rat salivary glands.

Renin-angiotensin system (RAS) systemically or locally collaborates with tissue homeostasis, growth and development, which has been extensively studied for its pharmacological implications. This study was primarily aimed at finding and characterizing local RAS in rat parotid, sublingual and submandibular glands. It was also hypothesized that vasoactive drugs could affect the expression of RAS targets, as well as saliva flow and its composition. Therefore, another objective of this study was to compare the effects of losartan (angiotensin II receptor blocker) and isoproterenol (β-adrenergic receptor agonist). Forty-one Wistar rats were divided into three groups and administered a daily intraperitoneal dose of saline, losartan or isoproterenol solutions for one week. The following RAS targets were studied using qPCR: renin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), ACE-2, elastase-2 (ELA-2), AT1-a and MAS receptors, using RPL-13 as a reference gene. Morphology of glands was analyzed by immunohistochemistry using REN, ACE, ACE-2, AT1, AT2 and MAS antibodies. The volume and total protein content of saliva were measured. Our results revealed that ACE, ACE-2, AT1-a, AT2 and MAS receptors were expressed in all salivary gland samples, but REN and ELA-2 were absent. Losartan decreased mRNA expression of RAS targets in parotid (MAS) and submandibular glands (ACE and both AT receptors), without affecting morphological alterations, and significantly decreased saliva and total protein secretions. Isoproterenol treatment affected gene expression profiles in parotid (ACE, ACE-2, AT1-a, MAS, AGT), and submandibular (ACE, AT2, AGT) glands, thus promoting acinar hypertrophy in serous acini, without significant changes in salivary flow or total protein content. These drugs affected mainly acini, followed by duct systems and myoepithelial cells, whereas blood vessels were not affected. In conclusion, there is a local RAS in major rat salivary glands and losartan, an angiotensin II receptor blocker, affected not only the RAS-target gene expression but also decreased salivary flow and total protein content.