Ventricular mRNA Research Articles

Abstract 18258: Leucine Supplementation Improves Myocardial Function in a HFpEF Rat Model

Introduction: We previously documented that leucine supplementation protects from skeletal muscle atrophy by increasing skeletal muscle mass and force during hind limb immobilization. One of the proposed underlying mechanisms was the suppression of HDAC4 protein expression and increased nuclei accretion. Hypothesis: The present study aimed to examine whether the protective effects of leucine supplementation are also observed in the myocardium in an animal model of heart failure with preserved ejection fraction (HFpEF) and to assess the role of HDAC4 modulation. Methods: Female ZSF1 lean (control, con) and obese (HFpEF) rats were randomized at an age of 20 weeks to one of three groups: lean rats receiving standard chow (con), obese rats receiving standard chow (HFpEF), and obese rats receiving leucine-supplemented chow (3% w/w) for 12 weeks (HFpEF/leu). All animals underwent echocardiography at baseline and after 6 and 12 weeks. Prior to organ harvest left ventricular (LV) hemodynamics were measured invasively, blood samples were taken, cardiac mitochondrial function was assessed and LV tissue was collected for molecular and histological analyses. Results: Assessment of diastolic function revealed a 20% reduction of E/é in the HFpEF/leu group compared to untreated HFpEF. While no alterations of left ventricular hemodynamics were observed between the groups, stiffness factor β was significantly reduced in leucine-treated rats. Left ventricular mRNA levels of Collagen type I a1 and matrix metalloproteinase 2 were reduced by 25% and 22%, respectively. Assessment of mitochondrial oxidative phosphorylation revealed a significant improvement of complex-I- and complex-II mediated respiration in leucine-treated rats compared to HFpEF. Finally, HDAC4 protein expression levels were reduced by 40% in the HFpEF/leu group compared to HFpEF. Conclusions: This study presents beneficial effects of leucine supplementation on cardiac function, remodeling, and metabolic parameters in an HFpEF rat model. These benefits may be attributed to suppressed HDAC4 expression.

Developmental differences in myocardial transmembrane Na+ transport: implications for excitability and Na+ handling.

Little is currently known about possible developmental changes in myocardial Na+ handling, which may have impact on cell excitability and Ca2+ content. Resting intracellular Na+ concentration ([Na+ ]i ), measured in freshly isolated rat ventricular myocytes with CoroNa green, was not significantly different in neonates (3-5days old) and adults, but electrical stimulation caused marked [Na+ ]i rise only in neonates. Inhibition of L-type Ca2+ current by CdCl2 abolished not only systolic Ca2+ transients, but also activity-dependent intracellular Na+ accumulation in immature cells. This indicates that the main Na+ influx pathway during activity is the Na+ /Ca2+ exchanger, rather than voltage-dependent Na+ current (INa ), which was not affected by CdCl2 . In immature myocytes, INa density was two-fold greater, inactivation was faster, and the current peak occurred at less negative transmembrane potential (Em ) than in adults. Na+ channel steady-state activation and inactivation curves in neonates showed a rightward shift, which should increase channel availability at diastolic Em , but also require greater depolarization for excitation, which was observed experimentally and reproduced in computer simulations. Ventricular mRNA levels of Nav 1.1, Nav 1.4 and Nav 1.5 pore-forming isoforms were greater in neonate ventricles, while a decrease was seen for the β1subunit. Both molecular and biophysical changes in the channel profile may contribute to the differences in INa density and voltage-dependence, and also to the less negative threshold Em , in neonates compared to adults. The apparently lower excitability in immature ventricle may confer protection against the development of spontaneous activity in this tissue. KEY POINTS: Previous studies showed that myocardial preparations from immature rats are less sensitive to electrical field stimulation than adult preparations. Freshly isolated ventricular myocytes from neonatal rats showed lower excitability than adult cells, e.g. less negative threshold membrane potential and greater membrane depolarization required for action potential triggering. In addition to differences in mRNA levels for Na+ channel isoforms and greater Na+ current (INa ) density, Na+ channel voltage-dependence was shifted to the right in immature myocytes, which seems to be sufficient to decrease excitability, according to computer simulations. Only in neonatal myocytes did cyclic activity promote marked cytosolic Na+ accumulation, which was prevented by abolition of systolic Ca2+ transients by blockade of Ca2+ currents. Developmental changes in INa may account for the difference in action potential initiation parameters, but not for cytosolic Na+ accumulation, which seems to be due mainly to Na+ /Ca2+ exchanger-mediated Na+ influx.

Changes in myocardial myosin heavy chain isoform composition with exercise and post-exercise cold-water immersion

This study investigated the changes in myocardial myosin heavy chain (MHC) isoforms, MHC-α and MHC-β composition in young healthy rodents following endurance training, with and without post-exercise cold-water immersion (CWI). Male rats were either trained on a treadmill for 10weeks with (CWI) or without (Ex) regular CWI after each running session, or left sedentary (CON). Left ventricular mRNA of MHC-α, MHC-β, thyroid receptor α1 (TR-α1) and β (TR-β) were analyzed using rt-PCR and semiquantitative PCR analysis. MHC isoform protein composition was determined using SDS-PAGE electrophoresis. MHC-α isoform protein was predominant in all groups. The relative expression of MHC-β (%MHC-β) protein was not different between groups (CWI 34.7 ± 6.9%; Ex 32 ± 5.3%; CON 35.5 ± 10%; P = 0.7). MHC-β mRNA was reduced in Ex (0.7 ± 0.3-fold) compared to CWI (1.3 ± 0.2-fold; P < 0.001) and CON (1.01 ± 0.2-fold; P = 0.03). TRα1 mRNA was lower in CWI (0.4 ± 0.05-fold) than Ex (1.02 ± 0.3-fold) and CON (1.01 ± 0.2-fold) (P < 0.001 for both). CWI exhibited greater %MHC-β mRNA (56.8 ± 4.1%) than Ex (44.4 ± 7.7%; P = 0.001) and CON (48.5 ± 7.8%; P = 0.03). Neither exercise nor post-exercise CWI demonstrated a distinct effect on myocardial MHC protein isoform composition. However, CWI increased the relative expression of MHC-β mRNA compared with Ex and CON. Although this implicates a potential negative long-term impact of post-exercise CWI, future studies should include measures of cardiac function to better understand the effect of such isoform mRNA shifts following regular use of CWI.

ZSF1 rat as animal model for HFpEF: Development of reduced diastolic function and skeletal muscle dysfunction.

AimsThe prevalence of heart failure with preserved ejection fraction (HFpEF) is still increasing, and so far, no pharmaceutical treatment has proven to be effective. A key obstacle for testing new pharmaceutical substances is the availability of suitable animal models for HFpEF, which realistically reflect the clinical picture. The aim of the present study was to characterize the development of HFpEF and skeletal muscle (SM) dysfunction in ZSF1 rats over time.Methods and resultsEchocardiography and functional analyses of the SM were performed in 6‐, 10‐, 15‐, 20‐, and 32‐week‐old ZSF1‐lean and ZSF1‐obese. Furthermore, myocardial and SM tissue was collected for molecular and histological analyses. HFpEF markers were evident as early as 10 weeks of age. Diastolic dysfunction, confirmed by a significant increase in E/e′, was detectable at 10 weeks. Increased left ventricular mRNA expression of collagen and BNP was detected in ZSF1‐obese animals as early as 15 and 20 weeks, respectively. The loss of muscle force was measurable in the extensor digitorum longus starting at 15 weeks, whereas the soleus muscle function was impaired at Week 32. In addition, at Week 20, markers for aortic valve sclerosis were increased.ConclusionsOur measurements confirmed the appearance of HFpEF in ZSF1‐obese rats as early as 10 weeks of age, most likely as a result of the pre‐existing co‐morbidities. In addition, SM function was reduced after the manifestation of HFpEF. In conclusion, the ZSF1 rat may serve as a suitable animal model to study pharmaceutical strategies for the treatment of HFpEF.

Oligonucleotide-based Preconditioning of DCD Cardiac Donors and Its Impact on Cardiac Viability.

While clinical donation after circulatory death (DCD) cardiac transplantation is being implemented with increasing frequency to address the supply/demand mismatch of donor grafts, no research to date has examined a strategy of donor preconditioning to optimize the viability of DCD hearts for transplantation. In our rat model of the DCD protocol, we investigate the impact of pretreating donors with phosphorothioate-linked cytosine and guanine rich oligodeoxynucleotides (CpG ODN) and their effects on cardiac function, injury, and a novel left ventricular (LV) mRNA biomarker panel. DCD rats were subjected to a withdrawal protocol, followed by 20 minutes of warm acirculatory standoff, representing a group of severely injured hearts as previously demonstrated. Beating heart controls and DCD rats were pretreated with vehicle or stimulatory CpG ODN (beating heart control and DCD stimulated with CpG ODN, BST and DST). Hearts were harvested for ex situ heart perfusion (ESHP), where LV function, histochemical injury, and differences in gene expression were characterized between groups. Donor pretreatment with CpG ODN doubled the number of functional DCD hearts at ESHP. Pretreatment was associated with improved systolic and diastolic LV function, a reduction in histological injury, and markedly reduced elaboration of cardiac troponin-I in coronary effluent during ESHP. Pretreatment was also associated with a reduction in mRNA biomarkers associated with myocardial injury. A single dose of CpG ODN was associated with reduced biomarkers of cardiac injury and a 100% increase in cardiac viability in this rodent model of marginal DCD cardiac donation.

Abstract P140: The Role of Pumpkin Flesh in Reducing Mean Arterial Pressure in a Genetic Rat Model for Hypertension

Objective: ACE inhibitors (ACEi) remain the most commonly prescribed medication; lowering blood pressure, increasing bradykinin levels and decreasing cardiac hypertrophy. A demographic-specific bradykinin sensitivity renders African-American patients on ACEi with elevated risk of angioedema. This increased risk has led a search for natural ACEi from functional foods or natural resources, one of which being pumpkin. In Sprague Dawley rats with L-NAME induced hypertension, pumpkin seed oil significantly reduced systolic blood pressure. As a promising source of β-carotene, phenolics and a beneficial fatty acid profile, this study aimed to examine the potential of pumpkin flesh and seed in regulating blood pressure management in a genetic model for hypertension. Methods: Spontaneously Hypertensive Rats (SHR) and Wistar-Kyoto rats (WKY), aged 6 weeks, were fed one of the following diets for 8 weeks: 4% pumpkin flesh (PF), 4% pumpkin seed (PS), control diet (Ctrl), and an SHR-Captopril treated group (Cap). Non-invasive blood pressure monitoring was employed for weekly measuring of mean arterial pressure (MAP). Upon 8 weeks of treatment, animals were euthanized for structural analysis of cardiac hypertrophy (Heart weight to tibial length ratio). Left ventricular mRNA expression of ANP and BNP was quantified using quantitative RT-PCR techniques. Results: At 8 weeks, MAP in SHR-PF and SHR-PS groups was significantly reduced by 23% (p=0.0135) and 27% (p=0.0018) respectively, compared to SHR-Ctrl. Heart weight ratios showed no significant difference between SHR-Ctrl and SHR-PF or SHR-PS, while SHR-Cap heart weight significantly decreased by 15% (p=0.0028) in comparison to SHR-Ctrl. Additionally, increased mRNA expression of both ANP and BNP was observed in the SHR-PF group in comparison to WKY-Ctrl. Conclusions: The reduction of blood pressure observed in response to pumpkin-based diets suggests an alternative pathway of blood pressure control independent of ACE inhibition. This potential new pathway elucidates the health promoting capabilities of pumpkin as an alternative blood pressure treatment for all demographics.

Increased Left Ventricular mRNA Levels of the Inflammatory Biomarkers Pentraxin‐3 and Interleukin 1 Receptor‐Like 1 are Correlated with Diastolic Dysfunction in a Pre‐Clinical Swine Model of HFpEF

Approximately 50% of heart failure cases are diagnosed as heart failure with preserved ejection fraction (HFpEF). HFpEF is challenging to accurately diagnose given traditional biomarkers, such as natriuretic peptide levels, have shown reduced prognostic value for HFpEF patients. Thus, the purpose of this study was to identify biomarkers considered potentially relevant to HFpEF in a new pre‐clinical Ossabaw swine model of heart failure. We hypothesized female Ossabaw swine fed a Western Diet and subject to chronic cardiac pressure‐overload would display physiologic features and biomarkers relevant to HFpEF. Animals were assigned into control (CON, n=5) and Western Diet‐fed aortic‐banded heart failure (WD‐AB, n=4) groups. WD‐AB animals were fed a Western Diet for 10 months started at 2 months of age, and subject to aortic‐banding for 6 months beginning at 6 months of age. Terminal studies were conducted at 12 months of age. Echocardiography was used to examine left ventricular (LV) mechanics (2‐dimensional speckle tracking) the week of terminal studies. RNA‐seq was performed on LV samples and Module Enrichment Analysis was performed on all expressed and varying genes, with networks generated using Ingenuity Pathway Analysis (IPA). qRT‐PCR was used to confirm RNA‐seq data, and group comparisons were made using a student's t‐test with significance reported at the P &lt; 0.10 and P &lt; 0.05 levels. IPA found enrichment of inflammatory and immune gene networks with significance to heart failure in WD‐AB animals. Gene interactions within these lists indicated activation of Pentraxin‐3 (PTX3) and interleukin 1 receptor‐like 1 (IL1RL1), which were chosen for validation given their potential relevance to HFpEF as inflammatory biomarkers. The approximate 5‐fold increase in PTX3 reported by RNA‐seq in WD‐AB compared to CON animals was confirmed separately using qRT‐PCR, as was an increase in IL1RL1 mRNA level. Both PTX3 and IL1RL1 mRNA levels (X‐axis) showed a significant negative correlation with LV apical early diastolic rotation rate (i.e. LV untwisting; Y‐axis), a 2D speckle tracking measure of diastolic function. Group means reflected a right and downward shift along the regression line for both PTX3 and IL1RL1 versus LV untwisting in the WD‐AB group compared to CON, indicating an increase in LV levels of these biomarkers is associated with a decline in LV diastolic mechanical function. In conclusion, WD‐AB animals express increased LV mRNA levels of the inflammatory biomarkers PTX3 and IL1RL1 that are significantly correlated with diastolic function. These data suggest studies in this Ossabaw pre‐clinical model may be useful for elucidating and testing biomarkers with diagnostic value potentially relevant to HFpEF.Support or Funding InformationHL112998 (CAE); VA‐Merit I01BX003271 (RSR); HL125503 (JP); HL136292 (TLD); HL122737, HL123295, &amp; HL129639 (YW).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A single systemic injection of AAV9-hIGFBP2 prevents left ventricular hypertrophy and dysfunction in metabolic syndrome

Metabolic syndrome (MetS) is a known risk factor for cardiovascular events. It is characterized by central obesity plus any two of dyslipidemia, insulin resistance, and hypertension. Obese and diabetic patients with MetS have low plasma insulin-like growth factor binding protein 2 (IGFBP2). Our first aim was to investigate IGFBP2 cardiac expression in MetS patients and in a mice model of MetS. The second aim was to assess if gene therapy with adeno-associated virus 9 carrying human IGFBP2 (AAV9-hIGFBP2) could reduce MetS associated left ventricular hypertrophy in mice. We measured plasma IGFBP2 by ELISA and cardiac mRNA IGFBP2 expression in MetS patients by RT-qPCR. Both plasma levels and heart expression of IGFBP2 were decreased in patients with MetS vs. control patients. Further, in a C57BL/6J mouse model of diet-induced MetS, found similar left ventricular mRNA IGFBP2 expression. Finally, we demonstrated for the first time that in MetS mice with decreased cardiac IGFBP2 mRNA levels, human IGFBP2 can be induced by a single AAV9-hIGFBP2 injection, and that the increased IGFBP2 prevents left ventricle wall thickening, hypertrophy and dysfunction. Human plasma and cardiac IGFBP2 are decreased in MetS patients. In mice, restoration of cardiac IGFBP2 expression level prevents MetS associated left ventricular dysfunction and hypertrophy. These clinical and animal data suggest that IGFBP2 is a new cardiac marker and therapeutic target in MetS to prevent heart remodeling consistent with heart failure.

Persistence of risk factors associated with maternal cardiovascular disease following aberrant inflammation in rat pregnancy

Introduction Pre-eclampsia is associated with increased risk of subsequent cardiovascular and metabolic disease in the affected mothers. While aberrant inflammation contributes to the pathophysiology of pre-eclampsia, it is unclear whether maternal inflammation contributes to the increased risk of disease. Here, we determined the effect of aberrant inflammation in pregnancy on cardiovascular and metabolic disease risk factors. Methods Wistar rats were administered low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5 to induce inflammation. Controls included pregnant rats treated with saline and nonpregnant rats treated with LPS or saline. We previously showed that LPS-treated pregnant rats exhibit key features of pre-eclampsia. Echocardiographic parameters, heart weight, blood pressure, blood lipids, pulse-wave velocity, and glucose tolerance, were assessed at 16 weeks postpartum. Messenger RNA levels of transcription factors associated with cardiac growth were measured in left ventricular tissue; histone modifications and global DNA methylation were determined in hearts and livers at GD 17.5 and at 16 weeks postpartum. Results Compared with saline-treated pregnant rats and nonpregnant rats treated with LPS or saline, LPS-treated pregnant rats exhibited left ventricular hypertrophy and increased blood cholesterol and low-density lipoprotein levels at 16 weeks postdelivery. LPS-treated rats had increased left ventricular mRNA levels of hypertrophy-associated transcription factors at GD 17.5 and increased levels of modified histones in hearts and livers at GD 17.5 and 16 weeks postpartum. Other parameters remained unchanged. Conclusion Aberrant inflammation during pregnancy results in persistent alterations in maternal physiological parameters and epigenetic modifications that could contribute to the pathophysiology of cardiovascular disease.

Persistence of risk factors associated with maternal cardiovascular disease following aberrant inflammation in rat pregnancy.

Pre-eclampsia is associated with increased risk of subsequent cardiovascular and metabolic disease in the affected mothers. While aberrant inflammation contributes to the pathophysiology of pre-eclampsia, it is unclear whether maternal inflammation contributes to the increased risk of disease. Here, we determined the effect of aberrant inflammation in pregnancy on cardiovascular and metabolic disease risk factors. Wistar rats were administered low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5 to induce inflammation. Controls included pregnant rats treated with saline and nonpregnant rats treated with LPS or saline. We previously showed that LPS-treated pregnant rats exhibit key features of pre-eclampsia. Echocardiographic parameters, heart weight, blood pressure, blood lipids, pulse-wave velocity, and glucose tolerance, were assessed at 16 weeks postpartum. Messenger RNA levels of transcription factors associated with cardiac growth were measured in left ventricular tissue; histone modifications and global DNA methylation were determined in hearts and livers at GD 17.5 and at 16 weeks postpartum. Compared with saline-treated pregnant rats and nonpregnant rats treated with LPS or saline, LPS-treated pregnant rats exhibited left ventricular hypertrophy and increased blood cholesterol and low-density lipoprotein levels at 16 weeks postdelivery. LPS-treated rats had increased left ventricular mRNA levels of hypertrophy-associated transcription factors at GD 17.5 and increased levels of modified histones in hearts and livers at GD 17.5 and 16 weeks postpartum. Other parameters remained unchanged. Aberrant inflammation during pregnancy results in persistent alterations in maternal physiological parameters and epigenetic modifications that could contribute to the pathophysiology of cardiovascular disease.

Mesenchymal Stromal Cells Modulate Monocytes Trafficking in Coxsackievirus B3-Induced Myocarditis.

Mesenchymal stromal cell (MSC) application in Coxsackievirus B3 (CVB3)‐induced myocarditis reduces myocardial inflammation and fibrosis, exerts prominent extra‐cardiac immunomodulation, and improves heart function. Although the abovementioned findings demonstrate the benefit of MSC application, the mechanism of the MSC immunomodulatory effects leading to a final cardioprotective outcome in viral myocarditis remains poorly understood. Monocytes are known to be a trigger of myocardial tissue inflammation. The present study aims at investigating the direct effect of MSC on the mobilization and trafficking of monocytes to the heart in CVB3‐induced myocarditis. One day post CVB3 infection, C57BL/6 mice were intravenously injected with 1 x 106 MSC and sacrificed 6 days later for molecular biology and flow cytometry analysis. MSC application reduced the severity of myocarditis, and heart and blood pro‐inflammatory Ly6Chigh and Ly6Cmiddle monocytes, while those were retained in the spleen. Anti‐inflammatory Ly6Clow monocytes increased in the blood, heart, and spleen of MSC‐treated CVB3 mice. CVB3 infection induced splenic myelopoiesis, while MSC application slightly diminished the spleen myelopoietic activity in CVB3 mice. Left ventricular (LV) mRNA expression of the chemokines monocyte chemotactic protein‐1 (MCP)−1, MCP‐3, CCL5, the adhesion molecules intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, the pro‐inflammatory cytokines interleukin‐6, interleukin‐12, tumor necrosis factor‐α, the pro‐fibrotic transforming growth factorβ1, and circulating MCP‐1 and MCP‐3 levels decreased in CVB3 MSC mice, while LV stromal cell‐derived factor‐1α RNA expression and systemic levels of fractalkine were increased in CVB3 MSC mice. MSC application in CVB3‐induced myocarditis modulates monocytes trafficking to the heart and could be a promising strategy for the resolution of cardiac inflammation and prevention of the disease progression. Stem Cells Translational Medicine 2017;6:1249–1261

Small interfering RNA therapy against carbohydrate sulfotransferase 15 inhibits cardiac remodeling in rats with dilated cardiomyopathy

Carbohydrate sulfotransferase 15 (CHST15) is a sulfotransferase responsible for biosynthesis of chondroitin sulfate E (CS-E), which plays important roles in numerous biological events such as biosynthesis of proinflammatory cytokines. However, the effects of CHST15 siRNA in rats with chronic heart failure (CHF) after experimental autoimmune myocarditis (EAM) have not yet been investigated. CHF was elicited in Lewis rats by immunization with cardiac myosin, and after immunization, the rats were divided into two groups and treated with either CHST15 siRNA (2μg/week) or vehicle. Age matched normal rats without immunizations were also included in this study. After 7weeks of treatment, we investigated the effects of CHST15 siRNA on cardiac function, proinflammatory cytokines, and cardiac remodeling in EAM rats. Myocardial functional parameters measured by hemodynamic and echocardiographic studies were significantly improved by CHST15 siRNA treatment in rats with CHF compared with that of vehicle-treated CHF rats. CHST15 siRNA significantly reduced cardiac fibrosis, and hypertrophy and its marker molecules (left ventricular (LV) mRNA expressions of transforming growth factor beta1, collagens I and III, and atrial natriuretic peptide) compared with vehicle-treated CHF rats. CHF-induced increased myocardial mRNA expressions of proinflammatory cytokines [interleukin (IL)-6, IL-1β], monocyte chemoattractant protein-1, and matrix metalloproteinases (MMP-2 and -9), and CHST15 were also suppressed by the treatment with CHST15 siRNA. Western blotting study has confirmed the results obtained from mRNA analysis as CHST15 siRNA treated rats expressed reduced levels of inflammatory and cardiac remodeling marker proteins. Our results demonstrate for the first time, that CHST15 siRNA treatment significantly improved LV function and ameliorated the progression of cardiac remodeling in rats with CHF after EAM.

Heat acclimation memory: do the kinetics of the deacclimated transcriptome predispose to rapid reacclimation and cytoprotection?

Faster reinduction of heat acclimation (AC) after its decline indicates "AC memory." Our previous results revealed involvement of epigenetic mechanisms of transcriptional regulation. We hypothesized that the decline of AC (DeAC) is a period of "dormant memory" during which many processes are alerted to enable rapid reacclimation (ReAC). Using a genomewide approach we studied the AC, DeAC, and ReAC transcriptomes, to uncover hallmark pathways linked to "molecular memory" in the cardioacclimatome. Fifty rats subjected to heat acclimation [34°C for 2d (AC2d) or 30d (AC30)], DeAC (24°C, 30 days), ReAC (34°C, 2 days), and untreated controls were used. The GeneChip Rat Gene 1.0 ST Array was employed for left ventricular (cardiac) mRNA hybridization. Three independent bioinformatic analyses showed that 1) during AC2d enrichment of DNA impair/repair-linked genes is seen, and this is the molecular on-switch of acclimation; 2) genes activated in AC30 underlie the qualitative physiological adaptations of cardiac performance; 3) particular molecular programs encompassing constitutive upregulation of p38 MAPK, Jak/Stat, and Akt pathways and targets are specifically activated during DeAC and ReAC; and 4) epigenetic markers such as linker histones (histones H1 cluster), associated with nucleosome spacing, transcriptional chromatin modifiers, poly-(ADP-ribose) polymerase-1 (PARP1) linked to chromatin compaction, and microRNAs are only altered during DeAC/ReAC. The latter are newcomers to the AC/DeAC puzzle. We suggest that these transcriptional responses maintain euchromatin and proteostasis and enable faster physiological recovery upon ReAC by rapidly reestablishing the protected acclimated cardiophenotype. We propose that the cardiac AC model can be applied to acclimation processes in general.

Abstract 298: Low-dose Interleukin-18 is Necessary to Adapt to Pressure Overload in Mice

Background: We previously reported that circulatory Interleukin-18 (IL-18) level elevates in heart failure (HF) patients. However, the roles of circulatory IL-18 in HF have not been fully elucidated. Continuous infusion of IL-18 induces cardiomyocyte hypertrophy and fibrosis, whereas IL-18 null mice were fragile to pressure overload. We hypothesized that systemic (circulatory) and local (cardiac) IL-18 have distinct effects on HF and cardiac remodeling. Purpose: The purpose of this study was to elucidate the role of circulatory IL-18 in adaptation to pressure overload in IL-18 null mice. Methods: Wild type (WT) mice and IL-18 null (IL-18-/-) were subjected to transaortic constriction (TAC). Sequential serum IL-18 levels myocardial IL-18 mRNA expressions were determined by ELISA and qRT-PCR in WT mice. After two week of TAC, IL-18 null mice were administered either (IP) saline or recombinant IL-18 intraperitoneally (10ng/20g every other day). Cardiac function was assessed by transthoracic echocardiography. Two weeks after TAC, myocardial samples were obtained. Haematoxylin and eosin stained sections and Masson’s trichrome staining sections were prepared. Results: IL-18 concentration in serum and IL-18 expression in myocardial tissue increased gradually after TAC in WT mice. Forty-seven % (7/15) of TAC-operated IL-18-/- mice and 12% (2/17) of TAC-operated WT mice died of heart failure by 14days. TAC-operated IL-18-/- mice exhibited more severe left ventricular (LV) remodeling, characterized by cardiomyocyte hypertrophy, extensive interstitial fibrosis and elevation of fetal gene expressions compared with TAC-WT mice. Recombinant IL-18 given intraperitoneally improved the survival rate to 100% (10/10) following TAC operation in IL-18-/- mice. Furthermore, exogenous IL-18 administration suppressed ventricular ANP mRNA expression and myocardial cross-sectional area enlargement to non-TAC-operated level, whereas LV enlargement and contractile dysfunction were only partially suppressed in IL-18-/- mice following TAC. Conclusions: Circulatory IL-18 exerts opposing effects on cardiac hypertrophy under pressure-overload. IL-18 produced in the heart may have an effect such as preserving contractile function.

Abstract 297: Cardiac Transforming-growth-factor β-stimulated Clone 22 Gene Expression By Hypertrophic Stimuli

Objectives: Transforming-growth-factor β-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains largely unknown. The aim of the present study was to characterize the cardiac TSC-22 expression. Methods: Acute pressure overload was accomplished in conscious Sprague-Dawley (SD) rats by intravenous infusion of arginine 8 -vasopressin (AVP, 0.05 μg/kg/min) for 4 hours and subcutaneous infusion of angiotensin II (Ang II, 33 μg/kg/h) with and without Ang II receptor type 1 blocker losartan (400 μg/kg/h) by using osmotic minipumps for 2 weeks. Adenovirus-mediated intramyocardial gene transfer of TSC-22 was performed into left ventricle (LV) of SD rats. Experimental myocardial infarction (MI) was produced by ligation of the left anterior descending coronary artery. Cultured neonatal rat ventricular myocytes (NRVM) were treated with endothelin-1 (ET-1, 100 nM). Results: A significant 1.6-fold increase ( P &lt;0.05) in LV TSC-22 mRNA levels was noted already after 1 hour AVP infusion. Moreover, Ang II infusion markedly upregulated TSC-22 expression, LV mRNA levels being highest at 6 hours (11-fold, P &lt;0.001). Simultaneous infusion of losartan completely abolished Ang II-induced increase in TSC-22 mRNA levels. Adenovirus-mediated gene transfer of TSC-22 into LV resulted a 1.9-fold ( P &lt;0.001) increase in TSC-22 mRNA levels, accompanied by upregulated BNP mRNA levels (1.4-fold, P &lt;0.01). In response to experimental MI, TSC-22 mRNA levels were elevated 4.1-fold ( P &lt;0.001) at 1 day and 1.9-fold ( P &lt;0.05) at 4 weeks. In cultured NRVM, ET-1 treatment increased TSC-22 mRNA levels from 1 h to 24 h, the greatest increase being observed at 12 h (2.7-fold, P &lt;0.001). TSC-22 protein levels were upregulated from 4 h to 24 h with the highest increase at 24 h (4.7-fold, P &lt;0.01). Conclusion: These results indicate that TSC-22 expression is rapidly activated in response to pressure overload, MI and in ET-1 treated cultured NRVM. Moreover, adenovirus-mediated overexpression of TSC-22 mRNA was associated with elevated left ventricular BNP mRNA levels.

Is exercise really deleterious for the hypertensive heart?

Is exercise really deleterious for the hypertensive heart?

Microtubule proliferation in right ventricular myocytes of rats with monocrotaline-induced pulmonary hypertension

Microtubules are components of the cardiac cytoskeleton that can proliferate in response to pressure-overload in animal and human heart failure. We wished to test whether there was a proliferation of the microtubule cytoskeleton in the right ventricle of rats with pulmonary hypertension induced by monocrotaline (MCT) and whether this contributed to contractile dysfunction. Male Wistar rats were injected with 60mg/kg of MCT in saline or an equivalent volume of saline (CON). MCT produced clinical signs of heart failure within 4weeks of injection. Expression of right ventricular mRNA for α-tubulin was measured by real-time reverse transcription polymerase chain reaction. Free and polymerised fractions of β-tubulin protein were assessed using Western blot analysis and immunofluorescence microscopy was used to assess tyrosinated and acetylated (stabilized) microtubules. Right ventricular myocyte contraction was measured in response to the microtubule de-polymeriser colchicine (10μmol/l for at least 1h). Compared to CON, in MCT right ventricles there was a small but statistically significant increase in the expression of mRNA for α-tubulin (P<0.001); total (P<0.05) and polymerised fraction (P<0.01) of β-tubulin protein and level of acetylated tubulin (P<0.01). However colchicine treatment did not increase the contraction of MCT myocytes (P>0.05) or affect their response to increased stimulation frequency. Our observations support the hypothesis that microtubule proliferation is a common response to pulmonary hypertension in failing right ventricles but suggest that the effect this has on contraction depends upon the specific experimental or clinical conditions that prevail and the subsequent level of microtubule proliferation.

Adverse cardiac remodelling in spontaneously hypertensive rats: acceleration by high aerobic exercise intensity

In the present study it was hypothesized that voluntary aerobic exercise favours a pro-fibrotic phenotype and promotes adverse remodelling in hearts from spontaneously hypertensive rats (SHRs) in an angiotensin II-dependent manner. To test this, female SHRs at the age of 1 year were started to perform free running wheel exercise. Captopril was used to inhibit the renin-angiotensin system (RAS). Normotensive rats and SHRs kept in regular cages were used as sedentary controls. Training intensity, expressed as mean running velocity, was positively correlated with the left ventricular mRNA expression of TGF-β(1), collagen-III and biglycan but negatively correlated with the ratio of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)2a to Na(+)-Ca(2+) exchanger (NCX). A pro-fibrotic phenotype was verified by Picrosirius red staining. Sixty-seven per cent of SHRs performing free running wheel exercise died either spontaneously or had to be killed during a 6 month follow-up. In the presence of captopril, aerobic exercise did not show a similar positive correlation between training intensity and the expression of fibrotic markers. Moreover, in SHRs receiving captopril and performing free running wheel exercise, a training intensity-dependent reverse remodelling of the SERCA2a-to-NCX ratio was observed. None of these rats died spontaneously or had to be killed. In captopril-treated SHRs performing exercise, expression of mRNA for decorin, a natural inhibitor of TGF-β(1), was up-regulated. Despite these differences between SHR-training groups with and without captopril, positive training effects (lower resting heart rate and no progression of hypertension) were found in both groups. In conclusion, high aerobic exercise induces an angiotensin II-dependent adverse remodelling in chronic pressure overloaded hearts. However, high physical activity can potentially induce reverse remodelling in the presence of RAS inhibition.

Cardiac inflammation after local irradiation is influenced by the kallikrein-kinin system.

Radiotherapy of intrathoracic and chest wall tumors may lead to exposure of the heart to ionizing radiation, resulting in radiation-induced heart diseases (RIHD). The main manifestations of RIHD become apparent many years after treatment and include cardiomyopathy and accelerated atherosclerosis. This study examines the role of the kallikrein-kinin system (KKS) in RIHD by investigating the cardiac radiation response in a kininogen-deficient Brown Norway Katholiek (BN/Ka) rat model. BN/Ka rats and wild-type Brown Norway (BN) rats were exposed to local heart irradiation with a single dose of 18 Gy or 24 Gy and were observed for 3 to 6 months. Examinations included in vivo and ex vivo cardiac function, histopathology, gene and protein expression measurements, and mitochondrial swelling assays. Upon local heart irradiation, changes in in vivo cardiac function were significantly less in BN/Ka rats. For instance, a single dose of 24 Gy caused a 35% increase in fractional shortening in BN rats compared with a 16% increase in BN/Ka rats. BN rats, but not BN/Ka rats, showed a 56% reduction in cardiac numbers of CD2-positive cells, and a 57% increase in CD68-positive cells, together with a 52% increase in phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2). Local heart irradiation had similar effects on histopathology, mitochondrial changes, and left ventricular mRNA levels of NADPH oxidases in the two genotypes. These results suggest that the KKS plays a role in the effects of radiation on cardiac function and recruitment of inflammatory cells. The KKS may have these effects at least in part by altering Erk1/2 signaling.

A novel p38 MAPK target dyxin is rapidly induced by mechanical load in the heart

Dyxin is a novel LIM domain protein acting as a transcriptional cofactor with GATA transcription factors. Here, we characterized dyxin as a p38 mitogen-activated protein kinase (MAPK) regulated gene, since combined upstream MAPK kinase 3b and wild-type p38α MAPK gene transfer increased left ventricular dyxin mRNA and protein levels in vivo. We also studied cardiac dyxin expression in experimental models of pressure overload and myocardial infarction (MI) in vivo. Angiotensin II infusion increased left ventricular dyxin mRNA levels (9.4-fold, p<0.001) rapidly at 6 h followed by induction of protein levels. Furthermore, simultaneous administration of p38 MAPK inhibitor SB203580 abolished angiotensin II-induced activation of dyxin gene expression. During the post-infarction remodeling process, increased dyxin mRNA levels (7.7-fold, p<0.01) were noted at day 1 followed by the increase in proteins levels at 2 weeks after MI (1.5-fold, p<0.05). Moreover, direct wall stretch by using isolated rat heart preparation as well as direct mechanical stretch of cardiomyocytes in vitro activated dyxin gene expression within 1 h. Our results indicate that dyxin expression is rapidly upregulated in response to mechanical load, this increase being at least partly mediated by p38 MAPK. These results suggest that dyxin may play an important role in regulating hypertrophic process.