Chronic Administration Of Isoproterenol Research Articles

Abstract We106: Long Non-Coding RNA LIPTER Regulates Lipid Metabolism of Human Cardiomyocytes and Preserves Cardiac Function

Background: Recently, long non-coding RNAs (lncRNAs) have emerged as novel human-specific (i.e. non-conserved) regulators of cardiovascular development and disease, which possess diverse functions through interacting with other molecules, including DNA, RNA, protein, and lipid. We identified a human lncRNA, LIPTER, which mediates lipid droplet (LD) transport to regulate lipid metabilism of human cardiomyocytes (CMs). Mechanistically, LIPTER binds phospholipids PA and PI4P on the LD membranes and the MYH10 protein, connecting LDs to the cytoskeleton and facilitating LD transport to mitochondria. LIPTER deficiencies led to prominent LD accumulation, mitochondrial dysfunction, and death of human iPS cell-derived CMs. Since downregulation of LIPTER and enahnced LD accumulation are associated with cardiac dysfunction and heart failure in patients with metabolic disorders, such as obesity and diabetes mellitus, we thereby seek to explore the preclinical potential of LIPTER for the therapy of human metabolic disorder associated cardiac dysfunction and heart failure. Hypothesis: We hypothesize that transgenic overexpression of the human lncRNA LIPTER in mouse heart could attenuate metabolic syndrome-associated myocardium lipid accumulation and cardiac dysfunction, as well as prevent isoproterenol-induced heart failure through increasing the fatty acid oxidation (FAO) of mouse heart. Goals: To evaluate the preclinical potential of human lncRNA LIPTER for the therapy of cardiac dysfunction and heart failure by using mouse cardiac dysfunction and heart failure models. Methods: We established a LIPTER transgenic overexpression mouse line (LIPTER Tg ). Both WT and LIPTER Tg mice were fed with high fat diet for 10 months to induce cardiac lipid accumulation and dysfunction. Additionally, heart failure was induced by the chronic isoproterenol administration. After treatment, mice were subjected to histological and molecular analyses. Results: We found LIPTER Tg significantly enhanced FAO of mouse heart, reduced cardiac lipid accumulation and fibrosis, and alleviated cardiac dysfunction of high-fat-diet fed mice, as well as preserved cardiac function of mice post isoproterenol administration. Conclusions: We unveil a crucial role of human lncRNA LIPTER in regulating lipid metabolism of human CMs and testify its clinical potential for treating cardiac dysfunction and heart failure.

Macrophage OTUD1-CARD9 axis drives isoproterenol-induced inflammatory heart remodelling.

Chronic inflammation contributes to the progression of isoproterenol (ISO)-induced heart failure (HF). Caspase-associated recruitment domain (CARD) families are crucial proteins for initiation of inflammation in innate immunity. Nonetheless, the relevance of CARDs in ISO-driven cardiac remodelling is little explored. This study utilized Card9-/- mice and reconstituted C57BL/6 mice with either Card9-/- or Otud1-/- marrow-derived cells. Mechanistic studies were conducted in primary macrophages, cardiomyocytes, fibroblasts and HEK-293T cells. Here, we demonstrated that CARD9 was substantially upregulated in murine hearts infused with ISO. Either whole-body CARD9 knockout or myeloid-specific CARD9 deletion inhibited ISO-driven murine cardiac inflammation, remodelling and dysfunction. CARD9 deficiency in macrophages prevented ISO-induced inflammation and alleviated remodelling changes in cardiomyocytes and fibroblasts. Mechanistically, we found that ISO enhances the activity of CARD9 by upregulating ovarian tumour deubiquitinase 1 (OTUD1) in macrophages. We further demonstrated that OTUD1 directly binds to the CARD9 and then removes the K33-linked ubiquitin from CARD9 to promote the assembly of the CARD9-BCL10-MALT1 (CBM) complex, without affecting CARD9 stability. The ISO-activated CBM complex results in NF-κB activation and macrophage-based inflammatory gene overproduction, which then enhances cardiomyocyte hypertrophy and fibroblast fibrosis, respectively. Myeloid-specific OTUD1 deletion also attenuated ISO-induced murine cardiac inflammation and remodelling. These results suggested that the OTUD1-CARD9 axis is a new pro-inflammatory signal in ISO-challenged macrophages and targeting this axis has a protective effect against ISO-induced HF. Macrophage CARD9 was elevated in heart tissues of mice under chronic ISO administration. Either whole-body CARD9 knockout or myeloid-specific CARD9 deficiency protected mice from ISO-induced inflammatory heart remodeling. ISO promoted the assembly of CBM complex and then activated NF-κB signaling in macrophages through OTUD1-mediated deubiquitinating modification. OTUD1 deletion in myeloid cells protected hearts from ISO-induced injuries in mice.

L-Carvone from Mentha spicata L. Leaves Suppresses Oxidative Stress and Hypertrophy in the Isoproterenol-Induced Rat Model of Cardiac Hypertrophy

The effect of L-Carvone (a natural monoterpene from Mentha spicata L., leaves) in cardiac hypertrophy caused by isoproterenol administration was investigated. Male rats (Wistar) were divided into five groups: Control, diseased, diseased rats with losartan, diseased rats with low-dose L-Carvone, and high-dose L-Carvone. Rats were injected with isoproterenol (5 mg/kg) for 30 days to induce cardiac hypertrophy. Then, simultaneously with Losartan (15 mg/kg), L-Carvone was administered orally at a dosage of 25 mg/kg (low dose) and 100 mg/kg (high dose) treatment. The cardioprotective effect of L-Carvone was evaluated by examining the heart morphometric indices, and ECG analyses. Chronic isoproterenol administration resulted in changes in morphometric indices of the heart, ECG tracings, biochemical parameters such as tissue glucose, proteins, lipid profiles, serum cardiac markers, antioxidants, and histopathological integrity of the heart tissue. When compared with the isoproterenol group, L-Carvone administrated for 30 days ameliorated all these changes in rats significantly (p < 0.05). L-Carvone adequately averted chronic cardiac hypertrophy, most probably through its antioxidant potential.

Ivabradine curbs isoproterenol-induced kidney fibrosis

This study investigated whether chronic isoproterenol administration could induce kidney alterations and whether ivabradine, a heart rate (HR)-reducing substance exerting cardiovascular protection, is able to attenuate potential kidney damage. Twenty-eight Wistar rats were divided into non-diseased controls, rats treated with ivabradine, rats treated with isoproterenol, and rats treated with isoproterenol plus ivabradine. Six weeks of isoproterenol administration was associated with decreased systolic blood pressure (SBP) (by 25%) and glomerular, tubulointerstitial and vascular/perivascular fibrosis due to enhanced type I collagen volume (7-, 8-, and 4-fold, respectively). Ivabradine reduced HR (by 15%), partly prevented SBP decline (by 10%) and site-specifically mitigated kidney fibrosis by decreasing type I collagen volume in all three sites investigated (by 69, 58, and 67%, respectively) and the ratio of type I collagen-to-type III collagen in glomerular and vascular/perivascular sites (by 79 and 73%, respectively). We conclude that ivabradine exerts protection against kidney remodelling in isoproterenol-induced kidney damage.

Abstract P2028: A Role For Pannexin 1 In Heart Failure Induced By Acute And Chronic Isoproterenol Administration

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of cardiac dysfunction and fibrosis, we use an acute (14 day) and a chronic (28 day) model of adrenergic stimulation to induce heart failure in two novel mouse models: 1) We have generated mice with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP-flanked alleles of Panx1 to mice expressing Cre recombinase under the control of the cardiomyocyte specific MyHC6 promoter (Panx1 MyHC6-Cre ). 2) Using mice expressing the tamoxifen-inducible MerCreMer system under the control of the Tnnt2 promoter we generated mice with tamoxifen-inducible deletion of Panx1 in cardiomyocytes (Panx1 Tnnt2-MerCreMer ). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline as measured by echocardiography. However, after acute isoproterenol treatment (15 mg/kg/day, i.p.), Panx1 MyHC6-Cre mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy. Furthermore, after chronic administration of isoproterenol (15 mg/kg/day, osmotic pump) Panx1 MyHC6-Cre mice were still protected from cardiac hypertrophy compared to Panx1 fl/fl littermates. In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to Panx1-dependent release of ATP in a dose-dependent manner. This was demonstrated with both pharmacological blockade by spironolactone and siRNA-mediated knock-down of Panx1. Taken together, these data demonstrates that Panx1 deficiency in cardiomyocytes protects against isoproterenol-induced cardiac dysfunction and that ATP is released through Panx1 in response to isoproterenol. Further elucidating the role of Panx1 in cardiomyocytes in regulating cardiac function and fibrosis will identify Panx1 as a novel therapeutic target to prevent heart failure.

Minocycline Blunts Isoproterenol‐Induced Cardiac Hypertrophy

IntroductionExcess and chronic activation of the sympathetic nervous system often results in progressive adverse remodeling and heart failure development. Here we investigated whether chronic administration of isoproterenol (Iso), a β‐AR agonist, would promote hypertrophy, fibrosis, and dysfunction, and whether these maladaptive changes are prevented by minocycline, an FDA‐approved antibiotic, that has been shown to have cardioprotective effects outside of its antibacterial properties.MethodsWild‐type male C57BL/6J mice were subcutaneously implanted with osmotic minipumps, delivering either 30 mg/kg body weight per day of isoproterenol, or vehicle control. Following pump implantation, mice received daily intraperitoneal (I.P.) injections of either sterile saline or 50 mg/kg of minocycline. After 21 days, cardiac function was assessed by echocardiography, and hearts were collected for further analysis. A transverse slice of the ventricles was fixed for histology, and a portion of the left ventricle was used for RNA isolation, and mRNA and miRNA sequencing.ResultsEchocardiography revealed that isoproterenol increased heart rate, heart weight (hypertrophy), ejection fraction and fractional shortening, without leading to significant changes in fibrosis. Minocycline prevented the hypertrophic remodeling and changes in systolic function without affecting heart rate. While mRNA sequencing revealed that isoproterenol enriched gene networks associated with changes in inflammation, the miRNA sequencing showed that minocycline enhanced miR‐26b expression, which is reported to suppress GATA4‐mediated hypertrophy.ConclusionMinocycline mitigates isoproterenol‐induced cardiac hypertrophy while preserving contractility, as well as induces changes in RNA and miRNA pathways involved with inflammation and cardiac remodeling

A Critical Role for Pannexin 1 in Heart Failure Induced by Acute and Chronic Isoproterenol Administration

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of cardiac dysfunction and fibrosis, we use a model of chronic adrenergic stimulation to induce heart failure in two novel mouse models. We have generated mice with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP‐flanked alleles of Panx1 to mice expressing Cre recombinase under the control of the cardiomyocyte specific MyHC6 promoter (Panx1MyHC6‐Cre). Using the same approach with mice expressing the tamoxifen‐inducible MerCreMer system under the control of the Tnnt2 promoter we generated mice with tamoxifen‐inducible deletion of Panx1 in cardiomyocytes (Panx1Tnnt2‐MerCreMer). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline as measured by echocardiography. However, after acute isoproterenol treatment (15 mg/kg/day, i.p.), Panx1MyHC6‐Cre mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy. In vitro, treatment of H9c2 cardiomyocytes with isoproterenol led to Panx1‐dependent release of ATP. Furthermore, siRNA‐mediated knock down of Panx1 in H9c2 cells attenuated basal and maximal extracellular acidification rate (a measure of glycolysis) after 24 hours of isoproterenol treatment, indicating a role for Panx1 in regulation of cardiomyocyte metabolism. Taken together, these data demonstrates that Panx1 deficiency protects against isoproterenol‐induced cardiac dysfunction and pathological glycolytic shift in stressed cardiomyocytes. Further elucidating the role of Panx1 in cardiomyocytes in regulating cardiac function and fibrosis will identify a novel therapeutic target to prevent heart failure.

Metformin Prevents Low-dose Isoproterenol-induced Cardiac Dilatation and Systolic Dysfunction in Male Sprague Dawley Rats.

Myocardial metabolic abnormalities are well-recognized alterations in chronic heart failure, effects that may contribute to progressive cardiac dysfunction. However, whether metabolic alterations in-part mediate their deleterious effects by modifying the chronic impact of excess low-dose sympathetic stimulation on cardiac chamber dilatation is uncertain. We therefore aimed to determine the effect of metformin administration on cardiac function and mitochondrial architectural changes in a rat model of chronic sympathetic-induced left ventricular (LV) remodeling and systolic dysfunction [daily subcutaneous isoproterenol (ISO) injection at a low dose of 0.02 mg/kg for 7 months]. Echocardiography was used to assess in vivo LV dimensions and function, and mitochondrial and myofibril arrangement was assessed using transmission electron microscopy. Seven months of low-dose ISO administration increased LV diastolic diameter (in mm) [control (CONT): 7.29 ± 0.19 vs. ISO: 8.76 ± 0.21; P = 0.001], an effect that was attenuated by metformin (ISO + MET: 7.63 ± 0.29 vs. ISO: P = 0.001) administration. Similarly, ISO increased LV end-systolic diameter (CONT: 4.43 ± 0.16 vs. ISO: 5.49 ± 0.16: P < 0.0001), an effect prevented by metformin (ISO + MET: 4.04 ± 0.25 vs. ISO: P < 0.0001). Moreover, chronic ISO administration reduced LV endocardial fractional shortening (P = 0.0001), midwall fractional shortening (P = 0.0001), and ejection fraction (P = 0.0001), effects similarly prevented by metformin administration. Furthermore, changes in mitochondrial arrangement and relative mitochondrial area (CONT: 37.7 ± 2.2 vs. ISO: 28.1 ± 2.9; P = 0.05) were produced by ISO administration, effects prevented by metformin. In conclusion, metformin offers cardiac protection against chronic sympathetic-induced LV dilatation and systolic dysfunction. These data support a role for myocardial metabolic changes in mediating LV dilatation and LV dysfunction produced by chronic neurohumoral activation in cardiac disease.

Immune cell β2-adrenergic receptors contribute to the development of heart failure.

β-Adrenergic receptors (βARs) regulate normal and pathophysiological heart function through their impact on contractility. βARs are also regulators of immune function where they play a unique role depending on the disease condition and immune cell type. Emerging evidence suggests an important role for the β2AR subtype in regulating remodeling in the pathological heart; however, the importance of these responses has never been examined. In heart failure, catecholamines are elevated, leading to chronic βAR activation and contributing to the detrimental effects in the heart. We hypothesized that immune cell β2AR plays a critical role in the development of heart failure in response to chronic catecholamine elevations through their regulation of immune cell infiltration. To test this, chimeric mice were generated by performing bone marrow transplant (BMT) experiments using wild-type (WT) or β2AR knockout (KO) donors. WT and β2ARKO BMT mice were chronically administered the βAR agonist isoproterenol. Immune cell recruitment to the heart was examined by histology and flow cytometry. Numerous changes in immune cell recruitment were observed with isoproterenol administration in WT BMT mice including proinflammatory myeloid populations and lymphocytes with macrophages made up the majority of immune cells in the heart and which were absent in β2ARKO BMT animal. β2ARKO BMT mice had decreased cardiomyocyte death, hypertrophy, and interstitial fibrosis following isoproterenol treatment, culminating in improved function. These findings demonstrate an important role for immune cell β2AR expression in the heart's response to chronically elevated catecholamines.NEW & NOTEWORTHY Immune cell β2-adrenergic receptors (β2ARs) are important for proinflammatory macrophage infiltration to the heart in a chronic isoproterenol administration model of heart failure. Mice lacking immune cell β2AR have decreased immune cell infiltration to their heart, primarily proinflammatory macrophage populations. This decrease culminated to decreased cardiac injury with lessened cardiomyocyte death, decreased interstitial fibrosis and hypertrophy, and improved function demonstrating that β2AR regulation of immune responses plays an important role in the heart's response to persistent βAR stimulation.

A Dual Role for Death Receptor 5 in Regulating Cardiac Fibroblast Function.

The fibrotic response is involved in nearly all forms of heart failure and dysregulated responses can lead to enhanced cardiac dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor (DR) 5, are associated with multiple forms of heart failure, but their role in the heart is poorly defined. Our previous study identified DR5 expression on cardiac fibroblasts however, the impact of DR5 on fibroblast function remains unexplored. To investigate the role of DR5 in cardiac fibroblasts, a variety of fibroblast functions were examined following treatment with the endogenous ligand, TRAIL, or small molecule agonist, bioymifi. DR5 activation did not induce apoptosis in naïve fibroblasts but activated ERK1/2 signaling to increase proliferation. However, upon activation and differentiation to myofibroblasts, DR5 expression was elevated, and DR5 agonists induced caspase 3 activation resulting in myofibroblast apoptosis. To investigate the impact of DR5 regulation of fibroblasts in vivo, a chronic isoproterenol administration model of heart failure was used. Wild-type (WT) mice receiving isoproterenol had increased hypertrophy, cardiomyocyte death, and fibrosis and decreased contractility compared to vehicle treated animals. DR5 knockout (KO) mice had no overt baseline phenotype however, following isoproterenol infusion, increased cardiomyocyte death and hypertrophy in comparison to isoproterenol treated WT animals was observed. DR5KO mice had an augmented fibrotic response with isoproterenol treatment compared with WT, which corresponded with additional decreases in contractility. These findings identify a dual role for DR5 in cardiac fibroblast function through enhanced naïve fibroblast proliferation, which switches to a pro-apoptotic function upon differentiation to myofibroblasts. This is important in heart failure where DR5 activation suppresses maladaptive remodeling and may represent a novel therapeutic target for the treatment of heart failure.

Abstract 411: Absence of Sexual Dimorphism in Isoproterenol-induced Cardiac Dysfunction in C57BL/6 Mice

Sex-related differences in cardiovascular diseases are complex and highly context-dependent. The objective of this work was to comprehensively determine key sex differences in the response to acute and chronic adrenergic stimulation in C57Bl/6 mice. In the current work, there was no statistically significant difference in key echocardiographic parameters between male and female C57Bl/6 mice in response to acute adrenergic stimulation (a single sub-cutaneous dose of isoproterenol 10 mg/kg). After chronic adrenergic administration (sub-cutaneous injections of isoproterenol 10 mg/kg/day for 14 days), there was similar degree of cardiac dysfunction, cardiac hypertrophy, and myocardial fibrosis in male and female mice. Similarly, chronic isoproterenol administration induced hypertrophic and fibrotic genes in hearts of male and female mice to the same extent. Intriguingly, gonadectomy of male and female mice did not have a significant impact on isoproterenol-induced cardiac dysfunction as compared to sham-operated animals. In conclusion, the current work demonstrated lack of sex-related differences in isoproterenol-induced cardiac hypertrophy, dysfunction, and fibrosis in C57Bl/6 mice. This study challenges the conventional dogma of the detrimental cardiovascular effects of male sex hormones and the beneficial effects of female sex hormones.

CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation.

Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca2+/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart.

Abstract 12289: The Nuclear Receptor NR4A1 Regulates Transcriptional Programs Involved in Cardiac Fuel Metabolism and Hypertrophic Growth

The transcriptional coregulators, peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) alpha and beta, are inducible master regulators of mitochondrial biogenesis and metabolism. Studies in rodents and in humans have shown that expression and activity of PGC-1 coactivators is downregulated in the failing heart. Surprisingly, we found that adult mice with cardiac-specific deletion of the PGC-1alpha/beta genes exhibit relatively normal cardiac function despite a significant reduction in mitochondrial state 3 respiration, suggesting the existence of a compensatory energy metabolic pathway. Unbiased transcriptional profiling demonstrated upregulation of the orphan nuclear receptor NR4A1 (Nur77) and the related nuclear receptor NR4A3 (Nor1) in PGC-1 null hearts, consistent with an adaptive response to energetic stress. We found that expression of NR4A1 family members is induced in heart by a variety of physiological stress stimuli. In addition, activation of beta-adrenergic signaling with isoproterenol led to rapid and dramatic induction in the expression of all NR4A members in the mouse heart. To identify cardiac NR4A1 targets, total RNA-seq was performed in neonatal rat ventricular myocytes (NRVMs) overexpressing NR4A1. NR4A1 expression of genes involved in glucose uptake/utilization pathways and high energy phosphate transfer including Slc2a4 (Glut4), Eno3 (enolase 3), and Ckmt2 (mitochondrial creatinine kinase). Consistent with the transcriptomic changes, NR4A1 increased myocyte 2-deoxylglucose uptake and oxygen consumption rates (OCR) in these cells. Interestingly, the RNA-seq data also revealed that NR4A1 overexpression downregulated many cardiac pathologic hypertrophic markers such as ANF and BNP. NR4A1 overexpression also significantly inhibited phenylephrine-induced NRVM hypertrophy. Finally, NR4A1 knockout mice exhibit exuberant cardiac hypertrophy following chronic isoproterenol administration. Taken together, these data suggest NR4A1 serves as a stress-induced transcription factor that regulates fuel metabolism and cardiac remodeling. The long-term goal of this project is to determine if activation of this pathway holds therapeutic benefit in acute heart failure.

Abstract 18092: Nitroxyl (HNO) Improves Ventricular Relaxation and Ca2+-Handling in Rats with Induced Chronic Diastolic Dysfunction

Despite the morbidity and clinical burden of primary diastolic cardiac dysfunction, effective therapies for patients presenting with heart failure with preserved ejection fraction (HFpEF) are limited. Nitroxyl (HNO) produces cAMP-independent functional support, in part by improving myocardial calcium-handling. We hypothesize that HNO can rescue lusitropic function when acutely administered to rats with chronic diastolic dysfunction, mimicking HFpEF. Diastolic dysfunction, as demonstrated by decreased E/A ratios via echocardiography, was induced by either 4 weeks of chronic isoproterenol administration (via a mini-osmotic pump; ISO) or by renoprival hypertension (secondary to renal wrapping; RW) for 5 weeks. In vivo left-ventricular hemodynamics/pressure-volume relationships were assessed before/during a 30 minute IV administration of a HNO pro-drug (CXL-1020, 100 μg/kg/min). Ca2+ handling in isolated cardiomyocytes was also studied (CXL-1020, at 50 μM). In both cases, milrinone (mil) was used as a cAMP-dependent positive control (at a matched-efficacy in healthy rats, i.e., 10 μg/kg/min and 0.5 μM) In the setting of chronic diastolic dysfunction, HNO lowered the LV filling pressures in both models by a combined average of 46 ± 8% versus baseline, accelerated the time-constant (tau) of relaxation (-26 ± 3%) and improved compliance (flattened the end-diastolic PV relationship) (-61 ±3%). In myocytes isolated from dysfunctional hearts, HNO accelerated the time for Ca2+ to decline to 50% of its max amplitude (RT50) by -10 ± 2% and myocyte relengthening by -12 ± 3% in ISO rats. Despite eliciting comparable responses in healthy animals/myocytes, the HNO-mediated effects on both tau (in vivo) (-33 ± 3% vs mil -25 ± 2%) and Ca2+ decline (myocyte) (-10 ± 2% vs mil -6 ± 1%) were greater than those of milrinone in the ISO rats, which had greater impairment at baseline compared to RW rats. In conclusion, acute-therapy with HNO improved ventricular relaxation, compliance and Ca2+ handling, in the setting of chronic diastolic dysfunction. Moreover, these effects were greater than observed with the cAMP-dependent agent milrinone.

Abstract 232: A Genome-wide Association of Murine Echocardiographic Measures Implicates Muscle A-type Lamin-interacting Protein (MLIP) in Isoproterenol-induced Interventricular Septal Hypertrophy

Genome-wide association studies of heart failure in humans have yielded limited results, largely due to highly heterogeneous environmental and genetic backgrounds. The Hybrid Mouse Diversity Panel consists of inbred strains that are either fully sequenced or densely genotyped and as a whole displays significant natural variations. We assessed the hypothesis that precise environmental control, a well-defined genetic background, and a careful and uniform phenotyping scheme enable high-resolution mapping of cardiac remodeling in an isoproterenol-induced heart failure mouse model. Eight to ten-week old females from 105 inbred mouse strains (average N per strain = 6.7) were divided into control (average N per strain = 2.5) and treated (average N per strain =4.1) cohorts. Treated mice received 20 μg/g/day of isoproterenol through an abdominally implanted Alzet micropump for 3 weeks. All mice underwent echocardiography at baseline and at weekly time points. Phenotypic data were analyzed using the Efficient Mixed-Model Association (EMMA) algorithm to correct for population substructure. Our study showed that chronic isoproterenol administration resulted in marked inter-strain variations in echocardiographic measurements across the mouse panel. EMMA analysis of the data revealed many significant association peaks across echocardiographic measures. The most significant association signal is in the change of diastolic interventricular septal wall thickness between baseline and 1 week after isoproterenol treatment, a marker of early isoproterenol-induced left ventricular hypertrophy. The peak SNP rs13480288 (p-value of 4.0580e-09) and its surrounding SNPs (within correlation r2 &gt; 0.8) span across 3 genes. Of these, Mlip alone harbors a missence variant and a splice-site variant across our panel. Mlip, also known as muscle A-type lamin-interacting protein, is expressed abundantly in the heart and interacts directly with A-type lamins in the nuclear envelope. Mutations in A-type lamins have been shown to cause both hypertrophic and dilated cardiomyopathy. In conclusion, our study results provide strong evidence that genetic variations in Mlip contribute to differential responses in interverntricular septal hypertrophy to isoproterenol.

α‐Dystrobrevin functions to reinforce the interaction between dystrophin and the membrane, loss of α‐dystrobrevin results in significant loss of cardiac reserve

The C‐terminal region of dystrophin is highly conserved across vertebrates, but its function is largely unknown. α‐Dystrobrevin (DB) is one of several proteins that bind to this region of dystrophin. Mice lacking α‐DB demonstrate a mild myopathy in skeletal muscle, but the phenotype of the heart has not been fully characterized. Here we demonstrate that the loss of α‐DB significantly weakens the association of cardiac dystrophin with membrane glycoproteins. In the absence of α‐DB, wheat germ agglutinin pull down assay reveals a dystrophin:α‐dystroglycan ratio only 13% of that observed in wildtype mice. The physiological importance of the disruption of this interaction is evident in the marked pathology induced by chronic isoproterenol administration. This challenge protocol results in significant myocardial damage and mortality &gt;60% in mice lacking α‐DB, in contrast wild type mice survived with minimal injury. The lesions and mortality observed in the α‐DB null mice were not different from mice lacking dystrophin. These findings have significant implications on the design of truncated dystrophin constructs suitable for delivery via gene therapy, as many of these constructs lack the binding domain for α‐DB.

Age exacerbates chronic catecholamine-induced impairments in contractile reserve in the rat

Contractile reserve decreases with advancing age and chronic isoproterenol (ISO) administration is a well-characterized model of cardiac hypertrophy known to impair cardiovascular function. This study evaluated whether nonsenescent, mature adult rats are more susceptible to detrimental effects of chronic ISO administration than younger adult rats. Rats received daily injections of ISO (0.1 mg/kg sc) or vehicle for 3 wk. ISO induced a greater impairment in contractile reserve [maximum of left ventricular pressure development (Δ+dP/dt(max))] in mature adult ISO-treated (MA-ISO) than in young adult ISO-treated rats (YA-ISO) in response to infusions of mechanistically distinct inotropes (digoxin, milrinone; 20-200 μl·kg(-1)·min(-1)), while basal and agonist-induced changes in heart rate and systolic arterial pressure (SAP) were not different across groups. ISO decreased expression of the calcium handling protein, sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a, in MA-ISO compared with YA, YA-ISO, and MA rats. Chronic ISO also induced greater increases in cardiac hypertrophy [left ventricular (LV) index: 33 ± 3 vs. 22 ± 5%] and caspase-3 activity (34 vs. 5%) in MA-ISO relative to YA-ISO rats. Moreover, β-myosin heavy chain (β-MHC) and atrial natriuretic factor (ANF) mRNA expression was significantly elevated in MA-ISO. These results demonstrate that adult rats develop greater impairments in systolic performance than younger rats when exposed to chronic catecholamine excess. Reduced contractile reserve may result from calcium dysregulation, increased caspase-3 activity, or increased β-MHC and ANF expression. Although several studies report age-related declines in systolic performance in older and senescent animals, the present study demonstrates that catecholamine excess induces reductions in systolic performance significantly earlier in life.

Ovariectomy augments pressure overload-induced hypertrophy associated with changes in Akt and nitric oxide synthase signaling pathways in female rats

To elucidate the molecular mechanism underlying estrogen-mediated cardioprotection in left ventricular (LV) hypertrophy and remodeling, we analyzed myocardial hypertrophy as well as cardiac function and hypertrophy-related protein expression in ovariectomized, aortic-banded rats. Wistar rats subjected to bilateral ovariectomy (OVX) were further treated with abdominal aortic stenosis. Effects on LV morphology and function were assessed using echocardiography, and expression of protein levels was determined by Western blot analysis. The heart-to-body weight ratio was most significantly increased in the OVX-pressure overload (PO) group compared with the OVX group and in the PO group compared with sham. The LV weight-to-body weight ratio was also significantly increased in the OVX-PO group compared with the OVX group and in the PO group compared with sham. The most significant increases in LV end diastolic pressure, LV developed pressure, and +/-dp/dt(max) were observed in the OVX-PO group compared with the OVX group and represent compensatory phenotypes against hypertrophy. Both endothelial nitric oxide (eNOS) synthase expression and activity was markedly reduced in the OVX-PO group, and protein kinase B (Akt) activity was largely attenuated. Marked breakdown of dystrophin was also seen in hearts of OVX-PO groups. Finally, significantly increased mortality was observed in the OVX-PO group following chronic isoproterenol administration. Our results demonstrate that rats subjected to ovariectomy are unable to compensate for hypertrophy, showed deteriorated heart function, and demonstrated increased mortality. Simultaneous impairment of eNOS and Akt activities and reduced dystrophin by ovariectomy likely contribute to cardiac decompensation during PO-induced hypertrophy in ovariectomized rats.

Impact of chronic β-adrenoceptor activation on neurotensin-induced myocardial effects in rats

In heart failure chronic sympathetic activation results in contractile dysfunction in part through down-regulation of the β-adrenoceptor-cAMP system. However, the impact of chronic adrenergic activation on cardiac sympathetic neuromodulator systems is unclear. In this study, we sought to determine whether chronic adrenergic activation modifies myocardial norepinephrine release and contractile responses elicited by neurotensin, a neuropeptide found in cardiovascular system. Chronic administration of isoproterenol, a β-adrenoceptor agonist, to rats (0.05 mg/kg daily for 1 month, i.p.), produced cardiac hypertrophy with preserved baseline ventricular systolic function, but reduced contractile responses to exogenous norepinephrine as shown in isolated, isovolumically-contracting heart preparations. Neurotensin produced a marked increase in coronary effluent norepinephrine release, an effect abolished by SR 48692, a specific neurotensin receptor antagonist. In isoproterenol-treated rats, neurotensin has no significant impact on myocardial norepinephrine release. Consistently, concentration-dependent positive inotropic responses elicited by neurotensin in control rat hearts were blunted over a wide range of neurotensin concentrations (10 − 10 –10 − 5.5 M) in isoproterenol-treated rats. In conclusion, these data indicate that following chronic β-adrenoceptor activation, neurotensin-induced effects on norepinephrine release and subsequent contractile changes are markedly down-regulated.

Contractile responses to selective phosphodiesterase inhibitors following chronic β-adrenoreceptor activation

Contractile responses to phosphodiesterase (PDE) inhibitors are attenuated in heart failure, an effect limiting the clinical value of these agents. In this study, we sought to determine whether abnormalities in the beta-adrenoreceptor (beta-AR)-cyclic adenosine monophosphate (cAMP) signal transduction are sufficient to account for downregulation of PDE inhibitor-induced inotropic responses following chronic sympathetic activation. Sustained beta-AR activation produced by administration of isoproterenol (ISO) (50 microg kg(-1) day(-1) i.p. for 1 month) to rats resulted in cardiac hypertrophy, but did not affect baseline cardiac systolic function, as assessed in vivo by echocardiography and ex vivo under controlled loading conditions and heart rate (left ventricular systolic pressure-volume and stress-strain relations). Moreover, chronic ISO administration did not alter the baseline myocardial norepinephrine release or inotropic responses to incremental concentrations of Ca(2+) in isolated, perfused heart preparations. However, left ventricular contractile responses to ISO, the PDE III inhibitor amrinone, and the PDE IV inhibitor rolipram were attenuated following chronic beta-AR activation. Myocardial cAMP concentrations after stimulation with amrinone and rolipram were similar in ISO-treated and control rats. However, in ISO-treated rats, a marked decrease in contractile responsiveness to the cell-permeable, PDE-resistant cAMP analogue, 8-bromoadenosine cAMP, was noted. In conclusion, these data suggest that in cardiac disease, sustained beta-AR activation, without producing ventricular systolic dysfunction or enhanced myocardial norepinephrine release, is sufficient to account for the downregulation of contractile responses to PDE inhibitors. This effect appears to be largely mediated through abnormalities in signal transduction between cAMP and Ca(2+)-induced Ca(2+) release.