Large Animal Model Of Heart Failure Research Articles

Closed-chest occlusion of the left anterior descending artery in swine infarction model

Pigs have played a significant role in biological and medical research for many years. In the case of non-rodent models, pigs are the primary choices as experimental animals in the cardiovascular studies. Accumulating data indicate that the closed-chest coronary balloon-occlusion technique is one of the most successful method for creating ischemic heart failure (HF). However, consistent and thoroughly characterized large animal models of HF are a critical translational tool for drug development and toxicology. The knowledge of the different catheterization protocols is crucial to ensure a suitable animal model which can serve as a human-related preclinical validation process. Therefore it is essential to follow an optimized and standardized experimental protocol on a homogenous animal population, which help to obtain reliable and useful data for the translational large animal research programs.

Large and Small Animal Models of Heart Failure With Reduced Ejection Fraction.

Heart failure (HF) describes a heterogenous complex spectrum of pathological conditions that results in structural and functional remodeling leading to subsequent impairment of cardiac function, including either systolic dysfunction, diastolic dysfunction, or both. Several factors chronically lead to HF, including cardiac volume and pressure overload that may result from hypertension, valvular lesions, acute, or chronic ischemic injuries. Major forms of HF include hypertrophic, dilated, and restrictive cardiomyopathy. The severity of cardiomyopathy can be impacted by other comorbidities such as diabetes or obesity and external stress factors. Age is another major contributor, and the number of patients with HF is rising worldwide in part due to an increase in the aged population. HF can occur with reduced ejection fraction (HF with reduced ejection fraction), that is, the overall cardiac function is compromised, and typically the left ventricular ejection fraction is lower than 40%. In some cases of HF, the ejection fraction is preserved (HF with preserved ejection fraction). Animal models play a critical role in facilitating the understanding of molecular mechanisms of how hearts fail. This review aims to summarize and describe the strengths, limitations, and outcomes of both small and large animal models of HF with reduced ejection fraction that are currently used in basic and translational research. The driving defect is a failure of the heart to adequately supply the tissues with blood due to impaired filling or pumping. An accurate model of HF with reduced ejection fraction would encompass the symptoms (fatigue, dyspnea, exercise intolerance, and edema) along with the pathology (collagen fibrosis, ventricular hypertrophy) and ultimately exhibit a decrease in cardiac output. Although countless experimental studies have been published, no model completely recapitulates the full human disease. Therefore, it is critical to evaluate the strength and weakness of each animal model to allow better selection of what animal models to use to address the scientific question proposed.

Full-Length Transcriptome Sequencing: An Insight Into the Dog Model of Heart Failure.

Heart failure (HF) leads to a progressive increase in morbidity and mortality rates. This study aimed to explore the transcriptional landscape during HF and identify differentially expressed transcripts (DETs) and alternative splicing events associated with HF. We generated a dog model of HF (n = 3) using right ventricular pacemaker implantation. We performed full-length transcriptome sequencing (based on nanopore platform) on the myocardial tissues and analyzed the transcripts using differential expression analysis and functional annotation methods [Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses]. Additionally, we estimated the expression of the selected genes by quantitative real-time PCR (qRT-PCR) and detected the proportion of immune cells using flow cytometry. We found that increased B-type natriuretic peptide reduced ejection fraction, and apparent clinical signs were observed in the dog model of HF. We identified 67,458 transcripts using full-length transcriptome sequencing. A total of 785 DETs were obtained from the HF and control groups. These DETs were mainly enriched in the immune responses, especially Th1, Th2, and Th17 cell differentiation processes. Furthermore, flow cytometry results revealed that the proportion of Th1 and Th17 cells increased in patients with HF compared to controls, while the proportion of Th2 cells decreased. Differentially expressed genes in the HF and control groups associated with Th1, Th2, and Th17 cell differentiation were quantified using qRT-PCR. We also identified variable splicing events of sarcomere genes (e.g., MYBPC3, TNNT2, TTN, FLNC, and TTNI3). In addition, we detected 4,892 transcription factors and 406 lncRNAs associated with HF. Our analysis based on full-length transcript sequencing provided an analysis perspective in a dog model of HF, which is valuable for molecular research in an increasingly relevant large animal model of HF.

Increasing carbohydrate oxidation improves contractile reserves and prevents hypertrophy in porcine right heart failure

In heart failure, myocardial overload causes vast metabolic changes that impair cardiac energy production and contribute to deterioration of contractile function. However, metabolic therapy is not used in heart failure care. We aimed to investigate the interplay between cardiac function and myocardial carbohydrate metabolism in a large animal heart failure model. Using magnetic resonance spectroscopy with hyperpolarized pyruvate and magnetic resonance imaging at rest and during pharmacological stress, we investigated the in-vivo cardiac pyruvate metabolism and contractility in a porcine model of chronic pulmonary insufficiency causing right ventricular volume overload. To assess if increasing the carbohydrate metabolic reserve improves the contractile reserve, a group of animals were fed dichloroacetate, an activator of pyruvate oxidation. Volume overload caused heart failure with decreased pyruvate dehydrogenase flux and poor ejection fraction reserve. The animals treated with dichloroacetate had a larger contractile response to dobutamine stress than non-treated animals. Further, dichloroacetate prevented myocardial hypertrophy. The in-vivo metabolic data were validated by mitochondrial respirometry, enzyme activity assays and gene expression analyses. Our results show that pyruvate dehydrogenase kinase inhibition improves the contractile reserve and decreases hypertrophy by augmenting carbohydrate metabolism in porcine heart failure. The approach is promising for metabolic heart failure therapy.

P5999Heart failure rescue by high fat diet in a porcine model of hibernated myocardium

Abstract Background Extensive coronary artery disease without revascularization options results in ischemic hibernated myocardium and ultimately heart failure (HF). A metabolic reprogramming of the heart characterized by a shift from fatty acids (FFA) to glucose as the preferential metabolic substrate is frequent in HF and hibernated myocardium. Previous studies in mouse models of HF have shown that high fat diet (HFD) is able to reverse the metabolic reprogramming of the heart and improve cardiac function. Here we used a translational large animal model of ischemic HF evaluated by state-of-the-art imaging modalities Methods IHM was generated in Yucatan minipigs (N=50) by progressive stenosis of the proximal left anterior descending (LAD) after surgical insertion of a casein ameroid around the artery. Pigs underwent a serial multimodality imaging study including magnetic resonance imaging (MRI), multi-tracer PET/CT (18FDG & 14(R,S)-[18F]Fluoro-6-thia-heptadecanoic acid (18F-FTHA) 19F-palmitate in-house synthetized), and invasive coronary angiography (ICA). Once hibernated myocardium with metabolic switch was documented (complete occlusion of LAD, LVEF<50% on MRI, viable myocardium with dobutamine/MRI- evaluated contractile reserve, and metabolic substrate utilization shift towards glucose on PET/CT), animals were randomized to HFD or regular diet for 3 months Results The first part of the study was dedicated to establish the model in 42 pigs. Pigs were followed-up for 170±24 days with monthly ICA, MRI and PET/CT. Severe LAD stenosis was documented in all pigs 3–4 weeks after surgery. Mortality during follow-up was 57% (n=24) (15 peri-procedure, 2 between day 1 and 7, seven between day 7 and 45). Among those surviving beyond the sixth week, 90% (16/18) developed hibernated myocardium (mean LVEF 36±10%, absence of transmural delayed enhancement, contractile reserve and metabolic switch on PET/CT). In the second part of the study, 5 long-term survivors were allocated to receive HFD (20% extra lard), while 3 pigs served as controls. Mean LVEF before HFD initiation was 35±8%, and 36±5% in controls. Three months HFD was associated with a LVEF improvement in all treated animals (mean LVEF at the end of follow-up was 45±9%). Control animals on regular diet did not show any change in LVEF (35±5% at the end of follow-up) Conclusions We present a large animal model of HF due hibernated myocardium by placing an ameroid around the LAD. Mortality of this model is high (approx. 55%) but long-term survivors resemble all features seen in patients: LAD progressive stenosis with final occlusion but viable myocardium, LVEF deterioration and metabolic switch Feeding pigs with HFD to force cardiac reutilization of FFA, results in a consistent and large LVEF improvement in all animals Cardiac metabolic switch might be implicated in the pathogenesis of systolic dysfunction in hibernated myocardium and is a potential target for nutritional approaches

TCT-30 Renal Denervation Improves Cardiac Function in a Swine Model of Heart Failure

Introduction: Sympathetic nerve overactivity plays a critical role in the pathogenesis of heart failure (HF). Renal denervation (RDN) is currently under evaluation for the treatment of resistant hypertension and reduces blood pressure by disruption of sympathetic pathways that modulate cardiovascular function. We have demonstrated cardioprotective actions of RDN in rodent models of acute myocardial infarction and HF. We sought to determine if RDN treatment could improve cardiac function in a clinically relevant large animal model of HF. Methods: Yucatan miniswine (n=14) underwent 75 min of proximal left anterior descending coronary artery balloon-occlusion followed by 18 wks of reperfusion to generate large transmural infarcts and heart failure. Cardiac function was measured every 3 wks by 2-D echocardiography. HF was defined as left ventricular ejection fraction (LVEF) TM Multi-Electrode System (St. Jude Medical, St. Paul, MN). RDN was performed from distal to proximal in each renal artery, with at total of 24 ablation points separated both longitudinally and rotationally under fluoroscopic guidance. Results: At 3 wks of reperfusion following myocardial ischemia, LVEF was reduced in all pigs (SHAM RDN, 29.92.7%; RDN, 31.41.9%) with no further decline at the 6 wk treatment timepoint. At 6 wks of reperfusion, SHAM RDN and RDN treatments were performed without complication. Three wks after RDN treatment, LVEF was increased compared to SHAM (33.52.5 and 48.04.6%, respectively). LVEF was further increased 6 wks after RDN treatment compared to SHAM (31.51.5 vs. 62.00.6%, respectively). Interestingly, LVEF was higher 6 wks post-RDN compared to 3 wks post-RDN ( p =0.047). Conclusions: Preliminary results demonstrate that RDN significantly improves cardiac function in a clinically relevant swine model of HF.

Use of Adeno-Associated Virus Vector for Cardiac Gene Delivery in Large-Animal Surgical Models of Heart Failure.

The advancement of gene therapy-based approaches to treat heart disease represents a need for clinically relevant animal models with characteristics equivalent to human pathologies. Rodent models of cardiac disease do not precisely reproduce heart failure phenotype and molecular defects. This has motivated researchers to use large animals whose heart size and physiological processes more similar and comparable to those of humans. Today, adeno-associated viruses (AAV)-based vectors are undoubtedly among the most promising DNA delivery vehicles. Here, AAV biology and technology are reviewed and discussed in the context of their use and efficacy for cardiac gene delivery in large-animal models of heart failure, using different surgical approaches. The remaining challenges and opportunities for the use of AAV-based vector delivery for gene therapy applications in the clinic are also highlighted.

Ribonucleotide reductase-mediated increase in dATP improves cardiac performance via myosin activation in a large animal model of heart failure.

Heart failure remains a leading cause of morbidity, hospitalizations, and deaths. We previously showed that overexpression of the enzyme ribonucleotide reductase (RNR) in cardiomyocytes increased levels of the myosin activator, 2-deoxy-ATP, catalysed enhanced contraction, and improved cardiac performance in rodent hearts. Here we used a swine model of myocardial infarction (MI) to test preliminarily a novel gene therapy for heart failure based on delivery of the human RNR enzyme complex under the control of a cardiac-specific promoter via an adeno-associated virus serotype 6 vector--designated as BB-R12. We induced heart failure following MI in Yucatan minipigs by balloon occlusion of the left anterior descending artery. Two weeks, later, pigs received BB-R12 at one of three doses via antegrade coronary infusion. At 2 months post-treatment, LVEF and systolic LV dimension (measured by echocardiography) improved significantly in the high-dose group, despite further deterioration in the saline controls. Haemodynamic parameters including LV end-diastolic pressure, +dP/dt, and -dP/dt all trended towards improvement in the high-dose group. We observed no difference in the histopathological appearance of hearts or other organs from treated animals vs. controls, nor did we encounter any safety or tolerability concerns following BB-R12 delivery. These pilot results suggest cardiac-specific gene therapy using BB-R12 may reverse cardiac dysfunction by myosin activation in a large-animal heart failure model with no observed safety concerns. Thus further research into the therapeutic potential of BB-R12 for patients with chronic heart failure appears warranted.

Abstract 297: Paroxysmal And Sustained Atrial Fibrillation In A New Large Animal Model Of Nonischemic Heart Failure

Introduction: Atrial fibrillation is common in heart failure (HF). Understanding of the mechanisms of atrial fibrillation (AF) is limited by the paucity of large animal AF models, especially in the failing heart. We developed a large animal model of nonischemic heart failure (HF) in dogs by combined aortic insufficiency and aortic constriction and observed that a number of HF dogs developed paroxysmal AF on holter monitor. Here we characterize the spontaneously-occurring pAF in these HF dogs and perform electrophysiologic (EP) assessment of atrial refractoriness and AF inducibility along with echocardiographic imaging of left ventricle (LV) and left atrium (LA). Methods: HF was induced in dogs by aortic insufficiency and aortic constriction, and serial echocardiography (for LV fractional shortening (FS) and LA size) and Holter monitoring was performed. In control and HF dogs, EP study of atrial refractory period (AERP) and AF inducibility (duration and atrial cycle length (CL)) was performed. Results: By Holter monitoring, paroxysmal AF was noted in 5 dogs with episodes ranging from 15 to 94 beats long (mean of 49±27 beats, n=12). In EP studies, control dogs (N=3) exhibited AERP of 176±8 ms. Burst pacing resulted in AF of very brief duration (mean 32±24 sec) and a mean AF CL of 138±6 ms. LV FS averaged 37% and LA size averaged 4.3 cm2. HF dogs (N=5) exhibited RAERP of 150±8 (p=0.05 vs control). Two of these dogs had sustained AF with ventricular response up to 230 bpm on Holter monitor. In the other 3 HF dogs, burst pacing induced AF with a mean duration of 232±185 sec (at times with conversion to atrial flutter) and with a mean AF CL = 110±4 ms (p=0.002 vs control). Echo data showed LVFS averaged 30% and LA area of 14.9 cm2 (p=0.05 vs control). Conclusion: Thus we have developed a novel large animal model of HF that exhibits paroxysmal and sustained AF. This model will provide an opportunity for the study of underlying AF mechanisms, the progression of remodeling in HF hearts leading to AF, and the assessment of human-scale interventions to better treat and prevent this arrhythmia.

Large Animal Models for Left Ventricular Assist Device Research and Development

In vivo preclinical testing of left ventricular assist devices (LVADs) warrants a large animal model that faithfully simulates human etiology. Although LVAD recipients are in end-stage heart failure (HF), healthy, young animals have served as the experimental platform for most LVAD research and development (R&D) to demonstrate device safety, reliability, and biocompatibility. The rapidly growing HF epidemic, donor heart shortage, and clinical acceptance of LVAD for bridge-to-transplant therapy (BTT) has led to the expanded role of LVAD for destination therapy and bridge-to-recovery therapy. New paradigms for the clinical care of these emerging patient populations are needed. Clinically relevant, robust, and reproducible large animal models of HF are required to demonstrate efficacy, investigate physiologic responses, elucidate genetic, molecular, and cellular mechanism(s), and develop LVAD control strategies. The animal model must be comparable in size, anatomical structure, and phenotype; the technique used to initiate HF must reflect the clinical portrait, should be technically and financially feasible, result in predictable, stable, and irreversible HF, and demonstrate bidirectionality of the remodeling cascade. In this review, large animal species commonly used in cardiac research, techniques used to create chronic HF, and the combined applicability to preclinical LVAD R&D studies are presented.

Present and Future Perspectives on Cell Sheet-Based Myocardial Regeneration Therapy

Heart failure is a life-threatening disorder worldwide and many papers reported about myocardial regeneration through surgical method induced by LVAD, cellular cardiomyoplasty (cell injection), tissue cardiomyoplasty (bioengineered cardiac graft implantation), in situ engineering (scaffold implantation), and LV restrictive devices. Some of these innovated technologies have been introduced to clinical settings. Especially, cell sheet technology has been developed and has already been introduced to clinical situation. As the first step in development of cell sheet, neonatal cardiomyocyte sheets were established and these sheets showed electrical and histological homogeneous heart-like tissue with contractile ability in vitro and worked as functional heart muscle which has electrical communication with recipient myocardium in small animal heart failure model. Next, as a preclinical study, noncontractile myoblast sheets have been established and these sheets have proved to secrete multiple cytokines such as HGF or VEGF in vitro study. Moreover, in vivo studies using large and small animal heart failure model have been done and myoblast sheets could improve diastolic and systolic performance by cytokine paracrine effect such as angiogenesis, antifibrosis, and stem cell migration. Recently evidenced by these preclinical results, clinical trials using autologous myoblast sheets have been started in ICM and DCM patients and some patients showed LV reverse remodelling, improved symptoms, and exercise tolerance. Recent works demonstrated that iPS cell-derived cardiomyocyte sheet were developed and showed electrical and microstructural homogeneity of heart tissue in vitro, leading to the establishment of proof of concept in small and large animal heart failure model.

Abstract 19183: Insulin Resistance Develops after the Onset of Heart Failure in Dogs

Background: Heart failure (HF) is a condition associated with increased plasma norepinephrine levels. Norepinephrine is known to affect glucose homeostasis by decreasing insulin sensitivity. We have previously reported an increase in plasma norepinephrine levels in coronary microembolization-induced HF dogs. However, the status of plasma insulin and glucose levels is unknown in large animal models of HF. In the present study, we examined insulin and glucose levels in fasted plasma from dogs before and after the onset of HF. Methods: 12 healthy mongrel dogs weighing 20-30 kg underwent serial coronary microembolization to produce HF. Embolization was performed every 1-2 weeks until angiographic left ventricular ejection fraction (LVEF) was <35%. All procedures were performed during cardiac catheterization, under general anesthesia and sterile conditions. Fasted plasma was collected before and after inducing HF and was stored at -80 o C. Insulin and glucose levels were determined using commercially available ELISA kits for insulin and colorimetric assay kits for glucose, and expressed in microunits/mL and mg/dL, respectively. Homeostasis assessment model for insulin resistance (HOMA-IR) ([fasting insulin in microunits/mL * fasting glucose in mg/dL] * 0.5551/22.5) was calculated in all dogs at baseline and after inducing HF. Data were analyzed using paired t-tests. Results: Baseline LVEF was > 55% in all dogs. Once HF was established, mean LVEF was 31 ± 1%. Baseline mean fasting blood glucose and fasting plasma insulin were 84 ± 17 mg/dL and 9.7 ± 3.6 microunits/mL, respectively. Once HF was established, fasting blood glucose rose to 161 ± 44 mg/dL (p<0.0001) and fasting plasma insulin increased to 16.5 ± 4.0 microunits/mL (p=0.0008). HOMA-IR score increased from 2.0 ± 0.8 at baseline to 6.7 ± 3.0 with HF (p=0.0002). Conclusions: Hyperglycemia, hyperinsulinemia and insulin resistance develop after the onset of heart failure due to coronary microembolization in dogs consistent with observations previously reported in patients with chronic HF. This large animal model of HF can serve as a useful tool in exploring the underlying causes and consequences of insulin resistance in the setting of chronic HF.

Abstract 26: Myocardial Phosphodiesterase-2A Is Upregulated in Human and Experimental Heart Failure and Blunts Cardiac β-Adrenergic Inotropic Responsiveness

Augmented cGMP- and diminished cAMP-signaling within cardiomyocytes is characteristic for failing hearts. Cyclic nucleotide phosphodiesterases (PDEs) comprise a family of cyclic-nucleotide hydrolyzing enzymes, controlling cAMP and cGMP levels. Among them the PDE-2A isoform has the unique property to be stimulated by cGMP but primarily hydrolyzing cAMP. This appears to mediate a negative cross-talk between both signaling pathways. However, a potential role for PDE-2A in the failing heart has not been addressed yet. Here we show that PDE-2A protein levels were ∼2-fold higher in failing human hearts as well as in a large animal heart failure model from dog hearts subjected to rapid-pacing (n≥6, p<0.05). Intriguingly, PDE-2A protein levels were normal in hypertrophied hearts from patients with preserved cardiac function who underwent aortic valve replacement. Chronic beta-adrenergic stimulation by catecholamine infusions enhanced cAMP hydrolyzing activity of PDE-2A by four-fold (n≥6, p<0.05) in rat hearts in vivo and in isolated cardiomyocytes (measured by radioimmunoassay and FRET-based sensors, respectively) and correlated with blunted beta-adrenergic responsiveness. Consistent with this observation, overexpressed PDE-2A, which localized to the sarcomeric Z-line, blunted the rise in cAMP by 70% (n≥6, p<0.05) and abolished the positive inotropic effect after acute beta-adrenergic stimulation by 70% (n≥6, p<0.05) in isolated cardiomyocytes. Notably, those cardiomyocytes also showed marked protection from norepinephrine-induced hypertrophic responses, e. g. 40% less increase in cell surface area (n≥10, p<0.05). In summary, PDE-2A is markedly upregulated in human and experimental failing hearts. This may constitute an important defense mechanism during cardiac stress, by antagonizing the cAMP-mediated toxic effects. Thus, activating myocardial PDE-2A may represent a new intracellular anti-adrenergic therapeutic strategy in heart failure.

Highlights from the Literature

Highlights from the Literature

AAV6. ARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model

G protein-coupled receptor kinase 2 (GRK2), which is markedly upregulated in failing human myocardium, has been implicated as a contributing factor or consequence of heart failure (HF). Importantly, cardiac-specific GRK2 knockout mice have recently proved the pathological nature of GRK2 in HF. Targeted inhibition of GRK2 is possible using a peptide inhibitor known as the βARKct, which has rescued several disparate small animal HF models. This study was designed to evaluate long-term βARKct expression in a clinically relevant large animal HF model, using stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). A porcine model of HF subsequent to left ventricular (LV) myocardial infarction (MI) was used to study the effects of retrograde injection into the anterior interventricular vein of either AAV6.βARKct or AAV6.luciferase as a control 2 weeks after MI. Echocardiography and LV hemodynamics were performed before and 6 weeks after gene transfer. Robust and long-term βARKct expression was found after AAV6-mediated delivery, leading to significant amelioration of LV haemodynamics and contractile function in HF pigs compared with AAV6.luciferase-treated control animals that showed a continued decline in cardiac function. Interestingly, the neurohormonal axis was virtually normalized in AVV6.βARKct-treated HF animals, represented by reductions in plasma norepinephrine levels, whereas AAV6.luciferase-treated pigs showed further increases in plasma catecholamine levels. As a result, LV remodelling and foetal gene expression was reversed by AVV6.βARKct gene therapy. These data--showing sustained amelioration of cardiac function in a post-MI pig HF model--demonstrate the therapeutic potential of βARKct gene therapy for HF.

Augmentation of left ventricular mechanics by recirculation‐mediated AAV2/1–SERCA2a gene delivery in experimental heart failure

Down-regulation of sarcoplasmic reticulum calcium ATPase (SERCA2a) is a key molecular abnormality in heart failure (HF), which is not currently addressed by specific pharmacotherapy. We sought to evaluate, in detail, the impact of augmented SERCA2a expression on left ventricular (LV) mechanics in a large animal model of HF. Heart failure was induced in adult sheep by rapid pacing (180 b.p.m.) for 1 month, followed by delivery of adeno-associated virus (AAV) 2/1-SERCA, using a percutaneous, recirculating system for gene delivery over a 10 min period. Left ventricular mechanics was investigated by echocardiography and conductance catheter measurements in sheep receiving AAV2/1-SERCA2a after a further 4 weeks of pacing in comparison with untreated HF controls. Left ventricular function was significantly improved in the AAV2/1-SERCA2a-treated group, despite continued pacing, as measured by fractional shortening (delta absolute FS, control -4.2 ± 1.5% vs. treatment 4.4 ± 1.5%; P < 0.01) and conductance catheterization (delta Ees, control -1.22 ± 0.60 vs. treatment 0.65 ± 0.51; P < 0.05). Western blots showed an increase in SERCA protein in AAV2/1-SERCA2a-treated animals, and an analysis of gene delivery showed no evidence of regional myocardial heterogeneity in the distribution of AAV2/1-SERCA. In a large animal model, AAV2/1-mediated SERCA2a gene delivery using percutaneous, recirculating cardiac delivery leads to improved LV function.

Large Animal Models of Heart Failure

Congestive heart failure (HF) is a clinical syndrome, with hallmarks of fatigue and dyspnea, that continues to be highly prevalent and morbid. Because of the growing burden of HF as the population ages, the need to develop new pharmacological treatments and therapeutic interventions is of paramount importance. Common pathophysiologic features of HF include changes in left ventricle structure, function, and neurohormonal activation. The recapitulation of the HF phenotype in large animal models can allow for the translation of basic science discoveries into clinical therapies. Models of myocardial infarction/ischemia, ischemic cardiomyopathy, ventricular pressure and volume overload, and pacing-induced dilated cardiomyopathy have been created in dogs, pigs, and sheep for the investigation of HF and potential therapies. Large animal models recapitulating the clinical HF phenotype and translating basic science to clinical applications have successfully traveled the journey from bench to bedside. Undoubtedly, large animal models of HF will continue to play a crucial role in the elucidation of biological pathways involved in HF and the development and refinement of HF therapies.

Abstract 5340: Cardiac Resynchronization Therapy Corrects Dyssynchrony-induced Regional Gene Expression Changes on a Genomic Level

Purpose: Cardiac Resynchronization Therapy (CRT) improves symptoms and reduces mortality in patients with heart failure (HF). To characterize the molecular processes associated with functional improvement in CRT, we used a genomic approach in a large animal HF model. Methods: After creation of a left bundle branch block (LBBB), dogs in the HF group were subjected to either rapid atrial pacing with 200 bpm for 6 weeks (dyssynchronous HF, DHF, n=10), or 3 weeks of atrial pacing followed by 3 weeks of biventricular stimulation at 200bpm (CRT, n=9). Control animals without LBBB were not paced (NF, n=11). After 6 weeks, RNA from anterior and lateral regions of the LV was hybridized onto canine 44K arrays. Statistical Analysis of Microarrays (SAM) was used for data analysis. Results: Echocardiographically, CRT led to a significant increase in stroke volume (+27%, p=0.03) which translated into a non-significant increase in EF (DHF 25±4%; CRT 31±3% (p=0.15); NF 67±3%). A multiclass analysis of NF, DHF and CRT animals identified 1050 differentially expressed transcripts between anterior and lateral walls with a false discovery rate of 5%. For all these transcripts, dyssynchrony-induced expression changes were reversed by CRT to levels of NF hearts. As a result, CRT samples clustered with NF rather than DHF samples. Of particular interest were genes encoding for signal transduction pathways and contractile processes. Conclusions: By using a whole genome approach, we demonstrate a profound effect of electrical activation on the regional cardiac transcriptome. This is the first study showing that dyssynchrony-induced gene expression changes can be corrected by CRT on a genome-wide level.

Abstract 2883: Retroinfusion-facilitated Inotropic AAV9-S100A1 Gene Therapy Restores Global Cardiac Function In A Clinically Relevant Pig Heart Failure Model

Background: Cardiac expression of the Ca-dependent inotropic protein S100A1 is decreased in human end-stage heart failure (HF) and cardiomyocyte-targeted viral-based S100A1 gene transfer rescued failing myocardium in small animal models in vivo and in vitro via improved systolic and diastolic sarcoplasmic reticulum Ca-handling. We therefore hypothesized that cardioselective AAV9-S100A1 gene therapy will improve cardiac performance in a large animal experimental HF model under clinical conditions. Methods and Results: Left ventricular (LV) posterolateral myocardial infarction (MI) was induced in pigs by occlusion of the left coronary circumflex artery and resulted in LV failure (HF) 2 weeks post-MI reflected by a 40% and 27% reduction in LV +dp/dt max. and EF, respectively, as assessed by LV catheterization and echocardiography. Post-MI HF pigs were then randomized for retroinfusion of AAV9-luciferase (luc; n=6, 1.5×10 13 total viral particles, tvp) and AAV9-S100A1 (S100A1; n=6, 1.5×10 13 tvp) driven by a cardioselective promoter via the anterior cardiac vein while the left anterior descending artery was temporarily occluded. 14 weeks after cardiac gene transfer, the S100A1-treated HF group showed significantly enhanced S100A1 protein expression (+46.7±17.9%, P&lt;0.05 vs. control groups) in targeted remote LV myocardium and improved indices of cardiac function and remodeling (luc vs. S100A1: +dp/dtmax: 983±81 vs. 1526±83 mmHg/s, EF: 39±2.1 vs. 61±3.7 %, P&lt;0.05 S100A1 vs. luc, LV endsystolic diameter: luc 4.45±0.1 vs. S100A1 3.43 ±0.1 cm, P&lt;0.05 S100A1 vs. luc, HR: 72±4 vs. 69±2, beats/min, P=n.s. S100A1 vs. luc). Importantly, analyses of renal, hepatic and hematopoetic function showed no alteration as assessed by unchanged transaminases, retention values and white blood cell counts compared to sham pigs. Conclusions: Our translational study provides proof of concept that AAV9-S100A1 based HF gene therapy is feasible and restores cardiac function in a large animal HF model under clinical conditions. Next, certified toxicological analysis and different AAV9-S100A1 dosage protocols will be assessed to eventually advance to first phase I/II clinical studies determining therapeutic efficiency of cardiac S100A1 gene therapy in HF patients.

A novel alternative spliced variant of the brain natriuretic peptide gene is superior to BNP as a molecular marker of ventricular dysfunction

Background and Aim: Two mRNA isoforms of BNP (i.e., immature and mature transcripts) were found to be expressed in both normal and diseased porcine heart. Given that there were indications suggesting that heart failure (HF) may be associated with production of various isoforms of BNP, we searched for novel variants of alternative splicing of BNP in the porcine model of HF. Methods: HF was induced in 6-day-old neonatal piglets by a single i.v. injection of 2 mg/kg of Doxorubicin (Dox). To profile the cardiac gene expression changes associated with Dox-induced HF, a non-radioactive mRNA differential display (DDRT-PCR) was used. A novel alternative spliced variant of the BNP gene (designated E2-BNP) was identified, characterized, cloned and expressed. E2-BNP mRNA levels were determined by semi-quantitative and qRT-PCR. Results: On day 24 after Dox-administration, experimented piglets developed the features of diastolic HF. Using DDRT-PCR, the band corresponding to the E2-BNP was identified as being overexpressed in failing versus normal piglet myocardium. The sequence of this band displayed a 100% homology with exon 1 and exon 3 sequences of BNP. Further RT-PCR amplification of BNP (through exons 1-3) from the failing piglet myocardium cDNA produced three products, two of which were identical to the reported BNP immature and mature transcripts, whereas the third one was characterized by the precise deletion of the exon 2. Skipping of exon 2 ( E2) causes a frame-shift from the beginning of exon 3, generating a coding sequence for a new protein with no C-terminal homology to known natriuretic peptides. In newborn piglets (n=5), the E2-BNP mRNA was 15 fold more abundant in the left ventricle (LV) than in the right ventricle (RV). During development, the E2-BNP gene expression is rapidly downregulated in both ventricles being, at postnatal day 30, 10-20-fold lower as compared to newborn piglets. By contrast, in 30-day-old piglets with Dox-induced HF (n=12) the E2-BNP mRNA levels were found to be consistently and significantly augmented in both ventricles being much more up-regulated in the RV (40±2.6 fold) than in the LV (10±1.6 fold) as compared with age-matched controls. The average fold-increase of the BNP mRNA content in the failing porcine RV and LV was only 6.3±0.5 and 5.1±0.3, respectively. Conclusion: As such, variation in E2-BNP mRNA transcript abundance within the ventricular wall can be used to diagnose heart conditions in large-animal models of HF. In addition, the E2-BNP seems to be superior to BNP as a molecular marker for HF.