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Pulmonary hypertension (PH) is a devastating disease with high morbidity and mortality. Despite significant progress in the pharmacotherapy, current treatments only ameliorate the symptoms and cannot heal PH. Gene therapy may target the roots of the disease and holds evident promise. The current bottleneck for lung gene therapy is the delivery method. The requirements for the delivery mode are efficiency, safety, and the ability to target the anatomical site of interest, while avoiding off-target effects. Aerosolized gene delivery has been used in several studies and proven to be an efficient mode of administration for lung gene therapy. In this chapter, we describe a protocol of endobronchial aerosolization for PH gene therapy in a large animal model. Testing of a gene therapy in large animals is essential before clinical testing, since the lung anatomy and (patho)physiology differ immensely between humans and rodents, where most of the proof-of-concept studies are tested. The gene delivery vector is being aerosolized in the peripheral bronchi using a sprayer inserted through a flexible bronchoscope. This delivery mode results in efficient lung uptake and less off-target distribution relative to other airway delivery methods.

A disappointing number of new therapies for pulmonary hypertension (PH) have been successfully translated to the clinic. Adeno-associated viral (AAV) gene therapy has the potential to treat the underlying pathology of PH, but the challenge remains in efficient and safe delivery. The aims of this study were (1) to test the efficacy of endobronchial aerosolization delivery for AAV1-mediated sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) gene therapy in a PH pig model and (2) to identify the most efficient airway administration modality for in-lung gene therapy in PH. We hypothesized that delivery to the distal bronchi increases lung viral uptake and avoids virus loss in off-target compartments. In part 1 of the study, PH was induced in pigs by surgically banding the pulmonary veins. Two months postsurgery, 1 × 10(13) viral genomes (vg) of AAV1.SERCA2a or saline was endobronchially aerosolized using a bronchoscope. Two months after aerosolization, high vg copies (vgc) were detected in the lungs, accompanied by functional and morphometrical amelioration of PH. In part 2 of the study, we directly compared the endobronchial aerosolization gene delivery to the intratracheal aerosolization in PH pigs. Endobronchial delivery demonstrated higher viral expression (6,719 ± 927 vs. 1,444 ± 402 vgc/100 ng DNA, p = 0.0017), suggesting this delivery modality is a promising method for clinical AAV gene therapy for PH.

Left ventricular (LV) global longitudinal strain (GLS) has emerged as a significant prognostic marker in patients after myocardial infarction (MI). Although elevated LV filling pressure after MI might alter GLS, direct evidence for this is lacking. This study aimed to clarify the association between GLS and LV filling pressure in a large animal MI model. A total of 104 Yorkshire pigs underwent both echocardiographic and hemodynamic assessments 1–4 wk after induction of large anterior MI. GLS was measured in the apical four-chamber view using a semiautomated speckle-tracking software. LV pressure-volume relationship was invasively measured using a high-fidelity pressure-volume catheter. GLS >−14% was considered impaired. Compared with pigs with LV ejection fraction (LVEF) >40% and preserved GLS (n = 29), those with LVEF >40% and impaired GLS (n = 37) and those with LVEF ≤40% (n = 38) had significantly higher LV end-diastolic pressure (15.5 ± 5.5 vs. 19.7 ± 5.8 and 19.6 ± 6.6 mmHg; P = 0.008 and P = 0.026, respectively) and higher LV mean diastolic pressure (7.1 ± 2.9 vs. 10.4 ± 4.5 and 11.1 ± 5.4 mmHg; P = 0.013 and P = 0.002, respectively). GLS was modestly correlated with τ (r = 0.21, P = 0.039) and slope of LV end-diastolic pressure–volume relationship (r = 0.43, P < 0.001). Impaired GLS was associated with higher LV end-diastolic and mean-diastolic pressures after adjusting for LVEF and baseline characteristics (P = 0.026 and P = 0.001, respectively). Impaired GLS assessed by speckle-tracking echocardiography was associated with elevated LV filling pressure after MI. GLS has an incremental diagnostic value for detecting elevated LV filling pressure and may be particularly useful for evaluating post-MI patients with preserved LVEF. NEW & NOTEWORTHY Strain analysis was performed in 104 pigs after MI, and its relationship to invasive hemodynamic measurements was studied. Impaired longitudinal strain was associated with high ventricular filling pressure independent of LVEF in post-MI setting. Global longitudinal strain is a potential prognostic marker after MI.

Background: Sarcoplasmatic reticulum Ca++ ATPase (SERCA2a) is downregulated in the pulmonary vasculature of pulmonary hypertension (PH) patients. SERCA2a gene therapy seems a promising alternative for treating PH. The best gene delivery method has to be defined. Objective: a) compare two different airway delivery methods: bronchoscopic vs intratracheal sprayer, b) efficacy/safety of bronchoscopic delivered AAV1-SERCA2a Gene Therapy for treating PH. Methods and Results: Initially adeno-associated virus serotype1 (AAV1) encoding for reporter genes (luciferase, GFP) was injected in healthy pigs using a spray catheter inserted in a flexible bronchoscope or an intratracheal sprayer. Bronchoscopic delivery showed a denser distribution in the distal airways. We hypothesized that high concentrations of AAV1-SERCA2a would ameliorate PH. PH was induced in Yorkshire pigs (n=12) by pulmonary vein banding. 2 months after surgery the animals were randomized in 2 groups: treatment (injected with AAV1-SERCA2a) and saline. The animals were observed for 2 more months. 6 pigs reached the end timepoint. Mean PA pressure was ameliorated in the treatment vs saline injected animals (25.7 ± 5.8 vs 36.3 ± 3.0 mmHg, p Conclusion: Gene delivery using a bronchoscope is an efficient method for PH gene therapy. AAV1SERCA2 gene therapy using this delivery method ameliorates PH in a large animal model.

We conducted a meta-analysis of preclinical studies that tested left ventricular assist device (LVAD) therapy for reducing myocardial infarct size in experimental acute myocardial infarction (AMI). Twenty-six articles were included with a total of 488 experimental animal subjects. The meta-analysis showed that infarct size was significantly decreased by LVAD support compared to control animals (SDM, − 2.19; 95% CI, − 2.70 to − 1.69; P

International audience; Echocardiography offers rapid and cost-effective estimations of left ventricular (LV) mass, but its accuracy in patients with cardiac disease remains unclear. LV mass was measured by M-mode-based linear method and two-dimensional echocardiography (2DE)-based area-length method in pig models and correlation with actual LV weight was assessed. Twenty-six normal, 195 ischemic heart disease (IHD), and 33 non-IHD HF pigs were included. A strong positive linear relationship to the actual LV weight was found with 2DE-based area-length method (r = 0.82, p

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

Nebulization delivery of adeno-associated virus serotype 1 encoding sarcoplasmic reticulum Ca 2+ -ATPase2a (AAV1.SERCA2a) gene was examined in a Yukatan miniature swine model of chronic pulmonary hypertension (n = 13). Nebulization of AAV1.SERCA2a resulted in homogenous distribution of vectors, lower pulmonary vascular resistance, and a trend towards better long-term survival compared to control animals.

Background Left atrium (LA) physiology is influenced by changes in left ventricular (LV) performance and load. Objectives The purpose of this study was to define the effect of acute changes in LV loading conditions on LA physiology in subacute myocardial infarction (MI). Methods MI was percutaneously induced in 19 Yorkshire pigs. One to 2 weeks after MI, 14 pigs underwent acute LV unloading using a percutaneous LV assist device, Impella. The remaining 5 pigs underwent acute LV loading by percutaneous induction of aortic regurgitation. A pressure-volume catheter was inserted into the LA using a percutaneous transseptal approach, and LA pressure-volume loops were continuously monitored. Atrial arrhythmia inducibility was examined by burst-pacing of the right atrium. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) levels and ryanodine receptor phosphorylation were examined in LA tissues to study the potential effect of stretch-dependent oxidative stress. Results MI resulted in reduced LV ejection fraction and increased LV end-diastolic pressure with concomitant increase in LA pressure and volumes. Acute LV unloading resulted in a reduction of LV end-diastolic pressure, which led to proportional decreases in mean LA pressure and maximum LA volume. LA pressure-volume loops exhibited a pump flow-dependent, left-downward shift. This was associated with reduced LA passive stiffness, suggesting the alleviation of the LA stretch that was present after MI. Prior to acute unloading of the LV, 71% of the pigs were arrhythmia-inducible; LV unloading reduced this to 29% (p = 0.02). Time to spontaneous termination of atrial arrhythmias was decreased from median 55 s (range 5 to 300 s) to 3 s (range 0 to 59 s). In contrast, acute LV loading with aortic regurgitation increased LA pressure without a significant effect on arrhythmogenicity. Molecular analysis of LA tissue revealed that NOX2 expression was increased after MI, whereas acute LV unloading reduced NOX2 levels and diminished ryanodine receptor phosphorylation. Conclusions Acute LV unloading relieves LA stretch and reduces atrial arrhythmogenicity in subacute MI.

BACKGROUND: Non-invasive means of evaluating appropriate cardiac unloading remain to be established. We hypothesized that myocardial deformation assessed by echocardiographic speckle-tracking strain analysis can reliably estimate the degree of left ventricular (LV) unloading under mechanical circulatory support. METHODS AND RESULTS: A total of 24 Yorkshire pigs underwent Impella-mediated acute LV unloading 1–2 weeks after myocardial infarction (MI). Echocardiographic and invasive pressure-volume measurements were used to evaluate the degree of LV unloading. Pressure-volume analysis before and after LV unloading exhibited a significant decrease in stroke work (3,399±1,440 to 1,244±659 mmHg*mL, p

Coronary microembolization is one of the main causes of the "no-reflow" phenomenon, which commonly occurs after reperfusion of an occluded coronary artery. Given its high incidence and the fact that it has been proven to be an independent predictor of cardiac morbidity and mortality, there is an imperative need to study its underlying mechanisms and pathophysiology. Large animal models are essential to perform translational studies. Currently there is no animal model that recapitulates a clinical scenario of thrombogenic microembolism with preceding myocardial ischemia. Therefore, the goal of this study was to develop and characterize a novel pig model of coronary microembolization using autologous thrombus injection (CMET). Twenty-three pigs underwent myocardial infarction through percutaneous balloon occlusion of the left anterior descending artery (LAD). Each animal was enrolled in one of two groups: (1) the CMET group, in which the LAD occlusion was followed by delivery of autologous clotted blood in the LAD (distal to the balloon occlusion) and reperfusion; (2) the ischemic reperfusion (I/R) group, in which the LAD ischemia was followed by reperfusion. Surviving animals underwent functional and morphological characterization at 1-week post-procedure. Three sham operated animals were used as a control. CMET resulted in impaired left ventricular function compared to I/R pigs at 1 week. Three-dimensional echocardiography demonstrated reduced ejection fraction in the CMET group (CMET vs. I/R: 35.6 ± 4.2% vs. 47.6 ± 2.4%, p = 0.028). Invasive hemodynamic measurements by Swan-Ganz and left ventricular pressure-volume catheters revealed that CMET impaired left ventricular contractility and diastolic function. This was confirmed by both load-dependent indices including cardiac output (CMET vs. I/R: 2.7 ± 0.2 l/min, vs. 4.0 ± 0.1 l/min, p = 0.002) and load independent indices including preload-recruitable stroke work (CMET vs. I/R: 25.8 ± 4.0 vs. 47.5 ± 6.5 mmHg, p = 0.05) and end-diastolic pressure-volume relationship (slope, 0.68 ± 0.07 vs. 0.40 ± 0.11 mmHg/ml, p = 0.01). Our unique closed-chest model of coronary microembolization using autologous thrombus injection resembles the clinical condition of thrombogenic coronary microembolization in I/R injury. This model offers opportunities to conduct translational studies for understanding and treating coronary microembolization in myocardial infarction.

Increase in cardiac afterload as represented by hypertension is an established risk factor for cardiovascular diseases. Animal models of increased cardiac afterload offer studies aiming at identifying key molecular mechanisms and developing new therapeutic approaches. We have reported that banding of the ascending aorta in pigs results in significant cardiac hypertrophy and increased myocardial fibrosis at the chronic stages. These changes were accompanied by increased stiffness of the heart, but not by systolic dysfunction. In this chapter, we describe methods to surgically band the ascending aorta in pigs. After 3 months, animals develop systolic left ventricular pressure of200 mmHg with above described changes in the heart.

Despite enormous efforts in treating myocardial infarction (MI) and subsequent heart failure, the recent statistics from the American Heart Association evidently show that there still remains room for improvements. To develop and translate new therapeutics toward clinics, large animal models that allow us to test new therapies in human-like conditions are of extraordinary importance. In this chapter, we describe detailed protocols for the creation of a closed-chest MI model in pigs. The advantages of this model include high survival rate (90% after ischemia-reperfusion), adjustable MI size depending on coronary occlusion site, reproducible cardiac dysfunction, and relatively low invasive method. The temporary coronary occlusion method for ischemia-reperfusion injury as well as the permanent occlusion method, using clot injection or embolic coil implantation, are described. Furthermore, we describe the key steps needed for understanding, performing, and analyzing cardiac angiography and echocardiography in pigs.

Pulmonary hypertension (PH) is a pathophysiological condition defined as an increase in mean pulmonary arterial pressure ≥25 mmHg at rest assessed by right heart catheterization.Based on hemodynamic criteria, precapillary PH is characterized by a mean pulmonary capillary wedge pressure ≤15 mmHg as opposed to the postcapillary PH by15 mmHg. Postcapillary PH is one of the most common forms of PH, often caused by left ventricular dysfunction and heart failure.In this chapter, we describe protocols for creating a large animal model of postcapillary PH. It is induced by open chest surgery (lateral thoracotomy) to band the pulmonary veins. The model is characterized by low mortality, relatively easy surgical procedure with well reproducible results, and pulmonary and cardiac remodeling at the structural, functional, and molecular levels. The presence of right ventricular (RV) remodeling is of significant importance since right heart failure is the main cause of death in patients suffering from PH. One of the advantages of the model described in this chapter is that both adaptive and maladaptive forms of RV remodeling can be observed during the progression of the disease. This can help understand the progressive pathophysiology of RV failure in humans. Besides the description of the model, a detailed guidance of the RV functional assessment in pigs for both invasive (heart catheterization) and noninvasive (echocardiography) approaches is provided.

A wide range of approaches have been described to develop animal models of pulmonary vascular disease (PVD). Clinical heterogeneity in patients with pulmonary hypertension (PH) has prompted development of different techniques to create PH models in several animal species with the objective to recapitulate specific PH/PVD phenotypes. Chronic thromboembolic PH (CTEPH) is a clinically important phenotype of PH with a documented prevalence of 0.4-9.1% in patients with history of pulmonary embolism. A well-established large animal model of CTEPH is thus necessary for studying this disease in preclinical research. Different experimental protocols with inconsistent outcomes have been reported in the literature.We have focused on characterizing PH large animal models in a common framework; pulmonary hemodynamics, right ventricular (RV) function, and histological characterization of PVD. This research framework allows optimal evaluation of novel diagnostic tools, as well as new therapeutic strategies. The purpose of this protocol is to describe approaches to create experimental CTEPH models using recurrent pulmonary embolizations of dextran microspheres in swine. The key features of this experimental modeling approach are (1) nonsurgical, fully percutaneous techniques, (2) a minimum of four embolization procedures, with 1-2 month time period, (3) mild to moderate PH hemodynamics (mean PA pressure increase ~20-60%), (4) severe pulmonary vascular remodeling, (5) mild RV remodeling, and (6) a high reproducibility and low mortality (10%).

Mitral regurgitation (MR) is among the most common valvular heart diseases in clinics. MR induces volume overload of the heart and leads to heart failure (HF). Because physiological and molecular mechanisms in nonischemic HF are distinct from that of ischemic HF, a clinically relevant animal model of nonischemic HF is important for understanding the pathophysiology and developing new therapeutics targeting this HF phenotype. Additionally, the large animal model of MR provides opportunities to test new surgical and percutaneous approaches for correcting mitral valve insufficiency.In this chapter, we describe protocols for inducing MR in pigs using percutaneous approaches. Specifically, mitral valve chords are cut by a cardiac biopsy catheter inserted either antegrade (transseptal through venous access) or retrograde (arterial access) into the left ventricle. Both acute and chronic HF can be induced using this technique, and left atrial enlargement can be found at the chronic stage.

Increase in cardiac afterload as represented by hypertension is an established risk factor for cardiovascular diseases. Animal models of increased cardiac afterload offer studies aiming at identifying key molecular mechanisms and developing new therapeutic approaches. We have reported that banding of the ascending aorta in pigs results in significant cardiac hypertrophy and increased myocardial fibrosis at the chronic stages. These changes were accompanied by increased stiffness of the heart, but not by systolic dysfunction. In this chapter, we describe methods to surgically band the ascending aorta in pigs. After 3 months, animals develop systolic left ventricular pressure of >200 mmHg with above described changes in the heart.

A wide range of approaches have been described to develop animal models of pulmonary vascular disease (PVD). Clinical heterogeneity in patients with pulmonary hypertension (PH) has prompted development of different techniques to create PH models in several animal species with the objective to recapitulate specific PH/PVD phenotypes. Chronic thromboembolic PH (CTEPH) is a clinically important phenotype of PH with a documented prevalence of 0.4-9.1% in patients with history of pulmonary embolism. A well-established large animal model of CTEPH is thus necessary for studying this disease in preclinical research. Different experimental protocols with inconsistent outcomes have been reported in the literature.We have focused on characterizing PH large animal models in a common framework; pulmonary hemodynamics, right ventricular (RV) function, and histological characterization of PVD. This research framework allows optimal evaluation of novel diagnostic tools, as well as new therapeutic strategies. The purpose of this protocol is to describe approaches to create experimental CTEPH models using recurrent pulmonary embolizations of dextran microspheres in swine. The key features of this experimental modeling approach are (1) nonsurgical, fully percutaneous techniques, (2) a minimum of four embolization procedures, with 1-2 month time period, (3) mild to moderate PH hemodynamics (mean PA pressure increase ~20-60%), (4) severe pulmonary vascular remodeling, (5) mild RV remodeling, and (6) a high reproducibility and low mortality (

Background Delivering therapeutic materials, like stem cells or gene vectors, to the myocardium is difficult in the setting of ischemic heart failure because of decreased coronary flow and impaired microvascular perfusion ( MP ). The aim of this study was to determine if mechanical left ventricular ( LV ) unloading with the Impella increases coronary flow and MP in a subacute myocardial infarction. Methods and Results Anterior transmural myocardial infarction (infarct size, 26.0±3.4%) was induced in Yorkshire pigs. At 2 weeks after myocardial infarction, 6 animals underwent mechanical LV unloading by Impella, whereas 4 animals underwent pharmacological LV unloading using sodium nitroprusside for 2 hours. LV unloading with Impella significantly reduced end‐diastolic volume (−16±11%, P =0.02) and end‐diastolic pressure (EDP; −32±23%, P =0.03), resulting in a significant decrease in LV end‐diastolic wall stress ( EDWS ) (infarct: 71.6±14.7 to 43.3±10.8 kdynes/cm 2 [ P =0.02]; remote: 66.6±20.9 to 40.6±13.3 kdynes/cm 2 [ P =0.02]). Coronary flow increased immediately and remained elevated after 2 hours in Impella‐treated pigs. Compared with the baseline, MP measured by fluorescent microspheres significantly increased within the infarct zone (109±81%, P =0.003), but not in the remote zone. Although sodium nitroprusside effectively reduced LV ‐ EDWS , 2 (50%) of sodium nitroprusside–treated pigs developed profound systemic hypotension. A significant correlation was observed between the infarct MP and EDWS ( r 2 =0.43, P =0.03), suggesting an important role of EDWS in regulating MP during LV unloading in the infarcted myocardium. Conclusions LV unloading using an Impella decreased EDWS and increased infarct MP without hemodynamic decompensation. Mechanical LV unloading is a novel and efficient approach to increase infarct MP in patients with subacute myocardial infarction.

Pulmonary hypertension (PH) is a pathophysiological condition defined as an increase in mean pulmonary arterial pressure ≥25 mmHg at rest assessed by right heart catheterization.Based on hemodynamic criteria, precapillary PH is characterized by a mean pulmonary capillary wedge pressure ≤15 mmHg as opposed to the postcapillary PH by >15 mmHg. Postcapillary PH is one of the most common forms of PH, often caused by left ventricular dysfunction and heart failure.In this chapter, we describe protocols for creating a large animal model of postcapillary PH. It is induced by open chest surgery (lateral thoracotomy) to band the pulmonary veins. The model is characterized by low mortality, relatively easy surgical procedure with well reproducible results, and pulmonary and cardiac remodeling at the structural, functional, and molecular levels. The presence of right ventricular (RV) remodeling is of significant importance since right heart failure is the main cause of death in patients suffering from PH. One of the advantages of the model described in this chapter is that both adaptive and maladaptive forms of RV remodeling can be observed during the progression of the disease. This can help understand the progressive pathophysiology of RV failure in humans. Besides the description of the model, a detailed guidance of the RV functional assessment in pigs for both invasive (heart catheterization) and noninvasive (echocardiography) approaches is provided.

Mitral regurgitation (MR) is among the most common valvular heart diseases in clinics. MR induces volume overload of the heart and leads to heart failure (HF). Because physiological and molecular mechanisms in nonischemic HF are distinct from that of ischemic HF, a clinically relevant animal model of nonischemic HF is important for understanding the pathophysiology and developing new therapeutics targeting this HF phenotype. Additionally, the large animal model of MR provides opportunities to test new surgical and percutaneous approaches for correcting mitral valve insufficiency.In this chapter, we describe protocols for inducing MR in pigs using percutaneous approaches. Specifically, mitral valve chords are cut by a cardiac biopsy catheter inserted either antegrade (transseptal through venous access) or retrograde (arterial access) into the left ventricle. Both acute and chronic HF can be induced using this technique, and left atrial enlargement can be found at the chronic stage.

Despite enormous efforts in treating myocardial infarction (MI) and subsequent heart failure, the recent statistics from the American Heart Association evidently show that there still remains room for improvements. To develop and translate new therapeutics toward clinics, large animal models that allow us to test new therapies in human-like conditions are of extraordinary importance. In this chapter, we describe detailed protocols for the creation of a closed-chest MI model in pigs. The advantages of this model include high survival rate (>90% after ischemia-reperfusion), adjustable MI size depending on coronary occlusion site, reproducible cardiac dysfunction, and relatively low invasive method. The temporary coronary occlusion method for ischemia-reperfusion injury as well as the permanent occlusion method, using clot injection or embolic coil implantation, are described. Furthermore, we describe the key steps needed for understanding, performing, and analyzing cardiac angiography and echocardiography in pigs.

Gene therapy is recently attracting increased attention and cardiac gene therapy is not an exception. Advances in gene transfer vectors, development of new vector delivery methods, and discovery of new gene targets continue to fuel our motivation to use this approach in routine bedside care. In the past two years, we have witnessed important advances in the field, as the results of three recently completed cardiac gene therapy programs have been published. Unfortunately, none of the trials have met their primary efficacy endpoints, but sub-analysis demonstrated potential efficacy. Careful review and interpretation of these trial results will provide important insights and direct us to improve the future trial design. In this review, we update our previous overview with a specific focus on recent clinical trial results. We then contemplate future strategies towards successful application of gene therapy for treating clinical heart failure.

Aims Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, and effective treatment of AF still remains an unmet medical need. AF is associated with atrial conduction disturbances caused by electrical and/or structural remodelling. We hypothesized that AF suppresses expression of the gap junction protein connexin (Cx) 43 and that Cx43 gene transfer to both atria would prevent persistent AF. The first aim of this study was to assess whether AF is associated with connexin remodelling in a porcine model. A strategy to suppress persistent AF by gene therapy was then developed and evaluated in vivo . Methods and results AF was induced in domestic pigs via atrial burst pacing, causing a 62.4% reduction in atrial Cx43 protein. Adenoviruses encoding for Cx43 (AdCx43) or green fluorescent protein (AdGFP) were injected into both atria, followed by epicardial electroporation to enhance transgene expression. Combining direct injection of adenoviruses with electroporation achieved GFP reporter gene expression in ∼50% of atrial cells in vivo . AdCx43-treated animals exhibited a 2.5-fold increase in atrial Cx43 protein content and did not develop persistent AF during the observation period of 14 days. In contrast, control animals developed persistent AF within 7.4 ± 0.5 days. Rapid ventricular heart rates during AF led to deterioration of cardiac function in control pigs but not in pigs treated with AdCx43. Conclusion Our results highlight the contribution of Cx43 to the pathophysiology of AF and demonstrate the viability of gene therapy for prevention of atrial arrhythmias.

Aims Atrial fibrillation (AF) is linked to cardiomyocyte apoptosis, leading to atrial remodelling and reduction in electrical conduction velocity. We hypothesized that genetic suppression of an apoptotic key enzyme, caspase 3, would prevent the development of persistent AF by reducing apoptosis which may serve as an arrhythmogenic substrate. Methods and results Atrial fibrillation was induced in domestic pigs by atrial burst pacing via an implanted cardiac pacemaker. Study animals were then assigned to receive either Ad-siRNA-Cas3 gene therapy to inactivate caspase 3 or green fluorescent protein (Ad-GFP) as a control. Adenoviruses were applied using a hybrid technique employing right and left atrial virus injection followed by epicardial electroporation to increase expression of plasmid DNA. In pigs treated with Ad-siRNA-Cas3, the onset of AF was suppressed or significantly delayed compared with controls (10.3 ± 1.2 days vs. 6.0 ± 1.6 days; P = 0.04). Electrical mapping revealed prolonged atrial conduction in the control group that was prevented by Ad-siRNA-Cas3 gene therapy. On the molecular level, Ad-siRNA-Cas3 application resulted in down-regulation of caspase 3 expression and suppression of apoptotic activity. Conclusion Knockdown of caspase 3 by atrial Ad-siRNA-Cas3 gene transfer suppresses or delays the onset of persistent AF by reduction in apoptosis and prevention of intra-atrial conduction delay in a porcine model. These results highlight the significance of apoptosis in the pathophysiology of AF and demonstrate short-term efficacy of gene therapy for suppression of AF.

Patients with nonischemic cardiomyopathy (DCM) are at high risk for sudden cardiac death (SCD). However, the predictive value of prophylactic implantation of implantable cardioverter defibrillators (ICD) in this patient cohort is yet unclear. Whether NT pro BNP levels and/or reproducible non sustained ventricular tachycardias (NSVTs) are predictive for SCD was prospectively tested in 30 patients with DCM and LVEF ≤ 40%. All patients received Holter-recordings (HR) on three consecutive days and baseline NT-pro BNP levels were determined. Patients were followed for occurrence of ventricular tachyarrhythmias or unexplained syncope. A great degree of variability was found regarding the occurrence of NSVTs (10% had NSVTs in two consecutive HR, 10% in three consecutive HR, 30% in one HR and 50% had no NSVTs). Patients with NSVTs in more than one HR had significantly higher NT-pro BNP levels (first quartile: 715 pg/ml, median 2,176 pg/ml, third quartile 5,755 pg/ml vs. first quartile 273 pg/ml, median 566 pg/ml, third quartile 1,350 pg/ml, P = 0.0388). During a mean follow-up of 21.6 ± 1.2 months patients with an arrhythmic event had significantly higher NT-pro BNP levels than patients without event (first quartile: 1,002 pg/ml, median 4,075 pg/ml, third quartile 7,777 pg/ml vs. first quartile 173 pg/ml, median 267 pg/ml, third quartile 1,220 pg/ml, P = 0.0135). NT-pro BNP levels of 2,259 pg/ml were identified as optimal cut-off value for the prediction of arrhythmic events (P = 0.0313). In contrast reproducible NSVTs were not predictive for arrhythmic events (P = 0.0960). The present study demonstrates that in patients with DCM the value of reproducible NSVTs in predicting arrhythmic events is low. In contrast raised NT-pro BNP levels significantly correlated with occurrence of symptomatic ventricular arrhythmias. Larger prospective trials are required to confirm these results.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Gene therapy−dependent modulation of atrial electrophysiology may provide a more specific alternative to pharmacological and ablative treatment strategies. Objective We hypothesized that genetic inactivation of atrial repolarizing ether−a−go−go−related gene (ERG) K+ currents using a dominant−negative mutant would provide rhythm control in AF. Methods Ten domestic swine underwent pacemaker implantation and were subjected to atrial burst pacing to induce persistent AF. Animals were then randomized to receive either AdCERG−G627S to suppress ERG/IKr currents or green fluorescent protein (AdGFP) as control. Adenoviruses were applied using a novel hybrid technique employing atrial virus injection followed by epicardial electroporation to increase transgene expression. Results In pigs treated with AdCERG−G627S, the onset of persistent AF was prevented (n = 2) or significantly delayed compared to AdGFP controls (12 ± 2.1 vs. 6.2 ± 1.3 days; P < .001) during 14 day follow−up. Effective refractory periods were prolonged in the AdCERG−G627S group compared to AdGFP animals (221.5 ± 4.7 ms vs. 197.0 ± 4.7 ms; P < .006). Impairment of left ventricular ejection fraction (LVEF) during AF was prevented by AdCERG−G627S application (LVEFCERG−G627S = 62.1 ± 4.0% vs. LVEFGFP = 30.3 ± 9.1%; P < .001). Conclusion Inhibition of ERG function using atrial AdCERG−G627S gene transfer suppresses or delays the onset of persistent AF by prolongation of atrial refractoriness in a porcine model. Targeted gene therapy represents an alternative to pharmacological or ablative treatment of AF

Gene therapy-based modulation of atrioventricular (AV) conduction by overexpression of a constitutively active inhibitory Gα i protein effectively reduced heart rates in atrial fibrillation (AF). However, catecholamine stimulation caused an excessive increase in ventricular rate. We hypothesized that modest genetic suppression of a stimulatory G protein in the AV node would allow persistent rate control in acute AF and would prevent undesired heart rate acceleration during β-adrenergic activation. Atrial fibrillation was induced in 12 pigs by atrial burst pacing via an implanted cardiac pacemaker. Study animals were then assigned to receive either Ad-siRNA-Gαs gene therapy to inactivate Gαs protein or Ad-β-gal as control. Gαs protein inactivation resulted in a 20 % heart rate reduction (P

Die intraoperative Testung von implantierbaren Kardioverter/Defibrillatoren (ICD) ist personalintensiv und mit Komplikationen vergesellschaftet. In der vorliegenden Studie untersuchten wir, ob bei ICD-Implantationen mit unterschiedlicher maximaler Energieabgabe eine intraoperative Testung inklusive der Induktion von Kammerflimmern (DFT) notwendig gewesen ware. In einer retrospektiven Studie erhielten 111 Patienten (94 mannlich, 17 weiblich) einen ICD. Nach Auftreten von ventrikularen Tachyarrhythmien wurden im weiteren Verlauf alle Patienten mit antiarrhythmischen Substanzen aufgesattigt (Mexiletin, Amiodaron, Sotalol, Flecainid). DFT-Testungen wurden intraoperativ und nach Aufsattigung mit antiarrhythmischen Substanzen durchgefuhrt. In einem zweiten Schritt wurden die DFT-Resultate der Studienkohorte analysiert hinsichtlich virtueller ICDs mit unterschiedlicher maximaler Energieabgabe ≤ 30 J (LOD), 34 J (IOD) oder 36 J (HOD). Wahrend Implantation erreichten alle Patienten die Sicherheitsmarge von 10 J (10-J-SM) zwischen maximaler Energieabgabe und DFT. Nach Aufsattigung mit antiarrhythmischen Substanzen verpassten 6 Patienten (12%) mit LOD, 3 Patienten (11%) mit IOD und 3 Patienten (13%) mit HOD die 10-J-Sicherheitsmarge. Hatte man fur die Kohorte virtuelle ICDs mit LOD, IOD oder HOD implantiert, so hatten sechs (5,5%), ein (1%) bzw. kein Patient(en) (0%) die 10-J-SM nicht erreicht. Im Falle einer Aufsattigung der Studienkohorte mit antiarrhythmischen Substanzen hatten 18 (16%), 12 (10,8%) bzw. 9 (8%) der Patienten mit virtuellen ICDs (LOD/IOD/HOD) die 10-J-SM verpasst. Die vorliegende Studie zeigt, dass intraoperativ bei allen Patienten mit virtuellem HOD-ICD die 10-J-SM erreicht worden ware, jedoch nicht nach Aufsattigung mit antiarrhythmischen Substanzen. Eine intraoperative Testung aller Patienten mit HOD scheint entsprechend verzichtbar, solange keine Aufsattigung mit einem Antiarrhythmikum erfolgt.

We examined the costs and safety of follow-up of patients with an implantable cardioverter defibrillator (ICD). In a prospective study, a remote monitoring system was used to interrogate ICD devices via telephone. Twenty patients with an ICD were followed up conventionally (clinic visits) or remotely at 1, 3 and 6 months after implantation of the ICD. A total of 30 transmissions of ICD data were made via the remote monitoring system. Five transmissions (17%) were interrupted, mainly due to a loss of telemetry, but no data were lost. The duration of the remote follow-up was 12.7 min less than follow-up in clinic (25.8 min, P < 0.05). Five of the remote follow-up transmissions concerned arrhythmia episodes. These lasted significantly longer than those without arrhythmia (16.6 vs. 4.9 min, P < 0.05). In three patients an unscheduled visit to the outpatient clinic was necessary. The cost of remote follow-up for 100 ICD patients/year was calculated to be €44,267, or about 16% of the cost of conventional in-clinic follow-up.

Atrial fibrillation (AF) is associated with atrial conduction disturbances caused by electrical and structural remodeling. We hypothesized that expression of the gap junction protein connexin 43 is reduced during AF, and that atrial Cx43 gene transfer would prevent persistent atrial fibrillation. The first aim of this study was to assess whether atrial fibrillation (AF) is associated with connexin remodeling in a porcine model. Second, a strategy to suppress persistent AF by gene therapy was developed and evaluated in vivo.AF was inducedin domestic pigs via atrial burst pacing, causing a 62.4% reduction of atrial Cx43 protein. Adenoviruses encoding for connexin 43 (Ad-Cx43) or green fluorescent protein (Ad-GFP) were injected into both atria, followed by epicardial electroporation to enhance transgene expression. Ad-Cx43 treated animals did not exhibit persistent AF during the observation period of 14 days. In contrast, control animals developed persistent AF within 7.4 ± 0.5 days. Rapid ventricular heart rates during AF led to deterioration of cardiac function in control pigs but not in animals treated with Ad-Cx43.In conclusion, our results highlight the contribution of connexin 43 to atrial fibrillation and demonstrate the viability of electrophysiological gene therapy for prevention of atrial arrhythmias.