Swine Model of Myocardial Infarction Induced by Ischemia-Reperfusion and Embolization.

Acute myocardial infarction continues to account for a growing burden of heart failure worldwide. Despite existing therapies, new approaches for reducing the extent of damage and better managing heart failure progression are urgently needed. Preclinical large animal models are a critical step in the translation of scientific discoveries toward clinical trials and therapeutic application. In this chapter, we detail methods to induce swine models of myocardial infarction through catheter-mediated approaches involving either temporary (ischemia-reperfusion) or permanent (thrombus injection or embolic coil) occlusions. These techniques are relatively low in invasiveness, while infarct size with corresponding cardiac dysfunction can be controlled by adjusting the location of coronary occlusion. We also describe methods for cardiac angiography and echocardiography in pigs. This is the second edition of a previously published chapter with modifications.

The Sugen/Hypoxia Rat Model for Pulmonary Hypertension and Right Heart Failure.

Pulmonary hypertension (PH) is a devastating disease, characterized by complex remodeling of the pulmonary vasculature. PH is classified into five groups based on different etiology, pathology, as well as therapy and prognosis. Animal models are essential for the study of underlying mechanisms, pathophysiology, and preclinical testing of new therapies for PH. The complexity of the disease with different clinical entities dictates the necessity for more than one animal model to resemble PH, as a single model cannot imitate the broad spectrum of human PH.Here we describe a detailed protocol for creating a rat model of PH with right ventricular (RV) failure. Furthermore, we present how to characterize it hemodynamically by invasive measurements of RV and pulmonary arterial (PA) pressures. Animals subjected to this model display severe pulmonary vascular remodeling and RV dysfunction. In this model, rats undergo a single subcutaneous injection of Sugen (SU5416, a vascular endothelial growth factor inhibitor) and are immediately exposed to chronic hypoxia in a hypoxia chamber for 3-6 weeks. This Sugen/Hypoxia rat model resembles Group 1 PH.

[Genetic diagnostics and molecular approaches in pulmonary arterial hypertension]. / Genetische Diagnostik und molekulare Ansätze bei pulmonalarterieller Hypertonie.

The recently published new European guidelines for diagnosis and treatment of pulmonary hypertension now offer the so far most extensive description of genetic testing and counselling for pulmonary arterial hypertension patients. In addition, the importance of a clinical screening of healthy mutation carriers is highlighted as well as the genetic testing of patients with a suspicion of pulmonary veno-occlusive disease. We frame the respective parts of the guidelines on genetic testing and counselling in the context of recent data and provide comments. Finally, we give an outlook on novel molecular approaches starting from Sotatercept, addressing ion channels and novel therapeutic developments.

[Pulmonary hypertension associated with left heart disease (group 2)]. / Pulmonale Hypertonie assoziiert mit Linksherzerkrankungen (Gruppe 2).

Pulmonary hypertension associated with left heart disease (PH-LHD) corresponds to group two of pulmonary hypertension according to clinical classification. Haemodynamically, this group includes isolated post-capillary pulmonary hypertension (IpcPH) and combined post- and pre-capillary pulmonary hypertension (CpcPH). PH-LHD is defined by an mPAP > 20 mmHg and a PAWP > 15 mmHg, pulmonary vascular resistance (PVR) with a cut-off value of 2 Wood Units (WU) is used to differentiate between IpcPH and CpcPH. A PVR greater than 5 WU indicates a dominant precapillary component. PH-LHD is the most common form of pulmonary hypertension, the leading cause being left heart failure with preserved (HFpEF) or reduced ejection fraction (HFmrEF, HFrEF), valvular heart disease and, less commonly, congenital heart disease. The presence of pulmonary hypertension is associated with increased symptom burden and poorer outcome across the spectrum of left heart disease. Differentiating between group 1 pulmonary hypertension with cardiac comorbidities and PH-LHD, especially due to HFpEF, is a particular challenge. Therapeutically, no general recommendation for the use of PDE5 inhibitors in HFpEF-associated CpcPH can be made at this time. There is currently no reliable rationale for the use of PAH drugs in IpcPH, nor is therapy with endothelin receptor antagonists or prostacyclin analogues recommended for all forms of PH-LHD.

Endobronchial Gene Delivery for Pulmonary Hypertension in a Large Animal Model.

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.

Endobronchial Aerosolized AAV1.SERCA2a Gene Therapy in a Pulmonary Hypertension Pig Model: Addressing the Lung Delivery Bottleneck.

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 Ca2+ 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 × 1013 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.

Corrigendum: Novel Porcine Model of Coronary Dissection Reveals the Impact of Impella on Dissected Coronary Arterial Hemodynamics.

[This corrects the article DOI: 10.3389/fcvm.2020.00162.].

Left Ventricular Assist Devices for Acute Myocardial Infarct Size Reduction: Meta-analysis.

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 < 0.001). The meta-regression analysis demonstrated a high degree of heterogeneity associated with time from coronary artery occlusion to LVAD support, which correlated positively with infarct size. Subgroup analysis suggested smaller infarct size in LVAD therapies that withdrew blood from left heart than those from right heart. The proportion of left ventricular support relative to total cardiac output was positively correlated with infarct size reduction in Impella studies. Thus, early initiation of LVAD after ischemia and effective left ventricular venting may be important factors to reduce infarct size in AMI.

Impaired left ventricular global longitudinal strain is associated with elevated left ventricular filling pressure after myocardial infarction.

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.

Novel Porcine Model of Coronary Dissection Reveals the Impact of Impella on Dissected Coronary Arterial Hemodynamics.

Background: Coronary artery dissection (CAD) sometimes accompanies unstable hemodynamics and requires mechanical cardiac support. Meanwhile, mechanical cardiac support may influence coronary hemodynamics in CAD. No study has examined the impact of Impella left ventricular (LV) support on CAD. Materials and Methods: CAD was induced in eight Yorkshire pigs by injuring the left anterior descending artery (LAD) using a 0.018-in. stiff guidewire and/or deep engagement of a blunt-cut coronary guiding catheter. After the creation of CAD, hemodynamic parameters, coronary pressure, and flow as well as coronary angiograms were acquired before and after maximum LV support using the Impella CP. Result: CADs with a large flap were successfully created by deep engagement of a blunt-tip guiding catheter with forceful contrast injection. One animal (#8) exhibited thrombolysis in myocardial infarction (TIMI)-1 flow, while the others (animals #1-#7) showed TIMI-2/3 flow. In TIMI-2/3 animals, maximal Impella support increased mean coronary pressure (108.4 ± 22.5 to 124.7 ± 28.0 mmHg, P < 0.001) with unchanged mean coronary flow velocity (63.50 ± 28.66 to 48.32 ± 13.30 cm/s, P = 0.17) of the LAD distal to the dissection. The LV end-diastolic pressure (20.6 ± 6.6 vs. 12.0 ± 3.4 mmHg, P = 0.032), LV end-diastolic volume (127 ± 32 vs. 97 ± 26 ml, P = 0.015), stroke volume (68 ± 16 vs. 48 ± 14 ml, P = 0.003), stroke work (5,744 ± 1,866 vs. 4,424 ± 1,650 mmHg·ml, P = 0.003), and heart rate (71.4 ± 6.6 vs. 64.9 ± 9.3/min, P = 0.014) were all significantly reduced by Impella support, indicating effective unloading of the LV. In the TIMI-1 animal (animal #8), maximal Impella support resulted in further delay in angiographic coronary flow and reduced distal coronary pressure (22.9-17.1 mmHg), together with increased false-lumen pressure. Conclusion: Impella support effectively unloaded the LV and maintained the hemodynamics in a novel porcine model of CAD. Coronary pressure distal to the dissection was increased in TIMI-2/3 animals after Impella support but decreased in the animal with initial TIMI-1 flow.

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model.

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).

Echocardiographic Left Ventricular Mass Estimation: Two-Dimensional Area-Length Method is Superior to M-Mode Linear Method in Swine Models of Cardiac Diseases.

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 < 0.001), whereas a moderate relationship was found with M-mode method in the overall population (r = 0.68, p < 0.001). Two correlation coefficients were significantly different (p < 0.001), and were driven mainly by incremental overestimation of LV mass in heavier hearts using the M-mode method. IHD and LV dilation were the factors contributing to overestimation using M-mode method. 2DE-based area-length method provides a better estimation of LV weight in swine models of HF, particularly in those with IHD.

A Novel Large Animal Model of Thrombogenic Coronary Microembolization.

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.

Speckle-Tracking Echocardiographic Strain Analysis Reliably Estimates Degree of Acute LV Unloading During Mechanical LV Support by Impella.

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. 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 (3399 ± 1440 to 1244 ± 659 mmHg ml, p < 0.001), suggesting reduced external cardiac work. Both longitudinal strain (- 14.6 ± 4.1% to - 10.6 ± 2.3%, p < 0.001) and circumferential strain (- 18.7 ± 6.1% to - 9.3 ± 3.5%, p < 0.001) decreased after LV unloading, and there were linear relationships between stroke work and echocardiographic longitudinal (r = - 0.61, p < 0.001) as well as circumferential strains (r = - 0.75, p < 0.001). Echocardiographic LV strain analysis offers a non-invasive assessment of LV unloading in subacute MI.

Acute Left Ventricular Unloading Reduces Atrial Stretch and Inhibits Atrial Arrhythmias.

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.

Safety and long-term efficacy of AAV1.SERCA2a using nebulizer delivery in a pig model of pulmonary hypertension.

Nebulization delivery of adeno-associated virus serotype 1 encoding sarcoplasmic reticulum Ca2+-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.

A Pig Model of Myocardial Infarction: Catheter-Based Approaches.

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.

Swine Model of Mitral Regurgitation Induced Heart Failure.

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.

Pig Model of Increased Cardiac Afterload Induced by Ascending Aortic Banding.

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.