Negative Impact of Acute Reloading after Mechanical Left Ventricular Unloading.

Mechanical LV unloading for acute myocardial infarction (MI) is a promising supportive therapy to reperfusion. However, no data is available on exit strategy. We evaluated hemodynamic and cellular effects of reloading after Impella-mediated LV unloading in Yorkshire pigs. First, we conducted an acute study in normal heart to observe effects of unloading and reloading independent of MI-induced ischemic effects. We then completed an MI study to investigate optimal exit strategy on one-week infarct size, no-reflow area, and LV function with different reloading speeds. Initial studies showed that acute reloading causes an immediate rise in end-diastolic wall stress followed by a significant increase in cardiomyocyte apoptosis. The MI study did not result in any statistically significant findings; however, numerically smaller average infarct size and no-reflow area in the gradual reloading group prompt further examination of reloading approach as an important clinically relevant consideration.

Cardiac Gene Delivery in Large Animal Models: Antegrade Techniques.

Percutaneous antegrade coronary injection is among the least invasive cardiac selective gene delivery methods. However, the transduction efficiency of a simple bolus antegrade injection is quite low. In order to improve transduction efficiency in antegrade intracoronary delivery, several additional approaches have been proposed.In this chapter, we will describe the important elements associated with intracoronary delivery methods and present protocols for three different catheter-based antegrade gene delivery techniques in a preclinical large animal model. This is the second edition of this chapter, and it includes modifications we have made over the past several years that further enhance transduction efficacy.

Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

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.].

Effects of Therapeutic Hypothermia on Normal and Ischemic Heart.

Therapeutic hypothermia has been used for treating brain injury after out-of-hospital cardiac arrest. Its potential benefit on minimizing myocardial ischemic injury has been explored, but clinical evidence has yet to confirm positive results in preclinical studies. Importantly, therapeutic hypothermia for myocardial infarction is unique in that it can be initiated prior to reperfusion, in contrast to its application for brain injury in resuscitated cardiac arrest patients. Recent advance in cooling technology allows more rapid cooling of the heart than ever and new clinical trials are designed to examine the efficacy of rapid therapeutic hypothermia for myocardial infarction. In this review, we summarize current knowledge regarding the effect of hypothermia on normal and ischemic hearts and discuss issues to be solved in order to realize its clinical application for treating acute myocardial infarction.

Impaired Diastolic Function Predicts Improved Ischemic Myocardial Flow by Mechanical Left Ventricular Unloading in a Swine Model of Ischemic Heart Failure.

Background: Impact of mechanical left ventricular (LV) unloading on myocardial tissue perfusion and its regulating factors remain unclear. This study was conducted to elucidate the predictors of regional blood flow (RBF) improvement by mechanical LV unloading. Materials and Methods: One to four weeks after percutaneous induction of myocardial infarction (MI), Yorkshire pigs (n = 15) underwent mechanical LV unloading using Impella CP. Hemodynamic parameters were collected prior to LV unloading. RBF in infarct, border and remote myocardium were measured by fluorescent microsphere injections before and 120 min after LV unloading. Results: RBF showed variable responses to mechanical LV unloading. While infarct RBF improved in general (0.33 ± 0.13 to 0.42 ± 0.19 mL/min/g, p = 0.06), there were a few pigs that showed little improvement. Meanwhile, there were no clear trends in the border (1.07 ± 0.47 to 1.02 ± 0.65 mL/min/g, p = 0.73) and remote myocardial RBF (1.25 ± 0.52 to 1.23 ± 0.68 mL/min/g, p = 0.85). In the simple linear regression analysis, cardiac output, mean pulmonary arterial wedge pressure, mean left atrial pressure, minimum LV pressure, end-diastolic LV pressure, maximum dP/dt, slope of end-diastolic pressure-volume relationship (EDPVR) and end-diastolic wall stress were significantly associated with % change of infarct RBF. In the multiple regression model, slope of EDPVR and maximum dP/dt remained as independent predictors of infarct RBF change. Conclusion: Steeper EDPVR and lower maximum dP/dt were associated with increased blood perfusion in the infarct area after LV unloading. Our data suggests mechanical LV unloading is more beneficial in post-MI patients with high diastolic pressure associated with increased LV stiffness and in those with worse cardiac contractility.

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.

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.

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.

Consideration of clinical translation of cardiac AAV gene therapy.

Advancements in conventional cardiac care have significantly reduced mortality from coronary heart disease and acute myocardial infarction. However, the prevalence of heart failure continues to increase in an aging population with profound social and economic consequences. Cardiac gene therapy with adeno-associated virus (AAV) vectors is emerging as a potential modality for addressing this desperate clinical need. After showing initial promise in extensive preclinical studies and an early clinical trial, disappointing results of large-scale clinical trial derailed the progress of AAV-mediated cardiac gene therapy. However, it appears that knowledge gained from previous failures coupled with developments in targeted gene delivery have set the stage for a new frontier in cardiac AAV gene therapy.

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.

Contractile dysfunction of cardiomyopathic hamster myocytes is pronounced under high load conditions.

To understand the pathophysiology of hereditary cardiomyopathy, the contractile function of cardiomyopathic hamsters has been studied at the cellular level. However, most of the studies to date have described the cell shortening under the unloaded condition. Using a novel force-length measurement system for single cardiomyocytes, we studied the contractile function of cardiomyopathic hamster myocytes over a wide range of loading conditions. Cardiomyocytes were isolated from the ventricles of eight- to 10-week-old cardiomyopathic (CMP) hamsters (Bio TO-2 strain), as well as control (CTRL) Syrian hamsters. A pair of carbon fibers was attached to both ends of single cardiomyocytes and their contractile characteristics were recorded while changing the after-load by controlling the fiber motion. Under the unloaded condition, the shortening fraction (CMP 9.2+/-0.5% vs. CTRL 10.7+/-0.8%, P=0.06) and maximum shortening velocity (CMP 98.2+/-7.3 microm/s vs. CTRL 147.2+/-6.5 microm/s, P<0.05) were decreased in CMP hamster myocytes. The peak force under the isometric condition (CMP 35.8+/-2.2 mN/mm2 vs. CTRL 69.0+/-8.4 mN/mm2, P<0.05) and external work (CMP 898+/-130 J/m3 vs. CTRL 3058+/-576 J/m3, P<0.05) under physiologically loaded conditions were also decreased, but the differences were more pronounced under the loaded conditions. Calcium transients measured by Indo-1 revealed elevated diastolic level, decreased peak level, and slower diastolic decay in CMP myocytes thus being consistent with the observed contractile dysfunction. These results clearly indicate the importance of the loading conditions in evaluating the contractile function of CMP hamster myocytes, and may provide insights into the mechanism of contractile dysfunction in this disease.

Single cell mechanics of rat cardiomyocytes under isometric, unloaded, and physiologically loaded conditions.

One of the most salient characteristics of the heart is its ability to adjust work output to external load. To examine whether a single cardiomyocyte preparation retains this property, we measured the contractile function of a single rat cardiomyocyte under a wide range of loading conditions using a force-length measurement system implemented with adaptive control. A pair of carbon fibers was used to clamp the cardiomyocyte, attached to each end under a microscope. One fiber was stiff, serving as a mechanical anchor, while the bending motion of the compliant fiber was monitored for force-length measurement. Furthermore, by controlling the position of the compliant fiber using a piezoelectric translator based on adaptive control, we could change load dynamically during contractions. Under unloaded conditions, maximal shortening velocity was 106 +/- 8.9 microm/s (n = 13 cells), and, under isometric conditions, peak developed force reached 5,720 nN (41.6 +/- 5.6 mN/mm(2); n = 17 cells). When we simulated physiological working conditions consisting of an isometric contraction, followed by shortening and relaxation, the average work output was 828 +/- 123 J/m(3) (n = 20 cells). The top left corners of tension-length loops obtained under all of these conditions approximate a line, analogous to the end-systolic pressure-volume relation of the ventricle. All of the functional characteristics described were analogous to those established by studies using papillary muscle or trabeculae preparations. In conclusion, the present results confirmed the fact that each myocyte forms the functional basis for ventricular function and that single cell mechanics can be a link between subcellular events and ventricular mechanics.