Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.
Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.
Heart failure with reduced ejection fraction (HFrEF) accounts for about 50% of all heart failure cases, affecting an estimated 1 – 2% of people in the western world1. Its most prevalent cause is acute myocardial infarction (AMI). As acute mortality after AMI has declined significantly due to increased awareness and improved treatment options, emphasis has shifted towards its chronic sequelae; the most prominent being HFrEF2,3. Together with increasing health care costs4, the growing epidemic of heart failure stresses the need for novel diagnostics and therapies, which can be studied in a highly translational porcine model of adverse remodeling after AMI as previously described5.
Both, determinants (e.g., infarct size) and functional assessments (e.g., echocardiography) of adverse remodeling are often used for efficacy testing of new therapeutics, indicating the need for reliable and relatively inexpensive methods. The aim of the current paper is to address this need by introducing important and reliable outcome measures for efficacy testing in a pig model of acute myocardial infarction. These include infarct size (IS) in relation to area at risk (AAR), 3D transesophageal echocardiography (3D-TEE) and detailed admittance-based pressure-volume (PV) loop acquisition.
Infarct size is the main determinant of adverse remodeling and survival after AMI6. Although timely reperfusion of ischemic myocardium may salvage reversibly injured cardiomyocytes and limit infarct size, reperfusion itself causes additional damage through the generation of oxidative stress and a disproportionate inflammatory response (ischemia-reperfusion injury (IRI))7. Hence, IRI has been identified as a promising therapeutic target. The ability of novel therapeutics to decrease infarct size is quantified by assessing infarct size in relation to the area at risk (AAR). AAR quantification is mandatory to correct for inter-individual variability in coronary anatomy of animal models, as a larger AAR leads to a larger absolute infarct size. Since infarct size is directly related to cardiac performance and myocardial contractility, variations in AAR can influence study outcome measures irrespective of treatment modalities8.
Three-dimensional transesophageal echocardiography (3D-TEE) is a safe, reliable and, most importantly, clinically applicable inexpensive method to measure cardiac function non-invasively. Whereas transthoracic echocardiography (TTE) images are limited to 2D parasternal long- and short-axis views in pigs9, 3D-TEE can be used to obtain complete 3-dimensional images of the left ventricle. Therefore, it does not require mathematical approximations of left ventricular (LV) volumes such as the modified Simpson's rule10. The latter falls short of correctly estimating LV volumes after LV remodeling due to the lack of cylindrical geometry11. Moreover, 3D-TEE is preferable over epicardial echocardiography as it does not require surgical interventions, which have been observed to exert cardioprotective effects in the present model12. Although the use of 2D-TEE for the assessment of myocardial function has been described before13,14, limitations regarding ventricular geometry are similar to those observed in 2D-TTE and depend on the extent of LV remodeling. Hence, the larger the infarct (and thus the higher the probability of heart failure), the more likely 2D measurements become flawed by incorrect geometrical assumptions and the higher the need for 3D techniques.
Nonetheless, most imaging modalities are limited in their ability to assess intrinsic functional properties of the myocardium. PV loops provide such relevant additional information and their acquisition is therefore described in detail below.
All animal experiments were approved by the Ethical Committee on Animal Experimentation of the University Medical Center Utrecht (Utrecht, the Netherlands) and conform to the 'Guide for the care and use of laboratory animals'.
NOTE: The protocol to perform a closed-chest balloon occlusion is not part of the current manuscript and is described in detail elsewhere5. In short, pigs (60 – 70 kg) are subjected to 75 min transluminal balloon occlusion of the midportion of the left anterior descending artery (LAD).
Both, three-dimensional transesophageal echocardiography (3D-TEE) and pressure-volume (PV) loop measurements can be performed at baseline, short-term and long-term follow-up. Note that these measurements are considered unreliable in the first hours after myocardial infarction due to frequent arrhythmias in this phase. Infarct size (IS) and area-at-risk (AAR) measurements are preferably assessed at short-term follow-up (24 – 72 hr)15,16, since changes in microvasculature and secondary myocardial scar thinning culminate in less reliable results. Infarct size staining is performed using 2,3,5-triphenyltetrazolium chloride (TTC) (CAUTION, irritant), which is considered highly reproducible and relatively inexpensive. TTC is a white powder that colorlessly dissolves in saline. Upon contact with various dehydrogenases, it is converted to a brick red color. Thereby, it discriminates between viable (red) and dead myocardial tissue (white). For an overview on both invasive and non-invasive infarct size determination, readers are directed to a comprehensive review on this subject17.
Figure 1 shows the timeline including anesthesia, surgical preparation and primary outcome measurements used in this study.
1. Medication and Anesthesia
2. 3D Transesophageal Echocardiography (TEE)
3. Admittance-based Pressure-volume Loop Acquisition
4. Area At Risk (AAR) and Infarct Size (IS) Quantification
3D Transesophageal Echocardiography
3D transesophageal echocardiography (3D-TEE) can be used for the assessment of global cardiac function. After AMI, global cardiac function differs from healthy baseline values. In particular, left ventricular ejection fraction (LVEF) decreases from 59 ± 4% to 37 ± 6% after one week of reperfusion (n = 10) (GPJ van Hout, 2015). An increase in end-systolic volume (51 ± 7 to 82 ± 13 ml) and decrease in stroke volume (74 ± 11 to 47 ± 8 ml) is also observed, whereas end-diastolic volume does not differ between both time points (125 ± 14 to 129 ± 13 ml). Representative images one week after myocardial infarction (ischemia-reperfusion) are displayed in Figure 3. In our ample experience, we have not encountered any complications related to TEE.
Admittance-based Pressure-volume Loops
Pressure-volume (PV) loops can be used both to assess global cardiac function and specific intrinsic myocardial muscle properties. Outcome measurements of the former can be easily calculated from the graphs in Figure 4A and include EDV (lower right corner), ESV (upper left corner) and LVEF ((EDV – ESV)/EDV x 100%). Both, ESV and EDV provide important information on left ventricular geometry and LVEF is an important measure for determining left ventricular pump function. A previous study compared admittance-based PV loops to gold-standard cardiac magnetic resonance imaging (CMRI) in a pig model of AMI24. After eight weeks, PV loop measurements significantly overestimated both ESV and EDV. With regard to LVEF however, no significant difference was observed between PV loops and CMRI. In addition, both techniques showed a fairly good correlation of EDV and LVEF.
For intrinsic cardiac performance, different measurements can be derived from PV loops, such as end-systolic and end-diastolic pressure-volume relationship (ESPVR; EDPVR)25. Representative PV loop images with preload reduction and some examples of systolic and diastolic functional parameters are shown in Figure 4B. The ESPVR slope decreases, indicating decreased contractility. Additional valuable functional parameters that can be derived from PV loops are presented in Table 1.
Infarct Size/Area At Risk Quantification
In female Dalland landrace pigs (6 months; 60 – 70 kg), occlusion of the left anterior descending artery (LAD) directly distal to the first septal and first diagonal branch during 75 minutes leads to an area at risk (AAR) of 22 ± 2% of the left ventricle (LV) (n = 5) (GHJM Ellenbroek, 2015). Infarct size constitutes 16 ± 2% of the left ventricle and 73 ± 7% of the AAR. This fairly large IS/AAR has been chosen for as patients with a larger infarct size are more prone to the development of heart failure than patients with a smaller infarct size. In pigs, the greatest therapeutic benefit can therefore be gained when applying 75 minutes of ischemia. Moreover, due to greater infarct size, cardiac function deteriorates, which allows for functional improvement as well. When a shorter period of index ischemia is applied, cardiac infarct size is lower and function is only mildly impaired, which allows for only a very small window of functional improvement. Figure 5 shows a representative example of a TTC and Evans blue staining that allows clear identification of the 3 areas: 1) remote myocardium, 2) AAR, and 3) infarcted myocardium.
Figure 1. Timeline of the Experimental Protocol. This timeline provides an overview of the most important experimental steps in the used pig AMI model. Adequate induction of anesthesia is required prior to each measurement. Time indications can be observed under each proceeding. Infarct size is preferably assessed after 24 – 72 hours. 3D-TEE and PV loop data acquisition can be performed at baseline and at short- and long-term follow-up. The first hours after AMI, arrhythmias are frequent and can greatly interfere with cardiac hemodynamics and therefore prevent reliable data acquisition. AMI: acute myocardial infarction; 3D-TEE: three-dimensional transesophageal echocardiography; PV loop: pressure-volume loop. Please click here to view a larger version of this figure.
Figure 2. Positioning and Acquisition of 3D TEE Images. Anteroposterior (A) and mediolateral (B) X-ray images of the 3D-TEE probe positioning in the esophagus. Image acquisition follows upon correct visualization of the left atrium, left ventricle and aorta (C) and a perpendicular image of both left atrium and left ventricle (D). 3D-TEE: three-dimensional transesophageal echocardiography. Please click here to view a larger version of this figure.
Figure 3. Full Volume 3D-TEE Images of the Left Ventricle. 3D-TEE recordings of the left ventricle one week after acute myocardial infarction (75 minutes) and reperfusion. Perpendicular long-axis images of the left atrium and left ventricle can be observed in the upper half panel (A, B). A magnified example (C) of multiple cross-sectional images (D) of the left ventricle is displayed in the lower half panel. 3D-TEE: three-dimensional transesophageal echocardiography. Please click here to view a larger version of this figure.
Figure 4. Pressure-volume Loop Images at Baseline and After Myocardial Infarction. Representative PV loop images during apnea (i.e., pausing ventilation) at baseline (blue) and eight week after AMI (red) (A). Increases in EDV and ESV and a decrease in SV can be observed, indicating a decrease in LVEF (%). PV loop images with preload reduction are used to assess intrinsic myocardial function parameters (B). Compared to baseline, infarcted myocardium shows a decrease in contractility as derived from the ESPVR (straight blue and red lines). PV loop: pressure-volume loop; AMI: acute myocardial infarction; EDV: end-diastolic volume; SV: stroke volume; LVEF: left ventricular ejection fraction; ESPVR: end-systolic pressure-volume relationship. Please click here to view a larger version of this figure.
Figure 5. Infarct Size and Area-at-risk Staining. Representative image of infarct size and area-at-risk staining of the left ventricle after acute myocardial infarction (75 minutes) and subsequent reperfusion for three days. (Hemorrhagic) infarct tissue can be observed in rosy brown and gray-white, whereas the border zone is stained red. Surrounding blue-stained areas indicate remote myocardium. Please click here to view a larger version of this figure.
Volume parameters | Pressure parameters | Loading-independent parameters | Other | |||
Systolic | Diastolic | Systolic | Diastolic | Systolic | Diastolic | Other |
ESV | EDV | ESP | EDP | ESPVR | EDPVR | HR |
dP/dT | dP/dT | Ees | PRSW | SW | ||
Derivatives | τ (tau) | ESV100 | PRSW | |||
LVEF, SV, CO | PHT | Ea | dP/dV |
Table 1. Valuable Functional Parameters that can be Derived from Pressure-volume Loops. Categorized into volume, pressure and loading-independent parameters, this table describes the most commonly used (systolic and diastolic) parameters derived from PV loops. PV loops: pressure-volume loops; ESV: end-systolic volume; EDV: end-diastolic volume; LVEF: left ventricular ejection fraction; SV: stroke volume; CO: cardiac output; ESP: end-systolic pressure; dP/dT: derivative of pressure; τ (tau): isovolumic relaxation constant; PHT: pressure half-time; ESPVR: end-systolic pressure-volume relationship; Ees: end-systolic elastance; ESV100: end-systolic volume corrected for pressure (100 mmHg); Ea: arterial elastance; EDPVR: end-diastolic pressure-volume relationship; PRSW: preload recruitable stroke work; HR: heart rate; SW: stroke work; dP/dV: EDPVR slope (chamber stiffness).
Cardiac remodeling is largely depending on myocardial infarct size and the quality of myocardial infarct repair6,26. To assess the former in a standardized manner, the present manuscript provides an elegant method of in vivo infusion of Evans blue combined with ex vivo TTC staining, which has been validated and extensively used8,16,27,28. This method allows for quantification of the area at risk (AAR) and infarct size in relation to AAR16. The current approach reduces the risk of dye diffusion into the AAR, the infarct region or — with malpositioning — papillary muscle, as it does not require myocardial puncture. Moreover, there is no need for external ligation of the coronary artery, which may be imprecise, inaccurate and occasionally damage the myocardium. An alternative method, combining catheter-based Evans blue infusion into the LV and cross-clamping of the ascending aorta29, is undesirable for different reasons. Clamping occludes the left ventricular guiding catheter, hampering Evans blue infusion into the LV. Besides, compression and traction forces may lead to LCMA catheter and intracoronary balloon malpositioning and inaccurate AAR measurements. Moreover, since balloon occlusion of the LAD requires guiding catheter positioning in the LCMA, coronary filling from the LV is limited, preventing Evans blue entry from the LV into the coronary artery.
However, though superior to myocardial puncture and cross-clamping of the ascending aorta, the technique presented in this manuscript requires some precautions. Complete occlusion of (one of the) coronary arteries through an obstructing guiding catheter needs to be prevented. This can be controlled by monitoring wash-out rates and pressure, and can usually be avoided by slightly retracting the guiding catheter from of the coronary ostium. If inevitable, shorten the time the guiding catheter is positioned in the coronary artery as much as possible by preparation of other parts of the protocol. In addition, make sure the balloon completely occludes the target vessel before Evans blue infusion.
When Evans blue infusion is completed, induce VF and incise the caval vein to release blood pressure before balloon deflation and catheter withdrawal in order to prevent Evans blue diffusion into the AAR. Care should be taken to gently but firmly position the guiding catheter in the coronary ostium, allowing for diffusion of Evans blue in both the LAD and LCx. In addition, Evans blue infusion rates should not be too high since limited flow into the coronary arteries may lead to Evans blue wash-out into the systemic circulation. Although selectively infused into the coronary arteries, Evans blue diffusion into the systemic circulation cannot be completely prevented. Therefore, histologic analysis of other non-cardiac tissue (e.g., spleen, kidney) may still be problematic. Simultaneous TTC co-infusion into the AAR has been described before, but is undesirable in our opinion, as TTC does not reach the part of the AAR obstructed by the balloon. Moreover, previous analyses show that TTC might react with residual intravasal blood in the infarct area and overlap with the red color in the non-infarct AAR30. Future applications of this technique could be to preserve non-cardiac tissues by obstructing blood flow into the systemic circulation. This could be achieved by balloon obstruction of the descending thoracic aorta through a femoral approach.
Echocardiography to date remains a cornerstone for cardiac function assessment in both clinical care and various animal models in cardiovascular research. However, due to the thoracic shape of landrace pigs, transthoracic echocardiography (TTE) is limited to 2-dimensional long- and short-axis views of the LV9. Therefore, cardiac volume and LVEF have to be estimated by mathematical approximations such as the modified Simpson's rule, which assumes a cylindrical left ventricular morphology10. As a result of LV remodeling after MI however, cardiac dimensions change. Therefore, this particular geometric assumption cannot be made, reducing the accuracy and reliability of such measurements31.
This problem can be solved by using 3D echocardiography to obtain 3D images of the complete left ventricle. In pigs, LVEF assessment by epicardial 3D echocardiography shows excellent correlation with gold-standard CMRI24,32. However, this requires surgery prior to AMI induction for baseline measurements. Regardless of the approach, i.e., open chest vs. subxiphoidal approach, invasive surgery for epicardial echocardiography has been shown to be cardioprotective12,33,34. Concomitant adhesions hinder resternotomy, which renders epicardial echocardiography undesirable for baseline measurements in a closed-chest AMI model.To avoid these drawbacks, 3D images of the heart can be obtained through 3D transesophageal echocardiography (3D-TEE). This technique is portable, widely available and allows for serial measurements and visualization of the entire left ventricular volume. Moreover, it is reliable, relatively inexpensive and safe.
Note that it is important to gently insert the TEE probe into the mouth and esophagus, since ending up in the Zenker's diverticulum and applying too much pressure may lead to esophageal rupture. Moreover, since the anatomical relationship between stomach and heart differs from man, 3D-TEE in pigs does not allow for regional measurements (e.g., strain, tissue Doppler imaging) and is restricted to volume measurements. In the data presented in the manuscript, no increase of EDV 7 days after AMI was observed. A longer follow-up period is needed to extensively drive adverse remodeling, leading to an increased EDV at several weeks follow-up11.
In contrast to conventional echocardiography, admittance-based PV loops moderately overestimate LV volumes, both at baseline35 and after 8 weeks of follow-up24. Still, fairly good correlations and high degrees of agreement with CMRI have been found. Although PV loop measurements several weeks after AMI are less precise compared to baseline, LV dimensions and derivatives hereof (LVEF) are useful for the global assessment of cardiac function35.
In addition, PV loops provide specific information about intrinsic myocardial properties, such as ESPVR. Since regional functional measurements in TTE and TEE are limited and epicardial echocardiography is undesirable at baseline, PV loops provide an elegant and safe technique for the assessment of intrinsic myocardial function. Both, the decline in ESPVR slope and the typical shift in V0 can be used to compare different therapeutics. These classical features are validated in ex vivo canine heart suffering from pan-ischemia. Hence, in regional ischemia models, like AMI models, these specific characteristics are not always present, which can be attributed to many factors, of which ventricular remodeling and regional ischemia are the most important ones25,36,37.
For adequate data acquisition, it is critical to make sure no arrhythmias are present when converting conductance to volume and when acquiring PV loops. If arrhythmias are present, reposition the PV loop catheter so it does not irritate the myocardium. The administration of anti-arrhythmic drugs (e.g., 150 – 300 mg amiodarone) may also help. However, note that PV loop acquisition within several hours after acute myocardial infarction is not reliable due to frequent arrhythmias (e.g., premature ventricular complexes, bigemini).
Slightly advancing or retracting the PV loop catheter into the LV or from the muscular wall may also help to improve the shape of the PV loops. After changing PV loop catheter positioning, always double-check that the largest adequately placed segment is selected.
In conclusion, the current paper introduces three methods for cardiac assessment in a previously described pig AMI model with additional value for the evaluation of new therapeutics to decrease the burden of the ongoing heart failure epidemic.
The authors have nothing to disclose.
The authors gratefully acknowledge Marlijn Jansen, Joyce Visser, Grace Croft, Martijn van Nieuwburg, Danny Elbersen and Evelyn Velema for their excellent technical support during the animal experiments.
3-dimensional transesophageal echocardiography | |||
iE33 ultrasound device | Philips | – | |
X7-2t transducer | Philips | – | |
Aquasonic® 100 ultrasound transmission gel | Parker Laboratories Inc. | 01-34 | Alternative product can be used |
Battery handle type C (laryngoscope handle) | Riester | 12303 | |
Ri-Standard Miller blade MIL 4 (laryngoscope blade) | Riester | 12225 | |
Qlab 10.0 (3DQ Advanced) analysis software | Philips | – | |
Name | Company | Catalog Number | Comments |
Pressure-volume loop acquisition | |||
Cardiac defibrillator | Philips | ||
0.9% saline | Braun | ||
8F Percutaneous Sheath Introducer Set | Arrow | CP-08803 | Alternative product can be used |
9F Radifocus® Introducer II Standard Kit | Terumo | RS*A90K10SQ | Alternative product can be used |
8F Fogarty catheter | Edward Life Sciences | 62080814F | Alternative product can be used |
7F Criticath™ SP5107H TD catheter (Swan-Ganz) | Becton Dickinson (BD) | 680078 | Alternative product can be used |
Ultraview SL Patient Monitor and Invasive Command Module (external cardiac output device) | Spacelabs Healthcare | 91387 | Alternative product can be used |
ADVantage system™ | Transonic SciSense | – | |
7F tetra-polar admittance catheter (7.0 VSL Pigtail / no lumen) | Transonic SciSense | – | |
Multi-channel acquisition system (Iworx 404) | Iworx | – | |
Labscribe V2.0 analysis software | Iworx | – | Alternative product can be used |
Name | Company | Catalog Number | Comments |
Infarct size / area-at-risk quantification | |||
Diathermy | – | Alternative product can be used | |
Lebsch knife | – | Alternative product can be used | |
Hammer | – | Alternative product can be used | |
Bone marrow wax | Syneture | Alternative product can be used | |
Klinkenberg scissors | – | Alternative product can be used | |
Retractor | – | Alternative product can be used | |
Surgical scissors | – | ||
7F Percutaneous Sheath Introducer Set | Arrow | CP-08703 | Alternative product can be used |
8F Percutaneous Sheath Introducer Set | Arrow | CP-08803 | Alternative product can be used |
7F JL4 guiding catheter | Boston Scientific | H749 34357-662 | Alternative product can be used |
8F JL4 guiding catheter | Boston Scientific | H749 34358-662 | Alternative product can be used |
COPILOT Bleedback Control Valves | Abbott Vascular | 1003331 | Alternative product can be used |
BD Connecta™ | Franklin Lakes | 394995 | Alternative product can be used |
Contrast agent | Telebrix | ||
Persuader 9 Steerable Guidewire 9 (0.014", 180 cm, straight tip), hydrophilic coating | Medtronic Inc. | 9PSDR180HS | Alternative product can be used |
SAPPHIRE™ Coronary Dilatation Catheter (PTCA balloon suitable for the size of the particular coronary artery (2.75 – 3.25 mm)) | OrbusNeich | 103-3015 | Alternative product can be used |
Evans Blue | Sigma-Aldrich | E2129-100G | Toxic. Alternative product can be used |
2,3,5-triphenyl-tetrazolium chloride (TTC) | Sigma-Aldrich | T8877-100G | Irritant. Alternative product can be used |
9V battery | – | – | |
Ruler | – | – | |
Photocamera | Sony | – | |
ImageJ | National Institutes of Health | – | Alternative product can be used |