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Basic Science Aspects

ECHOCARDIOGRAPHY ASSESSMENT OF MYOCARDIAL FUNCTION AFTER BURN INJURY

Maass, David L.*; Naseem, R. Haris; Garry, Mary; Horton, Jureta W.*

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doi: 10.1097/01.shk.0000217814.93
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Abstract

INTRODUCTION

We have described previously that burns over 40% of the total body surface area (TBSA) alters myocardial systolic and diastolic function (1-7) and have proposed that this severe but transient myocardial dysfunction predisposes the injured subject to subsequent infectious complications (8-11). In both adult and pediatric burn injury, sepsis is the primary determinant of morbidity and mortality (12-14), and infection produces multiple organ dysfunction in 83% of patients with burn more than 20% TBSA. (15) Although the ability to characterize the time course of left ventricular (LV) dysfunction in a reproducible and noninvasive manner after injury has great clinical relevance, assessment of cardiac function requires invasive monitoring, and/or collection of ventricular muscle or myocytes. Echocardiography is widely used in clinical and experimental settings to assess cardiac systolic and diastolic function in a sequential and noninvasive manner (16-22). The use of transthoracic echocardiography (TE) to examine cardiac function in rodents has eliminated the need to sacrifice animals to prepare cardiomyocyte or LV muscle preparations for in vitro study (23-28). We have previously used TE to measure LV function in rats and mice with heart failure. For example, we have induced LV dysfunction with coronary artery ligation (29, 30), cryoinjury of the myocardium (31), and with hypoxia (32), demonstrating the sensitivity of this technique in the rodent under a variety of conditions. In addition, we have previously determined that measurements made with TE yield similar results when compared with invasive hemodynamic measurements (31).

Cutaneous burn injury produces several types of tissue injury, including increased capillary leak, the formation of platelet-derived thrombi and vessel occlusion, and tissue edema. Tissue injury, particularly chest wall edema, may complicate assessment of cardiac function using TE. Thus, this present study compared in vivo echocardiography and in vitro physiological measurements of cardiac function in adult C57/BL6 mice given a major burn injury. Specifically, TE was performed before and at several intervals for 8 days after burn over 40% TBSA. Parallel groups of mice were killed at these times, and LV function was studied in vitro using a Langendorff model. We hypothesized that application of TE in awake mice would provide in vivo assessment of myocardial performance that paralleled in vitro assessment of LV function using isolated perfused hearts (Langendorff).

MATERIALS AND METHODS

Animal model

Adult C57/BL6 mice, 9 to 10 weeks of age, were obtained from Jackson Laboratories (Bar Harbor, Me) and housed in the animal vivarium with a 12-h light/dark cycle. Mice were acclimatized for 7 to 9 days after arrival with food and water available at will. This study was reviewed and approved by the Institutional Review Board for Animal Research at the University of Texas Southwestern Medical School and was performed in accordance with National Institutes of Health guidelines for the use of laboratory animals. All mice were weighed immediately before anesthesia. After completing deep level inhalation anesthesia (isoflurane), the side and back of each mouse were closely clipped and then carefully shaved from the base of the tail to the base of the neck. Animals were then randomly assigned to sham burn or burn groups. In those animals designated for burn trauma, a cutaneous burn injury equal to 40% TBSA was produced by applying brass probes (1 × 2 cm with a 3-mm thickness), heated in 100°C boiling water, to the animals side and back for 5 seconds. The TBSA was calculated by using murine-specific date(33, 34); this calculation was verified by removing the animal pelt and measuring the actual burned area (34). The percentage of burned area was then calculated on the basis of the animal s measured TBSA. Animals designed for sham burn groups received identical regimens of anesthesia and handling, but no burn injury was given. After completing burn trauma, the mice were intraperitoneally administered lactated Ringer fluid resuscitation (4 mL/kg per % burn with half the volume administered during the first 8 h and the remaining volume given over the next 16 h postburn). All animals (both sham burnand burn) received analgesic (buprenorphine, 0.05 mg/kg s.c.) every 12 h after burn trauma. Animals were monitored closely for the first 8 h after burntrauma to determine adequate recovery from the anesthesia, animal responsiveness to external stimuli, the absence of pain, and the ability to consume food and water.

Determination of cardiac dysfunction by echocardiography

Echocardiograms to assess systolic function were performed using motion mode (M-mode) measurements. Hair was removed from the thorax and upper abdomen as described previously, and Nair (Princeton, NJ) hair remover was applied for 1 to 2 min; noninvasive transthoracic echocardiograms (General Electric Vivid7 Pro machine equipped with a 12-mHz transducer) were performed in conscious unsedated mice in a random and blinded manner (35). Images were obtained at baseline and 8, 12, 24, 48, 72 h, and 1 week after burn injury. Measurements on days 1, 2, 3, and 8 were performed at the same time of the day between 10 am and noon. M-mode and two-dimensional TE images were obtained in the parasternal short axis with a frame rate of 200 to 500 frames/s. The transducer was placed on the left hemithorax interfaced with a layer of US transmission gel (Aquasonic 100; Parker Laboratories, Fairfield, NJ). The two-dimensional parasternal short-axis imaging plane guided LV M-mode tracings close to the papillary muscle level. Depth was set at 2 cm with a sweep speed of 100 mm/s. Heart rate was calculated by multiplying number of beats in a second on a M-mode tracing by 60. Fractional shortening was calculated from M-mode images as the LV end-diastolic dimension (LVEDD) minus the LV end-systolic dimension (LVESD) divided by LVEDD.

M-mode measurements

Data represent the average of 9 consecutive cardiac cycles from at least 2 separate scans. End diastole was defined as the maximal LV diastolic dimension, and end systole was defined as the peak of posterior wall motion. FS %, a surrogate of systolic function, was calculated from LV dimensions as follows: FS % = (LVEDD − LVESD)/ LVEDD × 100, as shown in Figure 1, where LVEDD and LVESD at end diastole and end systole, respectively.

Fig. 1
Fig. 1:
Representative M-mode echocardiography of the same mouse at baseline, 12 h postburn, and 1 week postburn. Note increased LVEDD and decreased LVESD 12 h postburn as compared with baseline myocardial function. On postburn day 7, function returned to baseline level in the same mouse with no significant difference between baseline and postburn day 7 (P < 0.05). Lines represent LVEDD (DD) and LVESD (SD). Heart rate (HR) at baseline was 600 bpm (A), 12 h postburn HR was 360 bpm (B), whereas HR 7 days postburn was 720 bpm (C).

Langendorff perfused hearts

To examine cardiac contraction and relaxation in vitro, mice were anticoagulated with sodium heparin (200 U; Elkins-Sinn, Inc, Cherry Hill, NH) and cervically dislocated at designated intervals after burn injury. The heart was rapidly removed and placed in 4°C Krebs-Henseleit bicarbonate-buffered solution (pH, 7.4; PO2, 550 mmHg; PCO2, 38 mmHg). A cannula placed in the ascending aorta was used to maintain perfusion of the coronary artery (mL/min) by retrograde perfusion of the aortic stump cannula, as previously described for guinea pig, rat, rabbit, and mouse hearts (34, 36-39). Contractile function was assessed by measuring intraventricular pressure with a saline-filled latex balloon. Peak systolic LV pressure (LVP) was measured with a Statham pressure transducer (model P23ID; Gould Instruments, Inc, Oxnard, Calif) attached to the balloon cannula, and the rate of LVP rise (+dP/dt) and fall (−dP/dt) were obtained by using an electronic differentiator (model 7P20C; Grass Instruments, Inc, Quincy, Mass) and recorded (model 7DWL8P; Grass Recording Instruments). In addition, LVP and ±dP/dt responses to increases in either preload (LV volume) or perfusate calcium were examined. Data from the Grass recorder were input to a Dell Pentium computer (Dell Inc., Round Rock, Texas) and a Grass Poly VIEW Data Acquisition System (Grass Instruments, Quincy, Mass) was used to convert acquired data into digital form.

Statistical analysis

Cardiac function determined by the Langendorff preparation (including stabilization data) is expressed as mean ± SEM; separate analyses were performed for each parameter measured (e.g., LVP and +dP/dt max) as a function of treatment group and left ventricular volume using a repeated measures analysis of variance. A multiple comparison procedure using the Bonferroni method was used to determine significant differences between groups. Cardiac function determined by M-mode echocardiography is expressed as fractional shortening ± SEM and analyzed using a 1-way repeated-measure analysis of variance. Additional comparisons were performed using the Tukey test to determine significant differences between specific groups. Statistical significance for all analyses was defined as P < 0.05. All statistical analyses were performed using SigmaStat 2.03 (SPSS, Chicago, Ill) and Microsoft Excel (Microsoft, Seattle, Wash). Correlation between LV function (LV developed pressure) measured in the isolated Langendorff preparation and % fractional shortening measured by echocardiography was evaluated by Spearman Rho (SPSS version 14.0).

RESULTS

All mice survived for 8 days after burn injury. Mice resumed feeding within 30 min after recovering from anesthesia for the burn procedure, and all mice gained weight during the 8-day experimental period, as expected. Before the study, mice were handled on several occasions to condition or train them for the echocardiographic procedure in the absence of anesthesia. After several handling periods, the mice remained calm, and there was no evidence of bradycardia associated with the echocardiographic procedure.

Echocardiography in awake mice

A representative echocardiogram obtained from burned mice 12 h postburn is shown in Figure 1. The time course of burn-related changes in fractional shortening as measured by echocardiography in C57/BL6 mice during the 8-day study period is summarized in Figure 2. Burn injury altered ventricular performance in the adult mouse, but there were minimal chronotropic effects. There was a 26% ± 1% reduction in fractional shortening 12 h after burn, and fractional shortening gradually recovered over the first 3 days after burn injury. On day 8 postburn, there was no significant difference in fractional shortening measured in burned and in sham-burned animals. Heart rate measured by echocardiography was 640 ± 13 beats/min before burn injury; heart rate was 660 ± 13 beats/min 24 h postburn and 690 ± 13 beats/min 72 h postburn.

Fig. 2
Fig. 2:
Effects of burn injury (n = 10) or sham burn in controls (n = 10) on fractional shortening. All values are expressed as mean ± SEM. *Significant difference (P < 0.05) from baseline (preburn). †Significant difference from sham at P < 0.05. Analysis of variance and Student-Newman-Keuls test were used.

In vitro assessment of cardiac function using a langendorff model

Table 1 includes cardiac stabilization data collected in hearts, collected at several periods after burn injury and perfused at a constant LV end-diastolic volume and constant coronary flow rate. There was no change in cardiac function 6 h after burn injury; measures of cardiac contraction and relaxation tended to fall by 12 h postburn, but these changes did not achieve statistical significance. By 18 h postburn, there was a significant fall in LV developed pressure (peak systolic LVP minus LV end-diastolic pressure), and a significant reduction in ±dP/dt max (P < 0.05). In addition, LV developed pressure measured at 40 mmHg was significantly lower 18 h after burn injury compared with that measured in sham burns. LV contraction and relaxation defects measured during stabilization of hearts persisted in hearts collected 24 and 48 h after burn, whereas contractile function returned to values measured in shams 72 postburn. There was no significant difference in LVP and ±dP/dt measured in hearts collected day 8 postburn and compared with values measured in sham burns. LV function (LV developed pressure) measured in isolated hearts perfused in a Langendorff mode was positively correlated with % fractional shortening measured by echocardiography; Spearman Rho correlation coefficient was 0.417, P = 0.004. Linear regression analysis showed that the slope of the regression curves for both data sets (function measured in the Langendorff preparation and function measured by TE) were equivalent, indicating a direct linear relationship between these parameters.

Table 1
Table 1:
Cardiac Stabilization Data Collected in Hearts Perfused at Constant LV End-Diastolic Volume and Constant Coronary Flow Rate and Constant Heart Rate

To further explore the time-related effects of burn injury on myocardial contraction and relaxation, LV developed pressure (peak-systolic LVP minus LV end-diastolic pressure) was calculated for all hearts and plotted versus incremental increases in LV volume, preload (Fig. 3). Similarly, the rate of LVP rise (+dP/dt max), and the rate of LVP fall were calculated for all experimental groups as a function of increases in perfusate calcium. As shown in Figure 3, LVP and ±dP/dt max responses to increases in LV volume were significantly reduced 12, 24, and 48 h after burn injury. There was no significant difference in LVP and ±dP/dt responses to increases in preload 72 h or 8 days after burn injury compared with responses measured in sham burns. Similar patterns of inotropic responses to a nonadrenergic inotropic challenge (increases in perfusate calcium) were evident in burns compared with shams with significant contraction and relaxation defects confirmed 12, 24, and 48 h after burn injury (Fig. 4); systolic and diastolic responses to increases in perfusate calcium were identical in sham and burned animals 72 h and 8 days after injury.

Fig. 3
Fig. 3:
LV developed pressure (LVP = peak systolic LVP minus LV end-diastolic pressure) (A), +dP/dtmax (B), and −dP/dtmax (C) responses to incremental increases in LV volume or preload measured in subgroups of hearts harvested at several times postburn. Open symbols are sham groups; closed symbols are burns. All values are mean ± SEM. *Significant difference (P < 0.05) from sham. Analysis of variance and Student-Newman-Keuls test were used.
Fig. 4
Fig. 4:
LV developed pressure (LVP mmHg) (A), +dP/dtmax (B), and −dP/dtmax (C) responses to incremental increases in perfusate calcium. All values are mean ± SEM. *Significant difference (P < 0.05) from sham. Analysis of variance and Student-Newman-Keuls test were used.

DISCUSSION

Although we have previously reported that burn injury produces significant myocardial contraction and relaxation defects (35), those previous studies required animals to be killed; hearts or ventricular muscles were then used for in vitro assessment of contraction and relaxation responses to burn injury. The availability of a noninvasive, reproducible means of assessing cardiac function before burn injury as well as throughout the postburn period allowed each animal to serve as its own control. This present study confirmed that echocardiographic measurements of cardiac function after burn injury paralleled the in vitro Langendorff results and confirmed burn-related myocardial contractile dysfunction.

A novel aspect of this present study is that echocardiographic measurements over the postburn period were conducted in awake animals, eliminating the cardiodepressive effects of anesthesia. Intraperitoneal and inhaled anesthetic agents have been shown to alter heart rate, fractional shortening, and end-diastolic dimensions (24). Similarly, Yang and colleagues (23) compared echocardiography in nonanesthetized mice and in mice given sodium pentobarbital, xylazine, or inhaled isoflurane. These investigators confirmed the adverse effects of anesthesia on baseline cardiac function and confirmed that anesthetics complicated data interpretation (23). Training the animals by handling before injury and repeated measures of echocardiography studies eliminated the initial bradycardia noted with handling. In our study, animals were grasped by the nape of the neck and held firmly in the supine position in the palm of 1 hand as described by Yang and colleagues (23). Mice remained calm and did not struggle during the echocardiographic measurements.

In this present study, our assessment of cardiac function by echocardiography was limited to the measures of heart rate and measures of LVEDD and LVESD and calculation of fractional shortening. In our study, TE was shown to be reproducible and rapid and to provide easy yet reliable assessment of LV function. Echocardiography can be particularly susceptible to preload conditions. Fluid resuscitation therapies can confound results obtained via echocardiography by altering preload status in treated animals. In this study, however, we were able to corroborate our in vivo assessment of LV function with the in vitro Langendorff preparation, which is a reliable measure of cardiac contractility that is marginally influenced by preload or after load. Finally, in a separate study, we have verified our results using echocardiography with invasive hemodynamic measurements, which are not effected by preload and after load (31). Echocardiography has been used widely in the clinical arena to assess myocardial function, and the present study provided excellent correlation between measures of LV function as determined by in vitro Langendorff and in vivo echocardiography. Recent developments of new ultrasound-related technologies offer an additional objective and noninvasive means to assess left and right ventricular function in the clinical arena (21, 40, 41).

In summary, our measures of LV function in awake mice confirmed burn-related decreases in LV contractile performance as measured by either an in vitro Langendorff preparation or in vivo echocardiography. Our data suggest that echocardiography provides a noninvasive reliable means of detecting systolic and diastolic myocardial dysfunction after major injury such as burn trauma.

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Keywords:

Left ventricular performance; mice; dP/dt max; Langendorff model

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