Animals
27 healthy male C57BL6 mice, 10–12 weeks of age and with a body weight of 29 ± 3 g underwent DSE. The animals were housed under constant temperature (21–25°C) and in a 12 h light/dark cycle and maintained on water and food ad libitum. This experiment has been carried out with approval of the regional Animal Ethics Committee at Göteborg University, Göteborg, Sweden.
Anesthesia
The thorax was shaved mechanically and chemically and the mice were placed in a slight left decubitus position on an electrical heating pad (38.5–39°C) to maintain normothermia. All animals were weighed and lightly anesthetized with 1.1–1.2% isoflurane (Abbot Scandinavia AB, Solna) via a nose cone during the echocardiographic examination (Figure 1B).
Echocardiography
Echocardiography was performed at rest and during dobutamine stress before induction of MI. Examinations was performed using a high-frequency 15-MHz linear transducer (CL 15-7, Philip Medical System, Best) connected to a HDI 5000 ultrasound system (ATL, Philip Medical System, Best) see figure 1A and 1C. All data were stored and later evaluated off-line on an Echo-Pac (Vingmed, Horten) system by one investigator blinded to the animals' identity. For optimal orientation, the long-axis view was first performed, followed by a short-axis view of the pulmonary artery (PA) for pulsed-wave Doppler [10]. Cardiac output (CO) was calculated as CO = PA velocity time integral*π*(0.01/2)2 *HR. From an optimal parasternal short-axis view, two-dimensional targeted M-mode recordings were obtained at the level of the papillary muscles. End-diastolic (LVEDd), end-systolic (LVESd) LV dimensions and posterior wall thickness (LVPWd) were measured according to the leading-edge method of the American Society of Echocardiography [11]. Fractional shortening (FS, %) was calculated as FS = (LVEDd-LVESd)/LVEDd*100. Two-dimensionally guided pulsed Doppler recordings of LV outflow were obtained from the apical "four-chamber" view for measurements of heart rate and ejection time (ET, ms) [12]. Velocity of the circumferential fiber shortening (Vcf c, circ/s) was calculated as Vcf c = ((LVEDd-LVESd)/LVEDd)/ETc(s), where ETc is ET corrected for heart rate. All measurements were based on the average of at least three cardiac cycles.
Dobutamine stress
Dobutamine (Dobutrex, Eli Lilly Sweden AB, 250 mg) 1 μg/g body weight was given intraperitoneally. Cardiac reserve was investigated 8 min after dobutamine injection.
Induction of myocardial infarction
To keep the mice sedated and support breathing during the operation, the mice were anesthetized with isoflurane, orally intubated and connected to a respirator (SAR-830 small animal ventilator, GENEQ inc., Montreal, Canada) distributing a mixture of oxygen, air and 2–3% isoflurane. Electrodes were placed on the extremities and connected to an ECG-monitor in order to observe the cardiac rhythm during surgery. An incision was made between the 4th and 5th rib, revealing the upper part of the anterior LV wall and the lower part of the left atrium. An extensive MI was induced by ligation of the left anterior descending (LAD) coronary artery immediately after the bifurcation of the left coronary artery. The efficacy of the procedure was immediately verified by characteristic ECG-changes, and akinesis of the left ventricular anterior wall. After verification of MI the lungs were hyperinflated, positive end-expiratory pressure was applied and the chest was closed. The mice received an i.p. injection of 0.1 ml Temgesic to relieve postoperative pain and the mice recovered spontaneously when the isoflurane was turned off. For study design see figure 2.
Statistics
Student's t-test was used for statistical analysis of differences between the survivors and the deceased animals. ANOVA was used to evaluate differences among the three groups. Results are expressed as mean ± SD in tables and mean ± SEM in figures. Values of p < 0.05 were considered to be statistically significant. Data were analyzed using SPSS 11.0 for Windows (Chicago, Ill, USA)