Early myocardial dysfunction in streptozotocin-induced diabetic mice: a study using in vivo magnetic resonance imaging (MRI)

6–8 week old C57BL/6 female mice were obtained from Harlan (Indianapolis, IN) and housed at the MRI animal facility. All animals had free access to standard laboratory chow and water. Animal protocols were approved by the local Institutional Animal Care and Use Committee.

Mice were injected intraperitoneally with 100 mg/kg of STZ (in 0.05 mol/l citrate buffer, pH 4.5, Sigma, St. Louis, MO) or vehicle once a day for two consecutive days. Tail blood glucose (TBG) levels were measured using a glucose oxidase test strip (Lifescan, Milpitas, CA). Animals were considered to be diabetic if their TBG is > 13 mmol/l. The blood glucose levels and body weight of all mice were monitored biweekly. When their TBG is > 22 mmol/l, they were given intermittent low dosages of long acting insulin glargine [30] to maintain blood glucose at 19–25 mmol/l. Mitochondrial NADH oxidase activity was determined in ventricular tissues from diabetic mice, and MRI studies were performed on diabetic mice at 4 weeks. Age matched controls were included for comparisons.

For MRI studies, mice were anesthetized with inhaled isoflurane (1.5% at 1 L/min oxygen flow) via a nose cone. During the experiment, the mouse was positioned supine on a nonmagnetic warming pad to maintain constant body temperature throughout the MR study.

Experiments were performed on a 7 Tesla MR scanner (Bruker BioSpin, Ettlingen, Germany). For cardiac imaging the gradient echo sequence [31] with t_e = 4.0 ms, t_r = 100 ms, and a flip angle, θ = 85°–90° was used for all experiments. The heart was initially positioned by measurement to the center of the magnet and then further set by observing the position using fast imaging with steady state precession (FISP) [32] as the animal was moved. Once the heart was in the iso-center a gradient echo sequence with t_e = 4.0 ms and t_r = 50 ms was used to acquire single slice images in all three (axial, sagittal and coronal) planes. The slice position for the cine was set using this pilot scan. For cine and volume imaging a field of view, FOV = 2.56 × 2.56 cm was chosen and 256 each of phase encode and read steps were used to resolve the spatial distribution of excited spins. The image was made after zero filling in each direction to 512 × 512. Window functions were not used and some minor image quality enhancement may be achieved if window functions are used. For our purpose the signal contrast of the myocardium and "white" blood was sufficient to allow calculation of the relevant parameters.

Spectrometer triggering with combined respiratory and ECG triggers was used in all experiments. Thus, all MRI data are adjusted to accommodate for rate changes and the data are reflective of the ambient cardiac function and rate. The cardiac period, P, of mice used in these studies ranged from ~120–180 ms, and a respiratory rate of ~18–30 bpm. Trigger delays, t_d, were incremented at the spectrometer hardware in a chosen number of steps, n_steps, (n_steps = 10) according to t_d = (P-(P/n_steps))/P, where period is the time between the peaks of the R wave in the ECG trace. The images at each increment of the period were combined and displayed by cine in the mid-ventricular short axis slice. These provided data for assessment of left ventricular (LV) systolic and diastolic dynamics, and for calculations of fractional circumferential shortening and wall thickening after measuring the endocardial circumferential length and myocardial wall thickness at end diastole and end systole.

For volume calculations, a new set of images were collected with the slice position advanced by the slice thickness of 1.0 mm. In order to reduce the time that multi-slice experiments fit in to the repetition time (Tr) used for single slice experiments, the slice thickness was increased to 1.25 mm. Thus, in this case, the resolution in the slice shifting is compromised by 0.25 mm at most, and contributes an error of about 0.1% in total volume calculations compared with a 1 mm slice thickness. The total blood volume at end diastole and end systole was estimated by taking the sum of all cavity slice volumes assuming a uniform thickness of excitation across a chosen slice at the two trigger points.

For LV blood volume measurements, endocardial borders were manually delineated. An independent observer was used whenever a questionable or indistinct image was encountered. These measurements rarely changed the summed data obtained from a given data acquisition. The end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated as the sum of all cavity slice volumes at end diastole and end systole. For assessment of LV systolic and diastolic dynamics, the cavity slice volume was measured in all acquired images and was plotted against the time from onset of the QRS trigger, and a volume-time curve was established. Peak ejection rate and peak filling rate were calculated from the maximum slopes of the LV ejection and filling curve [33].

M-mode/Doppler echocardiography was performed using a 13 MHz 15L8 probe and an Acuson Sequoia 256 echocardiography system (Acuson, Mountain View, CA). The wall and cavity dimensions during diastole and systole were determined from long-axis views of the left ventricle [34]. The mitral flow velocities were assessed by Doppler, and the E wave and A wave were measured to provide an estimate of diastolic function [35].

Frozen and thawed mitoplasts from hearts were diluted into 10 mmol/l 3-(N-morpholino)propane-sulfonic acid (MOPS), 25 mmol/l KCl at pH 7.4 to a final concentration of 50 μg/ml [36]. NADH oxidase activity was measured as the rate of NADH disappearance at 340 nm upon addition 200 μM NADH [36]. Citrate synthase activity was monitored by detection of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reactive CoASH (412 nm, ε = 13,600) upon addition of 0.1 mmol/l DTNB, 0.3 mmol/l acetyl CoA, and 0.5 mmol/l oxaloacetate to frozen and thawed mitoplasts (20 μg/ml) [37]. All enzyme assays were performed at room temperature.

Data are expressed as mean ± SEM. An unpaired 2-tailed Student t test was used to make comparisons between control and diabetic groups. Significance was ascribed to P values < 0.05.

The mice generally developed significant elevation of blood glucose within a week following STZ injection (Figure 1). The STZ-treated mice showed blood glucose levels ranging from 17 to over 27 mmol/l as compared with 5.5–8.3 mmol/l for citrate-treated control mice. Diabetic mice with blood glucose levels of > 22 mmol/l were given low dosages of long acting insulin (1–1.5 IU s.c.) daily to maintain their blood glucose at 19–25 mmol/l. After 4 weeks of diabetes, the diabetic mice had a 13% lower body weight and an 11% lower heart weight than non-diabetic control mice. The ratio of heart weight to body weight was not significantly changed (Table 1).

The animals tolerated the anesthesia and procedure without difficulty. There were no post study deaths. The fast-gradient echo MR technique provides sufficient contrast between blood and myocardium to allow clear delineation of epicardial and endocardial borders (Figure 2). LV diameters and wall thickness were measured in mid-ventricular short-axis slices at end diastole and end systole. After 4 weeks of diabetes, no significant change of endocardial diameters at end diastole was observed (3.6 ± 0.1 vs. 3.4 ± 0.1 mm). However, endocardial diameters were significantly increased at end systole (2.4 ± 0.1 vs. 1.8 ± 0.1 mm, P < 0.001). Diabetic mice also showed significant thinning of the LV wall compared to control animals both at end diastole (0.85 ± 0.03 vs. 1.01 ± 0.05 mm, P < 0.01) and at end systole (1.19 ± 0.05 vs. 1.51 ± 0.06 mm, P < 0.001) (Table 2 & Figure 2).

The heart rates were similar between diabetic and control mice (446 ± 11 vs. 470 ± 11 bpm). To determine the effects of diabetes on LV systolic function, the LV blood volumes were measured from multiple short-axis slices that cover the whole heart at end diastole and end systole. The EDV was not significantly changed after 4 weeks of diabetes (44.8 ± 1.0 vs. 46.0 ± 1.1 μl). The ESV, on the other hand, was increased by 23%. Therefore, a significantly reduced myocardial contractile function was observed in diabetic mice. The stroke volume, ejection fraction and cardiac output were decreased by 20%, 18% and 16%, respectively. A similar decrease in LV fractional circumferential shortening was also seen in diabetic mice (Table 3).

To assess LV systolic and diastolic dynamics, peak ejection rate and peak filling rate were calculated from the systolic and diastolic limbs of the volume-time curve (Figure 3). In diabetic mice compared to controls, there was a significantly reduced maximum LV ejection rate (47 ± 5 vs. 62 ± 3 μl/s, n = 8, P < 0.05) and filling rate (33 ± 4 vs. 49 ± 3 μl/s, n = 8, P < 0.01) indicating a decrease of both contraction and relaxation (Figures 3 &4).

To compare the MRI techniques with conventional echocardiography, M-mode echocardiographic and Doppler flow studies were performed on control and 4-wk STZ-diabetic mice. The diabetic mice showed a decreased wall thickening and increased E/A ratio, indicating that STZ-induced diabetes affects both systolic and diastolic function (Figure 5).

To begin to assess the effects of diabetes on mitochondrial function, NADH oxidase activity, a measure of overall electron transport, was determined in mitoplasts prepared from hearts isolated from control and diabetic (4 weeks) mice. As shown in Figure 6, diabetes resulted in a decline in NADH oxidase activity of approximately 50% relative to control values. This was not due to an overall decline in mitochondrial protein content given that the yield of mitoplasts (not shown) and the activity of the Krebs cycle enzyme citrate synthase (Figure 6) did not vary between control and diabetic hearts. Therefore, this parameter of mitochondrial electron transport activity was severely compromised within 4 weeks of the onset of marked hyperglycemia. In contrast to the nearly 50% decline in cardiac NADH-linked electron transport chain activity (NADH oxidase) induced by diabetes, complex I activity declined 21.2% (P value ≤ 0.003). Complex I is the rate limiting step in electron transport chain. Therefore this decrement is, in part, responsible for the observed loss in NADH oxidase activity. Other deficits that contribute to the diabetes-induced declines in electron transport are under investigation.