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ORIGINAL RESEARCH
Assessment of Left Ventricular Myocardial Systolic Acceleration in Diabetic Rats Using Velocity Vector Imaging Haibin Zhang, MD, PhD, Zhangrui Wei, MD, Xiaoxing Zhu, MD, Hongling Li, MD, Ming Yu, MD, Yunyan Duan, MD, Ting Zhu, MD, Jun Zhang, MD, Xiaodong Zhou, MD, Miaozhang Zhu, MD Article includes CME test
Objectives—The purpose of this study was to investigate how the myocardial acceleration during isovolumic contraction changed in rats with diabetic cardiomyopathy and a normal left ventricular ejection fraction (LVEF) by using velocity vector imaging. Methods—Velocity vector imaging was performed in 12 control rats and 15 rats with streptozotocin-induced diabetic cardiomyopathy 12 weeks after streptozotocin injection. The segmental radial displacement, velocity, acceleration, and percent wall thickening were measured at the mid–left ventricular (LV) level.
Received June 12, 2013, from the Department of Ultrasound, PLA 210th Hospital, Dalian, China (H.Z.); Department of Ultrasound, PLA 117th Hospital, Hangzhou, China (Z.W.); Department of Ultrasound, Xijing Hospital (X.Z., H.L., M.Y., T.Z., J.Z., X.Z.), and Department of Physiology (M.Z.), Fourth Military Medical University, Xi’an, China; and Department of Medical Technology, Xi’an Medical University, Xi’an, China (Y.D.). Revision requested July 17, 2013. Revised manuscript accepted for publication September 1, 2013. Haibin Zhang, Zhangrui Wei, and Xiaoxing Zhu contributed equally to this work. Address correspondence to Haibin Zhang, MD, PhD, Department of Ultrasound, PLA 210th Hospital, 116011 Dalian, Liaoning, China. E-mail: [email protected] Abbreviations
EF, ejection fraction; LV, left ventricle; LVEF, left ventricular ejection fraction doi:10.7863/ultra.33.5.875
Results—Compared to control rats, rats with cardiomyopathy had a significant decrease in the peak radial acceleration during isovolumic contraction in most segments of the LV wall (including the anterior, anterolateral, inferolateral, and inferior segments; P < .05) but a similar LVEF, fractional shortening, and segmental displacement. Rats with cardiomyopathy also had a significant increase in LV end-diastolic and end-systolic diameters when corrected for body mass (P < .001; P = .003, respectively) and a significant decrease in the radial peak systolic velocities of the inferolateral and inferior wall segments (P < .05). In addition, rats with cardiomyopathy had a significant decrease in the peak radial diastolic acceleration in most segments of the LV wall (except for the anterolateral one; P < .05) but similar peak radial diastolic velocities in all LV wall segments compared to controls. Pathologic examination in rats with cardiomyopathy revealed ultrastructural impairment of the capillary and cardiocyte without any atherosclerotic lesion in the coronary artery compared to control rats. Conclusions—Myocardial acceleration during isovolumic contraction decreases in rats with diabetic cardiomyopathy and a preserved LVEF, suggesting the presence of regional LV systolic dysfunction. Key Words—basic science; diabetic cardiomyopathy; echocardiography; myocardial acceleration; velocity vector imaging
D
iabetic cardiomyopathy is defined as diabetes-associated changes in the structure and function of the myocardium in the absence of ischemic heart disease, hypertension, or other cardiac conditions. It is characterized by myocardial dilatation and hypertrophy, as well as a decrease in the systolic and diastolic function of the left ventricle (LV). These effects are related to myocardial damage, reactive hypertrophy, intermediary fibrosis, and changes in the small coronary vessels.1–6 Diabetic cardiomyopathy may be subclinical for a long time before the appearance of clinical symptoms.1–4 Hence, the use of appropriate diagnostic strategies for early detection of diabetic cardiomyopathy is imperative.
©2014 by the American Institute of Ultrasound in Medicine | J Ultrasound Med 2014; 33:875–883 | 0278-4297 | www.aium.org
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Early deteriorations of myocardial function in diabetic cardiomyopathy are characterized by a normal ejection fraction (EF) and a decrease in diastolic myocardial function.1–4 There is emerging evidence of the presence of reduced regional systolic function in patients with diabetic cardiomyopathy and a normal EF.1–3 However, classic 2-dimensional echocardiography may not be able to detect the early reduction in the LV systolic myocardial function.3,5,6 Sonographically derived myocardial acceleration during isovolumic contraction has been reported to be a good index for evaluation of left7–9 and right10,11 ventricular contractions and to be less preload and afterload dependent than other indices. We have demonstrated that myocardial acceleration can be derived from velocity vector imaging,12,13 which allows measurements of myocardial motion and deformation independently of the incident angle of the ultrasound beam. Instantaneous radial myocardial acceleration can be quantitatively measured from the LV shortaxis views by using velocity vector imaging.13 The purposes of this study were to investigate how myocardial acceleration changed and to clarify whether the regional systolic function was reduced in diabetic rats with a normal LVEF by using velocity vector imaging.
Materials and Methods Experimental Animals Twenty male Sprague Dawley rats (8 weeks old; mean weight ± SD, 255 ± 18 g) were administered streptozotocin at 65 mg/kg (1% streptozotocin solution diluted with 0.1 M citrate buffer, pH 4.4, before injection) through an intraperitoneal injection after a 12-hour fast, as previously described.14 Tail vein blood glucose samples were measured with an autoanalyzer (Surestep; Lifescan, Milpitas, CA) after 4 hours of fasting on days 3, 7, 28, 56, and 84 after injection. All rats that did not meet the criterion (fasting blood glucose >16.7 mM) were excluded; thus, 15 diabetic rats were entered into the diabetic cardiomyopathy group (2 rats died, and another 3 rats were excluded for streptozotocin tolerance). Another 12 ageand weight-matched male Sprague Dawley rats (8 weeks old; 252 ± 16 g) were selected for the control group and given the same dosage of sodium citrate buffer only. All rats had unlimited access to food and water and were maintained on a 12-hour light/dark cycle with the temperature and humidity kept at 20°C to 25°C and 68%, respectively, for 12 weeks. The protocol was approved by the local Institutional Animal Care and Use Committee.
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Echocardiographic Examination Twelve weeks after streptozotocin injection, an echocardiographic examination was performed with a 14-MHz linear array transducer (Acuson Sequoia 512C system; Siemens Medical Solutions, Mountain View, CA). After adequate anesthesia by intraperitoneal injection of 3% sodium pentobarbital (1 mL/kg), all rats were intubated in a supine position and ventilated with a rodent ventilator (CDW-2000, Shanghai, China). A thoracotomy was performed to obtain unrestricted visualization of LV walls. M-mode tracings were recorded at the mid-LV level. Left ventricular dimensions were measured from 3 consecutive cardiac cycles on the M-mode tracings. The LVEF was obtained by the Teichholz M-mode formula. Twodimensional echocardiographic cine loops of 3 consecutive beats were obtained from a short-axis view at the mid-LV level. The width of the ultrasound scan, imaging depth, and spatial temporal settings were optimized to achieve the highest possible frame rates. The temporal resolution was 9.4 ± 0.9 milliseconds with 103 to 125 frames per second (mean, 108 ± 7 frames per second) in all rats. Velocity Vector Imaging Velocity vector imaging was applied to the echocardiographic cine loops on an offline velocity vector imaging workstation (Sygno VVI; Siemens Medical Solutions). The LV wall at the midlevel was divided into 6 segments according to the standard 16-segment model of the American Society of Echocardiography (Morrisville, NC). Velocity vector imaging is a B-mode speckle-tracking algorithm to measure myocardial dynamics. The frameby-frame changes in the speckle position allow determination of its displacement. Velocity is estimated as the shift of each speckle divided by the time between successive frames. Similarly, acceleration is calculated as the difference between two sequential velocities divided by the frame-by-frame time interval.12,13 Thus, curves of myocardial displacement, velocity, and acceleration were obtained from each sampling point on the endocardial borders. Segmental displacement, velocity, and acceleration were calculated from the average of the sampling points in each segment (Figure 1). Acceleration was multiplied by the square root of the electrocardiographic R-R interval to compensate for the difference in the heart rate between the groups (corrected acceleration).15 Also, the instantaneous distance between the endocardium and epicardium (ie, instantaneous wall thickness) could be obtained by tracking speckles on the endocardium and epicardium. The instantaneous percent wall thickening was calculated according to the following
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formula: (instantaneous thickness – end-diastolic thickness)/end-diastolic thickness × 100%. Reproducibility of Data Six rats were randomly selected to determine the reproducibility of the velocity vector imaging measurements. Two-dimensional echocardiographic cine loops were analyzed by 2 independent observers (interobserver variability) and twice by the same observer (intraobserver variability) 10 days apart. The observers were both blinded to the other data for the rats and to each other’s readings.
Pathologic Examination After the echocardiographic examination, the rat hearts were excised, washed in a phosphate buffer solution, and cut into slices. Each slice was cut into serial 4-μm sections for hematoxylin-eosin staining to observe the coronary arteries and cardiocytes under light microscopy. Myocardial pieces from 5 rats in each group were prepared for ultrastructural observations under electron microscopy. The detailed methods were described in our previous study.14 Statistical Analysis All data were expressed as mean ± standard deviation unless otherwise stated. The normal distribution was tested
Figure 1. Comparison of LV wall movement between a normal rat and a rat with diabetic cardiomyopathy. A indicates anterior wall; AL, anterolateral wall; AS, anteroseptum; ECG, electrocardiogram; I, inferior wall; IL, inferolateral wall; IS, inferoseptum; and IVA, acceleration during isovolumic contraction.
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by the Shapiro-Wilk test. After checking for normality, the parameters between the control and cardiomyopathy groups were compared by a 2-tailed independent samples t test or a Mann-Whitney U test, when appropriate. All statistical analyses were performed with SPSS version 11.5 software (IBM Corporation, Armonk, NY). P < .05 was considered statistically significant.
Results General Characteristics of the Rats The general characteristics of the rats at 12 weeks after diabetes induction are listed in Table 1. The body mass and heart rate in the cardiomyopathy group were significantly lower than those in the control group (P < .001; P = .005, respectively). The blood glucose level was significantly higher in rats with cardiomyopathy than in controls (P < .001). A significant increase in the LV end-diastolic diameter and end-systolic diameter was found in the cardiomyopathy group when corrected for body mass (P < .001; P = .003, respectively). With regard to LV global systolic function, there were no significant differences in the LVEF and fractional shortening between the groups. No regional wall motion abnormalities were found on 2-dimensional echocardiography in any rat. Comparisons of Velocity Vector Imaging Parameters Between the Groups Velocity vector imaging was successfully performed in all segments of the rats. The peak percent wall thickening was measured from the percent wall thickening–time curves (Figure 1). For most of the LV wall segments, there was no significant difference in the peak percent wall thickening between the groups, except for the inferior wall, where the same measurement was significantly lower in the diabetic cardiomyopathy group than that in the control. The average
peak percent wall thickening of 6 wall segments was statistically similar between the groups (Table 2). No significant difference was found in either the peak radial displacement of any individual wall segment or in the average value of the same measurement for all 6 segments between the groups (Table 2). Peak radial systolic velocities of inferolateral and inferior wall segments were lower in rats with cardiomyopathy than in controls (Table 2; P = .008; P = .016, respectively). The average of 6 walls was also significantly decreased in the cardiomyopathy group (P= .002). Anterior, anterolateral, inferolateral, and inferior walls showed lower peak and corrected radial acceleration during isovolumic contraction in the cardiomyopathy group than in the control group (Table 2; all P < .05). The differences in the mean peak and corrected acceleration for the 6 walls were also significant between the groups (P < .05). As for diastolic parameters, peak radial diastolic velocities of all LV wall segments were statistically similar between the groups (Table 3; all P > .05). However, all LV wall segments except the anterolateral segment had lower peak radial diastolic acceleration and corrected diastolic acceleration in the cardiomyopathy group than in the control group (all P < .05). Rats with cardiomyopathy also had lower mean peak and corrected diastolic acceleration in the 6 walls (P < .05). Pathologic Findings There were no evident atherosclerotic plaques or stenoses in the coronary arteries under the epicardium in all specimens from both groups when observed by light microscopy (Figure 2). Ultrastructural impairments of the capillaries and cardiocytes were observed on electron microscopy in the cardiomyopathy group, such as microthrombi of the capillaries, swollen mitochondria, and destroyed sarcomere structures of the cardiocytes (Figure 3).
Table 1. General Characteristics of the Rats in the Control and Diabetic Cardiomyopathy Groups at 12 Weeks After Diabetes Induction Characteristic Fasting blood glucose, mM Body mass, g Heart rate, beats/min LV end-systolic diameter, mm LV end-systolic diameter corrected for body mass, mm/g × 10–3 LV end-diastolic diameter, mm LV end-diastolic diameter corrected for body mass, mm/g × 10–3 LVEF, % LV fractional shortening, %
Control (n = 12) 4.0 ± 0.3 524 ± 11 440 ± 40 2.4 ± 0.4 4.6 ± 0.7 4.7 ± 0.5 9.0 ± 0.9 80 ± 3 49 ± 3
Cardiomyopathy (n = 15) 25.2 ± 3.1a 232 ± 52a 377 ± 41a 2.5 ± 0.9 11.8 ± 5.3a 4.9 ± 0.6 22.3 ± 6.2a 79 ± 12 49 ± 13
Data are presented as mean ± SD. aP < .05 versus control.
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Reproducibility Intraobserver variability values were 5.3% ± 3.3% for displacement, 5.3% ± 3.9% for systolic velocity, 5.5% ± 3.7% for diastolic velocity, 5.9% ± 4.1% for acceleration during isovolumic contraction, 5.7% ± 3.6% for diastolic acceleration, and 9.6% ± 6.3% for percent wall thickening. The corresponding values for interobserver variability were 5.9% ± 4.0%, 6.0% ± 4.3%, 6.0% ± 3.9%, 6.4% ± 4.1%, 6.4% ± 3.9%, and 9.8% ± 6.1%, respectively.
Discussion Rat Model of Diabetic Cardiomyopathy Diabetic cardiomyopathy is a distinct primary disease process, independent of coronary artery disease and other macrovascular complications. Its pathologic substrate is characterized by the presence of myocardial damage, reactive hypertrophy, intermediary fibrosis, changes in the small coronary vessels, and cardiac autonomic neuropathy.1–6
Table 2. Segmental Peak Systolic Wall Thickening, Displacement, Velocity, and Acceleration During Isovolumic Contraction in the Control and Diabetic Cardiomyopathy Groups Wall Segment Anterior Anterolateral Inferolateral Inferior Inferoseptal Anteroseptal Average
Wall Thickening, % Control Cardiomyopathy 18.6 ± 7.5 20.7 ± 10.6 25.5 ± 15.7 26.7 ± 16.7 23.0 ± 11.9 20.0 ± 11.3 22.4 ± 11.8
15.2 ± 6.7 16.8 ± 10.3 18.5 ± 12.5 16.0 ± 9.1a 13.6 ± 4.9 14.4 ± 5.2 15.8 ± 7.0
Displacement, mm Control Cardiomyopathy 0.32 ± 0.08 0.35 ± 0.07 0.39 ± 0.08 0.35 ± 0.08 0.30 ± 0.07 0.29 ± 0.07 0.33 ± 0.06
Wall Segment
Acceleration, cm/s2 Control Cardiomyopathy
Anterior Anterolateral Inferolateral Inferior Inferoseptal Anteroseptal Average
33.2 ± 7.2 38.8 ± 7.2 44.7 ± 6.6 39.4 ± 6.7 31.0 ± 4.5 29.0 ± 8.3 36.0 ± 4.9
26.6 ± 2.5a 30.3 ± 5.8a 28.1 ± 4.0a 26.6 ± 4.4a 25.9 ± 7.5 23.9 ± 8.6 26.9 ± 2.8a
0.35 ± 0.10 0.39 ± 0.13 0.40 ± 0.16 0.37 ± 0.14 0.32 ± 0.10 0.30 ± 0.10 0.35 ± 0.10
Velocity, cm/s Control Cardiomyopathy 0.69 ± 0.16 0.76 ± 0.18 0.86 ± 0.14 0.76 ± 0.13 0.67 ± 0.10 0.65 ± 0.13 0.73 ± 0.09
0.57 ± 0.10 0.63 ± 0.14 0.66 ± 0.13a 0.62 ± 0.09a 0.55 ± 0.13 0.52 ± 0.14 0.59 ± 0.05a
Corrected Acceleration, cm/s2 Control Cardiomyopathy 13.3 ± 2.6 14.3 ± 2.4 16.5 ± 2.2 14.6 ± 2.4 11.5 ± 1.6 10.7 ± 3.0 13.3 ± 1.6
10.7 ± 1.2a 11.5 ± 2.3a 11.4 ± 2.2a 10.7 ± 1.8a 10.3 ± 2.5 9.4 ± 3.0 10.8 ± 1.1a
Date are presented as mean ± SD. aP < .05 versus control.
Table 3. Segmental Radial Peak Diastolic Velocity and Acceleration in the Control and Diabetic Cardiomyopathy Groups Wall Segment Anterior Anterolateral Inferolateral Inferior Inferoseptal Anteroseptal Average
Velocity, cm/s Control Cardiomyopathy 0.69 ± 0.17 0.75 ± 0.17 0.85 ± 0.19 0.76 ± 0.14 0.67 ± 0.08 0.65 ± 0.12 0.73 ± 0.11
0.66 ± 0.11 0.75 ± 0.19 0.72 ± 0.19 0.65 ± 0.10 0.58 ± 0.14 0.55 ± 0.16 0.65 ± 0.08
Acceleration, cm/s2 Control Cardiomyopathy 32.2 ± 6.9 33.2 ± 8.1 36.5 ± 7.2 33.3 ± 7.9 30.0 ± 4.2 31.0 ± 5.4 32.7 ± 4.0
24.8 ± 7.1a 28.0 ± 7.0 28.8 ± 6.5a 24.7 ± 3.4a 21.0 ± 6.4a 20.6 ± 7.8a 24.6 ± 3.5a
Corrected Acceleration, cm/s2 Control Cardiomyopathy 12.0 ± 2.6 12.3 ± 2.9 13.9 ± 2.6 12.3 ± 2.9 11.1 ± 1.6 11.5 ± 2.0 12.1 ± 1.4
9.9 ± 2.6a 11.3 ± 3.0 11.4 ± 2.6a 9.9 ± 1.2a 8.3 ± 2.2a 8.2 ± 2.9a 9.9 ± 1.3a
Date are presented as mean ± SD. aP < .05 versus control.
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In this study, the pathological findings in the cardiomyopathy group revealed the presence of ultrastructural impairments in the capillaries and cardiocytes, accompanied by normal coronary arteries under the epicardium. Although diabetic cardiomyopathy is independent of major epicardial coronary disease, metabolic pathway deregulation in diabetes can adversely affect the cardiocytes, as well as every cellular element within the vascular walls, thus decreasing myocardial function. We found destroyed basal laminas, slitshaped cavities, microthrombosis of the capillaries, opened intercalated disks, swollen mitochondria, and destroyed sarcomere structures of the cardiocytes (Figure 3). These findings were consistent with our previous study.14 Investigations in both animals and humans have shown cardiac structural changes in parallel with functional changes in diabetic cardiomyopathy.1–6 Left ventricular hypertrophy and dilatation appear as a result of hypertrophy of the myocardial cells, interstitial and perivascular fibrosis, greater thickening of the capillary basement membrane, and formation of microaneurysms in small capillary vessels.2 In our study, LV end-systolic and -diastolic diameters, when
corrected for body mass, were significantly increased in the cardiomyopathy group compared to the control group. Although streptozotocin-induced diabetes is a quite well-established model for investigating diabetic cardiomyopathy in small animals, and abnormal diastolic and systolic LV function can be demonstrated, the features of LV dysfunction in a rat model are variable and controversial. Several researchers have reported reduced LV fractional shortening and EF within 10 weeks after diabetes induction.16,17 However, this study demonstrated preservation of normal LV fractional shortening and EF within 12 weeks. Similar results have been previously reported.5,6 Yoon et al18 evaluated the natural course of streptozotocininduced diabetic Sprague Dawley rats. They found that diastolic dysfunction was prevalent in all diabetic rats 2 to 3 months after diabetes induction. The average time from induction of diabetes mellitus to development of both systolic and diastolic dysfunction was 9.2 months. Disagreements might be related to the differences in the rat strain, dose of streptozotocin, and age at which the streptozotocin was administrated.
Figure 2. Coronary arteries and cardiocytes of a normal rat (A) and a rat with diabetic cardiomyopathy (B) observed by light microscopy (hematoxylin-eosin, original magnification ×400). There are no evident atherosclerotic plaques or stenoses in the coronary arteries.
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In our previous study, rats with diabetic cardiomyopathy showed both reduced regional LV systolic function and ultrastructural impairments of the capillaries and cardiocytes 12 weeks after diabetes induction.14 In this study, under the same experimental conditions, rats with diabetic cardiomyopathy were examined at the same phase of diabetes development after the streptozotocin injection, as before. Myocardial Isovolumic Contraction Acceleration This study showed decreased acceleration during isovolumic contraction in LV anterior, anterolateral, inferolateral, and inferior wall segments in the cardiomyopathy group. Compared with percent wall thickening, displacement, and velocity, decreased acceleration was associated with more wall segments in the rats with cardiomyopathy. Given that myocardial displacement, velocity, and deformation could be dependent on changes in loading conditions,19–21 acceleration during isovolumic contraction may provide valuable knowledge on systolic dysfunction and be recommended as an index of regional contractile performance. Invasive studies have demonstrated that myocardial acceleration during
isovolumic contraction correlates well with myocardial contractility.8–10 It performs better than peak systolic myocardial velocity in diagnosing ischemia.11 As a measurement of LV contractile function, acceleration during isovolumic contraction is unaffected by preload and afterload changes within a physiologic range.7,8,10 On the contrary, several investigators reported that acceleration during isovolumic contraction might be load dependent.22,23 However, the methods used in these studies for changing the preload could have induced compensatory mechanisms, such as alterations in cardiac sympathetic activity and hormonal changes, thus leading to contradictory results. Dalsgaard et al7 excluded the potential influence of activated sympathetic mechanisms and found that acceleration during isovolumic contraction was unchanged after substantial increases in the preload in healthy subjects. The decreased myocardial acceleration in rats with diabetic cardiomyopathy and a normal LVEF might suggest the presence of reduced regional systolic function, which is in agreement with previous studies.5,6 Weytjens et al5 found that diabetic rats with preserved LV fractional shortening
Figure 3. Ultrastructural myocardial microcirculation and cardiocyte alterations in the diabetic cardiomyopathy myocardium observed by electron microscopy. A, Platelets attached to the inner surface of the capillary (white arrows) and red blood cell (black arrow; original magnification ×4000). B, Swollen mitochondria (1) and destroyed sarcomere structure (2; original magnification ×6000).
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and EF had a decrease in the radial systolic velocity and radial systolic strain rate of the anteroseptal wall. Similar results were found in humans. Several studies demonstrated subtle abnormalities in regional systolic function in diabetic patients with diastolic dysfunction and a normal LVEF.1–4 Different from the most segments of the LV, the anteroseptum and inferoseptum in rats with diabetic cardiomyopathy did not show significant decreases in acceleration during isovolumic contraction, probably due to factors such as the pressure inside the right ventricle, which might facilitate leftward movement of the septum during systole. The flow return to the right ventricle could also be influenced by intrathoracic pressure during respiration/ ventilation and further affected the motion of the septum. Diastolic Function in Rats With Diabetic Cardiomyopathy Diastolic dysfunction is a common finding and is thought to be the earliest detectable functional abnormality in diabetic cardiomyopathy.1–4 However, there was a close overlap between the periods of LV relaxation and left atrial contraction due to the high heart rate in rats (Figure 1). The single diastolic velocity wave on the velocity-time curve was an integration of the early diastolic velocity wave (e wave) and late diastolic velocity wave (a wave). This peak diastolic velocity resulted from both active and passive LV diastole and could not account for the changes in LV diastolic function. Our results showed that rats with diabetic cardiomyopathy and control rats had similar peak radial diastolic velocities in all LV wall segments. However, peak radial diastolic acceleration decreased significantly in all LV wall segments except the anterolateral segment in rats with diabetic cardiomyopathy. The diastolic acceleration wave, corresponding to descending limbs of systolic and diastolic velocity waves, occurred from late systole to early diastole. The peak diastolic acceleration appeared earlier than the peak diastolic velocity and could not be influenced by left atrial contraction. The peak early diastolic acceleration is regarded as a sensitive, preloadindependent marker for evaluation of LV diastolic function, which showed a good correlation with the peak drop in LV pressure (–dP/dtmin) and the time constant of LV isovolumic pressure decay (tau).9,24 The reduced diastolic acceleration suggested impaired myocardial relaxation in rats with diabetic cardiomyopathy. Limitations In a small-animal model such as rats, not only are their hearts very small, but also the heart rate is very high. The use of velocity vector imaging for regional quantification of LV function is a challenge. This work was a pilot
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study of velocity vector imaging–derived myocardial acceleration in rats. Optimal image quality is imperative for detecting tissue pixels and tracking the acoustic markers from frame to frame. A left thoracotomy was performed in all rats to eliminate the influences from the chest wall and lung tissue covering the heart in our first measurements of myocardial acceleration by velocity vector imaging. Intubation and thoracotomy might influence myocardial function and loading conditions in both control and diabetic rats. However, velocity vector imaging is a noninvasive cardiac imaging technology in itself, and myocardial acceleration measurements by transthoracic echocardiographic velocity vector imaging are available in the clinical setting.12,13 Considering the high heart rate of rats, a higher temporal resolution is necessary to analyze LV wall movement. We noticed that there was a large range of frame rates of imaging systems in previous studies of rats, which ranged from more than 200 frames per second25 to less than 100 frames per second.26–28 Although the frame rate in this study might have been relatively low (108 ± 7 frames per second), we and others have successfully measured the myocardial velocity, strain, and strain rate in rats with such a temporal resolution by using Doppler tissue imaging or speckle-tracking echocardiography.14,29,30 In addition, it was difficult to accurately determine the isovolumic contraction period according to Doppler spectra or Mmode tracings in this study. The peak systolic acceleration always corresponded to the QRS complex in all rats and was measured as the peak acceleration during isovolumic contraction (Figure 1). A difference in the heart rate between the normal rats and those with diabetic cardiomyopathy might have influenced our findings in theory because acceleration during isovolumic contraction could be influenced by changes in the heart rate. To compensate for variations in the heart rate, acceleration was multiplied by the square root of the electrocardiographic R-R interval. Conclusions Myocardial acceleration during isovolumic contraction is decreased in rats with diabetic cardiomyopathy and a preserved LVEF, suggesting the presence of regional LV systolic dysfunction. Myocardial acceleration could provide useful information for early detection of regional LV dysfunction.
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17. Westermann D, Van Linthout S, Dhayat S, et al. Cardioprotective and anti-inflammatory effects of interleukin converting enzyme inhibition in experimental diabetic cardiomyopathy. Diabetes 2007; 56:1834–1841. 18. Yoon YS, Uchida S, Masuo O, et al. Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation 2005; 111:2073–2085. 19. Borlaug BA, Melenovsky V, Redfield MM, et al. Impact of arterial load and loading sequence on left ventricular tissue velocities in humans. J Am Coll Cardiol 2007; 50:1570–1577. 20. Rösner A, Bijnens B, Hansen M, et al. Left ventricular size determines tissue Doppler-derived longitudinal strain and strain rate. Eur J Echocardiogr 2009; 10:271–277. 21. Donal E, Bergerot C, Thibault H, et al. Influence of afterload on left ventricular radial and longitudinal systolic functions: a two-dimensional strain imaging study. Eur J Echocardiogr 2009; 10:914–921. 22. Ruan Q, Nagueh SF. Usefulness of isovolumic and systolic ejection signals by tissue Doppler for the assessment of left ventricular systolic function in ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol 2006; 97:872–875. 23. Lyseggen E, Rabben SI, Skulstad H, Urheim S, Risoe C, Smiseth OA. Myocardial acceleration during isovolumic contraction: relation to contractility. Circulation 2005; 111:1362–1369. 24. Hashimoto I, Bhat AH, Li X, et al. Tissue Doppler-derived myocardial acceleration for evaluation of left ventricular diastolic function. J Am Coll Cardiol 2004; 44:1459–1466. 25. Pieper GM, Shah A, Harmann L, Cooley BC, Ionova IA, Migrino RQ. Speckle-tracking 2-dimensional strain echocardiography: a new noninvasive imaging tool to evaluate acute rejection in cardiac transplantation. J Heart Lung Transplant 2010; 29:1039–1046. 26. Brooks WW, Bing OH, Robinson KG, Slawsky MT, Chaletsky DM, Conrad CH. Effect of angiotensin-converting enzyme inhibition on myocardial fibrosis and function in hypertrophied and failing myocardium from the spontaneously hypertensive rat. Circulation 1997; 96:4002– 4010. 27. Liu W, Ashford MW, Chen J, et al. MR tagging demonstrates quantitative differences in regional ventricular wall motion in mice, rats, and men. Am J Physiol Heart Circ Physiol 2006; 291:H2515–H2521. 28. Loganathan R, Bilgen M, Al-Hafez B, Alenezy MD, Smirnova IV. Cardiac dysfunction in the diabetic rat: quantitative evaluation using high resolution magnetic resonance imaging. Cardiovasc Diabetol 2006; 5:7. 29. Prunier F, Gaertner R, Louedec L, Michel JB, Mercadier JJ, Escoubet B. Doppler echocardiographic estimation of left ventricular end-diastolic pressure after MI in rats. Am J Physiol Heart Circ Physiol 2002; 283:H346– H352. 30. Popović ZB, Benejam C, Bian J, et al. Speckle-tracking echocardiography correctly identifies segmental left ventricular dysfunction induced by scarring in a rat model of myocardial infarction. Am J Physiol Heart Circ Physiol 2007; 292:H2809–H2816. 883
ORIGINAL RESEARCH
Assessment of Left Ventricular Myocardial Systolic Acceleration in Diabetic Rats Using Velocity Vector Imaging Haibin Zhang, MD, PhD, Zhangrui Wei, MD, Xiaoxing Zhu, MD, Hongling Li, MD, Ming Yu, MD, Yunyan Duan, MD, Ting Zhu, MD, Jun Zhang, MD, Xiaodong Zhou, MD, Miaozhang Zhu, MD Article includes CME test
Objectives—The purpose of this study was to investigate how the myocardial acceleration during isovolumic contraction changed in rats with diabetic cardiomyopathy and a normal left ventricular ejection fraction (LVEF) by using velocity vector imaging. Methods—Velocity vector imaging was performed in 12 control rats and 15 rats with streptozotocin-induced diabetic cardiomyopathy 12 weeks after streptozotocin injection. The segmental radial displacement, velocity, acceleration, and percent wall thickening were measured at the mid–left ventricular (LV) level.
Received June 12, 2013, from the Department of Ultrasound, PLA 210th Hospital, Dalian, China (H.Z.); Department of Ultrasound, PLA 117th Hospital, Hangzhou, China (Z.W.); Department of Ultrasound, Xijing Hospital (X.Z., H.L., M.Y., T.Z., J.Z., X.Z.), and Department of Physiology (M.Z.), Fourth Military Medical University, Xi’an, China; and Department of Medical Technology, Xi’an Medical University, Xi’an, China (Y.D.). Revision requested July 17, 2013. Revised manuscript accepted for publication September 1, 2013. Haibin Zhang, Zhangrui Wei, and Xiaoxing Zhu contributed equally to this work. Address correspondence to Haibin Zhang, MD, PhD, Department of Ultrasound, PLA 210th Hospital, 116011 Dalian, Liaoning, China. E-mail: [email protected] Abbreviations
EF, ejection fraction; LV, left ventricle; LVEF, left ventricular ejection fraction doi:10.7863/ultra.33.5.875
Results—Compared to control rats, rats with cardiomyopathy had a significant decrease in the peak radial acceleration during isovolumic contraction in most segments of the LV wall (including the anterior, anterolateral, inferolateral, and inferior segments; P < .05) but a similar LVEF, fractional shortening, and segmental displacement. Rats with cardiomyopathy also had a significant increase in LV end-diastolic and end-systolic diameters when corrected for body mass (P < .001; P = .003, respectively) and a significant decrease in the radial peak systolic velocities of the inferolateral and inferior wall segments (P < .05). In addition, rats with cardiomyopathy had a significant decrease in the peak radial diastolic acceleration in most segments of the LV wall (except for the anterolateral one; P < .05) but similar peak radial diastolic velocities in all LV wall segments compared to controls. Pathologic examination in rats with cardiomyopathy revealed ultrastructural impairment of the capillary and cardiocyte without any atherosclerotic lesion in the coronary artery compared to control rats. Conclusions—Myocardial acceleration during isovolumic contraction decreases in rats with diabetic cardiomyopathy and a preserved LVEF, suggesting the presence of regional LV systolic dysfunction. Key Words—basic science; diabetic cardiomyopathy; echocardiography; myocardial acceleration; velocity vector imaging
D
iabetic cardiomyopathy is defined as diabetes-associated changes in the structure and function of the myocardium in the absence of ischemic heart disease, hypertension, or other cardiac conditions. It is characterized by myocardial dilatation and hypertrophy, as well as a decrease in the systolic and diastolic function of the left ventricle (LV). These effects are related to myocardial damage, reactive hypertrophy, intermediary fibrosis, and changes in the small coronary vessels.1–6 Diabetic cardiomyopathy may be subclinical for a long time before the appearance of clinical symptoms.1–4 Hence, the use of appropriate diagnostic strategies for early detection of diabetic cardiomyopathy is imperative.
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Early deteriorations of myocardial function in diabetic cardiomyopathy are characterized by a normal ejection fraction (EF) and a decrease in diastolic myocardial function.1–4 There is emerging evidence of the presence of reduced regional systolic function in patients with diabetic cardiomyopathy and a normal EF.1–3 However, classic 2-dimensional echocardiography may not be able to detect the early reduction in the LV systolic myocardial function.3,5,6 Sonographically derived myocardial acceleration during isovolumic contraction has been reported to be a good index for evaluation of left7–9 and right10,11 ventricular contractions and to be less preload and afterload dependent than other indices. We have demonstrated that myocardial acceleration can be derived from velocity vector imaging,12,13 which allows measurements of myocardial motion and deformation independently of the incident angle of the ultrasound beam. Instantaneous radial myocardial acceleration can be quantitatively measured from the LV shortaxis views by using velocity vector imaging.13 The purposes of this study were to investigate how myocardial acceleration changed and to clarify whether the regional systolic function was reduced in diabetic rats with a normal LVEF by using velocity vector imaging.
Materials and Methods Experimental Animals Twenty male Sprague Dawley rats (8 weeks old; mean weight ± SD, 255 ± 18 g) were administered streptozotocin at 65 mg/kg (1% streptozotocin solution diluted with 0.1 M citrate buffer, pH 4.4, before injection) through an intraperitoneal injection after a 12-hour fast, as previously described.14 Tail vein blood glucose samples were measured with an autoanalyzer (Surestep; Lifescan, Milpitas, CA) after 4 hours of fasting on days 3, 7, 28, 56, and 84 after injection. All rats that did not meet the criterion (fasting blood glucose >16.7 mM) were excluded; thus, 15 diabetic rats were entered into the diabetic cardiomyopathy group (2 rats died, and another 3 rats were excluded for streptozotocin tolerance). Another 12 ageand weight-matched male Sprague Dawley rats (8 weeks old; 252 ± 16 g) were selected for the control group and given the same dosage of sodium citrate buffer only. All rats had unlimited access to food and water and were maintained on a 12-hour light/dark cycle with the temperature and humidity kept at 20°C to 25°C and 68%, respectively, for 12 weeks. The protocol was approved by the local Institutional Animal Care and Use Committee.
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Echocardiographic Examination Twelve weeks after streptozotocin injection, an echocardiographic examination was performed with a 14-MHz linear array transducer (Acuson Sequoia 512C system; Siemens Medical Solutions, Mountain View, CA). After adequate anesthesia by intraperitoneal injection of 3% sodium pentobarbital (1 mL/kg), all rats were intubated in a supine position and ventilated with a rodent ventilator (CDW-2000, Shanghai, China). A thoracotomy was performed to obtain unrestricted visualization of LV walls. M-mode tracings were recorded at the mid-LV level. Left ventricular dimensions were measured from 3 consecutive cardiac cycles on the M-mode tracings. The LVEF was obtained by the Teichholz M-mode formula. Twodimensional echocardiographic cine loops of 3 consecutive beats were obtained from a short-axis view at the mid-LV level. The width of the ultrasound scan, imaging depth, and spatial temporal settings were optimized to achieve the highest possible frame rates. The temporal resolution was 9.4 ± 0.9 milliseconds with 103 to 125 frames per second (mean, 108 ± 7 frames per second) in all rats. Velocity Vector Imaging Velocity vector imaging was applied to the echocardiographic cine loops on an offline velocity vector imaging workstation (Sygno VVI; Siemens Medical Solutions). The LV wall at the midlevel was divided into 6 segments according to the standard 16-segment model of the American Society of Echocardiography (Morrisville, NC). Velocity vector imaging is a B-mode speckle-tracking algorithm to measure myocardial dynamics. The frameby-frame changes in the speckle position allow determination of its displacement. Velocity is estimated as the shift of each speckle divided by the time between successive frames. Similarly, acceleration is calculated as the difference between two sequential velocities divided by the frame-by-frame time interval.12,13 Thus, curves of myocardial displacement, velocity, and acceleration were obtained from each sampling point on the endocardial borders. Segmental displacement, velocity, and acceleration were calculated from the average of the sampling points in each segment (Figure 1). Acceleration was multiplied by the square root of the electrocardiographic R-R interval to compensate for the difference in the heart rate between the groups (corrected acceleration).15 Also, the instantaneous distance between the endocardium and epicardium (ie, instantaneous wall thickness) could be obtained by tracking speckles on the endocardium and epicardium. The instantaneous percent wall thickening was calculated according to the following
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formula: (instantaneous thickness – end-diastolic thickness)/end-diastolic thickness × 100%. Reproducibility of Data Six rats were randomly selected to determine the reproducibility of the velocity vector imaging measurements. Two-dimensional echocardiographic cine loops were analyzed by 2 independent observers (interobserver variability) and twice by the same observer (intraobserver variability) 10 days apart. The observers were both blinded to the other data for the rats and to each other’s readings.
Pathologic Examination After the echocardiographic examination, the rat hearts were excised, washed in a phosphate buffer solution, and cut into slices. Each slice was cut into serial 4-μm sections for hematoxylin-eosin staining to observe the coronary arteries and cardiocytes under light microscopy. Myocardial pieces from 5 rats in each group were prepared for ultrastructural observations under electron microscopy. The detailed methods were described in our previous study.14 Statistical Analysis All data were expressed as mean ± standard deviation unless otherwise stated. The normal distribution was tested
Figure 1. Comparison of LV wall movement between a normal rat and a rat with diabetic cardiomyopathy. A indicates anterior wall; AL, anterolateral wall; AS, anteroseptum; ECG, electrocardiogram; I, inferior wall; IL, inferolateral wall; IS, inferoseptum; and IVA, acceleration during isovolumic contraction.
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by the Shapiro-Wilk test. After checking for normality, the parameters between the control and cardiomyopathy groups were compared by a 2-tailed independent samples t test or a Mann-Whitney U test, when appropriate. All statistical analyses were performed with SPSS version 11.5 software (IBM Corporation, Armonk, NY). P < .05 was considered statistically significant.
Results General Characteristics of the Rats The general characteristics of the rats at 12 weeks after diabetes induction are listed in Table 1. The body mass and heart rate in the cardiomyopathy group were significantly lower than those in the control group (P < .001; P = .005, respectively). The blood glucose level was significantly higher in rats with cardiomyopathy than in controls (P < .001). A significant increase in the LV end-diastolic diameter and end-systolic diameter was found in the cardiomyopathy group when corrected for body mass (P < .001; P = .003, respectively). With regard to LV global systolic function, there were no significant differences in the LVEF and fractional shortening between the groups. No regional wall motion abnormalities were found on 2-dimensional echocardiography in any rat. Comparisons of Velocity Vector Imaging Parameters Between the Groups Velocity vector imaging was successfully performed in all segments of the rats. The peak percent wall thickening was measured from the percent wall thickening–time curves (Figure 1). For most of the LV wall segments, there was no significant difference in the peak percent wall thickening between the groups, except for the inferior wall, where the same measurement was significantly lower in the diabetic cardiomyopathy group than that in the control. The average
peak percent wall thickening of 6 wall segments was statistically similar between the groups (Table 2). No significant difference was found in either the peak radial displacement of any individual wall segment or in the average value of the same measurement for all 6 segments between the groups (Table 2). Peak radial systolic velocities of inferolateral and inferior wall segments were lower in rats with cardiomyopathy than in controls (Table 2; P = .008; P = .016, respectively). The average of 6 walls was also significantly decreased in the cardiomyopathy group (P= .002). Anterior, anterolateral, inferolateral, and inferior walls showed lower peak and corrected radial acceleration during isovolumic contraction in the cardiomyopathy group than in the control group (Table 2; all P < .05). The differences in the mean peak and corrected acceleration for the 6 walls were also significant between the groups (P < .05). As for diastolic parameters, peak radial diastolic velocities of all LV wall segments were statistically similar between the groups (Table 3; all P > .05). However, all LV wall segments except the anterolateral segment had lower peak radial diastolic acceleration and corrected diastolic acceleration in the cardiomyopathy group than in the control group (all P < .05). Rats with cardiomyopathy also had lower mean peak and corrected diastolic acceleration in the 6 walls (P < .05). Pathologic Findings There were no evident atherosclerotic plaques or stenoses in the coronary arteries under the epicardium in all specimens from both groups when observed by light microscopy (Figure 2). Ultrastructural impairments of the capillaries and cardiocytes were observed on electron microscopy in the cardiomyopathy group, such as microthrombi of the capillaries, swollen mitochondria, and destroyed sarcomere structures of the cardiocytes (Figure 3).
Table 1. General Characteristics of the Rats in the Control and Diabetic Cardiomyopathy Groups at 12 Weeks After Diabetes Induction Characteristic Fasting blood glucose, mM Body mass, g Heart rate, beats/min LV end-systolic diameter, mm LV end-systolic diameter corrected for body mass, mm/g × 10–3 LV end-diastolic diameter, mm LV end-diastolic diameter corrected for body mass, mm/g × 10–3 LVEF, % LV fractional shortening, %
Control (n = 12) 4.0 ± 0.3 524 ± 11 440 ± 40 2.4 ± 0.4 4.6 ± 0.7 4.7 ± 0.5 9.0 ± 0.9 80 ± 3 49 ± 3
Cardiomyopathy (n = 15) 25.2 ± 3.1a 232 ± 52a 377 ± 41a 2.5 ± 0.9 11.8 ± 5.3a 4.9 ± 0.6 22.3 ± 6.2a 79 ± 12 49 ± 13
Data are presented as mean ± SD. aP < .05 versus control.
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Reproducibility Intraobserver variability values were 5.3% ± 3.3% for displacement, 5.3% ± 3.9% for systolic velocity, 5.5% ± 3.7% for diastolic velocity, 5.9% ± 4.1% for acceleration during isovolumic contraction, 5.7% ± 3.6% for diastolic acceleration, and 9.6% ± 6.3% for percent wall thickening. The corresponding values for interobserver variability were 5.9% ± 4.0%, 6.0% ± 4.3%, 6.0% ± 3.9%, 6.4% ± 4.1%, 6.4% ± 3.9%, and 9.8% ± 6.1%, respectively.
Discussion Rat Model of Diabetic Cardiomyopathy Diabetic cardiomyopathy is a distinct primary disease process, independent of coronary artery disease and other macrovascular complications. Its pathologic substrate is characterized by the presence of myocardial damage, reactive hypertrophy, intermediary fibrosis, changes in the small coronary vessels, and cardiac autonomic neuropathy.1–6
Table 2. Segmental Peak Systolic Wall Thickening, Displacement, Velocity, and Acceleration During Isovolumic Contraction in the Control and Diabetic Cardiomyopathy Groups Wall Segment Anterior Anterolateral Inferolateral Inferior Inferoseptal Anteroseptal Average
Wall Thickening, % Control Cardiomyopathy 18.6 ± 7.5 20.7 ± 10.6 25.5 ± 15.7 26.7 ± 16.7 23.0 ± 11.9 20.0 ± 11.3 22.4 ± 11.8
15.2 ± 6.7 16.8 ± 10.3 18.5 ± 12.5 16.0 ± 9.1a 13.6 ± 4.9 14.4 ± 5.2 15.8 ± 7.0
Displacement, mm Control Cardiomyopathy 0.32 ± 0.08 0.35 ± 0.07 0.39 ± 0.08 0.35 ± 0.08 0.30 ± 0.07 0.29 ± 0.07 0.33 ± 0.06
Wall Segment
Acceleration, cm/s2 Control Cardiomyopathy
Anterior Anterolateral Inferolateral Inferior Inferoseptal Anteroseptal Average
33.2 ± 7.2 38.8 ± 7.2 44.7 ± 6.6 39.4 ± 6.7 31.0 ± 4.5 29.0 ± 8.3 36.0 ± 4.9
26.6 ± 2.5a 30.3 ± 5.8a 28.1 ± 4.0a 26.6 ± 4.4a 25.9 ± 7.5 23.9 ± 8.6 26.9 ± 2.8a
0.35 ± 0.10 0.39 ± 0.13 0.40 ± 0.16 0.37 ± 0.14 0.32 ± 0.10 0.30 ± 0.10 0.35 ± 0.10
Velocity, cm/s Control Cardiomyopathy 0.69 ± 0.16 0.76 ± 0.18 0.86 ± 0.14 0.76 ± 0.13 0.67 ± 0.10 0.65 ± 0.13 0.73 ± 0.09
0.57 ± 0.10 0.63 ± 0.14 0.66 ± 0.13a 0.62 ± 0.09a 0.55 ± 0.13 0.52 ± 0.14 0.59 ± 0.05a
Corrected Acceleration, cm/s2 Control Cardiomyopathy 13.3 ± 2.6 14.3 ± 2.4 16.5 ± 2.2 14.6 ± 2.4 11.5 ± 1.6 10.7 ± 3.0 13.3 ± 1.6
10.7 ± 1.2a 11.5 ± 2.3a 11.4 ± 2.2a 10.7 ± 1.8a 10.3 ± 2.5 9.4 ± 3.0 10.8 ± 1.1a
Date are presented as mean ± SD. aP < .05 versus control.
Table 3. Segmental Radial Peak Diastolic Velocity and Acceleration in the Control and Diabetic Cardiomyopathy Groups Wall Segment Anterior Anterolateral Inferolateral Inferior Inferoseptal Anteroseptal Average
Velocity, cm/s Control Cardiomyopathy 0.69 ± 0.17 0.75 ± 0.17 0.85 ± 0.19 0.76 ± 0.14 0.67 ± 0.08 0.65 ± 0.12 0.73 ± 0.11
0.66 ± 0.11 0.75 ± 0.19 0.72 ± 0.19 0.65 ± 0.10 0.58 ± 0.14 0.55 ± 0.16 0.65 ± 0.08
Acceleration, cm/s2 Control Cardiomyopathy 32.2 ± 6.9 33.2 ± 8.1 36.5 ± 7.2 33.3 ± 7.9 30.0 ± 4.2 31.0 ± 5.4 32.7 ± 4.0
24.8 ± 7.1a 28.0 ± 7.0 28.8 ± 6.5a 24.7 ± 3.4a 21.0 ± 6.4a 20.6 ± 7.8a 24.6 ± 3.5a
Corrected Acceleration, cm/s2 Control Cardiomyopathy 12.0 ± 2.6 12.3 ± 2.9 13.9 ± 2.6 12.3 ± 2.9 11.1 ± 1.6 11.5 ± 2.0 12.1 ± 1.4
9.9 ± 2.6a 11.3 ± 3.0 11.4 ± 2.6a 9.9 ± 1.2a 8.3 ± 2.2a 8.2 ± 2.9a 9.9 ± 1.3a
Date are presented as mean ± SD. aP < .05 versus control.
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In this study, the pathological findings in the cardiomyopathy group revealed the presence of ultrastructural impairments in the capillaries and cardiocytes, accompanied by normal coronary arteries under the epicardium. Although diabetic cardiomyopathy is independent of major epicardial coronary disease, metabolic pathway deregulation in diabetes can adversely affect the cardiocytes, as well as every cellular element within the vascular walls, thus decreasing myocardial function. We found destroyed basal laminas, slitshaped cavities, microthrombosis of the capillaries, opened intercalated disks, swollen mitochondria, and destroyed sarcomere structures of the cardiocytes (Figure 3). These findings were consistent with our previous study.14 Investigations in both animals and humans have shown cardiac structural changes in parallel with functional changes in diabetic cardiomyopathy.1–6 Left ventricular hypertrophy and dilatation appear as a result of hypertrophy of the myocardial cells, interstitial and perivascular fibrosis, greater thickening of the capillary basement membrane, and formation of microaneurysms in small capillary vessels.2 In our study, LV end-systolic and -diastolic diameters, when
corrected for body mass, were significantly increased in the cardiomyopathy group compared to the control group. Although streptozotocin-induced diabetes is a quite well-established model for investigating diabetic cardiomyopathy in small animals, and abnormal diastolic and systolic LV function can be demonstrated, the features of LV dysfunction in a rat model are variable and controversial. Several researchers have reported reduced LV fractional shortening and EF within 10 weeks after diabetes induction.16,17 However, this study demonstrated preservation of normal LV fractional shortening and EF within 12 weeks. Similar results have been previously reported.5,6 Yoon et al18 evaluated the natural course of streptozotocininduced diabetic Sprague Dawley rats. They found that diastolic dysfunction was prevalent in all diabetic rats 2 to 3 months after diabetes induction. The average time from induction of diabetes mellitus to development of both systolic and diastolic dysfunction was 9.2 months. Disagreements might be related to the differences in the rat strain, dose of streptozotocin, and age at which the streptozotocin was administrated.
Figure 2. Coronary arteries and cardiocytes of a normal rat (A) and a rat with diabetic cardiomyopathy (B) observed by light microscopy (hematoxylin-eosin, original magnification ×400). There are no evident atherosclerotic plaques or stenoses in the coronary arteries.
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In our previous study, rats with diabetic cardiomyopathy showed both reduced regional LV systolic function and ultrastructural impairments of the capillaries and cardiocytes 12 weeks after diabetes induction.14 In this study, under the same experimental conditions, rats with diabetic cardiomyopathy were examined at the same phase of diabetes development after the streptozotocin injection, as before. Myocardial Isovolumic Contraction Acceleration This study showed decreased acceleration during isovolumic contraction in LV anterior, anterolateral, inferolateral, and inferior wall segments in the cardiomyopathy group. Compared with percent wall thickening, displacement, and velocity, decreased acceleration was associated with more wall segments in the rats with cardiomyopathy. Given that myocardial displacement, velocity, and deformation could be dependent on changes in loading conditions,19–21 acceleration during isovolumic contraction may provide valuable knowledge on systolic dysfunction and be recommended as an index of regional contractile performance. Invasive studies have demonstrated that myocardial acceleration during
isovolumic contraction correlates well with myocardial contractility.8–10 It performs better than peak systolic myocardial velocity in diagnosing ischemia.11 As a measurement of LV contractile function, acceleration during isovolumic contraction is unaffected by preload and afterload changes within a physiologic range.7,8,10 On the contrary, several investigators reported that acceleration during isovolumic contraction might be load dependent.22,23 However, the methods used in these studies for changing the preload could have induced compensatory mechanisms, such as alterations in cardiac sympathetic activity and hormonal changes, thus leading to contradictory results. Dalsgaard et al7 excluded the potential influence of activated sympathetic mechanisms and found that acceleration during isovolumic contraction was unchanged after substantial increases in the preload in healthy subjects. The decreased myocardial acceleration in rats with diabetic cardiomyopathy and a normal LVEF might suggest the presence of reduced regional systolic function, which is in agreement with previous studies.5,6 Weytjens et al5 found that diabetic rats with preserved LV fractional shortening
Figure 3. Ultrastructural myocardial microcirculation and cardiocyte alterations in the diabetic cardiomyopathy myocardium observed by electron microscopy. A, Platelets attached to the inner surface of the capillary (white arrows) and red blood cell (black arrow; original magnification ×4000). B, Swollen mitochondria (1) and destroyed sarcomere structure (2; original magnification ×6000).
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and EF had a decrease in the radial systolic velocity and radial systolic strain rate of the anteroseptal wall. Similar results were found in humans. Several studies demonstrated subtle abnormalities in regional systolic function in diabetic patients with diastolic dysfunction and a normal LVEF.1–4 Different from the most segments of the LV, the anteroseptum and inferoseptum in rats with diabetic cardiomyopathy did not show significant decreases in acceleration during isovolumic contraction, probably due to factors such as the pressure inside the right ventricle, which might facilitate leftward movement of the septum during systole. The flow return to the right ventricle could also be influenced by intrathoracic pressure during respiration/ ventilation and further affected the motion of the septum. Diastolic Function in Rats With Diabetic Cardiomyopathy Diastolic dysfunction is a common finding and is thought to be the earliest detectable functional abnormality in diabetic cardiomyopathy.1–4 However, there was a close overlap between the periods of LV relaxation and left atrial contraction due to the high heart rate in rats (Figure 1). The single diastolic velocity wave on the velocity-time curve was an integration of the early diastolic velocity wave (e wave) and late diastolic velocity wave (a wave). This peak diastolic velocity resulted from both active and passive LV diastole and could not account for the changes in LV diastolic function. Our results showed that rats with diabetic cardiomyopathy and control rats had similar peak radial diastolic velocities in all LV wall segments. However, peak radial diastolic acceleration decreased significantly in all LV wall segments except the anterolateral segment in rats with diabetic cardiomyopathy. The diastolic acceleration wave, corresponding to descending limbs of systolic and diastolic velocity waves, occurred from late systole to early diastole. The peak diastolic acceleration appeared earlier than the peak diastolic velocity and could not be influenced by left atrial contraction. The peak early diastolic acceleration is regarded as a sensitive, preloadindependent marker for evaluation of LV diastolic function, which showed a good correlation with the peak drop in LV pressure (–dP/dtmin) and the time constant of LV isovolumic pressure decay (tau).9,24 The reduced diastolic acceleration suggested impaired myocardial relaxation in rats with diabetic cardiomyopathy. Limitations In a small-animal model such as rats, not only are their hearts very small, but also the heart rate is very high. The use of velocity vector imaging for regional quantification of LV function is a challenge. This work was a pilot
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study of velocity vector imaging–derived myocardial acceleration in rats. Optimal image quality is imperative for detecting tissue pixels and tracking the acoustic markers from frame to frame. A left thoracotomy was performed in all rats to eliminate the influences from the chest wall and lung tissue covering the heart in our first measurements of myocardial acceleration by velocity vector imaging. Intubation and thoracotomy might influence myocardial function and loading conditions in both control and diabetic rats. However, velocity vector imaging is a noninvasive cardiac imaging technology in itself, and myocardial acceleration measurements by transthoracic echocardiographic velocity vector imaging are available in the clinical setting.12,13 Considering the high heart rate of rats, a higher temporal resolution is necessary to analyze LV wall movement. We noticed that there was a large range of frame rates of imaging systems in previous studies of rats, which ranged from more than 200 frames per second25 to less than 100 frames per second.26–28 Although the frame rate in this study might have been relatively low (108 ± 7 frames per second), we and others have successfully measured the myocardial velocity, strain, and strain rate in rats with such a temporal resolution by using Doppler tissue imaging or speckle-tracking echocardiography.14,29,30 In addition, it was difficult to accurately determine the isovolumic contraction period according to Doppler spectra or Mmode tracings in this study. The peak systolic acceleration always corresponded to the QRS complex in all rats and was measured as the peak acceleration during isovolumic contraction (Figure 1). A difference in the heart rate between the normal rats and those with diabetic cardiomyopathy might have influenced our findings in theory because acceleration during isovolumic contraction could be influenced by changes in the heart rate. To compensate for variations in the heart rate, acceleration was multiplied by the square root of the electrocardiographic R-R interval. Conclusions Myocardial acceleration during isovolumic contraction is decreased in rats with diabetic cardiomyopathy and a preserved LVEF, suggesting the presence of regional LV systolic dysfunction. Myocardial acceleration could provide useful information for early detection of regional LV dysfunction.
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