Speckle-Tracking Echocardiography is Preferable Measurement for Detecting the Early and Subtle Alterations of Cardiac Dysfunction in Diabetic Rats


 BackgroundMyocardial damage caused by diabetic cardiomyopathy results in cardiac structural and functional abnormalities. Our previous studies have demonstrated that inhibiting RhoA/ROCK signaling pathway improved the structural abnormalities. The early detection of cardiac functional alterations accompanied by structural changes during therapy may facilitate better understanding on the pathophysiologic progress and guiding diabetic cardiomyopathy treatment. This study aimed to identify the optimal diagnostic measures for the early and subtle alterations of cardiac dysfunction in Type 2 diabetes mellitus rats.MethodsTwenty-four male Wistar rats were randomly divided into four groups and treated for 4 weeks: CON (control rats), DM (diabetic rats), DMF (DM + fasudil 10mg/kg/d) and CONF (CON + fasudil 10mg/kg/d). Left ventricular (LV) structure was quantified by histological staining and transmission electron microscopy. LV function and myocardial deformation were performed by high-frequency echocardiography.ResultsMyocardial hypertrophy and fibrosis were increased in diabetic rats and were remarkably alleviated in the DMF group. The diabetic rats had impaired LV performance evidenced by significant reduction of EF, FS and MV E/A, 26%, 34% and 20% respectively, while ROCK inhibition failed to improve the conventional ultrasonic parameters. However, the cardiac time intervals (CTI) parameters and speckle-tracking echocardiography (STE) parameters were significantly improved in the DMF group compared with the DM group (isovolumic contraction time, IVCT: P = 0.029; myocardial performance index, MPI: P = 0.037; fractional area change, FAC: P < 0.001; global circumferential strain, GCS: P = 0.003; global circumferential strain rate, GCSR: P = 0.021). Combining ROC curves with linear regression analysis, STE parameters were characterized by both the optimal predictions for cardiac damage [AUC (95% CI): FAC, 0.927 (0.744 to 0.993); GCS, 0.819 (0.610 to 0.945); GCSR, 0.899 (0.707 to 0.984)] and the strong correlations with cardiac fibrosis (FAC, r = -0.825; GCS, r = 0.772; GCSR, r = 0.829).ConclusionThe results suggest that STE strain and strain rate are preferable indicators of the early detection of subtle alterations in cardiac dysfunction and the quantitation of therapeutic efficacy in diabetic cardiomyopathy.


Abstract Background
Myocardial damage caused by diabetic cardiomyopathy results in cardiac structural and functional abnormalities. Our previous studies have demonstrated that inhibiting RhoA/ROCK signaling pathway improved the structural abnormalities. The early detection of cardiac functional alterations accompanied by structural changes during therapy may facilitate better understanding on the pathophysiologic progress and guiding diabetic cardiomyopathy treatment. This study aimed to identify the optimal diagnostic measures for the early and subtle alterations of cardiac dysfunction in Type 2 diabetes mellitus rats.

Methods
Twenty-four male Wistar rats were randomly divided into four groups and treated for 4 weeks: CON (control rats), DM (diabetic rats), DMF (DM + fasudil 10mg/kg/d) and CONF (CON + fasudil 10mg/kg/d). Left ventricular (LV) structure was quanti ed by histological staining and transmission electron microscopy. LV function and myocardial deformation were performed by high-frequency echocardiography.

Results
Myocardial hypertrophy and brosis were increased in diabetic rats and were remarkably alleviated in the DMF group. The diabetic rats had impaired LV performance evidenced by signi cant reduction of EF, FS and MV E/A, 26%, 34% and 20% respectively, while ROCK inhibition failed to improve the conventional ultrasonic parameters. However, the cardiac time intervals (CTI) parameters and speckle-tracking echocardiography (STE) parameters were signi cantly improved in the DMF group compared with the DM group (isovolumic contraction time, IVCT: P = 0.029; myocardial performance index, MPI: P = 0.037; fractional area change, FAC: P < 0.001; global circumferential strain, GCS: P = 0.003; global circumferential strain rate, GCSR: P = 0.021). Combining ROC curves with linear regression analysis, STE parameters were characterized by both the optimal predictions for cardiac damage [AUC (95% CI)

Conclusion
The results suggest that STE strain and strain rate are preferable indicators of the early detection of subtle alterations in cardiac dysfunction and the quantitation of therapeutic e cacy in diabetic cardiomyopathy.

Background
Diabetes mellitus (DM) remains a global prevalence with more than 450 million adult patients worldwide, and the number is expected to reach 693 million until 2045 [1]. Diabetic cardiomyopathy, acting as a serious cardiovascular complication induced by DM in particular type 2 diabetes mellitus (T2DM) [2], contributes to the incidence of the composite endpoint of worsening of heart failure (HF) or death. The term diabetic cardiomyopathy is de ned as a series of structural abnormalities, including a diffuse myocardial brosis, cardiomyocyte hypertrophy and coronary microvascular disorder, and cardiac dysfunction independent of the cardiovascular risk factors, such as hypertension, atherosclerosis or coronary artery disease [3,4].
With a growing understanding of the underlying mechanisms, RhoA/ROCK signaling pathway is proposed to contribute to the pathogenesis of diabetic cardiomyopathy [5][6][7]. Our previous studies have shown that inhibiting RhoA/ ROCK signaling pathway improved the structural abnormalities in diabetic hearts [8][9][10]. The changes of cardiac structure are usually accompanied with functional alterations. However, we have not observed the cardiac functional improvements in diabetic animals by traditional ultrasonic parameters. The early detection of cardiac performance during therapy may facilitate better understanding on the pathophysiologic progress and guiding diabetic cardiomyopathy treatment. Thus, there is an urgent need for diagnostic approaches for the early identi cation of cardiac functional alterations to predict diabetic cardiomyopathic process and therapeutic effect.
Transthoracic echocardiography is a standard non-invasive method of cardiac function assessment both in animal models and humans [11,12]. Conventional parameters, such as EF and FS, are widely used as surrogate markers of cardiac function [13]. However, the modest sensitivity and technical limitations make them fail to analyze the subtle changes of myocardial damage. Speckle-tracking echocardiography (STE) is a highly sensitive technique used for characterizing myocardium deformation by global and segmental strain parameters [14][15][16]. Previous studies have suggested that myocardial strain and strain rate are capable of detecting the subtle differences of pressure overload induced myocardial remodeling between young and adult rats [14]. Most notably, longitudinal strain (LS) parameters are well-accepted surrogates of putative subendocardial damage and may serve as diagnostic markers of microstructural remodeling [15]. Moreover, parameters derived from STE are valuable to describe the LV dynamic changes in active relaxation and contractility in T1DM and T2DM animal models [17]. Whereas, whether STE can be a sensitive diagnostic approach of diabetic cardiomyopathy to evaluate the subtle cardiac functional alterations and predict the therapeutic e cacy in the early stage is still lack of evidences.
In the present study, we aimed to investigate the subtle changes in cardiac structure and function in T2DM induced diabetic cardiomyopathy rat models after treated with a Rho-kinase inhibitor. By combining comprehensive STE analyses with several other measures, we sought to provide translational evidence for diabetic cardiomyopathy management. We tested the hypothesis that the early protective actions of ROCK inhibition on cardiac remodeling is detectable by assessing the subtle functional alterations by STE in diabetic cardiomyopathy rats.

Animals
Experimental protocols used in this study followed the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (1996) and were approved by the research ethic committee of Hebei Medical University. Male Wistar rats (Rattus norvegicus), 4 weeks old, were obtained from the Vital River Laboratory (Beijing, China). All rats were housed in a temperature-controlled room on a 12 h light/dark cycle with free access to water and standard rodent chow.
Following 1 week of acclimatization, the rats were randomly divided into two groups: control and diabetes. After 6 weeks of high-fat diet (HFD, 60 kcal% fat, D12492, Vital River Laboratory, Beijing, China) feeding, the rats were fasted for 8 h , and then were intraperitoneally injected with a single dose of Streptozocin (STZ, 35 mg/kg; S0130, sigma, USA) dissolved in 0.1 M citrate buffer (pH 4.5) to induce T2DM as previously described [18]. As controls, the rats received a single intraperitoneal injection of citrate buffer alone and were fed with normal diet as before. After 3 days, 1 weeks and 4 weeks of STZ-injection, fast blood glucose (FBG) level was measured by blood glucose meter (yuwell580, China) using tail vein blood sample. FBG > 11.1 mmol/l was considered as diabetes. All the diabetic rats were kept on HFD feeding for 24 weeks.

Experimental design
After 24 weeks for diabetes, the normal and diabetic rats, respectively and randomly, were divided into 4 groups (at least 6 rats in each group): the CON (control rats) group, the DM (diabetic rats) group, the DMF (diabetic rats + fasudil) group and the CONF (control rats + fasudil) group. The rats from the DMF group and CONF group were treated with fasudil (10 mg/kg/d; Hongri, Tianjin, China) by intraperitoneal injection. The CON group and DM group were intervened with an equal volume of saline intraperitoneally per day. After 4-week treatment, rats were sacri ced for the collections of heart tissues for further analysis (Fig.S1).

Histological analysis
LV samples of all rats were harvested and xed with 4% paraformaldehyde for 8 h at room temperature, and then embedded in para n. Subsequently, the heart samples were cut into 5-μm-thick sections, and separately stained with Hematoxylin-Eosin (HE) and Masson's trichrome as previously described [19]. Digital images were obtained by light microscopy (BX51T-PHD-J11, OLYMPUS, Tokyo, Japan). Image analysis was performed by using Image Pro Plus 6.0 software (Media Cybernetics, USA) to quantify the morphological changes and collagen content of the heart tissues. The data of cardiomyocyte cross sectional area (CSA) was collected from 30 representative cardiomyocytes per eld and 3 random elds in each slide. The volume fraction of collagen (CVF) was calculated by a formula (CVF %= average collagen area / area of total eld × 100).

Transmission electron microscopy (TEM)
Transmission electron microscopy was performed to detect the cardiac ultrastructure according to previous protocol [20,21]. LV samples were harvested and xed in 2.5% glutaraldehyde overnight at 4 °C, and post-xed in 1% osmic acid. After dehydration, the tissues were embedded with Epon 812 and baked at 60 °C for 36 h. Ultrathin sections (50~60 nm) were obtained and stained with 3% uranyl acetate-lead citrate, and then examined with a transmission electron microscope (H-7650, HITACHI, Japan). The Mitochondrial morphological parameters on each slide were calculated under six random elds, and the numbers of six slides were averaged for one rat. Mitochondrial morphological characteristics were quanti ed by Image Pro Plus 6.0 software (Media Cybernetics, USA): (1) mitochondrial area; (2) aspect ratio: de ned as the ratio between the major and minor axis of the mitochondria; (3) degree of branching: de ned as (Pm 2 ) / (4pAm), (Pm is the perimeter of the mitochondrial and Am is the area); (4) the number of mitochondrial vacuolization.

Echocardiography
Echocardiography was used to evaluate the cardiac function in all of the rats after 4-week drugs administration before sacri ced as described [22] . Ultrasound imaging was obtained with a 4 A VisualSonics high-resolution Vevo 2100 system (VisualSonics Inc., Toronto, Canada) with a 16-MHz linear transducer (MS-250). In brief, rats were anesthetized with 2% iso urane and placed in supine position on a heated platform (to maintain a body temperature at 37 °C) for continuous electrocardiogram monitoring. After the heart rate (HR) was stable, B-mode images were acquired in the parasternal long-axis and short-axis views, and M-mode images were performed in the parasternal short axis at the mid-papillary level. And then apical four-chamber view was performed to acquire the mitral valve ow via a pulsed wave (PW) Doppler method. All the parameters were assessed in 3 consecutive cardiac cycles at least; mean values from 3 measurements were calculated. And all the analyses were performed in a blinded manner.

Conventional echocardiography
The conventional parameters, including the left ventricular end-diastolic inner dimension (LVIDd), left ventricular end-systolic inner dimension and late (A) peak mitral valve ow velocity was analyzed using PW Doppler imaging in the apical four-chamber view to evaluate the diastolic function.

Cardiac time intervals (CTI)
Cardiac time intervals were measured by mitral valve ow Doppler imaging combined with an electrocardiogram through apical fourchamber view [23,24] . Isovolumic relaxation time (IVRT) was de ned as the time interval from aortic valve closing to mitral valve opening, which was used for diastolic function evaluation. Isovolumic contraction time (IVCT), indicating systolic function evaluation, was de ned as the time interval from mitral valve closure to aortic valve opening. Aortic ejection time (AET) de ned as the time interval from aortic valve opening to aortic valve closing. Myocardial performance index (MPI) was calculated as the sum of IVRT and IVCT divided by the AET: (IVRT + IVCT) / AET, which was a parameter of both systolic and diastolic function.

Speckle-tracking echocardiography (STE)
Speckle-tracking echocardiography was obtained in parasternal long-axis and short-axis views [16,25]. B-mode images with a frame rate above 200 frames/s were used, and then the LV endocardium and epicardium were traced manually in end diastole. For the long-axis view global longitudinal strain (GLS) and strain rate (GLRS) was calculated, and for the short-axis view global radial strain (GRS), global radial strain rate (GRRS), global circumferential strain (GCS) and global circumferential strain rate (GCRS) were obtained. Global strain and strain rate values were analyzed from the average of the 6 segments of the LV.

Quantitative real time PCR (qRT-PCR)
Total mRNA of the LV samples were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol [26].
Detailed methodology is available in the online-only Data Supplement.

Statistical analysis
The values are presented as means ± SD. Unpaired Student's t-tests were used for comparisons between two groups. Differences among three or more groups were analyzed using one-way ANOVA. Receiver operating characteristic (ROC) curves were studied to determine sensitivity and speci city of cardiac functional parameters to detect myocardial damage. Linear regression analysis was performed between continuous variables using Pearson's correlation coe cient (r). Statistical analysis was performed with GraphPad Prism software 8.0 (GraphPad Software Inc., USA) and IBM SPSS Statistics 26.0 (IBM Corporation, USA). P values < 0.05 were considered as statistically signi cant.

Evaluation of Cardiac Ultrastructure by Transmission Electron Microscopy
The mitochondrial morphology characteristics determined by stereology investigations directly reveal ultrastructure changes in cardiomyocytes. Remarkable changes in the morphological appearance of mitochondria were shown in diabetic rats, including the smaller mitochondrial area, the increased aspect ratio and degree of mitochondrial branching, and the higher number of distorted vacuous mitochondria (Fig.2), which indicated the progressive impairments in cardiac ultrastructure. Compared with the DM group, short-term ROCK inhibition signi cantly restored the mitochondrial area (DM vs DMF, 0.53 ± 0.05 vs 0.66 ± 0.09 μm 2 , P = 0.005; Fig.2b), reduced the mitochondrial aspect ratio (DM vs DMF, 2.48 ± 0.28 vs 1.83 ± 0.10, P < 0.001; Fig.2c ), reduced the degree of mitochondrial branching (DM vs DMF, 1.81 ± 0.12 vs 1.56 ± 0.07, P = 0.001; Fig.2d) and alleviated the vacuolization in mitochondria (DM vs DMF, 42.67 ± 5.24 vs 19.00 ± 10.30, P < 0.001; Fig.2e) in the DMF group. In addition, ROCK inhibition improved the imbalance of mitochondrial division/fusion by upregulating Mfn1 and Mfn2 mRNA transcriptions and downregulating Drp1 and Fis1 mRNA transcriptions (Fig.S2).

Evaluation of Cardiac Function by Conventional Echocardiography
The diabetic rats showed a noteworthy decline in HR, from 354 bmp to 295 bmp, and an enlargement in LV characterized by the increased LVIDd and LVEDV, compared with the CON group (Table S2). However, ROCK inhibition had no signi cant effect on the HR in diabetic rats. The diabetic rats had impaired LV systolic performance evidenced by the reductions of EF and FS, 26% and 34% respectively (Fig.3 & Table   S2). Meanwhile, the CO was signi cantly depressed in the DM group, however the SV had no change (Fig.3 & Table S2). In the DMF group, CO and SV were signi cantly restored (CO: DM vs DMF, 76.67 ± 15.64 vs 95.90 ± 11.49 ml/min, P = 0.034; SV: DM vs DMF, 259 ± 34 vs 312 ± 46 μl, P = 0.034; Fig.3), but EF and FS did not show the same trend. In addition, cardiac diastolic dysfunction was evidenced by a reduced mitral valve E/A in the DM group compared to the controls (CON vs DM, 1.53 ± 0.23 vs 1.22 ± 0.10, P = 0.043; Fig.3g). Interestingly, a 4-week treatment by Fasudil failed to improve the mitral valve E/A in diabetic rats (Fig.3g).

Evaluation of Myocardial Deformation by Speckle-Tracking Echocardiography
Myocardial strain parameters were measured by highly sensitive STE which had been demonstrated as a reliable approach to detect myocardial deformation and cardiac impairments (Fig.5 & Table S3). Compared with the CON group, GLS and GLSR were markedly decreased in diabetic rats, re ecting the damage of myocardial deformation in the longitudinal direction (GLS: CON vs DM, P < 0.001; GLSR: CON vs DM, P < 0.001; Fig.5c & d). These impairments were prevented in the DMF group, whereby GLS exhibited a markedly increase but GLSR showed no signi cance (GLS: DM vs DMF, P < 0.001; Fig.5c). A similar pattern was found in the circumferential direction where GCS and GCSR were reduced in diabetic rats (GCS: CON vs DM, P < 0.001; GCSR: CON vs DM, P < 0.001; Fig.5e & f), meanwhile, both of them were signi cantly ameliorated in the DMF group (GCS: DM vs DMF, P = 0.003; GCSR: DM vs DMF, P = 0.021; Fig.5e & f). In the radial direction, though GRS and GRSR were reduced in diabetic rats, only the strain rate revealed a signi cant change (CON vs DM, P = 0.024, Fig.5h). However, ROCK inhibition had no signi cant effect on both GRS and GRSR in the DMF group (Fig.5g & h). Additionally, FAC was attenuated in the diabetic cardiomyopathy animals (CON vs DM, P < 0.001, Fig.5i) and subsequently increased in the DMF group (DM vs DMF, P < 0.001, Fig.5i).
Diagnostic Assessment of Myocardial Damage Figure 6 summarized the relative cardiac function alterations induced by diabetes and intervention compared with the control group by a spider's web plot. Healthy controls were shown as a blue circle, which was normalized to 1. Changes in the DM group were marked by the red circle and in the DMF group were marked by the green one. Similar deviations were observed in the treated and untreated diabetic rats. Noteworthily, treatment in the DMF group tried to drive the green circle to the healthy shape via in uencing several functional parameters and structural parameters (Fig.6).
To assess the validity of the parameters as the predictors of diabetic cardiomyopathy, ROC curves were displayed in Table 2. EF, FS and MV E/A had superior sensitivity and speci city for diabetic cardiomyopathy among the conventional parameters, at a cutoff value of 65.62%, 35.07% and 1.25, respectively (Fig.7a & Table 2). Similarly, the signi cant area under the ROC curves and the prominent sensitivity and speci city in the CTI parameters, including IVCT, IVRT and MPI, also made them possible to distinguish subtle myocardial damages induced by diabetes (Fig.7b). From this analysis, the cutoff values of IVCT, IVRT and MPI were proposed at 23.33 ms, 25.56 ms, and 0.71 (Table 2) respectively. Among STE parameters, FAC, GCS and GCSR were the superior predictors for the detection of myocardial damage at a cutoff value of 48%, -17.14% and -4.03 1/s, respectively (Fig.7c & Table 2). GLSR and GRSR also allowed to differentiate the damage and nondamage cardiomyocytes induced by diabetes ( Table 2). Other STE parameters, GLS and GRS failed to distinguish the myocardial damage due to the lack of the signi cance in the ROC curves analysis. Notably, the ability to predict myocardial damage were comparable among the CTI parameters (MPI, IVRT) and the STE parameter (GLSR), which all indicated the cardiac diastolic function (Table 3). Furthermore, the parameters characterized the cardiac systolic function, including MPI, IVCT, FAC, GCR, GCSR and GRSR were demonstrated the nearly equivalent validity according to our study (Table 3).
Linear regression analysis was performed to clarify the associations between echocardiographic parameters and cardiac structural parameters (Table S4). MPI and FAC were strongly associated with the diabetes-induced cardiac hypertrophy and brosis ( Fig.8 & Table S4).
GCS and GCSR were relevant intensively to cardiac brosis but moderately to the CSA of cardiomyocytes (Fig.8 & Table S4). The conventional parameters (EF and FS), the CTI parameters (IVCT and IVRT) and the STE parameters (GLS, GLSR and GRSR) all showed the moderate correlations with cardiac hypertrophy and brosis respectively (Table S4). Besides, CO and MV E/A showed the low correlations with both two cardiac structural parameters, and SV and GRS showed no signi cant relationships according to the analysis (Table S4).

Discussion
The major ndings in our study were that: ROCK inhibition improved diabetes-induced myocardial damage both in microstructure and ultrastructure. The early alterations of cardiac dysfunction accompanied by structure changes could be detected both by CTI and STE parameters in diabetic hearts. According to our data, parameters derived from STE were more sensitive to identify the early changes of myocardial damages and quantify the subtle cardiac dysfunction. Moreover, FAC, GCS and GCSR were demonstrated to be noninvasively potential predictors for diabetic cardiomyopathy diagnosis, and valuable and feasible measures to evaluate the therapeutic effect.
Diabetic cardiomyopathy causes severe myocardial damage resulting in the abnormalities of cardiac function and structure. Some diabetic animal models have been described for the study of DM and its complications, predominantly in rodents [3,27]. In our study, we used STZhigh-fat diet T2DM rat models combined with a longer amount of time to mimic the common structural and functional phenotype of the diabetic human hearts [3,28]. Experimental and clinical researches have shown that cardiac hypertrophy and brosis both contribute to LV remodeling [2,28]. In line with previous studies, we found the signi cant augmentation of myocardial hypertrophy and brosis in diabetic rat heart, which indicated that STZ-induced diabetic rats in our study developed into cardiac remodeling under the long-term in uence of diabetic state.
Numerous molecular signaling pathways have involved in the pathophysiological process of diabetic cardiomyopathy [2,3,28]. Recent studies revealed the signi cant role of RhoA/ROCK signaling pathway in the underlying mechanisms of diabetic cardiomyopathy [5,6]. The Rho-associated coiled-coil containing kinases (ROCKs) are members of the AGC serine/threonine kinase family, and are downstream effectors of RhoA, a member of small GTP-binding protein. There are two isoforms of ROCKs, known as ROCK1 and ROCK2, encoded by two different genes [29]. Activated ROCK contributes to the cardiomyocyte apoptosis, cardiac hypertrophy and brosis [5,30,31], and mediates vascular smooth muscle cell contraction and several proin ammatory, thrombogenic and brogenic molecules [5]. Notably, recent study showed that T2DM patients treated with glucose-lowering drugs, antihypertensive treatment and statins were of signi cant increase in ROCK activation, suggesting the additional role of ROCK inhibition in the prevention of T2DM [32]. These highlight the importance of RhoA/ROCK signaling pathway in diabetic heart therapy. Moreover, our previous studies have shown ROCK inhibition improved the cardiac structure abnormalities in diabetic hearts [8][9][10]. In the present study, we used Fasudil, a ROCK inhibitor [33], to inhibit RhoA/ ROCK signaling pathway in diabetic animal models. After 4-week treatment, ROCK inhibition prominently alleviated cardiac hypertrophy and brosis, suggesting a cardioprotective effects against cardiac remodeling in diabetic rats. The cardiac ultrastructure in diabetic rats was con rmed by TEM detection, which characterized by dumbbell-shaped mitochondria for disturbed fusion, smaller fragment and even the distorted vacuous mitochondria for exacerbated ssion. Mitochondrial dysfunction and impaired energetics characterize the diabetic heart ultrastructural damage [2,4,28,34]. Previous studies have proved that RhoA/Rho kinase pathway is involved in the regulation of the mitochondrial dynamics in some cases [35][36][37][38]. In our study, ROCK inhibition improved the disturbed mitochondrial ssion/fusion in diabetic rats and facilitated mitochondrial fusion and uncoupling to restore the cardiac ultrastructural abnormalities. These indicated that ROCK inhibition improved the abnormalities both in cardiac microstructure and ultrastructure.
Accompanied by the changes in structure, it is the cardiac dysfunction. Correspondingly, we found the impaired LV systolic and diastolic performance in diabetic rats. Our animal models shared numerous features with diabetic cardiomyopathy including the enlarged LV, remarkable myocardial hypertrophy and brosis and obvious dysfunction. Interestingly, ROCK inhibition increased the SV and CO to a certain degree, possibly relevant to an integrated consequence of the slightly elevated LVEDV and HR. However, the classical parameters, EF, FS and MV E/A, derived from the conventional echocardiography were almost not changed in the presence of 4-week treatment with fasudil. Notably, Lai [6] have observed signi cant improvements of cardiac dysfunction in diabetic rats treated with fasudil for 8 weeks via modulation of Ca 2+ handling and actin remodeling. That suggesting that the duration of administration might be a key factor for the different results, and the traditional detection methods of the cardiac function in the early stage of treatment might be another. Accordingly, EF, FS and MV E/A, the conventional parameters, might be insensitive to the early and subtle alterations in cardiac dysfunction along with the improvements in structure in diabetic rats.
Because of the results in our study and others have demonstrated the limitations of EF, FS and MV E/A in evaluating the early alterations of cardiac dysfunction [17,39], we used CTI and STE to characterize the diabetic myocardial damage and assess the therapeutic effect respectively. CTI parameters de ned by the opening and closing of the heart valves have been suggested and investigated in kinds of cardiovascular disease [23,40]. Previous studies have shown that IVCT was prolonged and AET was shortened in HF [23,41,42]. In addition, AET has been proved to be a predictor of the outcome in primary pulmonary hypertension and ischemic heart disease [42]. Furthermore, MPI is demonstrated a superior marker of cardiovascular morbidity and mortality than conventional parameters [43]. In diabetic populations or animals, MPI has been found increased with a prolonged IVCT and IVRT and a shortened AET [43][44][45][46][47]. Similar ndings were obtained in our study, where the diabetic rats had signi cantly increased IVCT, IVRT and MPI. Noticeably, the diabetic rats with worsen LV systolic and diastolic dysfunction were improved by inhibiting RhoA/ROCK pathway. The early and subtle changes of cardiac structure could be detected by IVCT and MPI. CTI parameters, in particular the IVCT and MPI, are feasible in the early detection of cardiac function alterations and might be predictive of the therapeutic effect in diabetic cardiomyopathy.
Further, the present study showed the bene ts of STE in predicting diagnosis and therapy in diabetic cardiomyopathy. Myocardial deformation measured by STE is broadly governed by myocardial microstructure. The LV longitudinal mechanics are predominantly determined by subendocardium, while the circumferential and radial mechanics are decided by the mid-wall and the subepicardium [48,49]. Myocardial strain is well known to account for ventricular vascular coupling, as decreased myocardial strain is known to attenuate myocardial e ciency [50]. Damage in different myocardial layers leads to distinct phenotypes in cardiac dysfunction [48], which make it possible to classify heart disease according to layer-speci c alterations. Damage mainly in subendocardium may be accompanied by diastolic dysfunction but preserved EF together with reduced longitudinal but unaltered radial and circumferential mechanics in patients and animals [15,51]. Thus, GLS is well-accepted marker of subendocardial damage and linked to subendocardial brosis [52]. For another, an acute transmural insult involving in the subepicardial and midmyocardial dysfunction results in a reduction in LV circumferential, radial and twist mechanics as well as a decrease in EF [48]. Consistent with this theory, Niu [14] fount the reduced longitudinal and radial mechanics in pressure overload-induced adult rats along with a severely impaired contractile function, which was featured with the LV stiffness in the whole layers [14]. In diabetic humans and animal models, the LV function is obviously impaired with reduced myocardial strain and strain rate [17,53]. This is in accordance with our ndings, where global strain and strain rate were markedly decreased in diabetic rats in the longitudinal, circumferential and radial directions, suggesting a transmural cardiac remodeling. Inhibiting RhoA/ROCK signaling pathway signi cantly ameliorated GLS, GCS and GCSR, which manifested the improvements of the cardiac hypertrophy and brosis in the global heart. Additionally, mitochondria are the powerhouse of the cell, which continuously provide large amounts of adenosine triphosphate (ATP) to cardiomyocytes to maintain cardiac function [34]. The improvements of cardiac ultrastructure also contributed to restore the global strain and strain rate in the DMF group. Moreover, FAC, re ecting the degree of thickening in the radial direction [48], was attenuated in diabetic rats and subsequently increased by ROCK inhibition. Accordingly, it might be suggested that the changes of cardiac microstructure and ultrastructure could be detected by STE parameters at the early stage of the treatment compared with conventional parameters. STE parameters could be suitable markers for predicting diagnosis and therapeutic effect and providing the quantitative assessment of layerspeci c cardiac function alterations.
The present study has demonstrated that ROCK inhibition improved diabetes-induced myocardial hypertrophy and brosis, and the alterations of cardiac dysfunction accompanied by structure changes were detected both by CTI and STE parameters ( Figure 6). However, what kind of parameters is the optimal diagnostic approach to assess the subtle cardiac damage and predict the therapeutic effect in the early stage of diabetic cardiomyopathy remains to reveal. Hence, we demonstrated the validity of parameters as predictors for diabetic cardiomyopathy by ROC curves and linear regression analyses. Numerous studies have been identi ed the sensitivity and speci city of conventional parameters for cardiovascular disease diagnosis [3,4,12].Similar ndings were con rmed in our study. Whereas, the mild ability of indicating diabetic myocardial hypertrophy and brosis made the conventional parameters fail to detect the subtle alterations of cardiac function. CTI parameters, such as IVCT, IVRT, MPI, showed prominent sensitivity and speci city of cardiac systolic and diastolic function respectively, which are consistent with previous data [43]. For the strong correlations with cardiac microstructure, MPI was a best index to distinguish diabetes-induced myocardial damage, compared with IVCT and IVRT. As previously reported, MPI is a superior predictor of cardiovascular morbidity and mortality in the general population, and can provide prognostic information of cardiovascular risk factors [40].The ndings in our study also con rmed that MPI offers the therapeutic information on the early alterations both of cardiac systolic and diastolic dysfunction.
Parameters derived from STE, including FAC, GCS and GCS, were demonstrated superior predictors for the detection of diabetes-induced myocardial damage in our study. According to analysis, FAC, GCS and GCSR were sensitive and speci c for detecting the early and subtle alterations in cardiac dysfunction. Previous literature has shown that GLS parameters are detectable in corresponding microstructural and functional changes in subendocardium, and are associated with the subendocardial brosis [15]. However, in our study GLS and GLSR just exhibited a moderate relationship with cardiac brosis and hypertrophy in the whole ber layers, which made it a lower signi cance in differentiating damage and non-damage in cardiomyocytes in comparison with the other STE parameters. Nevertheless, GLS was obviously enhanced in the DMF group, suggesting the probable improvements in subendocardium and the high potential to predict the cardiac diastolic function alterations in the early stage of treatment. We further compared the area under the ROC curve for distinguishing the most suitable diagnostic markers for diabetic cardiomyopathy. Interestingly, CTI parameters (MPI and IVRT) and STE parameters (GLS, FAC, GCS and GCSR) shared the equal capacity for the diagnosis of diabetic cardiomyopathy. Moreover, CTI parameters were mainly exhibited the speci city in predicting the cardiac dysfunction, but STE parameters the sensitivity. As the understanding of layer-speci c structure changes to the myocardial deformation helps in estimating the transmural disease burden correctly, and provides pathophysiologic insight into the mechanisms of LV dysfunction [48]. The advantage that STE parameters could provide the additional information on the layer-speci c pathologies in diabetic heart made it superior to CTI parameter.

Limitations
Several possible limitations should be considered. Firstly, the drug for diabetes treatment is a major limitation in the present study. Though RhoA/ROCK signaling pathway is of increasing importance in diabetic heart therapy, ROCK inhibition is not the classic way for diabetic cardiomyopathy therapy compared with glucose control by Metformin. However, STE is valuable and feasible in detection of the early and subtle cardiac functional alterations, which still has provided new insight into diabetic cardiomyopathy management. Further we will improve our animal models to challenge more useful methods for the diagnosis of diabetic cardiomyopathy and provide more preclinical data for translational medicine. Secondly, the current number of the investigated rats may serve as a limitation. Nevertheless, the sample size is based on statistical calculations according to previous observations, in addition to the reasonability in experimental studies. So, the sample size should be expanded to solve this problem, which must be a part of future translational studies. Finally, despite that we discussed the ability of STE parameters for indicating the damage in different ber layers on the basis of prior studies, we did not correlate the functional parameters with structural parameters, including myocardial hypertrophy and brosis, in different myocardial ber layers respectively. Even so, that does not exert a signi cant impact on the way of drawing to the conclusion based on our data in scienti c manners.

Conclusion
In conclusion, our data suggesting that the early and subtle alterations of cardiac function in diabetic hearts are detectable by CTI and STE parameters simultaneously. The comparison between CTI and STE parameters has identi ed STE strain and strain rate to be preferable diagnostic measures for the early detection and quanti cation of layer-speci c functional alterations. Furthermore, FAC, GCS and GCSR are proved to be potential predictive indexes of cardiac dysfunction in diabetic cardiomyopathy. Our study provides translational evidence for a better understanding on the pathophysiologic progress of diabetic cardiomyopathy during management.  Values are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs CON group; # P < 0.05, ## P < 0.001, ### P < 0.001 vs DM group. CON: control rats, DM: diabetic rats, DMF: diabetic rats and fasudil, CONF: control rats and fasudil. FBG: fasting blood glucose, BW: body weight, HW: heart weight, LVW: left ventricle weight, TL: tibia length, HW/BW: heart weight / body weight, HW/TL: heart weight / tibia length, LVW/HW: left ventricle weight / heart weight.   Values are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs CON group; #P < 0.05, ##P < 0.001, ###P < 0.001 vs DM group.
CON: control rats, DM: diabetic rats, DMF: diabetic rats and fasudil, CONF: control rats and fasudil.    Cardiac function changes. The relative values of the treated and untreated diabetic rats in comparison to the healthy ones were shown.
Healthy controls were normalized to one. CON: control rats, DM: diabetic rats, DMF: diabetic rats and fasudil, CONF: control rats and fasudil.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. LYJSupplementaryMaterials2021327.pdf