The major findings 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 STZ-high-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 fibrosis both contribute to LV remodeling[2, 28]. In line with previous studies, we found the significant augmentation of myocardial hypertrophy and fibrosis in diabetic rat heart, which indicated that STZ-induced diabetic rats in our study developed into cardiac remodeling under the long-term influence of diabetic state.
Numerous molecular signaling pathways have involved in the pathophysiological process of diabetic cardiomyopathy[2, 3, 28]. Recent studies revealed the significant 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. Activated ROCK contributes to the cardiomyocyte apoptosis, cardiac hypertrophy and fibrosis[5, 30, 31], and mediates vascular smooth muscle cell contraction and several proinflammatory, thrombogenic and fibrogenic molecules. Notably, recent study showed that T2DM patients treated with glucose-lowering drugs, antihypertensive treatment and statins were of significant increase in ROCK activation, suggesting the additional role of ROCK inhibition in the prevention of T2DM. 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[8-10]. In the present study, we used Fasudil, a ROCK inhibitor, to inhibit RhoA/ ROCK signaling pathway in diabetic animal models. After 4-week treatment, ROCK inhibition prominently alleviated cardiac hypertrophy and fibrosis, suggesting a cardioprotective effects against cardiac remodeling in diabetic rats. The cardiac ultrastructure in diabetic rats was confirmed by TEM detection, which characterized by dumbbell-shaped mitochondria for disturbed fusion, smaller fragment and even the distorted vacuous mitochondria for exacerbated fission. 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[35-38]. In our study, ROCK inhibition improved the disturbed mitochondrial fission/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 fibrosis 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 have observed significant improvements of cardiac dysfunction in diabetic rats treated with fasudil for 8 weeks via modulation of Ca2+ 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 defined 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. Furthermore, MPI is demonstrated a superior marker of cardiovascular morbidity and mortality than conventional parameters. In diabetic populations or animals, MPI has been found increased with a prolonged IVCT and IVRT and a shortened AET[43-47]. Similar findings were obtained in our study, where the diabetic rats had significantly 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 benefits 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 efficiency. Damage in different myocardial layers leads to distinct phenotypes in cardiac dysfunction, which make it possible to classify heart disease according to layer-specific 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 fibrosis. 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. Consistent with this theory, Niu 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. 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 findings, 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 significantly ameliorated GLS, GCS and GCSR, which manifested the improvements of the cardiac hypertrophy and fibrosis 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. The improvements of cardiac ultrastructure also contributed to restore the global strain and strain rate in the DMF group. Moreover, FAC, reflecting the degree of thickening in the radial direction, 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 layer-specific cardiac function alterations.
The present study has demonstrated that ROCK inhibition improved diabetes-induced myocardial hypertrophy and fibrosis, 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 identified the sensitivity and specificity of conventional parameters for cardiovascular disease diagnosis[3, 4, 12].Similar findings were confirmed in our study. Whereas, the mild ability of indicating diabetic myocardial hypertrophy and fibrosis made the conventional parameters fail to detect the subtle alterations of cardiac function. CTI parameters, such as IVCT, IVRT, MPI, showed prominent sensitivity and specificity of cardiac systolic and diastolic function respectively, which are consistent with previous data. 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.The findings in our study also confirmed 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 specific 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 fibrosis. However, in our study GLS and GLSR just exhibited a moderate relationship with cardiac fibrosis and hypertrophy in the whole fiber layers, which made it a lower significance 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 specificity in predicting the cardiac dysfunction, but STE parameters the sensitivity. As the understanding of layer-specific structure changes to the myocardial deformation helps in estimating the transmural disease burden correctly, and provides pathophysiologic insight into the mechanisms of LV dysfunction. The advantage that STE parameters could provide the additional information on the layer-specific pathologies in diabetic heart made it superior to CTI parameter.
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 fiber layers on the basis of prior studies, we did not correlate the functional parameters with structural parameters, including myocardial hypertrophy and fibrosis, in different myocardial fiber layers respectively. Even so, that does not exert a significant impact on the way of drawing to the conclusion based on our data in scientific manners.