In this animal model of induced cardiac hypertrophy, we demonstrated that continuous cardiac hypertrophy leads to significant myocardial fibrosis and necrosis. Berberine inhibited the progression of cardiac fibrosis and the deterioration of myocardial GCS after the formation of cardiac hypertrophy. We have shown that the layered strain derived from speckle tracking, particularly endocardial strains, are more closely associated with changes in myocardial interstitial fibrosis than the left ventricular ejection fraction derived from 2D echocardiography.
To the best of our knowledge, there is no report in the literature on the evaluation of left ventricular systolic function in an animal model of myocardial hypertrophy by layered strain imaging technology. Based on the previous series of experiments10–12, the animal model of myocardial hypertrophy in this study was made by intraperitoneal injection of isoproterenol without opening the chest. Pathological staining confirmed that the myocardial cross-sectional area of rats in the myocardial hypertrophy group increased and myocardial fibrosis was observed. Weighing with electronic balance, the calculated LVMI of rats in the myocardial hypertrophy group increased (2.93 vs. 1.99; P < 0.05). Conventional transthoracic echocardiography showed that the thickness of the interventricular septum increased (4.56 vs. 2.18; P < 0.05), the relative wall thickness increased (1.11 vs. 0.78; P < 0.05), and the EF response to systolic function was normal (94.65 vs. 94.86; P > 0.05). These findings are consistent with early cardiac changes in patients with myocardial hypertrophy, which indicates that our method for preparing a rat model of myocardial hypertrophy is feasible.
Echocardiography is still a recognized non-invasive examination method, which could provide reliable and accurate evaluation of cardiac function13. LVEF is a commonly used index for clinical evaluation of left ventricular systolic function, and it can reflect left ventricular pumping function. It cannot reflect the inherent contractile characteristics of the myocardium, so it has no advantage in identifying early myocardial contractile dysfunction and evaluating the efficacy of short-term drug intervention14. The left ventricular myocardium of a normal person is composed of three layers of myocardium, the endocardial myocardium, the middle myocardium and the epicardial myocardium. The movement of left ventricular myocardium can be divided into longitudinal, radial and circular movements15. The layered strain technology is developed on the basis of the two-dimensional speckle tracking imaging technology(2D-STI). Which automatically tracks the three-layer myocardial acoustic speckles to obtain the strain values of different myocardial layers, and quantitatively evaluate the influence of the disease on the strain of each layer of the left ventricle. To understand the degree of myocardial involvement in detail16. In myocardial ischemia, the degree of damage of the inner, middle and outer myocardium of the ventricular wall is different, which becomes the theoretical basis for evaluating the function of myocardial stratification. The layered strain technology has the advantages of accurate and rapid assessment of myocardial movement without angle dependence, and has become a new emerging technology in recent years to assess myocardial function, especially systolic function under pathological conditions17.
We demonstrated that in our animal model, when evaluating myocardial hypertrophy early myocardial injury, the circumferential strain was more sensitive than the left ventricular ejection fraction, which was confirmed by histopathological examination. In our study, compared with the Control group, the ISO group had no statistical difference in the EF value of the two groups, while the ISO group had a lower CS absolute value. The possible reason for this change was that in the early stage of cardiac remodeling, myocardial cell hypertrophy and thickening of the ventricular wall were conducive to preserving the contractility of the left ventricle in order to compensatively maintain the left heart pump function18. Hypertrophic ventricles were definitely thicker, which reduced the cavity volume more than normal-walled ventricles. LVEF might overestimate the systolic function of hypertrophic ventricles19. We also found that the ISO group GSCendo and GSCmid were significantly lower than the control group, while GSCepi did not significantly decrease. This may be due to the fact that the subendocardial myocardium is most susceptible to myocardial ischemia and other factors. The abnormal deposition of collagen matrix occurs first, the collagen fibers are increased and arranged disorderly, and the subepicardial myocardium is relatively less affected20. Histopathological results confirmed. Our results confirmed that with the process of myocardial hypertrophy, circumferential strain might be more prone to subtle changes in left ventricular mechanics. Previous studies had shown that when the heart was under hemodynamic burden, the heart could use the Frank-Starling mechanism to increase muscle mass and use the neurohormonal mechanism to compensate21. The layered strain results showed that the CS of the two groups showed a decreasing trend from subendocardial myocardium to subepicardial myocardium, and the interlayer strain values were statistically different. This may be because the width of myocardial cells increased from the subendocardial to the middle layer, and the myosin phenotypes of the subendocardial and middle layers were also different from the myosin phenotypes of the subepicardium. Not only the structure was heterogeneous, but the myocardial mechanical state was also heterogeneous. The mechanical state of each layer of myocardium was different during systole, and the subepicardial myocardium produced positive torsional deformation. At the same time, the three layers of myocardium had different contributions to the circumferential strain. The subendocardial myocardium bear greater wall tension and the relative movement amplitude was greater, while the subepicardial myocardium wall tension was smaller and its relative movement was less20,22,23. In addition, we found that the changes of GCS and GSCendo had a significant correlation with myocardial interstitial fibrosis, indicating that circumferential strain could accurately assess the severity of myocardial fibrosis. In summary, the circumferential strain of the left ventricular endocardium might be more sensitive to changes in myocardial function and could reflect myocardial dysfunction earlier. Compared with traditional imaging methods, the layered strain technology based on 2D-STI could provide patients with more comprehensive subclinical myocardial dysfunction information earlier.
ISO-induced myocardial hypertrophy is a reliable, reproducible and well-characterized model of myocardial hypertrophy, which is related to apoptosis, fibrosis, arrhythmia, myocardial cell loss and myocardial ischemia24–26. The RhoA/ROCK pathway is involved in a variety of cardiovascular diseases, such as heart failure, and it may also be involved in ISO-induced cardiac hypertrophy to a large extent27,28. However, the mechanism by which ROCK inhibitors lead to beneficial effects in cardiac hypertrophy remains unclear. Guo, LL, etc. proved that the ROCK inhibitor berberine had a myocardial protective effect in myocardial infarction model, and was considered to be a new type of adjuvant therapy to reduce myocardial injury29. Guo, J et al. through observations before and after reperfusion therapy in myocardial injury rats, found that berberine might inhibit myocardial cell apoptosis and inflammation by inhibiting the Rho/ROCK pathway, and had a significant cardioprotective effect on myocardial I/R30. Due to the relationship between ROCK and cardiomyocyte remodeling, we conducted this study to evaluate the effect of berberine on ISO-induced cardiomyocyte hypertrophy in rats with cardiac hypertrophy. In this study, berberine hydrochloride was administered to rats with myocardial hypertrophy for 1 week, STI-layered strain imaging technology was used to monitor the contraction of left ventricular myocardium, and H&E staining and Masson staining were used to observe myocardial pathological changes. The results showed that after berberine treatment, compared with group C, the structural and functional indexes of conventional echocardiography in group A and group B did not change significantly, but GSCendo was increased, group B was more significant, and the difference was statistically significant. Significance, proved that berberine could effectively improve the contractile function of the left ventricular subendocardial myocardium in rats with myocardial hypertrophy. The mechanism may be related to the greater ratio of the endocardium to myocardial work, and the myocardial oxygen consumption is positively correlated with the myocardial work. In addition, H&E staining and Masson staining of myocardial tissue showed that the inflammation and fibrosis of myocardial tissue were alleviated after berberine treatment, which further proved that berberine could reduce myocardial inflammation and myocardial fibrosis. The possible mechanism was that berberine hydrochloride could act on the Rho/ROCK signal pathway to inhibit the abnormal expression of AngⅡ and SOD, inhibit oxidative stress, and improved myocardial hypertrophy, and the high dose was more obvious. STI-layered could recognize these subtle layer changes.
In this experiment, rats with myocardial hypertrophy were used as the research object. The single medication mode was used to observe the improvement of heart function, eliminating the influence of confounding factors in the combination therapy on the evaluation of drug efficacy. At the same time, accurate and sensitive layered strain imaging technology improved the reliability of experimental results. Animal models play a key role in preclinical research, but the difference between animals and humans is always a problem. Using a chronic mouse model of LV dysfunction, the accuracy and applicability of the strain parameters have been verified by standard conductance catheter measurements31. STI has also been applied to various animal models, such as diabetic cardiomyopathy and myocardial infarction models32,33. In summary, the stratified strain technique can quantitatively evaluate the changes in the myocardial function of each layer of the left ventricle in patients with myocardial hypertrophy,which is expected to be a new method for clinical quantitative evaluation of left ventricular systolic dysfunction.
Critique of Study
This study had several limitations. (1) The doses of berberine used in this experiment were 5 mg/kg/day and 10 mg/kg/day, and the duration of the medication was relatively short. Therefore, it was necessary to increase the observation of drug course, and at the same time to investigate whether the reduction of EF value occurred in the rats with reduced layered strain value;This should be explored in future experiments; (2) The rat was small and cannot accurately divide the three parts of the short axis of the left ventricle. To ensure the maximum possible accuracy of the measurement, only the most obvious left ventricle was selected Image of papillary muscle level. (3) The number of experimental cases was small, and the correlation between stratified strain and clinical application of berberine need to be further confirmed in large-scale clinical trials in the future.