Effects Of Diabetic Autonomic Neuropathy On Cardiac Electrophysiology And Its Intervention Study

Backgrounds:Diabetes (diabetes mellitus, DM) is one of the most challenging diseases for human health at present. As the incidence of diabetes increases, its harmfulness is becoming more and more prominent, following with the serious social and economic burden. There are many complications of diabetes which can almost cause damages to each viscera and organization of the body. In these complications of diabetes mellitus, diabetic cardiac autonomic neuropathy (DCAN) is one of the most serious and hidden complications. Epidemiological studies have shown that about half of diabetic patients complicated with DCAN. Moreover, the incidence of the DCAN will obviously increased with the extension of the course. This eventually lead to the incidence of malignant arrhythmias, myocardial infarction, heart failure and even sudden cardiac death, which cause serious threat to the diabetic patient’s life. Recent studies revealed that the incidence of ventricular arrhythmias (VAs) increased obviously in those patients with DCAN, and often accompanied by obviously long QT, even lead to sudden death, but less researchs were found about its mechanisms and effective intervention treatments.Cardiac autonomic nerve contains the sympathetic nerve and vagus nerve, they complement each other and antagonistic to each other, and regulate the activity of the heart intensively. In diabetic individuals, hyperglycemia can cause ischemia, hypoxia; oxidative stress; polyol pathway; advanced glycosylation end products (AGE) and the depletion of the nerve growth factor(NGF), these comprehensive factors lead to cardiac autonomic nerve damages. Sympathetic nerves and vagus nerves suffer different degrees of damage. The vagus nerves suffer dominantly in the early stage, and both the sympathetic nerves and vagus nerves are involved later. But there are uneven damages in different regions, lead to the heterogenous denervation of the heart. Any uneven cardiac autonomic denervation (which may contain regional uneven distribution between sympathetic nerves and imbalances between the sympathetic and vagus nerves) can all cause the increased electrophysiological heterogeneity of these regions, and will increase the incidence of ventricular arrhythmias obviously.Heterogenous cardiac sympathetic innervations has been found in diabetic individuals who was diagnosed with cardiac autonomic neuropathy (CAN) by radionuclide imaging techniques or laboratory examination, especially in the proximal and the distal of the left ventricular (LV). With longer duration of DM, the sympathetic denervation is more significant in the distal of LV than in the proximal, which form the uneven sympathetic nerve injury. The depletion of NGF is similar to the sympathetic denervation of the same region, which indicate that the depletion of NGF may be involved in the mechanisms of DCAN. In the first part of this study, we intend to:explore the relationship between the depletion of NGF and the sympathetic denervation in the proximal and distal of LV in diabetic rats with DCAN; to explore the relationship between the heterogenous cardiac sympathetic innervations and the ventricular arrhythmias(VAs). By infusing the exogenous NGF to the left stellate ganglion (LSG) in diabetic rats, we intend to observe whether it can repair the heterogenous cardiac sympathetic innervations caused by DCAN in LV; to observe whether it can decrease the incidence of VAs, by this method, we intend to explore new therapeutic measures on DCAN and the VAs second to it.DCAN can lead to the imbalance of cardiac sympathetic innervation which may increase the incidence of VAs. The sensitive degree to hyperglycemia injury is different in sympathetic and vagus nerves, so the DCAN can lead to the disequilibrium between the sympathetic and the vagus nerves, which will increase the instability of cardiac electrophysiology and the incidence of VAs. Our idea is to achieve the balance between the sympathetic and vagus nerves in rats with DCAN through intervention therapy, thus reducing the incidence of VAs. Plasticity of researchs on mesenchymal stem cells(MSCs) have laid a solid foundation for the application of MSCs in multiple organs and multiple diseases. As a kind of autologous stem cell, MSCs has strong ability of self-renewal, proliferation and multi-directional differentiation potential. MSCs can be differentiated into neurons in specific induction conditions, it can also secrete a variety of nutritional factors through paracrine, so it can be used as ideal seed cells for the treatment of central and peripheral nervous system injury. MSCs treatment for DCAN may have a broad application prospect, but there is still no related report. In the second part of this study, we assume MSCs can repair or reverse the diabetic cardiac autonomic nerve damage, improve the imbalance between the sympathetic and the vagus nerves, reduce the incidence of VAs caused by DCAN through its multi-directional differentiation potential and paracrine effects. We hope to offer a new and reliable treatment and theoretical foundation for DCAN.As mentioned above, based on the previous researchs, our studies intend to explore the relationship between the depletion of NGF in the left ventricle and the cardiac sympathetic innervations in diabetic rats; to explore the relationship between the VAs and DCAN; to intervene the DCAN through different methods; to find new treatment ideas for preventing the DCAN and the VAs second to it. This topic is divided into the following two parts:Part I:Effects of exogenous nerve growth factor on heterogenous cardiac sympathetic innervation and ventricular arrhythmias in diabetic ratsObjectives:The main objectives of this study are:(1) To explore the characteristics of diabetic cardiac autonomic neuropathy, to explore the relationship between the depletion of NGF and the cardiac sympathetic denervation, to explore the relationship between the diabetic cardiac autonomic neuropathy and VAs.(2) To explore the new therapeutic measures which can prevent the diabetic autonomic neuropathy and VAs second to it.Material and Methods:All male wistar rats were randomly divided into3groups:control group, DM with saline infused into the LSG group (DM), DM with NGF infused into the LSG group (DN). Diabetes was induced by intraperitoneal injection of streptozotocin(STZ,50mg/kg; Sigma, USA). The animals were subsequently maintained for24weeks, then the DN group received an NGF (20ug/kg; Jinlujie, Wuhan, China) injection to the LSG whereas in the DM group the rats received an equivalent volume of saline. After28weeks all rats received electrophysiological experiments in vivo to induce the VAs, then sympathetic innervations and the expression of tyrosine hydroxylase(TH) and NGF were studied respectively at the proximal and distal of LV by immunostaining, RT-PCR or western blot analysis.Results:Rats of DM group has significantly prolonged corrected QT interval and higher incidence of inducible VAs than that of control group(P<0.01). The density of tyrosine hydroxylase-positive fibers and corresponding mRNA, together with the NGF protein content in the proximal and the distal of the LV were significantly decreased in DM group respectively compared to the control group(all P<0.01). And there were significant proximal-distal heterogeneities about TH and NGF expression in DM group(all P<0.01). After NGF being infused to the LSG, both the TH and NGF expression were increased in DN group compared to the DM group(all P<0.01), and the significant proximal-distal heterogeneities of TH and NGF expression were both reduced markedly in DN group than in DM group(P>0.05). Moreover, the corrected QT interval and QT dispersion were significantly shortened in the DN group than in the DM group(all P<0.01), and the incidence of VAs in the DN group was much lower than that in the DM group.Conculsions:Heterogeneous cardiac sympathetic denervation in the left ventricle could result in electrophysiological unbalance of these regions in diabetic rats, leading to prolonged corrected QT interval and higher incidence of ventricular arrhythmias; NGF intervention can arouse the cardiac sympathetic nerve regeneration, reduce not only the heterogeneity of cardiac sympathetic innervations but also the incidence of ventricular arrhythmias in diabetic rats. Part Ⅱ:Mesenchymal stem cell therapy improves diabetic cardiac autonomic neuropathy and decreases the inducibility of ventricular arrhythmiasObjectives::In this part of the study, we investigated the effect of MSCs therapy on DCAN in diabetic rats, to explore the new therapeutic measures which can prevent the diabetic autonomic neuropathy and VAs second to it.Material and Methods:Forty wistar rats were divided into normal control, diabetes mellitus (DM) control, MSC treatment (6×106MSCs via direct myocardial injection) and MSC-conditioned medium group (100μl via direct myocardial injection). Immunohistochemistry was used to measure choline acetyltransferase (ChAT, a marker for parasympathetic nerves), growth-associated protein-43(GAP-43, a marker for new autonomic nerves) and tyrosine hydroxylase (TH, a marker for sympathetic nerves) positive nerve fibers in the ventricular myocardium. Heart rate variability was used to evaluate the changes between the sympathetic and vagus nerves. Programmed electrical stimulation was used to assess the inducibility of ventricular arrhythmias in animals. Inorder to confirm that MSCs promoting cardiac autonomic nerve regeneration mediated partly by its paracrine function, MSC-conditioned medium was prepared as described. Briefly, MSCs were cultured in FBS-supplemented DMEM medium, then in serum-free DMEM. At24h, the medium was collected. The concentrations of neurotrophin-3(NT-3) and nerve growth factor (NGF) in the MSC-conditioned medium were measured by ELISA kits (Millipore, Billerica, MA, USA). Serum-free DMEM was used as a control.Results:Two weeks after MSCs treatment, the density of ChAT, TH and GAP43-positive nerve fibers in MSCs and MSC-conditioned medium group was higher than in DM control group (P<0.05or P<0.01). The ChAT/TH ratio in MSCs group was higher than in DM control group (0.37±0.014vs0.27±0.020, P<0.01). The standard deviation of normal-to-normal R-R intervals in MSCs (5.13±0.69) and MSC-conditioned medium group (4.30±0.56) was higher than in DM control group (3.45±0.60, P<0.05). The inducibility of VAs in the MSCs group was lower than in the DM control group(P<0.01). We also found that MSCs can synthesize and secrete detectable neurotrophic factors such as NT-3and NGF. The concentrations of NT-3and NGF were higher in MSC-conditioned medium than DMEM alone(P<0.01). During the whole experiment, we found no tumorigenic phenomenon after MSCs transplantated to the myocardium.Conculsions:MSC therapy may promote cardiac nerve sprouting and increase the ratio of vagus to sympathetic nerve fibers. It may also suppress the inducibility of ventricular arrhythmias in the diabetic rats.