Study And Intervene On The Relationship Between Cardiac Autonomic Nerve Remodeling And Ventricular Arrhythmias

Background:The normal physiological activity of the heart is reglulated by the autonomic nerve system, which comprises the sympathetic nerve system and parasympathetic nerve system. Generally, stimulating the sympathetic system cause accelerated heart rate, more rapid conduction and strengthened contraction, which is called positive chronotropic, positive chronotropic and positive inotropic action respectively, while stimulating the parasympathetic nerve leads to the opposite condition. Under the mutual adjustment of the two systems, the heart is electrically and mechanically controlled as a syncytium. However, under some pathological conditions, the cardiac autonomic nerve could be damaged and cause abnormal heart behavior. Diabetes mellitus and myocardial infarction are two common diseases that cause cardiac autonomic neuropathy.According to the data published by the International Diabetes Federation in 2015, there were 415 million diabetes patients globally, and another 318 million people were at the risk of diabetes mellitus (DM). Every 6 seconds, someone would die of DM, and the mortality is higher than the total number of AIDS, tuberculosis and malaria. DM is a kind of metabolic disease, and its main feature is high blood glucose or hyperglycemia. Permanent existence of hyperglycemia will cause many damages and dysfunctionsof the body, such as the heart, kidney, vessels and the eyes. In these complications of DM, diabetic cardiac autonomic neuropathy (DCAN) is one of the most hidden and serious complications. Epidemiological studies have shown that about one third of diabetes patients are complicated with DCAN. Moreover, the incidence of the DCAN will obviously increase with the extension of the diabetes course. This eventually leads to the high incidence of malignant arrhythmias and even sudden cardiac death, which lead to serious threat to the diabetic patients’ life.Clinical studies have demonstrated that the parasympathetic component can be impaired in diabetes, which is the earliest sign of DCAN. Besides, heterogeneous cardiac sympathetic innervations has been found in diabetic individuals those be complicated with cardiac autonomic neuropathy (CAN) by radionuclide imaging techniques. Collectively, these findings have led to the notion of imbalanced sympatho-parasympathetic innervations in the diabetic heart, and it may be relevant with the high occurrence of ventricular arrhythmias in diabetic patients, but direct neuroanatomical evidence supporting this idea is lacking. In the first part of this study, histologic and molecular biological technique were used to investigate the influence of diabetes mellitus on cardiac autonomic innervations and its relationship with the inducibility of ventricular arrhythmias in diabetic rats.Cardiac innervation is highly plastic and changed over time at different stages of cardiovascular disease. MI induces nerve reinnervations, including sympathetic nerve and cholinergic nerve fibers, and infarction induced nerve sprouting is mainly sympathetic nerve fibers. The autonomic imbalance is characterized by increased sympathetic and decreased parasympathetic activity. Therefore, infarction-induced cardiac autonomic abnormalities might lead to sympathetic overactivation and subsequent VAs. Cardiac innervation is sculpted by growth factors during infarction. Nerve growth factor (NGF), a chemoattractive factor, plays a key role in sympathetic nerve sprouting and hyperinnervation. Besides, cardiac reinnervation is also modulated by chemorepulsive factors such as semaphoring 3A (sema 3A). Sema 3A is a secreted protein that regulates axon/dendrite growth and neuronal migration. It initiates growth cone collapse, inhibits axonal outgrowth, and plays crucial roles in neural, cardiac and peripheral vascular patterning. In the second part of this study, the effects of sema 3A were investigated by regulating cardiac sema 3A expression via the local intramyocardial injection of lentiviral-mediated sema 3A shRNA and the intravenous injection of recombinant sema 3A. Our data revealed that silencing sema 3A augmented sympathetic hyperinnervation, increased myocardial NE content and inducible VAs. Conversely, the administration of exogenous sema 3A attenuated those abnormalities and protected infarcted hearts from inducible VAs.DM is associated with a marked increase (by a factor of 2 to 4) in the risk of coronary heart disease and is considered as a "coronary heart disease equivalent". Moreover, epidemiological studies have shown higher mortality after myocardial infarction (MI) in diabetic than nondiabetic patients. Ventricular arrhythmias (VAs) is one of the most important causes of death in diabetic patients with MI, but the exact mechanism is unclear. Our previous study indicated that DM causes cardiac nerve denervation, relative sympathetic hyperinnervation and inhomogeneous neural innervations, which may be associated with an increase in the induction of ventricular arrhythmia in diabetic rats. DM predisposes to MI and previous studies have demonstrated that MI results in sympathetic nerve sprouting, which increases arrhythmia events. To our knowledge, few studies have evaluated the remodeling of sympathetic nerves after MI in diabetic models, so the exact mechanism of remodeling and whether this peculiar remodeling is associated with the high incidence of VAs in diabetic patients with MI is unclear. In the third part of this study, we investigate the risk of ventricular arrhythmias after myocardial infarction with diabetes associated with sympathetic neural remodeling in rabbits.Part Ⅰ:The influence of diabetes mellitus on cardiac autonomic innervations and its relationship with the inducibility of ventricular arrhythmias in diabetic ratsObjectives:Diabetic cardiac autonomic neuropathy (DCAN) may cause fatal ventricular arrhythmias and increase mortality in diabetics. However, few data are available regarding the exactly changed character of cardiac autonomic denervation after diabetes. In this study, we dynamically observed the progression of DCAN and its relationship with the inducibility of ventricular arrhythmias in diabetic rats.Methods:Rats were randomly divided into normal control and diabetes mellitus (DM) groups. Rats were sacrificed on month 3 and 6 post-treated. Heart rate variability and programmed electrical stimulation were used to assess the electrophysiological characters and the inducibility of ventricular arrhythmias in the animals. Immunohistochemistry and real-time RT-PCR were used to measure choline acetyltransferase (ChAT, a marker for parasympathetic nerves) and tyrosine hydroxylase (TH, a marker for sympathetic nerves) positive nerve fibers and corresponding mRNA expressions in the proximal and distal regions of the left ventricle.Results:Short-term of diabetes resulted in distal myocardial parasympathetic denervation with sparing of the proximal myocardium. By 6 months, both parasympathetic and sympathetic denervation further aggravated. Moreover, electrophysiological studies showed sympatho-parasympathetic imbalance and the increased inducibility of ventricular arrhythmias in diabetic rats.Conclusions:DM causes cardiac nerve denervation and causes relatively sympathetic hyperinnervation and inhomogeneous neural innervations, which may be associated with the increased inducibility of ventricular arrhythmias in the diabetic rats.Part Ⅱ:Effects of semaphorin 3A on cardiac autonomic regulation and subsequent ventricular arrhythmias in post-infarcted heartsBackground:To investigate the effects of semaphorin 3A (sema 3A) on cardiac autonomic regulation and subsequent ventricular arrhythmias (VAs) in post-infarcted hearts.Method and results:In order to explore the functions of sema 3 A in post-infarcted hearts, lentivirus-Sema 3A-shRNA and negative control vectors were delivered to the peri-infarcted myocardium rats respectively. Meanwhile, recombinant sema 3A and control (0.9% NaCl solution) were injected intravenously into infarcted rats to test the therapeutic potential of sema 3 A. Results indicated that levels of sema 3A were higher in post-infarcted hearts compared with sham rats. However, sema 3A silencing leaded to sympathetic hyperinnervation, increased myocardial norepinephrine (NE) content and inducible VAs. Conversely, the intravenous administration of sema 3A to infarcted rats reduced sympathetic nerve sprouting, improved cardiac autonomic regulation, and decreased the incidence of inducible VAs. However, both infarct size and cardiac function were similar among infarcted hearts.Conclusions:The upregulation and administration of sema 3A exerted beneficial effects on infarction-induced cardiac autonomic disorders by increasing cardiac electrical stability and reducing VAs. Sema 3A might be a potential therapeutic agent for cardiac autonomic abnormalities induced arrhythmias.Part Ⅲ:Risk of ventricular arrhythmias after myocardial infarction with diabetes associated with sympathetic neural remodeling in rabbitsObjective:In this study, we aimed to explore sympathetic neural remodeling after MI in diabetic rabbits and its relationship with VAs.Methods:Rabbits were randomly assigned to 4 groups:control, DM, MI and diabetic myocardial infarction (DI). After electrophysiological experiments in vivo, immunohistochemistry was used to measure tyrosine hydroxylase (TH, a marker for sympathetic nerves) positive nerve fibers in the infarcted border and the non-infarcted left ventricle free wall. The mRNA levels of nerve growth factor (NGF) and TH were examined by quantitative real-time RT-PCR.Results:The corrected QT interval and QT dispersion were significantly more prolonged with DI than other conditions. The density of TH-positive fibers and corresponding mRNA expression were significantly higher with DI than with DM and under control conditions, but was lower than with the MI group. Moreover, the distribution and structure of regenerated nerve was heterogeneous in DI rabbits.Conclusions:MI results in sympathetic neural remodeling in diabetic rabbits, which may be responsible in part for the increased occurrence of VAs.