The Effect Of The Applied Weak Steady Direct Current Electrical Fields On Renovation Of Injured And Ischemic Rabbit Myocardium

1. Background and ObjectiveAs change of living style, coronary artery disease is more and more becoming predominant disease which has being threatening the human health since 1980s. The therapic methods of coronary artery disease have drug, surgical intervention and percutaneous coronary intervention, but the prostecdtive efficacy has not been ideal yet. At present, there is still not effective therapeutic choice for the treatment of coronary artery disease which is at stage of myocardial infarction,ischemic cardiomyopathy and diffuse vessel disease. Recently, therapeutic angiogenesis provided a new method for treatment of coronary artery disease and gained preliminary therapeutic effect on renovation of necrotic and apoptosis myocardium.In 1843, the great German physiologist Emil Du-Bois Reymond used a galvanometer to measure ~1μA flowing out of a cut in his own finger. The injury potentials are due to the short-circuiting of the transepithelial potential difference that occurs at a skin lesion are the endogenous electrical fields (EEFs) which play important roles in controlling several aspects of the cell biology of wound healing. These include directed migration into the wound of epithelial cells, the proliferation of epithelial cells, the axis of division of epithelial cells, endothelial cell remodeling with associated angiogenesis, the proportion of nerves sprouting at the wound, and the directional growth of nerve sprouts towards the wound edge.Additionally, these three endothelial cell behaviors, ie reorientation, elongation, and directed migration, are all forerunners of angiogenesis. Each is regulated by a physiological EF, and angiogenesis frequently takes place within a steady EF; vascular sprouting directed towards a wound. Endogenous EFs therefore may regulate angiogenesis in vivo. It may also be worth testing the notion of using an applied EF to direct blood vessel growth in vivo. Proliferation of vascular endothelial cells (VECs) is a key event in angiogenesis, and full maturation of new vessels into fully formed collateral circulation requires a vessel wall that contains SMCs and fibroblasts.The applied direct current electrical fields (DC EFs) of 150 to 400 mV/mm showed significant effects in directing the 3 types of vascular cells migration, orientation, and elongation. Injured and ischemic tissue such as myocardium also are polarized electically, and this can produce a DC EF of 5.8 mV/mm across an 8-mm zone at the boundary with undamaged tissue. It is unlikely that such a small EF will have significant effects on angiogenesis in this particular situation. An insufficient angiogenic response occurs in ischemic cardiomyopathy, especially in complicated coronary artery disease because angiogenesis and vasculogenesis are critical process in wound healing.Therefore, the applied steady direct current electrical fields (DC EFs) of 1.5 to 4.0V/cm is a promising approach for therapeutic angiogenesis in complicated coronary artery disease. Recent work showed that electrical stimulation induced significant angiogenesis in vivo, through enhanced vascular endothelial growth factor (VEGF) production by muscle cells and vascular endothelial cells, and improved ischemic rat limbs. Applied EFs with a wide variety of stimulation protocols have been used clinically to treat nonhealing skin wounds, and many studies have claimed success. In addition, nearly every clinical trial using electric fields to stimulate healing in mammalian wounds reported a significant increase in the rate of healing from 13 to 50%.Howere, if applied EFs would been used to treat ischemic cardiomyopathy, may angiogenesis and collateral circulation be enhanced? May injured and ischemic myocardium be repaired and regenerated? Morever, the passage of electric current through tissue can produce electrothermal, electrochemical or electrophysical effects. In do so, is applying a DC EFs range from 1.5 to 4.0V/cm to a heart safe?The study was to apply the weak steady DC EFs range from 1.5 to 4.0V/cm to a normal or injuried and chronic ischemic (infarcted) heart in rabbits in order to investigate if their electrothermal, electrochemical or electrophysical effects in a normal heart would be safe, if angiogenesis and collateral circulation will be enhanced, and if injured and ischemic myocardium would be repaired and regenerated. 2. MethodsIn part one, the safety to apply a DC EFs range from 1.5 to 4.0V/cm to a normal heart was explored .Firstly, the WYJ voltage and current flow-sdtabilizing direct current power supply was Modified, and the Pt electrodes was self-made.Secondly, the purpose of study was to investigate the electrochemical action of the applied weak steady DC EFs on the interstitial fluid. The heparinized arterial blood directly drawn from the left ventricles of hearts in 10 rabbits was added into the self-made Hittorf measuring device for determination of transference numbers instead of the Interstitial fluid, followed by which the direct current of 4.5V(n=5) and 6.0V (n=5)was applied to electrolyse for 1h, respectively. The blood pH value and the ion concentrations of potassium (K+), sodium(Na+), chlorinum(Cl?), calcium (Ca2+)and bicarbonate(HCO3?) were measured in the Hittorf device's anode cell, cathode cell and in-between cell, and the normal control. Thirdly, the effect of the applied weak steady DC EFs on electrocardiogram(ECG) and hemodynamics was investigate.. A pair of Pt electrodes was parallelly placed on the cardiac apex epicardium at the two sides of the main branch of the left coronary artery in the rabbit hearts by thoracotomy without endotracheal intubation, following by which a DC EFs range of 0 to 6.0V/cm was applied. The parameters about the limb-lead ECGs and the arterial and left ventriculal blood pressures by continuously invasive monitor were measured and analyzed .Finally, the cardiac electrical injuries caused by the applied weak steady DC EFs was evaluated. Ensued from the previous study, the epicardial temperature in the anelectrode was measured, the myocardial histologic structures were also observed under the light microscope and the transmission electron microscope.In part two, the aim of this study was to approach the therapeutic efficacy of both plane and transmural DC EFs of 2V or 3V which were used to treat ischemic cardiomyopathy in rabbits.In the first place, a myocardial infarction model in rabbits without endotracheal intubation was establish. The sternum was carefully incised along the midline to avoid injury to the parietal pleura. The major branch of the left coronary artery of 10 rabbits used as coronary artery ligation group was then ligated. Five rabbits sham ligating the coronary artery were used as control. After 4 weeks, cardiac functional analyses by noradrenalin challenge and histopathological analyses were performed. In the second place, this study was to apply both plane and transmural DC EFs of 2V or 3V to injuried and chronic ischemic (infarcted) heart in rabbits in order to investigate the effect on renovation of injured and ischemic rabbit myocardium. Fourty Chinese rabbits were randomly divided into five groups:the myocardial infarction (MI)group, the plane electrical fields 2V(PEF2V) group, the plane electrical fields of 3V (PEF3V)group, the transmural electrical fields 2V(TEF2V) group , the transmural electrical fields 3V(TEF3V) group. Accordingly, the various DC EFs were applied on the surface of ischemic myocardium of each group rabbit models of chronic myocardial infarction. After 4 weeks, cardiac functional analyses by noradrenalin challenge and histopathological analyses were performed. The wet organ weight, dry organ weight, and body weight were measured to calculate, as parameters of organ edema following congestive heart failure. Infarct size was measured in macrospecimens stained with Masson's trichrome reagent. As an index of myocardial necrosis, the extent of fibrosis was measured in images at magnification of 400. The number of capillaries was counted in images stained by CD31 immunostaining. Collagen fiber biochemistric remodeling was appraisaled by using TypeⅠand typeⅢcollagen's protein levels determined from immunostaining and Western blots.In the last place, ensued from the previous study, the purpose of study was to investigate myocardial cells'proliferation and apoptosis in ischemic myocardium. After 4 weeks, proliferating myocyte nuclei of heart tissue labeled by Ki-67 nuclear antigen in rabbit model of myocardial infarction were determined from immunohistochemical staining at a magnification of×400. TUNEL assay for cardiomyocyte apoptosis of heart tissue are also analyzed for infarcted area, border zone infarcted area, and noninfarcted area, respectively. Eventually, expression of Bcl-2 /Bax related to apoptosis in rabbit model of myocardial infarction was determined from immunohistochemical staining and Western blots.3. Results(1) The voltage output of the modified WYJ voltage and current flow-sdtabilizing direct current power supply was adjusted within range between 0.0 to 5.0V precisely. The maximum current density of the self-made Pt electrodes at DC EFs of 5.0V /cm was only 0.089mA/cm, and naked eyes failed to view electrical burn in the ventricular wall when DC EFs of 5.0V /cm were applied on on the epicardium of a normal pig heart for 1h.(2) The electrochemical effects of the applied weak steady DC EFs (≤6.0V) on the interstitial fluid may be neglected. As far as the effect on ECG and hemodynamics is concerned, the weak steady EFs (≤3.0V/cm) is safe.And the weak steady EFs(≤3.5V/cm) failed to cause the cardiac electrical injuries obviously.(3) Compared with the coronary artery sham-lagation group, the coronary artery ligation group all presented to myocardial infarction through ECG and histologic characteristics. Cardiac functional analyses by noradrenalin after 4 weeks showed the parameters on cardiac systolic function in the coronary artery ligation group got worse significantly, that is to say LVSP and +dp/dtmax increased, while t-dp/dtmax shorten. Meanwhile, cardiac diastolic function got worse significantly, too. LVEDP raised, and -dp/dtmax dropped.(4) All parameters of organ edema following congestive heart failure which include heart weight, ratio of wet heart weight to body weight,wet lung weight, ratio of wet lung weight to body weight, ratio of wet lung weight to dry lung weight, wet liver weight, ratio of wet liver weight to body weight,and ratio of wet liver weight to dry liver weight were not significantly different among the five groups. Cardiac functional analyses by noradrenalin demonstrated that cardiac systolic and diastolic function in PEF2V, TEF2V and TEF3V groups were significantly improved more than that in MI and PEF3V groups. Significant improvement was found in minimizing infarct area and lowering the extent of fibrosis among the 4 received DC EFs groups, and the transmural electrical fields is superior to the plane electrical fields. Additionally, changes in typeⅠand typeⅢcollagen's protein levels determined from immunostaining and Western blots indicated two transmural electrical fields were also preferable to collagen fiber biochemistric remodeling. Finally, More CD 31-immunostained vessels were also found in infarcted area, border zone infarcted area, and noninfarcted area in TEF3Vgroup than that in the other groups, but predominant in noninfarcted area.(5) In noninfarcted area, myocardial cells'proliferation in PEF2V and TEF3V groups was significantly higher than MI group; still in border zone infarcted area, myocyte proliferation among the 4 received DC EFs groups increased more than MI group, and two transmural EFs groups were also superior to two plane EFs groups.Compared with MI group, cardiomyocyte apoptosis only in border zone infarcted area in PEF2V and TEF3V groups was significantly restrained. Furthermore, Bcl-2 /Bax related to apoptosis mainly expressed in noninfarcted area and especially border zone infarcted area among 5 groups. Expression of Bcl-2 in noninfarcted area and border zone infarcted area was gradually increased in sequence among MI , PEF2V, PEF3V, TEF2V and TEF3V groups , and two transmural EFs groups were also superior to two plane EFs groups.Howere, Bax expressed counter to Bcl-2.4. Conclusions(1) the Modified WYJ voltage and current flow-sdtabilizing direct current power supply and the self-made Pt electrodes can satisfy the requirement of this study. Effect of the applied weak steady DC EFs (3.0V/cm) on ECG, hemodynamics, electrochemical action and cardiac electrical injuries is not obvious, thus is safe.(2) A myocardial infarction model in rabbits without endotracheal intubation was establish according to the thoracic cavity anatomic characteristic peculiar to a rabbit efficiently, conveniently and operationally. It establish a support for further experiments technologically.(3) The study to approach the therapeutic efficacy of both plane and transmural DC EFs of 2V or 3V which were used to treat chronic myocargial infarction in rabbits suggests:①The suitably weak steady DC EFs induce angiogenesis and possibly collateral circulation in ischemic myocardium, minimize infarct area, lower the extent of myocardial fibrosis, lessen ventricular remodeling, thereby protect the myocardium.②The suitably weak steady DC EFs may promote cardiomyocyte proliferation and restrain myocyte apoptosis in rabbit myocardial infarction model. Maybe myocyte renewal and regeneration after myocardial infarction benefit from it.③Overall transmural electrical fields, especial at 3V between endocardium and epicardium in a infarcted rabbit heart is superior to plane electrical fields on the epicardium of a infarcted rabbit heart.(4) Howere, this study using a rabbit model was still preliminary and vague because of the very small heart of the rabbit.Large animal models, in particular dogs, can present infarct size limitation experiments as normalized by the border zone infarcted area versus the infarctedarea. This also may prevent obfuscation by variable anatomy and coronary collateral flow.