Experimental Study On Effects Of Electric Stimulations With Different Amplitude During Absolute Refractory Period On Cardiac Function Of Rabbits With Heart Failure.

Major advances have been made over the past two decades in the pharmacologic treatment of chronic heart failure (HF). Angiotensin-converting enzyme inhibitors, beta-blockers, and aldosterone antagonists have had a substantial impact on reducing mortality and morbidity in patients with HF and low left ventricular ejection fraction. These treatments delayed the progression toward advanced intractable HF but did not arrest progressive worsening of the disease. Patients on optimal medical therapy continued to deteriorate, albeit at a much slower pace, ultimately requiring further intervention. This gave rise to a host of device-based therapies that emerged in recent years to address this unmet need. Device therapies such as cardiac resynchronization, the CorCap cardiac support device (Acorn Cardiovascular, Inc., St. Paul, MN), and the OPTIMIZER System (Impulse Dynamics USA, Inc., Orangeburg, NY) are a few examples. According to the development and implementation of cardiac contractility modulation (CCM) as a device-based therapy for the treatment of patients with advanced HF. Treatment of patients with HF using CCM electrical signals is at present an investigational form of therapy. CCM had no effect on heart rate and induced no arrhythmia in short time. It is concluded that electric currents delivered during the ARP could significantly enhance the contractility of myocardium safely, suggesting that CCM stimulation is a novel potent method for contractility modulation. CCM signals are non excitatory signals applied during the absolute refractory period(absolute refractory period electrical stimulation, ARPES) and have been shown to enhance the strength of left ventricular contraction in studies performed in animals and humans with heart failure, regardless of the presence of dyssynchrony and QRS prolongation. In patients with congestive heart failure, improvement of exercise tolerance and quality of life have been shown. Recent studies from myocardial biopsies demonstrate that CCM treatment normalizes expression of many genes that are aBNPrmally expressed in heart failure, including proteins involved with calcium cycling. These findings suggest that CCM might be an alternative or even additional electrical treatment option for patients with heart failure.Heart failure (HF) is a term for a syndrome caused by disease of the heart muscle or by extracardiac influences which impair the work of the heart as a pump.Heart failure is defined as a defect in cardiac function when, despite sufficient filling of the chambers, cardiac output decreases and the heart is not able to maintain the metabolic needs of tissues, whilst activation of neurohumoral substances, apoptosis and adaptive changes of the myocardium at the molecular level develop. Neurohumoral systems help to maintain circulatory homeostasis by increasing inotropy and chronotropy of the failing myocardium and by modulating vascular reaction. Vascular tension is thus regulated by mediators which either dilate or contract blood vessels in various parts of the organism in order to ensure blood flow through active organs (metabolic vasodilatation) while maintaining adequate perfusive pressure by vasoconstriction.Sympathoadrenal system (SAS) decrease in activity of mechanoreceptors in heart failure results in the release of catecholamines into the bloodstream. The positively chronotropic effect of the catecholamines increases heart rate and their positively inotropic effect increases the power and speed of myocardial contraction. Vasoconstriction of the systemic blood vessels, renal blood vessels and the splanchnic and skin veins caused by noradrenaline increases afterload and preload. Renin-angiotensin-aldosterone system (RAAS) is activated, and PRA is increased in patients with manifest heart failure and it rises progressively during decompensation.The effect of angiotensin II is direct peripheral vasoconstriction and further stimulation of the sympathetic system. It also stimulates the adrenal cortex to secrete aldosterone, stimulates the production of arginine vasopressin, causes hypertrophy of the myocardium and vascular wall (remodelling) and increases the volume of collagen in the myocardium and vascular wall.In this study, expressions level of serum NE, Renin, Ang-II and ALD was observed. effects of ARPES and the safety of this method were investigated. And the neuroendocrine mechanism was studied.BNP correlation with NYHA grading of cardiac function is the most important diagnosis index of HF, whose sensitivity is 90% and specificity 76%. Increased levels of NT-proBNP were the most prominent independent predictor of congestive heart failure, irrespective of renal function. The level of NT-proBNP was also the most prominent indicator of death within 60 days. Furthermore, measuring NT-proBNP levels could help in deciding when to discharge the patient and whether the treatment for heart failure should be intensified. In a study of the BNP (Breathing Not Properly), 1,586 patients were examined for dyspnoea on emergency admission. The highest plasma concentrations of BNP were found in patients suffering from dyspnoea caused by heart failure, medium concentrations were found in patients with non-cardiac dyspnoea and a history of left ventricle dysfunction, and the lowest concentrations were found in patients without heart failure or left ventricle function disorder signs.Chronic or subclinical inflammation are common pathologic foundation in HF. which results in the production and release of proinflammatory cytokines, activation of the complement system, and production of autoantibodies. Thus, it is important to consider HF as a systemic illness, not just a disease of th"epump". Of these agents, cellular inflammatory factor may play a important role in the development and progression of this disorder.Cytokines are produced by immune system cells, the formation could be induced by a neurohormonal activation. TNF alpha causes muscle loss, reduces muscular contractility and induces myocardial hypertrophy. Thus TNF causes endothelial dysfunction and at the same time impaired synthesis and increased skeletal muscle catabolism. An increase in TNF, which is observed especially in NYHA IV patients, may lead to a serious limitation of exercise capacity. Interleukin 6 is associated with a worse NYHA class, prolonged hospitalization and a poor function of the left ventricle. Its role as a prognostic marker has not been clarified as yet and the mechanism of its harmful effect is not completely clear. There is, however, some scientific knowledge of its being involved in the development of ventricular hypertrophy through the stimulation of glycoprotein receptor 130 which is present on heart myocytes.Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40. Remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in the distribution of gap junctions and the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models.In cardiomyocytes Ca²+ is stored in an intracellular vesicular network called the sarcoplasmic reticulum (SR); and is available for immediate release into the cytosol, where it binds to Troponin C and enables actin–myosin binding and sliding of the myofilaments that results in sarcomere shortening and myocardial contraction. Accommodation aBNPrmal of Ca²+ occurs in heart failure and correlation with heart failure, arrhythmia, myocardial infarction, ischemia-reperfusion injury, catecholaminergic polymorphic ventricular tachycardia, arrhythmogenic right ventricular dysplasia and sudden cardiac death.Acute efficacy of ARPES was evaluated by measuring the time derivative of left ventricular pressure(dP/dt), cardiac function hemodynanmics, myocardium CX43, RyR2 and blood serum BNP, the effect of ARPES on heart failure was observed. In the summary, to investigate mechanism of action effects on cardiac function from the aspects of SAS, RAAS, cellular inflammatory factor, myocardium RyR2 and CX43.Part 1 Effects of electric stimulations with different amplitude during absolute refractory period on cardiac function of rabbits with heart failureObjective: To study effects of electric stimulations with different amplitude on cardiac function during absolute refractory period, hemodynanmics, blood serum BNP. To investigate whether ARPES can improve cardiac function and play an important role in cardioprotection.Methods: Thirty rabbits were randomly assigned equally to 3 groups: sham-operated(SHAM) group, heart failure(HF) group, and absolute refractory period electric stimulations(ARPES) group. Thoracotomy was performed on all the rabbits. Electric pulses were delivered during the ARP on the posterior wall of left ventricle in ARPES group for 12 hours. Chronic HF was induced in 20 rabbits by the transverse ascending aorta constriction (TAC). Cardiac function parameters were measured and serum specimen was prepared at preoperation, postoperation and after ARPES. Hemodynamics of rabbits was observed of preoperation and treatment of ARPES treatment. The biochemical indicator of serum was examined by biochemistry technique. Expression of BNP was detected by double antibody sandwich ELISA. Limb lead electrocardiogram was routinely monitored during operation. Standard 2-dimensional images were obtained using a 3.5-MHz transducer and digitally stored in cine-loop format by ultrachocardiograph in SHAM, HF and ARPES groups. Inter-ventricular septum thickness at end diastole (IVSd), left ventricular posterior wall thickness at end diastole (LVPWd), left ventricular end-systolic dimension(LVESD), left ventricular end-diastolic dimension (LVEDD), Left ventricular ejection fraction(LVEF), left ventricular fractional shortening(LVFS), left ventricular end-diastolic volume(EDV) and end-systolic volume(ESV) were measured by M-ultrasoundcardiogram from the standard left ventricular long axis view. From the standard apical views (5-chamber), aortic valve orifice velocity (AV) was obtained. Mitral regurgitation was observed and mitral valve orifice blood flow velocity(MV) was obtained from the standard apical views (4-chamber). Maximum positive left ventricular pressure change(+LVdP/dtmax) or minimum negative left ventricular pressure change(-LVdP/dtmin) were measured by MPA2000 cardio function meter. Left ventricular systolic pressure (LVSP),left ventricular end-diastolic pressure (LVEDP),mean left ventricular pressure(LVPmean),heart rate (HR) were all measured by MPA2000 cardio function meter。Results:1 Basic characteristics: There was no difference in three groups in weight, heart rate, respiratory frequency before operation. Heart rate, respiratory frequency in HF group and ARPES group were increased obviously compared with that of SHAM group (P<0.05), and the weight decreased but there is no statistical significance. Heart rate, respiratory frequency of HF group and ARPES group after operation were markedly higher than that before operation (P<0.05), whereas the body weight was not changed before or after operation. Heart rate and respiratory frequency in SHAM group after operation were similar as that before operation.2 Changes of biochemical indicators in serum: There was no statistical significance in three groups in biochemical indicator before operation. Liver function indicator such as ALT and AST and myocardium zymogram CK, CK-MB, LDH in ARPES group and HF group were enhanced significantly compared with that of SHAM group(P < 0.05). The above-mentioned indicators in ARPES group were decreased but with no statistical significance compared with that of HF group. ALT, AST ,urea, creatinine, K⁺, Na⁺, Cl^-, Ca²⁺, Mg²⁺ , very low density lipoprotein(VLDL) and blood glucose were not changed in three groups. Total cholesterol(TC) and low-density lipoprotein (LDL) in HF group and ARPES group were lower significantly than those of SHAM group(P<0.05). ALT,AST,CK,CK-MB and LDH were significantly increased while TG and TC were lower after operation compared with those before operation(P<0.05); other biochemical indicators were not changed. ALL the biochemical indicators in SHAM group were not changed compared with after peration. 3 Effects of electric stimulations applied with different amplitude during absolute refractory period on cardiac function: LVEDD, LVESD, EDV, ESV and AV in HF group were increased compared with that in SHAM group (P<0.01), EF, FS, MV were decreased obviously(P<0.05),whereas LVPWd, IVSd, LVEDD and EDV in ARPES group were dropped down compared with that in HF group but were not statistically changed. LVESD, ESV decreased remarkably (P<0.01), EF, FS, AV, MV increased notablely(P<0.01), while LVPWd and IVSd had no statistical significance. LVEDD, LVESD, ESV, AV in ARPES group were increased compared with that in SHAM group (P<0.05); while EF, MV and FS were decreased notably(P<0.01). Effects of electric stimulations in different amplitude during absolute refractory period on cardiac function are not statistically different. LVEDD, LVESD, ESV, AV in HF group and ARPES group of preoperation were higher markedly than those of postoperation(P<0.01); EF, MV and FS were lower obviously (P<0.05); while LVPWd, IVSd and EDV had no statistical significance. ALL the cardiac echocardiogram indicators after operation in SHAM group were not changed compared with those before operation.Correlation analysis: It is suggested that LVEDD, LVESD were negatively correlated with LVEF.4 Effects of electric stimulations with different amplitude applied during absolute refractory period on cardiac hemodynamics of rabbits: LVSP, LVEDP, LVPmean, +LVdP/dtmax and absolute value of -LVdP/dtmin in ARPES group and HF group were decreased obviously compared with those in SHAM group(P<0.01). The above-mentioned parameters in ARPES group were enhanced markedly compared with that in HF group (P<0.01). In addition, Effects of electric stimulations with different amplitude applied during absolute refractory period on cardiac hemodynamics are no statistical difference.5 Effects of electric stimulations with different amplitude applied during absolute refractory period on serum BNP of rabbits: There was no statistical significance in three groups in expression level of serum BNP before operation. The expressions level of serum BNP in ARPES group and HF group were enhanced significantly ly compared with that in SHAM group(P<0.01). The expression level of serum BNP in ARPES group were decreased markedly compared with that in HF group (P<0.01). Electric stimulations with different amplitude applied during absolute refractory period had no significant effect on expression level of serum BNP. The expression level of serum BNP after toperation in HF group and ARPES group were markedly higher than those before operation(P<0.01), whereas the expression level of serum BNP of postoperation in SHAM group were not changed compared with that of preoperation.Conclusions:Chronic HF could be produced in rabbits by coarctation of aorta. ARPES treatment in short period can enhance the left ventricular cardiac contractility in rabbits with heart failure and it can improve cardiac function, stabilize cardiac hemodynamics of rabbits with heart failure. Electric stimulations with different amplitude applied during absolute refractory period had no significant effect on cardiac contractility cardiac function and hemodynamics.Part 2 Effects of electric stimulations with different amplitude during absolute refractory period on sympathetic nervous system and renin- angiotensin-aldosterone system in rabbits with heart failureObjective: To study effects of electric stimulations during absolute refractory period on sympathetic nervous system and renin -angiotensin-aldosterone system by observing expression level of serum norepinephrine(NE), rennin, Angâ…¡(Angâ…¡) and aldosterone (ALD). To study whether the effects of electric stimulations with different amplitude(ARPES-L, ARPES-H) during absolute refractory period on sympathetic nervous system and renin-angiotensin-aldosterone system is different or not.Methods: Blood was collected and serum specimens were prepared for measurement at preoperation, postoperation and after ARPES. The expression level of serum NE, rennin, Angâ…¡and ALD were examined by double antibody sandwich ELISA. Results:1 Genenal conditions and biochemical indicators in serum were the same as those in Part 1.2 Effects of electric stimulations in different amplitude applied during absolute refractory period on sympathetic nervous system and renin-angiotensin-aldosterone system of rabbits: There was no statistical significance in three groups in expression level of serum NE, Renin, Angâ…¡and ALD before operation. The expressions level of serum NE, Renin, Angâ…¡and ALD in ARPES group and HF group were enhanced obviously compared with those in SHAM group(P<0.01). The expression level of serum NE, Renin and Angâ…¡in ARPES group were decreased markedly compared with those in HF group (P<0.01), but the expression level of serum ALD had no statistical significance. The expression level of serum NE, Renin, Angâ…¡and ALD in HF group and ARPES group after opreation were markedly higher than those before operation(P<0.01), whereas the above-mentioned indicators in SHAM group were not changed after operation compared with those before operation. Electric stimulations with different amplitude applied during absolute refractory period had no statistical effect on expression level of serum NE, Renin, Angâ…¡and ALD.Correlation analysis: It is suggested that serum NE, Renin, Angâ…¡and ALD were positively correlated with NE(r=0.8642, 0.8329, 0.6581). In addition, ALD and Angâ…¡were positively correlated with Renin(0.6911, 0.8141); ALD was positive correlation with Angâ…¡(0.6895).Conclusions:The expression level of serum NE, Renin, Angâ…¡and ALD were increased markedly in rabbits with heart failure. Sympathetic nervous system and RAAS was activated, suggesting that two systems may play a role in the compensation mechanism and progression of heart failure. Electric stimulations with different amplitude applied during absolute refractory period had no statistical effect on sympathetic nervous system and RAAS.Part 3 Effects of electric stimulations with different amplitude on cellular inflammatory factor(TNF-α, IL-6) in rabbits with heart failureObjective: To study the effects of electric stimulations with different amplitude during absolute refractory period on cellular inflammatory factor by observing the expression level of TNF-α, IL-6. To investigate whether ARPES is cardioprotective by anti-inflammatory mechanism. To study the effect of ARPES on inflammatory factor.Methods: Blood was collected and serum specimens were prepared in the same way as in Part 1. The expression level of serum TNF-αand IL-6 was examined by double antibody sandwich ELISA.Results:1 There was no statistical significance in three groups in the expression level of serum TNF-αand IL-6 before operation. The expression level of serum TNF-αand IL-6 in ARPES group and HF group were enhanced obviously compared with that in SHAM group(P < 0.01). The above-mentioned indicators in ARPES group were decreased markedly compared with those in HF group(P<0.01). The expression level of serum TNF-αand IL-6 in HF group and ARPES group after operation were markedly higher than those before operation (P< 0.01), whereas the above-mentioned indicators in SHAM group were not changed after operation. Electric stimulations with high amplitude during absolute refractory period decreased the expression level of serum TNF-αand IL-6 compared with that in low amplitude, whereas there was no statistical significance.Correlation analysis: It is suggested that the expression level of serum TNF-αwas positively correlated with IL-6, Renin, Angâ…¡, ALD (r=0.8582, 0.8676, 0.7691, 0.6986) but not correlated with NE. Moreover the expression level of serum IL-6 was positively correlated with NE, Renin, Angâ…¡, ALD (0.8004, 0.8457, 0.8208, 0.7299).Conclusions:Cellular inflammatory factors, sympathetic nervous system and renin -angiotensin-aldosterone system activated are together involved in generation and development of heart failure. The ARPES play an important role in anti-inflammatory mechanism in rabbits with heart failure. However, effects of ARPES with high amplitude on cellular inflammatory factor is not different from those of ARPES with low amplitude. The benefits of effects on improving cardiac function of ARPES were decreased by activating neuro-endocrine System.Part 4 Effects of electric stimulations with different amplitude during absolute refractory period on the expression of myocardium Ryanodine Receptor type-2 (RyR2) and gap junctions connexin(CX43) of rabbits with heart failureObjective: To study effects of electric stimulations with different amplitude during absolute refractory period on left ventricular myocardium excitation contraction coupling by observing the expression level of left ventricular RyR2, CX43. To investigate the molecular level mechanism of the effects of Non-Excitatory Currents(NEC).Methods: The expression level of left ventricular myocardium RyR2, CX43 was measured by fluorescent quantitation polymerase chain reaction(PCR).Results:1 Changes of the expression level of left ventricular myocardium RyR2, CX43: The expression level of left ventricular myocardium RyR2, CX43 in ARPES group and HF group were decreased obviously compared with those in SHAM group(P<0.01). The above-mentioned indicators in ARPES group were enhanced markedly compared with those in HF group(P<0.01). Electric stimulations with high amplitude during absolute refractory period increased the expression level of RyR2, CX43 in left ventricular myocardium compared with that in low amplitude but had no statistical significance.2 Correlation analysis: It is suggested that the expression level of left ventricular myocardium RyR2 was positively correlated with CX43, EF, LVSP, +LVdP/dtmax(r=0.5636, 0.6707, 0.5480, 0.6745). Moreover, the expres- sion level of CX43 was positively correlated with CX43, EF, LVSP, +LVdP/dtmax (0.8350, 0.5174, 0.7629). Conclusions:The expression level of left ventricular myocardium RyR2 and CX43 were decreased in rabbits with heart failure, and cardiac contractility was decreased, which is involved in the pathogenesis of heart failure. The ARPES in short time elevate the expression level of RyR2 and CX43, especially CX43, in left ventricular myocardium, enhance cardiac contractility, and improve cardiac function in rabbits with heart failure. Effects of ARPES with high amplitude is not different from that of ARPES with low amplitude on the expression level of left ventricular myocardium RyR2 and CX43.