Enhanced L-type Calcium Channels, Sodium-calcium Exchange And Sodium Pump In Order To Improve The Experimental Study Of The Myocardial Inotropic

Background: Positive inotropic agents are an efficacious drug in the short-term treatment of patients with severe left ventricular dysfunction and decompensated HF(heart failure). At the moment,however, most of the inotropic agents clinically evaluated have not proved beneficial effect on impaired diastolic function. Therefore a new agent which can improve both systolic and diastolic function of patient with HF has been increasingly demanded, especially to the patients with low-output and reduced diastolic function. We present a new inotropic stratage which provides the use of antibody against synthetical peptides to stimulate L-type Ca channel(LCC), Na-Ca exchanger(NCX) and Na pump simultaneously. The activation of LCC is a efficient pathway to increase the intracellular Ca2+ concentration directly, so the agents mediated by stimulating LCC can enhance myocardial contractile force. Ca is transported out of cell mainly via NCX, the enhancement of NCX can contribute to improve diastolic function and the ability of NCX to extrude Ca is controlled by intracellular [Na],which depends on Na pump function.Aim:(1) sequence alignment of the key amino acid of LCC,NCX and Na pump was done using Clustal W and according to the result ,We designed the sequence of peptides.Then the synthetic peptides was used as antigen to do active immunization in rat. IgGs in the positive sera was purified .We observed the effects of the antibodies on the fundamental ion channel and transport protein of cardiac myocytes in rat/guinea pig,and hoped to find the ones which can stimulate LCC,NCX and Na pump simultaneously.(2) To observe the effects of the purified IgGs on the hemodynamic indexes of the isolated rat heart and on the vascular smooth muscle activity (3) To observe the effects of the purified IgGs on the calcium transient and contraction of single cardiomyocyte in order to analysis the mechanisms underlying effects of antibodies on inotropism of myocardium. (4)To observe the functional effects of the purified IgGs on the acute heart failure modes.Materials and methods: (1) The peptides were synthesized by MeiLian (Xi'an) biotechnological Co. We used these peptides as antigen to immunize rat, and monitored the titer of the antibodies by ELISA. IgGs in the positive sera was purified by affinity chromatography with a MabTrap Kit .(2) The effects of the purified IgGs on the ion channel and transporter was observed by whole cell voltage clamp technique.(3) The effects of the purified IgGs on the hemodynamic indexes were observed by Langendorff isolated heart perfusing. (4) The effects of the purified IgGs on the vascular smooth muscle activity were observed by isolated rat thoracic aorta ring .(5)The calcium trantient and contraction of single cardiomyocyte was measured by CCD ionic imaging system. (6) The acute heart failure model was made by Langendorff perfusing with pentobarbital sodium. Then we observed the effect of the purified IgGs on the...