RhNRG-1 Improves Cardiac Function Of Infarcted Rats By Increasing The Expression Of Cardiac Specific MLCK

The ErbB family of receptor tyrosine kinases and their ligands, neuregulins(NRGs), are critical involved not only in cardiac development but also in themaintenance of structural and functional integrity of the adult heart, rhNRG-1(recombinant human NRG-1)improves cardiac function and survival in models ofischemic, dilated, and viral cardiomyopathy with the ErbB-activation. But themechanism is still unknown.Genechips, followed by confirmation via Real time PCR and Western blot, wereused to study the mechanism. The main result was that rhNRG-1 can increase theexpression of a predict gene: Similar to Myosin light chain kinase 2, skeletal/cardiacmuscle (MLCK2) (GenBank accession number XM₂14645), which was proved to berat's cardiac specific MLCK by Northern blot, Western blot, Immunofluorescence andmlc-2v-p detection in this thesis, rhNRG-1 can recovery the disorderedcardiomyocytes' sarcomeres, but the RNA interfering of cardiac MLCK can disturbthe recovery. So, rhNRG-1 improves cardiac function of infracted rats by increasingthe expression of cardiac specific myosin light chain kinase.There are five parts in this thesis.Partâ… rhNRG-1 improves the cardiac function of infarcted rats. Rat's left ventricle was infarcted by ligation of the left anterior descending coronaryartery (LAD). Sham operated animals were similarly treated except that the suturearound the LAD was left untied, rhNRG-1(10μg/kg/2h x 7days, ivgtt.) or vehicle wasadministered by implanted osmotic minipumps. Sham-operated animals received notreatment. Echocardiography was performed before and after treatments. Results:LVEDD, LVEDS decreased 9.1%, 18.8%, EF, FS increased 62.5%, 75.1%(P＜0.01,n＞10) after treated with rhNRG-1, compared with vehicle. RhNRG-1 improvescardiac function of infarcted rats obviously.Partâ…¡rhNRG-1 increases the expression of similar MLCK, discovered byGenechip and confirmed by Real time PCR and Western blot.The RNA samples in different groups were extracted from the non-infarction area ofleft ventricle coming from the first part experiment, reversed to cDNA, labeled cRNA,hybridized with Rat Genome 230 2.0 Arrays (Affymetrix). Result: rhNRG-1 increased the expression of a predict gene: Similar to Myosin light chain kinase 2,skeletal/cardiac muscle (MLCK2)(GenBank accession number XM₂14645). Thevalues in sham, vehicle and rhNRG-1 groups were 1.000, 0.677 and 1.652 separately,rhNRG-1/vehicle was 2.441(P＜0.01, n=3).This result was confirmed by Real time PCR and Western blot. The 1400bp in the Nterminal of similar MLCK is the specific segment decided by NCBI blast. So, thespecific segment (1018-1317) was amplified in the Real time PCR; Similar MLCK(487-885)-GST fusion protein, coming from the Similar MLCK-GST/pGEX-2Tplasmid, was used to immune rabbit, get specific antibody of similar MLCK, andconfirm the Genechip's result by Western blot and so on.Partâ…¢Similar MLCK distributes in the cardiomyocytes specifically.The probe of similar MLCK (238-885), labeled withα-³²PdCTP, was hybridized withrat's multiple tissue northern blots, the single band (about 4.3kb) was shown on theheart tissue lane. Similar MLCK (about 86kd) was only expressed in the heart tissue,confirmed by multiple tissue western blots in which the similar MLCK specificalantibody, produced in the second part experiment, was used.The cultured neonatal rat cardiomyocytes and the frozen slides of adult rat heart werestained withα-actinin, smooth muscle actin, similar MLCK and smooth MLCKantibodies. Result: similar MLCK limited in the cardiomyocytes, smooth MLCKlocated in the smooth muscle within the heart tissure. Similar MLCK and smoothMLCK were distributed on cardiomyocyte and vascular separately.Partâ…£Similar MLCK, which can phosphorylate the MLC-2v, is a cardiacspecific MLCK.Cos7 cells were transferred with cardiac MLCK /pCDNA3, lysised, mixed withMLC-2v, Ca2+,CaM, ATP and so on. The phosphorylation of MLC-2v was detectedby western blot to confirm that similar MLCK is a cardiac specific MLCK.Result: Cardiac MLCK phosphorylated the mlc-2v dependent on the Ca2+, CaM. Thereaction times longer, the dose of cardiac MLCK larger, the phosphorylation ofmlc-2v was stronger, but the temperature had no evident effect on this reaction. Thesimilar MLCK, distributed on the cardiomyocytes specifically, can phosphorylate themlc-2v, so similar MLCK is proved to be a cardiac specific MLCK.Partâ…¤rhNRG-1 benefits cardiomyocytes by increasing the expression ofcardiac MLCK. RNAiâ… ,â…¡,â…¢plasmids were co-transferred into cos7 with cardiac MLCK/pCDNA3 plasmid separately, detect the cardiac MLCK by western blot. Result: RNAiâ… ,â…¡,â…¢plasmids decreased the expression of cardiac MLCK about 80%, 50%, 20%respectively. RNAiâ… was the best one and chosen to do the followed expriment.Cultured rat neonatal cardiomyocytes were transferred with control or RNAiâ… plasmids, and divided into five groups. The sarcomere arrangements of transferredcells, in which green fluor can be seen because the expression of GFP, were observedbyα-actinin staining. The disordered sarcomeres can be divided into four grades byimaging analysis system. The cell numbers in different grades were calculated. Result:In the normal, normal+RNAiâ… , serum free+control, serum free+rhNRG-1+control,and serum free+rhNRG-1+RNAiâ… groups, the percent of cells, whose disorderedareas≥50%, were 2%,80%,60%,28%,80% separately. The sarcomeres of normalcardiomyocytes were disturbed when the RNA of cardiac MLCK was silenced.rhNRG-1 can recover the disordered sarcomere with serum free culture, but thisfunction disappeared when cardiac MLCK RNA was interfered. So, rhNRG-1 benefitscardiomyocytes by increasing the expression of cardiac MLCK.Conclusions:1. Similar MLCK (GenBank accession number XM₂14645) is a cardiac specificMLCK.2. rhNRG-1 improves cardiac function of infarcted rats.3. rhNRG-1 increases the expression of cardiac specific MLCK.4. The function of rhNRG-1 (recovering the damaged cardiaomyocytes) disappearedwhen the RNA of cardiac MLCK was interfered.5. rhNRG-1 benefits cardiomyocytes by increasing the expression of cardiac MLCK.The potential application and novelty of this project:1. Similar MLCK (GenBank accession number XM₂14645) is a cardiac specificMLCK, potential original medical target for the new drugs about enhancing thecardiac contraction.2. rhNRG-1 improves cardiac function of infracted rats and increases the expressionof cardiac specific MLCK.3. rhNRG-1 benefits the damaged cardiomyocytes depending on the expression ofcardiac MLCK.