Time Course Proteomic Profile And Differentially Expressed Genes Of Rat Acute Myocardial Ischemia

Chapter 1 Potential biomarkers discorvery of acute myocardial ischemia using SELDI-TOF MSBackground The increasing prevalence of Coronary Artery Disease (CAD) has high mortality and morbidity. Besides, CAD constructs the major death cause of sudden death in forensic science. About 1/4 CAD patients have not pectoral syndrome and half of them do not show electrocardiogram changes. Forensic pathologist found that when the important area is ischemia, such as ischemia of ventricular septal, could lead to sudden cardiac death (SCD). Troponin (cTn) is widely used as ancillary clinical diagnosis of CAD. As a marker of myocardial infarction, cTn could be detected 2-4 h in the plasma after the onset of the clinical syndrome. The earlier biomarkers, such as CRP and HABP, had to be used for earlier diagnose of cardiac ischemia for their poor specificity. SELDI-TOF MS was novel proteomic tool. According to its principle, the proteins were captured by ProteinChips which can bind with different proteins or peptides for their different chemistry properties, after that, the proteins or peptides were bound with energy molecules and run in the mass spectrometry when inspired by laser. The peptides were separated by their flying time in the MS. This equipment was widely used to find disease biomarkers. In this study, SELDI TOF MS were used to detect the pepitides profile and biomarkers of cardiac ischemia in the sera and myocardium.Methods Sprague-Dawley rats were divided as three groups: operation group, sham operation group, non-operation group. The sera and ischemia myocardium were collected 5 min-6 h after the coronary artery ligation. CM10 ProteinChip was selected as the experiment chip. The sera and myocardium protein extracts were processed by CM10 ProteinChip and analyzed by SELDI-TOF MS. CKMB, cTnI and histological change were detected too.Result The peaks detected in the operation groups and nearly seen in the sham operation groups were called as specific peaks. Those peaks emerged in the operation groups and sham operation groups but not in the non-operation groups were called as non-specific groups. Three peaks with m/z 7564Da, 7586 Da and 9583 Da were only found in the operation groups' sera. The peaks 7564Da and 7586 Da were seen simultaneously had the sensitivity of 97.0% and specificity of 97.0% in the>15 min operation groups. Some non-specific peaks in response to injury have also been seen, they are the peaks with the m/z 4983 Da, 5140 Da, 8075 Da, 9423 Da. Four non-specifc peaks with marked peak height changes: 4400 Da, 4542 Da, 8400 Da(increased), and 7666 Da(decreased). Two kinds of peaks were found in the myocardium protein extraction. One is the three novel specific peaks which were only found in operation groups with the 100% specificity: 6304Da, 8337Da, 8376Da. Peak 6304Da, 8337Da were found with a high peak height >5 min operation group, while peak height of 8376Da was low in 5 min group but increased in the >15min groups. Another kind of peaks was specific peaks with increased peak height. Peak 6658 Da, 6876 Da increased in>15min groups, peak 8577 Da increased in >5 min groups. While the significant increase of CKMB and cTnI in the serum could be detected until 4 hours after the ligation. For the HE staining of the myocardium, contraction band was observed after 1 h of the ligation, inflammatory cells were found in the ischemia area 4-6 h after the ligation.Conclusion Whether in the serum or the myocardium, the change peaks found by SELDI-TOF MS were the earliest changes after cardiac ischemia by now. These changes may reflect the modified peptides or novel proteion. They may be used as the biomarker of cardiac ischemia for clinical purpose or ancillary ischemia sigh for SCD.Chaper 2 Screening expreesed genes in a rat model during cardiac ischemia by suppression subtractive hybridizationBackground Understanding the molecular changes after the cardiac ischemia is pivotal for the diagnosis of myocardial infarction and therapy of the ischemia cardiac diseases. By now, The main approch used for research on myocardial ischemia transcription is microarray detection. This method can only detect the known genes and ESTs and limited by the molecule transcription level. Suppression Subtractive Hybridization (SSH) is a approch to detect the different transcription gene between two groups. The SSH is complementary to cDNA microaray. We detected the transcription changes after cardiac ischemia by SSH in this study.Method Eight Sprague-Dawley rats were sacrificed after the coronary artery ligation for 1 h and the myocardium were collected. After the mRNA was extracted, a PCR-Select cDNA Substraction Kit was used as the manufacture's protocol. Two hybridizations were established: the forward hybridization: ischemia myocardium cDNA were used as the tester, and non-ischemia cDNA as the driver; the reverse hybridization: non-ischemia myocardium cDNA as the tester, and ischemia cDNA as the driver. The subtractive cDNA library were sequenced and identified by dot blot.Result Sixteen known genes were sequenced after forward hybridization: they can be classified as the following: (1) Myocardium gene: myoglobin, cardiac troponin T2, tropomyosin 1, Rattus norvegicus beta-glo. (2)Translation related gene: ribosomal protein L39, ribosomal protein S11 (Rps11),eukaryotic translation elongation factor 1 alpha 1. (3) Metabolism and enzymes: ATPase 3 (Psmc3), malate dehydrogenase 1, NAD, lactate dehydrogenase B (Ldhb), UDP-glucose pyrophosphorylase 2 (Ugp2).(4) Myocardium remodeling: cathepsin L (Cts1). (5) Others: Sjogren syndrome antigen B (Ssb), basigin (Bsg), unc-45 homolog A (Unc45a). Only one known gene, NADH dehydrogenase (ubiquinone) Fe-S protein 2, was found in the reverse hybridization. Sixteen unknown clones were detected: Nine of them can be aligned in the REFRNA databank, including 7 sequences which were predicted similar to ribosomal protein, and two others were telethonin and actin; Seven of them were totally unknown about there function. Among them, Ssb, basigin, unc-45 homolog A (Unc45a),cathepsin L (Cts1) and Telethonin were first reported to be associated with acute myocardial ischemia.Conclusion We established the SSH library of rat ischemia (ischemia duration for 1 h). Five genes including Ssb, basigin, unc-45 homolog A (Unc45a), cathepsin L (Cts1) and Telethonin were first found to be associated with acute myocardial ischemia. Seven totally new ESTs were found.