Effect And Mechanism Of Novel Cardiac-specific Kinase TNNI3K On Cardiomyocytes Injury Induced By Hypoxia/ischemia

While constructing human adult heart and aorta cDNA libraries and making large-scale ESTs(Expressed Sequence Tags) sequencing, our research group members identified a novel gene localized on 1p31.1 based on bioinformatics analyses which might interact with cytoskeleton regulating signal pathway. A yeast two-hybrid screen proved it could interect with a series of sarcomeric regulating protein among which the most frequent candidate was cTnI and then was named TNNI3K (cTnI-interacting kinase) (GenBank accession no. AF116826), results that were further confirmed by co-immunoprecipitation in vivo. Multiple fetal and adult northern blot and 76-tissue array suggest TNNI3K is cardiac-specific gene and was undetectable in other tissues. In vitro kinase assays using immunoprecipitates of the series of FLAG-tagged TNNI3K mutants which found that TNNI3K not only autophosphorylated but also phosphorylated tyrosine substrate myelin basic protein (MBP) demonstrated that TNNI3K is a functional protein kinase. Phylogenetic parsimony analysis further clearly indicates TNNI3K belong to MAPKKKs superfamily in the evolutionary tree.A great deal pre-work indicated that TNNI3K, as a novel cardiac-specific protein kinase, has functional activities. But its downstream targets, acting mechanism and biological signal pathway under pathological stress still remain poorly understood. So further study in this work will be carried out from molecular cytological level (primary cultured neonatal rat cardiomyocytes transducted with adenovirus carrying/ non-carrying TNNI3K),animal model and histological level (treating with TNNI3K modified stem cells),and then clinical serological level to clarify its biological significance under hypoxia/ischemia intervention.All researches in this work are original novel and never been reported.Part I: Effects and mechanism of TNNI3K on hypoxia induced cardiocyte injuryObjective:First time to prove whether the TNNI3K levels could be changed with different hypoxia time periods(0h,3h,6h,12h and 24h)in vitro cultured primary cardiomyocytes. Then the effects of TNNI3K over-expression as a result of adenovirally mediated gene transfer on neonatal rat cardiac myocyte injury induced by hypoxia were investigated and its relative signal transduction pathway was elucidated.Methods:Primary ventricular cardiomyocytes were prepared from neonatal rats by collagenase digest method. Hypoxia was achieved by using an anaerobic jar (AnaeroPack Series, Mitsubishi Gas Chemical Co, Inc) equipped with an AnaeroPack, disposable O2-absorbing and CO2-generating agent, and an indicator to monitor oxygen depletion. Whole experiment contains three parts:1.Cell injury induced by hypoxia/serum free culture was observed at different time periods (0h,3h,6h,12h,24h). The cardiomyocytes beating rate and rhythm in each group were documented under inverted microscope and lactate dehydrogenase(LDH) in cultured cell medium was assayed using automatic biochemical analysis instrument(Beckman,USA) in order to confirm the successfully established cell injury model. Then western blot was used to semi-quantitate TNNI3K gene expression whose alteration is the base for all next work.2.Effect of TNNI3K on neonatal rat cardiac myocyte injury induced by hypoxia/serum-free. Primary cultured neonatal rat cardiomyocytes were transiently transducted with Ad-C (adenovirus-LacZ as control), Ad-TNNI3K(adenovirus carrying sense of TNNI3K) and Ad-TNNI3K-AS (adenovirus carrying anti-sense of TNNI3K) in which all were labelled with green fluorescent protein(GFP).The effects of TNNI3K gene overexpression on neonatal rat cardiac myocytes injury induced by hypoxia/serum-free were probed by evaluating LDH release,cells beating rate and rhythm and viability(assessed by MTT assay). Cell apoptosis was determined by both Hoechst 33258 staining and DeadEndTM Colorimetric TUNEL System.3.The action mechanism of TNNI3K overexpression on cardiomyocytes apoptosis induced by hypoxia/serum-free. Evaluation of the participative roles of three main classic MAPK pathways activation using semi-quantitating phosphorylated extracellular signal-related kinase( p-ERK), phosphorylated c-Jun N- terminal kinase(p-JNK) and phosphorylated p38(p-p38) by western blot. Furthermore,to explore the mitochondrial apoptotic signal pathway effects of TNNI3K at 12h exposing to hypoxia through measuring Caspase-3 activity by Microplate Reader, the release of cytochrome C from mitochondrial to cytoplasm using immunochemical method combining laser scanning confocal microscope(LSCM),change of cell mitochondrial membrane potential( m) which staining with Rhodamine 123 assessing by FACS(flow cytometry) and proapoptotic protein Bax expression by western blot.Results:1.The AnaeroPack jar is capable of depleting the concentration of O2 down to PO2<30mmHg or SO2 about 20% in 1 hour which successfully meets our experimental requirement in this study.After 6hs of hypoxia,cardiomyocytes beated slower than before exposing to hypoxia and the beating rhythm became irregular which still worsen and almost no-beating for majority of the cells after 24hs hypoxia.LDH level in medium increased significantly at 6hs post hypoxia(P<0.05)and much dramatically at the 24h which suggest the injury of the cells. The elevation of TNNI3K protein expression occurred at early hypoxia time(3h) and peaked at 12h then went down slowly at 24h post hypoxia.2.All Adenovirus-mediated transduction cells divided into Ad-C,Ad-TNNI3K and Ad-TNNI3K-AS groups. After exposing to hypoxia 12 h and 24h, cell viability decreased in three groups but this was much better in Ad-TNNI3K and Ad-TNNI3K- AS groups than that in Ad-C group while there was no significant difference between the former two. Furthermore,12h- and 24h- hypoxia increased medium LDH levels in all three groups but those were relatively much lower in group Ad-TNNI3K than those in groups Ad-C and Ad-TNNI3K-AS even though when compared between them those in Ad- TNNI3K-AS group showd significant lower than in Ad-C group. Overexpress- ion of TNNI3K in Ad-TNNI3K group also decreased apoptosis at 12h and 24h by Hoechst 33258 staining which showed less chromatin condensation and aggregation under the nuclear membrane and less nuclear fragmentation and apoptosis body formation. Apoptosis index calculation by DeadEndTM Colorimetric TUNEL System assay was also in accordance with this result. All above revealed the very important cytoprotective and anti-apoptotic effect of TNNI3K gene on hypoxia induced injury. The paradoxical results of Ad-TNNI3K-AS group whose activity was sometimes between group Ad-TNNI3K and Ad-C whereas sometimes no significant difference compared with Ad- TNNI3K group possibly due to the species difference becouse the anti-sense TNNI3K deriving from human sequence might not inhibit the homologous gene of TNNI3K in rat ventricular myocyte or probably due to the unpredictable problems of anti-sense nucleotide technique per se.So,in all next studies on mechanism, we only focused in Ad-C and Ad- TNNI3K groups.3. Based on western blot analysis,we delineate the MAPKs signaling pathway roles in hypoxia induced apoptosis. The results indicted that (1) only ERK1/2 and JNK signal pathways involve in TNNI3K functional effect responding to hypoxia treatment. There were low expression levels of p-JNK and p-ERK1/2 in both Ad-C and Ad- TNNI3K groups at 0h hypoxia. After 3h hypoxia, p-ERK1/2 in Ad-C group signifiantly increased and still higher at 6h of hypoxia while which couldnot be seen in Ad-TNNI3K group(p<0.05),then followed by a decline in both groups and made no difference between them at 12 or 24h.For p-JNK, the pattern of change was considerably different to that seen for p- ERK1/2.Levels in Ad-C group initially slightly increased at 3h and then maintained very low throughout the time course while that in Ad- TNNI3K group increased at 3h and 6h and peaked at 12h making significant difference between two groups at 6 and 12h(P<0.01) and then kept quite low levels in both groups at 24h.(2)High expression of TNNI3K could inhibit activation of caspase-3 and alleviate the release of cytochrom C from mitochondria to cytoplasm and reduce the expression of proapoptotic protein Bax.All above results suggest that the antiapoptotic effect of TNNI3K might be through mitochondria dependent signal pathway.Conclusion:In vitro primary cultured neonatal rat cardiomyocytes experiment,we first approved that cardiac-specific gene TNNI3K has the protective effect on cell injury and apoptosis induced by hypoxia.This function might be through inhibiting the early stage and trensient activation of p- ERK1/2 and increasing the expression of p- JNK in MAPK cascade signal pathway.The anti-apoptotic mechanism was motochondria dependent through inhibiting the activation of caspase-3 and reducing the release of cytochrom C from mitochondria to cytoplasm and the expression of proapoptotic protein Bax. Part II: Effect of Intramyocardial Administration of TNNI3K on Rabbit Post- myocardial infarction Cardiac FunctionObjective:To observe the effect of intramyocardial injection of TNNI3K on cardiac function in a rabbit model of infarction.Methods:P19CL6,a derivation of mouse embryonal carcinoma(EC) P19 cells which is an undifferentiated stem cell derived from murine teratocarcinoma, is morphologically similar to P19 cells but does not express SSEA-1 (stage specific embryonic antigen-1) antigen, a specific EC marker.It is known that P19CL6 cells efficiently differentiate into beating cardiomyocytes expressing cardiac- specific genes after culture in the presence of an inducer, dimethylsulfoxide (DMSO). The vector pcDNA6-FLAG /TNNI3K was obtained from our prophase work and the stable transfected P19CL6 cells with pcDNA6-FLAG(FLAG only) or pcDNA6-TNNI3K were endowed by our Japanese cooperator.Briefly, the vectors pcDNA6-FLAG (FLAG only) and pcDNA6- FLAG/TNNI3K were transfected into P19CL6 cells using electropoie 2000 (Invitro- gen) and maintained in medium containing 500μg/ml blasticidin (Invitrogen). Acute myocardial infarction (AMI) was produced in rabbits using ligation of left ventricular branch of left circumflex coronary.After 5 days being induced by 1%DMSO, early differentiated P19CL6 cells with or without high expression of TNNI3K were collected respectively and resuspended with normal saline. 24 experimental rabbits were randomly divided into four groups(i) Sham operation control group,n=6(C) ; (ii)AMI+ intramyocardial normal saline injection group,n=6 (NaCl);(iii) AMI+ intramyocardial pcDNA6-FLAG injection group, n=6 (FLAG); (iiii) AMI+ intramyocardial pcDNA6-FLAG/ P19CL6 injection group,n=6 (P19CL6). 4 weeks after operation ,left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular wall thickness and interventricular septum thickness were measured by Color Doppler Eechocardiography and then left ventricular ejection (LVEF) was calculated. Haemodynamics parameters including left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP),maximum LV pressure rising velocity(+dp/dtmax) and maximum LV pressure decreasing velocity(-dp/dtmax) were recorded with an electrophysiolograph and then the hearts were excised. Specimens were coded and fixed in 10% buffered formalin for subsequent histopathological analyses using hematoxylin and eosin stain (HE stain).Results:Around 10 to 12 days differentiation induced by 1%DMSO, P19CL6-TNNI3K and P19CL6-FLAG cells which seeded at 4 x105 per 60mm dish contracted first at a restricted area then on days 12 to 16 almost all of the sheet beated synchronously. Myocardial infarction was successfully produced in rabbits using coronary artery ligation. NaCl group received an intramyocardial injection of totally 200μl of normal saline at the border zone between infarcted and normal myocardial,and the same amount volume injection containing 4x105 P19CL6-TNNI3K and P19CL6-FLAG cells designated as TNNI3K and FLAG group. Sham operation group was set as control. Ventricular function was evaluated by echocardiography 4 weeks later.There were no significant difference in heart rate among all groups before and 4 weeks after AMI. LVESD or LVEDD were less expanded and cardiac function was less impaired in TNNI3K group than those in FLAG and NaCl groups even though for all post- MI groups ,including TNNI3K, FLAG and NaCl ,there were more deteriorative cardiac function than those in control. TNNI3K group still had thicker (p<0.05)ventricular which similar to control compared with the NaCl or FLAG groups at 4 weeks. Hemodynamics study using four-channel physiologic recorder also confirmed even there were higher LVEDP and lower LVESP or±dp/dtmax in all MI groups while which were better in TNNI3K than in FLAG and NaCl groups.We could not find significant difference for all above parameters between FLAG and NaCl groups. Further histological analyses revealed less myocardial fibrosis in TNNI3K group than that in FLAG and NaCl groups.Conclusion:Intramyocardial injection of high-expression TNNI3K gene afterAMI can effectively improve ventricular function by means of attenuating cardiac remodelling and myocardial fibrosis.Part III. Clinical Observation of TNNI3K Serum Levels in Ischemic Heart DiseaseObjective:To establish an ELISA (Enzyme-linked immunosorbent assay) with high veracity, reliability and repeatability system and detect serum TNNI3K levels in normal humans, acute myocardial infarction (AMI),heart failure caused by post-myocardial infarction remodelling or ischemic cardiomyopathy(CHD/HF)(EF≤45%)and the heart failure patients with dilated cardiomyopathy (DCM).Then discrepancies were compared among groups so as to explore the significance of TNNI3K on ischemic heart disease from clinical point of view. Methods:Set up a standard curve of purified TNNI3K protein first and establish a high veracity, reliability and repeatability ELISA system by repeatitive intra- or inter- batch experiment. The healthy blood donators were enrolled as the Control and the definite diagnosis of patients with AMI, CHD/HF(EF≤45%)and DCM were maken through patients'history of chief complaint, symptoms, signs, lab examination, radiography,electrocardiogram(ECG),echocardiography,coronary angiography,et al. Serum TNNI3K levels in controls or different group patients were detected using above direct ELISA system. Results:TNNI3K can be detected in the serum of both normal control and patients groups but the levels in CHD(including AMI and CHD/HF) and DCM were significantly higher than those in control(P<0.01)even though there was no significant difference between them.We couldnot find any significant difference either between AMI and CHD/HF groups as to different clinical stage or between the heart failure caused by CHD and DCM based on two-type mechanism.Conclusion:Serum level of TNNI3K elevated in coronary heart disease which suggests that it may play an important role in the occurrence and development of this disease.