| BACKGROUND Findings about regeneration capacity of zebrafish,newt and neonatal mouse heart have provided new research models and broaden our understanding of molecular mechanisms underlying heart regeneration.The application of lineage tracing technology both in zebrafish and neonatal mouse revealed that newly formed cadiomyocytes were mainly stemmed from preexisting cardiomyocytes by dedifferentiation and proliferation.Like most other adult mammals,human's cardiomyocytes are terminally differentiated and have limited regeneration capacity,which is far away enough to make up cell loss caused by myocardial infarction(MI).Therefore,once the underlying biology mechanisms of dedifferentiation and proliferation of preexisting cardiomyocytes were determined,the biggest problem in heart regeneration could probably got a chance to be settled.Gemberling et al showed that Neuregulin1(Nrg1)is a very potent mitogen of cardiomyocytes and can activate a regenerative program even in the absence of heart injury.The expression of erbb2 and erbb4 b was determined by q RT-PCR.Blocking Erbb2 by a chemical inhibitor inhibited cardiomyocyte proliferation during zerbrafish heart regeneration.Bersell et al showed that Nrg1 can promote adult mouse heart regeneration and recovery of cardiac function after MI by activating cell cycle re-entry in which Erb B4 is an indispensable receptor of Nrg1.In this study,it was demonstrated that Nrg1 can efficiently stimulate proliferation of mononucleated cardiomyocytes,which are the main cell type in zebrafish and also mainly present in neonatal mouse hearts.The neonatal mouse lost heart regeneration capacity after postnatal day 7(P7),which is coincidently the time window that cardiomyocytes withdrawing from cell cycle by undergoing bi-nucleation and become bi-or multi-nucleated.Interestingly,in another recent research,Erb B2 expression is significantly down regulated by P7 both in m RNA and protein level,Nrg1 induced cardiomyocytes proliferation decreased,suggesting that cardiomyocytes are less responsive to Nrg1 after P7.However,none of these studies have shown expression and functions of Erbb4 at protein level.Nrg1 belongs to epidermal growth factor(EGF)family,its tyrosine kinases receptors contain Erb B1(also named EGFR),Erb B2,Erb B3 and Erb B4.In canonical signaling,binding with Nrg1 will induce Erb B dimerization which will activate the tyrosine kinase domain,and function by phosphorylation of the intracellular domains.Nevertheless,among these four receptors,Erb B4 is the only one which can both bind directly to Nrg1 and activate the tyrosine kinase activity.As co-receptors,Erb B1 and Erb B2 can't bind directly to Nrg1.Erb B3 can bind to Nrg1,however,the homodimer doesn't show any catalytic activity,suggesting that the tyrosine kinase of Erb B3 is impaired.In addition,Erb B4 has another reaction pattern with Nrg1 referred as no-canonical signaling due to a special structure outside the transmembrane domain(TM),a relative longer “stalk” region than other receptors of its family,which make it easy to undergo ectodomain cleavage.The proteolytic cleavage of Erb B4 released a 120 KDa ectodomain fragment into extracellular medium and leaves an 80 KDa membrane-anchored fragment(cytoplasmic domain.ie.Erb B4-CTF)which will be further cleaved by ?-secretase and releases the Erb B4 intracellular domain(Erb B4-ICD).The cleavage ICD form can translocate into nucleus and function as a transcription factor.The significance of Nrg1/Erbb4 signaling on heart regeneration has been approved.However,the underlying mechanism of Nrg1 on reactivating cell cycle is still unclear.We still don't know how is the receptor Erbb4 involved in regulating cell cycle of cardiomyocytes and provoking heart regeneration.Therefore,exploring the expression pattern of Erbb4 during heart regeneration and among different model organisms with distinct regeneration capacities is important for understanding and disclosing the regeneration mechanisms.In our research,we will focus on the receptor Erbb4 and determine its different expression patterns between zebrafish and mouse.And explore the potential mechanism of Nrg1/Erb B4 involved in cell-cycle reentry by determining how Erb B4 responds to a specific overexpression of Nrg1 in cardiomyocytes with Cre-Loxp technology.METHODS 1.Animal usage and Experimental Manipulation 6-8 month-old wild type zebrafish were used for regeneration study and maintained with standard methods.The transgenic lines Tg(fli1?:EGFP),Tg(wt1:EGFP),Tg(cmlc2:Cre ER)and Tg(?-act2:lox P-m Tag-BFP-STOP-lox P-Nrg1)have been described before.Amputation procedure of zebrafish heart was performed as described before.Collect hearts at 3dpa,7dpa,14 dpa for RNA and protein extraction with uncut hearts used as control.Day 15 pregnant mice of ICR/CD-1 were used for mouse study.P1,P4,P8 mouse hearts were collected for protein extraction and 2 month old adult mouse were taken as control.All protocols used in the study were approved by IACUC of Children's Hospital Los Angeles.To induce specific overexpression of Nrg1 in cardiomyocytes of zebrafish,Tg(cmlc2:Cre ER)and Tg(?-act2:BSNrg1)were outcrossed and double transgenic embryos were raised up.Single transgenic Tg(?-act2:BSNrg1)with same age were used as negative control.GM6001,a chemical inhibitor of matrix metalloproteinases,were used to block the activity of TACE in targeting regulation of Erb B4 cleavage.Amputated zebrafish were processed from 2-3dpa for 24 hours in 10?M GM6001 fish water and hearts were collected for western blot.2.Quantitative RT-PCR(q RT-PCR)analysis Heart tissues at indicated stages were collected and homogenized in TRIzol? and total RNA were extracted according to the manufacture's instruction.RNA was converted to c DNA using Super Script? III First-Strand synthesis kits.Quantitative Real-time PCR was carried out using the Roche Light Cycler?480 Real-Time PCR System.Zebrafish ef1 a primer was used as internal reference.Cycle values were collected in triplicate for each time point.ef1a: F-AGCTCGTTGGAGTCAAC,R-TGCGCTGACTTCCTTGGTG;erbb4a: F-GTTGTGCCGTCGAACAATAG,R-CTGGCACTGATCCTCCTGA;erbb4b: F-ACTCAACGTGTTTCGCACTG,R-GCTCTGCCTCCAATAGTTGC.3.Protein extraction of nuclear fraction and cytoplasmic fraction Collect zebrafish or mouse ventricles and homogenize in Buffer A(10 m M HEPES,PH=7.5;10 m M KCl;1 m M EDTA;2 m M Mg Cl2,1% NP40).Then centrifuge at 500 g for 5 min,keep the supernatant of this step as cytoplasmic fraction.Wash once and resuspend the left pellet with 50?l Buffer B(Buffer A supplemented with 500 m M Na Cl and 25% Glycerol).Incubate on ice for 30 min and centrifuge at 12,000 g for 5 min.Keep the supernatant as nuclear fraction.4.Western Blot Heart tissues at the indicated stages were collected and homogenized in Tris-SDS lysis buffer(50 m M Tris p H7.4 and 1% SDS)and incubated in room temperature for 15 min,followed by centrifuge at 11,000 rcf for 8 min to get the supernatant.All protein samples(nuclear fraction and cytoplasmic fraction included)were mixed with 4 x Loading buffer nd 10 x Reducing reagent and boiled for 5 min.Proteins were resolved in 8% precast polyacrylamide gel and transferred onto a nitrocellulose membrane with iblot fast transfer system.The membranes were then blocked in Odyssey blocking solution at room temperature for 1 h.After that,the membranes were incubated in indicated primary antibodies diluted with Odyssey blocking solution overnight at 4°C.Then the membrane were washed and incubated with secondary antibodies diluted in same blocking solution for 1 h at room temperatue with light away.Signals were detected by Odyssey laser detecting system.Antibodies used for WB included: anti-Erb B4(Sc-283,Santa Cruz,1:1,000),antitropomyosin(CH1,DSHB,1:1,000),anti-GAPDH(Fitzgerald,1:1,000),IRDye? 800 CW Donkey anti-Mouse Ig G(Li-Co R,1:10,000),IRDye? 680 LT Donkey anti-Rabbit Ig G(LiCo R,1:10,000).5.Immunofluorescence staining(1)Immunofluorescence staining on sections Hearts were harvested at indicated stage.They were then fixed and sectioned from paraffin block.Antigen retrieval was performed under heat inducement in Vector antigen retrieval reagent.The sections were then blocked in 5% goat serum with 0.3% Triton X-100 at room temperature for 45 min.Incubate primary antibodies overnight at 4°C.Secondary antibodies were incubated for 1h at room temperature with light away.Zeiss LSM confocal were then used to take photos at lambda model.(2)Whole mount immunofluorescence staining Hearts were isolated from terminally anesthetized zebrafish and fixed in 4% PFA at 4°C for 24 h.Bleach in Dent's solution(25ml H2O2,10 ml DMSO,40 ml Me OH)for 24 h and fix in Dent's fix solution for another 24 h.Wash fix solution away and primary antibodies were incubated with heart for 5 days with rotating.Wash primary antibody away and incubate with secondary antibodies for another 5 days,clear with BABB(50ml benzyl alcohol,100 ml benzyl benzoate)and image with confocal under Z-stack.6.Statistical analysis Results of Western Blot were quantified with image J and expressed as mean± standard deviation(SD).Averages were calculated from ate least three repeat data.Differences were analyzed using two-tailed unpaired Student's test or ANOVA of which P'value below 0.05 ere taken as significant.RESULTS 1.q RT-PCR results during zebrafish heart regeneration To determine the expression pattern of erbb4 genes during early stages of zebrafish heart regeneration,we performed quantitative real time-PCR using wild type adult zebrafish hearts at baseline(uninjured)and during regeneration after injury(ventricular amputation).ef1 a was used as inner control.Both erbb4 a and erbb4 b were expressed in uninjured hearts.Expression levels of erbb4 a and erbb4 b are both significantly decreased by 7 dpa(days post amputation).2.Expression of Erbb4 in protein level during zabrafish heart regeneration.Western Blot data showed that there are two main forms of Erbb4 existing in zebrafish heart,a predominant 60 KDa trauncted form(Erbb4-ICD)and 180 KDa full length protein.Expression levels of Erbb4-ICD did not change significantly at 3 and 7 dpa,but by 14 dpa,we observed a significant decrease in Erbb4 protein levels.This change was consistent with the trend in decreased erbb4 m RNA levels.To determine the spatial expression of Erbb4,we performed immunostaining on both uninjured and injured heart,and further seprated nuclear fraction and cytoplasmic fraction for western blot.Results showed that Erbb4 is enriched in the nuclei of cardiomyocytes and expressed in both regenerating and non-regenerating areas.Western Blot data revealed that the truncated Erbb4 is mainly present in the nuclear fraction of both uninjured and regenerating hearts while residual expression of both truncated and full-length Erbb4 are detected in cytoplasmic and membrane fraction.In addition,immunostaining data showed that Erbb4 was expressed in cardiomyocytes of which mainly in nuclear area,and also in epicardium and endothelial cell of vessels in zebrafish hearts.3.Different expression pattern of Erb B4 in neonatal and adult mouse hearts.Western Blot data showed that Erb B4 also existed in mouse heart with mainly two forms, trauncted 60 KDa Erb B4-ICD and 180 KDa full length,which are also in the same location of cells as in zebrafish,ICD mainly in nucleus and full length in cytoplasm.Interestingly,compared with neonatal mouse,there was significant decreasing trend of Erb B4 in adult both for ICD and full length.In addition,there was also a decreasing trend with age up for neonatal mouse.4.Nrg1 induce expression of Erbb4 in nucleus of cardiomyocytes in juvenile zebrafish.Comparison of Erbb4 immunostaining on juvenile and adult zebrafish hearts showed that Erbb4 always expressed in endothelial cells with the development of hearts.Interestingly,it did not express in nucleus of cardiomyocytes in juvenile hearts and showed out at adult stage.After twice inducement with 4-HT on 55 dpf Tg(cmlc2:Cre ER;?-act2:BSNrg1)zebrafish(single transgene zebrafish was used as control),expression of Erbb4 in nuclear of cardiomyocytes up regulated significantly.5.Erbb4-ICD in zebrafish heart may come from direct translation of erbb4 TACE is required for Erbb4 cleavage and can be inhibited by GM6001,which is a broad range chemical inhibitor of matrix metalloprotease.After amputation,zebrafish were treated with 10?M of GM6001 for 24 hours from 2dpa to 3dpa.DMSO was used as a negative control.Hearts were then harvested and proteins were extracted for WB.Results showed that no obvious change was detected on Erbb4-ICD between GM6001 and DMSO control.After analysis of Erbb4's structure,we found that the “stalk” area of Erbb4 in zebrafish is as short as Jm-b of mammals.In addition,analyzing of erbb4's open reading frame showed that a 60 KDa protein could probably be translated directly in zebrafish.Comparison of Erbb4 immunostaining on juvenile and adult zebrafish hearts showed that Erbb4 always expressed in endothelial cells with the development of hearts.Interestingly,it did not express in nucleus of cardiomyocytes in juvenile hearts.However,overexpression of Nrg1 specificly in cardiomyocytes could significantly up regulate its expression in nucleus.CONCLUSION 1.In zebrafish and neonatal mouse hearts,which can be regenerated after injury,Erbb4 xist with mainly two forms,a 60 KDa ICD in nucleus and 180 KD full length in cytoplasm.2.In adult mouse hearts,which can't be regenerated after injury,Erb B4 also expressed with the ICD in nuclear and full length in cytoplasm.However,both of them were significantly lower than them at neonatal stage.3.In juvenile zebrafish,Erbb4 which expressed in nuclear of cardiomyocytes was much less than that in adult.Interestingly,overexpression of Nrg1 in cardiomyocytes could induce Erbb4's expression in nucleus.4.Chemical inhibition of TACE in zebrafish after amputation would not affect expression of Erbb4,indicating that Erbb4-ICD was probably not from cleavage of full length.In addition,analyzing of open reading form of erbb4 showed that Erbb4-ICD may come from direct translation of erbb4 by an alternative staring site. |
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