The Research Of Cardiac Hypertrophy Associated With Myeloproliferative Neoplasms In JAK2V617F Transgenic Mice

Myeloproliferative neoplasms (MPNs) are chronic hematopoieticmalignancies characterized by abnormal amplification of one or moremyeloid lineages. These diseases include polycythemia vera (PV),essential thrombocythemia (ET), and primary myelofibrosis (PMF). PVpatients have increased production of all three types of blood cells,whereas ET patients mainly show elevations of platelets. Patients withPMF develop fibrous (scar-like) tissues in the bone marrow as a result ofabnormal myeloid proliferation. MPNs mainly affect older people with anaverage onset of55years. So far, there is no effective cure for the diseases.Complications associated with MPNs include thrombosis, hemorrhage,heart attacks, strokes, myeloid metaplasia, and acute leukemia. Strokesand heart attacks caused by these diseases are usually fatal. In fact,cardiovascular events represent the leading cause of morbidity andmortality in the course of PV and ET. However, how the bloodabnormality associated with MPNs leads to cardiac changes is not wellunderstood.JAK2V617F, a mutant form of tyrosine kinase JAK2, represents amajor molecular defect in MPNs. It is found in over95%of PV and over50%of ET and PMF cases. In early studies, we have generatedJAK2V617F transgenic mice by using the vav-1promoter which drivestransgene expressions in the hematopoietic system. JAK2V617Ftransgenic mice display MPN-like phenotypes with increased levels of redblood cells, platelets, and white blood cells. These mice thus represent amodel system to study MPNs and associated complications. In the present study, we investigated cardiac changes and remodeling during the courseof MPN phenotype development in these mice.Materials and methodsAnimalsJAK2V617F transgenic mice were generated as previously described.The JAK2V617F transgene is under the control of the vav-1promoterwhich drives gene expression only in hematopoietic cells. These mice havebeen crossed with wild type C57BL/6mice for over10generations.Homozygous line A JAK2V617F mice were used in this study. These micecarry26copies of the JAK2V617F transgene. Wild type C57BL/6werepurchased from The Jackson Laboratory and used as control together withnon-transgenic siblings of JAK2V617F transgenic mice. At least10micewith about equal male and female representations were used for eachcontrol or experimental group. Animals were housed in ventilated cagesunder standard conditions. This study was carried out in strict accordancewith the recommendations in the Guide for the Care and Use of LaboratoryAnimals of the National Institutes of Health.Mouse tissue collection, fixation, and sectioningMice were weighed and put under deep anesthesia through inhalationof isoflurane. Terminal blood collection was performed by removing theeyeball from the socket. Hearts were then removed, blotted free of blood,and weighed. This is followed by fixation in10%neutral bufferedformalin overnight at room temperature and subsequent embedding inparaffin. Tissue sections (5μm) were cut from various positions. Histochemical staining, image acquisition, and digitalquantificationTissue sections were deparaffinized and then subjected to H&E,Masson’s trichrome, reticulin, and toluidine blue staining by usingreagents and kits from Sigma-Aldrich following standard protocols. Slideswere viewed with an Olympus BX-51upright microscope equipped withU Plan Fluorite objectives. Images were acquired using a DP71digitalcamera with the DP-BSW-V3.1camera control software (Olympus) andwere processed with the Adobe Photoshop software. Quantification ofhistochemical stain images was done by using the NIH ImageJ program.Cardiac sections from at least10mice per group were analyzed. Thethickness at the obtuse margin of the left ventricle of each heart wasmeasured, excluding trabeculations. Cardiomyocyte size was assessed bymeasuring cross-sectional area of cardiomyocytes from H&E-stainedfields randomly selected.Total RNA isolation and real time PCR analysisTotal RNAs were isolated from mouse tissues by using the RNeasyMini kit (Qiagen), and single strand cDNAs were synthesized with equalamounts of total RNAs by using the QuantiTect reverse transcription kitfrom Qiagen. Real time PCR was performed with iQ SYBR GreenSupermix (Bio-Rad) and primers specific for endogenous mouse Jak2,transgenic human JAK2V617F, and mouse glyceral-dehyde-3-phosphatedehydrogenase (GAPDH) as previously described.Results1.JAK2V617F transgenic mice displayed cardiomegalyWe employed mice of40–45weeks to characterize the cardiomegaly further. Microscopic examinations of hematoxylin and eosin(H&E)-stained cardiac sections under low magnification revealedsignificant thickening of the left ventricular wall in the JAK2V617Ftransgenic mice (P <0.01). Further exanimation under high magnificationdemonstrated enlarged cardiomyocytes (P <0.001). On average, the cellsfrom the transgenic mice were30%bigger in area than the control.2. JAK2V617F transgenic mice developed fibrosis in the heartwe performed Masson’s trichrome staining, a commonly usedmethod to detect collagen fibers.As indicated by the bright blue staining,prominent collagen fibrosis was observed in the left ventricle of transgenicmouse hearts. The fibrosis occurred in both interstitial and perivascularregions. In contrast, trichrome staining in the cor respondent regions ofcontrol mouse hearts was marginal.We further employed reticulin stainingto investigate type III collagen. veryintensive and thick interstitial reticulinfibers were observed with the transgenic mice. Reticulin staining in thecell-cell junction of control mouse hearts was also apparent but were muchthinner.3.Coronary artery thrombosis and inflammatory cell infiltrationwere found in the heart of JAK2V617F transgenic miceWe further examined at least5selected H&E-stained cardiac sectionsfrom each of20control and20JAK2V617F transgenic mice of40–70weeks of age. We found occurrence of thrombosis in the coronary arteryof4transgenic, but not a single control mice. We also noticed that cardiacsections from5JAK2V617F transgenic mice but not at all from controlmice had infiltration of inflammatory cells. Trichrome and reticulinstaining revealed collagen fibrosis in the affected regions, toluidine blue staining showed that mast cells were among the infiltrated inflammatorycells.4. Real time PCR analyses revealed a minimal expression oftransgenic JAK2V167F in the mouse heartTo determine the expression of endogenous Jak2and transgenicJAK2V617F in the heart, we performed real time PCR analysis withspecific primers as described previously. Expression of these genes inhematopoietic tissues including bone marrow, peripheral blood, andspleen were included for comparison. However, expression of transgenicJAK2V617F in the heart was much lower, about200-fold below the levelof mouse Jak2in the heart or of JAKV617F in the hematopoietic tissues.The data thus demonstrate a minimal expression of JAK2V617F transgenein mouse heart cells. Although we cannot totally rule out the contributionof JAK2V617F expression in cardiomyocytes to the development ofcardiac hypertrophy phenotypes, the possibility for this is very low.ConclusionsWe have demonstrated that JAK2V617F-induced MPNs can furtherlead to cardiac hypertrophy in mice. This has major implications for ourunderstanding of complications that occur in MPN patients. Our data helpto explain the high morbidity and mortality of MPN patients due tocardiovascular events. We have further demonstrated that the cardiacremodeling in JAK2V617F transgenic mice is manifest in fibrosisinvolving not only type I collagen but also type III reticulin fibers. Bylinking a hematological malignancy with heart diseases, JAK2V617Ftransgenic mice thus represent a unique model system to studycardiovascular diseases as well as blood disorders.