Active Wnt signaling in response to cardiac injury

Martinus I. F. J. Oerlemans 0 1 2 3 Marie-Jose Goumans 0 1 2 3 Ben van Middelaar 0 1 2 3 Hans Clevers 0 1 2 3 Pieter A. Doevendans 0 1 2 3 Joost P. G. Sluijter 0 1 2 3 0 H. Clevers Hubrecht Institute , Utrecht, The Netherlands 1 M.-J. Goumans Department of Molecular Cell Biology, Leiden University Medical Centre , Leiden, The Netherlands 2 M. I. F. J. Oerlemans B. van Middelaar P. A. Doevendans J. P. G. Sluijter Department of Cardiology, University Medical Centre Utrecht , Utrecht, The Netherlands 3 P. A. Doevendans J. P. G. Sluijter Interuniversity Cardiology Institute of the Netherlands (ICIN) , Utrecht, The Netherlands Although the contribution of Wnt signaling in infarct healing is suggested, its exact role after myocardial infarction (MI) still needs to be unraveled. We evaluated the cardiac presence of active Wnt signaling in vivo following MI, and investigated in which cell types active Wnt signaling was present by determining Axin2 promoterdriven LacZ expression. C57BL/6 Axin2-LacZ reporter mice were sacrificed at days 0, 1, 3, 7, 14, and 21 after LAD ligation. Hearts were snap-frozen for immunohistochemistry (IHC) or enzymatically digested to obtain a single cell suspension for flow cytometric analysis. For both FACS and IHC, samples were stained for b-galactosidase and antibodies against Sca-1, CD31, ckit, and CD45. - Cardiovascular disease is one of the leading causes of morbidity and mortality in the western world [23]. Following myocardial infarction (MI), damaged myocardium is replaced with scar tissue, which may result in heart failure for which limited therapeutic options are available. One potential important signal transduction pathway involved in regulating cardiac repair and potentially stem cell maintenance and differentiation is the Wnt pathway, which plays an essential role in normal cardiac development [8]. Wnt signaling has been shown to be a key regulator of stem cell growth, differentiation, and proliferation in both normal homeostasis and diseased state [10, 34]. Wnt proteins form a family of highly conserved secreted signaling molecules in which the canonical Wnt/b-catenin pathway is mostly studied. Upon binding of Wnt to the seven-transmembrane domain spanning frizzled (Fzd) receptor and the co-receptor lipoprotein receptor-related 5/6 (Lrp5/6) proteins GSK3b is inactivated, thereby preventing the breakdown of b-catenin. After stabilization and accumulation, b-catenin enters the nucleus, where it binds to LEF/TCF transcription factors to activate the transcription of Wnt target genes. Although extensively studied in cardiac development, the exact role of Wnt signaling after MI still needs to be unraveled. Infarct size reduction was achieved by both stimulation and inhibition of Wnt/b-catenin signaling [2, 14], and both downregulation [4] and upregulation [32] of b-catenin gene levels were observed in cardiac hypertrophy. Moreover, epicardium-derived progenitor cells lacking b-catenin displayed impaired coronary artery formation [41], while b-catenin depletion in cardiac progenitor cells enhanced differentiation during cardiac remodeling [42]. Recent data demonstrates that b-catenin is involved in expansion of resident cardiac progenitor cells, but its role in differentiation of these cells remains controversial [20, 22, 33, 42]. Most studies investigating Wnt signaling during pathological conditions (e.g. myocardial infarction) focused on proteins participating in Wnt signal transduction such as Dishevelled-1 [9, 38] and b-catenin [7, 40]. However, this not necessarily means that active Wnt/b-catenin signaling, including transcription of Wnt target genes, is present. Considering its potency to act as a therapeutic target, fundamental insights on Wnt time-dependency and cell specificity upon injury in the adult injured myocardium are necessary. Therefore, we evaluated the presence of active Wnt signaling in vivo in the heart following MI. Moreover, we investigated in which cell types active Wnt signaling was present during different phases following MI. After binding of b-catenin to LEF/TCF transcription factors in the nucleus, several Wnt target genes are activated, including the Axin2 gen [21]. Axin2 is able to downregulate b-catenin and acts as a negative regulator of Wnt signaling [16]. We used Axin2?/LacZ reporter mice in which the LacZ gene is under the control of the Axin2 promoter, providing a reliable way to detect Wnt activity by visualizing LacZ-reporter-positive cells [24]. Materials and methods Male and female C57BL/6 Axin2?/LacZ reporter mice were bred and used at 810 weeks of age. For generation of the Axin2-lacZ mouse [24], the b-galactosidase (NLS-lacZ) gene was introduced in frame to the endogenous Axin2 promoter by homologous recombination, thereby replacing most of exon 2 (MGI Ref ID J:74286) but leaving the Axin2 promoter intact. All experiments were approved by the Animal Experimentation Committee of the Utrecht University and were in accordance with the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The MI was induced by ligation of the left anterior descending (LAD) coronary artery under isoflurane anesthesia, as described previously [37, 39]. Briefly, mice were anesthetized with isoflurane, orally intubated, and ventilated. A left thoracotomy was performed at the third intercostal space, and muscles and pericardium were dissected. LAD ligation was performed with an 8-0 nonabsorbable ethilon suture. After verification that coronary occlusion had occurred by the change of color and kinesis of the apex and anterior-lateral wall, the thorax was closed in layers. After detubation, mice were kept warm until fully recovered. Mice were sacrificed at baseline (0 day, controls) and at 1, 3, 7, 14, and 21 days after MI. The hearts were flushed with phosphate-buffered saline (PBS) and dissected. The left ventricle was cut in two halves through the center of the infarct along the longitudinal axis. One half was snap-frozen in liquid nitrogen and stored at 80 C; the other half was kept in PBS for further processing. Remote area was defined as the non-infarcted part of the interventricular septum. Cell isolation and flow cytometry analysis Freshly dissected hearts, containing the infarcted and noninfarcted area, were perfused with PBS and washed, minced into 12 mm2 pieces, digested for 45 min at 37 C with 10 mg/ml collagenase A (Roche), and passed through a 70-lm filter. Remaining cells were plated on a 12-well plate DMEM with 10% fetal bovine serum (FBS) as described previously [36] or aliquoted after centrifugation for flow cytometric analysis. After centrifugation, cells were resuspended in PBS containing 4% FBS, and aliquots containing 1.0 9 106 cells were stained. Single cell suspensions were s (...truncated)