| Background. Despite the frequent clinical use of adult bone marrow mononuclear cells (BMMNCs) for cardiac repair, whether noncanonical Wnt signaling is capable of inducing cardiomyogenic differentiation in vitro remains uncertain. In addition, it is unknown whether intramyocardial transplantation of these "preprogrammed" cells into infarcted myocardium can improve cardiac function. Methods and results. BMMNCs were isolated from adult C57BL/6 wild-type (WT) mice via Ficoll-Paque density-gradient centrifugation and cultured in the presence of Wnt11 or empty-vector (control). In addition, vehicle treated, Wnt11-treated BMMNCs (preprogrammed) or untreated control BMMNCs from heterozygous-mutant green fluorescent protein (GFP) expressing transgenic mice were injected into the pen-infarct region of infarcted sib-matched WT mouse hearts (recipient mice receiving preprogrammed or unprogrammed BMMMNCs or vehicle are designated as WTWnt11, WTBMMNC and WTvehicle, respectively) 48 h following left ventricular infarction; 30 min arterial occlusion, 48 h reperfusion. In vitro, when BMMNCs were exposed to ectopically expressed Wnt11, expression of Oct-4 and Nanog (markers of pluripotency) was markedly decreased; whereas, induction of mRNA expression (quantitative real-time reverse-transcription polymerase chain reaction) of cardiac-specific genes (Nkx2.5, GATA-4, atrial natriuretic peptide, alpha- and beta-myosin heavy chain, and cardiac troponin T) was observed by day 3 with subsequent progression to a pattern characteristic of the cardiac fetal gene program. After 21 days, 27.6+/-0.6% and 29.6+/-1.4% of BMMNCs expressed the cardiac-specific antigens cardiac myosin heavy chain and cardiac troponin T, respectively (immunocytochemistry), indicating cardiomyogenic lineage commitment. Wnt11-induced cardiac-specific expression was completely abolished by the protein kinase C inhibitor bisindolylmaleimide I, partially abolished by the c-Jun-N-terminal kinase inhibitor SP600125, and attenuated by the Wnt inhibitor Dickkopf-1. Infarcted hearts from with WtWnt11 treated mice showed significantly more improvement in systolic LV function when compared to WTBMMNC mice (n=5) and WT vehicle mice following intramyocardial injection (P<0.01); though compared to WTvehicle treated mice, WTBMMNC showed slightly improved left ventricular (LV), indicating the existence of intrinsic reparative properties of unprogrammed BMMNCs. Conclusions. In adult density-gradient separated BMMNCs, noncanonical signaling via Wnt11 induces robust cardiomyogenic differentiation in a protein kinase C- and c-Jun-N-terminal kinase-dependent manner. Moreover, cardiomyogenic preprogramming of BMMNCs with Wnt11 before their myocardial transplantation enhances the in vivo cardiac reparative potential of these cells. |
