Abstract 1072: Green Fluorescent Protein-Based Myoblast Tagging: A Potential Confounder of Outcome Measurements.
Green Fluorescent Protein (GFP) and its variants are commonly used in cell transplantation for tracking purposes. Using a rat model of myocardial infarction, we recently found that eGFP-transduced skeletal myoblasts yielded a significantly poorer echocardiographic recovery of LV function than their non-transduced counterparts (-14% and +9% from baseline LV ejection fraction, respectively, p<0.05). These data laid the grounds for the present study designed to identify the molecular mechanisms linking eGFP expression to contractile dysfunction and, more specifically, to test the hypothesis that eGFP could inhibit actin-myosin interactions. To this end, we assessed the mechanical (in vitro motility assay), enzymatic (actin-activated ATPase rate), biochemical (co-precipitation) and structural properties of myosin in the presence of eGFP and F-actin. In vitro motility assays and actin-activated ATPase activity were performed at 1:0.5, 1:1 and 1:3 myosin:eGFP molar ratios. As eGFP concentration increased relatively to myosin, the percentage of moving filaments decreased by 27±5% and 90±4% at 1:1 and 1:3 myosin:eGFP ratios, respectively, compared to controls (p<0.001). The maximum ATPase rate decreased by 62±11% and 97±1% at 1:1 and 1:3 myosin:eGFP ratios, respectively (n=5/group, p<0.001). Using HIS-select nickel affinity assay, we further found that myosin co-precipitated with eGFP. Electrophoresis and western-blot analysis using anti-myosin antibody revealed that myosin specifically bound to eGFP. Finally, crystal structures of myosin (2MYS), actin filament (1ATN) and GFP (1EMA) obtained from the PDB indicated that GFP and actin filament exhibit similar electrostatic surface patterns and the ClusPro docking model showed that at least 6 segments of the GFP could bind to the myosin head. In conclusion, our data demonstrate that the cytoplasmic expression of eGFP within muscle cells results in the binding of eGFP to myosin, thereby disturbing the actin-myosin interaction and in turn, the contractile function of the transduced cells. This adverse effect of eGFP should be kept in mind when using this marker to track cells following transplantation since the induced changes in cellular function may confound interpretation of functional data.