Background: Genetic manipulation of cardiac (myo-)fibroblasts (cFB) in vitro and in vivo is challenging and, so far, not efficient. Based on promising data using the Moloney murine leukemia virus (MMlv) for targeting of non-myocytes in embryonic mouse hearts, we have investigated the transduction efficiency of cFB and non-cFB by the MMlv in vitro and in vivo. Transduction rate was further enhanced by complexing MMlv with magnetic nanoparticles (MNP); this approach was probed in vitro and in vivo under a magnetic field. To improve electrical conduction, the gap junction protein Connexin43 (Cx43) was overexpressed in the cells.
Method: Noncardiac 3T3 FB lacking Cx43 were transduced in vitro using either the lentivirus or MMlv encoding for Cx43 and transduction efficiency was investigated. The FRAP technique was used to examine the functionality of the newly formed gap junctions in vitro. In vivo, MMlv/MNP complexes encoding Cx43/mCherry or only mCherry (control) were injected into the lesion area using a magnet 3 days after myocardial infarction (MI). Cardiac function was monitored by echocardiography. Electrophysiological testing (EPT) was performed 2 weeks postsurgery. The Cx43 content of the scar as well as of the remote myocardium was determined by Western blot (WB) analysis.
Results: In vitro analyses revealed a significantly higher transduction rate of 3T3 FB and cFB when using MMlv/MNP compared with lentivirus/MNP complexes (72 vs. 26%). FRAP experiments showed the formation of functional gap junctions between Cx43-expressing FB. In vivo, EPT 2 weeks after MI and MMlv/MNP-based gene therapy of the lesion area showed a significantly reduced incidence of ventricular tachycardias (VT) in mice transduced with MMlv-Cx43/mCherry compared with mice treated only with the control virus (31.3%, n = 16 vs. 76.5%, n = 17). Also, the average duration of the VT was lower in the Cx43 group (Cx43: 0.7 s vs. control: 1.1 s). WB analysis showed a significantly increased Cx43 content in MMlv-Cx43-transduced cardiac scars. MMlv/MNP transduction or the increase of the Cx43 content in the lesion area had no significant effects on cardiac pump function.
Conclusion: MMlv-based combined with magnet MNP-based gene transfer is highly efficient in cardiac and noncardiac FB. MMlv-based Cx43 gene transfer in acute MI results in strongly increased protein expression in the fibrotic scar area and hence significant anti-VT protection 2 weeks after cryoinjury and gene therapy.
