Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling.

BACKGROUND: Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions. Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling. METHODS AND RESULTS: High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (>80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation. Transients from adjacent myotubes displayed different excitation thresholds, indicating an absence of coupling between cells, consistent with skeletal muscle biology. In contrast, simultaneous transduction of HDFs with MyoD and connexin43-encoding vectors resulted in the appearance of transients in adjacent myotubes with identical thresholds, indicative of electrical coupling. Notably, dye transfer studies confirmed gap junctional intercellular communication. CONCLUSIONS: Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. These observations strengthen the prospect of developing gene-based strategies for repairing cardiac conduction defects.

Control of somite patterning by Sonic hedgehog and its downstream signal response genes.

In the avian embryo, previous work has demonstrated that the notochord provides inductive signals to activate myoD and pax1 regulatory genes, which are expressed in the dorsal and ventral somite cells that give rise to myotomal and sclerotomal lineages. Here, we present bead implantation and antisense inhibition experiments that show that Sonic hedgehog is both a sufficient and essential notochord signal molecule for myoD and pax1 activation in somites. Furthermore, we show that genes of the Sonic hedgehog signal response pathway, specifically patched, the Sonic hedgehog receptor, and gli and gli2/4, zinc-finger transcription factors, are activated in coordination with somite formation, establishing that Sonic hedgehog response genes play a regulatory role in coordinating the response of somites to the constitutive notochord Sonic hedgehog signal. Furthermore, the expression of patched, gli and gli2/4 is differentially patterned in the somite, providing mechanisms for differentially transducing the Sonic hedgehog signal to the myotomal and sclerotomal lineages. Finally, we show that the activation of gli2/4 is controlled by the process of somite formation and signals from the surface ectoderm, whereas upregulation of patched and activation of gli is controlled by the process of somite formation and a Sonic hedgehog signal. The Sonic hedgehog signal response genes, therefore, have important functions in regulating the initiation of the Sonic hedgehog response in newly forming somites and in regulating the patterned expression of myoD and pax1 in the myotomal and sclerotomal lineages following somite formation.