The amount of jetPEI solution mixed with the plasmid DNA resulted in an N/P ratio of 5. Thus, 3 mg of DNA and 6 mL of jetPEI were added to each well (of a six-well plate) where the fibroblasts were seeded at 50 – 70% confluence.

AIMS: Optogenetics approaches, utilizing light-sensitive proteins, have emerged as unique experimental paradigms to modulate neuronal excitability. We aimed to evaluate whether a similar strategy could be used to control cardiac-tissue excitability. METHODS AND RESULTS: A combined cell and gene therapy strategy was developed in which fibroblasts were transfected to express the light-activated depolarizing channel Channelrhodopsin-2 (ChR2). Patch-clamp studies confirmed the development of a robust inward current in the engineered fibroblasts following monochromatic blue-light exposure. The engineered cells were co-cultured with neonatal rat cardiomyocytes (or human embryonic stem cell-derived cardiomyocytes) and studied using a multielectrode array mapping technique. These studies revealed the ability of the ChR2-fibroblasts to electrically couple and pace the cardiomyocyte cultures at varying frequencies in response to blue-light flashes. Activation mapping pinpointed the source of this electrical activity to the engineered cells. Similarly, diffuse seeding of the ChR2-fibroblasts allowed multisite optogenetics pacing of the co-cultures, significantly shortening their electrical activation time and synchronizing contraction. Next, optogenetics pacing in an in vitro model of conduction block allowed the resynchronization of the tissue's electrical activity. Finally, the ChR2-fibroblasts were transfected to also express the light-sensitive hyperpolarizing proton pump Archaerhodopsin-T (Arch-T). Seeding of the ChR2/ArchT-fibroblasts allowed to either optogentically pace the cultures (in response to blue-light flashes) or completely suppress the cultures' electrical activity (following continuous illumination with 624 nm monochromatic light, activating ArchT). CONCLUSIONS: The results of this proof-of-concept study highlight the unique potential of optogenetics for future biological pacemaking and resynchronization therapy applications and for the development of novel anti-arrhythmic strategies.