Cardiac Optogenetics: Enhancement by All-trans-Retinal.

All-trans-Retinal (ATR) is a photosensitizer, serving as the chromophore for depolarizing and hyperpolarizing light-sensitive ion channels and pumps (opsins), recently employed as fast optical actuators. In mammalian optogenetic applications (in brain and heart), endogenous ATR availability is not considered a limiting factor, yet it is unclear how ATR modulation may affect the response to optical stimulation. We hypothesized that exogenous ATR may improve light responsiveness of cardiac cells modified by Channelrhodopsin2 (ChR2), hence lowering the optical pacing energy. In virally-transduced (Ad-ChR2(H134R)-eYFP) light-sensitive cardiac syncytium in vitro, ATR supplements ≤2 µM improved cardiomyocyte viability and augmented ChR2 membrane expression several-fold, while >4 µM was toxic. Employing integrated optical actuation (470 nm) and optical mapping, we found that 1-2 µM ATR dramatically reduced optical pacing energy (over 30 times) to several µW/mm(2), lowest values reported to date, but also caused action potential prolongation, minor changes in calcium transients and no change in conduction. Theoretical analysis helped explain ATR-caused reduction of optical excitation threshold in cardiomyocytes. We conclude that cardiomyocytes operate at non-saturating retinal levels, and carefully-dosed exogenous ATR can enhance the performance of ChR2 in cardiac cells and yield energy benefits over orders of magnitude for optogenetic stimulation.

Nanopatterned Human iPSC-based Model of a Dystrophin-Null Cardiomyopathic Phenotype.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer unprecedented opportunities to study inherited heart conditions in vitro, but are phenotypically immature, limiting their ability to effectively model adult-onset diseases. Cardiomyopathy is becoming the leading cause of death in patients with Duchenne muscular dystrophy (DMD), but the pathogenesis of this disease phenotype is not fully understood. Therefore, we aimed to test whether biomimetic nanotopography could further stratify the disease phenotype of DMD hiPSC-CMs to create more translationally relevant cardiomyocytes for disease modeling applications. We found that anisotropic nanotopography was necessary to distinguish structural differences between normal and DMD hiPSC-CMs, as these differences were masked on conventional flat substrates. DMD hiPSC-CMs exhibited a diminished structural and functional response to the underlying nanotopography compared to normal cardiomyocytes at both the macroscopic and subcellular levels. This blunted response may be due to a lower level of actin cytoskeleton turnover as measured by fluorescence recovery after photobleaching. Taken together these data suggest that DMD hiPSC-CMs are less adaptable to changes in their extracellular environment, and highlight the utility of nanotopographic substrates for effectively stratifying normal and structural cardiac disease phenotypes in vitro.