Abstract 11931: Optogenetic Control of Arrhythmogenic Myofibroblast-Cardiomyocyte Interactions
INTRODUCTION: Cardiac myofibroblasts (MFBs) are thought to elicit arrhythmogenic slow conduction and ectopic activity by causing partial depolarization of cardiomyocytes (CMCs) following establishment of heterocellular gap junctional coupling. In order to provide direct evidence that depolarizing current flow from moderately polarized MFBs to electrically coupled CMCs is the primary cause of these conditions, we investigated whether switching MFBs to a hyperpolarized state using the light-driven Cl- pump Halorhodopsin causes normalization of membrane polarization of CMCs coupled to MFBs.
METHODS: Based on pLenti-CaMKIIa-eNpHR 3.0-EYFP (Addgene), we created CMV-eNpHR3.0-eYFP lentivirus. Halorhodopsin expressed by the transduced myofibroblasts (HaloMFBs) was targeted to the membrane. Halorhodopsin function was evaluated using patch clamp experiments in single HaloMFBs and HaloMFB-CMC cell pairs.
RESULTS: Virus transduction had no effects on the morphology and the membrane potential (Vm) of MFBs measured in the dark. When exposed to orange light (LED, CW = 590 nm), HaloMFBs showed a light-intensity dependent membrane hyperpolarization. At maximal illumination intensities (~ 1mW/mm2), HaloMFBs hyperpolarized from -28.3 ± 9.8 mV to -74.2 ± 18.1 mV (n=9). Photocurrents ranged from 0.17 to 1.09 pA/pF (average: 0.42 ± 0.3 pA/pF, n=9) and activated/deactivated with time constants of 9.6 ± 3.1 msec and 11.0 ± 3.0 msec, respectively. Photocurrents showed no voltage dependence and no desensitization with multiple stimulations. Gap junctional coupling of HaloMFB-CMC cell pairs was not affected by light stimulation and was similar to coupling of MFB-CMC pairs (range: 67 to 74 nS). Light stimulation of CMC-HaloMFB cell pairs induced a rescue of CMC resting potentials back to normal values (light-off: - 54.1 ± 10.9 mV; light-on: - 72.1 ± 13.7 mV; n = 7).
CONCLUSION: This novel use of light-driven pumps in primary cardiac cells provides direct evidence that arrhythmogenic CMC depolarization in MFB-CMC cell pairs is due to the depolarized phenotype of MFBs. Moreover, the results indicate that optogenetic methods may represent ideal tools to systematically investigate adverse electrotonic interactions between stromal and parenchymal cells in the heart.
- © 2012 by American Heart Association, Inc.