Abstract 11749: Cell-Specific Expression of Voltage-Sensitive Protein Confirms Cardiac Myocyte to Non-Myocyte Electrotonic Coupling in Healed Murine Infarct Border Tissue
Introduction: Heterocellular electrotonic coupling of cardiac myocytes and non-myocytes is common in vitro, yet its presence, extent and role in vivo are debated. Optogenetic tools provide a unique means for cell-specific assessment of electrophysiology. Here, we utilise the genetically-encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential (Vm) in myocytes or fibroblasts of isolated murine hearts.
Methods: Transgenic mice expressing VSFP2.3 under the control of either alpha-myosin heavy chain promoter (αMHC; myocytes) or Wilm’s tumor promoter (WT1; fibroblasts) were generated by Cre-LoxP recombination. Infarcts (left ventricular freewall) were caused by cryoablation, followed by 8 weeks recovery. Vm was measured in mechanically-uncoupled (10μM blebbistatin) Langendorff-perfused hearts by simultaneous collection of VSFP2.3 donor (mCerulean; CFP) and acceptor (citrine; YFP), or of VSFP2.3 YFP and voltage-sensitive dye (di-4-ANBDQPQ) signals, using an EMCCD camera (128х128 pixels, 511Hz) on an upright microscope (10х water-immersion lens). Cell-specificity of transgene expression was confirmed post hoc by immunohistochemistry (myomesin for myocytes, vimentin for fibroblasts) using confocal microscopy.
Results: Cardiomyocyte action potentials (AP) were successfully measured in αMHC-VSFP2.3 mice by CFP/YFP ratiometry. After adding di-4-ANBDQPQ to these hearts, signal comparison reconfirmed slower AP upstroke and repolarisation kinetics of VSFP2.3 (Fig.A). Strikingly, myocyte-like AP were also recorded, in (dye-free) WT1-VSFP2.3 hearts, from fibroblasts at the infarct border zone (Fig.B).
Conclusions: Results demonstrate the utility of VSFP2.3 for cell-specific cardiac electrophysiology research. In addition, observation of AP-like potentials in non-excitable cells confirms in vivo electrotonic coupling of cardiac myocytes and non-myocytes in healed murine infarct border tissue.
Author Disclosures: T. Quinn: None. P. Camelliti: None. U. Siedlecka: None. T. Poggioli: None. L.M. Loew: None. T. Knöpfel: None. P. Kohl: None.
- © 2014 by American Heart Association, Inc.