Electrical Coupling Between Cardiomyocytes and Unexcitable Cells: The Effect of Cardiac Fibroblasts and Genetically Engineered HEK-293 Cells on Cardiac Action Potential Shape and Propagation
Excess cardiac myofibroblasts in fibrotic heart diseases as well as cell-based therapies involving implantation of stem cells or genetically engineered somatic cells in the heart may all lead to a situation where a cardiomyocyte becomes electrically coupled to an unexcitable cell. In these settings, electrotonic loading of cardiomyocytes by unexcitable cells can affect cardiac action potential generation, propagation, and repolarization depending on the properties of both cardiomyocytes and unexcitable cells. The objective of this dissertation was to advance our understanding of the electrical interactions between cardiomyocytes and unexcitable cells using a variety of electrophysiological, molecular, and cell culture techniques.
First, we utilized aligned cardiomyocyte monolayers covered with unexcitable cardiac fibroblasts or human embryonic kidney-293 (HEK) cells that expressed similar levels of the gap junction protein connexin-45. These cells weakly coupled to cardiomyocytes and marginally slowed cardiac conduction only at high coverage density, while producing no other measurable electrophysiological changes in cardiomyocytes. In contrast, unexcitable HEK cells genetically engineered to stably express the more conductive connexin-43 channels (Cx43 HEK) strongly coupled to cardiomyocytes, depolarized cardiac resting membrane potential, significantly slowed impulse propagation, decreased maximum capture rate, and increased action potential duration (APD) at high coverage density. None of the studied unexcitable cells significantly altered conduction velocity anisotropy ratio or the relatively low incidence of pacemaking activity of cardiac monolayers at any coverage density.
Next, we utilized individual micropatterned cell pairs consisting of a cardiomyocyte and an unexcitable Cx43 HEK cell with or without stably overexpressed inward rectifier potassium channels (Kir2.1+Cx43 HEK). By systematically varying the relative sizes of micropatterned cells, we showed that Cx43 HEK cells significantly depolarized cardiomyocytes, reduced maximum upstroke velocity and action potential amplitude, prolonged APD, and modulated beating rate as a function of HEK:CM area ratio. In contrast, in cell pairs formed between cardiomyocytes and Kir2.1+Cx43 HEK cells we observed significant reduction in cardiomyocyte action potential amplitude, duration, and maximum upstroke velocity, but no change in other measured parameters.
Finally, we utilized a hybrid dynamic clamp setting consisting of a live micropatterned cardiomyocyte coupled in real time to a virtual model of capacitive and/or ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells. We found that coupling of cardiomyocytes to the ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells was sufficient to reproduce the dependence of cardiomyocyte maximal diastolic potential and pacemaking behavior on HEK:CM area ratio observed in micropatterned cell pairs, but did not replicate the observed changes in action potential upstroke or duration. The pure capacitance model with no ionic current, on the other hand, significantly decreased cardiomyocyte maximum upstroke velocity and prolonged cardiomyocyte APD as function of HEK:CM area ratio without affecting maximal diastolic potential or pacemaking behavior. When the unexcitable cell model containing both capacitive and ionic currents was connected to cardiomyocytes, all changes in action potential shape observed in micropatterned cell pairs were accurately reproduced.
These studies describe how coupling of unexcitable cells to cardiomyocytes can alter cardiomyocyte electrophysiological properties dependent on the unexcitable cell connexin isoform expression, ion channel expression, and cell size. This knowledge is expected to aid in the design of safe and efficient cell and gene therapies for myocardial infarction, fibrotic heart disease, and cardiac arrhythmias.
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