Investigation into the mechanisms determining spontaneous activity in human-induced pluripotent stem cell-derived cardiomyocytes

da Silva Costa, Ana Filipa (2019) Investigation into the mechanisms determining spontaneous activity in human-induced pluripotent stem cell-derived cardiomyocytes. PhD thesis, University of Glasgow.

Abstract

Background: Human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an alternative to adult primary cells for scientific and commercial research, but they have an embryonic rather than adult electrophysiological phenotype. In particular: low expression of inward rectifying channel (Ik1) contributes towards the unstable resting membrane potential and results in spontaneous electrical activity.
Purpose: This study examines contribution of Ik1 and other ionic currents to the spontaneous electrical activity of hiPSC-CMs and tests the concept that additional external Ik1 activity can be added via co-culture with Ik1-expressing HEK293 cells.
Methods: hiPSC-CM’s ionic currents and channels were investigated using different ion channel blockers. Ik1 was externally added to hiPSC-CM culture via co-culture with I¬k1-overexpressing HEK at different densities and ratios. The effects on contractility and voltage (action potentials) were investigated using two in-house platforms: CellOPTIQ and MUSCLEMOTION.
Results: Based on the sensitivity to the If blocker, the pacemaker current (If) is either absent or not a dominant contributor to pacemaking in hiPSC-CMs. Blockade of Ik1 with BaCl2 and PA-6 did not affect interval time, thus showing that there is low availability of Ik1. Co-culture with Ik1-HEK at 1:1 HEK:hiPSC led to a prolongation of mean interval (from 963.524.5ms to 1516.079.3ms, n=15, p<0.0001). The cause of this is unknown. Co-culture using fibroblasts and hiPSC-CMs was done to study the effects of a different cell type in co-culture. These indicated that the amplitude of the contraction signals might be affected by different cell types and their elasticity, rather than effects within the hiPSC-CMs. To improve cell-to-cell coupling, IK1-HEK were transfected with Cx43 and introduced into co-culture. Under these conditions, the electrophysiological behaviour was similar to co-culture with Ik1-HEK cells. The signal to noise ratio (SNR) of membrane dye (FluoVolt) was used to estimate the degree of coupling of iPSC-CMs to Ik1-HEK.
Conclusions: hiPSC-CMs have low contribution from Ik1, but the spontaneous activity was not dominated by If. Co-culture with Ik1-HEK rather than wild-type HEK led to prolongation of interval between APs, suggesting that hiPSC-CMs were successfully coupled to Ik1-expressing HEK cells. Increased expression of Cx43 to potentially further develop this effect - by improving electrical linkage - has no overall further effects. In summary, enhancing Ik1 pharmacologically was not possible, but co-culture with specific ion channel cell lines appeared to successfully add to the channels contributing to the AP in hiPSC-CMs.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from EPSRC.
• Keywords: hiPSC-CMs, cardiomyocytes, Ik1 fibroblast, cardiac stem cell, maturation.
• Subjects: Q Science > QP Physiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health > Cardiovascular & Metabolic Health
• Supervisor's Name: Smith, Professor Godfrey and Burton, Dr. Francis
• Date of Award: 2019
• Depositing User: Miss Ana Da Silva Costa
• Unique ID: glathesis:2019-41143
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Apr 2019 14:02
• Last Modified: 25 Jan 2023 11:43
• Thesis DOI: 10.5525/gla.thesis.41143
• URI: https://theses.gla.ac.uk/id/eprint/41143

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