Abstract
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current (Ik1). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking Ik1-like currents with BaCl2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with Ik1’s role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized.
Highlights
The growing use of human induced pluripotent stem cellderived cardiomyocytes provides genetically diverse human cardiac models to study cardiac arrhythmia patients with different phenotypes and to assess proarrhythmic risk of new drugs in vitro [36, 39]
We investigate the role of cell density on KCNJ2/Kir2.1 and related hiPSC-CMs electrophysiology (Fig. 1) using mRNA and protein quantification, all-optical cardiac electrophysiology, pharmacological probing, and computational analysis
Previous voltage-clamp studies in isolated hiPSC-CMs have reported the lack of or insufficient levels of Ik1 compared with adult cardiomyocytes [31], with differences at the single-cell level on the order of 10–100 times
Summary
The growing use of human induced pluripotent stem cellderived cardiomyocytes (hiPSC-CMs) provides genetically diverse human cardiac models to study cardiac arrhythmia patients with different phenotypes and to assess proarrhythmic risk of new drugs in vitro [36, 39]. Several cardiomyopathy phenotypes have been successfully recapitulated in vitro, including dilated cardiomyopathy [40], hypertrophic cardiomyopathy [26], and arrhythmogenic right ventricle cardiomyopathy [5] These experimental cardiac models are used for discovering new drug therapies [46] and for cardiotoxicity screening [13]. It has been shown that the spontaneous activity of hiPSC-CMs may be partially influenced by low levels of the inward rectifier potassium current (Ik1) [27, 42]. Insufficient number or low conductance of functional Kir2.1 ion channels can cause lower membrane resistance [37], with a net inward current during rest, a relatively depolarized resting membrane potential, increased spontaneous rates [33], and slower upstroke (through the effects of the resting membrane potential on sodium channel availability) and potentially can affect the action potential duration of hiPSC-CMs
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