Abstract
The ultrarapid delayed rectifier K+ current (IKur), mediated by Kv1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of Kv1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout. This model as well as isogenic wild-type control hiPSCs could selectively be differentiated into ventricular or atrial cardiomyocytes at high efficiency, based on the specific manipulation of retinoic acid signaling. Investigation of electrophysiological properties in Kv1.5-deficient cardiomyocytes compared to isogenic controls revealed a strictly atrial-specific disease phentoype, characterized by cardiac subtype-specific field and action potential prolongation and loss of 4-aminopyridine sensitivity. Atrial Kv1.5-deficient cardiomyocytes did not show signs of arrhythmia under adrenergic stress conditions or upon inhibiting additional types of K+ current. Exposure of bulk cultures to carbachol lowered beating frequencies and promoted chaotic spontaneous beating in a stochastic manner. Low-frequency, electrical stimulation in single cells caused atrial and mutant-specific early afterdepolarizations, linking the loss of KCNA5 function to a putative trigger mechanism in familial AF. These results clarify for the first time the role of Kv1.5 in atrial hiPSC-cardiomyocytes and demonstrate the feasibility of cardiac subtype-specific disease modeling using engineered hiPSCs.
Highlights
Atrial fibrillation (AF) constitutes the most prevalent cardiac arrhythmia and accounts for about one third of all hospitalizations related to heart rhythm disturbances (Fuster et al, 2011)
A previously described approach based on a self-replicating RNA vector expressing four reprogramming factors was employed to generate integration-free wild-type human induced pluripotent stem cells (hiPSCs) from fetal human fibroblasts (Figure 1A) (Yoshioka et al, 2013)
Results revealed that a stimulation with 0.5 μM retinoic acid (RA) during CM induction was sufficient for robustly inducing atrial markers including KCNA5 while strongly suppressing ventricular ones like IRX4 and MYL2. This variant of the protocol was applicable both to WT and KCNA5fs/fs hiPSCs suggesting that the model as well as its isogenic control may efficiently be converted into the desired cardiac subtype
Summary
Atrial fibrillation (AF) constitutes the most prevalent cardiac arrhythmia and accounts for about one third of all hospitalizations related to heart rhythm disturbances (Fuster et al, 2011). A familial history of AF has been associated with a 40% risk increase for developing it and over the last years, a number of AF-related gene loci and mutations have been identified (Lubitz et al, 2010; Hucker et al, 2016) These comprise several developmental regulators such as PITX2 as well as ion channel genes involved in cardiac action potential (AP) generation, suggesting a direct causative role of the latter group (Kirchhof et al, 2011; Christophersen and Ellinor, 2016). QTc interval prolongation predisposes for arrhythmia-inducing triggering events such as early afterdepolarizations (EADs) and is considered a risk factor in particular for lone AF cohorts (Johnson et al, 2008; Nielsen et al, 2013) In this context, we here report an isogenic KCNA5 knockout model in hiPSCs-CMs, to investigate basic as well drug-induced phenotypes in a cardiac subtype-specific manner
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