LacZ Adeno-associated virus(AAV Serotype 6)

Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are an evolving platform for understanding molecular disease mechanisms and evaluating cardiovascular drugs. A major limitation of this system is that they represent a heterogeneous mixture of ventricular-, atrial-, and nodal-like CMs. By expressing voltage-sensitive fluorescent proteins under the control of lineage-specific promoters, researchers have developed a convenient system that allows high-throughput subtype-specific optical action potential (AP) imaging in these cells. This not only allows for quantification of electrical phenotypes in patient-specific CMs, but also allows for the study of subtype-specific drug effects, which may aid drug development and safety pharmacology in the cardiovascular field.

Here, wild-type (wt) KCNQ1 ion channel subunits were overexpressed in hiPSC-derived LQT1R190Q CMs via adeno-associated virus (AAV6)-mediated gene transfer (Figure 1A). The mutated KCNQ1 gene encodes a transport-deficient ion channel subunit that interacts with the wild-type subunit and interferes with its integration into the plasma membrane, resulting in a dominant-negative effect, which might be overcome by overexpression of wild-type subunits. AP was optically recorded seven days after LQT1R190Q CM infection with MLC2v-VSFP lentivirus, followed by AAV6 virus encoding wt KCNQ1 fused to a hemagglutinin (HA) epitope tag (wt KCNQ1-HA AAV6) or a control virus encoding LacZ (LacZ-AAV6) infected cells. Three days later (day 10), AP was recorded again in the same cells, and post hoc staining for the HA epitope and β-gal transgene was performed to confirm viral transduction of the study cells (Figure 1A). Indeed, wt KCNQ1 overexpression significantly shortened the AP of LQT1R190Q CM (Figure 1B and D ), reaching APD values ​​similar to those measured in LQT1corr cells. In contrast, no changes in APD were detected in CM infected with LacZ-AAV6 (Figure 1C and D). Therefore, sequential AP imaging in the same cells using the MLC2v-VSFP sensor can detect successful LQT1 phenotype rescue in patient-derived hiPSC-CMs.

Figure 1. Measurements of dynamic changes in action potential duration in single cells over time. (Chen Z, et al., 2017)