Identification of the optimal reference genes for atrial fibrillation model established by iPSC-derived atrial myocytes

Abstract

Background: Atrial fibrillation (AF) stands as a prevalent and detrimental arrhythmic disorder, characterized by intricate pathophysiological mechanisms. The availability of reliable and reproducible AF models is pivotal in unraveling the underlying mechanisms of this complex condition. Unfortunately, the researchers are still confronted with the absence of consistent in vitro AF models, hindering progress in this crucial area of research.

Methods: Human induced pluripotent stem cells derived atrial myocytes (hiPSC-AMs) were generated based on the GiWi methods and were verified by whole-cell patch clamp, immunofluorescent staining, and flow cytometry. Then hiPSC-AMs were employed to establish the AF model by HS. Whole-cell patch clamp technique and calcium imaging were used to identify the AF model. The stability of 29 reference genes was evaluated using delta-Ct, GeNorm, NormFinder, and BestKeeper algorithms; RESULTS: HiPSC-AMs displayed atrial myocyte action potentials and expressed the atrial-specific protein MLC-2 A and NR2F2, about 70% of the cardiomyocytes were MLC-2 A positive. After HS, hiPSC-AMs showed a significant increase in beating frequency, a shortened action potential duration, and increased calcium transient frequency. Of the 29 candidate genes, the top five most stably ranked genes were ABL1, RPL37A, POP4, RPL30, and EIF2B1. After normalization using ABL1, KCNJ2 was significantly upregulated in the AF model; Conclusions: In the hiPSC-AMs AF model established by HS, ABL1 provides greater normalization efficiency than commonly used GAPDH.

Keywords: Atrial fibrillation; Atrial fibrillation model; Electrical stimulation; Induced pluripotent stem cells; Quantitative real-time polymerase chain reaction; Reference gene.

Fig. 1

Cultivation of stem cells and differentiation of atrial myocytes. A. Immunofluorescence demonstrates the expression of pluripotency markers OCT4 and SSEA4 in hiPSCs. Scale bars: 50 μm. B. The timeline of drug addiction and cell differentiation stage for hiPSCs differentiating into atrial myocytes. C. Cellular state changes during the differentiation of hiPSCs into atrial myocytes

Fig. 2

Characteristics of hiPSC and hiPSC-derived cardiomyocytes. A. Representative confocal micrographs immunofluorescence images of CM marker cTnT and atrial myocyte maker MLC-2 A and NR2F2. Scale bars: 20 μm. B. Flow cytometry analysis showing the percentage of cTnT positive cells and MLC-2 A positive cells after 30d of differentiation. C. Patch clamp experiments showing representative action potentials from the sinus node cell, atrial myocytes, and ventricular myocytes

Fig. 3

Establishing an atrial fibrillation model in hiPSC-ACM using high-frequency electrical stimulation. A. Brief Process and Diagram of Electrical Stimulation Program. B. A 20-second Ca⁺ transient fluorescent image and statistic analysis of Ca2 + transients F/F0, n = 15 for each group. C. Patch clamp shows the AF model and control group’s action potential morphology and beating rate. D. Statistic analysis of APD₉₀. E. The figure demonstrates the number of beats recorded for hiPSC-AMs within 10 s under light microscopy, n = 11 for each group. **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Expression levels of candidate reference genes between the control group and atrial fibrillation samples. A. The range of reference values for each reference gene in 18 samples. B. The relative expression differences of each reference gene, represented as log (Foldchange,2)

Fig. 5

The stability of reference genes as demonstrated by the NormFinder algorithm and the stability verification of three genes. A. Pairwise variation (V) results indicate that normalization can be performed using only two reference genes, without the need for three or more. B. The stability of each reference gene is indicated, with greater stability indicated by closer proximity to the right-hand side of the chart. C. Demonstrated the effects of ABL1, GAPDH, and CDKN1A in the normalization of RT-PCR for KCNJ2(n = 3 for the control group, n = 8 for the HS group)