Fatty Acids Enhance the Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

Abstract

Although human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a novel platform for heart regeneration, disease modeling, and drug screening, their immaturity significantly hinders their application. A hallmark of postnatal cardiomyocyte maturation is the metabolic substrate switch from glucose to fatty acids. We hypothesized that fatty acid supplementation would enhance hPSC-CM maturation. Fatty acid treatment induces cardiomyocyte hypertrophy and significantly increases cardiomyocyte force production. The improvement in force generation is accompanied by enhanced calcium transient peak height and kinetics, and by increased action potential upstroke velocity and membrane capacitance. Fatty acids also enhance mitochondrial respiratory reserve capacity. RNA sequencing showed that fatty acid treatment upregulates genes involved in fatty acid β-oxidation and downregulates genes in lipid synthesis. Signal pathway analyses reveal that fatty acid treatment results in phosphorylation and activation of multiple intracellular kinases. Thus, fatty acids increase human cardiomyocyte hypertrophy, force generation, calcium dynamics, action potential upstroke velocity, and oxidative capacity. This enhanced maturation should facilitate hPSC-CM usage for cell therapy, disease modeling, and drug/toxicity screens.

Keywords: cardiomyocyte maturation; embryonic stem cells; fatty acids; induced pluripotent stem cells; metabolism.

Figure 1

Fatty Acid Treatment Leads to hPSC-CM Morphological and Molecular Changes Representative control (A) and fatty acid-treated (B) cells were stained with α-actinin antibody (green), phalloidin (F-actin in red), and Hoechst 33342 for nuclei (blue). Scale bar, 25 μm. Compared with control hPSC-CMs, fatty acid-treated cells exhibited significant changes in cell area (C), circularity index (D), and sarcomere length (E). n > 150 from three different cardiomyocyte differentiation runs. Data are presented as mean ± SEM. ^∗p < 0.05. See also Figure S4.

Figure 2

Fatty Acid-Treated hPSC-CMs Exhibit Improved Calcium Transient Kinetics Calcium transients were evaluated by loading the hPSC-CMs with the intracellular calcium ratiometric indicator Fura-2 AM and were stimulated at 1 Hz. (A) Representative transients from control and fatty acid-treated hPSC-CMs. Note the higher amplitude, faster upstroke, and decay of the Ca²⁺ transient in the treated cells. (B–D) Calcium transient amplitude magnitudes were significantly higher in fatty acid-treated hPSC-CM (B), as indicated by increases in maximal upstroke (C) and decay (D) velocities. n = 10–16 cells from three separate cardiomyocyte differentiation runs. Data are presented as mean ± SEM. F/F₀ is the ratio of F₃₄₀/F₃₈₀. ^∗p < 0.05 versus control hPSC-CMs.

Figure 3

Fatty Acid Treatment Significantly Increases hPSC-CM Contractile Force (A–C) Representative force traces generated by control and fatty acid-treated hPSC-CMs (A). The statistical analysis results are shown in (B). Control and fatty acid-treated cardiomyocyte area on microposts is shown in (C). n = 91 for control hPSC-CMs and n = 91 for fatty acid-treated hPSC-CMs from four different cardiomyocyte differentiation runs. Data are presented as mean ± SEM. ^∗p < 0.05 versus control hPSC-CMs. See also Figure S4.

Figure 4

Fatty Acid Treatment Improves Cardiomyocyte Action Potential Maximum Upstroke Velocity and Increases Cardiomyocyte Membrane Capacitance (A) Representative action potential traces from control and fatty acid-treated cells. (B) Statistical analysis of action potential maximum upstroke velocity. (C) Analysis of cardiomyocyte membrane capacitance. Data were obtained from three different cardiomyocyte differentiation runs. n = 24 for control group and n = 25 for fatty acid-treated group. ^∗p < 0.05 versus control hPSC-CMs. Data are presented as mean ± SEM. See also Table S2.

Figure 5

The Effect of Fatty Acids on Mitochondrial Function (A) Representative traces for control and fatty acid-treated hPSC-CMs responding to the ATP synthase inhibitor oligomycin, the respiratory uncoupler FCCP, and the respiratory chain blockers rotenone and antimycin A. Note the higher maximal OCR in the fatty acid-treated cells. (B) Statistical analysis of the differences in respiratory reserve capacity. The OCR values were normalized to the number of cells present in each well, as described in Experimental Procedures. Data were from six different cardiomyocyte differentiation runs. ^∗p < 0.05 versus control hPSC-CMs. Data are presented as mean ± SEM.

Figure 6

Genome-wide Effects of Fatty Acid Treatment Assessed by RNA Sequencing (A) Volcano plot shows the differential expressed genes after fatty acid treatment. (B and C) Gene ontology (GO) enrichment analysis illustrating the upregulated and downregulated pathways after fatty acid treatment. (D) qPCR verified some of the candidate genes in RNA sequencing that involved fatty acid transportation, and also the genes involved in glucose metabolism. ^∗p < 0.05 versus control hPSC-CMs. See also Table S1.

Figure 7

Fatty Acid Treatment Activates Multiple Intracellular Signal Pathways Western blots for the phosphoprotein level in control, 15-min, and 30-min fatty acid-treated hPSC-CMs.