Targeting HIF-1α in combination with PPARα activation and postnatal factors promotes the metabolic maturation of human induced pluripotent stem cell-derived cardiomyocytes

Abstract

Immature phenotypes of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) limit the utility of these cells in clinical application and basic research. During cardiac development, postnatal cardiomyocytes experience high oxygen tension along with a concomitant downregulation of hypoxia-inducible factor 1α (HIF-1α), leading to increased fatty acid oxidation (FAO). We hypothesized that targeting HIF-1α alone or in combination with other metabolic regulators could promote the metabolic maturation of hiPSC-CMs. We examined the effect of HIF-1α inhibition on the maturation of hiPSC-CMs and investigated a multipronged approach to promote hiPSC-CM maturation by combining HIF-1α inhibition with molecules that target key pathways involved in the energy metabolism. Cardiac spheres of highly-enriched hiPSC-CMs were treated with a HIF-1α inhibitor alone or in combination with an agonist of peroxisome proliferator activated receptor α (PPARα) and three postnatal factors (triiodothyronine hormone T3, insulin-like growth factor-1 and dexamethasone). HIF-1α inhibition significantly increased FAO and basal and maximal respiration of hiPSC-CMs. Combining HIF-1α inhibition with PPARα activation and the postnatal factors further increased FAO and improved mitochondrial maturation in hiPSC-CMs. Compared with mock-treated cultures, the cultures treated with the five factors had increased mitochondrial content and contained more cells with mitochondrial distribution throughout the cells, which are features of more mature cardiomyocytes. Consistent with these observations, a number of transcriptional regulators of mitochondrial metabolic processes were upregulated in hiPSC-CMs treated with the five factors. Furthermore, these cells had significantly increased Ca²⁺ transient kinetics and contraction and relaxation velocities, which are functional features for more mature cardiomyocytes. Therefore, targeting HIF-1α in combination with other metabolic regulators significantly improves the metabolic maturation of hiPSC-CMs.

Keywords: Calcium transients; Cardiomyocyte; Fatty acid; Metabolism; Stem cell.

Figure 1.

Microscale tissue engineering generates enriched hiPSC-CMs and maturation treatments do not alter hiPSC-CM purity. (A) Schematic diagram of experimental design and cell morphology of hiPSCs and cardiac spheres (CSs). hiPSCs were treated with activin A (100 ng/mL) at day 0 and BMP4 (10 ng/mL) at day 1. Cells at differentiation day 4 were aggregated into cardiac spheres and the cardiac spheres at day 21 were treated with maturation factors or DMSO control for 1 week. (B) Immunostaining of differentiation cultures for cardiomyocyte markers. At day 28, cells were dissociated, replated and stained for NKX2-5 (red), α-actinin (green), cardiac troponin I (red) and cardiac troponin T (green). Cell nuclei were counterstained with Hoechst 33342 (blue). FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone. (C) Quantitative analysis of NKX2-5 by high-content imaging using ArrayScan. Data are presented as mean ± SEM (n=5).

Figure 2.

Treatments of 3D cardiac spheres with a HIF-1α inhibitor, a PPARα activator and the postnatal factors (T3, IGF-1 and dexamethasone) improve β-oxidation and other substrate oxidation. (A) Representative traces of real-time measurement of mitochondrial oxygen consumption rate (OCR) following addition of etomoxir (ETO, 100 μM). Quantification of amount of OCR derived from (B) fatty acid β-oxidation and (C) oxidation of non-fatty acid substrates in hiPSC-CMs treated with maturation factors or DMSO control. Data were normalized to cell counts and presented as mean ± SEM (n=8). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; by one-way ANOVA. FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone.

Figure 3.

Treatments of 3D cardiac spheres with a HIF-1α inhibitor, a PPARα activator and the postnatal factors (T3, IGF-1 and dexamethasone) improve mitochondrial function in hiPSC-CMs. (A) Representative traces showing the OCR of hiPSC-CMs following sequential addition of oligomycin (Oligo, 2 μM), FCCP (1 μM), and rotenone/antimycin A (Rot/Ant, 0.5 μM). (B) Quantification of basal respiration, maximal respiration, ATP production, proton leak, non-mitochondrial respiration and reserve capacity. All measurements were normalized to cell counts and presented as mean ± SEM (n=8). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; by one-way ANOVA. FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone.

Figure 4.

Treatment of 3D cardiac spheres with a HIF-1α inhibitor, a PPARα activator and the postnatal factors (T3, IGF-1 and dexamethasone) alters distribution and content of mitochondria in hiPSC-CMs. (A) Representative images showing the locations of mitochondria in hiPSC-CMs using MitoTracker dye (red), myofibrils with α-actinin (green), and nuclei with Hoechst (blue). (B)The overall mitochondrial distribution among FM+WY+TID-treated and control cells was categorized into 3 different levels: Score 1 cells with mitochondria clustered near the nucleus; Score 2 cells with mitochondria near the nucleus and at a low level in other areas; and Score 3 cells with mitochondria distributed throughout the cytoplasm. n=240 cells/group. (C) Representative dot plots of flow cytometric analysis of mitochondrial content of hiPSC-CMs using MitoTracker Red. (D) Representative histograms of MitoTracker Red fluorescence intensity and the relative fluorescence intensity of treated and control hiPSC-CMs. Data are represented as mean ± SEM (n=3). (E) Quantification of the ratio of mitochondria-encoded complex I ND1 or mt-CO₂ to nuclear-encoded complex II LPL or SHDA DNA in treated compared to control cells. Data are presented as mean ± SEM (n=3). *P<0.05, ***P<0.001; by Student’s t test. FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone.

Figure 5.

Treatments of 3D cardiac spheres with a HIF-1α inhibitor, a PPARα activator and the postnatal factors (T3, IGF-1 and dexamethasone) improve calcium transient kinetics and contractility. Calcium transients were assessed by loading hiPSC-CMs with the intracellular calcium dye fluo-4 AM. (A) Representative transient traces from control and FM+WY+TID-treated hiPSC-CMs. (B) The calcium transient amplitude magnitudes, (C) maximal upstroke velocity, (D) time to 50% peak, (E) maximal decay velocity, (F) time to 50% decay, and (G) beating frequency were recorded at the stimulation frequency of 1 Hz. Results were mean ± SEM (n=30). *P<0.05, **P<0.01; by Student’s t test. Representative image and heat map (top) depicting time-averaged magnitude of all motion and tracing (bottom) of average beating speed vs. time of hiPSC-CMs treated with (G) DMSO or (H) FM+WY+TID. Contractions are denoted in blue by the first peak and relaxations are denoted in red by the second peak of the duplex. (I) Contraction velocity and (J) relaxation velocity of control and FM+WY+TID-treated hiPSC-CMs. Data are represented as mean ± SEM (n=14). *P<0.05, **P<0.01; by Student’s t test. FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone.

Figure 6.

Treatments of 3D cardiac spheres with a HIF-1α inhibitor, a PPARα activator and the postnatal factors (T3, IGF-1 and dexamethasone) alter gene expression. Volcano plots portray log₂ fold change (FC) vs. −log₁₀(P value) for differentially expressed genes in hiPSC-CMs treated with (A) FM+WY+TID and (B) tissues from human left ventricle (LV) compared with DMSO-treated hiPSC-CMs. The red dots denote significantly regulated genes (P<0.02); N indicates the number of differentially expressed genes. (C) Venn diagram showing the number of significantly up-regulated genes in FM+WY+TID-treated hiPSC-CMs vs. DMSO-treated hiPSC-CMs and LV vs. DMSO-treated hiPSC-CMs (P<0.002). (D) Bubble plot showing the enrichment analysis of differentially expressed genes in hiPSC-CMs treated with FM+WY+TID and LV compared with DMSO-treated hiPSC-CMs. Colors of displayed circles indicate the levels of significant enrichment of the gene ontology terms (GO terms) according to negative log₁₀(P value) (NLP). The area of displayed circles corresponds to Gene_ratio which is the ratio of genes enriched within each GO term. (E) Genes within the identified GO terms that are commonly upregulated in hiPSC-CMs treated with FM+WY+TID and LV compared with DMSO-treated hiPSC-CMs. FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone.

Figure 7.

Fold changes of significantly upregulated targets that are common in both FM+WY+TID-treated hiPSC-CMs vs. DMSO-treated hiPSC-CMs and LV vs. DMSO-treated hiPSC-CMs. Heat map showing upregulated genes that are categorized according to biological processes; (A) fatty acid oxidation, (B) oxidation-reduction, and (C) mitochondrial transport. Data are presented as Log₂ fold change expression relative to mean for all conditions (n=3). (D) Quantitative measurement of gene expression in FM+WY+TID-treated hiPSC-CMs relative to those in DMSO-treated hiPSC-CMs was validated with qRT-PCR. Data are presented as mean ± SEM (n=3). FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone.

Figure 8.

Downregulated genes/biological processes. (A) Venn diagram of the numbers of genes downregulated genes in FM+WY+TID-treated hiPSC-CMs and LV as detected by RNA-Seq analysis. (B) Top 10 significant biological processes of the commonly downregulated genes with corresponding genes in both FM+WY+TID-treated hiPSC-CMs and LV compared with DMSO-treated hiPSC-CMs. FM, FM19G11; WY, WY-14643; TID, T3 + IGF-1 + dexamethasone.