Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy

Abstract

Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategies.

Keywords: PCCB; iPSC; iPSC-derived cardiomyocytes; microRNAs; propionic acidemia.

Figure 1

Expression of cardiac and PCCB proteins in isogenic control and PCCB iPSC-CMs. (a) Flow cytometry analysis for cTnT cardiac marker. A representative experiment for cTnT expression is shown. (b) Immunofluorescence analysis for cTnT, GATA-4, SMA, and α-ACT in iPSC-derived cardiomyocytes; scale bar: 100 µm. (c) Representative blot of the analysis of PCCB protein in the cardiomyocytes generated from both iPSC lines. GADPH was used as the loading control. (d) Acylcarnitine analysis by tandem mass spectrometry. (1) free carnitine; (2) deuterated free carnitine (internal standard); (3) propionylcarnitine; (4) deuterated propionylcarnitine (internal standard); (*) culture medium, interfering compound.

Figure 2

Analysis of cardiac markers and miRNAs expression in isogenic control and PCCB iPSC-CMs. (a) Relative mRNA expression of MYH6, MYH7, ACTN2, SERCA2, RyR2, CACNA1C and KCNQ1 genes by qRT-PCR. (b) Relative expression levels of miR-1a, miR-30c, miR-133a, miR-199a, miR-199b, miR-208a, and miR-378 were evaluated by qRT-PCR in iPSC-derived cardiomyocytes. In (a,b), data represent mean ± standard deviation of three independent cardiomyocyte differentiations, at least each for triplicate. Statistical significance was determined by Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Analysis of oxidative stress and mitochondrial respiration in isogenic control and PCCB iPSC-CMs. (a) Detection of intracellular ROS level by flow cytometry using H₂DCFDA as a fluorescence probe under basal conditions. Data represent mean values ± SD from at least three independent experiments. Statistical significance was determined by Student’s t-test. * p < 0.05. A representative experiment of ROS level in isogenic control iPSC-CMs (control in blue) and PCCB iPSC-CMs (PCCB in red) is shown. (b) Expression analysis of catalase protein by Western Blot. A representative blot is shown, and GADPH was used as a loading control. At least three experiments were performed. The corresponding quantification of proteins by laser densitometry is not shown because of the total absence of catalase enzyme in isogenic control iPSC-CMs. (c) Bioenergetic profile of WT and PCCB iPSC-CMs. Representative profile of basal OCR in isogenic control and PCCB iPSC-CMs after the addition of oligomycin, FCCP, rotenone, and antimycin A. The results shown are mean ± standard deviation of 3 to 5 experiments.

Figure 4

Analysis of cell ultrastructure and autophagy process in the iPSC-CMs. Representative images of EM were shown of isogenic control (a) and PCCB (b) iPSC-CMs at 3000x magnification. Black arrows show degradation vesicles (a). LD: lipid droplets (b). N: cell nucleus (a,b). Scale bar: 1 µm. (c) Representative blot of the protein analysis of LAMP1, ATG5-ATG12 conjugate, ATG5, p62, S6, and P-S6 (its phosphorylated form). GADPH was used as the loading control. (d) The corresponding quantification by laser densitometry is shown as the mean ± standard deviation of at least three experiments. In S6 and its phosphorylated form analysis, two experiments were performed (mean 0.9 and 2.06, respectively). Statistical significance was determined by Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Expression analysis of proteins involved in MAMs, ER stress, apoptosis, and mitochondrial biogenesis in the iPSC-CMs. (a) Representative blots of the analysis of MFN2, SIG-1R, GRP75, HERP, GRP78, BCL2, and CASPASE 3 protein levels. GADPH was used as the loading control. (b) The corresponding quantification by laser densitometry is shown as the mean ± standard deviation of at least three experiments. Statistical significance was determined by Student’s t-test. (c) Relative mRNA expression of PPARGC1A, PPARD, and PPARG genes by qRT-PCR. Data represent the mean ± standard deviation of at least three experiments. Statistical significance was determined by Student’s t-test. * p < 0.05; ** p < 0.01.