Activation of AMPK promotes cardiac differentiation by stimulating the autophagy pathway

Abstract

Autophagy, a critical catabolic process for cell survival against different types of stress, has a role in the differentiation of various cells, such as cardiomyocytes. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is an energy-sensing protein kinase involved in the regulation of autophagy. In addition to its direct role in regulating autophagy, AMPK can also influence other cellular processes by regulating mitochondrial function, posttranslational acetylation, cardiomyocyte metabolism, mitochondrial autophagy, endoplasmic reticulum stress, and apoptosis. As AMPK is involved in the control of various cellular processes, it can influence the health and survival of cardiomyocytes. This study investigated the effects of an AMPK inducer (Metformin) and an autophagy inhibitor (Hydroxychloroquine) on the differentiation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). The results showed that autophagy was upregulated during cardiac differentiation. Furthermore, AMPK activation increased the expression of CM-specific markers in hPSC-CMs. Additionally, autophagy inhibition impaired cardiomyocyte differentiation by targeting autophagosome-lysosome fusion. These results indicate the significance of autophagy in cardiomyocyte differentiation. In conclusion, AMPK might be a promising target for the regulation of cardiomyocyte generation by in vitro differentiation of pluripotent stem cells.

Keywords: AMPK; Autophagy; Cardiomyocyte differentiation; Hydroxychloroquine; Metformin.

Fig. 1

a Schematic representation of the cardiac differentiation protocol in a 2D culture. hESCs were induced to cardiac mesoderm using a cocktail of the small molecules (SMs) including CHIR (CHIR99021): Wnt inducer and to cardiac progenitor cells using SB431542: Inhibitor of transforming growth factor-β, Pur (Purmorphamine): a sonic hedgehog (SHH) agonist and IWP2: Wnt antagonist. Samples were collected at days 0, 1, 2, 4, 5 and 13 for differentiation day matching. b Immunofluorescence staining of cardiac markers during differentiated days in the presence of autophagy inducer and inhibitor. T-(red) Mesoendoderm marker on day one of differentiation, MESP1-(green) Mesoderm marker on day two of differentiation, GATA4-(red) CPC marker on day four of differentiation, cTNT-(red) cardiomyocyte-specific cytoskeletal protein on day 13 of differentiation. Nuclear staining was performed using DAPI. Scale bar, 100 μm. The data was quantified by Image J software. Data were presented as mean ± SEM, **P < 0.01, ***P < 0.001, ****P < 0.0001. c Assessment of α-MHC gene expression in heart cells in the presence of autophagy inducer and inhibitor at day 13 of differentiation. The relative expressions were compared to day 0. All experiments were performed in three biological replicates, and data were presented as mean ± SEM, ** P < 0.01. d Beating rate per minute in the HCQ-12.5 µM, Met-100 µM, and the control group. e Percentage of beating cells in the HCQ-12.5 µM, Met-100 µM, and the control group. All data were presented as Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

a Expression of specific autophagy markers during cardiac differentiation by qPCR method. All data were normalized to GAPDH gene relative to day zero. RH6-CM data are displayed as Mean ± SEM of 3 biological replications. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The autophagy markers expression between days were assessed, and each group has been compared with its own group day 2 during differentiation (d: P < 0.05, c: P < 0.01, b: P < 0.001, a: P < 0.0001) b Expression of LC3 autophagy marker in RH6-cardiomyocytes in the presence of autophagy inducer and inhibitor. Nuclear staining was performed using DAPI. Scale bar, 20 μm. Data were quantified with three biological replications in Image J software and reported as a percentage of LC3 marker expression. c Ultrastructural analysis in RH6-cardiomyocytes in the presence of inhibitor and inducer of autophagy. The accumulation of initiative autophagosomes (purple arrow) in the state of autophagy inhibition and destructive autophagosomes (orange arrow) in the induction of autophagy were observed. Scale bar, 1 μm. Scale bar, 300 nm. M: Mitochondrion, LD: Lipid droplet

Fig. 3

A summary of bioinformatics workflow from gene selection to analysis. A gene lists associated with autophagy, apoptosis, and cardiac differentiation terms were collected from GO database. The gene lists were submitted to TRUST and RegNetwork databases. Finally, the common genes between the autophagy, apoptosis and cardiac differentiation pathways were selected for the next analysis in Friedman et al. scRNA-seq dataset

Fig. 4

a UMAP plot for the days of differentiation, UMAP plot in terms of gene clusters, and Cell type assignment of clusters. b Heatmap of specific autophagy genes, cardiac differentiation, and genes involved in all three pathways of autophagy, apoptosis, and cardiac differentiation on cardiac differentiation days. c Dot plot diagram according to the days of differentiation for specific autophagy genes (yellow diagram), apoptosis genes (blue diagram), and heart differentiation days (red diagram). d Gene regulatory relationships in 48 single genes of apoptotic genes (blue), autophagic genes (yellow), specific heart differentiation genes (red), common between three pathways (purple), autophagy and apoptosis (green), autophagy and cardiac differentiation (brown), apoptosis and cardiac differentiation (gray), and not categorized (white). The size of the genes is directly related to the degree of nodes (input and output of their edges). e Among the genes shown in section d, the top 10 degree genes were examined for expression during cardiac differentiation days. f and g The expression pattern of TP53 and MYC as the two possible factors in regulating all three pathways of autophagy, apoptosis, and cardiac differentiation during specific days of cardiac differentiation by qPCR in RH6 line in the presence of inhibitor (HCQ) and inducer (Met) of autophagy. As can be seen, the expression of these two genes decreases as cardiac cell differentiation progresses. Expression of these two genes was always observed negative during differentiation after day zero (in all three groups). Expression data were normalized to the GAPDH gene relative to day zero. The relative expressions of all stages were compared to day 0. All experiments were performed in three biological replicates, and data were presented as mean ± SEM. The significance in each group was compared with the control of that day. Significance levels are *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001