AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling

Li, Dong; Armand, Lawrence C.; Sun, Fangxu; Hwang, Hyun; Wolfson, David; Rampoldi, Antonio; Liu, Rui; Forghani, Parvin; Hu, **n; Yu, Wen-Mei; Qu, Cheng-Kui; Jones, Dean P.; Wu, Ronghu; Cho, Hee Cheol; Maxwell, Joshua T.; Xu, Chunhui

doi:10.1186/s13287-023-03554-7

AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling

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Published: 08 November 2023

Volume 14, article number 322, (2023)
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AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling

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Dong Li¹,
Lawrence C. Armand¹,
Fangxu Sun²,
Hyun Hwang¹,
David Wolfson^1,3,
Antonio Rampoldi¹,
Rui Liu¹,
Parvin Forghani¹,
**n Hu⁴,
Wen-Mei Yu¹,
Cheng-Kui Qu¹,
Dean P. Jones⁴,
Ronghu Wu²,
Hee Cheol Cho^1,3,
Joshua T. Maxwell¹ &
…
Chunhui Xu ORCID: orcid.org/0000-0003-1748-0648^1,3

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Abstract

Background

Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation.

Methods

hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli.

Results

Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features—including enhanced Ca²⁺ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells.

Conclusion

AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

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Background

Heart disease is the number one cause of death and has become a significant issue to public health. Due to differences in physiology among species, animal models have limitations in modeling human pathological conditions—thus encouraging the exploration of more human relevant resources. Following the breakthrough in the development of human-induced pluripotent stem cells (hiPSCs), hiPSC-derived cardiomyocytes (hiPSC-CMs) have emerged as an unprecedented opportunity to overcome the limitations of current models in modeling human diseases and drug discovery. However, compared with primary human cardiomyocytes, hiPSC-CMs resemble fetal cardiomyocytes more closely in structure and function, imposing limitations in the use of hiPSC-CMs [1, 2]. Therefore, further enhancement of hiPSC-CMs maturity in a scalable manner has become paramount to improve applications of these cells. We and others have demonstrated that combining HIF-1α inhibition with molecules that target key pathways involved in energy metabolism could significantly promote hiPSC-CMs to be both structurally and functionally more mature [3, Full size image

GO term analysis was performed using the differentially expressed proteins. Notably, in E10-treated hiPSC-CMs, top upregulated GO terms of biological process (BP) were associated with mitochondrial function and fatty acid metabolism (Fig. 6B). Based on cellular component (CC) and molecular function (MF), top GO terms were also related to mitochondrial metabolism (Fig. 6C). KEGG pathway analysis of the upregulated proteins in the E10-treated cells indicated upregulation of pathways associated with oxidative phosphorylation, metabolic pathways and citrate cycle (Fig. 6C). In addition, fatty acid degradation, cardiac contraction and PPAR signaling were also present in the upregulated KEGG pathways.

The String protein–protein interaction analysis identified a large number of differentially expressed proteins that are involved in the oxidative phosphorylation pathway. The high connectivity among these proteins further indicated their important roles in oxidative phosphorylation (Fig. 6D).

Detailed examination of the upregulated differentially expressed proteins identified the key proteins associated with mitochondrial biogenesis, TCA cycle and oxidative phosphorylation (Fig. 6E). These upregulated proteins included eight enzymes that catalyze the TCA cycle—CS, ACO2, IDH3A, IDH3B (isocitrate dehydrogenase (NAD(+)) 3 non-catalytic subunit beta), IDH3G (isocitrate dehydrogenase (NAD(+)) 3 non-catalytic subunit gamma), OGDHL (oxoglutarate dehydrogenase L), OGDH, DLST, DLD, SUCLG1 (succinate-CoA ligase GDP/ADP-forming subunit alpha), SUCLA2 (succinate-CoA ligase ADP-forming subunit beta), SDHA, SDHB, FH and MDH2 (malate dehydrogenase 2). In agreement with RNA-seq, E10 treatment upregulated SLC2A4, MT-CO2, CKMT2, PDHA1, PDHX, DLAT, DLD and another PDH E1 subunit PDHB.

In agreement with the observation in RNA-seq data, many important proteins in mitochondrial electron transport chains were upregulated in E10-treated cells. Specifically, we observed upregulated proteins in complex I—NADH dehydrogenase MT-ND2 and MT-ND5—and several NADH:ubiquinone oxidoreductase subunits—NDUFAF3, NDUFA12, NDUFA4, NDUFS2, NDUFA9, NDUFA5, NDUFAB1, NDUFA1, NDUFA3, NDUFS5, NDUFA8, NDUFC2, NDUFAF4, NDUFS8, NDUFB9, NDUFB1, NDUFB8, NDUFB11, NDUFS4, NDUFB3, NDUFB5, NDUFA2, NDUFA7, NDUFA11, NDUFA10 and NDUFS7. Upregulation of SDHA and SDHB in complex II was detected with upregulated SDHB being detected by RNA-seq as well. Additionally, UQCR subunits—UQCR10, UQCRB, UQCRC1 and UQCRH—in complex III and terminal components of the respiratory chain—COA6, COX6B1, COX6C, COX7A2 and COX7B—were upregulated. In line with our RNA-seq data, several subunits of ATP synthase F1 and F0 complex were upregulated, including ATP5A1, ATP5B, ATP5C1, ATP5O (ATP5PO, ATP synthase F1 subunit OSCP), ATP5H (ATP5PD, mitochondrial F0 complex, subunit D), ATP5I (ATP5ME, mitochondrial F0 complex, subunit E), ATP5L (ATP5MG, mitochondrial F0 complex, subunit G) and ATP5J (ATP5PF, mitochondrial F0 complex, subunit F). Also, E10-treated cells had increased expression of several solute carriers (SLC) family 25 members, including SLC25A24, SLC25A11, SLC25A5, SLC25A13 and SLC25A4. These results indicated an increased capacity of electron translocation for respiration and ATP synthesis in E10-treated cells.

Upregulation of many important proteins involved in fatty acid metabolism was also indicated. These included fatty acid-binding proteins FABP3 and FABP7, which are the intracellular fatty acid-binding proteins that regulate the uptake and transport of long-chain fatty acids into cells. The expression of SLC27A4 also increased, suggesting enhanced capacity of fatty acid uptake in E10-treated cells upon entry in cells and within cells. E10-treated cells had increased expression of acyl-CoA synthases (ACSs, which catalyze the process of fatty acid activation into fatty acyl-CoAs), including long-chain family members ACSL1, ACSL3 and short-chain family members ACSS2, ACSS3. Intriguingly, we found the upregulation of CPT1B (carnitine palmitoyltransferase 1B), carnitine acyl-carnitine translocase SLC25A20 and CPT2 (carnitine palmitoyltransferase 2). All of which are essential proteins in transporting long-chain fatty acyl-CoAs from the cytoplasm into mitochondria. Notably, E10-treated cells had upregulated MYLCD (malonyl-CoA decarboxylase, which catalyzes the breakdown of malonyl-CoA that inhibits CPT1, thus increasing the rate of CPT1-mediated fatty acid transport). E10 treatment also resulted in the upregulation of several enzymes which are essential to FAO process, including ACOX1 (acyl-CoA oxidase 1), ACADM, HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha), HADHB and HADH (hydroxyacyl-CoA dehydrogenase). In addition, CROT (carnitine O-octanoyltransferase) and CRAT (carnitine O-acetyltransferase), BDH1 (3-hydroxybutyrate dehydrogenase 1) and ACAA1 (acetyl-CoA acyltransferase 1) involved in peroxisomal fatty acid oxidation as well as the key enzymes EC1 and EC2 in β-oxidation of unsaturated fatty acids were also upregulated. Collectively, the upregulation of the above essential proteins in E10-treated cells demonstrated increased consumption of fatty acid for the cell energy demand. Furthermore, comparison of the RNA-seq with proteomic data revealed a subset of overlap** genes and proteins that were differentially expressed in E10-treated cells. Specifically, 90 of them were upregulated and 25 were downregulated, respectively (Fig. 6G).

Together, these results revealed that E10 upregulated key regulators of mitochondrial function and FAO, which was consistent with improved metabolic maturation in E10-treated 3D hiPSC-CMs.

Metabolomics identified enhanced energy-related metabolic pathways in E10-treated hiPSC-CMs

To gain additional understanding of the metabolic differences associated with AMPK activation, we performed untargeted metabolomic analysis using HILIC (ultra-high-resolution mass spectrometry with hydrophilic interaction liquid chromatography) to compare the metabolic profile of E10-treated hiPSC-CMs with their DMSO-treated counterparts. Of the 9903 features analyzed, 917 features significantly changed in E10-treated hiPSC-CMs from DMSO controls (P < 0.05). Principal component analysis (PCA) of the metabolic profiles between E10-treated hiPSC-CMs and DMSO controls showed a clear separation of metabolome between the two cultures (Fig. 7A). Unsupervised two-way hierarchical analysis of the significant features confirmed the distinction of metabolic profiles between the E10-treated hiPSC-CMs and DMSO controls (Fig. 7C, Metabolic feature table in Additional file 3).

To identify the significantly affected pathways, we performed pathway enrichment analysis on the 917 metabolic features that were different between the groups and identified 12 pathways that were enriched in E10-treated hiPSC-CMs. Top pathways included multiple mitochondria and energy metabolism-related activities, including lysine metabolism, carnitine shuttle, vitamin B12 (cyanocobalamin) metabolism, glycerophospholipid metabolism, ubiquinone biosynthesis, arginine and proline metabolism, pentose phosphate pathway, TCA cycle, phosphatidylinositol phosphate metabolism, urea cycle/amino group metabolism, androgen and estrogen biosynthesis and metabolism, pentose and glucuronate interconversions (Fig. 7D).

In E10-treated cells, two intermediates of the glycolysis pathway, D-glucose-6-phosphate and fructose-1,6-bisphosphate, were significantly decreased, while the level of D-glucose and pyruvate was not significantly different compared with DMSO-treated cells (Fig. 7E). In addition, the level of lipoamide was significantly increased in E10-treated cells (Fig. 7F). Lipoamide acts as an essential cofactor for mitochondrial 2-ketoacid dehydrogenases that are key enzymes in TCA cycle, which include PDH, OGDH and branched-chain ketoacid dehydrogenase (BCKDH) [20,21,22]. Combined results in RNA-seq and Proteomics on upregulated PDH, OGDH and BCKDH, the increased level of lipoamide further suggested an enhanced TCA cycle in E10-treated hiPSC-CMs based on our findings in GO and KEGG pathway analysis.

Among the upregulated pathways, carnitine shuttle system is responsible for the transport of fatty acids into mitochondria for subsequent β-oxidation. Upon cellular entry, fatty acids are activated into fatty acyl-CoAs by ACSs followed by conjugation to carnitine forming fatty acyl-carnitines catalyzed by CPT1B and located in the outer mitochondrial membrane. Fatty acyl-carnitines then diffuse across the mitochondrial membrane by action of carnitine acyl-carnitine translocase (SLC25A20) located on inner mitochondrial membrane. Once within mitochondria, carnitine is released from fatty acyl-carnitines by CPT2 and transferred back to cytoplasm, while fatty acyl-CoAs enter β-oxidation. Interestingly, E10 treatment significantly reduced the level of fatty acyl-carnitines in E10-treated cells compared to DMSO control (Fig. 7G and H). Specifically, we found marked, lower levels of short-chain fatty acyl-carnitine propionyl-carnitine, two medium-chain fatty acyl-carnitines (octadecenoyl-carnitine and hexadecenoyl-carnitine) and five long-chain fatty acyl-carnitines (L-palmitoyl-carnitine, linoelaidyl-carnitine, stearoyl-carnitine, arachidyl-carnitine and myristoyl-carnitine). Together, they imply elevated fatty acid β-oxidation in E10-treated cells.

Overall, these findings suggested AMPK activation-induced metabolic changes and promoted metabolic flux in energy-related metabolisms in hiPSC-CMs. These results demonstrate consistency with our findings in RNA-seq and proteomics analyses and reveal the upregulated expression profile of key regulators in carnitine shuttle system which remarkably facilitated fatty acids transportation and β-oxidation.

AMPK activator-treated hiPSC-CMs enabled pathological modeling

Based on aforementioned assessments of multiple features associated with metabolic maturation and extensive omic analyses, we concluded that E10 treatment could drive significantly increased FAO and metabolic maturation in 3D hiPSC-CMs. In addition, the expression of ADRB1 (adrenergic receptor B1) was significantly upregulated in AMPK-activated hiPSC-CMs (Fig. 8A). Given that the metabolic switch from FAO to glycolysis is associated with heart failure [23], we examined if E10-treated 3D hiPSC-CMs could model pathological response and recapitulate some of the changes related to heart failure including glycolysis enhancement, lipid accumulation and apoptosis. Three-dimensional hiPSC-CMs were first treated with E10 for 7 days to promote cardiomyocyte maturation. The cells were then treated with isoproterenol (100 µM), a small molecule adrenergic agonist to simulate pathological conditions for 6 days in hypoxic environment and low-glucose medium. The treatment with pathological stimuli increased glycolysis, glycolytic capacity and glycolytic reserve compared with no pathological stimuli in E10-treated cells, as detected by the extracellular acidification rate (ECAR; index of glycolytic activity) using the Seahorse assay (Fig. 8B). In contrast, the treatment of pathological stimuli did not alter glycolysis, glycolytic capacity and glycolytic reserve in immature cells (without E10 treatment) (Fig. 8B). Consistently, E10-treated cells but not immature cells had significant upregulation of the glycolysis-related genes including SLC2A1, GAPDH, LDHA and PKM2 under the treatment with pathological stimuli compared with cells without the treatment with pathological stimuli (Fig. 8C). These results indicate that E10-treated hiPSC-CMs had increased glycolysis when challenged with pathological stimuli, a pathological response associated with heart failure [7].

The pathological stimuli also induced the upregulation of triacylglycerol synthesis-associated gene CD36 (cluster of differentiation 36) and PLIN2 (Perilipin 2; a marker of lipid accumulation) in E10-treated 3D hiPSC-CMs but not in immature cells (Fig. 8D). In addition, the expression of HSL (LIPE; Lipase E, hormone-sensitive type) that hydrolyzes stored triglycerides to free fatty acids was downregulated in E10-treated hiPSC-CMs when challenged with pathological stimuli (Fig. 8D). Consistently, an increased accumulation of lipid droplets was observed in E10-treated hiPSC-CMs when challenged with pathological stimuli as detected by ArrayScan analysis of Nile red staining (Fig. 8E). Increased accumulation of lipid droplets is also a pathological response associated with heart failure [24].

In addition to the change in glycolysis and lipid accumulation, the pathological stimuli resulted in significantly increased cell death (Fig. 8F–I). Following the treatment of pathological stimuli, E10-treated cells had twofold decreased ATP content while immature hiPSC-CMs had 29% decreased ATP content compared with parallel cultures without the treatment of pathological stimuli. Similarly, the treatment of pathological stimuli resulted in higher magnitude of decrease in cell viability and cell counts in E10-treated cells compared with immature cells. In addition, increased caspase 3/7 activity was observed in E10-treated cells when challenged with the pathological stimuli.

These results suggest that E10-treated hiPSC-CMs responded to pathological stimuli, which was not achievable in immature hiPSC-CMs. Thus, E10-treated hiPSC-CMs have advantages for pathological modeling.

Discussion

In this study, we demonstrated pharmacological agent-induced AMPK activation could drive hiPSC-CMs to a more mature stage with improved metabolic, structural and functional properties. The maturation treatment was conducted in size-controlled cardiac spheres which are scalable and suitable for high-throughput assays. Three-dimensional hiPSC-CMs treated with AMPK activators had increased ATP content, mitochondria content, mitochondrial membrane potential and mitochondrial function, as well as increased fatty acid uptake and FAO, suggesting that these cells were metabolically more mature. In contrast, inhibition of AMPK by pharmacological treatment and genetic modification decreased mitochondrial function and abolished the AMPK activator improved mitochondrial function in hiPSC-CMs. In addition, we observed an improved structural development after AMPK activator treatment including the increased sarcomere length, cell area, perimeter and length to width ratio. AMPK activator-treated hiPSC-CMs also had enhanced calcium transient kinetics and higher velocities of maximum contraction and relaxation, which are functional features of more mature cardiomyocytes. RNA-seq, proteomic and metabolomic analyses revealed a distinct molecular signature associated with more mature cardiomyocytes in AMPK activator-treated 3D hiPSC-CMs. Furthermore, the resulting more mature hiPSC-CMs enable pathological modeling that was not achievable in immature hiPSC-CMs. Together, this study provides valuable insights into the strategies that help to achieve hiPSC-CM maturation through metabolic regulation.

During our study, other groups reported that the treatment of differentiated cells in 2D cultures with AMPK activator AICAR increased metabolic maturation of hiPSC-CMs [25, 26]. Compared with the previous publications, our study highlights the following new findings. (1) We found that AMPK activation enabled pathological modeling. (2) We identified an AMPK activator that is more potent than the one used in the previous publications. (3) We found that AMPK activation not only promoted metabolic maturation but also functional maturation. (4) We characterized the molecular action of AMPK activation through comprehensive transcriptomics, proteomics and metabolomics analyses. (5) We used human heart tissue as a control for molecular characterization. (6) We confirmed the role of AMPK by inhibition of AMPK through both pharmacological and genetic methods. (7) Three-dimensional cultures of cardiac spheres generated highly enriched cardiomyocytes which allow more rigorous investigation of the effect of AMPK activation on metabolic maturation.

Our cardiac differentiation approach and 3D culture produced hiPSC-CMs at high purity, serving as excellent starting materials for examining the effect of AMPK activators on cardiomyocyte maturation. We believe that this is a critical and rigorous step before evaluating culture conditions for promoting cardiomyocyte maturation, since if the assessment of mitochondrial function is done in bulk cultures, it is possible that changes in mitochondrial function simply reflect changes in cardiomyocyte purity in cultures (cardiomyocytes have higher mitochondrial content than non-cardiac cells). The cardiac spheres used in our study were generated by microscale tissue engineering [27]. The advantage of this technology is that it allows for the generation of 3D multicellular aggregates with controlled sphere sizes. An optimal sphere size is important to overcome the limitation on molecule diffusion into 3D cultures to allow cells to receive molecules in the medium effectively. Typically, cardiac spheres generated by microscale tissue engineering do not contain necrotic centers suggesting sufficient diffusion of nutrient molecules to the center of spheres [12].

Our approach for screening AMPK activators for promoting metabolic maturation is effective by using assays of mitochondrial features including ATP content, mitochondrial membrane potential and mtDNA:nDNA ratio. These three high-throughput assays complemented each other and ensured the selection of optimal drugs and doses for our study. Among 4 AMPK activators, we selected A-769662 and EX229 with each activator at two doses for further evaluation in 3D hiPSC-CMs and found consistent outcomes compared with our initial screening. We also found the outcomes of ATP content, mitochondrial membrane potential and mtDNA:nDNA ratio correlated well with mitochondrial functional measurement by Seahorse Mito Stress test.

Both A-769662 and EX229 have been reported as a potent and specific pharmacological activator of AMPK. A-769662 can allosterically activate AMPK containing the β1 isoform and inhibit dephosphorylation of Thr172 on α subunits [28, 29]. A-769662 binds to the cleft between the catalytic α subunits and the regulatory β subunits and its function highly depends on the autophosphorylation site Ser108 within the β subunit carbohydrate-binding module (CBM). Mutation of the Ser108 on β1 subunit can almost completely abolish the activation of AMPK [30,31,32,33]. EX229 is a cyclic benzimidazole derivative that binds to the same site of AMPK as A-769662 but is five to tenfold more potent than A-769662 in cell-free assays [34]. Consistently, we also found that EX229 was more potent in promoting hiPSC-CM maturation than A-769662.

Treatment of 3D hiPSC-CMs with EX229 also increased fatty acid uptake and FAO rates, which are key features of metabolic maturation of cardiomyocytes since immature cardiomyocytes rely on glycolysis as the primary energy source. As cardiomyocytes mature, mitochondrial content significantly increases to meet the high energetic demands, and consequently, FAO becomes as the dominant energy provider due to the increased mitochondrial function and oxidative capacity. The increased FAO in hiPSC-CMs is consistent with our findings on mitochondrial maturation induced by the treatment with EX229 based on comprehensive and complementary measurements. High-content imaging by ArrayScan for TMRM and TOM20 together with qPCR analysis of the mtDNA/nDNA in AMPK activator-treated hiPSC-CMs demonstrated that AMPK activation significantly increased mitochondrial membrane potential and content of hiPSC-CMs, which was further confirmed by flow cytometry analysis of MitoTracker Red and TOM20. Furthermore, the elevated mitochondrial function was confirmed using the Seahorse Mito Stress assay, which displayed higher level of basal and maximal oxygen consumption, and ATP production. Such increased mitochondrial function is likely to satisfy the increased energy demands indicating a higher respiratory capacity and oxidative potential occurred in AMPK activator-treated hiPSC-CMs; consistently, higher level of ATP content was detected in these cells. In addition, transcriptomic and proteomic profiling also identified a large number of differentially expressed genes and proteins linked to metabolic maturity of cardiomyocytes including upregulation of proteins associated with mitochondrial biogenesis and oxidative phosphorylation, indicating the robustness of metabolic modulation via AMPK activation. Since mitochondrial dynamics can regulate mitochondrial function, it will be of interest to investigate the effect of AMPK activators on mitochondrial morphology using transmission electron microscope.

The role of AMPK activation in mitochondrial maturation of hiPSC-CMs was also confirmed through the treatment with a pharmacological inhibitor of AMPK, Compound C, or with siRNA that targets AMPK. Both Compound C or siRNA knockdown of AMPK decreased key mitochondrial features including ATP content, mitochondrial membrane potential and mtDNA:nDNA ratio, as well as mitochondrial function detected by the Seahorse Mito Stress test. In addition, both Compound C and siRNA knockdown AMPK abolished the positive effect of AMPK activators.

We also found that metabolic maturation induced by AMPK activators was correlated with higher levels of structural and functional maturation. A hallmark of cardiomyocyte functional maturation is the development of Ca²⁺ handling and contractility. Immature cardiomyocytes differ in many ways in Ca²⁺ handling compared with mature cardiomyocytes, such as lower calcium transient amplitudes and slower rise/decay kinetics [3]. AMPK activator-treated hiPSC-CMs exhibited significantly improved calcium handling properties including significantly faster rate of rise and decay of Ca²⁺ transients. RNA-seq also revealed that AMPK activator-treated hiPSC-CMs had increased expression of several genes that are important in regulating Ca²⁺ handling and electrophysiology, such as PLN, KCNJ2, ATP2A2, SCN1B and CACNG4. KCNJ2 is the major inward-rectifier type potassium channel that establishes and maintains resting membrane potential and cell excitability. PLN plays a crucial role in regulating Ca²⁺ cycling in cardiomyocytes [35]. Many DEGs and GO terms identified in AMPK activator-treated hiPSC-CMs were also identified in pediatric heart tissues when these samples were compared with DMSO-treated hiPSC-CMs. However, we also note that a large number of genes were differentially expressed when AMPK activator-treated hiPSC-CMs were compared with pediatric heart tissues, suggesting that the maturation level of AMPK activator-treated hiPSC-CMs differed from that of mature cardiomyocytes in pediatric heart. To reach higher levels of maturation, further study is needed to examine the effect of combining AMPK activation with other methods such as electromechanical stimulation [36] and co-culture with non-cardiac cells [37] on hiPSC-CM maturation. In addition, DEGs identified in E10-treated cells are likely related to the effects of E10 treatment on cellular processes such as metabolism, signaling pathways or cell differentiation. Further analysis and experimentation would be helpful to understand molecular mechanisms underlying E10-promoted cardiomyocyte maturation.

The resulting more mature hiPSC-CMs could serve as a valuable model for understanding the underlying mechanisms of cardiac pathological conditions in humans. hiPSC-CMs have drawn extensive attention in in vitro disease modeling due to their remarkable advantages over animal models in overcoming physiological difference among species, recapitulating complexities of human cardiomyocytes. More mature hiPSC-CMs with improved metabolic maturation are particularly helpful for the study of cardiac metabolic remodeling during disease. Under normal conditions, FAO by mitochondria provides a main energy source in mature cardiomyocytes. Under pathological conditions, cardiomyocytes undergo metabolic remodeling by increasing glycolysis. Such disease phenotype is difficult to model using immature hiPSC-CMs since these cells have limited capacity of FAO, and instead, they use glycolysis as a main energy source. Indeed, we found that AMPK activator-treated hiPSC-CMs were able to respond to pathological stimuli and to produce disease phenotypes including glycolysis enhancement, lipid accumulation and cell death, which were not achievable in untreated immature hiPSC-CMs. In our pathological modeling experiments, we used isoproterenol to induce disease phenotypes in hiPSC-CMs. Although isoproterenol can induce many aspects of the pathophysiology of heart failure and is commonly used to study cardiac defects in animal models, this model has limitations to fully recapitulate clinical outcomes. In addition, future study can help establish if the maturation treatment also improves the use of hiPSC-CMs in modeling other cardiac diseases such as hypertrophic cardiomyopathy and dilated cardiomyopathy [38]. Nevertheless, our results suggest that higher levels of metabolic maturity rendered hiPSC-CMs a unique capacity of modeling the key events in cardiomyocytes during heart failure, highlighting the importance of cardiomyocyte maturation.

Conclusions

In summary, our study demonstrated that hiPSC-CMs could be driven to a more mature stage by activating AMPK using a pharmacological agent-induced strategy. The AMPK activator-treated hiPSC-CMs allowed us to conduct pathological modeling study that was not achievable in non-modulated hiPSC-CMs. In addition, more mature hiPSC-CMs will also advance broad applications of these cells in regenerative medicine, modeling of other cardiac diseases and cardiotoxicity. Our approaches used in screening AMPK activators paved an effective way for the future drug and dose identification in pharmacological drug-induced hiPSC-CM maturation. Our findings will also facilitate future efforts on large-scale production of mature hiPSC-CMs, given that our cardiac sphere culture system has advantage for scale-up suspension culture [39].

Availability of data and materials

The RNA-seq data were deposited at the GEO database under accession code GSE203480. The proteomic data were deposited in the public MassIVE database with the identifier MSV000087351.

Abbreviations

3D:: Three-dimensional
A100:: A-769662 at 100 µM
A200:: A-769662 at 200 µM
E10:: EX229 at 10 µM
E50:: EX229 at 50 µM
ECAR:: Extracellular acidification rate
FAO:: Fatty acid oxidation
GO:: Gene Ontology
HILIC:: Ultra-high-resolution mass spectrometry with hydrophilic interaction liquid chromatography
hiPSC:: Human-induced pluripotent stem cell
hiPSC-CMs:: Cardiomyocytes derived from human-induced pluripotent stem cells
LC–MS–MS:: Liquid chromatography with tandem mass spectrometry
LV:: Heart tissue samples from pediatric left ventricle
OCR:: Oxygen consumption rate
RNA-seq:: RNA-sequencing
siRNA:: Small interfering RNA

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Acknowledgements

We thank the flow cytometry core facility of the Emory and Children's Pediatric Research Center for their shared equipment, services and expertise. The graphical abstract was created with BioRender.com.

Funding

This study was supported in part by grants from the National Institutes of Health (R01HL136345 and R01AA028527) and the National Science Foundation and the Center for Advancement of Science in Space (CBET 1926387). Role of funding body: The funding body played no role in the design of the study and collection, analysis and interpretation of data and in writing the manuscript.

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Authors and Affiliations

Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA, USA
Dong Li, Lawrence C. Armand, Hyun Hwang, David Wolfson, Antonio Rampoldi, Rui Liu, Parvin Forghani, Wen-Mei Yu, Cheng-Kui Qu, Hee Cheol Cho, Joshua T. Maxwell & Chunhui Xu
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, 30332, USA
Fangxu Sun & Ronghu Wu
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA
David Wolfson, Hee Cheol Cho & Chunhui Xu
Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
**n Hu & Dean P. Jones

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Dong Li
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Lawrence C. Armand
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Fangxu Sun
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Hyun Hwang
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David Wolfson
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Antonio Rampoldi
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Rui Liu
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Parvin Forghani
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**n Hu
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Wen-Mei Yu
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Cheng-Kui Qu
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Dean P. Jones
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Ronghu Wu
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Hee Cheol Cho
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Joshua T. Maxwell
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Chunhui Xu
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Contributions

DL and CX designed experiments. DL, LCA, FS, HH, DW, AR, RL, PF, XH and W-MY performed experiments and analyzed data. C-KQ, DPJ, RW and HCC provided new analytical tools. DL and CX wrote the manuscript. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Chunhui Xu.

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Ethics approval and consent to participate

The investigation conformed to the principles outlined in the Declaration of Helsinki. The Institutional Review Board at Emory University granted a waiver of the informed consent for the use of the anonymized and discarded heart tissues obtained from pediatric patients during cardiac repair surgery. (1) Title of the approved project: Tissue Study. (2) Name of the institutional approval committee or unit: Institutional Review Board at Emory University. (3) Approval number: IRB00005500. (4) Date of approval: 8/24/2007 (initial approval); 12/16/2022 (effective).

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1.

Supplemental results, methods, tables and figures.

Additional file 2. Beating cardiac spheres.

Additional file 3.

Metabolic feature table.

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Li, D., Armand, L.C., Sun, F. et al. AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling. Stem Cell Res Ther 14, 322 (2023). https://doi.org/10.1186/s13287-023-03554-7

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Received: 21 February 2023
Accepted: 30 October 2023
Published: 08 November 2023
DOI: https://doi.org/10.1186/s13287-023-03554-7

AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling