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A transcriptional enhancer regulates cardiac maturation

Nature Cardiovascular Research volume 3, pages 666–684 (2024)Cite this article

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Abstract

Cardiomyocyte maturation is crucial for generating adult cardiomyocytes and the application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, regulation at the cis-regulatory element level and its role in heart disease remain unclear. Alpha-actinin 2 (ACTN2) levels increase during CM maturation. In this study, we investigated a clinically relevant, conserved ACTN2 enhancer’s effects on CM maturation using hPSC and mouse models. Heterozygous ACTN2 enhancer deletion led to abnormal CM morphology, reduced function and mitochondrial respiration. Transcriptomic analyses in vitro and in vivo showed disrupted CM maturation and upregulated anabolic mammalian target for rapamycin (mTOR) signaling, promoting senescence and hindering maturation. As confirmation, ACTN2 enhancer deletion induced heat shock protein 90A expression, a chaperone mediating mTOR activation. Conversely, targeting the ACTN2 enhancer via enhancer CRISPR activation (enCRISPRa) promoted hPSC-CM maturation. Our studies reveal the transcriptional enhancer’s role in cardiac maturation and disease, offering insights into potentially fine-tuning gene expression to modulate cardiomyocyte physiology.

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Fig. 1: ACTN2 enh del impairs the morphological and functional maturation of hPSC-CMs.
Fig. 2: Single-cell transcriptomics of ACTN2 enh del hPSC-CMs reveals disrupted sarcomeric and metabolic pathways.
Fig. 3: ACTN2 enh del disrupts mitochondria morphology and function.
Fig. 4: Mechanistic analysis of ACTN2 enhancer transcription regulation.
Fig. 5: Actn2 enh del in vivo impairs cardiac maturation and function in adult mice.
Fig. 6: Actn2 enh del in vivo disrupts early postnatal CM maturation.
Fig. 7: mTOR pathway is activated in Actn2 enh del hearts and hPSC-CMs.
Fig. 8: Leveraging the ACTN2 conserved enhancer region to promote the maturation of hPSC-CMs.

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Data availability

The bulk RNA-seq data and single-cell RNA-seq data are available at the Gene Expression Omnibus (GSE266306 and GSE266307). Source Data are also provided with this manuscript.

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Acknowledgements

The authors would like to thank all members of the Tampakakis, Kwon and Kass laboratories and M. Ranek at Johns Hopkins University for insightful comments and recommendations. We would also like to thank A. Cammarato for kindly providing Drosophila melanogaster flies. E.T. is supported by grants from the National Heart, Lung, and Blood Institute (NHLBI) (HL-145135), the American Heart Association (AHA) (CDA34660077), the W. W. Smith Charitable Trust, the Magic That Matters Fund, the Johns Hopkins University Catalyst Award and the Maryland Stem Cell Research Fund (MSCRF) (2023-MSCRFL-5984). S.M., E.C. and C.K. were supported by grants from the National Institutes of Health (NIH)/NHLBI (R01HL156947, T32HL007227), the AHA (TPA1058685) and the MSCRF (MSCRFD-6139). M.A. was supported by the NIH/NHLBI (K08 HL166690). B.L.L was supported by the NIH/NHLBI (K99 HL15584).

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Authors and Affiliations

  1. Department of Medicine, Division of Cardiology, Johns Hopkins University, Baltimore, MD, USA

    Myo Htet, Sheetal Bajpayi, Harshi Gangrade, Marios Arvanitis, Sean Murphy, Navid Koleini, Chulan Kwon & Emmanouil Tampakakis

  2. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Shunyao Lei, Marios Arvanitis, Asimina Zoitou, Elaine Zhelan Chen, Chulan Kwon & Emmanouil Tampakakis

  3. Department of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA

    Marios Arvanitis & Emmanouil Tampakakis

  4. Department of Cell Biology, Neurobiology, and Anatomy and Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

    Brian Leei Lin

  5. Institute of Cell Engineering, Johns Hopkins University, Baltimore, MD, USA

    Chulan Kwon

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Contributions

M.H. designed, carried out and supervised this work and wrote the manuscript. H.G., S.L., S.B. and A.Z. assisted with experimental work. M.A. performed bioinformatics analyses. B.L.L. assisted with cell isolation and functional analyses. S.M. performed bioinformatics analyses. E.C. assisted with in vitro free fatty acid treatments and bioinformatics. N.K. assisted with mouse cardiomyocyte isolation, Seahorse analyses and co-immunoprecipitation. C.K. assisted with experimental design. E.T. designed and supervised this work, assisted with experimental work and wrote the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Emmanouil Tampakakis.

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The authors declare no competing interests.

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Nature Cardiovascular Research thanks Kailong Li, Sean Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 An evolutionary conserved ACTN2 genetic variant linked with heart failure.

A genetic variant linked with heart failure (rs535411) is within a highly evolutionary conserved region of the ACTN2 transcriptional enhancer. A second variant (rs663798) also associated with heart failure is upstream of the conserved region.

Extended Data Fig. 2 ACTN2 enhancer deletion does not disrupt the differentiation of hPSC-CMs or gene splicing.

a. Gating strategy used to detect differentiated hPSC-CMs. b. Flow cytometry analysis of ACTN2 enh del hPSC-CMs vs isogenic controls after immunostaining for cardiac Troponin T. The gating strategy used to detect hPSC-CMs for flow cytometry analysis is represented. The differentiation efficiency of both hPSC lines is similar. c. Map illustrating the alternatively spliced ACTN2 variants, and the RT-PCR products based on the different primer sets. d. RT-PCR using cDNA generated from ACTN2 enh del hPSC-CMs vs isogenic controls showing no difference in the size of alternatively spliced ACTN2 transcripts. Similar results were obtained after repeating the experiment 4 independent times.

Extended Data Fig. 3 ACTN2 enhancer deletion increases the transcriptomic entropy of hPSC-CMs.

Entropy score calculated from hPSC-CM our single cell RNA-Seq data (one differentiation batch) shows increased entropy in ACTN2 enh del cardiomyocytes suggestive of disrupted maturation. The center line of the box corresponds to the 50th percentile. The lower and upper bounds of the boxes correspond to the 25th and the 75th percentiles. The upper whisker marks the largest value within 1.5 times interquartile range above the 75th percentile. The lower whisker marks the smallest value within 1.5 times interquartile range below the 25th percentile. The interquartile range is defined as the distance between the first and the third quartiles.

Extended Data Fig. 4 Luciferase assay to validate the ACTN2 enhancer conserved region.

a. Illustration of the different luciferase vectors tested. b. Luciferase analysis of hPSC-CMs transfected with minimal promoter vs the conserved ACTN2 enhancer sequence with and without the rs535411 variant, showing increased luciferase activity in the presence of ACTN2 enhancer. This effect was not observed in cells transfected with the mutant variant. c-d. Overexpression of MEF2A and MEF2C transcription factors through modified mRNA transfection in hPSC-CMs resulted in markedly increased luciferase activity in both enhancer vectors. However, this induction was significantly lower in cells with ACTN2 rs535411 enhancer variant. All graphs report Luciferase compared to minimal promoter. (4 independent experiments were analyzed). Data are presented as mean values +/− SEM. All replicates are biological. The Shapiro-Wilk test was performed to assess normal distribution, and one-way ANOVA with Bonferroni’s multiple comparisons test or Kruskal-Wallis with Dunn’s multiple comparisons test were used as appropriate. Only P values < 0.1 are reported.

Extended Data Fig. 5 Temporal ACTN2 enhancer activation in mice.

a. Analysis of ENCODE ChIP-Seq datasets for H3K27Ac and H3K4me1 from mouse hearts at different developmental stages showed increased ACTN2 enhancer activation in older mice supporting a role in the temporal regulation of ACTN2 gene expression. b. Analysis of P300 occupancy data from mouse hearts at different developmental stages, similarly, showed an increased in P300 peaks in postnatal hearts. c. Mef2a ChIP-Seq data showing binding of Mef2a in the ACTN2 enhancer region in adult mouse hearts.

Extended Data Fig. 6 Temporal activation of Actn enhancer regions in Drosophila melanogaster.

a. H3K27Ac ChIP-Sequencing analysis in Drosophila melanogaster reveals an extra active enhancer region upstream of the actinin (Actn) gene promoter that is only present in adult flies. b. Alternatively spliced Actn gene variants (Actn-RB, RC, RD) are expressed at different developmental stages (larva vs fly), likely temporarily regulated by different transcriptional enhancers such as the one revealed by H3K27Ac ChIP-Seq. (5 independent experiments were analyzed). Data are presented as mean values +/− SEM. All replicates are biological.

Extended Data Fig. 7 Actn2 expression is reduced in Actn2 enh del mouse hearts.

Actn2 protein is decreased in both heterozygous and homozygous Actn2 enh del hearts. Data are presented as mean values +/− SEM. The Shapiro-Wilk test was performed to assess normal distribution, and one-way ANOVA with Bonferroni’s multiple comparisons test was used to compare all groups with control. (4 samples per group were analyzed). All replicates are biological. Only P values < 0.1 are reported.

Source data

Extended Data Fig. 8 Suppression of mTORC1 improves the maturation of ACTN2 enh del hPSC-CMs.

a. Gene expression of RAPTOR and 3-phosphoinositidine-dependent protein kinase-1 (PDK1) as part of mTORC1 pathway were upregulated, while RICTOR levels which is part of the mTORC2 complex remained unchanged. (3 independent samples per group). b. Both ACTN2 enh del hPSC-CMs and isogenic controls showed very similar cell size after treatment with everolimus (total ~147 cells/group, 4 independent treatments). c. The morphology of ACTN2 enh del hPSC-CMs did not show any difference as compared to controls after treatment with everolimus. (4 independent samples per group) (bar graph: 25μm) d. ACTN2 enh del hPSC-CMs treated with everolimus (25 μM, mTORC1 inhibitor) normalized their calcium handling properties as compared to treated control hPSC-CMs. Time to peak, peak height and time to 50% to baseline are not different between control and ACTN2 enh del hPSC-CMs. (n = 8–10 batches per group). e. Seahorse analysis of cultured hPSC-CMs show reduced maximum oxygen consumption rate (OCR) and ECAR to OCR ratio (consistent with increased glycolysis and reduced oxidative phosphorylation) for ACTN2 enh del hPSC-CMs compared to isogenic controls. Treatment with everolimus, improved oxygen consumption for both for ACTN2 enh del hPSC-CMs and isogenic controls and normalized glycolysis for ACTN2 enh del hPSC-CMs. (4 independent samples per group). f. Both ACTN2 enh del hPSC-CMs and isogenic controls developed very similar gene expression of maturation related genes after everolimus treatment (3 independent samples per group). Data are presented as mean values +/− SEM. All replicates are biological. The Shapiro-Wilk test was performed to assess normal distribution, and the parametric student t-test or ANOVA with Bonferroni’s multiple comparisons test or the non-parametric Mann-Whitney (two-tailed) or Kruskal Willis with Dunn’s multiple comparisons tests were used as appropriate. Only P values < 0.1 are reported.

Extended Data Fig. 9 ACTN2 directly interacts with HSP90A.

a. Co-immunoprecipitation of ACTN2 with HSPA90A in both control and ACTN2 enh del hPSC-CMs, supporting the direct interaction between the two proteins. Despite the reduced ACTN2 levels in ACTN2 enh del hPSC-CMs, the ACTN2 immunoprecipitate appears more enriched with the inducible chaperone HSPA90A. b. Transfection of hPSC-CMs with HSP90AA1 siRNAs suppressed HSP90A protein levels in control hPSC-CMs. (3 independent experiments were analyzed) c. siRNA downregulation of HSP90A in ACTN2 enh del hPSC-CMs. (3 independent experiments were analyzed). Data are presented as mean values +/− SEM. All replicates are biological. The Shapiro-Wilk test was performed to assess normal distribution, and paired student parametric t-test (two-tailed) was used for all comparisons. Only P values < 0.1 are reported.

Source data

Extended Data Fig. 10 Leveraging enCRISPRa to upregulate ACTN2.

Transfection of hPSC-CMs with sgRNAs targeting the 5’ border of the conserved ACTN2 enhancer region, upregulated ACTN2 protein. Relative protein expression compared to sgRNA1 is presented. (3 independent experiments were analyzed). Data are presented as mean values +/− SEM. All replicates are biological. The Shapiro-Wilk test was performed to assess normal distribution, and paired student parametric t-test (two-tailed) was used for all comparisons.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

List of all gRNAs used in this study

Supplementary Table 2

List of all primers used for hPSC-CM qPCR

Supplementary Table 3

List of all primers used for mouse and Drosophila qPCR

Supplementary Table 4

List of primers used for ChIP qPCR

Supplementary Table 5

List of all antibodies

Source data

Source Data Fig.1

Unprocessed western blots for Fig. 1

Source Data

Statistical Source Data

Source Data

Source data for extended data figures

Source Data Fig. 7

Unprocessed western blots for Fig. 7

Source Data Extended Data Fig./Table 7

Source data for Extended Data Fig. 7

Source Data Extended Data Fig./Table 9

Source data for Extended Data Fig. 9

Source Data Extended Data Fig.10

Source data for Extended Data Fig. 10

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Htet, M., Lei, S., Bajpayi, S. et al. A transcriptional enhancer regulates cardiac maturation. Nat Cardiovasc Res 3, 666–684 (2024). https://doi.org/10.1038/s44161-024-00484-2

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