Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1

Jijun Huang^#^{1

2}, Josh Z Lee^#¹, Christoph D Rau^{1

3}, Arash Pezhouman^{4

5}, Tomohiro Yokota^{1

4

6}, Hiromi Miwa⁷, Matthew Feldman⁸, Tsz Kin Kong¹, Ziyue Yang⁹, Woan Ting Tay¹⁰, Ivan Pushkarsky¹¹, Kyungsoo Kim¹², Shan S Parikh¹², Shreya Udani¹⁰, Boon Seng Soh¹³, Chen Gao^{1

14}, Linsey Stiles¹⁵, Orian S Shirihai¹⁵, Bjorn C Knollmann¹², Reza Ardehali^{4

5}, Dino Di Carlo⁷, Yibin Wang^{1

10}

Affiliations

¹ Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles.
² Division of Endocrinology (J.H.), University of California, Los Angeles.
³ Department of Genetics and Computational Medicine, University of North Carolina, Chapel Hill (C.D.R.).
⁴ Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles.
⁵ Section of Cardiology, Department of Internal Medicine, Baylor College of Medicine, Houston, TX (A.P., R.A.).
⁶ Department of Medicine, Greater Los Angeles VA Healthcare System, CA (T.Y.).
⁷ Department of Bioengineering, Samueli School of Engineering (H.M., S.U., D.D.), University of California, Los Angeles.
⁸ School of Medicine, Meharry Medical College, Nashville, TN (M.F.).
⁹ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX (Z.Y.).
¹⁰ Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.).
¹¹ Forcyte Biotechnologies, Inc, Los Angeles, CA (I.P.).
¹² Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.).
¹³ Institute of Molecular and Cell Biology, The Agency for Science, Technology and Research (A*STAR), Singapore (B.S.S.).
¹⁴ Department of Pharmacology and System Physiology, University of Cincinnati, OH (C.G.).
¹⁵ Department of Medicine, David Geffen School of Medicine (L.S., O.S.S.), University of California, Los Angeles.

^# Contributed equally.

PMID: 37844148
PMCID: PMC10593507
DOI: 10.1161/CIRCULATIONAHA.122.061602

Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1

Jijun Huang et al. Circulation. 2023.

. 2023 Oct 17;148(16):1263-1266.

doi: 10.1161/CIRCULATIONAHA.122.061602. Epub 2023 Oct 16.

Authors

Affiliations

¹ Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles.
² Division of Endocrinology (J.H.), University of California, Los Angeles.
³ Department of Genetics and Computational Medicine, University of North Carolina, Chapel Hill (C.D.R.).
⁴ Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles.
⁵ Section of Cardiology, Department of Internal Medicine, Baylor College of Medicine, Houston, TX (A.P., R.A.).
⁶ Department of Medicine, Greater Los Angeles VA Healthcare System, CA (T.Y.).
⁷ Department of Bioengineering, Samueli School of Engineering (H.M., S.U., D.D.), University of California, Los Angeles.
⁸ School of Medicine, Meharry Medical College, Nashville, TN (M.F.).
⁹ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX (Z.Y.).
¹⁰ Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.).
¹¹ Forcyte Biotechnologies, Inc, Los Angeles, CA (I.P.).
¹² Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.).
¹³ Institute of Molecular and Cell Biology, The Agency for Science, Technology and Research (A*STAR), Singapore (B.S.S.).
¹⁴ Department of Pharmacology and System Physiology, University of Cincinnati, OH (C.G.).
¹⁵ Department of Medicine, David Geffen School of Medicine (L.S., O.S.S.), University of California, Los Angeles.

^# Contributed equally.

PMID: 37844148
PMCID: PMC10593507
DOI: 10.1161/CIRCULATIONAHA.122.061602

No abstract available

Keywords: RNA, messenger; myocytes, cardiac; sequence analysis, RNA.

PubMed Disclaimer

Conflict of interest statement

Disclosures None.

Figures

**Figure 1:. RBFox1 promotes rodent neonatal and human induced pluripotent stem cell–derived cardiomyocyte maturation at molecular, morphologic, and functional levels.**
A) Top BioPlanet pathway terms enriched with up or down regulated DEGs between adult versus neonatal rat heart based on RNA-seq; B) Volcano Plot showing *Rbfox1* and mRNA splicing pathway genes among all the DEGs in the above RNA-seq data set. DEGs cutoff, |log2FC| > 0.5 and padj <0.05, Bonferroni correction is used to calculate the adjusted p-values. The insert bar graph shows expression of *Rbfox1* measured by RT-qPCR. n = 3, with 6-8 hearts pooled together for each n in the fetal samples, *, p < 0.05; unpaired Welch’s t-test; C) left, UMAP distribution of single cell RNA-seq data showing P1 to P14 postnatal mouse cardiomyocytes labeled by sub-clusters (CM1-5). CM: cardiomyocytes; right, CM sub-cluster composition at each time point from P1 to P14; D) Violin Plot showing expression of *Rbfox1*, cell cycle genes (*Mki67*, *Aurka*), and cardiac genes (*Pln*, *Myh6*, *Myh7*) scaled across different CM sub-clusters. *significance* was labeled between CM5 and CM1-4; statistical analysis was performed by Kruskal-Wallis test followed by Dunn’s multiple comparison in R. E) Cell size comparison of Rbfox1 versus GFP expressing neonatal rat ventricular myocytes (NRVMs). ****, p < 0.0001, Wilcoxon rank-sum test, each dot represents one cell; **(F)** mRNA levels of Ca²⁺ handling genes in Rbfox1 vs. GFP expressing NRVMs, n = 7. *, p < 0.05, **, p < 0.01, two-way ANOVA with Sidak’s multiple comparisons test; G) Representative immunofluorescent staining of sarcomere protein ACTN2 in NRVMs expressing GFP (top) or Rbfox1 (bottom), scar bar = 20 μm. **H-I)** Ratio of NRVM with sarcomere score = 1 vs. total number of NRVM (H) and ratio of bi-nucleated vs. mono-nucleated cardiomyocytes (I) in NRVMs expressing GFP or Rbfox1. Sarcomere score = 1, defined as cardiomyocytes with 50% or more cell body covered with aligned ACTN2 staining. Each data point represents the average value from an imaging field, each field contained 7-21 cells (H) and 20-40 cells (I). ** p < 0.01, ****p < 0.0001 , unpaired student t-test, data passed normality test and variance is equal. **J-K).** Myocyte Contraction based on edge-tracing and beating frequency from NRVMs expressing GFP or Rbfox1 under electrical pacing at 0.6 Hz. Representative contraction curve from three region of interest (ROI) (J). Quantitative frequency in GFP versus Rbfox1 group (K). ****p < 0.0001, Wilcoxon rank-sum test, dot represent individual region of interest (ROI). L) Cytosolic Ca²⁺ transients in NRVMs expressing LacZ or Rbfox1 at a pacing frequency of 2.0 Hz measured with Fluo3-AM. **M-N)** Kinetics of calcium transients, time to peak (L) and Tau of decay (M). Data were summarized from two independent experiments. *, p < 0.05, ***, p < 0.001, unpaired Welch’s t-test; O) Action potential profile in LacZ (Control, top) and Rbfox1 expressing NRVMs (bottom) measured by whole field optical imaging. FluoVolt^™ Membrane Potential Kit was used as the action potential dye. P) Action potential duration as measured at time to 50% decay (APD₅₀), n = 50 ROIs from 3 independent sets. **** p < 0.0001, unpaired student t-test, data passed normality test and variance is equal. **Q).** Oxygen consumption rate (OCR) in NRVM expressing lacZ or increasing levels of Rbfox1 following adv-Rbfox1 infection at Low: 5.37x10⁷ opu/ml, Medium: 1.34x10⁸ opu/ml and High: 2.68x10⁸ opu/ml titers. *** p < 0.001, **** p < 0.0001, 5 replicates in each time point. Two-way ANOVA with Sidak’s correction of multiple comparison, two factors were Groups (LacZ, Adv-Rbfox1 groups) and Mito Status (Basal, Oligo, FCCP). R) Expression of Ca²⁺ handling and cardiomyocyte maturation genes in Rbfox1 vs. GFP treated hiPSC-CMs. *, p < 0.05, from 3 biological repeats, unpaired student t-test; S) Representative immunofluorescent image of hiPSC-CM expressing GFP (top) or Rbfox1 (bottom) for sarcomere protein ACTN2, scar bar = 20 μm; **T).** Ratio of sarcomere score =1 hiPSC-CM vs. total number of cells in each image field, each filed contained 5-25 cells. ****, p < 0.0001, unpaired student t-test, data passed normality test and variance is equal. U) Calcium transients in hiPSC-CM expressing LacZ (control) or Rbfox1. Frequency = 2.0 Hz; **V-W)** Kinetics of calcium transients, time to peak **(V)** and Tau of decay **(W)**. Data were summarized from two independent experiments. ***, p < 0.001, ****, p < 0.0001, Wilcoxon rank-sum test; X) Top enriched Gene Ontology (GO) terms in DEGs (left), and Rbfox1 regulated and cardiomyocyte maturation associated RNA splicing genes between neonatal versus adult rat heart and hiPSC-CMs expressing Rbfox1 versus GFP (right); Y) Representative traces of FLECS constructs under spontaneous contraction seeded with hiPSC-CM expressing LacZ (top) or Rbfox1 (bottom). Three individual regions of interest (ROI) were shown; Z) Fraction shortening of FLECS constructs seeded with hiPSC-CM expressing Rbfox1 versus LacZ. *, p < 0.05, Wilcoxon rank-sum test. Each dot represents an individual patterned contraction unit. All error bars represent standard deviation.

See this image and copyright information in PMC

References

1. Karbassi E, Fenix A, Marchiano S, Muraoka N, Nakamura K, Yang X and Murry CE. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nature reviews Cardiology. 2020;17:341–359. - PMC - PubMed
1. Guo Y and Pu WT. Cardiomyocyte Maturation: New Phase in Development. Circulation research. 2020;126:1086–1106. - PMC - PubMed
1. Gao C, Ren S, Lee JH, Qiu J, Chapski DJ, Rau CD, Zhou Y, Abdellatif M, Nakano A, Vondriska TM, Xiao X, Fu XD, Chen JN and Wang Y. RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure. The Journal of clinical investigation. 2016;126:195–206. - PMC - PubMed
1. Wang Y, Yao F, Wang L, Li Z, Ren Z, Li D, Zhang M, Han L, Wang SQ, Zhou B and Wang L. Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation. Nature communications. 2020;11:2585. - PMC - PubMed
1. Pushkarsky I. FLECS Technology for High-Throughput Single-Cell Force Biology and Screening. Assay Drug Dev Technol. 2018;16:7–11. - PMC - PubMed

Publication types

Actions
Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1

Affiliations

Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1

Authors

Affiliations

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources