Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current

Nature Cardiovascular Research volume 1, pages 1–13 (2022)Cite this article

Subjects

Abstract

Voltage-gated sodium channels (Nav channels) support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of NaV1.5 inactivation results in a small persistent sodium influx known as late sodium current (INa,L), which has emerged as a common pathogenic mechanism in both congenital and acquired cardiac arrhythmogenic syndromes. In the present study, using low-noise multichannel recordings in heterologous systems, LQTS3 patient-derived induced pluripotent stem cell cardiomyocytes and mouse ventricular myocytes, we demonstrate that the intracellular fibroblast growth factor homologous factors (FHF1–4) tune pathogenic INa,L in an isoform-specific manner. This scheme suggests a complex orchestration of INa,L in cardiomyocytes that may contribute to variable disease expressivity of NaV1.5 channelopathies. We further leverage these observations to engineer a peptide inhibitor of INa,L with a higher efficacy compared with a well-established small-molecule inhibitor. Overall, these findings lend insights into molecular mechanisms underlying FHF regulation of INa,L in pathophysiology and outline potential therapeutic avenues.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: ShRNA suppression of FHF2 upregulates INa,L in aMVMs.
Fig. 2: Bidirectional modulation of INa,L by FHF variants.
Fig. 3: Competition between protective and nonprotective FHFs continuously tunes INa,L.
Fig. 4: Generality of INa,L regulation by FHFs.
Fig. 5: FHF regulation of pathogenic INa,L in iPSC–CMs.
Fig. 6: A minimal FHF1A domain recapitulates potent INa,L inhibition.
Fig. 7: Cell-permeable FixR serves as a functional inhibitor of INa,L.

Similar content being viewed by others

Data availability

All data are available in the main text or the supplementary materials. Source data are available for this paper.

Code availability

Customized MATLAB scripts used for multichannel analysis are available at Github: https://github.com/manubenjohny/LateCurrent.

References

  1. Ahern, C. A., Payandeh, J., Bosmans, F. & Chanda, B. The hitchhiker’s guide to the voltage-gated sodium channel galaxy. J. Gen. Physiol. 147, 1–24 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Abriel, H. Cardiac sodium channel Nav1.5 and interacting proteins: physiology and pathophysiology. J. Mol. Cell Cardiol. 48, 2–11 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Clancy, C. E. et al. Deranged sodium to sudden death. J. Physiol. 593, 1331–1345 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wilde, A. A. M. & Amin, A. S. Clinical spectrum of SCN5A mutations: long QT syndrome, Brugada syndrome, and cardiomyopathy. JACC Clin. Electrophysiol. 4, 569–579 (2018).

    Article  PubMed  Google Scholar 

  5. Wan, E. et al. Aberrant sodium influx causes cardiomyopathy and atrial fibrillation in mice. J. Clin. Invest. 126, 112–122 (2016).

    Article  PubMed  Google Scholar 

  6. Horvath, B. & Bers, D. M. The late sodium current in heart failure: pathophysiology and clinical relevance. ESC Heart Fail. 1, 26–40 (2014).

    Article  PubMed  Google Scholar 

  7. Howard, T. et al. CaMKII-dependent late Na+ current increases electrical dispersion and arrhythmia in ischemia-reperfusion. Am. J. Physiol. Heart Circ. Physiol. 315, H794–H801 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Makielski, J. C. Late sodium current: a mechanism for angina, heart failure, and arrhythmia. Trends Cardiovasc. Med. 26, 115–122 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Bennett, P. B., Yazawa, K., Makita, N. & George, A. L. Jr. Molecular mechanism for an inherited cardiac arrhythmia. Nature 376, 683–685 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. Unudurthi, S. D. & Hund, T. J. Late sodium current dysregulation as a causal factor in arrhythmia. Expert Rev. Cardiovasc. Ther. 14, 545–547 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Horvath, B. et al. Dynamics of the late Na+ current during cardiac action potential and its contribution to afterdepolarizations. J. Mol. Cell Cardiol. 64, 59–68 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Hegyi, B. et al. beta-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII. J. Mol. Cell Cardiol. 123, 168–179 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. Matasic, D. S. et al. Modulation of the cardiac sodium channel Nav1.5 peak and late currents by NAD+ precursors. J. Mol. Cell Cardiol. 141, 70–81 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Maltsev, V. A., Kyle, J. W. & Undrovinas, A. Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its beta1 subunit. J. Physiol. Sci. 59, 217–225 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pei, Z., Xiao, Y., Meng, J., Hudmon, A. & Cummins, T. R. Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation. Nat. Commun. 7, 12035 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Abrams, J. M. et al. Fibroblast growth factor homologous factors tune arrhythmogenic late Nav1.5 current in calmodulin-binding-deficient channels. JCI Insight https://doi.org/10.1172/jci.insight.141736 (2020).

  17. Thompson, C. H., Hawkins, N. A., Kearney, J. A. & George, A. L. Jr. CaMKII modulates sodium current in neurons from epileptic Scn2a mutant mice. Proc. Natl Acad. Sci. USA 114, 1696–1701 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kang, P. W. et al. Elementary mechanisms of calmodulin regulation of Nav1.5 producing divergent arrhythmogenic phenotypes. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2025085118 (2021).

  19. Yan, H., Wang, C., Marx, S. O. & Pitt, G. S. Calmodulin limits pathogenic Na+ channel persistent current. J. Gen. Physiol. 149, 277–293 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Brink, P. A. et al. Phenotypic variability and unusual clinical severity of congenital long-QT syndrome in a founder population. Circulation 112, 2602–2610 (2005).

    Article  PubMed  Google Scholar 

  21. Verkerk, A. O., Amin, A. S. & Remme, C. A. Disease modifiers of inherited SCN5A channelopathy. Front. Cardiovasc. Med. 5, 137 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Antzelevitch, C. et al. The role of late INa in development of cardiac arrhythmias. Handb. Exp. Pharmacol. 221, 137–168 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Horvath, B. et al. Late sodium current inhibitors as potential antiarrhythmic agents. Front Pharmacol. 11, 413 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Musa, H. et al. SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia. Proc. Natl Acad. Sci. USA 112, 12528–12533 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wei, E. Q., Barnett, A. S., Pitt, G. S. & Hennessey, J. A. Fibroblast growth factor homologous factors in the heart: a potential locus for cardiac arrhythmias. Trends Cardiovasc. Med. 21, 199–203 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Park, D. S. et al. Ionic mechanisms of impulse propagation failure in the FHF2-deficient heart. Circ. Res. 127, 1536–1548 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Goldfarb, M. Fibroblast growth factor homologous factors: evolution, structure, and function. Cytokine Growth Factor Rev. 16, 215–220 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Smallwood, P. M. et al. Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development. Proc. Natl Acad. Sci. USA 93, 9850–9857 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gade, A. R., Marx, S. O. & Pitt, G. S. An interaction between the III-IV linker and CTD in Nav1.5 confers regulation of inactivation by CaM and FHF. J. Gen. Physiol. https://doi.org/10.1085/jgp.201912434 (2020).

  30. Wengert, E. R. & Patel, M. K. The role of the persistent sodium current in epilepsy. Epilepsy Curr. 21, 40–47 (2021).

    Article  PubMed  Google Scholar 

  31. Baker, E. M. et al. The novel sodium channel modulator GS-458967 (GS967) is an effective treatment in a mouse model of SCN8A encephalopathy. Epilepsia 59, 1166–1176 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cannon, S. C. Sodium channelopathies of skeletal muscle. Handb. Exp. Pharmacol. 246, 309–330 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Munoz-Sanjuan, I., Smallwood, P. M. & Nathans, J. Isoform diversity among fibroblast growth factor homologous factors is generated by alternative promoter usage and differential splicing. J. Biol. Chem. 275, 2589–2597 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Wang, C. et al. Fibroblast growth factor homologous factor 13 regulates Na+ channels and conduction velocity in murine hearts. Circ. Res. 109, 775–782 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tomek, J. et al. Development, calibration, and validation of a novel human ventricular myocyte model in health, disease, and drug block. eLife https://doi.org/10.7554/eLife.48890 (2019).

  36. Jiang, D. et al. Structure of the cardiac sodium channel. Cell 180, 122–134.e110 (2020).

    Article  CAS  PubMed  Google Scholar 

  37. Ashpole, N. M. et al. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel Nav1.5 gating by multiple phosphorylation sites. J. Biol. Chem. 287, 19856–19869 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Aiba, T. et al. Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes. Cardiovasc. Res. 85, 454–463 (2010).

    Article  CAS  PubMed  Google Scholar 

  39. Wang, C., Chung, B. C., Yan, H., Lee, S. Y. & Pitt, G. S. Crystal structure of the ternary complex of a Nav C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin. Structure 20, 1167–1176 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Aldrich, R. W., Corey, D. P. & Stevens, C. F. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature 306, 436–441 (1983).

    Article  CAS  PubMed  Google Scholar 

  41. Hennessey, J. A. et al. FGF12 is a candidate Brugada syndrome locus. Heart Rhythm 10, 1886–1894 (2013).

    Article  PubMed  Google Scholar 

  42. Dover, K., Solinas, S., D’Angelo, E. & Goldfarb, M. Long-term inactivation particle for voltage-gated sodium channels. J. Physiol. 588, 3695–3711 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Venkatesan, K., Liu, Y. & Goldfarb, M. Fast-onset long-term open-state block of sodium channels by A-type FHFs mediates classical spike accommodation in hippocampal pyramidal neurons. J. Neurosci. 34, 16126–16139 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Nguyen, Q. T. et al. Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival. Proc. Natl Acad. Sci. USA 107, 4317–4322 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Olson, E. S. et al. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc. Natl Acad. Sci. USA 107, 4311–4316 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hu, Z. et al. Regulation of blood pressure by targeting CaV1.2–Galectin-1 protein interaction. Circulation 138, 1431–1445 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Green, M. & Loewenstein, P. M. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55, 1179–1188 (1988).

    Article  CAS  PubMed  Google Scholar 

  48. Goldfarb, M. et al. Fibroblast growth factor homologous factors control neuronal excitability through modulation of voltage-gated sodium channels. Neuron 55, 449–463 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lou, J. Y. et al. Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels. J. Physiol. 569, 179–193 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Gabelli, S. B. et al. Regulation of the Nav1.5 cytoplasmic domain by calmodulin. Nat. Commun. 5, 5126 (2014).

    Article  CAS  PubMed  Google Scholar 

  51. Liu, C. J., Dib-Hajj, S. D., Renganathan, M., Cummins, T. R. & Waxman, S. G. Modulation of the cardiac sodium channel Nav1.5 by fibroblast growth factor homologous factor 1B. J. Biol. Chem. 278, 1029–1036 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Wittmack, E. K. et al. Fibroblast growth factor homologous factor 2B: association with Nav1.6 and selective colocalization at nodes of Ranvier of dorsal root axons. J. Neurosci. 24, 6765–6775 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Laezza, F. et al. The FGF14(F145S) mutation disrupts the interaction of FGF14 with voltage-gated Na+ channels and impairs neuronal excitability. J. Neurosci. 27, 12033–12044 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Niu, J. et al. Allosteric regulators selectively prevent Ca2+-feedback of Cav and Nav channels. eLife https://doi.org/10.7554/eLife.35222 (2018).

  55. Kellenberger, S., West, J. W., Scheuer, T. & Catterall, W. A. Molecular analysis of the putative inactivation particle in the inactivation gate of brain type IIA Na+ channels. J. Gen. Physiol. 109, 589–605 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wang, S. Y., Bonner, K., Russell, C. & Wang, G. K. Tryptophan scanning of D1S6 and D4S6 C-termini in voltage-gated sodium channels. Biophys. J. 85, 911–920 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Peters, C. H., Watkins, A. R., Poirier, O. L. & Ruben, P. C. E1784K, the most common Brugada syndrome and long-QT syndrome type 3 mutant, disrupts sodium channel inactivation through two separate mechanisms. J. Gen. Physiol. https://doi.org/10.1085/jgp.202012595 (2020).

  58. Clancy, C. E. & Rudy, Y. Na+ channel mutation that causes both Brugada and long-QT syndrome phenotypes: a simulation study of mechanism. Circulation 105, 1208–1213 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Sun, J. et al. FGF13 is a novel regulator of NF-kappaB and potentiates pathological cardiac hypertrophy. iScience 23, 101627 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. McCormack, K. et al. Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels. Proc. Natl Acad. Sci. USA 110, E2724–E2732 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Lee, S. R., Sang, L. & Yue, D. T. Uncovering aberrant mutant PKA function with flow cytometric FRET. Cell Rep. 14, 3019–3029 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Valencik, M. L. & McDonald, J. A. Codon optimization markedly improves doxycycline regulated gene expression in the mouse heart. Transgenic Res. 10, 269–275 (2001).

    Article  CAS  PubMed  Google Scholar 

  63. Hedon, C. et al. New role of TRPM4 channel in the cardiac excitation–contraction coupling in response to physiological and pathological hypertrophy in mouse. Prog. Biophys. Mol. Biol. 159, 105–117 (2021).

    Article  CAS  PubMed  Google Scholar 

  64. Huang, C. Y. et al. Enhancement of human iPSC-derived cardiomyocyte maturation by chemical conditioning in a 3D environment. J. Mol. Cell. Cardiol. 138, 1–11 (2020).

    Article  CAS  PubMed  Google Scholar 

  65. Hawthorne, R. N. et al. Altered electrical, biomolecular, and immunologic phenotypes in a novel patient-derived stem cell model of desmoglein-2 mutant ARVC. J. Clin. Med. https://doi.org/10.3390/jcm10143061 (2021).

  66. Schaniel, C. et al. A library of induced pluripotent stem cells from clinically well-characterized, diverse healthy human individuals. Stem Cell Rep. https://doi.org/10.1016/j.stemcr.2021.10.005 (2021).

  67. Limpitikul, W. B. et al. A precision medicine approach to the rescue of function on malignant calmodulinopathic long-QT syndrome. Circ. Res. 120, 39–48 (2017).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank H. Colecraft for valuable insight into and feedback on this work. The present study is supported by funding from the National Heart, Lung, and Blood Institute, the National Institute of Neurological Disorders and Stroke and the American Heart Association (grant no. R01 NS110672 to M.B.J., Postdoctoral Fellowship no. 836413 to N.C., grant nos. R01HL160089 and R01HL140934 to S.O.M., R01 HL128743 to G.F.T. and T32 HL120826 (Andrew Marks PI) to R.M.). Research supported in the present study was performed in the Columbia CCTI Flow cytometry core, supported in part by the Office of the Director, National Institutes of Health (NIH) under award no. S10RR027050. Images were collected and analyzed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by the NIH (grant no. P30 CA013696 (National Cancer Institute)). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author information

Authors and Affiliations

  1. Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA

    Nourdine Chakouri, Sharen Rivas, Johanna Diaz, Allen L. Hsu, Ryan Mahling & Manu Ben-Johny

  2. Department of Molecular Pharmacology and Therapeutics, Columbia University, New York, NY, USA

    Daniel Roybal & Steven O. Marx

  3. Division of Cardiology, Department of Medicine, Columbia University, New York, NY, USA

    Lin Yang, Bi-Xing Chen & Steven O. Marx

  4. Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA

    Josiah O. Owoyemi, Deborah DiSilvestre & Ivy E. Dick

  5. Division of Cardiology, Johns Hopkins Hospital, Baltimore, MD, USA

    Deborah DiSilvestre & Gordon F. Tomaselli

  6. Columbia Stem Cell Initiative, Stem Cell Core, Columbia University Irving Medical Center, New York, NY, USA

    Dario Sirabella & Barbara Corneo

  7. Department of Medicine, Albert Einstein College of Medicine, New York, NY, USA

    Gordon F. Tomaselli

Authors
  1. Nourdine Chakouri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sharen Rivas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Roybal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lin Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Johanna Diaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Allen L. Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ryan Mahling
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bi-Xing Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Josiah O. Owoyemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Deborah DiSilvestre
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Dario Sirabella
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Barbara Corneo
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Gordon F. Tomaselli
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Ivy E. Dick
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Steven O. Marx
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Manu Ben-Johny
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.C., S.O.M. and M.B.-J. conceptualized and designed the research. N.C., S.R., J.D., A.H., R.M., D.R., L.Y., B.-X.C., J.O.O., D.S., B.C., I.E.D. and M.B.-J. performed research and acquired the data. N.C., S.R., J.D. and M.B.-J. analyzed the data. N.C., S.R., J.D., D.R., L.Y., B.-X.C., J.O.O., D.S., B.C., I.E.D., G.T., S.O.M. and M.B.-J. contributed new reagents/analytic tools. M.B.-J., G.F.T. and S.O.M. acquired funds. N.C. and M.B.-J. created the figures and wrote the original draft. All the authors revised the manuscript.

Corresponding author

Correspondence to Manu Ben-Johny.

Ethics declarations

Competing interests

N.C., S.O.M. and M.B.-J. (inventors) filed a provisional patent (attorney docket no. CoU1046P/CU22077; filed 14 February 2022) for application of FixR for inhibiting late sodium current. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Cardiovascular Research thanks Mitchell Goldfarb, Christopher Ahern and Donald Bers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 shRNA suppression of FHF2 has no effect on INa,L in non-TG aMVM.

a, Epifluorescence and brightfield images of cultured aMVMs from non-TG mice transduced with FHF2 shRNA. Scale bar, 100 μm. (n = 3 mice) b, Exemplar multichannel NaV recordings from uninfected non-TG mice show minimal late channel openings. Format as in Fig. 1b. Population data are shown in Fig. 1h. c-d, Application of both FHF2 shRNA and scrambled shRNA revealed no change in INa,L openings for cultured aMVMs from non-TG mice. Population data are shown in Fig. 1h.

Extended Data Fig. 2 INa,L regulation by the FHF2-3 splice variants.

a, Schematic shows various FHF2 splice variants generated by alternate start sites. Conserved exons that encode the core domain are shown in red boxes labeled E2 to E5. b-e, Exemplar recordings of LQTS3-linked ΔKPQ mutant heterologously expressed in HEK293 cells in the presence of various FHF2 splice variants. f-i, Exemplar recordings of IQ/AA mutant in the presence of FHF2 splice variants. j, Bar graph summarizes changes in INa,L quantified as Rpersist upon co-expression of FHF splice variants. FHF2S, and FHF2VY yielded a partial reduction in INa,L for both the IQ/AA and the ΔKPQ mutant, while FHF2V and FHF2U resulted in no change. Data presented as mean ± SEM. For ΔKPQ mutant, + FHF2U, n = 10 cells (561 sweeps); + FHF2V, n = 10 cells (549 sweeps); +FHF2Y, n = 9 cells (512 sweeps); and + FHF2VY, n = 9 cells (499 sweeps). For IQ/AA mutant, + FHF2U, n = 9 cells (547 sweeps); + FHF2V, n = 10 cells (807 sweeps); + FHF2Y, n = 10 cells (564 sweeps); + FHF2VY, n = 9 cells (547 sweeps). Statistical Analysis, Kruskal-Wallis test followed by Dunn’s Multiple comparisons test, ***p < 0.001;**p = 0.026(ΔKPQ + FHF2Y) and *p = 0.039(ΔKPQ + FHF2VY), *p = 0.034(IQ/AA + FHF2S) and *p = 0.011 (IQ/AA + FHF2VY) when compared to no FHF control of each mutant channel. k, Exemplar multichannel recording from recombinant NaV1.5 wild-type in HEK293 cells shown minimal late channel openings in the presence of FHF3A. Population data is shown in Fig. 2m. l, Exemplar recordings show a partial reduction in late channel openings for NaV1.5 IQ/AA mutant channel in the presence of FHF3A. Population data are shown in Fig. 2m.

Source data

Extended Data Fig. 3 Generality of INa,L regulation by FHF1A.

a, Schematic illustrates the α-subunit of NaV1.5 channel showing missense (grey) and nonsense (red) mutations in disparate channel domains. b, Exemplar multichannel recordings of NaV1.5 F1759A in the absence (top) and presence of FHF1A (bottom). c-f. Exemplar recordings suggests that FHF1A inhibits E[1784]K, S[1904]L, Q[1909]R, and Δ1885 mutant.

Extended Data Fig. 4 Inhibition of phosphorylation-dependent INa,L by FHF1-2 isoforms.

a, Schematic shows NaV1.5 phosphorylation by PKA and CaMKII upregulates INa,L. b, FHF2S inhibits PKA-dependent INa,L of wild-type NaV1.5 (similar to the effects of FHF1A in Fig. 3d). e, FHF1B fails to inhibit PKA-dependent INa,L of wild-type NaV1.5. d, Exemplar multichannel recordings from wild-type NaV1.5 upregulated by co-expression of constitutively active CaMKII (CaMKIIT286D). e-f, Both FHF1A (panel e) and FHF2S (panel f) inhibits CaMKIIT286D-triggered INa,L of wild-type NaV1.5.

Extended Data Fig. 5 Elementary mechanisms underlying FHF1A regulation of INa,L.

a, FL distributions for NaV1.5 IFM/IQM at baseline (black line and gray shaded area; n = 231 sweeps from 3 one-channel recordings) and upon overexpression of FHF1A (red line and rose shaded area; n = 217 sweeps from 5 one-channel recordings). FL denotes the probability that the first opening occurred at time < t. FHF1A had minimal effect on the FL distribution for the IFM/IQM mutant. b, FL distributions for NaV1.5 LILA/WICW at baseline (gray shaded area; n = 176 sweeps from 3 one-channel recordings) and under overexpression of FHF1A (rose shaded area; n = 270 sweeps from 6 one-channel recordings). FHF1A decreased the pedestal value of FL for the LILA/WICW mutant suggesting that FHF1A increases closed-state inactivation. p < 0.001 by KS-test. c, Open-duration (OD) distribution for NaV1.5 IFM/IQM tallies the durations of a single sojourn to the open state. FHF1A shortens the OD distribution for the IFM/IQM mutant, hinting at potential increase in the rate constant for inactivation from the open state. For IFM/IQM, n = 7597 openings from 6 cells. For IFM/IQM + FHF1A, n = 1332 openings from 9 cells. p < 0.001 by KS-test. d, FHF1A has no effect on OD distribution of the LILA/WICW mutant. For LILA/WICW, n = 22272 openings from 8 cells. For LILA/WICW + FHF1A, n = 1361 openings from 6 cells. e, Conditional open-duration (OD) distribution for NaV1.5 ΔKPQ mutants, for single-level channel openings in the late phase, after 50 ms depolarization, (n = 12278 openings from 12 cells). FHF1A shortened the OD distribution (n = 5011 openings from 12 cells), while FHF1B evoked a minor increase in OD distribution (n = 2269 openings from 6 cells). f-k, conditional OD distributions confirms variable shortening of the OD distribution in the presence of FHF1A for E[1784]K (panel f; control, n = 9355 openings from 15 cells; +FHF1A, n = 1683 openings from 14 cells), S[1904]L (panel g; control, n = 7097 openings from 10 cells; +FHF1A, n = 10 cells and 1929 openings), Q[1909]R (panel h; control, n = 4406 openings from 13 cells; +FHF1A, n = 1658 openings from 11 cells), IQ/AA (panel i; control, n = 5345 openings from 13 cells; +FHF1A, n = 1266 openings from 13 cells; +FHF1B, n = 16360 openings from 13 cells), and Δ1810 (panel j; control, n = 1049 openings from 10 cells; +FHF1A, n = 576 openings from 9 cells), all consistent with the presence of open-state block. The Δ1885 mutant failed to show any appreciable change in OD distribution (panel k; control, n = 3955 openings from 10 cells; +FHF1A, n = 2195 openings from 9 cells). As FHF1A inhibits INa,L for this mutant, this effect likely modifies the closed state. l, Bar graph summary of mean OD calculated from OD distribution function in panels. e-k from sample sizes noted in each panel. Each bar, mean OD value. e-k, Statistical Analysis, p < 0.001 for comparisons of OD distribution absent FHF to with FHF1A for ΔKPQ, E[1784]K, S[1904]L, Q[1909]R, IQ/AA, and Δ1810 mutants by KS-test.

Source data

Extended Data Fig. 6 Relative expression profiles of FHF1-4 isoforms/splice-variants in iPSC-CMs.

a, Analysis of relative expression of FHF1-4 isoforms/splice-variants using RT-qPCR demonstrate FHF1A, FHF1B, and FHF2S as the major isoforms in healthy-donor iPSC-CMs. We observed that FHF2S expression was ~ 3-fold higher than FHF1B in the healthy-donor iPSC-CMs while FHF1A was ~ 45% lower than FHF1B. Each bar and error, mean ± SEM. n = 3 independent cultures. (see Supplementary Table 1). b, In the LQTS3 (ΔKPQ) iPSC-CMs, FHF1B is the major isoform/splice-variant with ~ 50% lower level of FHF1A and ~ 20% lower level of FHF2S. The relative prevalence of FHF1B in the LQTS3 (ΔKPQ) iPSC-CMs is consistent with the high baseline INa,L obtained in functional studies in Fig. 5. Each bar and error, mean ± SEM. n = 5 independent cultures. (see Supplementary Table 1).

Source data

Extended Data Fig. 7 Identifying a minimal FHF1A domain capable of inhibiting INa,L.

a, Sequence alignment of various FHF1A amino-terminal peptides utilized to identify a minimal effector domain. b, Schematic illustrates potential interaction between the peptides and NaV1.5 channel. c, Exemplar multichannel recordings from NaV1.5 IQ/AA mutant show increase late channel openings when co-expressed with 1-18 (panel c), 18-39 (panel d), 12-17 (panel e), or 9-32 (panel f) peptides. g, Ranolazine yields a modest inhibition of INa,L for NaV1.5 IQ/AA mutant. h-i, Both FixR (panel h) and ranolazine (panel i) inhibits INa,L for NaV1.5 S1904L mutant channel. j, Bar graph shows Rpersist for NaV1.5 S1904L mutant channel in the presence of FixR or ranolazine. Black dashed line, INa,L for NaV1.5 S1904L at baseline. Blue dashed line, INa,L for NaV1.5 S1904L co-expressed with FixR. Each bar and error, mean ± SEM. n = 10 cells (557 sweeps). Statistical Analysis, one-way ANOVA followed by Dunnet’s multiple-comparisons test ***p < 0.001. k, FixR also strongly inhibits INa,L for NaV1.5 Δ1885 mutant channel. l-m, Adenoviral expression of both GFP (panel l) and FixR (panel m) yielded no change in INa,L in iPSC-CMs derived from healthy donors (HD).

Source data

Extended Data Fig. 8 Mutations in the long-term inactivation particle preserves FHF1A regulation of INa,L.

a-b, Exemplar recordings of NaV1.5 IQ/AA mutant channel heterologously expressed in HEK293 cells in the presence of either FHF1A L9A mutant (panel a), or FHF1A R11A mutant (panel b). These conserved residues have been previously shown to be critical for long-term inactivation. c, Bar graph summarizes changes in Rpersist upon co-expression of the different FHF1A mutant variant. Each bar and error, mean ± SEM. For FHF1A L9A mutant, n = 8 cells (503 sweeps) and for FHF1A R11A mutant, n = 8 cells (494 sweeps). Statistical Analysis, Kruskal-Wallis test followed by Dunn’s multiple comparisons test: ***p < 0.001; for each mutant compared to no FHF1A.

Source data

Extended Data Fig. 9 Both FHF1A and FixR have minimal effect on peak current density and steady-state inactivation of mutant NaV1.5.

a, Exemplar current recordings for NaV1.5 IQ/AA mutant channels elicited in response to a family of voltage steps from −60 to +50 mV from a holding potential of −120 mV. b, Population data shows average peak current density (Jpeak) – voltage relationship for IQ/AA mutant. Each dot, mean ± SEM with n denoted in parenthesis. c, Steady-state inactivation curve (h∞(V) curve) elicited using step-depolarizations from a holding potential of −120 mV for IQ/AA mutant. Each dot, mean ± SEM with n denoted in parenthesis. d-f, Overexpression of FHF1A does not appreciably alter peak current density (panel e) and steady-state inactivation (panel f) compared to that measured at baseline. g-i, Overexpression of FixR does not appreciably alter peak current density (panel h) and steady-state inactivation (panel i).

Source data

Extended Data Fig. 10 Extended characterization of cell-permeable FixR.

Representative flow cytometric data shows the percentage of cellular uptake of FixR (FITC positive) into HEK293 cells and effect on cytotoxicity determined using a far red (APC) DEAD cell stain (Invitrogen). Cells are incubated with varying concentrations of FixR-cpp for 2 hours. Appreciable cellular uptake is observed between 5 and 25 μM. Minimal cell death is observed in the presence of up to 10 μM FixR-cpp. b, Additional epifluorescence and brightfield images show FixR-cpp uptake into freshly-dissociated cardiomyocytes from IQ/AAtg mice (n = 3 mice). Scale bar, 10 μm.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Tables 1–2.

Reporting Summary

Source data

Source Data Fig. 1

Statistical source.

Source Data Fig. 2

Statistical source.

Source Data Fig. 3

Statistical source.

Source Data Fig. 4

Statistical source.

Source Data Fig. 5

Statistical source.

Source Data Fig. 6

Statistical source.

Source Data Fig. 7

Statistical source.

Source Data Extended Data Fig. 2

Statistical source.

Source Data Extended Data Fig. 5

Statistical source.

Source Data Extended Data Fig. 6

Statistical source.

Source Data Extended Data Fig. 7

Statistical source.

Source Data Extended Data Fig. 8

Statistical source.

Source Data Extended Data Fig. 9

Statistical source.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chakouri, N., Rivas, S., Roybal, D. et al. Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current. Nat Cardiovasc Res 1, 1–13 (2022). https://doi.org/10.1038/s44161-022-00060-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s44161-022-00060-6

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing