FGF13 regulates cardiomyocyte impulse propagation via Cx43 trafficking independent of voltage-gated sodium channels

  1. Lala Tanmoy Das
  2. Mattia Malvezzi
  3. Aravind Gade
  4. Maiko Matsui
  5. Margaret McKay
  6. Eric Q. Wei
  7. Matea J. Zelich
  8. Keon Mazdisnian
  9. Jared Kushner
  10. Bi-xing Chen
  11. Isabella DiStefano
  12. Daniel Roybal
  13. Lin Yang
  14. Lisa Stoll
  15. James C. Lo
  16. Marian Kalocsay
  17. Fadi G. Akar
  18. Steven O. Marx
  19. Geoffrey S. Pitt
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Abstract

Background

Fibroblast growth factor homologous factor (FHF) variants associate with arrhythmias. Although FHFs are best characterized as regulators of voltage gated sodium channel (VGSC) gating, recent studies suggest broader, non-VGSC-related functions, including regulation of Cx43 gap junctions and/or hemichannels, mechanisms that have generally been understudied or disregarded.

Methods

We assessed cardiac conduction and cardiomyocyte action potentials in mice with constitutive cardiac-specific Fgf13 ablation (cFgf13KO ) while targeting Cx43 gap junctions and hemichannels pharmacologically. Using immunostaining and biochemistry, we characterized FGF13 regulation of Cx43 abundance and subcellular distribution. With proximity labeling proteomics, we investigated novel candidate mechanisms underlying FGF13 regulation of Cx43.

Results

FGF13 ablation prolonged the QRS and QT intervals on the surface electrocardiogram. Carbenoxolone, a Cx43 gap junction uncoupler, markedly prolonged the QRS duration leading to conduction system block in cFgf13KO but not in WT mice. Optical mapping revealed markedly decreased conduction velocity (CV) during ventricular pacing. Microscopy revealed markedly perturbed trafficking of Cx43, reduced localization in the intercalated disc, and suggested decreased membrane Cx43 but increased Cx43 hemichannels in cardiomyocytes from cFgf13KO mice. Resting membrane potential (RMP) was depolarized and APD50 was prolonged in cFgf13KO cardiomyocytes. Both were restored towards wildtype (WT) values with Gap19 (a Cx43 hemichannel inhibitor), expression of FGF13, or expression of a mutant FGF13 incapable of binding to VGSCs, emphasizing VGSC-independent regulation by FGF13. To assess the functional impact of RMP depolarization, hearts were subjected to hypokalemia, which had no effect in WT hearts but fully rescued CV in cFgf13KO hearts. Proteomic analyses revealed candidate roles for FGF13 in the regulation of vesicular-mediated transport. Biochemistry and immunocytochemistry showed that FGF13 ablation destabilized microtubules and reduced the expression of tubulins and MAP4, the major cardiac microtubule regulator.

Conclusions

FGF13 regulates microtubule-dependent trafficking and targeting of Cx43, thereby impacting cardiac impulse propagation via VGSC-independent mechanisms.

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