Contrasting Ionic Mechanisms of Impaired Conduction in FHF1- and FHF2-Deficient Hearts

Proper function of the cardiac sodium channel is essential for normal membrane excitability and conduction in the heart. FHFs (fibroblast growth factor homologous factors) 1–4 bind to the C-terminal domains of voltage-gated sodium channels, regulating channel trafficking and gating properties.¹ FHF2 is the predominant family member expressed in the mouse ventricle, with knockout mice displaying cardiac conduction disease.² In contrast, FHF1 is the predominant family member expressed in mouse atria (Figure [A through D]) and human atrial and ventricular myocardium³ (Figure [E]). Clinically, mutations in FHF1 have been linked to both idiopathic ventricular tachycardia⁴ and Brugada syndrome.⁵ Furthermore, reductions in FHF1 expression have been observed in diseased left atrial and left ventricular tissue,³ while FHF2–4 levels are not significantly changed (Figure [E]). Thus, dysregulated FHF1 activity may play a mechanistic role in both heritable and acquired arrhythmic disorders. Accordingly, using complementary in vivo murine models and human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), we sought to determine the distinct consequences of FHF1 deficiency on cardiac electrophysiology.

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All protocols conformed to the Association for the Assessment and Accreditation of Laboratory Animal Care and the New York University Grossman School of Medicine Animal Care and Use Committee. The data that support the findings of this study are available from the corresponding author on reasonable request.

We first determined the effects of Fhf1 deficiency on cardiac conduction parameters using electrocardiography. Adult Fhf1^KO (Fhf1 knockout) mice did not show differences in ECG parameters under baseline conditions. Previously, we showed that Fhf2^KO (Fhf2 knockout) mice demonstrate atrial and ventricular conduction abnormalities when challenged with hyperthermic stress.² At elevated temperatures, Fhf1^KO mice demonstrated P-wave prolongation while Fhf1^WT mice showed no change (Figure [F], top; Fhf1^KO, 18.24 [17.12–18.59] ms versus Fhf1^WT, 16.31 [15.87–16.83] ms). No atrial arrhythmias were observed. Fhf1^KO mice did not exhibit QRS prolongation with temperature elevation (Figure [F], bottom; Fhf1^KO, 11.89 [11.60–13.11] ms versus Fhf1^WT, 11.67 [11.08–12.10] ms). The susceptibility to atrial conduction slowing in both Fhf1^KO and Fhf2^KO mice suggests nonredundant functions of these 2 FHF family members on sodium channel behavior in atrial myocytes.

To explore distinct regulatory functions of FHF1 and FHF2 on the cardiac sodium channel in atrial myocytes, we performed whole-cell patch clamp. Fhf1^KO atrial myocytes showed diminished peak sodium current (I_Na) density at −40 mV compared with Fhf1^WT and Fhf2^KO atrial myocytes. There was no difference in peak I_Na density between Fhf1^WT and Fhf2^KO atrial myocytes (Figure [G]; Fhf1^KO, 32.07 pA/pF ±1.80 versus Fhf1^WT, 43.36 pA/pF ±2.97 versus Fhf2^KO, 41.26 pA/pF ±2.32; n=10–12 cells per genotype). Fhf2^KO atrial myocytes demonstrated a nonsignificant hyperpolarizing shift in V_1/2 steady-state inactivation compared with Fhf1^KO and Fhf1^WT atrial myocytes, reflecting the lower abundance of Fhf2 in atrial compared with ventricular myocytes.⁴ (Figure [H]; Fhf1^KO, −89.40 mV ±1.23 versus Fhf1^WT, −89.34 mV ±1.06 versus Fhf2^KO, −92.58 mV ±1.13; n=10 cells per genotype).

To investigate whether functional differences of FHF1 and FHF2 are conserved in human cardiomyocytes, we generated iPSC-CMs engineered with single and double knockout of FHF1 and FHF2. iPSC-CMs express both FHF1 and FHF2 before genetic modification (data not shown). Loss of FHF1 led to significantly reduced peak I_Na density at −25 mV compared with FHF^WT and Fhf2^KO, with no additional reduction in peak I_Na density in FHF1,2^KO iPSC-CMs (Figure [I]; Fhf1^KO, 77.90 pA/pF ±11.5 versus Fhf2^KO, 146.5 pA/pF ±20.2 versus FHF1,2^KO, 73.86 pA/pF ±7.57 versus FHF^WT, 160.7 pA/pF ±20.4; n=14–16 cells per genotype). Loss of FHF2 led to a hyperpolarizing shift in V_1/2 steady-state inactivation compared with FHF^WT and Fhf1^KO, with no additional shift in inactivation in FHF1,2^KO iPSC-CMs (Figure [J]; Fhf1^KO, −78.0 mV ±0.86 versus Fhf2^KO, −92.9 mV ±1.21 versus FHF1,2^KO, −93.1 mV ±0.85 versus FHF^WT, −80.8 mV ±1.08; n=14–16 cells per genotype). The absence of synergistic effects on I_Na in FHF1,2^KO iPSC-CMs indicates that FHF1 and FHF2 have functionally distinct effects on the cardiac sodium channel in both mouse and human cardiomyocytes.

In summary, our data indicate that FHF1 and FHF2 are key sodium channel regulatory proteins that influence excitability and conduction through different ionic mechanisms. FHF1 reduces sodium conductance through its effects on peak I_Na density, whereas FHF2 exerts its major effects on conduction through changes in sodium channel inactivation. These results indicate that species-specific differences in cardiac sodium channel function are influenced by which FHF family member is dominant. Further, given that FHF1 expression is significantly reduced in the failing human atrial and ventricular tissue, our data suggest that FHF1 deficiency, through its effects on sodium currents, may be clinically operative and contribute to conduction slowing and the heightened arrhythmia burden observed in diseased hearts.³

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Acknowledgments

The authors thank Dr Cynthia A. Loomis for her assistance with conducting RNA scope experiments. They would also like to thank Fang-Yu Liu and Jie Zhang for their technical assistance. The authors would also like to thank the Genome Technology Center (GTC) for expert library preparation and sequencing, and the Applied Bioinformatics Laboratories (ABL) for providing bioinformatics support and helping with the analysis and interpretation of the data. GTC and ABL are shared resources partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. This work has used computing resources at the NYU School of Medicine High Performance Computing (HPC) Facility.

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