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.
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 Fhf1KO (Fhf1 knockout) mice did not show differences in ECG parameters under baseline conditions. Previously, we showed that Fhf2KO (Fhf2 knockout) mice demonstrate atrial and ventricular conduction abnormalities when challenged with hyperthermic stress.2 At elevated temperatures, Fhf1KO mice demonstrated P-wave prolongation while Fhf1WT mice showed no change (Figure [F], top; Fhf1KO, 18.24 [17.12–18.59] ms versus Fhf1WT, 16.31 [15.87–16.83] ms). No atrial arrhythmias were observed. Fhf1KO mice did not exhibit QRS prolongation with temperature elevation (Figure [F], bottom; Fhf1KO, 11.89 [11.60–13.11] ms versus Fhf1WT, 11.67 [11.08–12.10] ms). The susceptibility to atrial conduction slowing in both Fhf1KO and Fhf2KO 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. Fhf1KO atrial myocytes showed diminished peak sodium current (INa) density at −40 mV compared with Fhf1WT and Fhf2KO atrial myocytes. There was no difference in peak INa density between Fhf1WT and Fhf2KO atrial myocytes (Figure [G]; Fhf1KO, 32.07 pA/pF ±1.80 versus Fhf1WT, 43.36 pA/pF ±2.97 versus Fhf2KO, 41.26 pA/pF ±2.32; n=10–12 cells per genotype). Fhf2KO atrial myocytes demonstrated a nonsignificant hyperpolarizing shift in V1/2 steady-state inactivation compared with Fhf1KO and Fhf1WT atrial myocytes, reflecting the lower abundance of Fhf2 in atrial compared with ventricular myocytes.4 (Figure [H]; Fhf1KO, −89.40 mV ±1.23 versus Fhf1WT, −89.34 mV ±1.06 versus Fhf2KO, −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 INa density at −25 mV compared with FHFWT and Fhf2KO, with no additional reduction in peak INa density in FHF1,2KO iPSC-CMs (Figure [I]; Fhf1KO, 77.90 pA/pF ±11.5 versus Fhf2KO, 146.5 pA/pF ±20.2 versus FHF1,2KO, 73.86 pA/pF ±7.57 versus FHFWT, 160.7 pA/pF ±20.4; n=14–16 cells per genotype). Loss of FHF2 led to a hyperpolarizing shift in V1/2 steady-state inactivation compared with FHFWT and Fhf1KO, with no additional shift in inactivation in FHF1,2KO iPSC-CMs (Figure [J]; Fhf1KO, −78.0 mV ±0.86 versus Fhf2KO, −92.9 mV ±1.21 versus FHF1,2KO, −93.1 mV ±0.85 versus FHFWT, −80.8 mV ±1.08; n=14–16 cells per genotype). The absence of synergistic effects on INa in FHF1,2KO 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 INa 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
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.
Sources of Funding
This study was supported by the National Institutes of Health grants (R01HL142498 to Dr Fishman and T32 GM066704 and F31 HL132438 to Dr Shekhar) and a Foundation Leducq Transatlantic Network of Excellence Award (Drs Fishman and Park).
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