B-po01-006 contrasting mechanisms of reduced membrane excitability in fhf1 and fhf2 deficient cardiomyocytes

Fibroblast homologous factors (FHFs) are potent regulators of sodium channels in excitable tissues. FHF gene mutations have been implicated in human diseases with altered sodium currents, including Brugada syndrome, idiopathic ventricular tachycardia, and febrile onset seizures. Although murine models of FHF2 deficiency have revealed its essential role in maintaining sodium channel availability in the mouse heart, the applicability of these findings to human cardiac biology is less clear as FHF1 is the dominant isoform expressed in the human heart. Characterizing FHF1 versus FHF2-dependent effects on the cardiac sodium channel in mouse and human cardiomyocytes is essential for therapeutic drug design. Here we set out to determine the biophysical effects of FHF1 versus FHF2 on sodium channels in murine and human cardiomyocytes. Sodium currents from Fhf1KO atrial cardiomyocytes were studied using a whole cell patch clamp configuration and compared to both wildtype and Fhf2KO atrial cardiomyocytes. In addition, human induced pluripotent stem cell (iPSC) derived cardiomyocytes with individual and combined FHF1 and FHF2 loss were created and similarly studied with whole cell patch clamp. Atrial myocytes taken from Fhf1KO hearts show diminished current density as compared to Fhf2KO and wildtype atrial myocytes, the latter of which do not differ from one another. Fhf2KO atrial myocytes demonstrated a hyperpolarizing shift in sodium channel steady-state inactivation consistent with previously published data. Our novel human iPSC derived cardiomyocytes demonstrated similar findings to murine cells, with isolated loss of FHF1 leading to reduced current density and loss of FHF2 leading to a left shift of steady state inactivation. Combined FHF1/FHF2 loss did not reveal synergistic effects but afforded a phenotype that was a summation of each of the individual genotypes. These data show the contrasting mechanisms by which FHF1 and FHF2 affect membrane excitability. While FHF2 exerts its effects on sodium channel inactivation, FHF1 is responsible for preserving current density with minimal effect on gating.