Differential regulation of cardiac sodium channels by intracellular fibroblast growth factors

Figure S1.. Targeted disruption of the Fgf13 locus and generation/validation of cardiac-specific Fgf13 targeted deletion mice. (A) Schematics of the endogenous Fgf13 locus (top), located on the X chromosome, and the floxed Fgf13 locus (middle). Following sequencing of the first and second generation of offspring, heterozygous Fgf13 floxed (Fgf13fl/+) female mice and hemizygous Fgf13 floxed (Fgf13fl/y) male mice were crossed to also generate homozygous Fgf13 floxed (Fgf13fl/fl) females. The Fgf13fl/y and Fgf13fl/fl animals were then crossed with transgenic mice expressing Cre recombinase driven by the cardiac-specific α-MHC promoter. Crossing the Cre recombinase positive, hemizygous floxed male (Fgf13fl/y) offspring with Fgf13fl/fl females (or Cre recombinase heterozygous Fgf13fl/+ females with Fgf13fl/y males) provided cardiac-specific Fgf13 targeted deletion hemizygous male and homozygous female animals, referred to collectively here as cardiac-specific Fgf13 knockouts, cFgf13KO. Three sets of primers were employed to detect the 5′ LoxP site, the 3′ LoxP site, and the α-MHC-driven Cre-recombinase (see Materials and methods). (B) Representative results from PCR analyses of homozygous…

Figure S2.. Loss of Fgf13 or Fgf12 does not measurably affect the expression levels of other Fgf transcripts in adult mouse LV. The expression levels of the Fgf11, Fgf12A, Fgf12B, Fgf13S(A), Fgf13VY, Fgf13U(B), Fgf14A, and Fgf14B transcripts in WT, cFgf13KO, and Fgf12KO LV samples were measured and normalized to Hprt expression in the same sample, as described in Materials and methods. These analyses revealed that the Fgf13 transcripts are undetectable in cFgf13KO LV. In addition, the mean ± SEM relative expression levels of the other Fgf transcripts in cFgf13KO and WT LV are indistinguishable.

Figure 1.. Cardiac-specific deletion of Fgf13 (cFgf13KO) alters inactivation, but not activation, of INa in LV myocytes. (A) Representative INa waveforms recorded from LV myocytes isolated from WT (black) and cardiac-specific Fgf13 deletion (cFgf13KO; orange) mice; the voltage-clamp protocol is illustrated below the current records. (B) Mean ± SEM normalized conductances (G) of INa activation and inactivation were plotted as a function of voltage and fitted to single Boltzmann functions (solid lines). There is a marked hyperpolarizing shift in the voltage-dependence of INa inactivation in cFgf13KO (orange) compared with WT (black) LV myocytes (P value <0.001; Table 1), whereas the voltage-dependences of INa activation are indistinguishable in cFgf13KO and WT LV myocytes (P value = 0.12; Table 1). Control experiments revealed that the voltage-dependences of INa activation and inactivation in Fgf13 floxed (grey) LV myocytes are indistinguishable from WT LV myocytes (P values are 0.11 and 0.97, respectively; Table 1). It was not possible, however, to conduct additional control experiments to independently determine if there are any of the previously reported cardiotoxic effects (Pugach et al.,…

Figure S3.. The amplitudes/densities of INa in mouse LV myocytes are not affected by the loss of iFGF13, and the properties of INa in mouse LV myocytes are not affected by the loss of iFGF12. (A) The cardiac-specific deletion of Fgf13 does not affect peak INa densities in LV myocytes: peak INa densities at all voltages in WT, Fgf13 floxed, and cFgf13KO mouse LV myocytes are not significantly different (P values >0.15 at all voltages). (B and C) Consistent with the negligible expression of Fgf12 transcripts in adult mouse LV (see Fig. S2), peak INa densities at all voltages (B) and the voltage-dependences of INa activation and inactivation (C) determined in adult Fgf12KO LV myocytes are indistinguishable (P values >0.13) from INa in WT LV myocytes at all voltages.

Figure S4.. The time-dependence of AAV9-mediated expression of iFGF12B and eGFP in adult mouse LV and the validation of the anti-iFGF12 antibody. (A and B) Western blots of fractionated protein lysates prepared from cFgf13KO ventricles 2, 3, or 4 wk following retro-orbital injections of a 1:1 mixture of the hFGF12B-expressing and eGFP-expressing AAV9 viruses, probed with an anti-EGFP (A) or anti-iFGF12 (B) antibody, as described in Materials and methods. (C) To validate the anti-iFGF12 antibody used in the Western blot in B, protein lysates prepared from WT and Fgf12KO adult mouse left and right atria were fractionated, transferred, and the membranes were probed with an anti-iFGF12 antibody, as described in Materials and methods. A prominent band at ∼20 kD was detected with the anti-iFGF12 antibody in the WT, but not in the Fgf12KO, atrial protein samples, consistent with the elimination of iFGF12 proteins and validating the anti-iFGF12 antibody for Western blot analyses of iFGF12 protein expression. Source data are available for this figure: SourceData FS4.

Figure 2.. Expression of iFGF12B in cFgf13KO LV myocytes alters the voltage-dependence of INa activation and inactivation. (A) Representative INa waveforms recorded from cFgf13KO LV myocytes without (orange) and with (blue) iFGF12B expression; the voltage-clamp protocol is illustrated below the current records. (B) The expression of iFGF12B in cFgf13KO LV myocytes resulted in depolarizing shifts in INa activation and inactivation (blue) compared with cFgf13KO LV myocytes (orange; P values are <0.001 for both G-V activation and inactivation; Table 1). (C) Expression of iFGF12B in cFgf13KO LV myocytes also accelerated INa recovery from inactivation compared with the currents in both WT and cFgf13KO LV myocytes (P value = 0.04; Table 1). (D) The decay phases of the currents in cFgf13KO cells expressing iFGF12B (blue), however, are indistinguishable from cFgf13KO (orange) LV myocytes (inset). In addition, in 13 (of 19) cFgf13KO + iFGF12B-expressing LV myocytes, the decay phases of INa were best described by single exponentials with Ƭ values indistinguishable from Ƭf determined in WT cells, i.e., the slow component of INa decay was undetectable in these 13 cells. In the remaining (6 of 19)…

Figure 3.. Coexpression of iFGF12B modulates the time- and voltage-dependent properties of NaV1.5-encoded currents in Xenopus oocytes. (A) Representative human NaV1.5-encoded (INa) waveforms recorded from Xenopus oocytes expressing NaV1.5 alone (black) or together with iFGF12B (blue); the voltage-clamp protocol is illustrated below the current records. (B) The mean ± SEM normalized conductance-versus-voltage plots reveal marked depolarizing shifts in the voltage-dependences of activation and inactivation of the currents with iFGF12B coexpression (blue), relative to NaV1.5 expressed alone (black; P values are 0.012 and <0.001, respectively; Table 2). (C) The rate of INa recovery from inactivation is also accelerated with iFGF12B coexpression (P value = 0.04; Table 2). (D and E) Inset: Overlaying representative INa waveforms, recorded on membrane depolarizations to −20 mV, illustrates the difference in the decay phases of the currents recorded from Xenopus oocytes expressing NaV1.5 alone (black) or NaV1.5 with iFGF12B (blue). Analyses of the decay phases of the currents revealed that the time constants of the fast and slow components of INa decay (Ƭf and Ƭs, respectively; D) are…

Figure 4.. iFGF12B regulates the activation of the voltage sensor in repeat IV of NaV1.5. VCF recordings were obtained as described in Materials and methods. Representative fluorescence signals recorded from Xenopus oocytes expressing NaV1.5 alone (black) or with iFGF12B (blue) in response to 50-ms depolarizing voltage steps to −140, −80, −20, and 40 mV, are presented; only four fluorescence traces are shown for clarity. The mean ± SEM normalized steady-state fluorescence signals were plotted and fitted with a single Boltzmann function (solid lines). Coexpression of iFGF12B induced a hyperpolarizing shift in the VSD-IV F-V curve (P value <0.001, Table 3), but caused no change in the F-V curves of the other VSDs (see Table 3).

Figure 5.. Coexpression of iFGF13VY affects the voltage dependence of steady-state inactivation and the inactivation kinetics of NaV1.5-encoded currents in Xenopus oocytes. (A) Representative INa waveforms recorded from Xenopus oocytes expressing NaV1.5 alone (black) or with iFGF13VY (orange); the voltage-clamp protocol is illustrated below the current records. (B) The mean ± SEM conductance versus voltage plots reveal marked depolarizing shifts in the voltage dependence of inactivation with iFGF13VY coexpression compared with NaV1.5 expressed alone (P value <0.001; Table 2). The voltage dependence of INa activation, however, is not affected by iFGF13VY (P value = 0.3; Table 2). (C and D) Coexpression of iFGF13VY accelerated INa recovery from inactivation (C; P value <0.001; Table 2) and overlay of representative INa waveforms (D, inset), evoked at −20 mV, illustrates the difference in the decay phases of INa in recordings from Xenopus oocytes expressing NaV1.5 without (black) and with (orange) iFGF13VY. (E) Analyses of the decay phases of the currents revealed that the time constants, Ƭf and Ƭs, of the fast and slow components of INa decay, respectively, (D) are indistinguishable for the…

Figure 6.. Distinct modulation of NaV1.5 voltage sensor conformations with iFGF13VY coexpression. The F-V curves for all four VSDs of NaV1.5 recorded from Xenopus oocytes expressing NaV1.5 alone (black) or with iFGF13VY (orange) show that iFGF13VY affects the voltage-dependences of activation of VSD-I and VSD-IV. For VSD-I, iFGF13VY coexpression shifts the F-V curve toward depolarizing potentials (P value = 0.002; Table 3). For VSD-IV, the F-V curves show that iFGF13VY results in a steeper slope and a small shift toward more hyperpolarized membrane potentials relative to NaV1.5 expressed alone (P values are 0.022 and <0.001, respectively; Table 3). The effect on the VSD-IV F-V curve with iFGF13VY coexpression is distinct from that observed with iFGF12B coexpression (compare with Fig. 4).

Figure 7.. Analysis of iFGF12B, iFGF13VY, and iFGF12B/13VY chimeras reveals a strong correlation between the slopes of the VSD-IV F-V curves and the slow component of INa inactivation. (A) Alignment of the iFGF12B and iFGF13VY sequences highlights the prominent difference in N-termini. (B–D) Several iFGF12B/13VY chimeras were constructed by swapping the N-termini, C-termini, and both C- and N-termini domains. Each chimera was coexpressed with NaV1.5, and effects on the time-and voltage-dependent properties of INa and VSD-IV activation were determined. Correlation and linear regression analyses were performed on these results obtained from iFGF chimeras, iFGF12B, and iFGF13VY. (B and C) The analyses revealed that the slow time constants of INa decay (Ƭs; B) and the fractional amplitudes of the slow component (%Aslow; C), determined for currents evoked at 0 mV, are both strongly (negatively [B] or positively [C]) correlated with the VSD-IV F-V k values. (D) The k and V1/2 values, determined from the VSD-IV F-V curves, can be used to predict %Aslow.

Figure S5.. Switching N-terminal domains between iFGF12B and iFGF13VY is not sufficient to replicate the iFGF-specific modulation of NaV1.5 gating and function. (A) Schematics of iFGF12B, iFGF13VY, and the iFGF chimeras generated with the iFGF12B and iFGF13VY N-termini exchanged (iFGF12B/13 and iFGF13VY/12). (B) Normalized inactivation, G-V, and VSD-IV F-V curves for NaV1.5 expressed alone (grey dashed line), with iFGF12B (blue dashed line), iFGF13VY (orange dashed line), or one of the iFGF chimeras, FGF12B/13 (blue triangle) or FGF13VY/12 (orange diamond). The effects of the two iFGF chimeras on the G-V and F-V curves are distinct from those observed with either iFGF12B or iFGF13VY, implying that the N-termini of iFGF12B and iFGF13VY alone do not confer iFGF-specific regulation of NaV1.5.