Fibroblast Growth Factor Homologous Factors Modulate Cardiac Calcium ChannelsNovelty and Significance
Rationale: Fibroblast growth factor (FGF) homologous factors (FHFs; FGF11–14) are intracellular modulators of voltage-gated Na+ channels, but their cellular distribution in cardiomyocytes indicated that they performed other functions.
Objective: We aimed to uncover novel roles for FHFs in cardiomyocytes, starting with a proteomic approach to identify novel interacting proteins.
Methods and Results: Affinity purification of FGF13 from rodent ventricular lysates followed by mass spectroscopy revealed an interaction with junctophilin-2, a protein that organizes the close apposition of the L-type Ca2+ channel CaV1.2 and the ryanodine receptor 2 in the dyad. Immunocytochemical analysis revealed that overall T-tubule structure and localization of ryanodine receptor 2 were unaffected by FGF13 knockdown in adult ventricular cardiomyocytes but localization of CaV1.2 was affected. FGF13 knockdown decreased CaV1.2 current density and reduced the amount of CaV1.2 at the surface as a result of aberrant localization of the channels. CaV1.2 current density and channel localization were rescued by expression of an shRNA-insensitive FGF13, indicating a specific role for FGF13. Consistent with these newly discovered effects on CaV1.2, we demonstrated that FGF13 also regulated Ca2+-induced Ca2+ release, indicated by a smaller Ca2+ transient after FGF13 knockdown. Furthermore, FGF13 knockdown caused a profound decrease in the cardiac action potential half-width.
Conclusions: This study demonstrates that FHFs not only are potent modulators of voltage-gated Na+ channels but also affect Ca2+ channels and their function. We predict that FHF loss-of-function mutations would adversely affect currents through both Na+ and Ca2+ channels, suggesting that FHFs may be arrhythmogenic loci, leading to arrhythmias through a novel, dual-ion channel mechanism.
- arrhythmias, cardiac
- cardiac electrophysiology
- fibroblast growth factors
- ion channels/membrane transport
Despite an ever-growing understanding of ion channel structure, function, and regulation, many components of the macromolecular complexes anchored by ion channels are not yet known or well characterized. Identification of these channel-interacting proteins and discovery of their functions within the channelsome provide important insight into physiological and pathological function. Mutations in newly defined channel-interacting proteins often explain genetic causes of arrhythmias in cases in which mutations in known arrhythmia loci are not found. Fibroblast growth factor (FGF) homologous factors (FHFs), a subfamily of FGF proteins (FGF11–14) expressed predominantly in excitable cells,1 are prime examples of channel-interacting proteins for which cardiac functions are not well understood. Although part of the FGF superfamily, FHFs do not function as growth factors and are incapable of activating FGF receptors.2 Rather, FHFs remain intracellular and have been shown to bind and modulate voltage-gated Na+ channels.3,4 Their roles as Na+ channel regulators have been studied most extensively in the brain, driven in large part by the identification of FGF14 as the locus for spinocerebellar ataxia and by observations that Fgf14−/− mice display an ataxia phenotype that correlates with decreased Na+ channel function and diminished neuronal excitability.5,6
FHFs are also expressed in cardiomyocytes, but their roles in regulating cardiac function have heretofore received less attention. We showed that FGF13, the most highly expressed FHF in murine heart, directly binds NaV1.5, the predominant cardiac Na+ channel, and participates in trafficking NaV1.5 to the sarcolemmal membrane and modulating Na+ channel kinetics.7–9 Consistent with these regulatory roles for Na+ channels, knockdown of FGF13 led to a reduction in conduction velocity and maximum capture rate (the ability of the cells to recover from a stimulus of a specific speed) in a neonatal rat ventricular cardiomyocyte monolayer.7
Several lines of evidence suggest that the effects of FHFs in excitable cells extend beyond Na+ channel modulation. For example, the complex changes in synaptic physiology in Fgf14−/− mice are not consistent with a defect limited to Na+ channel dysfunction.10 Indeed, we recently found that FGF14 knockdown in cerebellar granule cells reduced presynaptic Ca2+ currents and synaptic transmission at the granule cell to Purkinje cell synapse.11 Moreover, as we reported,7 the cellular distribution of FHFs in ventricular cardiomyocytes extended beyond the distribution of NaV1.5 channels. We therefore aimed to determine novel roles for FHFs in cardiomyocytes by looking for new FHF interactors. Here, we report the discovery that junctophilin-2 (JPH2) interacts with FGF13 in rodent ventricular myocytes. JPH2 is a protein responsible for coordinating the interaction of the sarcolemma and sarcoplasmic reticulum in the dyad. We therefore investigated the role of FGF13 in regulating ionic currents in the dyad and showed that FGF13 has essential roles in regulating the L-type, voltage-gated Ca2+ channel (CaV1.2) currents in adult ventricular myocytes. These results provide, to the best of our knowledge, the first evidence that FHFs modulate ion channels other than voltage-gated Na+ channels in cardiomyocytes and highlight previously unknown modulatory roles for FHFs in cardiac physiology such as regulation of Ca2+-induced Ca2+ release (CICR) and the integrated electric activity of the ventricular action potential. Our results lead to the hypothesis that loss-of-function mutations in FHFs could underlie inherited cardiac arrhythmias.
Detailed Methods are provided in the Online Data Supplement.
The adenoviruses expressing FGF13 shRNA or scrambled shRNA with green fluorescent protein (GFP) have been previously described.7 FGF13 rescue viruses and the shRNA virus with the GFP removed were generated similarly using the AdEasy System (Agilent).
Animals were handled according to National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study was approved by Duke University Animal Care and Welfare Committee. Cardiomyocytes were isolated from 6- to 8-week-old C57/Bl6 mice or Sprague-Dawley rats and cultured as previously described.7
Immunocytochemistry and T-Tubule Staining
Immunocytochemistry methods have been previously described.7 Imager/analyzer was blinded to the manipulation, and all cells imaged were used for analysis. For T-tubule staining, cells were plated onto glass-bottom plates (MatTek Corp) and cultured as above. Cells were then incubated in 0.5 µmol/L di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS, Life Technologies) and were imaged live.
Image Processing and Fast Fourier Transform
The experimenter analyzing images was blinded to treatment. Stacks were deconvolved using Hyugens software (Scientific Volume Imaging) and exported as Tiff files. Voxel colocalization was performed on deconvolved images using the Pearson correlation coefficient. Channel localization analysis was performed in ImageJ (National Institutes of Health) and fast Fourier transformed using OriginLab software.
Simultaneous Patch Clamping and Ca2+ Transient Recording
Cardiomyocytes were plated onto glass-bottom plates (MatTek Corp) and cultured as above. Viruses expressing GFP were not used as GFP interferes with the Fura-2 emission. Whole-cell patch clamping was performed as above with the addition of 150 µmol/L Fura-2 pentapotassium salt (Life Technologies) and the removal of EGTA in the pipette solution. For Ca2+ transient peak measurement, autofluorescence and background emissions were first subtracted and the peak was measured as the difference from the baseline using IonWizard software (IonOptix). Excitation-contraction coupling gain was defined as the Ca2+ transient peak divided by the Ca2+ current peak.
Sarcoplasmic Reticulum Load Measurements
Sarcoplasmic reticulum (SR) load was measured using 0.25 µmol/L Fura-2 AM–loaded cardiomyocytes as previously described.14
Fresh adult mouse ventricular heart lysate was prepared by homogenizing tissue and immunoprecipitating FGF13 as previously described.7 After immunoprecipitation, samples were subjected to SDS-PAGE, and coimmunoprecipitation was verified by Western blot.
Antibody (20 µg anti-FGF13 or control IgG) was irreversibly crosslinked to protein A/G agarose beads (Santa Cruz Biotechnology), and ventricular tissue lysate (≈23 mg total protein) was added to crosslinked beads. The bound proteins were eluted in 400 μL of 0.2% Rapigest SF Surfactant (Waters) in 50 mmol/L ammonium bicarbonate and subjected to an in-solution tryptic digestion at the Duke Proteomics Core Peptide. Identifications were determined using liquid chromatography/tandem mass spectrometry; after data acquisition, all spectra were searched against the SwissProt database with the mouse taxonomy selected.
Biotinylation and Western Blotting
Surface biotinylation and Western blotting were performed as previously described.7
Results are presented as mean±SE of the mean. Statistical significance of differences between groups was assessed using 1-way ANOVA and was set at P<0.05.
JPH2 Is in Complex With FGF13
Although we previously showed that FGF13 is an intracellular modulator of voltage-gated Na+ channels,7 close analysis of FGF13 immunocytochemistry in adult rat cardiomyocytes revealed an overall distribution that extended beyond what has been reported for NaV1.5 in heart.15 Specifically, we observed a striated pattern similar to a T-tubule distribution (Figure 1A, inset), in addition to the sarcolemmal and nuclear distribution we previously reported.7 This led us to hypothesize that FGF13 may have roles in cardiomyocyte physiology beyond NaV1.5 regulation. To identify other potential FGF13 interactors, we performed immunoprecipitation using a previously validated FGF13 antibody or an IgG (as a control) from adult ventricular tissue and liquid chromatography/mass spectrometry analysis of the immunoprecipitated FGF13 protein complex. One interesting candidate we identified was JPH2. As shown in Figure 1B, we identified 4 unique peptides spanning all soluble domains of JPH2. We found this candidate to be of interest because it is a protein responsible for properly juxtaposing CaV1.2 and the ryanodine receptor 2 (RyR2) in the T tubule (Figure 1B), consistent with the T-tubular distribution of FGF13. The specificity of the mass spectrometry approach was confirmed through the identification of 3 FGF13 peptides after FGF13 immunoprecipitation but none with the control IgG and 8 peptides (but none in the control IgG sample) from the known FGF13-binding partner NaV1.5 (see Online Table I for all peptide data). We further validated that FGF13 was in complex with JPH2 through coimmunoprecipitation, detecting JPH2 in FGF13 immunoprecipitates but not in the IgG control (Figure 1C).
FGF13 Knockdown Perturbs Dyad Organization by Affecting CaV1.2 Localization
JPH2 is a protein responsible for coordinating the interactions between the sarcolemma and the SR. JPH2 has a C-terminal transmembrane domain in the SR, and its N terminus interacts with the inner leaflet of the sarcolemma, providing a means to juxtapose the sarcolemma and SR so as to promote efficient CICR by apposing CaV1.2 and RyR2.16 On the basis of our discovery that FGF13 was a component of the JPH2 macromolecular complex, we tested whether FGF13 participated in dyad ion channel targeting. We performed immunocytochemistry on mouse ventricular myocytes that were uninfected (control) or had been infected with a FGF13 shRNA adenovirus targeting all FGF13 splice variants (FGF13 knockdown) or a scrambled shRNA adenovirus (SCR; Figure 2). As we previously showed7 (repeated in Figure 4), the knockdown adenovirus reduced FGF13 protein by ≈90%, and the SCR virus had no effect. In the control and SCR, we observed α1C, the CaV1.2 pore-forming subunit, colocalized with RyR2 in a striated pattern. Little α1C or RyR2 was observed between the striations. After FGF13 knockdown, however, we saw a loss of colocalization, and a large portion of α1C was now present between the RyR2 striations (Figure 2A; Pearson correlation coefficient of 0.52±0.03 versus 0.65±0.03 and 0.62±0.03 in control and SCR cardiomyocytes, respectively; P<0.05 for FGF13 knockdown versus control or SCR cardiomyocytes; n=8 cells per group).
We then performed quantitative analysis on the pattern of CaV1.2. In control adult mouse cardiomyocytes (Figure 2A) or after infection with the SCR virus (Figure 2B), α1C displayed the expected striated pattern with a periodicity of ≈2 µm, consistent with a T-tubular distribution.17 In contrast, the pattern in FGF13 knockdown cells was discontinuous, with multiple punctae found between the residual striations (Figure 2B). With the analyzer blinded to treatment status, we quantified the change in distribution with intensity profiles of α1C in confocal z stacks of nonnuclear areas, as shown in Figure 2B. In SCR cardiomyocytes, we detected a regular pattern of intense α1C staining at ≈2 µm intervals (analyzed by fast Fourier transform; Figure 2D), with almost no signal in the intervening intervals. In contrast, after FGF13 knockdown, the peak amplitude of α1C staining at the ≈2-μm interval was reduced by >50% (fast Fourier transform amplitudes [in arbitrary units]: control, 0.284±0.017 (n=8); SCR, 0.234±0.009 (n=8); FGF13 knockdown, 0.116±0.020 (n=12); P<0.001 for FGF13 knockdown versus SCR and control), and significant α1C signal was observed in between the peaks (Figure 2B and 2D).
We tested whether this altered α1C distribution was secondary to a general defect in T-tubule architecture or was more specific to α1C using 2 separate analyses. First, we performed immunocytochemistry for the sodium-calcium exchanger, another T-tubule–localized protein, and observed that FGF13 knockdown did not affect sodium-calcium exchanger distribution (Figure 2C and 2D). Second, the membrane-binding dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate revealed that T-tubular distribution was grossly unaffected by FGF13 knockdown (Figure 2E).
The specific change in CaV1.2 localization after FGF13 knockdown led us to hypothesize that FGF13, as a member of the JPH2 macromolecular complex anchored by JPH2, affected CaV1.2 targeting T tubules. We therefore tested whether CaV1.2 was a component of the JPH2 complex. Indeed, by coimmunoprecipitation, we were able to pull down the α1C, the pore-forming subunit of CaV1.2, with an antibody to JPH2 but not a control IgG (Figure 2F). These data are consistent with a previous observation that JPH2 interacts with Cav1.1 in skeletal muscle18 but are, to the best of our knowledge, the first demonstration of an interaction between JPH2 and an L-type Ca2+ channel in cardiac muscle. Together, these data indicated that FGF13 knockdown did not affect T-tubular or SR architecture, but FGF13 has a specific role in targeting CaV1.2 to its proper T-tubular location.
FGF13 Modulates CaV1.2 Trafficking to the Surface and Current Density
We hypothesized that this aberrant CaV1.2 localization would lead to a decrease in CaV1.2 at the surface. We therefore quantified the relative amount of CaV1.2 at the sarcolemma after FGF13 knockdown by labeling surface proteins with biotin and capturing them with avidin beads after cell lysis. Both the surface fraction and the whole-cell lysate of adult mouse ventricular myocytes were then probed with an antibody against the pore-forming α1C subunit of CaV1.2. These biotinylation experiments revealed a decrease in the amount of α1C at the surface after FGF13 knockdown (Figure 3A and 3B). The total amount of α1C (in the cellular lysate) was unchanged. To test whether this decreased CaV1.2 at the surface led to physiological changes, we measured Ca2+ channel current density using whole-cell patch clamp. In FGF13 knockdown cardiomyocytes but not the SCR cardiomyocytes, Ca2+ channel current density was reduced by 35% compared with control cardiomyocytes (Figure 3C and 3D). Channel availability, voltage dependence of activation, and steady-state inactivation were unaffected (Table). Together, these results suggest that FGF13 knockdown reduced Ca2+ current density by affecting the number of channels at the sarcolemma.
Human FGF13-VY Can Rescue CaV1.2 Current Density and Localization
To confirm a role for FGF13 in targeting CaV1.2 to the T tubules and regulating L-type Ca2+ channel current density, we performed rescue experiments on adult rat ventricular cardiomyocytes treated with FGF13 shRNA by coexpressing human shRNA-insensitive and Hisx6-tagged FGF13-VY (hFGF13-VY). FGF13 is differentially spliced at its N terminus,1 and we chose this FGF13 splice variant for rescue because it is the most highly expressed isoform in mouse and rat cardiomyocytes.7 The adenovirus expressing FGF13 shRNA also expressed GFP (via a cytomegalovirus promoter), and the adenovirus expressing hFGF13-VY also expressed monomeric red fluorescent protein, allowing us to identify cells in which hFGF13-VY was expressed in the context of endogenous FGF13 knockdown (Figure 4A). Immunostaining for endogenous FGF13 (with an anti-FGF13 antibody) and hFGF13-VY (with an anti-Hisx6 antibody) demonstrated not only effective knockdown of endogenous FGF13 but also that the expressed hFGF13-VY recapitulated the overall cellular distribution of endogenous FGF13 (Figure 4A), although the striated pattern was even more obvious when compared with endogenous FGF13. We suspected that the subtle differences in pattern reflected the specific distribution of hFGF13-VY compared with the distribution of all endogenous splice variants recognized by the antibody in control cells.
Having established the efficacy of the hFGF13-VY rescue, we examined CaV1.2 Ca2+ current density in adult rat ventricular myocytes using whole-cell patch clamp. As in mouse cardiomyocytes (Figure 3C), FGF13 knockdown reduced CaV1.2 Ca2+ channel current density (Figure 4B; Table). Expression of hFGF13-VY restored Ca2+ current density to wild-type levels (Figure 4B). We also noted a hyperpolarizing shift in steady-state inactivation compared with control cells (Table). The reasons for this are unclear but may result from the overexpression of the specific FGF13-VY splice variant in the context of the knockdown of other FGF13 splice variants, thus altering any counterbalancing effects imparted by these absent variants. Additionally, the cellular distribution of α1C was restored with FGF13-VY overexpression, as indicated by the overall pattern and by Fourier transform (Figure 4C and 4D). This rescue strategy therefore firmly established a role for FGF13 in targeting CaV1.2 to T tubules and provided a potent confirmation of the specificity of the FGF13 knockdown virus.
FGF13 Affects Ca2+ Transients But Preserves Excitation-Contraction Coupling Gain
The mislocalization of CaV1.2 after FGF13 knockdown, the newly described interaction between FGF13 and JPH2, and previous studies that defined clear roles for JPH2 in CICR14 prompted us to query whether FGF13 influenced excitation-contraction coupling gain. We, therefore, simultaneously recorded Ca2+ currents and Ca2+ transients in control, SCR, and FGF13 knockdown rat ventricular myocytes (Figure 5). Following a previously described protocol to ensure steady-state CaV1.2 function,14 we held the cells at −40 mV to inactivate Na+ channels, gave a 500-millisecond pulse to 0 mV to induce influx of Ca2+ through CaV1.2, and recorded it via whole-cell patch clamp. We simultaneously recorded Ca2+ transients using Fura-2 in the pipette internal solution. Representative traces are shown in Figure 5A. Knockdown of FGF13 not only reduced Ca2+ current through CaV1.2 (Figure 5B), as previously shown, but also led to decreased Ca2+ release from the SR (Figure 5C). Interestingly, it appeared that those channels that were at the surface were coupling with RyR2 appropriately because excitation-contraction coupling gain was not different between the groups. These changes were not due to decreased SR load as measured by rapid application of caffeine in Fura-2–loaded cells (peak height: 0.28±0.05 Δ340/380 (n=5), 0.32±0.03 Δ340/380 (n=12), and 0.29±0.03 Δ340/380 (n=14) for control, SCR, and knockdown, respectively; P=0.66). These data indicated that decreased Ca2+ transients after FGF13 knockdown were due at least partially to decreased current through CaV1.2 and are consistent with its discovered interaction with JPH2, a known regulator of this function.
FGF13 Affects Multiple Phases of the Cardiac Action Potential
Having established that FGF13 modulates not only voltage-gated Na+ channels7 but also CaV1.2 Ca2+ channels and Ca2+ transients, we hypothesized that loss of FGF13 would have measurable effects on the cardiac action potential. Therefore, using current clamp, we recorded evoked action potentials in control, SCR, or knockdown adult rat ventricular cardiomyocytes (Figure 6A–6D). In FGF13 knockdown cells, the action potential peak amplitude decreased by ≈20% (Figure 6A and 6B), consistent with the previously defined effects of FGF13 on the cardiac Na+ channel current.7 We also observed a shortening of the action potential half-width (Figure 6A and 6C; control, 18.60±3.34 milliseconds [n=9]; scrambled shRNA, 17.41±3.40 milliseconds [n=5]; FGF13 shRNA, 9.85±1.34 milliseconds [n=7]; P<0.05 for FGF13 knockdown versus SCR and control). These data are consistent with changes in phase 2 of the cardiac action potential that is mediated predominantly by CaV1.2 and implicate FGF13 as a potent regulator of Na+ and Ca2+ channels in cardiac myocytes, leading to changes in the cardiac action potential.
Since the initial identification of FHFs,1 their complete physiological roles have remained shrouded. Originally hypothesized to act as extracellular growth factors similar to canonical FGFs, these nonsecreted FHF proteins do not appear capable of activating FGF receptors.2 The subsequent discovery that FHFs are binding partners for the intracellular C terminus of voltage-gated Na+ channels3 provided context to appreciate how knockout of FGF14 might reduce neuronal excitability and cause ataxia in mice19 and to hypothesize mechanisms for spinocerebellar ataxia 27, for which FGF14 was identified as the genetic locus.5 Our recent identification of a role for FHFs in the regulation of cardiac Na+ channel function and conduction7 demonstrated cardiac-specific roles. Still, the cellular distribution of FGF13 in cardiomyocytes hinted at additional functions distinct from Na+ channel regulation.
Here, we show for the first time that FHFs regulate cardiac ion channels other than voltage-gated Na+ channels. Thus, these results fit well with our recent demonstration that FGF14 regulates presynaptic Ca2+ channels in cerebellar neurons.11 Specifically, we have demonstrated here that FHFs are part of the dyad macromolecular complex with JPH2 and CaV1.2. Through electrophysiological and molecular biological methods, we have demonstrated that FGF13 affects Ca2+ current density and targeting of CaV1.2 to T tubules. This effect on targeting is specific to CaV1.2, not a general effect on T-tubule structure, because sodium-calcium exchanger and RyR localization were not altered, nor was overall T-tubule structure perturbed by FGF13 knockdown. We hypothesize that the abnormal localization of CaV1.2 after FGF13 knockdown represents a defect in intracellular sorting of these proteins in cardiomyocytes. Missorting of CaV1.2 was rescued by overexpression of a human FGF13 splice variant, demonstrating the specificity of the FGF13 knockdown shRNA. These data not only provide the first evidence that FGF13 is required for proper targeting of CaV1.2 to the T tubule in cardiac muscle but also show that FGF13 knockdown had profound physiological effects on Ca2+ current density and Ca2+ cycling, culminating in a decrease in cardiac action potential amplitude and duration and a reduced Ca2+ transient amplitude in parallel with its reduction in CaV1.2 Ca2+ channel current.
The identification of JPH2 as an FGF13-interacting protein provides mechanistic insight to this effect. JPH2 is a structural protein found in the cardiac dyad, where the T tubule is juxtaposed to 1 terminal cisterna of the SR. The structure of JPH2 fixes the distance between the plasma membrane and SR for efficient CICR (Figure 1C). JPH2 contains a cytosolic alpha helical domain that is capped on either side by membrane interaction motifs. On the N terminus are multiple membrane occupation and recognition nexus motifs that interact with the inner leaflet of the sarcolemma. At the C terminus is a transmembrane domain that anchors JPH2 to the SR.16 Reminiscent of our results after FGF13 knockdown, JPH2 knockdown in cardiomyocytes affects CICR. This provides further support that the interaction between JPH2 and FGF13 that we observed is functionally relevant. Moreover, analogous to loss-of-function mutations in JPH2, we hypothesize that FGF13 mutations may lead to orphaned ryanodine receptors that are no longer apposed to CaV1.2 and thus may be associated with heart failure.20 Further studies in the appropriate model are necessary to test that hypothesis.
Nevertheless, the phenotypes after FGF13 and JPH2 knockdown are not identical. JPH2 knockdown did not decrease CaV1.2 Ca2+ current and has no reported effect on voltage-gated Na+ currents. Moreover, JPH2 is observed only in a striated pattern in cardiomyocytes, whereas we observed additional FGF13 throughout the cytoplasm and in the nucleus. Thus, the fraction of FGF13 that interacts with JPH2 likely represents only 1 component of the overall FGF13 pool and is likely also distinct from the fraction interacting with NaV1.5. Other potential FGF13 interactors such as microtubules in neurons have previously been reported,21 likely indicating additional FGF13 pools. The tubulin interaction site on FGF13 maps to a region in close proximity to where NaV1.5 interacts in our recent crystal structure.9 Thus, we predict that any FGF13 interacting with microtubules would be unable to bind NaV1.5 simultaneously, further underlining the concept of distinct FGF13 pools. Although we have so far been unable to observe high affinity binding between tubulin and FGF13, the possibility of such an interaction in cardiomyocytes is attractive, particularly in light of the demonstration that CaV1.2 is trafficked along microtubules via BIN1 in ventricular myocytes.22
In summary, this study identified FHFs as novel modulators of the cardiac L-type CaV1.2 Ca2+ channel and thereby significantly expands our understanding of the roles of these proteins. Our data have important clinical implications. FGF12, the dominant FHF in human heart (data not shown), is >60% homologous with mouse FGF13. Our data suggest that FGF12 loss-of-function mutations would decrease both Ca2+ and Na+ channel currents. Because loss-of-function mutations in NaV1.5 or CaV1.2 have been reported in Brugada syndrome,23,24 we specifically hypothesize that FGF12 should be explored as a candidate locus for Brugada syndrome. Only ≈30% of Brugada syndrome patients have an identified mutation,25 so loss-of-function mutations in FGF12 may underlie the mechanism in at least some of the remaining cases by nature of its ability to affect both Na+ and Ca2+ currents.
We thank the staff at the Duke Proteomics Core Facility for their help with the affinity purification/mass spectroscopy and G. Vann Bennett (Duke University) for the antibody to sodium-calcium exchanger.
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) R01 HL71165 and R01 HL112918 (G.S. Pitt), by the Gertrude Elion Mentored Medical Student Award and NHLBI F30 HL112540-01 (J.A. Hennessey), and by an American Heart Association predoctoral fellowship award (E.Q.W.).
In May 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.113.301215/-/DC1.
- Nonstandard Abbreviations and Acronyms
- voltage-gated Ca2+ channel
- Ca2+-induced Ca2+ release
- fibroblast growth factor
- fibroblast growth factor homologous factor
- green fluorescent protein
- ryanodine receptor 2
- scrambled shRNA adenovirus
- sarcoplasmic reticulum
- Received February 19, 2013.
- Revision received June 25, 2013.
- Accepted June 26, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Fibroblast growth factor (FGF) homologous factors bind to and modulate the voltage-gated Na+ channels.
FGF13 is the predominant adult murine cardiac FGF homologous factor that modulates NaV1.5 channel current density and kinetics.
FGF13 has widespread subcellular distribution within ventricular cardiac myocytes.
What New Information Does This Article Contribute?
FGF13 is in complex with junctophilin-2, and a pool of FGF13 localizes to the T tubules in adult ventricular cardiac myocytes.
Knockdown of FGF13 in mouse and rat ventricular cardiac myocytes leads to mislocalization of the CaV1.2 L-type Ca2+ channel, but it does not affect the localization of the sodium-calcium exchanger or ryanodine receptor or the T-tubule structure.
FGF13 knockdown decreases CaV1.2 current density (ICa) in adult mouse and rat ventricular cardiac myocytes as a result of a decrease in the number of channels at the surface, leading to reduced Ca2+-induced Ca2+ release.
FGF13 knockdown reduces peak amplitude and half-width of the action potential in adult rat ventricular cardiac myocytes.
FGF13 modulates voltage-gated Na+ channels in cardiac myocytes, but its widespread distribution, specifically in a striated pattern, indicates that it might also regulate other ion channels. We discovered that FGF13 resides in the cardiac dyad in complex with the structural protein junctophilin-2. Without disturbing the T-tubule structure or other ion channels in the dyad, FGF13 knockdown specifically reduces CaV1.2 channel density at the sarcolemma as a result of mislocalization, leading to reduced ICa and decreased Ca2+-induced Ca2+ release. Cardiac myocytes from FGF13 knockdown show a decrease in the peak amplitude and the half-width of the action potential. These findings reveal a novel modulatory pathway through which FGF13 regulates Ca2+ channel function and suggest that mutations in fibroblast growth factor homologous factors could underlie cardiac arrhythmias characterized by the loss of function of Na+ or Ca2+ channels.