Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current Ludovic Gillet, PhD,* Jean-Sébastien Rougier, PhD,* Diana Shy, MSc,* Stephan Sonntag, PhD,† Nathalie Mougenot, PhD,‡ Maria Essers, BSc,* Doron Shmerling, PhD,† Elise Balse, PhD,§¶║ Stéphane N. Hatem, MD, PhD,§¶║ Hugues Abriel, MD, PhD* From the *Department of Clinical Research, University of Bern, Bern, Switzerland, †PolyGene AG, Rümlang, Switzerland, ‡Plateau d’Expérimentation Coeur, Muscle, Vaisseaux, Université Pierre et Marie Curie, Paris, France, §Institut National de la Santé et de la Recherche Médicale (INSERM), UMR S1166, Institut de Recherche Sur Les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition, Paris, France, ¶Sorbonne Universités, UPMC Univ Paris 06, UMR S1166, Paris, France, and ║Institute of Cardiometabolism & Nutrition, ICAN, Pitié-Salpêtrière Hospital, Paris, France. BACKGROUND Membrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization. OBJECTIVE The purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes.
controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97fl/fl mice. CONCLUSION These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.
METHODS SAP97fl/fl mice were generated by inserting loxP sequences flanking exons 1–3 of the SAP97 gene. SAP97fl/fl mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97fl/fl mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase–polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice.
ABBREVIATIONS AP ¼ action potential; APD ¼ action potential duration; ECG ¼ electrocardiogram; IK1 ¼ inward rectifier potassium current; IKur ¼ ultrarapid delayed rectifier potassium current; INa ¼ sodium current; Iss ¼ sustained potassium current; Ito ¼ transient outward potassium current; QTc ¼ corrected QT interval; RT-PCR ¼ reverse transcriptase–polymerase chain reaction; SAP97 ¼ synapse-associated protein-97
RESULTS Action potential duration was greatly prolonged in αMHC-Cre/SAP97fl/fl cardiomyocytes compared to SAP97fl/fl
(Heart Rhythm 2015;12:181–192) I 2015 Heart Rhythm Society. All rights reserved.
The research leading to these study results received funding from the European Community’s Seventh Framework Program FP7/2007–2013 under grant agreement No. HEALTH-F2-2009-241526, from EUTrigTreat (to Drs. Abriel and Shmerling), and from the Swiss National Science Foundation to Dr. Abriel (310030B_14706035693). We are grateful to the Berne University Research Foundation. Address reprint requests and correspondence: Dr. Ludovic Gillet, University of Bern, Department of Clinical Research, Murtenstrasse 35, 3010 Bern, Switzerland. E-mail address: [email protected]. or Dr. Hugues Abriel, University of Bern, Department of Clinical Research, Murtenstrasse 35, 3010 Bern, Switzerland. E-mail address: [email protected].
Introduction
1547-5271/$-see front matter B 2015 Heart Rhythm Society. All rights reserved.
KEYWORDS SAP97; Sodium channel; Potassium channel; Action potential; Tissue-specific knockout
Proteins of the membrane-associated guanylate kinase (MAGUK) family are characterized by numerous protein– protein interaction domains, including the PDZ domains.1 PDZ is an acronym combining the first letters of 3 proteins: postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 (ZO-1), which were first discovered to share this domain.2 The PDZ domains have common structures with 80–90 amino acids. http://dx.doi.org/10.1016/j.hrthm.2014.09.057
182 MAGUKs regulate the function and localization of many ion channels in neurons and epithelial cells, mostly at cell-to-cell junctions.1 However, little is known about their function in the heart.3 SAP97 (synapse-associated protein-97) and ZO-1 (zonula occludens-1) are the main MAGUK proteins expressed in cardiomyocytes.3 SAP97 (also called Dlg1 or Dlgh1, for Disks large homolog 1, encoded by the DLG1 gene) is involved in the targeting of receptor and channel proteins to specialized domains of the plasma membranes and in the modulation of ion channel activity.1,4 SAP97 is expressed in cardiomyocytes,3,5 in which it has been shown to associate with and regulate several ion channels. Leonoudakis et al6 first identified a direct association of the inwardly rectifying potassium channel Kir2.2 with SAP97 in rat cardiac ventricular myocytes. Using a truncated form of the C-terminal GST-Kir2.2 fusion protein, they showed that the last 3 amino acids (SEI) were essential for association with SAP97.6 Using affinity pull-down experiments, they also demonstrated that each of the Kir2.1, Kir2.2, Kir2.3, and Kir4.1 channels can interact with distinct scaffolding proteins (dystrophin-associated complex, SAP97, CASK, Veli), and that there is channel specificity in the interaction.7 Subsequently, Vaidyanathan et al8 reported that silencing SAP97 in adult rat ventricular myocytes reduces the whole-cell density of the inward rectifier potassium current IK1 by reducing the number of Kir2.x channels. They also presented evidence that silencing SAP97 blunted the β1-adrenergic receptor-mediated regulation of IK1, suggesting that this scaffolding protein is important for assembling a macromolecular signaling complex. Other cardiac potassium channels have also been shown to interact with SAP97. Godreau et al3 reported that SAP97 is abundantly expressed in human atrial myocardium and that immunoprecipitation of SAP97 co-precipitated KV1.5 channels and vice versa. It was also shown that adenovirus-mediated SAP97 overexpression in rat cardiac myocytes resulted in the clustering of endogenous KV1.5 subunits at myocyte–myocyte contacts and in an increase in both the maintained component of the outward potassium current IKur and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches.9 In another study using rat ventricular or human atrial lysates for pull-down experiments with GST fusion proteins comprising the last 50 residues of KV4.2 and KV4.3 channels, our groups found that SAP97 also interacts with these channels, which account for a large part of the outward potassium current Ito in the heart.10 These findings suggest that SAP97 and cardiac KV4.x channel subunits interact directly via the C-terminus of the channels. Moreover, it was observed that SAP97 and KV4.3 channels co-localize at the plasma membrane of rat atrial myocytes and that SAP97 modulates cardiac Ito. In a recent study, our groups demonstrated a specific interaction between the PDZ domain-binding motif of NaV1.5 (SIV) and SAP97 in mouse ventricles and human atria.11 Immunostainings performed on rat heart sections
Heart Rhythm, Vol 12, No 1, January 2015 showed that both NaV1.5 and SAP97 are localized at intercalated discs, and electrophysiologic studies demonstrated that the sodium current INa was reduced in rat atrial myocytes that were infected with SAP97 shRNA-containing lentiviruses. More recently, Milstein et al12 found that NaV1.5, Kir2.1, and SAP97 are parts of the same macromolecular complex. Moreover, they found a reciprocal modulation between NaV1.5 and Kir2.1. Although different experimental models have been used to study cardiac ion channel regulation by SAP97, thus far no data are available regarding the role of SAP97 in regulating heart function in vivo. Two total knockout SAP97 mouse models have been previously generated (13,14), and in both cases, severe organ malformations led to early death right after birth.13,14 No cardiac malformation was reported. Because ion channel interacting proteins are considered important actors in ion channel physiology and pathophysiology,15 we investigated the in vivo significance of SAP97 in the mouse heart by generating a genetically modified mouse model in which SAP97 expression was constitutively suppressed in cardiomyocytes, and we studied the consequences on cardiac ion channel function and heart activity.
Methods and materials All experiments involving animals were performed according to the Swiss Federal Animal Protection Law and were approved by the Cantonal Veterinary Administration, Bern. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Generation of conditional SAP97 knockout mice A conditional mouse model was generated for SAP97/Dlg1, which allows tissue or time-point specific inactivation of the Dlg1 gene by use of the Cre-/loxP-system (Figure 1; see Online Supplementary Data for a detailed description).
RNA extraction and quantitative reverse transcriptase–polymerase chain reaction RNA extraction and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) procedures are described in detail in the Online Supplementary Data.
Protein extraction and western blot These procedures were performed as previously reported (see Online Supplementary Data for a detailed description).16
Immunohistochemistry of mouse ventricular sections Immunostainings were performed on heart cryosections as previously described.16
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Figure 1 Generation of the conditional SAP97-knockout mouse. Homologous recombination of the Dlg1 locus (Dlg1-WT) with the targeting vector pDlg1TV and the resulting targeted Dlg1 alleles are shown. Two loxP sites are inserted into the Dlg1 locus, flanking a region of 3.6 kb that contains exons 1 to 3 of Dlg1 as well as the promoter region of the gene. Directly downstream of the 50 loxP site, a neomycin resistance cassette (neo) is inserted that is flanked by FRT sites, the recognition sites for the Flp recombinase (Dlg1-neoflox allele). Upon breeding to Flp deleter mice, the neomycin resistance cassette is deleted, and only a single FRT site remains in the Dlg1-flox allele. The short (SA) and long homology regions (LA) are indicated as gray boxes. Positions of primers and size of products for genotyping polymerase chain reaction (PCR), as well as restriction sites and the probe for southern blot analyses, are indicated (A). Representative result of southern blot analysis with genomic DNA from Dlg1 wt/wt and Dlg1 neoflox/wt ES cells that are cut with KpnI and probed, using a sequence following the long homology region in the Dlg1 locus. This resulted in a 16-kb signal for the wild-type allele and an 8-kb signal for the Dlg1-neoflox allele (B). PCR analysis of all 3 genotypes using the primer depicted in A. The wild-type allele resulted in a PCR product of 586 bp, whereas a 633-bp fragment was generated for the Dlg1-flox allele (C). Levels of SAP97 mRNA transcripts in SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes (D). Representative western blots showing decreased levels of SAP97 in αMHC-Cre/SAP97fl/fl hearts (E).
Antibodies
Measurement of cardiac parameters
A detailed description is given in the Online Supplementary Data.
Echocardiography and ECG measurement procedures are described in detail in the Online Supplementary Data.
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Isolation of mouse ventricular myocytes Ventricular myocyte isolations were performed as previously described,16 according to a modified procedure of established enzymatic methods.17
cardiomyocytes compared to SAP97fl/fl controls (n ¼ 4, P o .05; Figure 1D). Western blots, performed on wholeheart lysates, showed a 71% ⫾ 4% decrease in SAP97 protein levels from αMHC-Cre/SAP97fl/fl hearts (n ¼ 4, P o .05; Figure 1E).
Electrophysiology Ionic currents and action potential recordings were performed in the whole-cell configuration at room temperature (221–231C) using materials and solutions previously described (see the Online Supplementary Data for details).16
Statistical analysis Data are presented as mean ⫾ SEM. The Student t test was used to compare the means between the SAP97fl/fl and αMHC-Cre/SAP97fl/fl groups. P o .05 was considered significant.
Results Generation of a mouse line with a tissue-specific deletion of SAP97
SAP97fl/fl mice were generated as described in the Methods and materials section and were crossed with αMHC-Cre transgenic mice in which the cardiac-specific α-myosin heavy-chain (Myh6) promoter drives expression of Cre recombinase. The offspring were αMHC-Cre/SAP97fl/fl mice with a cardiomyocyte-specific deletion of SAP97. SAP97fl/fl and αMHC-Cre/SAP97fl/fl mice were born at the expected mendelian frequency, were viable throughout adulthood, and showed no apparent illnesses or increased mortality. Experiments were performed on 3- to 4-month-old adult mice. Body weight was similar between αMHC-Cre/SAP97fl/fl and SAP97fl/fl control mice (30 ⫾ 1 g and 31 ⫾ 1 g, respectively). Echocardiography showed no differences in cardiac morphological and functional parameters (Table 1). Quantitative RT-PCR analysis of RNA extracted from isolated cardiomyocytes showed that SAP97 mRNA transcript levels were decreased by 80% ⫾ 2% in αMHC-Cre/SAP97fl/fl Table 1
Action potential duration is increased in αMHC-Cre/SAP97fl/fl mouse cardiomyocytes To assess the functional consequences of SAP97 deletion on cardiac cell electrical activity, action potential (AP) recordings were performed on isolated cardiomyocytes (Figure 2 and Online Supplemental Table 1). Although AP maximal upstroke velocity dV/dtmax (reflecting sodium channel availability), AP amplitude, and time to peak were not different between αMHC-Cre/SAP97fl/fl and control myocytes (Figures 2B–2D), action potential duration (APD) was greatly prolonged in αMHC-Cre/SAP97fl/fl cardiomyocytes (1.9 ⫾ 0.3, 2.3 ⫾ 0.4, and 2.1 ⫾ 0.2 folds for APD30, APD50, and APD90, respectively, n ¼ 10, P o .05; Figure 2E). This finding suggests that mainly potassium channel function rather than sodium channel function is altered by the reduction in SAP97 expression. Moreover, the resting membrane potential of αMHC-Cre/SAP97fl/fl cardiomyocytes was slightly depolarized compared to control cardiomyocytes (þ2.4 ⫾ 0.9 mV, n ¼ 10, P o .05; Figure 2F).
NaV1.5 function and distribution are not altered in αMHC-Cre/SAP97fl/fl mouse heart To determine which ionic conductances were altered by SAP97 deletion in cardiomyocytes, cardiac sodium and potassium currents were studied in isolated ventricular cardiomyocytes using the patch-clamp technique in wholecell configuration. Cell capacitance was similar between SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes (170 ⫾ 10 pF, n ¼ 23; and 168 ⫾ 7 pF, n ¼ 27, respectively, P ¼ NS). INa density analysis showed no differences between
Echocardiographic data
n Heart rate (bpm) IVSd (mm) LVEDD (mm) LVPWd (mm) IVSs (mm) LVESD (mm) LVPWs (mm) Cardiac output (mL/min) Ejection fraction (%) Fractional shortening (%)
SAP97fl/fl
αMHC-Cre/SAP97fl/fl
8 536 ⫾ 0.6 ⫾ 4.1 ⫾ 0.7 ⫾ 1.0 ⫾ 2.6 ⫾ 1.1 ⫾ 12 ⫾ 71.83 ⫾ 35.95 ⫾
523 0.6 4.2 0.8 1.0 2.7 1.1 13 71.46 35.88
30 0.01 0.1 0.02 0.02 0.1 0.03 0.8 2.26 1.81
7 ⫾ 49NS ⫾ 0.02NS ⫾ 0.1NS ⫾ 0.04NS ⫾ 0.05NS ⫾ 0.1NS ⫾ 0.06NS ⫾ 1.0NS ⫾ 3.10NS ⫾ 2.46NS
P value
.652 1 .490 .762 .818 .670 .820 .438 .923 .983
Data are given as mean ⫾ SEM. IVSd ¼ interventricular septum in diastole; IVSs ¼ interventricular septum in systole; LVEDD ¼ left ventricular internal end-diastolic diameter; LVESD ¼ left ventricular internal end-systolic diameter; LVPWd ¼ left ventricular posterior wall in diastole; LVPWs ¼ left ventricular posterior wall in systole; NS ¼ not significant.
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fl/fl
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Figure 2 Action potential duration (APD) is increased in αMHC-Cre/SAP97fl/fl cardiomyocytes. Representative APs recorded from cardiomyocytes of SAP97fl/fl mice (black line) and αMHC-Cre/SAP97fl/fl mice (red line) (A). Maximal AP upstroke velocity (B), AP amplitude (C), and time to peak (D) remain unchanged in αMHC-Cre/SAP97fl/fl cardiomyocytes (white squares) compared to controls (black squares). Increased APD at 30, 50, and 90% repolarization in alphaMHC-Cre/SAP97fl/fl cardiomyocytes (E). Resting membrane potential is slightly depolarized in alphaMHC-Cre/SAP97fl/fl cardiomyocytes (F). *P o .05.
αMHC-Cre/SAP97fl/fl and control myocytes (Figure 3A). Voltage dependence of INa activation and inactivation also was not significantly different (Figure 3B). Kinetics of INa fast inactivation were unchanged (see Online Supplemental Figure 1A). In one of our recent studies, we observed that NaV1.5 co-localized with SAP97 at the intercalated discs of cardiomyocytes.11 To examine the consequence of SAP97 deletion on the distribution of NaV1.5 in cardiac tissue, immunostainings of cardiac ventricular sections were performed. As shown in Figure 3C, NaV1.5 expression is present in both lateral membranes and intercalated discs of SAP97fl/fl ventricular sections, and this localization was not altered in αMHC-Cre/SAP97fl/fl hearts.
IK1, Ito, and IKur potassium currents are decreased in αMHC-Cre/SAP97fl/fl cardiomyocytes In previous studies, SAP97 has been found to interact with and regulate different cardiac potassium channels such as Kir2.x channels (Kir2.1, Kir2.2, Kir2.3), which are responsible for the inward rectifier IK1 potassium current, KV4.2 and KV4.3 channels, which are responsible for the transient outward potassium current Ito, and KV1.5 channels, which are responsible for the ultrarapid, outwardly rectifying potassium current IKur.7–10,12,18 In our study, these different potassium currents were recorded in isolated cardiomyocytes from SAP97fl/fl and αMHC-Cre/SAP97fl/fl mouse hearts. IK1, Ito, and IKur were all found to be decreased in
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Figure 3 Whole-cell INa is not modified in αMHC-Cre/SAP97fl/fl cardiomyocytes. Current density–voltage relationship of INa in SAP97fl/fl and αMHC-Cre/ SAP97fl/fl mice (A). Steady-state activation and inactivation curves from SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes. Boltzmann curves of individual cells were fitted to steady-state activation (SAP97fl/fl V1/2 ¼ –29.6 ⫾ 0.7 mV, K ¼ 5.5 ⫾ 0.1; αMHC-Cre/SAP97fl/fl V1/2 ¼ –30 ⫾ 1.5 mV, K ¼ 5.8 ⫾ 0.2) and inactivation data (SAP97fl/fl V1/2 ¼ –75.9 ⫾ 1.5 mV, K ¼ 6.6 ⫾ 0.3; αMHC-Cre/SAP97fl/fl V1/2 ¼ –76.9 ⫾ 2.1 mV, K ¼ 5.8 ⫾ 0.2) (B). NaV1.5 immunostaining in SAP97fl/fl and αMHC-Cre/SAP97fl/fl ventricular sections (C).
αMHC-Cre/SAP97fl/fl mouse hearts (Figures 4A–4C). Peak currents were decreased by 53% ⫾ 6%, 45% ⫾ 9%, and 53% ⫾ 6% for IK1, Ito, and IKur, respectively (P o .05). On the
contrary, the ISS potassium current, which has not been reported to be regulated by SAP97, remained unchanged (Figure 4D).
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Figure 4 Whole-cell IK1, Ito, and IKur are decreased in αMHC-Cre/SAP97 cardiomyocytes. Representative traces of whole-cell current and current density– voltage relationships for IK1 (A), Ito (B), IKur (C), and Iss (D) recorded in SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes. *P o.05.
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Figure 5 SAP97-associated ion channel expression is not modified in αMHC-Cre/SAP97fl/fl mouse hearts. Representative western blots showing expression of SAP97, Cre recombinase, and SAP97-associated ion channels in SAP97fl/fl and alpha-MHC-Cre/SAP97fl/fl mouse hearts (A). Quantitative comparison of protein expression levels from multiple western blots similar to that shown in (A). Band intensities for each mouse heart were normalized to the averaged control (SAP97fl/fl) per experimental day before pooling results for statistical testing *P o. 05 (B).
Ion channel expression in αMHC-Cre/SAP97fl/fl mouse hearts In order to investigate whether expression of the different cardiac ion channels was modified subsequent to SAP97 reduction, NaV1.5, Kir2.1, Kir2.2, KV4.2, KV4.3, and KV1.5 whole-cell expression was analyzed by western blot (Figure 5). After quantification of band densities, no significant differences were found in potassium channel protein levels between SAP97fl/fl and αMHC-Cre/SAP97fl/fl mouse hearts (Figure 5B). Unexpectedly, NaV1.5 expression was slightly increased by 14% (P o.05) in αMHC-Cre/SAP97fl/fl mouse hearts.
Surface ECGs show a prolonged QT interval in αMHC-Cre/SAP97fl/fl mice To investigate the in vivo functional consequences of deletion of SAP97 in the mouse heart in relation to the concomitant decrease in potassium currents, surface echocardiography and ECG analysis were performed on lightly anesthetized mice. Echocardiography revealed no major differences in functional
parameters between SAP97fl/fl and αMHC-Cre/SAP97fl/fl mice (Table 1). On ECG, the P wave, PR interval, and QRS duration were not different between αMHC-Cre/SAP97fl/fl mice and controls. However, the corrected QT interval (QTc) was significantly prolonged in αMHC-Cre/SAP97fl/fl mice compared to SAP97fl/fl control mice (58.3 ⫾ 2.0 ms vs 49.1 ⫾ 1.2 ms, respectively; Table 2 and Figure 6).
Discussion In this study, we generated and characterized for the first time a mouse line with cardiomyocyte-specific deletion of SAP97 and obtained evidence for in vivo regulation of cardiac cell electrical activity by SAP97. The main findings can be summarized as follows: (1) IK1, Ito, and IKur were reduced in ventricular myocytes that had reduced expression of SAP97; (2) unexpectedly, NaV1.5 function and localization were not altered in αMHC-Cre/SAP97fl/fl mice; and (3) SAP97-deficient mice displayed a prolonged QT interval on ECG.
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ECGs parameters SAP97fl/fl
n Heart rate (bpm) P duration (ms) PR interval (ms) QRS duration (ms) QTc (ms)
7 416.8 ⫾ 19.0 ⫾ 43.4 ⫾ 20.1 ⫾ 49.1 ⫾
29.7 0.5 1.9 1.6 1.2
αMHC-Cre/SAP97fl/fl
P value
6 394.2 ⫾ 20.7 ⫾ 40.3 ⫾ 22.8 ⫾ 58.3 ⫾
.664 .077 .313 .178 .001
48.7NS 0.9NS 2.5NS 1.3NS 2.0*
Data are given as mean ⫾ SEM. NS ¼ not significant; QTc ¼ corrected QT interval. * P o .05.
Cardiac-specific deletion of SAP97 is not lethal The in vivo role of SAP97 had already been studied in the past, but no data were available regarding its role in cardiac development or function.13,14 This protein appears to be essential for the development of different organs, and SAP97 total knockout mice do not survive after birth. In the present study, we observed that mice with cardiac-specific reduced SAP97 expression can survive until adult age. Echocardiography did not reveal any morphological alterations, thus suggesting that SAP97 is not crucial for cardiac development. Moreover, SAP97 is also known to be expressed in
neurons and plays a role in the organization of synaptic macromolecular complexes. Thus, it would not be surprising if sympathetic or parasympathetic regulation of the heart could be modulated by SAP97; however, this question remains to be further investigated.
Role of SAP97 in regulation of potassium currents is confirmed We observed that at least 3 ventricular potassium currents were altered in αMHC-Cre/SAP97fl/fl cardiomyocytes, leading to increased APD and concomitant prolongation of the
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Figure 6 QT interval is prolonged in αMHC-Cre/SAP97fl/fl mice. Typical examples of ECG traces recorded in SAP97fl/fl and αMHC-Cre/SAP97fl/fl mice (A). ECG recordings show prolongation of the QT interval in αMHC-Cre/SAP97fl/fl mice compared to SAP97fl/fl (instead of Cre/SAP97fl/fl) mice (B).
190 QT interval on ECG. However, the mechanisms underlying these decreased whole-cell potassium current densities still have to be determined. The mechanism does not involve regulation of total channel expression since western blots did not show any differences in Kir2.1, Kir2.2, KV4.2, KV4.3, or KV1.5 protein levels between αMHC-Cre/SAP97fl/fl and control hearts. These results differ from those obtained by Vaidyanathan et al,8 who found that expression levels of Kir2.1, Kir2.2, and Kir2.3 were decreased in the 4 days after SAP97 silencing by shRNAs in adult rat ventricular myocytes that were kept in culture. It was previously demonstrated that overexpression of SAP97 in HEK293 cells increases the whole-cell current density of Kir2.3 through multiple mechanisms.18 One of these mechanisms was an increase in the number of functional channels at the membrane, and the other was an increase in the unitary conductance amplitude. However, results obtained in adult rat ventricular myocytes did not show any change in unitary conductance amplitude.8 SAP97 has also been reported to regulate the properties of KV channels by modulating channel surface expression.9,10 Previous data indicated that silencing of SAP97 also accelerated the time-dependent inactivation of Ito, possibly because of loss of interaction with calmodulin kinase II.10 We measured the kinetics of Ito inactivation, but we did not find any difference between αMHC-Cre/SAP97fl/fl and control cells (see Online Supplemental Figure 1B). A reason for this discrepancy could be the difference in free Ca2þ concentrations in the intrapipette medium used to perform Ito measurements (0.5 mM in El-Haou et al10 vs 20 nM in the present study). Thus, whether SAP97 regulates potassium channel trafficking toward the sarcolemma (and thus surface expression) unitary conductance, or kinetics of inactivation in vivo should be the topic of further study.
Are SAP97 and NaV1.5 interacting in the mouse heart? Regulation of the cardiac NaV1.5 sodium channel by interacting proteins is an active field of research.19 Indeed, NaV1.5 has been shown to be part of distinct macromolecular complexes that interact with different partner proteins, depending on its location in the cardiomyocyte.16 At least 2 pools of NaV1.5 have been proposed: one population of channels at the lateral sarcolemma that is interacting with the syntrophin/dystrophin complex and another population at the intercalated discs that may interact with SAP97, as neither syntrophin proteins nor dystrophin is expressed at the discs. In a previous work, our group obtained molecular and functional evidence that SAP97 interacts with the PDZ domain-binding motif of NaV1.5 in the mouse heart.11 Silencing SAP97 expression in rat myocytes in culture or in HEK293 cells expressing NaV1.5 led to reduced INa. Subsequently, Milstein et al12 were able to coimmunoprecipitate NaV1.5 and SAP97 in rat ventricle lysates. In addition, it was suggested that SAP97 allows NaV1.5 to interact with the Kir2.1 potassium channel and that
Heart Rhythm, Vol 12, No 1, January 2015 expression of both channels is co-regulated. In our recent study that addressed the in vivo significance of the NaV1.5PDZ domain-binding motif in mice, we found that deleting this motif (ΔSIV mice) led to decreased expression of NaV1.5 at the lateral membrane of cardiomyocytes but did not alter NaV1.5 localization at the intercalated discs.16 This observation challenged the question of the role of NaV1.5 and SAP97 interaction in cardiac cells. In the present study, we did not find any changes in NaV1.5 localization and function when SAP97 expression was suppressed. One of the main differences between these two mouse models16 and the previous studies is that these mice are constitutive knockin or knockout mice. In other words, genetic modification is present early in development, whereas in the other studies,11,12 SAP97 expression was silenced in adult myocytes that were kept in culture. Thus, it may not be ruled out that in an inducible SAP97 knockout mouse model, the consequences on INa could be different. In addition, NaV1.5 has been found to interact with other regulatory proteins at the intercalated discs, such as connexin-43,20–22 plakophilin2,23 and ankyrin G.24 Such a complex organization of NaV1.5 channels at the intercalated discs may suggest that they play a crucial role in this specific region of cardiac myocytes, and that perhaps the consequences of a constitutive deletion of one of these regulatory proteins could be counteracted by modifications in expression or organization of the other partner proteins. Interestingly, in a recent study, Asimaki et al25 investigated the effect of the human 2057del2 mutation in the gene encoding plakoglobin, in the context of arrhythmogenic cardiomyopathy. By using different models such as zebrafish, neonatal rat ventricular myocytes, and cardiac myocytes derived from human induced pluripotent stem cells taken from arrhythmogenic cardiomyopathy probands with mutations in the gene encoding plakophilin-2, they found that decreased expression of plakoglobin or plakophilin-2 was associated with altered distribution of connexin-43, NaV1.5, and SAP97. Note that this altered distribution of SAP97 was accompanied by a large increase in APD with concomitant reduction of potassium current. In summary, the role of SAP97dependent regulation of NaV1.5 remains an open question. Although it seems likely that both proteins are interacting, it may be postulated that SAP97 is not directly involved in targeting and stabilizing NaV1.5 at the intercalated discs. Alternative roles for this interaction, such as providing a scaffold for signaling proteins such as calmodulin kinase II,10 will have to be investigated in the future.
Study limitations One of the main limitations of this study is that we have not been able to investigate SAP97 protein distribution within murine cardiomyocytes. SAP97 has been proposed to be mainly expressed at the intercalated discs of cardiac myocytes8,11,12 but also at the lateral membrane10 and T-tubules.8,12 However, none of these studies presented an appropriate negative control (ie, cardiac tissue in which
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SAP97 expression is suppressed). It is worth noting that we performed immunostaining on heart sections and on isolated cardiomyocytes of αMHC-Cre/SAP97fl/fl mice using more than 10 different commercial anti-SAP97 antibodies, but the signal was never different from control tissues. Another comment could be made on the approach that we used to determine the in vivo role of SAP97. It is known that compensatory mechanisms can develop when a protein is constitutively suppressed. This is the case with utrophin (a homolog of dystrophin), which has been shown to be upregulated in the heart of dystrophin-deficient mice.26 This utrophin upregulation could partially compensate for dystrophin deletion and the subsequent INa decrease. Thus, by using an inducible knockout mouse model instead of a constitutive model, our findings could have been different.
Conclusion We generated a cardiac-specific SAP97-deficient mouse line and provided, for the first time, evidence for in vivo regulation of cardiac ion channels by SAP97. Surprisingly, INa was not altered in SAP97-deficient cardiomyocytes, whereas regulation of potassium currents was confirmed. Whether SAP97 regulates potassium channel surface expression or unitary conductance still has to be elucidated. However, based on the important role of SAP97 in regulating cardiac potassium currents, it could be proposed that DLG1 may be a susceptibility gene that should to be investigated in patients with congenital long QT syndrome.
Appendix Supplementary data Supplementary material cited in this article is available online at doi:10.1016/j.hrthm.2014.09.057.
References 1. Funke L, Dakoji S, Bredt DS. Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annu Rev Biochem 2005;74:219–245. 2. Kennedy MB. Origin of PDZ (DHR, GLGF) domains. Trends Biochem Sci 1995;20:350. 3. Godreau D, Vranckx R, Maguy A, Rucker-Martin C, Goyenvalle C, Abdelshafy S, Tessier S, Couetil JP, Hatem SN. Expression, regulation and role of the MAGUK protein SAP-97 in human atrial myocardium. Cardiovasc Res 2002;56: 433–442. 4. Fujita A, Kurachi Y. SAP family proteins. Biochem Biophys Res Commun 2000;269:1–6. 5. Godreau D, Vranckx R, Maguy A, Goyenvalle C, Hatem SN. Different isoforms of synapse-associated protein, SAP97, are expressed in the heart and have distinct effects on the voltage-gated Kþ channel Kv1.5. J Biol Chem 2003;278: 47046–47052. 6. Leonoudakis D, Mailliard W, Wingerd K, Clegg D, Vandenberg C. Inward rectifier potassium channel Kir2.2 is associated with synapse-associated protein SAP97. J Cell Sci 2001;114:987–998.
191 7. Leonoudakis D, Conti LR, Anderson S, Radeke CM, McGuire LM, Adams ME, Froehner SC, Yates JR 3rd, Vandenberg CA. Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins. J Biol Chem 2004;279:22331–22346. 8. Vaidyanathan R, Taffet SM, Vikstrom KL, Anumonwo JM. Regulation of cardiac inward rectifier potassium current (I(K1)) by synapse-associated protein97. J Biol Chem 2010;285:28000–28009. 9. Abi-Char J, El-Haou S, Balse E, Neyroud N, Vranckx R, Coulombe A, Hatem SN. The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes. Am J Physiol Heart Circ Physiol 2008;294: H1851–H1861. 10. El-Haou S, Balse E, Neyroud N, Dilanian G, Gavillet B, Abriel H, Coulombe A, Jeromin A, Hatem SN. Kv4 potassium channels form a tripartite complex with the anchoring protein SAP97 and CaMKII in cardiac myocytes. Circ Res 2009;104: 758–769. 11. Petitprez S, Zmoos AF, Ogrodnik J, et al. SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes. Circ Res 2011;108:294–304. 12. Milstein ML, Musa H, Balbuena DP, Anumonwo JM, Auerbach DS, Furspan PB, Hou L, Hu B, Schumacher SM, Vaidyanathan R, Martens JR, Jalife J. Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia. Proc Natl Acad Sci U S A 2012;109:E2134–E2143. 13. Mahoney ZX, Sammut B, Xavier RJ, Cunningham J, Go G, Brim KL, Stappenbeck TS, Miner JH, Swat W. Discs-large homolog 1 regulates smooth muscle orientation in the mouse ureter. Proc Natl Acad Sci U S A 2006;103: 19872–19877. 14. Iizuka-Kogo A, Ishidao T, Akiyama T, Senda T. Abnormal development of urogenital organs in Dlgh1-deficient mice. Development 2007;134:1799–1807. 15. Ackerman MJ, Mohler PJ. Defining a new paradigm for human arrhythmia syndromes: phenotypic manifestations of gene mutations in ion channel- and transporter-associated proteins. Circ Res 2010;107:457–465. 16. Shy D, Gillet L, Ogrodnik J, et al. PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function. Circulation 2014;130:147–160. 17. Wolska BM, Solaro RJ. Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry. Am J Physiol 1996;271: H1250–H1255. 18. Vikstrom KL, Vaidyanathan R, Levinsohn S, O'Connell RP, Qian Y, Crye M, Mills JH, Anumonwo JM. SAP97 regulates Kir2.3 channels by multiple mechanisms. Am J Physiol Heart Circ Physiol 2009;297:H1387–H1397. 19. Abriel H. Cardiac sodium channel Na(v)1.5 and interacting proteins: physiology and pathophysiology. J Mol Cell Cardiol 2010;48:2–11. 20. Malhotra JD, Thyagarajan V, Chen C, Isom LL. Tyrosine-phosphorylated and nonphosphorylated sodium channel beta1 subunits are differentially localized in cardiac myocytes. J Biol Chem 2004;279:40748–40754. 21. Jansen JA, Noorman M, Musa H, et al. Reduced heterogeneous expression of Cx43 results in decreased Nav1.5 expression and reduced sodium current that accounts for arrhythmia vulnerability in conditional Cx43 knockout mice. Heart Rhythm 2012;9:600–607. 22. Rhett JM, Ongstad EL, Jourdan J, Gourdie RG. Cx43 associates with Na(v)1.5 in the cardiomyocyte perinexus. J Membr Biol 2012;245:411–422. 23. Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patino GA, Taffet SM, Isom LL, Delmar M. Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res 2009;105:523–526. 24. Mohler PJ, Rivolta I, Napolitano C, LeMaillet G, Lambert S, Priori SG, Bennett V. Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes. Proc Natl Acad Sci U S A 2004;101:17533–17538. 25. Asimaki A, Kapoor S, Plovie E, et al. Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Sci Transl Med 2014;6:240ra74. 26. Albesa M, Ogrodnik J, Rougier JS, Abriel H. Regulation of the cardiac sodium channel Nav1.5 by utrophin in dystrophin-deficient mice. Cardiovasc Res 2011;89:320–328.
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CLINICAL PERSPECTIVES Genetic cardiac channelopathies such as congenital long QT syndrome and Brugada syndrome are caused by mutations in genes coding for ion channel subunits or ion channel regulatory proteins. These pathologies are characterized by a broad genetic heterogeneity involving several dozens of genes. However, in many patients and families, the culprit genes are still unknown. In this study, we investigated the consequences of specific ablation of SAP97 in mouse cardiac cells a scaffolding protein that has been shown to interact with and regulate the surface expression of cardiac potassium and sodium channels. It was observed that SAP97-deficient cardiac cells had prolonged APD due to decreased repolarizing potassium current. ECGs of these SAP97-deficient mice displayed prolonged QT intervals. These findings suggest that genetic variants such as functional polymorphisms and mutations in DLG1, the gene coding for SAP97, that lead to reduced expression or loss of function of SAP97 may delay ventricular repolarization and hence lead to QT-interval prolongation. This study should stimulate the search for genetic variants of DLG1 in patient cohorts with long QT syndrome.
controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97fl/fl mice. CONCLUSION These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.
METHODS SAP97fl/fl mice were generated by inserting loxP sequences flanking exons 1–3 of the SAP97 gene. SAP97fl/fl mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97fl/fl mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase–polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice.
ABBREVIATIONS AP ¼ action potential; APD ¼ action potential duration; ECG ¼ electrocardiogram; IK1 ¼ inward rectifier potassium current; IKur ¼ ultrarapid delayed rectifier potassium current; INa ¼ sodium current; Iss ¼ sustained potassium current; Ito ¼ transient outward potassium current; QTc ¼ corrected QT interval; RT-PCR ¼ reverse transcriptase–polymerase chain reaction; SAP97 ¼ synapse-associated protein-97
RESULTS Action potential duration was greatly prolonged in αMHC-Cre/SAP97fl/fl cardiomyocytes compared to SAP97fl/fl
(Heart Rhythm 2015;12:181–192) I 2015 Heart Rhythm Society. All rights reserved.
The research leading to these study results received funding from the European Community’s Seventh Framework Program FP7/2007–2013 under grant agreement No. HEALTH-F2-2009-241526, from EUTrigTreat (to Drs. Abriel and Shmerling), and from the Swiss National Science Foundation to Dr. Abriel (310030B_14706035693). We are grateful to the Berne University Research Foundation. Address reprint requests and correspondence: Dr. Ludovic Gillet, University of Bern, Department of Clinical Research, Murtenstrasse 35, 3010 Bern, Switzerland. E-mail address: [email protected]. or Dr. Hugues Abriel, University of Bern, Department of Clinical Research, Murtenstrasse 35, 3010 Bern, Switzerland. E-mail address: [email protected].
Introduction
1547-5271/$-see front matter B 2015 Heart Rhythm Society. All rights reserved.
KEYWORDS SAP97; Sodium channel; Potassium channel; Action potential; Tissue-specific knockout
Proteins of the membrane-associated guanylate kinase (MAGUK) family are characterized by numerous protein– protein interaction domains, including the PDZ domains.1 PDZ is an acronym combining the first letters of 3 proteins: postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 (ZO-1), which were first discovered to share this domain.2 The PDZ domains have common structures with 80–90 amino acids. http://dx.doi.org/10.1016/j.hrthm.2014.09.057
182 MAGUKs regulate the function and localization of many ion channels in neurons and epithelial cells, mostly at cell-to-cell junctions.1 However, little is known about their function in the heart.3 SAP97 (synapse-associated protein-97) and ZO-1 (zonula occludens-1) are the main MAGUK proteins expressed in cardiomyocytes.3 SAP97 (also called Dlg1 or Dlgh1, for Disks large homolog 1, encoded by the DLG1 gene) is involved in the targeting of receptor and channel proteins to specialized domains of the plasma membranes and in the modulation of ion channel activity.1,4 SAP97 is expressed in cardiomyocytes,3,5 in which it has been shown to associate with and regulate several ion channels. Leonoudakis et al6 first identified a direct association of the inwardly rectifying potassium channel Kir2.2 with SAP97 in rat cardiac ventricular myocytes. Using a truncated form of the C-terminal GST-Kir2.2 fusion protein, they showed that the last 3 amino acids (SEI) were essential for association with SAP97.6 Using affinity pull-down experiments, they also demonstrated that each of the Kir2.1, Kir2.2, Kir2.3, and Kir4.1 channels can interact with distinct scaffolding proteins (dystrophin-associated complex, SAP97, CASK, Veli), and that there is channel specificity in the interaction.7 Subsequently, Vaidyanathan et al8 reported that silencing SAP97 in adult rat ventricular myocytes reduces the whole-cell density of the inward rectifier potassium current IK1 by reducing the number of Kir2.x channels. They also presented evidence that silencing SAP97 blunted the β1-adrenergic receptor-mediated regulation of IK1, suggesting that this scaffolding protein is important for assembling a macromolecular signaling complex. Other cardiac potassium channels have also been shown to interact with SAP97. Godreau et al3 reported that SAP97 is abundantly expressed in human atrial myocardium and that immunoprecipitation of SAP97 co-precipitated KV1.5 channels and vice versa. It was also shown that adenovirus-mediated SAP97 overexpression in rat cardiac myocytes resulted in the clustering of endogenous KV1.5 subunits at myocyte–myocyte contacts and in an increase in both the maintained component of the outward potassium current IKur and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches.9 In another study using rat ventricular or human atrial lysates for pull-down experiments with GST fusion proteins comprising the last 50 residues of KV4.2 and KV4.3 channels, our groups found that SAP97 also interacts with these channels, which account for a large part of the outward potassium current Ito in the heart.10 These findings suggest that SAP97 and cardiac KV4.x channel subunits interact directly via the C-terminus of the channels. Moreover, it was observed that SAP97 and KV4.3 channels co-localize at the plasma membrane of rat atrial myocytes and that SAP97 modulates cardiac Ito. In a recent study, our groups demonstrated a specific interaction between the PDZ domain-binding motif of NaV1.5 (SIV) and SAP97 in mouse ventricles and human atria.11 Immunostainings performed on rat heart sections
Heart Rhythm, Vol 12, No 1, January 2015 showed that both NaV1.5 and SAP97 are localized at intercalated discs, and electrophysiologic studies demonstrated that the sodium current INa was reduced in rat atrial myocytes that were infected with SAP97 shRNA-containing lentiviruses. More recently, Milstein et al12 found that NaV1.5, Kir2.1, and SAP97 are parts of the same macromolecular complex. Moreover, they found a reciprocal modulation between NaV1.5 and Kir2.1. Although different experimental models have been used to study cardiac ion channel regulation by SAP97, thus far no data are available regarding the role of SAP97 in regulating heart function in vivo. Two total knockout SAP97 mouse models have been previously generated (13,14), and in both cases, severe organ malformations led to early death right after birth.13,14 No cardiac malformation was reported. Because ion channel interacting proteins are considered important actors in ion channel physiology and pathophysiology,15 we investigated the in vivo significance of SAP97 in the mouse heart by generating a genetically modified mouse model in which SAP97 expression was constitutively suppressed in cardiomyocytes, and we studied the consequences on cardiac ion channel function and heart activity.
Methods and materials All experiments involving animals were performed according to the Swiss Federal Animal Protection Law and were approved by the Cantonal Veterinary Administration, Bern. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Generation of conditional SAP97 knockout mice A conditional mouse model was generated for SAP97/Dlg1, which allows tissue or time-point specific inactivation of the Dlg1 gene by use of the Cre-/loxP-system (Figure 1; see Online Supplementary Data for a detailed description).
RNA extraction and quantitative reverse transcriptase–polymerase chain reaction RNA extraction and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) procedures are described in detail in the Online Supplementary Data.
Protein extraction and western blot These procedures were performed as previously reported (see Online Supplementary Data for a detailed description).16
Immunohistochemistry of mouse ventricular sections Immunostainings were performed on heart cryosections as previously described.16
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183
kDa 130
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Calnexin
Figure 1 Generation of the conditional SAP97-knockout mouse. Homologous recombination of the Dlg1 locus (Dlg1-WT) with the targeting vector pDlg1TV and the resulting targeted Dlg1 alleles are shown. Two loxP sites are inserted into the Dlg1 locus, flanking a region of 3.6 kb that contains exons 1 to 3 of Dlg1 as well as the promoter region of the gene. Directly downstream of the 50 loxP site, a neomycin resistance cassette (neo) is inserted that is flanked by FRT sites, the recognition sites for the Flp recombinase (Dlg1-neoflox allele). Upon breeding to Flp deleter mice, the neomycin resistance cassette is deleted, and only a single FRT site remains in the Dlg1-flox allele. The short (SA) and long homology regions (LA) are indicated as gray boxes. Positions of primers and size of products for genotyping polymerase chain reaction (PCR), as well as restriction sites and the probe for southern blot analyses, are indicated (A). Representative result of southern blot analysis with genomic DNA from Dlg1 wt/wt and Dlg1 neoflox/wt ES cells that are cut with KpnI and probed, using a sequence following the long homology region in the Dlg1 locus. This resulted in a 16-kb signal for the wild-type allele and an 8-kb signal for the Dlg1-neoflox allele (B). PCR analysis of all 3 genotypes using the primer depicted in A. The wild-type allele resulted in a PCR product of 586 bp, whereas a 633-bp fragment was generated for the Dlg1-flox allele (C). Levels of SAP97 mRNA transcripts in SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes (D). Representative western blots showing decreased levels of SAP97 in αMHC-Cre/SAP97fl/fl hearts (E).
Antibodies
Measurement of cardiac parameters
A detailed description is given in the Online Supplementary Data.
Echocardiography and ECG measurement procedures are described in detail in the Online Supplementary Data.
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Isolation of mouse ventricular myocytes Ventricular myocyte isolations were performed as previously described,16 according to a modified procedure of established enzymatic methods.17
cardiomyocytes compared to SAP97fl/fl controls (n ¼ 4, P o .05; Figure 1D). Western blots, performed on wholeheart lysates, showed a 71% ⫾ 4% decrease in SAP97 protein levels from αMHC-Cre/SAP97fl/fl hearts (n ¼ 4, P o .05; Figure 1E).
Electrophysiology Ionic currents and action potential recordings were performed in the whole-cell configuration at room temperature (221–231C) using materials and solutions previously described (see the Online Supplementary Data for details).16
Statistical analysis Data are presented as mean ⫾ SEM. The Student t test was used to compare the means between the SAP97fl/fl and αMHC-Cre/SAP97fl/fl groups. P o .05 was considered significant.
Results Generation of a mouse line with a tissue-specific deletion of SAP97
SAP97fl/fl mice were generated as described in the Methods and materials section and were crossed with αMHC-Cre transgenic mice in which the cardiac-specific α-myosin heavy-chain (Myh6) promoter drives expression of Cre recombinase. The offspring were αMHC-Cre/SAP97fl/fl mice with a cardiomyocyte-specific deletion of SAP97. SAP97fl/fl and αMHC-Cre/SAP97fl/fl mice were born at the expected mendelian frequency, were viable throughout adulthood, and showed no apparent illnesses or increased mortality. Experiments were performed on 3- to 4-month-old adult mice. Body weight was similar between αMHC-Cre/SAP97fl/fl and SAP97fl/fl control mice (30 ⫾ 1 g and 31 ⫾ 1 g, respectively). Echocardiography showed no differences in cardiac morphological and functional parameters (Table 1). Quantitative RT-PCR analysis of RNA extracted from isolated cardiomyocytes showed that SAP97 mRNA transcript levels were decreased by 80% ⫾ 2% in αMHC-Cre/SAP97fl/fl Table 1
Action potential duration is increased in αMHC-Cre/SAP97fl/fl mouse cardiomyocytes To assess the functional consequences of SAP97 deletion on cardiac cell electrical activity, action potential (AP) recordings were performed on isolated cardiomyocytes (Figure 2 and Online Supplemental Table 1). Although AP maximal upstroke velocity dV/dtmax (reflecting sodium channel availability), AP amplitude, and time to peak were not different between αMHC-Cre/SAP97fl/fl and control myocytes (Figures 2B–2D), action potential duration (APD) was greatly prolonged in αMHC-Cre/SAP97fl/fl cardiomyocytes (1.9 ⫾ 0.3, 2.3 ⫾ 0.4, and 2.1 ⫾ 0.2 folds for APD30, APD50, and APD90, respectively, n ¼ 10, P o .05; Figure 2E). This finding suggests that mainly potassium channel function rather than sodium channel function is altered by the reduction in SAP97 expression. Moreover, the resting membrane potential of αMHC-Cre/SAP97fl/fl cardiomyocytes was slightly depolarized compared to control cardiomyocytes (þ2.4 ⫾ 0.9 mV, n ¼ 10, P o .05; Figure 2F).
NaV1.5 function and distribution are not altered in αMHC-Cre/SAP97fl/fl mouse heart To determine which ionic conductances were altered by SAP97 deletion in cardiomyocytes, cardiac sodium and potassium currents were studied in isolated ventricular cardiomyocytes using the patch-clamp technique in wholecell configuration. Cell capacitance was similar between SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes (170 ⫾ 10 pF, n ¼ 23; and 168 ⫾ 7 pF, n ¼ 27, respectively, P ¼ NS). INa density analysis showed no differences between
Echocardiographic data
n Heart rate (bpm) IVSd (mm) LVEDD (mm) LVPWd (mm) IVSs (mm) LVESD (mm) LVPWs (mm) Cardiac output (mL/min) Ejection fraction (%) Fractional shortening (%)
SAP97fl/fl
αMHC-Cre/SAP97fl/fl
8 536 ⫾ 0.6 ⫾ 4.1 ⫾ 0.7 ⫾ 1.0 ⫾ 2.6 ⫾ 1.1 ⫾ 12 ⫾ 71.83 ⫾ 35.95 ⫾
523 0.6 4.2 0.8 1.0 2.7 1.1 13 71.46 35.88
30 0.01 0.1 0.02 0.02 0.1 0.03 0.8 2.26 1.81
7 ⫾ 49NS ⫾ 0.02NS ⫾ 0.1NS ⫾ 0.04NS ⫾ 0.05NS ⫾ 0.1NS ⫾ 0.06NS ⫾ 1.0NS ⫾ 3.10NS ⫾ 2.46NS
P value
.652 1 .490 .762 .818 .670 .820 .438 .923 .983
Data are given as mean ⫾ SEM. IVSd ¼ interventricular septum in diastole; IVSs ¼ interventricular septum in systole; LVEDD ¼ left ventricular internal end-diastolic diameter; LVESD ¼ left ventricular internal end-systolic diameter; LVPWd ¼ left ventricular posterior wall in diastole; LVPWs ¼ left ventricular posterior wall in systole; NS ¼ not significant.
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fl/fl
0
SAP97 fl/fl +/SAP97 Cre
20 mV 10 ms
-80
Figure 2 Action potential duration (APD) is increased in αMHC-Cre/SAP97fl/fl cardiomyocytes. Representative APs recorded from cardiomyocytes of SAP97fl/fl mice (black line) and αMHC-Cre/SAP97fl/fl mice (red line) (A). Maximal AP upstroke velocity (B), AP amplitude (C), and time to peak (D) remain unchanged in αMHC-Cre/SAP97fl/fl cardiomyocytes (white squares) compared to controls (black squares). Increased APD at 30, 50, and 90% repolarization in alphaMHC-Cre/SAP97fl/fl cardiomyocytes (E). Resting membrane potential is slightly depolarized in alphaMHC-Cre/SAP97fl/fl cardiomyocytes (F). *P o .05.
αMHC-Cre/SAP97fl/fl and control myocytes (Figure 3A). Voltage dependence of INa activation and inactivation also was not significantly different (Figure 3B). Kinetics of INa fast inactivation were unchanged (see Online Supplemental Figure 1A). In one of our recent studies, we observed that NaV1.5 co-localized with SAP97 at the intercalated discs of cardiomyocytes.11 To examine the consequence of SAP97 deletion on the distribution of NaV1.5 in cardiac tissue, immunostainings of cardiac ventricular sections were performed. As shown in Figure 3C, NaV1.5 expression is present in both lateral membranes and intercalated discs of SAP97fl/fl ventricular sections, and this localization was not altered in αMHC-Cre/SAP97fl/fl hearts.
IK1, Ito, and IKur potassium currents are decreased in αMHC-Cre/SAP97fl/fl cardiomyocytes In previous studies, SAP97 has been found to interact with and regulate different cardiac potassium channels such as Kir2.x channels (Kir2.1, Kir2.2, Kir2.3), which are responsible for the inward rectifier IK1 potassium current, KV4.2 and KV4.3 channels, which are responsible for the transient outward potassium current Ito, and KV1.5 channels, which are responsible for the ultrarapid, outwardly rectifying potassium current IKur.7–10,12,18 In our study, these different potassium currents were recorded in isolated cardiomyocytes from SAP97fl/fl and αMHC-Cre/SAP97fl/fl mouse hearts. IK1, Ito, and IKur were all found to be decreased in
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Figure 3 Whole-cell INa is not modified in αMHC-Cre/SAP97fl/fl cardiomyocytes. Current density–voltage relationship of INa in SAP97fl/fl and αMHC-Cre/ SAP97fl/fl mice (A). Steady-state activation and inactivation curves from SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes. Boltzmann curves of individual cells were fitted to steady-state activation (SAP97fl/fl V1/2 ¼ –29.6 ⫾ 0.7 mV, K ¼ 5.5 ⫾ 0.1; αMHC-Cre/SAP97fl/fl V1/2 ¼ –30 ⫾ 1.5 mV, K ¼ 5.8 ⫾ 0.2) and inactivation data (SAP97fl/fl V1/2 ¼ –75.9 ⫾ 1.5 mV, K ¼ 6.6 ⫾ 0.3; αMHC-Cre/SAP97fl/fl V1/2 ¼ –76.9 ⫾ 2.1 mV, K ¼ 5.8 ⫾ 0.2) (B). NaV1.5 immunostaining in SAP97fl/fl and αMHC-Cre/SAP97fl/fl ventricular sections (C).
αMHC-Cre/SAP97fl/fl mouse hearts (Figures 4A–4C). Peak currents were decreased by 53% ⫾ 6%, 45% ⫾ 9%, and 53% ⫾ 6% for IK1, Ito, and IKur, respectively (P o .05). On the
contrary, the ISS potassium current, which has not been reported to be regulated by SAP97, remained unchanged (Figure 4D).
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Figure 4 Whole-cell IK1, Ito, and IKur are decreased in αMHC-Cre/SAP97 cardiomyocytes. Representative traces of whole-cell current and current density– voltage relationships for IK1 (A), Ito (B), IKur (C), and Iss (D) recorded in SAP97fl/fl and αMHC-Cre/SAP97fl/fl cardiomyocytes. *P o.05.
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Figure 5 SAP97-associated ion channel expression is not modified in αMHC-Cre/SAP97fl/fl mouse hearts. Representative western blots showing expression of SAP97, Cre recombinase, and SAP97-associated ion channels in SAP97fl/fl and alpha-MHC-Cre/SAP97fl/fl mouse hearts (A). Quantitative comparison of protein expression levels from multiple western blots similar to that shown in (A). Band intensities for each mouse heart were normalized to the averaged control (SAP97fl/fl) per experimental day before pooling results for statistical testing *P o. 05 (B).
Ion channel expression in αMHC-Cre/SAP97fl/fl mouse hearts In order to investigate whether expression of the different cardiac ion channels was modified subsequent to SAP97 reduction, NaV1.5, Kir2.1, Kir2.2, KV4.2, KV4.3, and KV1.5 whole-cell expression was analyzed by western blot (Figure 5). After quantification of band densities, no significant differences were found in potassium channel protein levels between SAP97fl/fl and αMHC-Cre/SAP97fl/fl mouse hearts (Figure 5B). Unexpectedly, NaV1.5 expression was slightly increased by 14% (P o.05) in αMHC-Cre/SAP97fl/fl mouse hearts.
Surface ECGs show a prolonged QT interval in αMHC-Cre/SAP97fl/fl mice To investigate the in vivo functional consequences of deletion of SAP97 in the mouse heart in relation to the concomitant decrease in potassium currents, surface echocardiography and ECG analysis were performed on lightly anesthetized mice. Echocardiography revealed no major differences in functional
parameters between SAP97fl/fl and αMHC-Cre/SAP97fl/fl mice (Table 1). On ECG, the P wave, PR interval, and QRS duration were not different between αMHC-Cre/SAP97fl/fl mice and controls. However, the corrected QT interval (QTc) was significantly prolonged in αMHC-Cre/SAP97fl/fl mice compared to SAP97fl/fl control mice (58.3 ⫾ 2.0 ms vs 49.1 ⫾ 1.2 ms, respectively; Table 2 and Figure 6).
Discussion In this study, we generated and characterized for the first time a mouse line with cardiomyocyte-specific deletion of SAP97 and obtained evidence for in vivo regulation of cardiac cell electrical activity by SAP97. The main findings can be summarized as follows: (1) IK1, Ito, and IKur were reduced in ventricular myocytes that had reduced expression of SAP97; (2) unexpectedly, NaV1.5 function and localization were not altered in αMHC-Cre/SAP97fl/fl mice; and (3) SAP97-deficient mice displayed a prolonged QT interval on ECG.
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189
ECGs parameters SAP97fl/fl
n Heart rate (bpm) P duration (ms) PR interval (ms) QRS duration (ms) QTc (ms)
7 416.8 ⫾ 19.0 ⫾ 43.4 ⫾ 20.1 ⫾ 49.1 ⫾
29.7 0.5 1.9 1.6 1.2
αMHC-Cre/SAP97fl/fl
P value
6 394.2 ⫾ 20.7 ⫾ 40.3 ⫾ 22.8 ⫾ 58.3 ⫾
.664 .077 .313 .178 .001
48.7NS 0.9NS 2.5NS 1.3NS 2.0*
Data are given as mean ⫾ SEM. NS ¼ not significant; QTc ¼ corrected QT interval. * P o .05.
Cardiac-specific deletion of SAP97 is not lethal The in vivo role of SAP97 had already been studied in the past, but no data were available regarding its role in cardiac development or function.13,14 This protein appears to be essential for the development of different organs, and SAP97 total knockout mice do not survive after birth. In the present study, we observed that mice with cardiac-specific reduced SAP97 expression can survive until adult age. Echocardiography did not reveal any morphological alterations, thus suggesting that SAP97 is not crucial for cardiac development. Moreover, SAP97 is also known to be expressed in
neurons and plays a role in the organization of synaptic macromolecular complexes. Thus, it would not be surprising if sympathetic or parasympathetic regulation of the heart could be modulated by SAP97; however, this question remains to be further investigated.
Role of SAP97 in regulation of potassium currents is confirmed We observed that at least 3 ventricular potassium currents were altered in αMHC-Cre/SAP97fl/fl cardiomyocytes, leading to increased APD and concomitant prolongation of the
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Figure 6 QT interval is prolonged in αMHC-Cre/SAP97fl/fl mice. Typical examples of ECG traces recorded in SAP97fl/fl and αMHC-Cre/SAP97fl/fl mice (A). ECG recordings show prolongation of the QT interval in αMHC-Cre/SAP97fl/fl mice compared to SAP97fl/fl (instead of Cre/SAP97fl/fl) mice (B).
190 QT interval on ECG. However, the mechanisms underlying these decreased whole-cell potassium current densities still have to be determined. The mechanism does not involve regulation of total channel expression since western blots did not show any differences in Kir2.1, Kir2.2, KV4.2, KV4.3, or KV1.5 protein levels between αMHC-Cre/SAP97fl/fl and control hearts. These results differ from those obtained by Vaidyanathan et al,8 who found that expression levels of Kir2.1, Kir2.2, and Kir2.3 were decreased in the 4 days after SAP97 silencing by shRNAs in adult rat ventricular myocytes that were kept in culture. It was previously demonstrated that overexpression of SAP97 in HEK293 cells increases the whole-cell current density of Kir2.3 through multiple mechanisms.18 One of these mechanisms was an increase in the number of functional channels at the membrane, and the other was an increase in the unitary conductance amplitude. However, results obtained in adult rat ventricular myocytes did not show any change in unitary conductance amplitude.8 SAP97 has also been reported to regulate the properties of KV channels by modulating channel surface expression.9,10 Previous data indicated that silencing of SAP97 also accelerated the time-dependent inactivation of Ito, possibly because of loss of interaction with calmodulin kinase II.10 We measured the kinetics of Ito inactivation, but we did not find any difference between αMHC-Cre/SAP97fl/fl and control cells (see Online Supplemental Figure 1B). A reason for this discrepancy could be the difference in free Ca2þ concentrations in the intrapipette medium used to perform Ito measurements (0.5 mM in El-Haou et al10 vs 20 nM in the present study). Thus, whether SAP97 regulates potassium channel trafficking toward the sarcolemma (and thus surface expression) unitary conductance, or kinetics of inactivation in vivo should be the topic of further study.
Are SAP97 and NaV1.5 interacting in the mouse heart? Regulation of the cardiac NaV1.5 sodium channel by interacting proteins is an active field of research.19 Indeed, NaV1.5 has been shown to be part of distinct macromolecular complexes that interact with different partner proteins, depending on its location in the cardiomyocyte.16 At least 2 pools of NaV1.5 have been proposed: one population of channels at the lateral sarcolemma that is interacting with the syntrophin/dystrophin complex and another population at the intercalated discs that may interact with SAP97, as neither syntrophin proteins nor dystrophin is expressed at the discs. In a previous work, our group obtained molecular and functional evidence that SAP97 interacts with the PDZ domain-binding motif of NaV1.5 in the mouse heart.11 Silencing SAP97 expression in rat myocytes in culture or in HEK293 cells expressing NaV1.5 led to reduced INa. Subsequently, Milstein et al12 were able to coimmunoprecipitate NaV1.5 and SAP97 in rat ventricle lysates. In addition, it was suggested that SAP97 allows NaV1.5 to interact with the Kir2.1 potassium channel and that
Heart Rhythm, Vol 12, No 1, January 2015 expression of both channels is co-regulated. In our recent study that addressed the in vivo significance of the NaV1.5PDZ domain-binding motif in mice, we found that deleting this motif (ΔSIV mice) led to decreased expression of NaV1.5 at the lateral membrane of cardiomyocytes but did not alter NaV1.5 localization at the intercalated discs.16 This observation challenged the question of the role of NaV1.5 and SAP97 interaction in cardiac cells. In the present study, we did not find any changes in NaV1.5 localization and function when SAP97 expression was suppressed. One of the main differences between these two mouse models16 and the previous studies is that these mice are constitutive knockin or knockout mice. In other words, genetic modification is present early in development, whereas in the other studies,11,12 SAP97 expression was silenced in adult myocytes that were kept in culture. Thus, it may not be ruled out that in an inducible SAP97 knockout mouse model, the consequences on INa could be different. In addition, NaV1.5 has been found to interact with other regulatory proteins at the intercalated discs, such as connexin-43,20–22 plakophilin2,23 and ankyrin G.24 Such a complex organization of NaV1.5 channels at the intercalated discs may suggest that they play a crucial role in this specific region of cardiac myocytes, and that perhaps the consequences of a constitutive deletion of one of these regulatory proteins could be counteracted by modifications in expression or organization of the other partner proteins. Interestingly, in a recent study, Asimaki et al25 investigated the effect of the human 2057del2 mutation in the gene encoding plakoglobin, in the context of arrhythmogenic cardiomyopathy. By using different models such as zebrafish, neonatal rat ventricular myocytes, and cardiac myocytes derived from human induced pluripotent stem cells taken from arrhythmogenic cardiomyopathy probands with mutations in the gene encoding plakophilin-2, they found that decreased expression of plakoglobin or plakophilin-2 was associated with altered distribution of connexin-43, NaV1.5, and SAP97. Note that this altered distribution of SAP97 was accompanied by a large increase in APD with concomitant reduction of potassium current. In summary, the role of SAP97dependent regulation of NaV1.5 remains an open question. Although it seems likely that both proteins are interacting, it may be postulated that SAP97 is not directly involved in targeting and stabilizing NaV1.5 at the intercalated discs. Alternative roles for this interaction, such as providing a scaffold for signaling proteins such as calmodulin kinase II,10 will have to be investigated in the future.
Study limitations One of the main limitations of this study is that we have not been able to investigate SAP97 protein distribution within murine cardiomyocytes. SAP97 has been proposed to be mainly expressed at the intercalated discs of cardiac myocytes8,11,12 but also at the lateral membrane10 and T-tubules.8,12 However, none of these studies presented an appropriate negative control (ie, cardiac tissue in which
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SAP97 expression is suppressed). It is worth noting that we performed immunostaining on heart sections and on isolated cardiomyocytes of αMHC-Cre/SAP97fl/fl mice using more than 10 different commercial anti-SAP97 antibodies, but the signal was never different from control tissues. Another comment could be made on the approach that we used to determine the in vivo role of SAP97. It is known that compensatory mechanisms can develop when a protein is constitutively suppressed. This is the case with utrophin (a homolog of dystrophin), which has been shown to be upregulated in the heart of dystrophin-deficient mice.26 This utrophin upregulation could partially compensate for dystrophin deletion and the subsequent INa decrease. Thus, by using an inducible knockout mouse model instead of a constitutive model, our findings could have been different.
Conclusion We generated a cardiac-specific SAP97-deficient mouse line and provided, for the first time, evidence for in vivo regulation of cardiac ion channels by SAP97. Surprisingly, INa was not altered in SAP97-deficient cardiomyocytes, whereas regulation of potassium currents was confirmed. Whether SAP97 regulates potassium channel surface expression or unitary conductance still has to be elucidated. However, based on the important role of SAP97 in regulating cardiac potassium currents, it could be proposed that DLG1 may be a susceptibility gene that should to be investigated in patients with congenital long QT syndrome.
Appendix Supplementary data Supplementary material cited in this article is available online at doi:10.1016/j.hrthm.2014.09.057.
References 1. Funke L, Dakoji S, Bredt DS. Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annu Rev Biochem 2005;74:219–245. 2. Kennedy MB. Origin of PDZ (DHR, GLGF) domains. Trends Biochem Sci 1995;20:350. 3. Godreau D, Vranckx R, Maguy A, Rucker-Martin C, Goyenvalle C, Abdelshafy S, Tessier S, Couetil JP, Hatem SN. Expression, regulation and role of the MAGUK protein SAP-97 in human atrial myocardium. Cardiovasc Res 2002;56: 433–442. 4. Fujita A, Kurachi Y. SAP family proteins. Biochem Biophys Res Commun 2000;269:1–6. 5. Godreau D, Vranckx R, Maguy A, Goyenvalle C, Hatem SN. Different isoforms of synapse-associated protein, SAP97, are expressed in the heart and have distinct effects on the voltage-gated Kþ channel Kv1.5. J Biol Chem 2003;278: 47046–47052. 6. Leonoudakis D, Mailliard W, Wingerd K, Clegg D, Vandenberg C. Inward rectifier potassium channel Kir2.2 is associated with synapse-associated protein SAP97. J Cell Sci 2001;114:987–998.
191 7. Leonoudakis D, Conti LR, Anderson S, Radeke CM, McGuire LM, Adams ME, Froehner SC, Yates JR 3rd, Vandenberg CA. Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins. J Biol Chem 2004;279:22331–22346. 8. Vaidyanathan R, Taffet SM, Vikstrom KL, Anumonwo JM. Regulation of cardiac inward rectifier potassium current (I(K1)) by synapse-associated protein97. J Biol Chem 2010;285:28000–28009. 9. Abi-Char J, El-Haou S, Balse E, Neyroud N, Vranckx R, Coulombe A, Hatem SN. The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes. Am J Physiol Heart Circ Physiol 2008;294: H1851–H1861. 10. El-Haou S, Balse E, Neyroud N, Dilanian G, Gavillet B, Abriel H, Coulombe A, Jeromin A, Hatem SN. Kv4 potassium channels form a tripartite complex with the anchoring protein SAP97 and CaMKII in cardiac myocytes. Circ Res 2009;104: 758–769. 11. Petitprez S, Zmoos AF, Ogrodnik J, et al. SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes. Circ Res 2011;108:294–304. 12. Milstein ML, Musa H, Balbuena DP, Anumonwo JM, Auerbach DS, Furspan PB, Hou L, Hu B, Schumacher SM, Vaidyanathan R, Martens JR, Jalife J. Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia. Proc Natl Acad Sci U S A 2012;109:E2134–E2143. 13. Mahoney ZX, Sammut B, Xavier RJ, Cunningham J, Go G, Brim KL, Stappenbeck TS, Miner JH, Swat W. Discs-large homolog 1 regulates smooth muscle orientation in the mouse ureter. Proc Natl Acad Sci U S A 2006;103: 19872–19877. 14. Iizuka-Kogo A, Ishidao T, Akiyama T, Senda T. Abnormal development of urogenital organs in Dlgh1-deficient mice. Development 2007;134:1799–1807. 15. Ackerman MJ, Mohler PJ. Defining a new paradigm for human arrhythmia syndromes: phenotypic manifestations of gene mutations in ion channel- and transporter-associated proteins. Circ Res 2010;107:457–465. 16. Shy D, Gillet L, Ogrodnik J, et al. PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function. Circulation 2014;130:147–160. 17. Wolska BM, Solaro RJ. Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry. Am J Physiol 1996;271: H1250–H1255. 18. Vikstrom KL, Vaidyanathan R, Levinsohn S, O'Connell RP, Qian Y, Crye M, Mills JH, Anumonwo JM. SAP97 regulates Kir2.3 channels by multiple mechanisms. Am J Physiol Heart Circ Physiol 2009;297:H1387–H1397. 19. Abriel H. Cardiac sodium channel Na(v)1.5 and interacting proteins: physiology and pathophysiology. J Mol Cell Cardiol 2010;48:2–11. 20. Malhotra JD, Thyagarajan V, Chen C, Isom LL. Tyrosine-phosphorylated and nonphosphorylated sodium channel beta1 subunits are differentially localized in cardiac myocytes. J Biol Chem 2004;279:40748–40754. 21. Jansen JA, Noorman M, Musa H, et al. Reduced heterogeneous expression of Cx43 results in decreased Nav1.5 expression and reduced sodium current that accounts for arrhythmia vulnerability in conditional Cx43 knockout mice. Heart Rhythm 2012;9:600–607. 22. Rhett JM, Ongstad EL, Jourdan J, Gourdie RG. Cx43 associates with Na(v)1.5 in the cardiomyocyte perinexus. J Membr Biol 2012;245:411–422. 23. Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patino GA, Taffet SM, Isom LL, Delmar M. Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res 2009;105:523–526. 24. Mohler PJ, Rivolta I, Napolitano C, LeMaillet G, Lambert S, Priori SG, Bennett V. Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes. Proc Natl Acad Sci U S A 2004;101:17533–17538. 25. Asimaki A, Kapoor S, Plovie E, et al. Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Sci Transl Med 2014;6:240ra74. 26. Albesa M, Ogrodnik J, Rougier JS, Abriel H. Regulation of the cardiac sodium channel Nav1.5 by utrophin in dystrophin-deficient mice. Cardiovasc Res 2011;89:320–328.
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CLINICAL PERSPECTIVES Genetic cardiac channelopathies such as congenital long QT syndrome and Brugada syndrome are caused by mutations in genes coding for ion channel subunits or ion channel regulatory proteins. These pathologies are characterized by a broad genetic heterogeneity involving several dozens of genes. However, in many patients and families, the culprit genes are still unknown. In this study, we investigated the consequences of specific ablation of SAP97 in mouse cardiac cells a scaffolding protein that has been shown to interact with and regulate the surface expression of cardiac potassium and sodium channels. It was observed that SAP97-deficient cardiac cells had prolonged APD due to decreased repolarizing potassium current. ECGs of these SAP97-deficient mice displayed prolonged QT intervals. These findings suggest that genetic variants such as functional polymorphisms and mutations in DLG1, the gene coding for SAP97, that lead to reduced expression or loss of function of SAP97 may delay ventricular repolarization and hence lead to QT-interval prolongation. This study should stimulate the search for genetic variants of DLG1 in patient cohorts with long QT syndrome.
