A common co-morbidity modulates disease expression and treatment efficacy in inherited cardiac sodium channelopathy

A common co-morbidity modulates disease expression and treatment efficacy in inherited cardiac... Abstract Aims Management of patients with inherited cardiac ion channelopathy is hindered by variability in disease severity and sudden cardiac death (SCD) risk. Here, we investigated the modulatory role of hypertrophy on arrhythmia and SCD risk in sodium channelopathy. Methods and results Follow-up data was collected from 164 individuals positive for the SCN5A-1795insD founder mutation and 247 mutation-negative relatives. A total of 38 (obligate) mutation-positive patients died suddenly or suffered life-threatening ventricular arrhythmia. Of these, 18 were aged >40 years, a high proportion of which had a clinical diagnosis of hypertension and/or cardiac hypertrophy. While pacemaker implantation was highly protective in preventing bradycardia-related SCD in young mutation-positive patients, seven of them aged >40 experienced life-threatening arrhythmic events despite pacemaker treatment. Of these, six had a diagnosis of hypertension/hypertrophy, pointing to a modulatory role of this co-morbidity. Induction of hypertrophy in adult mice carrying the homologous mutation (Scn5a1798insD/+) caused SCD and excessive conduction disturbances, confirming a modulatory effect of hypertrophy in the setting of the mutation. The deleterious effects of the interaction between hypertrophy and the mutation were prevented by genetically impairing the pro-hypertrophic response and by pharmacological inhibition of the enhanced late sodium current associated with the mutation. Conclusion This study provides the first evidence for a modulatory effect of co-existing cardiac hypertrophy on arrhythmia risk and treatment efficacy in inherited sodium channelopathy. Our findings emphasize the need for continued assessment and rigorous treatment of this co-morbidity in SCN5A mutation-positive individuals. SCN5A, Sudden death, Cardiac hypertrophy, Hypertension, Conduction delay, Ventricular arrhythmias Translational perspectives SCN5A mutations present with a broad spectrum of clinical phenotypes, including sudden cardiac death (SCD). Disease penetrance and severity varies between individuals carrying the same SCN5A mutation, and common co-morbidities may modulate arrhythmia and SCD risk with potential implications for antiarrhythmic treatment efficacy. This study conducted in patients and mice, provides strong evidence for a modulatory role of hypertension/hypertrophy in modulating arrhythmic risk of the SCN5A-1795insD mutation. Our findings support the concept that SCN5A mutation-positive patients should be carefully monitored for the development of cardiac hypertension and hypertrophy during follow up, and that their clinical management should be adjusted where necessary to also include rigorous management of this co-morbidity. Introduction The genetic basis of the Mendelian cardiac rhythm disorders associated with sudden cardiac death (SCD) has been brought into focus over the last 20 years with the discovery of a large spectrum of causal mutations in genes encoding components of cardiac ion channels.1 Although this enabled genetic testing and consequently improvements in clinical care, patient management is still hindered by the reduced penetrance and substantial variability in disease severity and SCD risk among mutation-positive individuals.1 Although co-morbidities are expected to modulate disease severity, these remain unexplored. Identification of disease modifiers in these disorders is however hindered by the substantial genetic heterogeneity across patients, as different mutations may be associated with different effects and thus also contribute to inter-individual variability.2 Studies in large families that harbour founder mutations (where all affected individuals carry the same familial mutation) circumvent this limitation and therefore offer opportunities for the identification of modulatory factors. We have previously described a large Dutch family harbouring a founder mutation, p.Tyr1795_Glu1796insAsp (previously called ‘SCN5A-1795insD’), in the SCN5A gene, which encodes the major sodium channel isoform in heart (NaV1.5).3,4 This mutation displays multiple biophysical defects and causes ‘overlap’ sodium channelopathy with features of long QT syndrome, Brugada syndrome, and conduction disease.5,6 While variability in disease severity among mutation-positive individuals in this family is long recognized,3 predictors of arrhythmia and SCD risk have remained elusive. By combining clinical observations in the family with experimental studies in Scn5a1798insD/+ mice carrying the mouse homolog of the mutation, here, we uncovered a modulatory effect of hypertension and cardiac hypertrophy on disease severity and expression. In particular, the co-occurrence of cardiac hypertrophy was found to exacerbate cardiac conduction slowing and arrhythmia risk in the setting of the mutation, and was associated with a decreased efficacy of pacemaker treatment in preventing SCD. Our findings furthermore provide evidence for a potential therapeutic role of pharmacological late sodium current inhibition. Methods Details on study approval, SCN5A-1795insD mutation study population, clinical data collection, pathology reports, generation of Scn5a1798insD/+ and Scn5a1798insD/+-Nfatc2-/- mice, transverse aortic constriction (TAC) and chow experiments, electrophysiological assessments in Langendorff-perfused hearts, quantitative PCR assay and analysis and whole-mount in situ hybridization, and statistical analysis are provided in the Supplementary Methods. Data are presented as mean ± standard deviation or median (interquartile range), as appropriate. Results Hypertension and hypertrophy in older SCN5A-1795insD mutation-positive patients who died suddenly Through an extensive genealogical search, we reconstructed the pedigree of the family with the SCN5A-1795insD mutation back to the 18th century, linking 164 mutation-positive individuals and 247 mutation-negative relatives (Figure 1A). Mutation-positive patients displayed (atrio-)ventricular conduction slowing, sinus node dysfunction, excessive ventricular repolarization abnormalities at slow heart rates, and SCD occurring predominantly at night.3,4 A total of 38 individuals died suddenly or suffered life-threatening ventricular arrhythmia (13 males, 25 females; average age at event 38 ± 18 years, range 13–76) (Figure 1A and B). Twenty-five of these individuals were confirmed or obligate mutation-positive; genetic testing was not possible in the rest as these were deceased individuals from past generations, the majority of which died more than 50 years ago. Eighteen individuals who suffered SCD or a life-threatening arrhythmia were older than 40 years at the time of the event (4 males, 14 females, average age 54 ± 9 years, range 41–76) (Figure 1A and B). Recent clinical information was available for 10 of these (all confirmed mutation-positive); nine of them had a clinical diagnosis of hypertension and/or evidence for the presence of left ventricular hypertrophy (LVH) on magnetic resonance imaging (MRI), echocardiography, or post-mortem examination (including increased heart weight; Table 1), suggesting a modulatory effect of cardiac hypertrophy on arrhythmia risk. Post-mortem analysis furthermore indicated the additional presence of slight interstitial fibrosis (potentially hypertension-related) in three out of four patients (Table 1). Evidence for coronary artery disease, with hypertension as a major risk factor, was found in two patients. In one patient, small foci of subendocardial necrosis were found, but this was not associated with critically stenosing or thrombosed coronary lesions. The other patient displayed a fibrotic scar consistent with an old myocardial infarction without signs of recent-onset ischaemia. Table 1 Characteristics of SCN5A-1795insD mutation-positive patients suffering a serious cardiac event above the age of 40 with available recent clinical information Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  DCM, dilated cardiomyopathy; ICD, implantable cardioverter-defibrillator; LVH, left ventricular hypertrophy; PM, pacemaker; SCD, sudden cardiac death; VF, ventricular fibrillation; VT, ventricular tachycardia. a Patient did not have documented hypertension prior to VF, but developed clinically relevant hypertension 1 year later. Table 1 Characteristics of SCN5A-1795insD mutation-positive patients suffering a serious cardiac event above the age of 40 with available recent clinical information Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  DCM, dilated cardiomyopathy; ICD, implantable cardioverter-defibrillator; LVH, left ventricular hypertrophy; PM, pacemaker; SCD, sudden cardiac death; VF, ventricular fibrillation; VT, ventricular tachycardia. a Patient did not have documented hypertension prior to VF, but developed clinically relevant hypertension 1 year later. Figure 1 View largeDownload slide Life-threatening cardiac events in SCN5A-1795insD mutation-positive patients. (A) The SCN5A-1795insD founder population pedigree. Circles indicate SCN5A-1795insD mutation-positive patients suffering sudden cardiac death, ventricular fibrillation, and/or ventricular tachycardia in the presence or absence of pacemaker treatment and hypertension and/or left ventricular hypertrophy. (B) Survival from sudden cardiac death and/or ventricular arrhythmias was significantly reduced in SCN5A-1795insD mutation-positive patients (n = 164) vs. mutation-negative relatives (n = 247) (P = 1.3026E−18). (C) Overall survival from sudden cardiac death and/or ventricular arrhythmias in SCN5A-1795insD mutation-positive patients was significantly higher in those with (n = 87) as compared to without (n = 77) pacemaker (P = 0.000003), but pacemaker efficacy was significantly reduced in mutation-positive patients above the age of 40 years (P = 0.028; Cox regression model). (D) Pacemaker readout (A, atrial lead; V, ventricular lead; paper speed 25 mm/s) of a 58-year female mutation-positive patient showing a nocturnal, fast (up to 300/min) and apparently polymorphic ventricular tachycardia despite adequate anti-bradycardia treatment. Figure 1 View largeDownload slide Life-threatening cardiac events in SCN5A-1795insD mutation-positive patients. (A) The SCN5A-1795insD founder population pedigree. Circles indicate SCN5A-1795insD mutation-positive patients suffering sudden cardiac death, ventricular fibrillation, and/or ventricular tachycardia in the presence or absence of pacemaker treatment and hypertension and/or left ventricular hypertrophy. (B) Survival from sudden cardiac death and/or ventricular arrhythmias was significantly reduced in SCN5A-1795insD mutation-positive patients (n = 164) vs. mutation-negative relatives (n = 247) (P = 1.3026E−18). (C) Overall survival from sudden cardiac death and/or ventricular arrhythmias in SCN5A-1795insD mutation-positive patients was significantly higher in those with (n = 87) as compared to without (n = 77) pacemaker (P = 0.000003), but pacemaker efficacy was significantly reduced in mutation-positive patients above the age of 40 years (P = 0.028; Cox regression model). (D) Pacemaker readout (A, atrial lead; V, ventricular lead; paper speed 25 mm/s) of a 58-year female mutation-positive patient showing a nocturnal, fast (up to 300/min) and apparently polymorphic ventricular tachycardia despite adequate anti-bradycardia treatment. Decreased pacemaker treatment efficacy in SCN5A-1795insD mutation-positive patients older than 40 Mutation-positive patients died suddenly predominantly during the night, and excessive QT-prolongation during (nocturnal) bradycardic episodes was frequently documented on Holter recordings.3,4,7 Hence, a bradycardia-dependent trigger for arrhythmia was originally suspected, and pacemaker implantation has been routinely employed in the family to prevent SCD.4 While this approach initially proved successful,4 in the last decade seven mutation-positive individuals suffered ventricular tachyarrhythmias and/or SCD despite pacemaker implantation (Figure 1A). All of them were older than 40 (age range 41–58 years), and in the majority, ventricular fibrillation, or tachycardia was documented (Table 1), in addition to ventricular extrasystoles recorded during Holter monitoring or exercise testing in several cases. Figure 1D shows a pacemaker readout displaying a nocturnal, fast ventricular tachycardia (presumably polymorphic) in a 58-year-old female mutation-positive patient; following this episode, her pacemaker was replaced with an implantable cardioverter-defibrillator. These observations suggested an age-dependent shift towards a different arrhythmia triggering mechanism at older age with arrhythmias occurring despite prevention of bradycardia. Indeed, comparing the occurrence of SCD/life-threatening arrhythmia between individuals with and without pacemaker showed that while pacemaker implantation was highly protective in young mutation-positive patients, its efficacy in preventing arrhythmias and SCD was significantly decreased above the age of 40 years (P = 0.028; proportional hazard assumption of pacemaker implantation tested using the Schoenfeld residuals from a Cox regression model) (Figure 1C). Importantly, in six of the seven mutation-positive patients who suffered a life-threatening event despite pacemaker treatment, a history of hypertension was confirmed and/or the presence of LVH documented on MRI, echocardiography, or post-mortem examination (Table 1). These observations led us to hypothesize that the co-occurrence of the mutation with cardiac hypertrophy, developing with age secondary to for instance hypertension, plays a pivotal role in modulating arrhythmia risk. The co-occurrence of cardiac hypertrophy is pro-arrhythmic in aged Scn5a1798insD/+ mice We further explored the modulatory role of cardiac hypertrophy in Scn5a1798insD/+ mice carrying the exact mouse homolog of the human SCN5A-1795insD mutation.5,8 We have previously generated two distinct mouse lines harbouring the Scn5a1798insD/+ mutation, with respectively the FVB/N and 129P2 inbred genetic background, enabling investigation of the effect of the mutation on different genetic backgrounds. In young adult mice, we previously demonstrated strain-dependent variable disease severity, with more pronounced conduction slowing and prolongation of repolarization in mutant mice of the 129P2 strain.8 We now studied aged mice wild-type for the Scn5a gene (WT) and Scn5a1798insD/+ mutant for the Scn5a gene (MUT) of both strains. The 129P2 strain was found to develop more cardiac hypertrophy with age as compared to the FVB/N strain. This feature was intrinsic to the 129P2 strain and independent of the mutation, since both aged WT- and MUT-129P2 mice displayed greater heart weight and higher expression levels of pro-hypertrophic markers as compared to aged WT- and MUT-FVB/N mice (Figure 2A and B). These intrinsic strain-dependent differences in susceptibility to hypertrophy enabled a comparison of the pro-arrhythmic effect of the mutation in the presence (i.e. aged 129P2-MUT mice) and absence (i.e. aged FVB/N-MUT mice) of hypertrophy. Therefore, we conducted electrocardiogram (ECG) studies in anaesthetized mice and electrophysiological studies in Langendorff-perfused hearts, comparing aged (8–22 months old) WT and MUT mice of both strains. This indeed uncovered a genotype-strain interaction, where aged 129P2-MUT mice displayed significantly more pronounced ventricular conduction slowing in vivo (QRS-duration on ECG analysis; Figure 2C and D; Supplementary material online, Table S1) and ex vivo (left ventricular activation time in isolated Langendorff-perfused hearts; Figure 2F and G). Moreover, 129P2-MUT mice exhibited significantly more spontaneous ventricular extrasystoles and arrhythmias in vivo (Figure 2C and E) and an increased inducibility of ventricular arrhythmias ex vivo (Figure 2H and I). While these observations do not provide a causal link, they are in line with a pro-arrhythmic interaction between hypertrophy and the mutation, similar to our observations in the SCN5A-1795insD family. Figure 2 View largeDownload slide Pro-arrhythmic effects of cardiac hypertrophy in aged Scn5a1798insD/+ mice. Mice of the 129P2 strain develop more severe cardiac hypertrophy with age (WT, wild type and MUT, Scn5a1798insD/+ to a similar extent) than mice of the FVB/N strain, as indicated by increased heart weight to body weight ratio (A) and Anf expression on in situ hybridization (B). (C) Typical examples of surface electrocardiograms and arrhythmias. (D and E) Aged 129P2-MUT mice show significantly increased QRS-duration and more spontaneous arrhythmias (Pearson χ2 overall P = 0.000039) on electrocardiogram than aged 129P2-WT, FVB/N-WT, and FVB/N-MUT mice. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms) obtained by optical mapping. (G) Aged 129P2-MUT mice display significantly longer left ventricular activation times, indicating more pronounced conduction slowing. (H) Typical example of a ventricular tachycardia induced by one short-coupled extra stimulus (S1) in an isolated aged 129P2-MUT heart, and non-inducibility in an aged FVB/N-MUT heart with up to three extra stimuli (S1–S3). (I) Isolated aged 129P2-MUT hearts display significantly higher inducibility of ventricular arrhythmias (Pearson χ2 overall P = 0.03). Data in panel D are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. AVB: atrioventricular block; SND: sinus node dysfunction; VPBs: ventricular premature beats; VT: ventricular tachycardia. Figure 2 View largeDownload slide Pro-arrhythmic effects of cardiac hypertrophy in aged Scn5a1798insD/+ mice. Mice of the 129P2 strain develop more severe cardiac hypertrophy with age (WT, wild type and MUT, Scn5a1798insD/+ to a similar extent) than mice of the FVB/N strain, as indicated by increased heart weight to body weight ratio (A) and Anf expression on in situ hybridization (B). (C) Typical examples of surface electrocardiograms and arrhythmias. (D and E) Aged 129P2-MUT mice show significantly increased QRS-duration and more spontaneous arrhythmias (Pearson χ2 overall P = 0.000039) on electrocardiogram than aged 129P2-WT, FVB/N-WT, and FVB/N-MUT mice. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms) obtained by optical mapping. (G) Aged 129P2-MUT mice display significantly longer left ventricular activation times, indicating more pronounced conduction slowing. (H) Typical example of a ventricular tachycardia induced by one short-coupled extra stimulus (S1) in an isolated aged 129P2-MUT heart, and non-inducibility in an aged FVB/N-MUT heart with up to three extra stimuli (S1–S3). (I) Isolated aged 129P2-MUT hearts display significantly higher inducibility of ventricular arrhythmias (Pearson χ2 overall P = 0.03). Data in panel D are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. AVB: atrioventricular block; SND: sinus node dysfunction; VPBs: ventricular premature beats; VT: ventricular tachycardia. Chronic pressure overload elicits conduction delay and sudden death in Scn5a1798insD/+ mice To provide direct evidence for a modulatory effect of hypertrophy in the setting of the mutation, we subjected adult FVB/N-WT and FVB/N-MUT mice (10–12 weeks old) to TAC (duration of 2 weeks), an intervention which leads to chronic pressure overload and consequent development of cardiac hypertrophy. Transverse aortic constriction induced similar extent of hypertrophy in WT and MUT mice, as illustrated by equal increases in heart mass and up-regulation of hypertrophic genes (Figure 3B and C; Supplementary material online, Table S2). However, approximately 35% of MUT-TAC mice died suddenly during the 2-week post-TAC period, while all WT-TAC and sham mice survived (Figure 3A). Continuous 24 h telemetric ECG recordings in a subset of MUT-TAC mice revealed progressive bradycardia and excessive (atrio-)ventricular conduction abnormalities prior to SCD (Supplementary material online, Figure S2). Electrocardiogram analysis in surviving MUT mice post-TAC uncovered a more pronounced increase in QRS-duration compared to WT (Supplementary material online, Table S3). Moreover, ex vivo measurements in isolated Langendorff-perfused hearts post-TAC showed atrioventricular delay and exacerbated ventricular conduction slowing in MUT but not WT mice (Figure 3D–G; Supplementary material online, Table S2). Hence, TAC elicited SCD and conduction abnormalities in MUT mice only, indicating a synergistic, deleterious interaction between cardiac hypertrophy and the mutation. Figure 3 View largeDownload slide Cardiac hypertrophy induced by transaortic constriction causes sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of the pro-hypertrophic marker Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT mice. (D) Typical examples of atrioventricular delay measurements (atrial stimulation, 120 ms). (E) MUT-TAC mice display more severe atrioventricular conduction delay vs. WT-TAC. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms). (G) MUT-TAC mice display increased left ventricular activation time. Data in panel G are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. Additional data is presented in Supplementary material online, Table S2. Figure 3 View largeDownload slide Cardiac hypertrophy induced by transaortic constriction causes sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of the pro-hypertrophic marker Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT mice. (D) Typical examples of atrioventricular delay measurements (atrial stimulation, 120 ms). (E) MUT-TAC mice display more severe atrioventricular conduction delay vs. WT-TAC. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms). (G) MUT-TAC mice display increased left ventricular activation time. Data in panel G are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. Additional data is presented in Supplementary material online, Table S2. Transverse aortic constriction-induced conduction abnormalities and SCD in Scn5a1798insD/+ mice are attenuated by decreasing the hypertrophic response through genetic inhibition of the calcineurin-Nfat pathway Activation of the calcineurin-Nfat (Nuclear Factor of Activated T-cells) signaling pathway is known to play a major role in mediating the pro-hypertrophic consequences of chronic pressure overload of the heart. To investigate whether the more severe electrophysiological abnormalities in FVB/N-MUT mice post-TAC are the direct consequences of cardiac hypertrophy and not to other (indirect) effects of pressure overload, we abrogated the hypertrophic response by inducing genetic deletion of the main downstream effector of the calcineurin-Nfat pathway by crossing Scn5a1798insD/+ mice with mice lacking Nfatc2 (Nfatc2-/-).9,10 WT and MUT (Scn5a1798insD/+) mice deficient for Nfatc2 (Nfatc2-/-), and littermate WT and MUT animals with unaltered Nfatc2 expression (Nfatc2+/+), were subjected to TAC for a period of 2 weeks. As expected, cardiac hypertrophy in response to pressure overload was attenuated in WT and MUT mice deficient for Nfatc2 (WT-Nfatc2-/- and MUT-Nfatc2-/-), as illustrated by lower heart weights and lower expression of hypertrophic genes when compared with WT-Nfatc2+/+ and MUT-Nfatc2+/+ (Figure 4B and C, Supplementary material online, Table S4). No SCD was observed in MUT-Nfatc2-/- mice subjected to TAC (Figure 4A), and the (atrio-)ventricular conduction abnormalities secondary to TAC observed in MUT mice with intact Nfatc2 expression were rescued in MUT-Nfatc2-/- mice (Figure 4D–F; Supplementary material online, Table S4). Hence, blocking the downstream, pro-hypertrophic signaling cascade prevented the TAC-induced conduction abnormalities and SCD in MUT mice, providing support for a direct interaction between the mutation and cardiac hypertrophy. Figure 4 View largeDownload slide Rescue of transaortic constriction induced sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice by genetic deletion of Nfatc2 or late sodium current inhibition by Ranolazine. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice (Nfatc2-/-: with genetic deletion of Nfatc2; Ranolazine fed Ranolazine chow) subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT (+/- Ranolazine chow) mice, but lower in WT and MUT mice on a Nfatc2-/- background. (D) Typical examples of atrioventricular delay measurements (atrial stimulation at 120 ms). The atrioventricular conduction delay induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. (E) Typical examples of left ventricular activation maps in isolated hearts. (F) Increased left ventricular activation time induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. In panels B, C, D, and F, data are presented as ratio for transaortic constriction vs. Sham (mean ± standard deviation); actual values are in Supplementary material online, Tables S2 and S4. Figure 4 View largeDownload slide Rescue of transaortic constriction induced sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice by genetic deletion of Nfatc2 or late sodium current inhibition by Ranolazine. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice (Nfatc2-/-: with genetic deletion of Nfatc2; Ranolazine fed Ranolazine chow) subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT (+/- Ranolazine chow) mice, but lower in WT and MUT mice on a Nfatc2-/- background. (D) Typical examples of atrioventricular delay measurements (atrial stimulation at 120 ms). The atrioventricular conduction delay induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. (E) Typical examples of left ventricular activation maps in isolated hearts. (F) Increased left ventricular activation time induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. In panels B, C, D, and F, data are presented as ratio for transaortic constriction vs. Sham (mean ± standard deviation); actual values are in Supplementary material online, Tables S2 and S4. Chronic late sodium current inhibition prevents transverse aortic constriction-induced conduction abnormalities and SCD in Scn5a1798insD/+ mice We have previously demonstrated that the SCN5A-1795insD mutation is associated with multiple biophysical defects including a gain of channel function due to sustained (late) inward sodium current.5,6 This mutation-induced enhanced late sodium current (INa,late) can be blocked through pharmacological inhibition. We, therefore, explored whether blocking this biophysical defect of the mutation would prevent the exacerbation of electrophysiological abnormalities associated with TAC in MUT mice. For this, we administered the INa,late inhibitor Ranolazine by feeding WT and MUT mice either control or Ranolazine chow for the 2 weeks period of TAC or sham (starting 2 days after the TAC or sham procedure). Food intake and body weights were constant throughout the duration of the experiment and did not differ between groups (Supplementary material online, Figure S1). INa,late inhibition decreased QTc-duration in sham mice, and moreover prevented TAC-induced QTc-prolongation (Supplementary material online, Table S3). Blocking INa,late did not affect the magnitude of TAC-induced cardiac hypertrophy, as indicated by a similar increase in pro-hypertrophic markers in WT-TAC and MUT-TAC mice that were fed Ranolazine chow (Figure 4B and C; Supplementary material online, Table S2). Yet, INa,late blockade prevented SCD and attenuated (atrio-)ventricular conduction abnormalities in MUT mice subjected to TAC (Figure 4D–F, Supplementary material online, Table S2). Hence, pharmacological inhibition of the mutation-induced enhanced INa,late rescued the TAC-induced deleterious interaction between hypertrophy and the Scn5a1798insD/+ mutation. Discussion Our findings point to a modulatory effect of hypertension and consequent cardiac hypertrophy on age-dependent risk for sudden arrhythmic death and pacemaker treatment efficacy in the SCN5A-1795insD mutation-positive patients. This is supported by our observations in Scn5a1798insD/+ mice carrying the homologous mutation, where cardiac hypertrophy (either occurring with age or induced by TAC) led to severe conduction disturbances and an increased risk for ventricular arrhythmias and/or SCD. This study for the first time provides evidence for a modulatory role of co-morbidity in modulating disease severity of an inherited arrhythmic disease, demonstrating a pro-arrhythmic gene-environment interaction. Modulatory effect of cardiac hypertrophy on (age-dependent) arrhythmic phenotype Cardiac hypertrophy, which commonly occurs as a consequence of hypertension, develops over time and progressively remodels the myocardium. Its impact is therefore expected to increase with age, potentially altering disease severity in mutation-positive patients in an age-dependent manner. The observed age-dependent shift from bradycardia-induced (prevented by pacemaker therapy) to apparent bradycardia-independent arrhythmias and SCD in older SCN5A-1795insD mutation-positive patients moreover indicates a modulatory effect on disease expression. Our clinical findings are further supported by our observations in Scn5a1798insD/+ mice, where mice from the strain most prone to age-dependent hypertrophy (i.e. 129P2-Scn5a1798insD/+ mice) developed a more pronounced arrhythmic phenotype with significantly more spontaneous and inducible ventricular arrhythmias. Although other factors besides hypertrophy, that differ between the patients and between the two mouse strains may also contribute, collectively this human and mouse data support the concept that age-dependent development of cardiac hypertrophy interacts with sodium channel dysfunction to predispose the heart to ventricular arrhythmias and SCD. Data obtained in the TAC studies in young-adult Scn5a1798insD/+ mice provide direct proof of the modulatory role of hypertrophy on disease expression. Despite the fact that wild type and Scn5a1798insD/+ mice developed similar extent of cardiac hypertrophy secondary to TAC, SCD and conduction abnormalities were observed only in Scn5a1798insD/+ mice with chronic pressure overload. In contrast to aged 129P2-Scn5a1798insD/+ mice however, no spontaneous or inducible ventricular arrhythmias were observed in mutant mice following TAC (data not shown). The young-adult age of the mice subjected to TAC, the relatively abrupt development of hypertrophy secondary to TAC (in contrast to gradual progression with age), and the short duration of TAC (two weeks) may underlie this apparent discrepancy. One should note however that as for aged Scn5a1798insD/+ mice, young-adult Scn5a1798insD/+ mice developed ventricular conduction slowing post-TAC, a phenomenon that is well established to promote ventricular arrhythmias. Potential mechanisms underlying modulatory role of hypertrophy The mechanisms underlying the modulatory, pro-arrhythmic effects of hypertension and consequent hypertrophy may be numerous and complex. Hypertrophy is associated with progressive electrical (alterations in sodium current and other ion channels), homeostatic (dysregulation of intracellular calcium homeostasis) and structural (collagen deposition) remodelling.11–14 These alterations may be further exacerbated in the setting of an SCN5A mutation, acting synergistically with the biophysical defects caused by the SCN5A mutation in creating a highly arrhythmogenic environment.15,16 In support of this, we found that similar levels of hypertrophy induced a pro-arrhythmic phenotype in Scn5a1798insD/+ but not wild type mice, indicating a synergistic interaction between hypertrophy and the mutation. Moreover, TAC-induced conduction disturbances and SCD were prevented by either blocking the downstream pro-hypertrophic response (by genetic deletion of Nfatc2) or by pharmacological inhibition of the detrimental consequences of the mutation (i.e. through INa,late inhibition). Enhanced INa,late increases sodium influx, which may secondarily lead to increased intracellular calcium concentrations. The latter is a well-established pro-arrhythmic feature of hypertrophy and heart failure, and may be exacerbated in SCN5A-1795insD mutation-positive patients in the presence of hypertrophy.17,18 The involvement of the enhanced INa,late defect in mediating, at least in part, the observed interaction with hypertrophy is supported by the fact that targeting this molecular defect by INa,late blockade prevented SCD and attenuated (atrio-)ventricular conduction abnormalities in mutant mice post-TAC. While enhanced INa,late is a well-established pro-arrhythmic consequence of hypertrophy,18 one might speculate that other hypertension-associated pathways (including the renin angiotensin aldosterone system) may also be involved in mediating the role of hypertrophy in increasing arrhythmic propensity, with potential relevance for patient management. Implications for age-dependent treatment efficacy The observed age-dependent change in arrhythmia phenotype had crucial consequences for treatment efficacy in SCN5A-1795insD mutation-positive patients: while pacemaker implantation remained 100% efficacious in preventing SCD in young mutation-positive individuals, it no longer afforded complete protection over the age of 40. These findings therefore underline the necessity for additional treatment strategies in older SCN5A mutation-positive patients. Our combined observations in the family and in mice implicate a role for hypertension and consequent hypertrophy in disease expressivity with consequences for treatment efficacy, although other co-morbidities may also play an as yet unidentified modulatory role. Our findings indicate a pro-arrhythmic interaction between hypertrophy and the mutation, and prevention of either hypertrophy (by genetic deletion of Nfatc2) or the effect of the mutation (by Ranolazine) was sufficient to prevent SCD in mutant mice subjected to TAC. Hence, clinical management of affected patients with either INa,late inhibition or anti-hypertensive treatment (aimed at preventing LVH) is potentially beneficial, since either of these approaches would prevent the deleterious interaction between cardiac hypertrophy and the mutation. While INa,late inhibition may have additional (long-term) benefits aside from restoration of repolarization, including the prevention of intracellular sodium/calcium dysregulation, it is as yet unclear whether this therapeutic approach will be clinically applicable in the near future. Ranolazine may have pro-arrhythmic side-effects due to its (limited) IKr blocking properties, and the development of more selective INa,late inhibitors (including Eleclazine) was discontinued.19,20 Thus, while awaiting (further) development of novel compounds targeting INa,late,20 current clinical management should focus on carefully monitoring SCN5A-1795insD mutation-positive patients for co-morbidities such as hypertension. In addition, hypertension should be aggressively treated early on to prevent LVH development, which should be regularly monitored by echocardiography. In particular, drugs targeting the renin-angiotensin system may be beneficial.21 This approach is in line with the 2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death,22 which state that appropriate therapy should also take into account underlying diseases, co-morbidities, and associated medical conditions that may contribute to and/or exacerbate arrhythmia. Limitations While the homogeneity in genetic cause in the extended SCN5A-1795insD family allowed for detection of this interaction, it is associated with the limitation that transferability of these observations to other SCN5A mutations, although considered likely, will need to be explored. It is possible that our current findings are specific for ‘overlap syndrome’ mutations associated with both a loss and a gain of sodium channel function. Furthermore, while hypertension and/or hypertrophy was observed in the majority of older SCN5A-1795insD mutation-positive patients who suffered a life-threatening event despite pacemaker treatment, we currently do not know the exact prevalence of these co-morbidities in all mutation-positive and -negative individuals in the family. Conclusion Our findings show that a common co-morbidity (e.g. hypertension) may significantly affect arrhythmia risk and survival in inherited sodium channelopathy. The impact of such unrelated co-morbidities varies with age, leading to potential age-dependent changes in arrhythmia mechanism. As a consequence, efficacy of treatment strategies to prevent SCD in the setting of inherited arrhythmic disease may vary over time, underscoring the need for continuous diagnosis and monitoring of relevant co-morbidities and their rigorous treatment. Supplementary material Supplementary material is available at European Heart Journal online. Acknowledgements The authors thank Prof. Leon de Windt (Maastricht University Medical Center, The Netherlands) for kindly providing the Nfatc2-/- mice, and Dr Jan Ruijter (Department of Medical Biology, Academic Medical Center, Amsterdam, The Netherlands) for expert assistance with quantitative RT–PCR data analysis. Funding This work was supported by an Innovational Research Incentives Scheme Vidi grant (91714371 to C.A.R.) and Vici grant (016150610 to C.R.B.) from the Netherlands Organisation for Health Research and Development (ZonMw); a ZonMw Priority Medicines (PM-Rare) (113303006 to C.A.R./A.A.M.W.); the Division for Earth and Life Sciences (ALW; 836.09.003 to C.A.R.) with financial aid from the Netherlands Organization for Scientific Research (NWO); the InterUniversity Cardiology Institute of the Netherlands (061.02 to C.A.R. and C.R.B); the Netherlands CardioVascular Research Initiative CVON (Dutch Heart Foundation, Dutch Federation of University Medical Centres, ZonMw, and the Royal Netherlands Academy of Sciences) (projects PREDICT CVON2012-10 to J.P.T./A.A.M.W./M.P.B./T.A.B.V./C.R.B., and DOSIS CVON2014-40 to J.P.T.); and the Dutch Heart Foundation (NHS2010/B201 to C.A.R.). Conflict of interest: S.R. and L.B. are former employees of Gilead Sciences. A.A.M.W. serves on the scientific advisory board of Lilanova. C.A.R. has previously received research grants from Gilead Sciences. All other authors declared no conflict of interest. References 1 Bezzina CR, Lahrouchi N, Priori SG. Genetics of sudden cardiac death. Circ Res  2015; 116: 1919– 1936. Google Scholar CrossRef Search ADS PubMed  2 Shimizu W, Moss AJ, Wilde AAM, Towbin JA, Ackerman MJ, January CT, Tester DJ, Zareba W, Robinson JL, Qi M, Vincent GM, Kaufman ES, Hofman N, Noda T, Kamakura S, Miyamoto Y, Shah S, Amin V, Goldenberg I, Andrews ML, McNitt S. Genotype-phenotype aspects of type 2 long QT syndrome. J Am Coll Cardiol  2009; 54: 2052– 2062. Google Scholar CrossRef Search ADS PubMed  3 Bezzina C, Veldkamp MW, van den Berg MP, Postma AV, Rook MB, Viersma JW, Langen IM, van Tan-Sindhunata G, Bink-Boelkens MT, van Der Hout AH, Mannens MM, Wilde AA. A single Na(+) channel mutation causing both long-QT and Brugada syndromes. Circ Res  1999; 85: 1206– 1213. 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Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease. Circulation  2012; 125: 3079– 3091. Google Scholar CrossRef Search ADS PubMed  7 Tobé TJ, Langen CD, de Bink-Boelkens MT, Mook PH, Viersma JW, Lie KI, Wesseling H. Late potentials in a bradycardia-dependent long QT syndrome associated with sudden death during sleep. J Am Coll Cardiol  1992; 19: 541– 549. Google Scholar CrossRef Search ADS PubMed  8 Remme CA, Scicluna BP, Verkerk AO, Amin AS, Brunschot S, van Beekman L, Deneer VHM, Chevalier C, Oyama F, Miyazaki H, Nukina N, Wilders R, Escande D, Houlgatte R, Wilde AAM, Tan HL, Veldkamp MW, Bakker JMT, de Bezzina CR. Genetically determined differences in sodium current characteristics modulate conduction disease severity in mice with cardiac sodium channelopathy. Circ Res  2009; 104: 1283– 1292. 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Ann N Y Acad Sci  2007; 1099: 339– 348. Google Scholar CrossRef Search ADS PubMed  14 Díez J, González A, López B, Querejeta R. Mechanisms of disease: pathologic structural remodeling is more than adaptive hypertrophy in hypertensive heart disease. Nat Clin Pract Cardiovasc Med  2005; 2: 209– 216. Google Scholar CrossRef Search ADS PubMed  15 Coronel R, Casini S, Koopmann TT, Wilms-Schopman FJ, Verkerk AO, de Groot JR, Bhuiyan Z, Bezzina CR, Veldkamp MW, Linnenbank AC, van der Wal AC, Tan HL, Brugada P, Wilde AA, de Bakker JM. Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome: a combined electrophysiological, genetic, histopathologic, and computational study. Circulation  2005; 112: 2769– 2777. Google Scholar CrossRef Search ADS PubMed  16 Hummel YM, Wilde AA, Voors AA, Bugatti S, Hillege HL, van den Berg MP. Ventricular dysfunction in a family with long QT syndrome type 3. Europace  2013; 15: 1516– 1521. Google Scholar CrossRef Search ADS PubMed  17 Remme CA, Wilde AA. Late sodium current inhibition in acquired and inherited ventricular (dys)function and arrhythmias. Cardiovasc Drugs Ther  2013; 27: 91– 101. Google Scholar CrossRef Search ADS PubMed  18 Coppini R, Ferrantini C, Yao L, Fan P, Lungo M, Del Stillitano F, Sartiani L, Tosi B, Suffredini S, Tesi C, Yacoub M, Olivotto I, Belardinelli L, Poggesi C, Cerbai E, Mugelli A. Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy. Circulation  2013; 127: 575– 584. Google Scholar CrossRef Search ADS PubMed  19 Wilde AA, Remme CA. Therapeutic approaches for Long QT syndrome type 3: an update. Europace  2018; 20: 222– 224. Google Scholar CrossRef Search ADS PubMed  20 Portero V, Casini S, Hoekstra M, Verkerk AO, Mengarelli I, Belardinelli L, Rajamani S, Wilde AA, Bezzina CR, Veldkamp MW, Remme CA. Anti-arrhythmic potential of the late sodium current inhibitor GS-458967 in murine Scn5a-1798insD+/- and human SCN5A-1795insD+/- iPSC-derived cardiomyocytes. Cardiovasc Res  2017; 113: 829– 838. Google Scholar CrossRef Search ADS PubMed  21 Dahlöf B, Devereux RB, Kjeldsen SE, Julius S, Beevers G, Faire U, de Fyhrquist F, Ibsen H, Kristiansson K, Lederballe-Pedersen O, Lindholm LH, Nieminen MS, Omvik P, Oparil S, Wedel H; LIFE Study Group. Cardiovascular morbidity and mortality in the Losartan Intervention For Endpoint reduction in hypertension study (LIFE): a randomised trial against atenolol. Lancet  2002; 359: 995– 1003. Google Scholar CrossRef Search ADS PubMed  22 Priori SG, Blomström-Lundqvist C, Mazzanti A, Blom N, Borggrefe M, Camm J, Elliott PM, Fitzsimons D, Hatala R, Hindricks G, Kirchhof P, Kjeldsen K, Kuck K-H, Hernandez-Madrid A, Nikolaou N, Norekvål TM, Spaulding C, Veldhuisen DJV. 2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death: the Task Force for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death of the Europe. Eur Heart J  2015; 36: 2793– 2867. Google Scholar CrossRef Search ADS PubMed  Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com. 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Abstract

Abstract Aims Management of patients with inherited cardiac ion channelopathy is hindered by variability in disease severity and sudden cardiac death (SCD) risk. Here, we investigated the modulatory role of hypertrophy on arrhythmia and SCD risk in sodium channelopathy. Methods and results Follow-up data was collected from 164 individuals positive for the SCN5A-1795insD founder mutation and 247 mutation-negative relatives. A total of 38 (obligate) mutation-positive patients died suddenly or suffered life-threatening ventricular arrhythmia. Of these, 18 were aged >40 years, a high proportion of which had a clinical diagnosis of hypertension and/or cardiac hypertrophy. While pacemaker implantation was highly protective in preventing bradycardia-related SCD in young mutation-positive patients, seven of them aged >40 experienced life-threatening arrhythmic events despite pacemaker treatment. Of these, six had a diagnosis of hypertension/hypertrophy, pointing to a modulatory role of this co-morbidity. Induction of hypertrophy in adult mice carrying the homologous mutation (Scn5a1798insD/+) caused SCD and excessive conduction disturbances, confirming a modulatory effect of hypertrophy in the setting of the mutation. The deleterious effects of the interaction between hypertrophy and the mutation were prevented by genetically impairing the pro-hypertrophic response and by pharmacological inhibition of the enhanced late sodium current associated with the mutation. Conclusion This study provides the first evidence for a modulatory effect of co-existing cardiac hypertrophy on arrhythmia risk and treatment efficacy in inherited sodium channelopathy. Our findings emphasize the need for continued assessment and rigorous treatment of this co-morbidity in SCN5A mutation-positive individuals. SCN5A, Sudden death, Cardiac hypertrophy, Hypertension, Conduction delay, Ventricular arrhythmias Translational perspectives SCN5A mutations present with a broad spectrum of clinical phenotypes, including sudden cardiac death (SCD). Disease penetrance and severity varies between individuals carrying the same SCN5A mutation, and common co-morbidities may modulate arrhythmia and SCD risk with potential implications for antiarrhythmic treatment efficacy. This study conducted in patients and mice, provides strong evidence for a modulatory role of hypertension/hypertrophy in modulating arrhythmic risk of the SCN5A-1795insD mutation. Our findings support the concept that SCN5A mutation-positive patients should be carefully monitored for the development of cardiac hypertension and hypertrophy during follow up, and that their clinical management should be adjusted where necessary to also include rigorous management of this co-morbidity. Introduction The genetic basis of the Mendelian cardiac rhythm disorders associated with sudden cardiac death (SCD) has been brought into focus over the last 20 years with the discovery of a large spectrum of causal mutations in genes encoding components of cardiac ion channels.1 Although this enabled genetic testing and consequently improvements in clinical care, patient management is still hindered by the reduced penetrance and substantial variability in disease severity and SCD risk among mutation-positive individuals.1 Although co-morbidities are expected to modulate disease severity, these remain unexplored. Identification of disease modifiers in these disorders is however hindered by the substantial genetic heterogeneity across patients, as different mutations may be associated with different effects and thus also contribute to inter-individual variability.2 Studies in large families that harbour founder mutations (where all affected individuals carry the same familial mutation) circumvent this limitation and therefore offer opportunities for the identification of modulatory factors. We have previously described a large Dutch family harbouring a founder mutation, p.Tyr1795_Glu1796insAsp (previously called ‘SCN5A-1795insD’), in the SCN5A gene, which encodes the major sodium channel isoform in heart (NaV1.5).3,4 This mutation displays multiple biophysical defects and causes ‘overlap’ sodium channelopathy with features of long QT syndrome, Brugada syndrome, and conduction disease.5,6 While variability in disease severity among mutation-positive individuals in this family is long recognized,3 predictors of arrhythmia and SCD risk have remained elusive. By combining clinical observations in the family with experimental studies in Scn5a1798insD/+ mice carrying the mouse homolog of the mutation, here, we uncovered a modulatory effect of hypertension and cardiac hypertrophy on disease severity and expression. In particular, the co-occurrence of cardiac hypertrophy was found to exacerbate cardiac conduction slowing and arrhythmia risk in the setting of the mutation, and was associated with a decreased efficacy of pacemaker treatment in preventing SCD. Our findings furthermore provide evidence for a potential therapeutic role of pharmacological late sodium current inhibition. Methods Details on study approval, SCN5A-1795insD mutation study population, clinical data collection, pathology reports, generation of Scn5a1798insD/+ and Scn5a1798insD/+-Nfatc2-/- mice, transverse aortic constriction (TAC) and chow experiments, electrophysiological assessments in Langendorff-perfused hearts, quantitative PCR assay and analysis and whole-mount in situ hybridization, and statistical analysis are provided in the Supplementary Methods. Data are presented as mean ± standard deviation or median (interquartile range), as appropriate. Results Hypertension and hypertrophy in older SCN5A-1795insD mutation-positive patients who died suddenly Through an extensive genealogical search, we reconstructed the pedigree of the family with the SCN5A-1795insD mutation back to the 18th century, linking 164 mutation-positive individuals and 247 mutation-negative relatives (Figure 1A). Mutation-positive patients displayed (atrio-)ventricular conduction slowing, sinus node dysfunction, excessive ventricular repolarization abnormalities at slow heart rates, and SCD occurring predominantly at night.3,4 A total of 38 individuals died suddenly or suffered life-threatening ventricular arrhythmia (13 males, 25 females; average age at event 38 ± 18 years, range 13–76) (Figure 1A and B). Twenty-five of these individuals were confirmed or obligate mutation-positive; genetic testing was not possible in the rest as these were deceased individuals from past generations, the majority of which died more than 50 years ago. Eighteen individuals who suffered SCD or a life-threatening arrhythmia were older than 40 years at the time of the event (4 males, 14 females, average age 54 ± 9 years, range 41–76) (Figure 1A and B). Recent clinical information was available for 10 of these (all confirmed mutation-positive); nine of them had a clinical diagnosis of hypertension and/or evidence for the presence of left ventricular hypertrophy (LVH) on magnetic resonance imaging (MRI), echocardiography, or post-mortem examination (including increased heart weight; Table 1), suggesting a modulatory effect of cardiac hypertrophy on arrhythmia risk. Post-mortem analysis furthermore indicated the additional presence of slight interstitial fibrosis (potentially hypertension-related) in three out of four patients (Table 1). Evidence for coronary artery disease, with hypertension as a major risk factor, was found in two patients. In one patient, small foci of subendocardial necrosis were found, but this was not associated with critically stenosing or thrombosed coronary lesions. The other patient displayed a fibrotic scar consistent with an old myocardial infarction without signs of recent-onset ischaemia. Table 1 Characteristics of SCN5A-1795insD mutation-positive patients suffering a serious cardiac event above the age of 40 with available recent clinical information Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  DCM, dilated cardiomyopathy; ICD, implantable cardioverter-defibrillator; LVH, left ventricular hypertrophy; PM, pacemaker; SCD, sudden cardiac death; VF, ventricular fibrillation; VT, ventricular tachycardia. a Patient did not have documented hypertension prior to VF, but developed clinically relevant hypertension 1 year later. Table 1 Characteristics of SCN5A-1795insD mutation-positive patients suffering a serious cardiac event above the age of 40 with available recent clinical information Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  Gender  Age  PM  Event  Hypertension  Hypertension-related cardiac findings  Comments/other findings  Male  45  Yes  SCD  Yes  Postmortem: HW 490 g, concentric LVH, slight interstitial myocardial fibrosis  SCD (VF documented)  Postmortem: <50% stenosing coronary atherosclerosis; small foci of subendocardial necrosis; no valvar stenosis  Female  55  Yes  SCD  Borderline  MRI: LVH  SCD at night; previously documented VT  Female  46  Yes  SCD  Yes  Postmortem: HW 550 g, concentric LVH, interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  49  Yes  SCD  Yes  Postmortem: HW 470 g, concentric LVH, slight interstitial fibrosis  SCD at night  Postmortem: minimal coronary atherosclerosis; no valvar stenosis  Female  41  Yes  VF  Noa  Echo: marginal LVH  VF (resuscitated successfully)  Female  50  Yes  SCD  Yes  Echo: possible DCM  SCD (VF documented)  Female  58  Yes  VT  Yes  Echo: diastolic dysfunction  VT documented at night necessitating ICD implantation  Female  69  No  VT/VF  Yes  Unknown  Multiple episodes of syncope; documented polymorphic VTs  Male  60  No  SCD  Yes  Unknown  Normal echo 1 year prior to SCD  Male  69  No  SCD  Yes  Postmortem: HW 530 g, concentric LVH  SCD (VF documented)  Postmortem: ischaemic scar; no signs of recent-onset ischaemia; no valvar stenosis  DCM, dilated cardiomyopathy; ICD, implantable cardioverter-defibrillator; LVH, left ventricular hypertrophy; PM, pacemaker; SCD, sudden cardiac death; VF, ventricular fibrillation; VT, ventricular tachycardia. a Patient did not have documented hypertension prior to VF, but developed clinically relevant hypertension 1 year later. Figure 1 View largeDownload slide Life-threatening cardiac events in SCN5A-1795insD mutation-positive patients. (A) The SCN5A-1795insD founder population pedigree. Circles indicate SCN5A-1795insD mutation-positive patients suffering sudden cardiac death, ventricular fibrillation, and/or ventricular tachycardia in the presence or absence of pacemaker treatment and hypertension and/or left ventricular hypertrophy. (B) Survival from sudden cardiac death and/or ventricular arrhythmias was significantly reduced in SCN5A-1795insD mutation-positive patients (n = 164) vs. mutation-negative relatives (n = 247) (P = 1.3026E−18). (C) Overall survival from sudden cardiac death and/or ventricular arrhythmias in SCN5A-1795insD mutation-positive patients was significantly higher in those with (n = 87) as compared to without (n = 77) pacemaker (P = 0.000003), but pacemaker efficacy was significantly reduced in mutation-positive patients above the age of 40 years (P = 0.028; Cox regression model). (D) Pacemaker readout (A, atrial lead; V, ventricular lead; paper speed 25 mm/s) of a 58-year female mutation-positive patient showing a nocturnal, fast (up to 300/min) and apparently polymorphic ventricular tachycardia despite adequate anti-bradycardia treatment. Figure 1 View largeDownload slide Life-threatening cardiac events in SCN5A-1795insD mutation-positive patients. (A) The SCN5A-1795insD founder population pedigree. Circles indicate SCN5A-1795insD mutation-positive patients suffering sudden cardiac death, ventricular fibrillation, and/or ventricular tachycardia in the presence or absence of pacemaker treatment and hypertension and/or left ventricular hypertrophy. (B) Survival from sudden cardiac death and/or ventricular arrhythmias was significantly reduced in SCN5A-1795insD mutation-positive patients (n = 164) vs. mutation-negative relatives (n = 247) (P = 1.3026E−18). (C) Overall survival from sudden cardiac death and/or ventricular arrhythmias in SCN5A-1795insD mutation-positive patients was significantly higher in those with (n = 87) as compared to without (n = 77) pacemaker (P = 0.000003), but pacemaker efficacy was significantly reduced in mutation-positive patients above the age of 40 years (P = 0.028; Cox regression model). (D) Pacemaker readout (A, atrial lead; V, ventricular lead; paper speed 25 mm/s) of a 58-year female mutation-positive patient showing a nocturnal, fast (up to 300/min) and apparently polymorphic ventricular tachycardia despite adequate anti-bradycardia treatment. Decreased pacemaker treatment efficacy in SCN5A-1795insD mutation-positive patients older than 40 Mutation-positive patients died suddenly predominantly during the night, and excessive QT-prolongation during (nocturnal) bradycardic episodes was frequently documented on Holter recordings.3,4,7 Hence, a bradycardia-dependent trigger for arrhythmia was originally suspected, and pacemaker implantation has been routinely employed in the family to prevent SCD.4 While this approach initially proved successful,4 in the last decade seven mutation-positive individuals suffered ventricular tachyarrhythmias and/or SCD despite pacemaker implantation (Figure 1A). All of them were older than 40 (age range 41–58 years), and in the majority, ventricular fibrillation, or tachycardia was documented (Table 1), in addition to ventricular extrasystoles recorded during Holter monitoring or exercise testing in several cases. Figure 1D shows a pacemaker readout displaying a nocturnal, fast ventricular tachycardia (presumably polymorphic) in a 58-year-old female mutation-positive patient; following this episode, her pacemaker was replaced with an implantable cardioverter-defibrillator. These observations suggested an age-dependent shift towards a different arrhythmia triggering mechanism at older age with arrhythmias occurring despite prevention of bradycardia. Indeed, comparing the occurrence of SCD/life-threatening arrhythmia between individuals with and without pacemaker showed that while pacemaker implantation was highly protective in young mutation-positive patients, its efficacy in preventing arrhythmias and SCD was significantly decreased above the age of 40 years (P = 0.028; proportional hazard assumption of pacemaker implantation tested using the Schoenfeld residuals from a Cox regression model) (Figure 1C). Importantly, in six of the seven mutation-positive patients who suffered a life-threatening event despite pacemaker treatment, a history of hypertension was confirmed and/or the presence of LVH documented on MRI, echocardiography, or post-mortem examination (Table 1). These observations led us to hypothesize that the co-occurrence of the mutation with cardiac hypertrophy, developing with age secondary to for instance hypertension, plays a pivotal role in modulating arrhythmia risk. The co-occurrence of cardiac hypertrophy is pro-arrhythmic in aged Scn5a1798insD/+ mice We further explored the modulatory role of cardiac hypertrophy in Scn5a1798insD/+ mice carrying the exact mouse homolog of the human SCN5A-1795insD mutation.5,8 We have previously generated two distinct mouse lines harbouring the Scn5a1798insD/+ mutation, with respectively the FVB/N and 129P2 inbred genetic background, enabling investigation of the effect of the mutation on different genetic backgrounds. In young adult mice, we previously demonstrated strain-dependent variable disease severity, with more pronounced conduction slowing and prolongation of repolarization in mutant mice of the 129P2 strain.8 We now studied aged mice wild-type for the Scn5a gene (WT) and Scn5a1798insD/+ mutant for the Scn5a gene (MUT) of both strains. The 129P2 strain was found to develop more cardiac hypertrophy with age as compared to the FVB/N strain. This feature was intrinsic to the 129P2 strain and independent of the mutation, since both aged WT- and MUT-129P2 mice displayed greater heart weight and higher expression levels of pro-hypertrophic markers as compared to aged WT- and MUT-FVB/N mice (Figure 2A and B). These intrinsic strain-dependent differences in susceptibility to hypertrophy enabled a comparison of the pro-arrhythmic effect of the mutation in the presence (i.e. aged 129P2-MUT mice) and absence (i.e. aged FVB/N-MUT mice) of hypertrophy. Therefore, we conducted electrocardiogram (ECG) studies in anaesthetized mice and electrophysiological studies in Langendorff-perfused hearts, comparing aged (8–22 months old) WT and MUT mice of both strains. This indeed uncovered a genotype-strain interaction, where aged 129P2-MUT mice displayed significantly more pronounced ventricular conduction slowing in vivo (QRS-duration on ECG analysis; Figure 2C and D; Supplementary material online, Table S1) and ex vivo (left ventricular activation time in isolated Langendorff-perfused hearts; Figure 2F and G). Moreover, 129P2-MUT mice exhibited significantly more spontaneous ventricular extrasystoles and arrhythmias in vivo (Figure 2C and E) and an increased inducibility of ventricular arrhythmias ex vivo (Figure 2H and I). While these observations do not provide a causal link, they are in line with a pro-arrhythmic interaction between hypertrophy and the mutation, similar to our observations in the SCN5A-1795insD family. Figure 2 View largeDownload slide Pro-arrhythmic effects of cardiac hypertrophy in aged Scn5a1798insD/+ mice. Mice of the 129P2 strain develop more severe cardiac hypertrophy with age (WT, wild type and MUT, Scn5a1798insD/+ to a similar extent) than mice of the FVB/N strain, as indicated by increased heart weight to body weight ratio (A) and Anf expression on in situ hybridization (B). (C) Typical examples of surface electrocardiograms and arrhythmias. (D and E) Aged 129P2-MUT mice show significantly increased QRS-duration and more spontaneous arrhythmias (Pearson χ2 overall P = 0.000039) on electrocardiogram than aged 129P2-WT, FVB/N-WT, and FVB/N-MUT mice. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms) obtained by optical mapping. (G) Aged 129P2-MUT mice display significantly longer left ventricular activation times, indicating more pronounced conduction slowing. (H) Typical example of a ventricular tachycardia induced by one short-coupled extra stimulus (S1) in an isolated aged 129P2-MUT heart, and non-inducibility in an aged FVB/N-MUT heart with up to three extra stimuli (S1–S3). (I) Isolated aged 129P2-MUT hearts display significantly higher inducibility of ventricular arrhythmias (Pearson χ2 overall P = 0.03). Data in panel D are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. AVB: atrioventricular block; SND: sinus node dysfunction; VPBs: ventricular premature beats; VT: ventricular tachycardia. Figure 2 View largeDownload slide Pro-arrhythmic effects of cardiac hypertrophy in aged Scn5a1798insD/+ mice. Mice of the 129P2 strain develop more severe cardiac hypertrophy with age (WT, wild type and MUT, Scn5a1798insD/+ to a similar extent) than mice of the FVB/N strain, as indicated by increased heart weight to body weight ratio (A) and Anf expression on in situ hybridization (B). (C) Typical examples of surface electrocardiograms and arrhythmias. (D and E) Aged 129P2-MUT mice show significantly increased QRS-duration and more spontaneous arrhythmias (Pearson χ2 overall P = 0.000039) on electrocardiogram than aged 129P2-WT, FVB/N-WT, and FVB/N-MUT mice. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms) obtained by optical mapping. (G) Aged 129P2-MUT mice display significantly longer left ventricular activation times, indicating more pronounced conduction slowing. (H) Typical example of a ventricular tachycardia induced by one short-coupled extra stimulus (S1) in an isolated aged 129P2-MUT heart, and non-inducibility in an aged FVB/N-MUT heart with up to three extra stimuli (S1–S3). (I) Isolated aged 129P2-MUT hearts display significantly higher inducibility of ventricular arrhythmias (Pearson χ2 overall P = 0.03). Data in panel D are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. AVB: atrioventricular block; SND: sinus node dysfunction; VPBs: ventricular premature beats; VT: ventricular tachycardia. Chronic pressure overload elicits conduction delay and sudden death in Scn5a1798insD/+ mice To provide direct evidence for a modulatory effect of hypertrophy in the setting of the mutation, we subjected adult FVB/N-WT and FVB/N-MUT mice (10–12 weeks old) to TAC (duration of 2 weeks), an intervention which leads to chronic pressure overload and consequent development of cardiac hypertrophy. Transverse aortic constriction induced similar extent of hypertrophy in WT and MUT mice, as illustrated by equal increases in heart mass and up-regulation of hypertrophic genes (Figure 3B and C; Supplementary material online, Table S2). However, approximately 35% of MUT-TAC mice died suddenly during the 2-week post-TAC period, while all WT-TAC and sham mice survived (Figure 3A). Continuous 24 h telemetric ECG recordings in a subset of MUT-TAC mice revealed progressive bradycardia and excessive (atrio-)ventricular conduction abnormalities prior to SCD (Supplementary material online, Figure S2). Electrocardiogram analysis in surviving MUT mice post-TAC uncovered a more pronounced increase in QRS-duration compared to WT (Supplementary material online, Table S3). Moreover, ex vivo measurements in isolated Langendorff-perfused hearts post-TAC showed atrioventricular delay and exacerbated ventricular conduction slowing in MUT but not WT mice (Figure 3D–G; Supplementary material online, Table S2). Hence, TAC elicited SCD and conduction abnormalities in MUT mice only, indicating a synergistic, deleterious interaction between cardiac hypertrophy and the mutation. Figure 3 View largeDownload slide Cardiac hypertrophy induced by transaortic constriction causes sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of the pro-hypertrophic marker Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT mice. (D) Typical examples of atrioventricular delay measurements (atrial stimulation, 120 ms). (E) MUT-TAC mice display more severe atrioventricular conduction delay vs. WT-TAC. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms). (G) MUT-TAC mice display increased left ventricular activation time. Data in panel G are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. Additional data is presented in Supplementary material online, Table S2. Figure 3 View largeDownload slide Cardiac hypertrophy induced by transaortic constriction causes sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of the pro-hypertrophic marker Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT mice. (D) Typical examples of atrioventricular delay measurements (atrial stimulation, 120 ms). (E) MUT-TAC mice display more severe atrioventricular conduction delay vs. WT-TAC. (F) Typical examples of left ventricular activation maps (stimulation at 120 ms). (G) MUT-TAC mice display increased left ventricular activation time. Data in panel G are presented as median (interquartile range) and all other data are presented as mean ± standard deviation. Additional data is presented in Supplementary material online, Table S2. Transverse aortic constriction-induced conduction abnormalities and SCD in Scn5a1798insD/+ mice are attenuated by decreasing the hypertrophic response through genetic inhibition of the calcineurin-Nfat pathway Activation of the calcineurin-Nfat (Nuclear Factor of Activated T-cells) signaling pathway is known to play a major role in mediating the pro-hypertrophic consequences of chronic pressure overload of the heart. To investigate whether the more severe electrophysiological abnormalities in FVB/N-MUT mice post-TAC are the direct consequences of cardiac hypertrophy and not to other (indirect) effects of pressure overload, we abrogated the hypertrophic response by inducing genetic deletion of the main downstream effector of the calcineurin-Nfat pathway by crossing Scn5a1798insD/+ mice with mice lacking Nfatc2 (Nfatc2-/-).9,10 WT and MUT (Scn5a1798insD/+) mice deficient for Nfatc2 (Nfatc2-/-), and littermate WT and MUT animals with unaltered Nfatc2 expression (Nfatc2+/+), were subjected to TAC for a period of 2 weeks. As expected, cardiac hypertrophy in response to pressure overload was attenuated in WT and MUT mice deficient for Nfatc2 (WT-Nfatc2-/- and MUT-Nfatc2-/-), as illustrated by lower heart weights and lower expression of hypertrophic genes when compared with WT-Nfatc2+/+ and MUT-Nfatc2+/+ (Figure 4B and C, Supplementary material online, Table S4). No SCD was observed in MUT-Nfatc2-/- mice subjected to TAC (Figure 4A), and the (atrio-)ventricular conduction abnormalities secondary to TAC observed in MUT mice with intact Nfatc2 expression were rescued in MUT-Nfatc2-/- mice (Figure 4D–F; Supplementary material online, Table S4). Hence, blocking the downstream, pro-hypertrophic signaling cascade prevented the TAC-induced conduction abnormalities and SCD in MUT mice, providing support for a direct interaction between the mutation and cardiac hypertrophy. Figure 4 View largeDownload slide Rescue of transaortic constriction induced sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice by genetic deletion of Nfatc2 or late sodium current inhibition by Ranolazine. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice (Nfatc2-/-: with genetic deletion of Nfatc2; Ranolazine fed Ranolazine chow) subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT (+/- Ranolazine chow) mice, but lower in WT and MUT mice on a Nfatc2-/- background. (D) Typical examples of atrioventricular delay measurements (atrial stimulation at 120 ms). The atrioventricular conduction delay induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. (E) Typical examples of left ventricular activation maps in isolated hearts. (F) Increased left ventricular activation time induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. In panels B, C, D, and F, data are presented as ratio for transaortic constriction vs. Sham (mean ± standard deviation); actual values are in Supplementary material online, Tables S2 and S4. Figure 4 View largeDownload slide Rescue of transaortic constriction induced sudden cardiac death and conduction disturbances in Scn5a1798insD/+ mice by genetic deletion of Nfatc2 or late sodium current inhibition by Ranolazine. (A) The Kaplan–Meier survival curves of wild type (WT) and Scn5a1798insD/+ (MUT) mice (Nfatc2-/-: with genetic deletion of Nfatc2; Ranolazine fed Ranolazine chow) subjected to Sham or transaortic constriction (TAC). (B and C) Magnitude of cardiac hypertrophy [measured by heart weight/tibia length ratio (B) and mRNA expression levels of Rcan1-4 (C)] secondary to transaortic constriction is similar in WT and MUT (+/- Ranolazine chow) mice, but lower in WT and MUT mice on a Nfatc2-/- background. (D) Typical examples of atrioventricular delay measurements (atrial stimulation at 120 ms). The atrioventricular conduction delay induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. (E) Typical examples of left ventricular activation maps in isolated hearts. (F) Increased left ventricular activation time induced by transaortic constriction in MUT mice is reversed by genetic inhibition of Nfatc2 and by Ranolazine. In panels B, C, D, and F, data are presented as ratio for transaortic constriction vs. Sham (mean ± standard deviation); actual values are in Supplementary material online, Tables S2 and S4. Chronic late sodium current inhibition prevents transverse aortic constriction-induced conduction abnormalities and SCD in Scn5a1798insD/+ mice We have previously demonstrated that the SCN5A-1795insD mutation is associated with multiple biophysical defects including a gain of channel function due to sustained (late) inward sodium current.5,6 This mutation-induced enhanced late sodium current (INa,late) can be blocked through pharmacological inhibition. We, therefore, explored whether blocking this biophysical defect of the mutation would prevent the exacerbation of electrophysiological abnormalities associated with TAC in MUT mice. For this, we administered the INa,late inhibitor Ranolazine by feeding WT and MUT mice either control or Ranolazine chow for the 2 weeks period of TAC or sham (starting 2 days after the TAC or sham procedure). Food intake and body weights were constant throughout the duration of the experiment and did not differ between groups (Supplementary material online, Figure S1). INa,late inhibition decreased QTc-duration in sham mice, and moreover prevented TAC-induced QTc-prolongation (Supplementary material online, Table S3). Blocking INa,late did not affect the magnitude of TAC-induced cardiac hypertrophy, as indicated by a similar increase in pro-hypertrophic markers in WT-TAC and MUT-TAC mice that were fed Ranolazine chow (Figure 4B and C; Supplementary material online, Table S2). Yet, INa,late blockade prevented SCD and attenuated (atrio-)ventricular conduction abnormalities in MUT mice subjected to TAC (Figure 4D–F, Supplementary material online, Table S2). Hence, pharmacological inhibition of the mutation-induced enhanced INa,late rescued the TAC-induced deleterious interaction between hypertrophy and the Scn5a1798insD/+ mutation. Discussion Our findings point to a modulatory effect of hypertension and consequent cardiac hypertrophy on age-dependent risk for sudden arrhythmic death and pacemaker treatment efficacy in the SCN5A-1795insD mutation-positive patients. This is supported by our observations in Scn5a1798insD/+ mice carrying the homologous mutation, where cardiac hypertrophy (either occurring with age or induced by TAC) led to severe conduction disturbances and an increased risk for ventricular arrhythmias and/or SCD. This study for the first time provides evidence for a modulatory role of co-morbidity in modulating disease severity of an inherited arrhythmic disease, demonstrating a pro-arrhythmic gene-environment interaction. Modulatory effect of cardiac hypertrophy on (age-dependent) arrhythmic phenotype Cardiac hypertrophy, which commonly occurs as a consequence of hypertension, develops over time and progressively remodels the myocardium. Its impact is therefore expected to increase with age, potentially altering disease severity in mutation-positive patients in an age-dependent manner. The observed age-dependent shift from bradycardia-induced (prevented by pacemaker therapy) to apparent bradycardia-independent arrhythmias and SCD in older SCN5A-1795insD mutation-positive patients moreover indicates a modulatory effect on disease expression. Our clinical findings are further supported by our observations in Scn5a1798insD/+ mice, where mice from the strain most prone to age-dependent hypertrophy (i.e. 129P2-Scn5a1798insD/+ mice) developed a more pronounced arrhythmic phenotype with significantly more spontaneous and inducible ventricular arrhythmias. Although other factors besides hypertrophy, that differ between the patients and between the two mouse strains may also contribute, collectively this human and mouse data support the concept that age-dependent development of cardiac hypertrophy interacts with sodium channel dysfunction to predispose the heart to ventricular arrhythmias and SCD. Data obtained in the TAC studies in young-adult Scn5a1798insD/+ mice provide direct proof of the modulatory role of hypertrophy on disease expression. Despite the fact that wild type and Scn5a1798insD/+ mice developed similar extent of cardiac hypertrophy secondary to TAC, SCD and conduction abnormalities were observed only in Scn5a1798insD/+ mice with chronic pressure overload. In contrast to aged 129P2-Scn5a1798insD/+ mice however, no spontaneous or inducible ventricular arrhythmias were observed in mutant mice following TAC (data not shown). The young-adult age of the mice subjected to TAC, the relatively abrupt development of hypertrophy secondary to TAC (in contrast to gradual progression with age), and the short duration of TAC (two weeks) may underlie this apparent discrepancy. One should note however that as for aged Scn5a1798insD/+ mice, young-adult Scn5a1798insD/+ mice developed ventricular conduction slowing post-TAC, a phenomenon that is well established to promote ventricular arrhythmias. Potential mechanisms underlying modulatory role of hypertrophy The mechanisms underlying the modulatory, pro-arrhythmic effects of hypertension and consequent hypertrophy may be numerous and complex. Hypertrophy is associated with progressive electrical (alterations in sodium current and other ion channels), homeostatic (dysregulation of intracellular calcium homeostasis) and structural (collagen deposition) remodelling.11–14 These alterations may be further exacerbated in the setting of an SCN5A mutation, acting synergistically with the biophysical defects caused by the SCN5A mutation in creating a highly arrhythmogenic environment.15,16 In support of this, we found that similar levels of hypertrophy induced a pro-arrhythmic phenotype in Scn5a1798insD/+ but not wild type mice, indicating a synergistic interaction between hypertrophy and the mutation. Moreover, TAC-induced conduction disturbances and SCD were prevented by either blocking the downstream pro-hypertrophic response (by genetic deletion of Nfatc2) or by pharmacological inhibition of the detrimental consequences of the mutation (i.e. through INa,late inhibition). Enhanced INa,late increases sodium influx, which may secondarily lead to increased intracellular calcium concentrations. The latter is a well-established pro-arrhythmic feature of hypertrophy and heart failure, and may be exacerbated in SCN5A-1795insD mutation-positive patients in the presence of hypertrophy.17,18 The involvement of the enhanced INa,late defect in mediating, at least in part, the observed interaction with hypertrophy is supported by the fact that targeting this molecular defect by INa,late blockade prevented SCD and attenuated (atrio-)ventricular conduction abnormalities in mutant mice post-TAC. While enhanced INa,late is a well-established pro-arrhythmic consequence of hypertrophy,18 one might speculate that other hypertension-associated pathways (including the renin angiotensin aldosterone system) may also be involved in mediating the role of hypertrophy in increasing arrhythmic propensity, with potential relevance for patient management. Implications for age-dependent treatment efficacy The observed age-dependent change in arrhythmia phenotype had crucial consequences for treatment efficacy in SCN5A-1795insD mutation-positive patients: while pacemaker implantation remained 100% efficacious in preventing SCD in young mutation-positive individuals, it no longer afforded complete protection over the age of 40. These findings therefore underline the necessity for additional treatment strategies in older SCN5A mutation-positive patients. Our combined observations in the family and in mice implicate a role for hypertension and consequent hypertrophy in disease expressivity with consequences for treatment efficacy, although other co-morbidities may also play an as yet unidentified modulatory role. Our findings indicate a pro-arrhythmic interaction between hypertrophy and the mutation, and prevention of either hypertrophy (by genetic deletion of Nfatc2) or the effect of the mutation (by Ranolazine) was sufficient to prevent SCD in mutant mice subjected to TAC. Hence, clinical management of affected patients with either INa,late inhibition or anti-hypertensive treatment (aimed at preventing LVH) is potentially beneficial, since either of these approaches would prevent the deleterious interaction between cardiac hypertrophy and the mutation. While INa,late inhibition may have additional (long-term) benefits aside from restoration of repolarization, including the prevention of intracellular sodium/calcium dysregulation, it is as yet unclear whether this therapeutic approach will be clinically applicable in the near future. Ranolazine may have pro-arrhythmic side-effects due to its (limited) IKr blocking properties, and the development of more selective INa,late inhibitors (including Eleclazine) was discontinued.19,20 Thus, while awaiting (further) development of novel compounds targeting INa,late,20 current clinical management should focus on carefully monitoring SCN5A-1795insD mutation-positive patients for co-morbidities such as hypertension. In addition, hypertension should be aggressively treated early on to prevent LVH development, which should be regularly monitored by echocardiography. In particular, drugs targeting the renin-angiotensin system may be beneficial.21 This approach is in line with the 2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death,22 which state that appropriate therapy should also take into account underlying diseases, co-morbidities, and associated medical conditions that may contribute to and/or exacerbate arrhythmia. Limitations While the homogeneity in genetic cause in the extended SCN5A-1795insD family allowed for detection of this interaction, it is associated with the limitation that transferability of these observations to other SCN5A mutations, although considered likely, will need to be explored. It is possible that our current findings are specific for ‘overlap syndrome’ mutations associated with both a loss and a gain of sodium channel function. Furthermore, while hypertension and/or hypertrophy was observed in the majority of older SCN5A-1795insD mutation-positive patients who suffered a life-threatening event despite pacemaker treatment, we currently do not know the exact prevalence of these co-morbidities in all mutation-positive and -negative individuals in the family. Conclusion Our findings show that a common co-morbidity (e.g. hypertension) may significantly affect arrhythmia risk and survival in inherited sodium channelopathy. The impact of such unrelated co-morbidities varies with age, leading to potential age-dependent changes in arrhythmia mechanism. As a consequence, efficacy of treatment strategies to prevent SCD in the setting of inherited arrhythmic disease may vary over time, underscoring the need for continuous diagnosis and monitoring of relevant co-morbidities and their rigorous treatment. Supplementary material Supplementary material is available at European Heart Journal online. Acknowledgements The authors thank Prof. Leon de Windt (Maastricht University Medical Center, The Netherlands) for kindly providing the Nfatc2-/- mice, and Dr Jan Ruijter (Department of Medical Biology, Academic Medical Center, Amsterdam, The Netherlands) for expert assistance with quantitative RT–PCR data analysis. Funding This work was supported by an Innovational Research Incentives Scheme Vidi grant (91714371 to C.A.R.) and Vici grant (016150610 to C.R.B.) from the Netherlands Organisation for Health Research and Development (ZonMw); a ZonMw Priority Medicines (PM-Rare) (113303006 to C.A.R./A.A.M.W.); the Division for Earth and Life Sciences (ALW; 836.09.003 to C.A.R.) with financial aid from the Netherlands Organization for Scientific Research (NWO); the InterUniversity Cardiology Institute of the Netherlands (061.02 to C.A.R. and C.R.B); the Netherlands CardioVascular Research Initiative CVON (Dutch Heart Foundation, Dutch Federation of University Medical Centres, ZonMw, and the Royal Netherlands Academy of Sciences) (projects PREDICT CVON2012-10 to J.P.T./A.A.M.W./M.P.B./T.A.B.V./C.R.B., and DOSIS CVON2014-40 to J.P.T.); and the Dutch Heart Foundation (NHS2010/B201 to C.A.R.). Conflict of interest: S.R. and L.B. are former employees of Gilead Sciences. 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European Heart JournalOxford University Press

Published: Apr 27, 2018

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