Na_v1.5 mutations linked to dilated cardiomyopathy phenotypes

Abstract

Na_v1.5 dysfunctions are commonly linked to rhythms disturbances that include type 3 long QT syndrome (LQT3), Brugada syndrome (BrS), sick sinus syndrome (SSS) and conduction defects. Recently, this channel protein has been also linked to structural heart diseases such as dilated cardiomyopathy (DCM).

So far, 17 mutations on Na_v1.5 gene have been linked to a particular clinical phenotype grouping mixed cardiac arrhythmias and DCM. Most of these mutations are localized on the highly conserved voltage sensitive domain (VSD) of Na_v1.5 channel. Mutations in the center of these VSDs create a conduction pathway, known as gating pore, through which cations can permeate. Here, we discuss the hypothesis that DCM linked mutations of the Na_v1.5 channel create gating pores similar to the one observed as a result of the R219H mutation.⁷ Using the structural model of the Na_v1.4 channel’s VSDs,¹⁷ we show that most of the Na_v1.5 DCM linked mutations could induce gating pores. This implies that cations could permeate through the gating pores and may underlay the mixed arrhythmias and DCM phenotype.

Several Na_v1.5 mutations are linked to dilated cardiomyopathy phenotypes, but their pathogenic mechanism remains to be elucidated

Dilated cardiomyopathy (DCM) is a common disorder characterized by a contraction defect of the left ventricle which is the result of its dilatation. The prevalence of DCM is estimated at 1 case out of 2500 individuals.¹ DCM patients often suffer from various forms of arrhythmias as well as heart failure. Its high prevalence, incidence, and mortality make it an important health concern.

Nowadays, genetically inherited dilated cardiomyopathies are evaluated to account for 30% to 48% of all DCM cases.² Mutations of the Na_v1.5 channel are estimated to account for 1.7% of all familial dilated cardiomyopathy cases³ to 3% of all DCM cases² making it a major cause of inherited DCM. Interestingly, mutations of Na_v1.5 are usually linked to pure arrhythmic disorders such as Brugada syndrome⁴ or Long QT syndrome.⁵ Moreover, Na_v1.5 linked DCM phenotypes are often severe as they usually feature various forms of arrhythmias such as bundle branch block, atrio-ventricular blocks, atrial fibrillation, and ventricular tachycardia (Table 1).

Table 1. Characteristics of Na_v1.5 DCM linked mutations.

	Mutation	Localization	Biophysical defect				Clinical Phenotype						Reference
			Current density	Activation	Inactivation	Persistent Current	AFL	AFib	Tach	AVB and/or BBB	PVC	TdP
Gain of function	S216L	S3-S4 / DI	≈	≈	+	1,17%		x					2 , 26 - 28
	R814W	R3 – S4 /DII	≈	-	≈	≈	x		x				6 , 29
	D1595H	S3 / DIV	≈	-	≈	ND		x					6 , 29
	P2005A	C-term	ND	≈	+	0,54%				x			2 , 28
Intermediate	R219H	R1 – S4 / DI	≈	≈	≈	ND	x		x	x	x		7
	R222Q	R2 – S4 / DI	↓,≈	-	-	≈	x	x	x	x	x		2 , 30 - 33
	R225W	R3 – S4 /DI	↓	+	+	ND			x	x			24
	R814Q	R3 – S4 /DII	ND	-	-	ND			x	x			16 , 34
	D1275N	S3 / DIII	≈, ↓	≈, +	≈, -, +	↑	x	x	x	x	x		29 , 35 - 40
	V1279I	S3 / DIII	ND	ND	ND	≈		x	x	x			3
	F1520L	DIII - DIV loop	ND	ND	ND	ND				x	x		3
Loss of function	T220I	S4 / DI	↓	≈	-	ND		x	x	x			29 , 35 , 41
	E446K	DI - DII loop	↓	≈	-	0,22%						x	3 , 42
	A1180V	DII-DIII loop	≈	≈	-	0,50%		x		x			43
	R1193Q	DII-DIII loop	≈	≈	-	~1%						x	44 , 45
	ΔQKP 1507–1509	DIII - DIV loop	≈	+	≈	~2%				x		x	46 , 47
	I1835T	C-term	↓	≈	-	≈							30

↑, Increase; ↓, Decrease; ≈, No impact; +, shifted toward more positive values; -, shifted toward more negative values; AFL, atrial flutter; AFib, atrial fibrillation; AVB, first, second, or third degree atrio-ventricular block; BBB, incomplete right or left bundle branch block; ND, not determined; PVC, premature ventricular contractions; Tach, tachycardia; TdP, Torsades de Pointes

A total of 17 Na_v1.5 mutations have been reported to be associated to the development of DCM (Table 1). These mutations are located either in the intracellular loops of the protein or in its voltage sensitive domains (VSDs) (Fig. 1). Recently, it has been reported that some of these mutations are linked to very similar familial phenotypes although they induce divergent biophysical defects⁶ (Table 1). Our investigation of the Na_v1.5/R219H mutation revealed that it induces a cationic leak in the usually non-conductive VSD of the channel.⁷ Such leak currents have already been observed in similar proteins.^8-10 They have been named gating pore currents or omega currents. In the cardiomyocyte, it is possible that gating pore currents have deleterious effects, which could lead to DCM. However, Na_v1.5 mutations outside the VSD motifs are not though to generate such currents. This means that the generation of gating pore currents alone cannot explain the development of the pathology.

Figure 1. Two-dimensional structure of the voltage-gated sodium channels. The location of the Na_v1.5 mutations associated with the mixed arrhythmias and DCM phenotype are shown in blue while location of the Na_v1.4 mutations associated with periodic paralysis are shown in green.

Thus, despite recent advances in understanding of the link between Na_v1.5 mutations and DCM, the pathological mechanism linking the mutations and the phenotype is still a subject of debate. Here we discuss our hypothesis on how Na_v1.5 and DCM are linked based on the data available from studies on Na_v1.5 and the similar Na_v1.4 channel expressed in skeletal muscle.

Mutations in the channel’s intracellular loops all seem to generate persistent current

Table 1 shows that some mutations that occur on the intracellular loops of the channel could be classified either as gain-of-function mutations (ex: ΔQKP 1507–1509) or loss-of-function mutations (ex: A1180V). However, these DCM linked mutations induce persistent sodium current. This persistent current could be the single common characteristic which links all the mutations located in the intracellular loops. Indeed, it is known that persistent sodium current can lead to sodium overload and calcium overload.¹¹ Both phenomena are known to decrease the sarcomere’s sensitivity to calcium.¹² Incidentally, investigation of the known gene mutations linked to DCM shows that diminution in the sarcomere’s sensitivity to calcium are often linked to DCM.¹³

It is yet unclear why some mutations which induce persistent current are linked to DCM while others are not.⁵ The amplitude of the persistent sodium current may account for these differences.

Mutations in the channel’s VSDs could be linked to DCM via the induction of gating pores

The remaining Na_v1.5 DCM linked mutations are all located in the VSDs (Fig. 1). Interestingly, very similar mutations in Na_v1.4 channel have been shown to cause periodic paralysis phenotypes. For example, the Na_v1.4 mutations, R675Q and R675W have all been shown to cause periodic paralysis^14,15 via the induction of a gating pore. The mutation of the corresponding residues in Na_v1.5 (R814Q and R814W) are linked to DCM phenotypes.^2,6,16 The similar localization of the periodic paralysis linked mutations in Na_v1.4 and DCM linked mutations in Na_v1.5 (Fig. 1) prompted us to further examine the spatial localization of those mutations.

We recently reported that the Na_v1.5/R219H mutation was linked to a familial DCM phenotype.⁷ This mutation did not change the classical biophysical properties of the channel which indicated that neither activation, inactivation nor recovery defects of the channel were linked to the pathology. However, the mutation did induce a proton-selective gating pore. This confirms that the induction of a gating pore might be a common characteristic linking all VSD located Na_v1.5 mutations to DCM.

Using the structural model of the VSDs that we recently published,¹⁷ we examined more closely the environment of the DCM linked mutations. As shown by Figure 2, most VSD located mutations are located in close proximity to the VSD’s hydrophobic septum either in the channel’s resting or activated state. This septum maintains a separation between the extracellular and the intracellular media. The collapse of the hydrophobic septum in either of these states gives rise to the gating pore, which would be implicated in the pathogenesis of periodic paralysis and DCM.

Figure 2. Structural model of the Na_v1.4 channel’s VSDs in their resting and activated conformations. The protein is displayed as a gray ribbon. The water crevices are shown in blue and the residues which correspond to the DCM linked mutations in Na_v1.5 are displayed as red spheres.

To this day, most mutations known to induce gating pores occur on the highly conserved positively charged amino acids of the S4 segment. It is postulated that such mutations disrupt the interaction between the charged amino acid on the S4 segment and a motif formed by residues in the S1, S2, and S3 segments of the VSD called the gating charge transfer center (GCTC).^17,18 Thus, it is possible that mutations affecting residues in the GCTC would generate a similar gating pore. Indeed, the Na_v1.5/D1275N and Na_v1.5/F1520L could disrupt the S4-GCTC interactions thus inducing gating pores. Similarly, mutations located in close proximity to key amino acids may affect the structure of the helice, which may in turn disrupt the GCTC-S4 interaction. Such a mechanism could occur for the Na_v1.5/T220I and Na_v1.5/V1279I mutations.

Both the gating pore mechanism and the persistent current mechanism may lead to ionic homeostasis imbalance

Gating pores have been shown to be permeable to monovalent ions such as proton, sodium, and potassium. Both proton and sodium permeation through these pores would lead to acidosis and calcium overload which would diminish the sensitivity of sarcomeres to calcium. Similar consequences could be expected as the result of the persistent sodium current. Thus, this mechanism could unify all the DCM linked Na_v1.5 mutations.

Moreover, ionic homeostasis imbalance and alterations to the biophysical properties of Na_v1.5 could explain the arrhythmias experienced by the patients. Acidosis in the heart could induce cardiac conduction disorders through the block of connexins 40/43¹⁹ and affect the function of ion channels.²⁰ Most importantly, proton-selective gating pores could change sodium homeostasis through the activation of the sodium-proton antiport exchanger.²¹ This sodium overload could lead to an elevation of the myocytes’ resting potential which would facilitate the onset of cardiac arrhythmias. Furthermore, excess in intracellular sodium could lead the activation of the sodium-calcium exchanger in its reverse mode.¹¹ This would increase the intracellular calcium concentration leading to cardiomyocyte apoptosis²² as well as major dysfunctions of heart rhythm or cardiac contractile function.¹²

Ionic homeostasis imbalance is the most likely mechanism to explain the association between Na_v1.5 mutations and DCM

It should be mentioned that other hypothesis linking Na_v1.5 to DCM has been put forward. Indeed, some may argue that DCM is the result of severe arrhythmic phenotypes.²³ Such a hypothesis would be congruent with the severity of the various Na_v1.5 linked DCM familial phenotypes. However, this hypothesis would not account for the observations of Bezzina et al. who have reported that a 1-y-old patient with Na_v1.5 mutation was affected by DCM even though arrhythmia episodes before death were of short duration.^24,25

Thus, we suggest that a mechanism involving ionic homeostasis imbalance via the induction of either gating pore currents or persistent sodium current is the only mechanism likely to provide the link between Na_v1.5 mutations and DCM. Of course, further investigations are warranted to confirm this. We suggest that studies on Na_v1.5 linked DCM mutations should focus on determining whether mutations located in the VSDs yield gating pores. Then, it would be interesting to determine if persistent sodium currents generated by Na_v1.5 mutant channels could lead to sodium and/or calcium overload. Finally, it should be shown that sodium and calcium overload can, if experienced chronically, lead to DCM.

Gosselin-Badaroudine P, Keller DI, Huang H, Pouliot V, Chatelier A, Osswald S, Brink M, Chahine M. A proton leak current through the cardiac sodium channel is linked to mixed arrhythmia and the dilated cardiomyopathy phenotype. PLoS One. 2012;7:e38331. doi: 10.1371/journal.pone.0038331.

Gosselin-Badaroudine P, Delemotte L, Moreau A, Klein ML, Chahine M. Gating pore currents and the resting state of Nav1.4 voltage sensor domains. Proc Natl Acad Sci U S A. 2012;109:19250–5. doi: 10.1073/pnas.1217990109.