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. 2017 Aug 1;113(10):1256-1265.
doi: 10.1093/cvr/cvx122.

The Expression of the Rare caveolin-3 Variant T78M Alters Cardiac Ion Channels Function and Membrane Excitability

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Free PMC article

The Expression of the Rare caveolin-3 Variant T78M Alters Cardiac Ion Channels Function and Membrane Excitability

Giulia Campostrini et al. Cardiovasc Res. .
Free PMC article

Abstract

Aims: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability.

Methods and results: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect.

Conclusion: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.

Keywords: Arrhythmia; Caveolin; Electrophysiology; Genetic diseases; Ion channels.

Figures

Figure 1
Figure 1
WT and T78M cav-3 proteins are expressed at the plasma membrane and form caveolae. (A) Representative confocal images of MEF-KO cells co-transfected with either the WT-EGFP cav-3 (green, top left) or the T78M-EGFP cav-3 (green, bottom left) and the membrane-targeted CFP (pm, red, centre) (exp = 3). Right panels show both signals overlapped (yellow). Scale bar = 20 µm. (B) Representative transmission electron micrographs of thin-sections from non-transfected (NT) MEF-KO cells (left) showing absence of any visible caveola, and from MEF-KO cells transfected with either the WT-EGFP cav-3 (centre) or the T78M-EGFP cav-3 (right) in which typical caveolar structures (arrows) can be seen at the plasma membrane (pm). The inset in the first panel shows a magnified region in which a clathrin-coated vesicle (Clv) is visible. rer, rough endoplasmic reticulum; N, nucleus; G, golgi apparatus; M, mitochondrion; n/exp = 25/3). Scale bar =150 nm.
Figure 2
Figure 2
The T78M cav-3 interacts with hHCN4 and hKv1.5 channels and targets them to lipid rafts. (A) Blots showing that hHCN4, hKV1.5, and cav-3 transfected in MEF-KO cells localize into lipid rafts isolated by discontinuous sucrose gradient (n ≥ 2). Lys, lysate; LR lipid raft fractions; NLR non-lipid raft fractions. (B, C) Co-immunoprecipitation (co-IP) experiments from MEF-KO cells co-transfected with either HCN4 (B) or V5-Kv1.5 (C) and WT cav-3 EGFP or T78M cav-3 EGFP (n = 3). An aliquot of the input (in) and of the co-IP eluate (+) were tested by western blot. A negative control (–) was performed by omitting the cav-3 antibody in the IP procedure. The cav-3 signal appear at around 50 kDa because it is a fusion protein with EGFP. The band just above the caveolin signal represents the heavy chain of the mouse IgG used during the immunoprecipitation, since it can be directly recognized by the anti-mouse IgG secondary antibody used in the cav3 western blots (data not shown).
Figure 3
Figure 3
The T78M cav-3 affects hKv1.5 channel properties. Representative current traces recorded from MEF-KO cells co-transfected with hKv1.5 and WT cav-3 (top), T78M cav-3 (centre), or both (bottom) during activation (A) and inactivation voltage protocols (B). Insets in A show, on an expanded scale, tail currents recorded at –50 mV used for activation curve analysis. (C) Mean activation curves (top; V1/2 values were: WT 1.41 ± 0.70 mV, n/exp = 27/13; T78M –2.63 ± 1.43*, n/exp = 23/11; WT/T78M –2.21 ± 1.15*, n/exp = 17/5), inactivation curves (centre; WT –5.9 ± 0.9 mV, n/exp = 24/12; T78M –10.9 ± 0.9*, n/exp = 24/11; WT/T78M –11.5 ± 0.6*, n/exp = 18/5) and current density-voltage relations (bottom, WT n/exp = 22/12, T78M n = 26/11, WT/T78M n = 21/5) in the three groups (WT filled circles, T78M open circles, WT/T78M half-filled circles,) *P < 0.05 by nested and One-way ANOVA with Fisher’s test.
Figure 4
Figure 4
The T78M cav-3 alters hHCN4 channel voltage-dependence. (A) Representative current traces elicited by a double hyperpolarizing step protocol to –85 and –125 mV (holding potential –35 mV) recorded from MEF-KO cells expressing hHCN4 and the WT, T78M or both forms of cav-3, as indicated; traces were normalized and overlapped for comparison. (B) Mean hHCN4 activation curves (V1/2 values: WT –86.2 ± 1.1 mV, n/exp = 26/15; T78M –78.3 ± 1.1* mV, n/exp =30/11; WT/T78M –77.4 ± 1.0* mV, n/exp =29/10. *P < 0.05 by nested and One-Way ANOVA with Fisher’s test), obtained from the different groups (WT filled circles, T78M open circles; WT/T78M half-filled circles). (C) Mean V1/2 values from day-matched recordings of hHCN4 currents in control (CTRL, empty circles) or in the presence of 10 µM cAMP in the pipette solution (+cAMP, black circles) in MEF-KO cells transfected with hHCN4 and WT, T78M or both forms of cav-3, as indicated (WT, control: –84.1 ± 1.5 mV n/exp = 6/4, cAMP: –73.4 ± 1.5 mV n/exp = 11/4; T78M, control: –79.8 ± 1.5 mV n/exp = 8/3, cAMP: –71.9 ± 1.0 mV n/exp = 16/3; WT/T78M, control: –77.8 ± 1.5 mV n/exp = 8/3, cAMP: –72.5 ± 1.6 mV n/exp = 8/3). *P < 0.05 by Student’s t-test. (D) Mean current density-voltage relations are shown for comparison (WT, n/exp = 20/10; T78M, n/exp = 14/9; WT/T78M, n = 20/10).
Figure 5
Figure 5
The electrical properties of NRVCs are altered in the presence of T78M cav-3. (A) Time course of the inter-beat interval (IBI) in neonatal cardiomyocytes transfected with the empty vector (EV, top), the WT cav-3 (centre) or the T78M cav-3 (bottom) vectors; insets show 5 s stretches of the original action potential recordings. Scatter plots showing single values (empty circles) and mean values (black circles) of (B) The IBI (EV 0.55 ± 0.04 s, n/exp = 7/2; WT 0.61 ± 0.04 s, n/exp = 18/3; T78M 0.58 ± 0.05 s, n/exp = 9/3), (C) Coefficient of variation of the IBI (CVIBI; EV 0.14 ± 0.04; WT 0.12 ± 0.01; T78M 0.29 ± 0.03), (D) Maximum diastolic potential (MDP; EV –57.9 ± 5.2 mV; WT –52.6 ± 1.6 mV; T78M –55.1 ± 2.5 mV) and of (E) the MDP standard deviation (SDMDP, EV 0.85 ± 0.21; WT 1.13 ± 0.12; T78M 2.41 ± 0.35). *P < 0.05 vs. all other conditions by nested and one-way ANOVA with Fisher’s test.
Figure 6
Figure 6
Mathematical models of both atrial and sinoatrial cells show an arrhythmic contribution of the T78M cav-3. Atrial (top) and sinoatrial (bottom) action potentials generated using the Grandi-Bers human atrial cell model and the Severi-DiFrancesco rabbit sinoatrial cell model,, respectively. Thin line, basal conditions (WT); thick line, after insertion of the T78M cav-3-dependent alterations.

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