Echocardiographic findings in patients carrying the genetic variant associated with DCM.

<p>Gender: M, male; F, female. Symptoms: P, palpitations; T, transplanted; p-T, Pre-transplant stage; p-S, Presyncope; S, Syncope; SHF, sudden heart failure; (-) means no dyspnea. New York Heart Association Functional Classification is indicated by a rank between 1 and 4. LVEF indicates left ventricle ejection fraction; TDLVD, telediastolic left ventricle diameter; LAA1, left atrial volume; E/A ratio, relation between E-wave and A-wave in Doppler. Knock in myocardium is indicated by presence (1) or absence (0).</p

Microscopic images of post-mortem tissue.

<p>Pictures showed myofibrillar loss (A and C) and mild interstitial fibrosis (B and D). Nuclear pleomorphism and cellular atrophy is also observed (C). Hematoxylin-eosin labeling (A and C), and picro-Sirius Red labeling (B and D). Scale: A and B (200x) C and D (400x).</p

Pedigree of the family with variants that did not show segregation (ID#18–19).

<p>Pedigree of the family with variants that did not show segregation (ID#18–19).</p

I890T markedly decreases peak <i>I</i>_Na.

<p>Voltage dependence of sodium currents measured from WT and I890T cells. Whole cell currents were elicited by depolarizing potentials as shown in the inset. (A) Representative whole cell sodium current density traces recorded from WT and I890T cells. (B) Current-voltage (<i>I</i>–<i>V</i>) relationship. <i>I</i>_Na amplitude was normalized to the cell capacitance to obtain current density (<i>I</i>_Na density) values. Experimental points represent the peak-amplitude of current density at each given voltage, for WT (filled circles) and I890T (open circles). Values are expressed as mean ± SE.</p

Biophysical parameters of WT and I890T channels.

<p>Activation and steady-state inactivation parameters were calculated by data fitting to Boltzmann functions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053220#s2" target="_blank">Methods</a>). <i>V_1/2</i> is the voltage for half-maximal activation or steady-state inactivation and <i>k</i> is the slope factor. Slow inactivation and recovery from inactivation data were fitted to mono-exponential functions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053220#s2" target="_blank">Methods</a>) to obtain the time constant <i>τ</i>. Values are expressed mean ± SE. *<i>p</i><0.05; **<i>p</i><0.01.</p

Pedigrees of the families with variants showing complete segregation (ID#1–4).

<p>Pedigrees of the families with variants showing complete segregation (ID#1–4).</p

I890T modifies Na_v1.5 channel activation kinetics.

<p><i>I</i>_Na voltage-dependence of activation and steady-state inactivation for WT and I890T cells. Conductance values for the activation curve were obtained from the peak current values taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053220#pone-0053220-g002" target="_blank">Figure 2</a>. Symbols represent experimental data plotted against the given depolarizing voltage values for WT (filled circles) and I890T (open circles). Steady-state inactivation protocol is shown in the inset on the left. Relative current values were determined using 500 ms pre-pulses to different potentials followed by a test pulse to −20 mV. Symbols represent experimental data plotted against preconditioning pulse values for WT (filled squares) and I890T (open squares). Values are expressed as mean ± SE. Solid lines represent the Boltzmann fit of the experimental points.</p

Clinical and genetic characterization of the proband and his family.

<p>(A) Family pedigree with corresponding ECGs. Open symbols indicate clinically normal subjects and filled symbols mark clinically affected individuals. Plus signs indicate the carriers of the mutation I890T and minus signs, non-carriers. The arrow identifies the proband. Basal ECG of the proband and ECGs at the time of the ajmaline test of the family members are presented. (B) Detail of the electropherograms obtained after <i>SCN5A</i> sequence analysis. The arrow indicates the nucleotide position 2669 of <i>SCN5A</i>, where a double peak (T to C heterozygote change, <i>c</i>.2669 T>C) was identified in the proband’s DNA.</p

I890 is a conserved aminoacid, located in the intramembrane pore region of Na_v1.5 DII.

<p>(A) Sequence alignment of the pore modules of human Na_v1.5 channel (DII) and Na_v<i>Ab</i>. Identical aminoacids are highlighted in grey. Isoleucine-890 is marked with a dark box. Similar aminoacids are included inside light boxes and dots identify insertions (lower panel). Sequence alignment of human voltage-gated sodium channel α-subunit family members and of Na_v1.5 channels of different species, upper and middle panels, respectively. The position of the first amino acid of each sequence is indicated on the left side, and the reference for each protein according to Uniprot is shown at the right side. (B) Partial view of the CPHmodel showing the pore module of DII of Na_v1.5 channel (in green), based on the coordinates of Na_v<i>Ab</i> channel (in red). I890_Nav1.5 and T169_Nav<i>Ab</i> are located in the middle of P1-helix and highlighted in blue and magenta, respectively. View from the interior side of the pore. (C) Na_v1.5 channel scheme. The relative position of the I890T mutation in the S5–S6 loop of domain II (DII) is indicated with an arrow.</p

I890T does not affect the time course of inactivation, slow inactivation, or recovery from inactivation.

<p>(A) Experimental data obtained for the current-voltage relationship (Fig. 2) was used to determine inactivation time constants in the voltage range between −40 and 20 mV. Current decay after the peak <i>I</i>_Na was fitted to a mono-exponential function (from −40 to −25 mV) and a bi-exponential function (from −20 to 20 mV), and the resulting time constants (<i>τ</i>) were plotted <i>versus</i> the applied voltage for WT and I890T. (B) Voltage dependence of slow inactivation for WT and I890T were studied by applying the double protocol pulse shown in the inset. A 20–970 ms conditioning pre-pulse to −20 (P1) was followed by a 20 ms hyperpolarization to −120 mV, to recover fast-inactivated channels, and then a 20 ms test pulse to −20 mV (P2). The peak current ratio P2/P1 was plotted against the P1 prepulse duration, and data was fitted to mono-exponential functions (solid lines). (C) Recovery from inactivation properties for WT and I890T were studied by applying the double pulse protocol shown in the inset. A 50 ms depolarizing pulse to −20 mV (P1) was followed by a hyperpolarizing pulse to −120 mV of increasing duration (1–30 ms), that preceded a test pulse to −20 mV (P2). The P2/P1 ratio values plotted against the recovery interval times were fitted to mono-exponential functions (solid lines). A, B and C: Values are expressed as mean ± SE. Symbols represent values for WT (filled symbols) and I890T (open symbols).</p