Voltage-Gated Ion Channel Dysfunction Precedes Cardiomyopathy Development in the Dystrophic Heart

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is associated with severe cardiac complications including cardiomyopathy and cardiac arrhythmias. Recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. It is, however, unknown if the ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiac pathology.To address this question, we first investigated sodium channel impairments in cardiomyocytes derived from dystrophic neonatal mice prior to cardiomyopahty development, by using the whole cell patch clamp technique. Besides the most common model for DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying an utrophin mutation. In neonatal cardiomyocytes, dystrophin-deficiency generated a 25% reduction in sodium current density. In addition, extra utrophin-deficiency significantly altered sodium channel gating parameters. Moreover, also calcium channel inactivation was considerably reduced in dystrophic neonatal cardiomyocytes, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. To assess developmental changes, we also studied sodium channel impairments in cardiomyocytes derived from dystrophic adult mice, and compared them with the respective abnormalities in dystrophic neonatal cells. Here, we found a much stronger sodium current reduction in adult cardiomyocytes. The described sodium channel impairments slowed the upstroke of the action potential in adult cardiomyocytes, and only in dystrophic adult mice, the QRS interval of the electrocardiogram was prolonged.Ion channel impairments precede pathology development in the dystrophic heart, and may thus be considered potential cardiomyopathy triggers

Action potential properties in wt and dystrophic adult cardiomyocytes.

<p>Typical AP of a wt cardiomyocyte (top left) elicited by a 4-ms current injection at 125% threshold. On the top right, the upstrokes of typical APs recorded from wt and dystrophic cardiomyocytes are overlayed on an expanded time scale. In the lower panel, maximum upstroke velocity (max. dV/dt), and the maximum depolarisation value reached by the AP (abs. amplitude) are plotted against the membrane potential. * and ** indicate significant differences (ANOVA; p<0.05 and <0.01; n-values: 5–8) between the three tested groups, respectively. Tukey's Post Hoc test revealed no significant differences between mdx-utr and mdx.</p

Surface ECG recordings on wt and dystrophic neonatal and adult mice.

<p>(Top) Typical original ECG signals (lead 1) from neonatal and adult wt mice, and the corresponding averages over 100 beats (insets). (Bottom right) Overlay of representative QRS-complexes (lead 2) in adult wt and mdx mice. In the graph, the QRS intervals of neonatal and adult ECGs are compared between wt and dystrophic mice. *** significant difference (ANOVA, p<0.001, n-values: 6–15) between the three tested groups. No significant differences exist between mdx-utr and mdx.</p

Sodium and barium current parameters in wt and dystrophic cardiomyocytes.

<p>The given parameters were obtained by the analysing procedures described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020300#s4" target="_blank">Materials and Methods</a>. V_0.5 is the voltage at which half-maximum activation or inactivation occurred, and k represents the slope factor. V_rev is the reversal potential, and n gives the number of experiments. Values represent means ± SEM. Data in italic indicate that ANOVA revealed a significant difference (p<0.05) between the respective parameters of the three different groups (wt, mdx, and mdx-utr). Data in bold indicate that ANOVA revealed p<0.01. Statistical comparisons between two groups were made using Tukey's Post Hoc test. Here,</p>&<p>p<0.05 indicates a significant difference between mdx and wt.</p>§<p>p<0.05 and</p>§§§<p>p<0.001 indicate significant differences between mdx-utr and wt.</p>$<p>p<0.05 and</p>$$<p>p<0.01 indicate significant differences between mdx-utr and mdx.</p

Calcium channel properties in wt and dystrophic neonatal cardiomyocytes.

<p><b>A.</b> Original barium current traces (five episodes displayed) of a wt and mdx-utr cardiomyocyte, elicited by the pulse protocol on top. The prepulse was used to eliminate currents through T-type calcium channels. <b>B.</b> Current-voltage relationships derived from a series of experiments on wt and dystrophic cardiomyocytes as shown in A. <b>C.</b> Comparison of the current decay kinetics between wt and dystrophic cardiomyocytes at various membrane voltages. τ –values were derived from single exponential fits of the current decay after channel activation. * indicates a significant difference (ANOVA, p<0.05) between the three tested groups. Tukey's Post Hoc test revealed a significant difference (p<0.05) between mdx-utr and wt, but not between mdx-utr and mdx. <b>D.</b> Voltage-dependency of steady-state inactivation in wt and dystrophic cardiomyocytes. For n-values see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020300#pone-0020300-t001" target="_blank">Table 1</a>.</p

Sodium channel properties in wt and dystrophic adult cardiomyocytes.

<p><b>A.</b> Original current traces of a typical wt, mdx, and mdx-utr cardiomyocyte, elicited by the pulse protocol in B. <b>B.</b> Current-voltage relationships derived from a series of experiments as shown in A. <b>C.</b> Maximum current densities in wt and dystrophic cardiomyocytes. *** indicates that ANOVA revealed a significant difference (p<0.001) between the three tested groups. <b>D.</b> Comparison of the voltage-dependencies of activation and steady-state inactivation between wt and dystrophic cardiomyocytes. For detailed statistics and n-values see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020300#pone-0020300-t001" target="_blank">Table 1</a>.</p

Sodium channel properties in wt and dystrophic neonatal cardiomyocytes.

<p><b>A.</b> Original current traces of a typical wt, mdx, and mdx-utr cardiomyocyte, elicited by depolarising voltage-steps. <b>B.</b> Current-voltage relationships derived from a series of experiments as shown in A. The Inset (bar graph) shows a comparison of the maximum current densities between wt and dystrophic cardiomyocytes. <b>C.</b> Comparison of the current decay kinetics between wt and dystrophic cardiomyocytes at different membrane voltages. Decay half-times represent the time periods between the current peak and the time point at which the current had decayed to 50%. * indicates that ANOVA revealed a significant difference (p<0.05) between the respective parameters of the three tested groups. Tukey's Post Hoc test for comparison between two groups revealed significant differences (p<0.05) between mdx-utr and wt, and between mdx-utr and mdx. <b>D.</b> Voltage-dependencies of activation and steady-state inactivation in wt and dystrophic cardiomyocytes. <b>E.</b> Recovery from inactivation in wt and dystrophic cardiomyocytes. Data were fit with a single exponential function. The pulse protocols are shown as insets in the respective graphs. Data points represent means ± SEM. Detailed statistics and n-values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020300#pone-0020300-t001" target="_blank">Table 1</a>.</p