Late Na+ current and protracted electrical recovery are critical determinants of the aging myopathy

The aging myopathy manifests itself with diastolic dysfunction and preserved ejection fraction. We raised the possibility that, in a mouse model of physiological aging, defects in electromechanical properties of cardiomyocytes are important determinants of the diastolic characteristics of the myocardium, independently from changes in structural composition of the muscle and collagen framework. Here we show that an increase in the late Na+ current (INaL) in aging cardiomyocytes prolongs the action potential (AP) and influences temporal kinetics of Ca2+ cycling and contractility. These alterations increase force development and passive tension. Inhibition of INaL shortens the AP and corrects dynamics of Ca2+ transient, cell contraction and relaxation. Similarly, repolarization and diastolic tension of the senescent myocardium are partly restored. Thus, INaL offers inotropic support, but negatively interferes with cellular and ventricular compliance, providing a new perspective of the biology of myocardial aging and the aetiology of the defective cardiac performance in the elderly.

shown as mean ± s.e.m. and scatter plots. *P<0.001 versus 3-5 m (Mann-Whitney rank sum test). (d) Quantitative measurements of interstitial fibrosis in male mice at 2-3 months (2-3 m, n = 12), 29-30 months (29-30 m, n = 11) are shown as mean ± s.e.m. and scatter plots. *P<0.01 versus 2-3 m (Mann-Whitney rank sum test). (e) Quantitative data for the transcript levels of transforming growth factor, beta1 (Tgfb1) and vimentin (Vim) in the LV of male mice at 3 months (3 m, n = 6) and 30-31 months (30-31 m, n = 6) are shown as mean ± s.e.m. and scatter plots. *P<0.05 versus 3 m (Mann-Whitney rank sum test). Figure 8. Blockade of the autonomous nervous system does not abrogate the electrical remodeling of the aging heart. Quantitative data for electrocardiographic parameters in anesthetized male mice 3-6 months (3-6 m, n = 12) and 30-33 months (30-33 m, n = 12) following complete block of the autonomic nervous system with atropine (0.5 mg kg -1 body weight, i.p.) plus propranolol (1 mg kg -1 body weight, i.p.). Data are as mean ± s.e.m. and scatter plots. *P<0.05 versus 3-6 m (Student's t-test). Figure 9. Aging is associated by electrical disturbances. (a) Monophasic action potentials (MAPs) during a protocol of programmed electrical stimulation (PES) in an old mouse. Reduced time interval for the stimulated premature beats (arrowheads) induces arrhythmia. VT: ventricular tachycardia. Scale bars: 500 ms, 2 mV. (b) Quantitative data for hearts obtained from male mice at ≤13 months (≤13 m, n = 25) and ≥24 months (≥24 m, n = 27) are expressed as percentage of organs displaying arrhythmic events following PES. *P<0.05 versus ≤13 m (Fisher exact test). Figure 10. Old myocytes present protracted repolarization of the action potential. (a) Quantitative data reported in Fig. 2g are shown as mean ± s.e.m and scattered plots. APs were recorded in myocytes isolated from male mice at 3 months (Young, n = 50 cells from 18 hearts) and 26-30 months (Old, n = 42 cells from 23 hearts) RMP: resting membrane potential. *P<0.05 versus young (Mann-Whitney rank sum test). Figure 11. Old myocytes present protracted repolarization of the action potential. (a,b) Action potential repolarization time of myocytes isolated from male mice at 3 months (Young, n = 14 cells from 7 hearts) and 30-35 months (Old, n = 14 cells from 8 hearts) obtained at 1 Hz (a) and 4 Hz (b) pacing rate. Data are shown as mean ± s.e.m and scattered plots. *P<0.05 versus Young (Mann-Whitney rank sum test). Supplementary Figure 13. The late Na + current I NaL is enhanced in old myocytes. (a) Whole-cell voltagegated Na + currents in a young (black traces) and an old (blue traces) LV myocyte. Scale bars: 100 ms, 2 pA/pF. The voltage-command protocol is shown in the lower traces. (b) Quantitative data for I NaL properties obtained in myocytes from male mice at 3 months (Young, n = 16-27 cells from 5 hearts) and 30-31 months (Old, n = 9-18 cells from 5 hearts) shown as mean ± s.e.m. *P<0.05 versus Young in I NaL -V Relation (Student's t-test and Mann-Whitney rank sum test). Conductance, activation, and inactivation plots were fitted with Boltzmann functions (Young, solid green line; Old, solid blue line). *P<0.0001 between fittings (Student's ttest). Parameters are reported in Supplementary Table 3.

Supplementary Figure 14. A slowly inactivating TTX-sensitive current is enhanced in old myocytes. (a)
Whole-cell voltage-gated currents recorded in voltage-clamp in an old myocyte with a depolarizing step from -80 mV to -40 mV before (Tyrode, black traces) and after exposure to 10 µM tetrodotoxin (TTX, red traces). Difference current is reported in the lower panel. Scale bars: 100 ms, 200 pA. (b) Quantitative data of the time dependent component of the TTX-sensitive difference current obtained in myocytes from male mice at 3-6 months (Young, n = 11 cells from 9 hearts) and 31-35 months (Old, n = 13 cells from 7 hearts) are shown as mean ± s.e.m. and scatter plots. *P<0.01 versus Young (Mann-Whitney rank sum test). With the protocol employed, peak I Na was reduced following TTX exposure. Due to specific amplifier gain settings, I Na was saturated in Tyrode solution, but its amplitude was not saturated and comparable between the two cell populations in TTX (74 ± 9 pA/pF in young and 64 ± 9 pA/pF in old myocytes). Figure 15. The fast Na + current I Na is not affected by aging. (a) Whole-cell voltage-gated Na + currents recorded in voltage-clamp in a young (black traces) and an old (blue traces) LV myocyte. Scale bars: 6 ms, 3 pA/pF. (b) I-V relations, voltage-dependency of conductance, steady state activation, and steady state inactivation, time dependency of reactivation for I Na in myocytes from male mice at 3 months (Young, n = 17-26 cells from 4 hearts) and 29-31 months (Old, n = 16-19 cells from 4 hearts) are shown as mean ± s.e.m. Conductance, activation, and inactivation plots were fitted with Boltzmann functions, and reactivation plots with an exponential function (Young, solid green line; Old, solid blue line). Parameters are reported in Supplementary Table 3.

Supplementary
Supplementary Figure 16. The L-type Ca 2+ current I CaL is not affected by aging. (a) Whole-cell voltagegated Ca 2+ currents recorded in voltage-clamp in a young (black traces) and an old (blue traces) LV myocyte. Scale bars: 100 ms, 1 pA/pF. (b) L-type Ca 2+ current properties obtained in myocytes from male mice at 3 months (Young, n = 19 cells from 5 hearts) and 27 months (Old, n = 17-20 cells from 5 hearts). I-V relations, voltage-dependency of conductance, steady state activation, and steady state inactivation for I CaL are shown as mean ± s.e.m. Fast and slow time constants () for I CaL were measured at 0 mV and are shown as mean ± s.e.m. and scatter plots. Conductance, activation, and inactivation plots were fitted with Boltzmann functions (Young, solid green line; Old, solid blue line). Parameters are reported in Supplementary Table 3. NP: nonparametric analysis.  Figure 4d. Data obtained in mice at 3-6 months (Young, n = 14 cells from 4 hearts) and 27-33 months (Old, n = 8 cells from 5 hearts) before (Tyr) and after exposure to 10 µM ranolazine (Ran) are shown as mean ± s.e.m. and scatter plots. *P<0.05 versus Tyr (paired t-test and Wilcoxon signed rank test); NP: non-parametric analysis.

Supplementary Figure 19. Inhibition of I NaL shortens local epicardial monophasic APs. (a) Monophasic
AP recorded before (KHB, black traces) and after perfusion of 10 µM mexiletine (Mex, red traces) in an old mouse heart. Scale bars: 500 ms, 2 mV. Traces are superimposed in the inset. Scale bar: 100 ms. (b) Quantitative data of repolarization times for hearts from female mice at 5 months (Young, n = 7) and male mice at 24-30 months (n = 9) before (KHB, Krebs-Henseleit buffer) and after exposure to the I NaL inhibitor mexiletine (Mex) are shown as median and IQR. *P<0.05 versus KHB (paired t-test).

Supplementary Figure 20. Inhibition of I NaL shortens the electrical recovery of the old heart. (a)
Quantitative data for electrocardiographic parameters obtained in male mice at 3 months (Young) and at 30 months (Old) at baseline (Base) and 1 hour after treatment with saline (Young, n = 12; Old n = 8) or mexiletine (5 mg kg -1 body weight, i.p.) (Young, n = 11; Old n = 10). Data are shown as mean ± s.e.m and scatter plots. *P<0.05 versus Base (paired t-test and Wilcoxon signed rank test); NP: non-parametric analysis. (b) Quantitative data for electrocardiographic parameters obtained in conscious male mice at 3-4 months (3-4 m, n = 8) and 23-25 months (23-25 m, n = 6) treated with mexiletine (5 mg kg -1 body weight) and followed for a twohour period. Data are shown as mean ± s.e.m and scatter plots. *P<0.05 versus baseline (before administration of the inhibitor, one-way ANOVA with Bonferroni's post hoc test); NP: non-parametric analysis. (a) Quantitative data for Ca 2+ transient and cell shortening properties in myocytes from male mice at 3 months, before (Tyr) and after exposure to 10 µM ranolazine (Ran) (Ca 2+ transients: n = 27 cells from 10 hearts; cell shortening n = 26 cells from 5 hearts); data are shown as mean ± s.e.m. and scatter plots. *P<0.05 versus Tyr (paired t-test and Wilcoxon signed rank test); NP: non-parametric analysis.

Supplementary Figure 23b and c. I NaL alters the amplitude and kinetics of Ca 2+ transients and contraction of LV myocytes. (b)
Quantitative data for Ca 2+ transient and cell shortening properties in myocytes from male mice at 3 months, before (Tyr) and after exposure to 10 µM mexiletine (Mex) (Ca 2+ transients: n = 11 cells from 2 hearts; cell shortening n = 12 cells from 2 hearts); data are shown as mean ± s.e.m. and scatter plots. *P<0.05 versus Tyr (paired t-test and Wilcoxon signed rank test); NP: non-parametric analysis. (c) Quantitative data for Ca 2+ transient and cell shortening properties in myocytes from male mice at 30-31 months, before (Tyr) and after exposure to 10 µM mexiletine (Mex) (Ca 2+ transients: n = 18 cells from 4 hearts; cell shortening n = 18 cells from 3 hearts); data are shown as mean ± s.e.m. and scatter plots. *P<0.05 versus Tyr (paired t-test). Figure 24. Prolongation of the AP with pharmacological interventions. (a) Quantitative AP repolarization properties in myocytes from female mice at 3 months, before (Tyr) and after exposure to 0.5 mM 4-aminopyridine (4-AP) (n = 7 cells from 3 hearts); data are shown as mean ± s.e.m. and scatter plots. *P<0.05 versus Tyr (paired t-test). (b) Quantitative AP repolarization properties in myocytes from mice at 3 months, before (Tyr) and after exposure to 1 nM anemonia toxin-II (ATX-II) (n = 5 cells from 4 hearts); data are shown as mean ± s.e.m. and scatter plots. *P<0.05 versus Tyr (paired t-test and Wilcoxon signed rank test); NP: non-parametric analysis. (c) Current traces in voltage-clamp mode showing the effects of 1 nM ATX-II on I NaL in a young myocytes. I NaL was elicited by a depolarizing step from V h -70 mV to -40 mV. Scale bars: 100 ms, 400 pA.