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. 2010 Dec 31;5(12):e15772.
doi: 10.1371/journal.pone.0015772.

Re-evaluation of the action potential upstroke velocity as a measure of the Na+ current in cardiac myocytes at physiological conditions

Géza Berecki  1 , Ronald Wilders, Berend de Jonge, Antoni C G van Ginneken, Arie O Verkerk
Affiliations

Re-evaluation of the action potential upstroke velocity as a measure of the Na+ current in cardiac myocytes at physiological conditions

Géza Berecki et al. PLoS One. .

Abstract

Background: The SCN5A encoded sodium current (I(Na)) generates the action potential (AP) upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of I(Na) with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of I(Na), which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC) technique might provide an alternative to traditional voltage clamp (VC) technique for the determination of I(Na) properties under physiological conditions.

Principal findings: We studied I(Na) under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak I(Na) during a depolarizing VC step or maximal upstroke velocity, dV/dt(max), during VC/CC served as an indicator of available I(Na). In HEK cells, biophysical properties of I(Na), including current density, voltage dependent (in)activation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied I(Na) in left ventricular myocytes isolated from control or failing rabbit hearts.

Conclusions: Our results demonstrate that the alternating VC/CC technique is a valuable experimental tool for I(Na) measurements under close-to-physiological conditions in cardiac myocytes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Activation of INa in HEK cells assessed with square-step voltage protocols in conventional VC experiments.
a, Top: Typical examples of Na+ current in response to depolarizing voltage steps from −140 mV. Bottom: Average fast and slow time constants of Na+ current inactivation. Note logarithmic ordinate scale. b, Average current-voltage relationship. c, Average steady-state activation. The solid line is the Boltzmann fit to the average data. d, Typical membrane depolarizations in response to a super- and subthreshold current pulse in a HEK cell during an alternating VC/CC experiment from a holding potential of −140 mV (top) and the first derivatives of the resulting membrane potential changes (dV/dt; bottom).
Figure 2
Figure 2. ‘Dynamic’ INa activation in HEK cells assessed with VC (a) and alternating VC/CC (b).
a, Typical example of Na+ current (bottom) during the upstroke phase of the ventricular action potential like waveform used as command potential in an action potential clamp experiment (top). b, Typical membrane depolarization from −85 mV (top) in response to a superthreshold current pulse and dV/dt (bottom). c, Average phase plane plots with current or dV/dt plotted against membrane potential. Note the similarity between the currrent densities of the VC and alternating VC/CC experiments. d, Average dynamic INa activation. Solid lines represent the Boltzmann fits to the average data.
Figure 3
Figure 3. Voltage-dependence of INa inactivation in HEK cells assessed with VC (a) and alternating VC/CC (b).
a and b, Top: voltage clamp (a) and VC/CC (b) protocols. Bottom: typical currents (a) and dV/dt's (b) measured after a 1-s prepulse (P1) to membrane potentials between −110 and −65 mV. c, Average voltage-dependence of inactivation. Solid lines represent the Boltzmann fits to the average data.
Figure 4
Figure 4. Recovery from INa inactivation in HEK cells assessed with VC (a) and alternating VC/CC (b).
a and b, Top: voltage clamp (a) and VC/CC (b) protocols with an interpulse interval of 1–1000 ms, as indicated. Bottom: typical examples of recovery from inactivation with an interpulse interval of 5 ms. c, Average recovery from inactivation. Inset, Average recovery from inactivation on a logarithmic time scale. Solid lines are double-exponential fits to the average data.
Figure 5
Figure 5. Slow inactivation properties in HEK cells assessed with VC (A) and alternating VC/CC (B).
a and b, Top: voltage clamp (A) and VC/CC (B) protocols, with a conditioning prepulse interval (P1) of 10–1000 ms, as indicated, and a 30-ms interval to remove fast inactivation. Bottom: typical examples of slow inactivation with a conditioning prepulse of 1000 ms. c, Average development of slow inactivation. Note the logarithmic time scale. Solid lines are double-exponential fits to the average data.
Figure 6
Figure 6. INa properties in control (CTRL) and heart failure (HF) rabbit ventricular myocytes assessed with alternating VC/CC, using the protocols shown in Figs. 1–5.
a, Typical examples of AP upstrokes (top) and their dV/dt's (bottom). b, Current-voltage relationships of the AP upstrokes. c, Dynamic INa activation curve with Boltzmann fits in solid lines. d, Steady-state voltage-dependence of inactivation with Boltzmann fits in solid lines. e, Recovery from inactivation with double-exponential fits in solid lines. f, Development of slow inactivation with double-exponential fits in solid lines.
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