Profile of L-type Ca(2+) current and Na(+)/Ca(2+) exchange current during cardiac action potential in ventricular myocytes

Abstract

Objective: The L-type Ca(2+) current (I(Ca,L)) and the Na(+)/Ca(2+) exchange current (I(NCX)) are major inward currents that shape the cardiac action potential (AP). Previously, the profile of these currents during the AP was determined from voltage-clamp experiments that used Ca(2+) buffer. In this study, we aimed to obtain direct experimental measurement of these currents during cardiac AP with Ca(2+) cycling.

Method: A newly developed AP-clamp sequential dissection method was used to record ionic currents in guinea pig ventricular myocytes under a triad of conditions: using the cell's own AP as the voltage command, using internal and external solutions that mimic the cell's ionic composition, and, importantly, not using any exogenous Ca(2+) buffer.

Results: The nifedipine-sensitive current (I(NIFE)), which is composed of I(Ca,L) and I(NCX), revealed hitherto unreported features during the AP with Ca(2+) cycling in the cell. We identified 2 peaks in the current profile followed by a long residual current extending beyond the AP, coinciding with a residual depolarization. The second peak and the residual current become apparent only when Ca(2+) is not buffered. Pharmacological dissection of I(NIFE) by using SEA0400 shows that I(Ca,L) is dominant during phases 1 and 2 whereas I(NCX) contributes significantly to the inward current during phases 3 and 4 of the AP.

Conclusion: These data provide the first direct experimental visualization of I(Ca,L) and I(NCX) during cardiac the AP and Ca(2+) cycle. The residual current reported here can serve as a potential substrate for afterdepolarizations when increased under pathologic conditions.

Fig.1

Panel A shows a representative current trace recorded with 0.1% (v/v) DMSO (used as solvent of channel blockers). The flat “zero current” indicate a lack of any DMSO effect on membrane currents (n=5 cells). Panel B and C are representative recordings of I_NIFE or I_NISO respectively. Panel D shows the statistical comparison of I_NIFE (n=7) and I_NISO (n=5), demonstrating no significant differences between the currents. Panel E shows the residual depolarization. Panel F shows a positive correlation between the residual depolarization and the residual current measured at 30 ms after −V_max.

Fig.2

Representative I_NIFE recorded under AP-clamp with 10 mM EGTA in pipette (n=10 cells). Notice an absence of the second dome, the residual current (A), and the residual depolarization (B). I_NIFE recorded with 2 mM EGTA (C, n=14 cells), the current-voltage relationship (D), and statistical comparison between different Ca²⁺ buffering conditions (E) show increased current density with more EGTA buffering. Panel F demonstrates that EGTA did not affect the voltage at −V_max but significantly reduced the residual depolarization at 30 ms after −V_max. (t-test, p<0.05*; p<0.01**)

Fig.3

Panel A shows a representative current recorded with 3 µM SEA0400 (I_SEA). When 10 µM nifedipine was added, I_SEA+NIFE displays similar characteristics (B). Panel C and D show the instant current-voltage relationship of I_SEA and I_SEA+NIFE and statistical comparison of data (n=7 cells, t-test, p<0.05*). Standard V-clamp protocols were used to measure the inhibitory effects of 3 µM SEA. Panel E and F show current traces and the I–V relationship of I_NCX before and after application of SEA. Panel G and H show representative current traces and the I–V relationship of I_Ca,L before and after application of SEA.

Fig.4

Column A and B show I_SEA and I_SEA+NIFE recorded in the absence and presence of EGTA, respectively. EGTA buffering of Ca²⁺ reversed I_SEA and amplified I_SEA+NIFE. Mathematical reconstruction of I_Ca,L and I_NCX assuming zero coupling (α=0, red line) or linear coupling (α=1, blue line) between I_Ca,L and I_NCX mark the boundaries of the actual currents. Black line shows an intermediate state (α=0.8), highlighting a probable profile of the currents.

Fig.5

Using 3 µM SEA0400 to treat cells shortened AP duration (A) and reduced I_NIFE (B). The residual current (C) and the residual depolarization (D) were significantly reduced (n=7 cells, t-test, p<0.05*), demonstrating a predominant influence of I_NCX on these features. Panel E shows a universal correlation (R=0.917) between the magnitude of residual current and residual depolarization under all experimental conditions tested.

Fig.6

These experiments were conducted at 36°C. Panel A shows a representative I_NIFE recorded under AP-clamp with Ca²⁺ cycling (n=19 cells). Notice the presence of the second dome and the residual current (A) and the residual depolarization (B). The insert shows the Ca²⁺ transient measured with Fura-2. Panel C shows a representative I_NIFE recorded with 10 mM EGTA (n=9 cells). Notice the absence of the residual current (C) and the residual depolarization (D). Panel E and F show the Mean ±SE and statistical comparison at the features points. (t-test p<0.01**, p<0.001***)