Role of sodium-calcium exchanger in modulating the action potential of ventricular myocytes from normal and failing hearts

Abstract

Increased Na+-Ca2+ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca2+ uptake, provide inotropic support through reverse-mode Ca2+ entry, and/or deplete intracellular Ca2+ stores. NCX is electrogenic and depends on Na+ and Ca2+ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na+]i on the AP in myocytes from normal and pacing-induced failing canine hearts and estimate the direction of the NCX driving force using simultaneously recorded APs and Ca2+ transients. AP duration shortened with increasing [Na+]i and was correlated with a shift in the reversal point of the NCX driving force. At [Na+]i > or =10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na+]i, NCX had a depolarizing effect, confirmed by partially inhibiting NCX with exchange inhibitory peptide. Exchange inhibitory peptide shortened the AP duration at 5 mmol/L [Na+]i and prolonged it at [Na+]i > or =10 mmol/L. With K+ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na+]i and became inward close to the predicted reversal point for the NCX driving force. The results were reproduced using a computer model. These results indicate that NCX plays an important role in shaping the AP of the canine myocyte, helping it to repolarize at high [Na+]i, especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na+]i.

Figure 1

Effect of [Na⁺]_i on AP and NCX driving force. Top, Representative APs (black trace) and Ca²⁺ transients (red trace) from myocytes isolated from normal hearts dialyzed with 5, 10, or 15 mmol/L [Na⁺]_i. Left panel illustrates the method for calculating the NCX driving force (E_m − E_NCX, magenta trace) from E_NCX (blue trace). Bottom, Analogous records for myocytes from failing hearts with 5, 10, or 15 [Na⁺]_i. RP is the potential at which E_m − E_NCX=0 (indicated with a dashed line in all panels).

Figure 2

[Na⁺]_i dependence of the APD, NCX driving-force RP, and TTRP in normal (solid square) and failing (open circle) myocytes. A and B, APD₉₀ shortened with increasing [Na⁺]_i (A), in correspondence with a shift in the NCX RP toward more hyperpolarized potentials (B). C, RP occurred later in the AP at higher [Na⁺]_i. TTRP/APD₉₀ is the TTRP normalized to APD₉₀.

Figure 3

[Na⁺]_i dependence of APD using paired pipette technique. [Na⁺]_i was varied within individual myocytes using sequential pipettes containing different levels of [Na⁺]_i. The direction and amplitude of the change in [Na⁺]_i are indicated by the arrows. Black and red arrows denote myocytes from normal and failing hearts, respectively. In one cell from a normal heart and four cells from failing hearts, a third pipette was used to sequentially patch the same cell with a different [Na⁺]_i.

Figure 4

Ratio of integrated reverse-mode to forward-mode NCX driving force during the AP. A, Int_rev represents the integral of the E_m−E_NCX curve from the upstroke of the AP to the NCX RP. Int_for is the integral from the driving-force RP to the point of AP repolarization (taken as the minimum of the NCX driving-force curve). B, Int_rev/Int_for increased as a function of [Na⁺]_i and was significantly higher in the failing group. C, Averaged running integrals for all data sets illustrate more rapid reverse-mode NCX activation in myocytes from failing (F) than normal (N) hearts at all levels of [Na⁺]_i. *P<0.05 for normal vs failing myocytes; †P<0.05 for myocytes from failing hearts at 10 mmol/L [Na⁺]_i vs myocytes from normal hearts at 5 mmol/L [Na⁺]_i.

Figure 5

Effect of XIP on APD. A, Block of net inward current at 5 [Na⁺]_i during the AP plateau would be expected to shorten the APD₉₀, whereas the predicted effect at 10 or 15 [Na⁺]_i would be AP prolongation, based on the net repolarizing influence of outward I_NCX. B, Paired pipette experiments confirmed the predicted effect of XIP on APD, supporting the validity of NCX driving-force estimates. C, Unpaired experiments show the change in mean APD₉₀ induced by 30 μmol/L XIP with 10 mmol/L [Na⁺]_i.*P<0.05 for normal vs failing myocytes; †P<0.05 for XIP vs control in normal myocytes. Myocytes were paced at 1 Hz in panel B and 0.25 Hz in panel C.

Figure 6

AP-clamp model simulations. A, Examples of model-derived Ca²⁺ transients and NCX driving forces using experimental AP recordings as inputs at varying levels of [Na⁺]_i. B, Correlation between model-derived and experimental NCX driving-force RPs. C and D, Influence of Ca²⁺ in the junctional subspace on the Ca²⁺ transient in AP-clamp mode (C) or free-running AP simulations (D).

Figure 7

AP-clamp currents with 10 mmol/L [Na⁺]_i and K⁺ currents blocked. Top panel shows AP waveform used to voltage-clamp a cardiomyocyte with Cs⁺ substitution of K⁺ in all solutions (to eliminate contamination by Ca²⁺ -activated Cl⁻ current; this experiment was carried out in a guinea pig cardiomyocyte). Under these conditions, the predominant membrane currents were I_Ca,L and I_NCX. The kinetics and direction of the membrane currents during the AP plateau corresponded to estimates of the NCX driving force (magenta trace) calculated from the simultaneously recorded [Ca²⁺]_i transient (red trace).