Cardiac IK1 underlies early action potential shortening during hypoxia in the mouse heart

Abstract

It is established that prolonged hypoxia leads to activation of K(ATP) channels and action potential (AP) shortening, but the mechanisms behind the early phase of metabolic stress remain controversial. Under normal conditions IK1 channels are constitutively active while K(ATP) channels are closed. Therefore, early changes in IK1 may underlie early AP shortening. This hypothesis was tested using transgenic mice with suppressed IK1 (AAA-TG). In isolated AAA-TG hearts AP shortening was delayed by approximately 24 s compared to WT hearts. In WT ventricular myocytes, blocking oxidative phosphorylation with 1 mM cyanide (CN; 28 degrees C) led to a 29% decrease in APD90 within approximately 3-5 min. The effect of CN was reversed by application of 100 microM Ba2+, a selective blocker of IK1, but not by 10 microM glybenclamide, a selective blocker of KATP channels. Accordingly, voltage-clamp experiments revealed that both CN and true hypoxia lead to early activation of IK1. In AAA-TG myocytes, neither CN nor glybenclamide or Ba2+ had any effect on AP. Further experiments showed that buffering of intracellular Ca2+ with 20 mM BAPTA prevented IK1 activation by CN, although CN still caused a 54% increase in IK1 in a Ca2+ -free bath solution. Importantly, both (i) 20 microM ruthenium red, a selective inhibitor of SR Ca2+ -release, and (ii) depleting SR by application of 10 microM ryanodine+1 mM caffeine, abolished the activation of IK1 by CN. The above data strongly argue that in the mouse heart IK1, not KATP, channels are responsible for the early AP shortening during hypoxia.

Fig. 1. Early MAP shorting induced by hypoxia is abolished in AAA-TG mice

A. A representative time course of MAP duration during hypoxia. In WT hearts, removal of O₂ from the perfusate (application of N₂; ) leads to an early, presumably I_K1-dependent, abbreviation of MAP (2; ) followed by a further rapid MAP shortening (3; ), presumably due to the activation of K_ATP channels. The time for early MAP shortening was defined as the time when MAPD75 falls by ∼10% of the Ctrl value measured just before application of N₂ (1; ). The early phase of MAP shortening is absent in AAA-TG hearts, and only the rapid phase is observed, which starts at the same time as the K_ATP-dependent phase of MAP shortening in WT hearts (3; ). B. Representative MAPs from WT (gray) and AAA-TG (black) hearts recorded at the times as indicated in A. MAP amplitudes were normalized to each other to highlight the differences in shape. C, D. MAPD75 measured at the times as indicated in A. At the time when the early MAP shortening has just begun in AAA-TG hearts (3), in WT hearts MAPD75 has been already reduced by 28%. Statistical significance is indicated compared to MAPD75 before application of N₂ (1). D. The time delay (D_t) between the application of N₂ and the time when MAPD75 is decreased by 10% in WT and AAA-TG hearts. n=4 for both WT AAA-TG hearts.

Fig. 2. CN-induced early AP shortening is abolished in isolated myocytes with suppressed I_K1

Examples of APs in WT (A) and AAA-TG (B) myocytes. A. In WT cells, 10 μM glybenclamide (Gb) does not significantly affect AP. Application of 1 mM CN in the presence of Gb leads to AP shortening within ∼3 min. Further addition of 100 μM Ba²⁺ completely reverses the effect of CN. (Insert) AP shortening caused by 100 μM pinacidil is not blocked by 100 μM Ba²⁺. B. Application of CN, Gb or Ba²⁺ exerts no effect on the AP in AAA-TG myocytes within the same period of time (∼3-5 min). C. Summarized data show that in WT myocytes 10 μM Gb has virtually no effect on APD90 while 1 mM CN reduces APD90 by nearly 30% within 3 min (n=10). Further application of 100 μM Ba²⁺ increases APD90 by ∼17% above the control level (n=10). In AAA-TG myocytes with suppressed I_K1, CN (3 min application), Gb or Ba²⁺ have no affect on APD90 (n=7). For each of the three conditions, a relative change in APD90 values is presented; therefore a dashed line at 100% represents control data.

Fig. 3. Time course of the CN-induced current at −60 mV in WT myocytes

An example of the time course of the whole-cell current amplitude measured at a −60 mV membrane potential during application of 1 mM CN at 28 °C. (Insert) −60 mV roughly corresponds to the peak of outward I_K1. (1) Currents are stable for a prolonged period of time until the application of CN. (2) CN leads to a quick increase in current amplitude which remains quasi-stable until (3) a sharp increase after >15 min due to activation of K_ATP channels.

Fig. 4. Application of CN leads to early upregulation of I_K1 in WT myocytes

A and B. Representative Ba²⁺-sensitive (A) and Ba²⁺-insensitive (B) currents recorded before and after a 3 min application of 1 mM CN at 28 °C. In A, the dotted trace represents a difference, or CN-activated, current. Currents were recorded in response to a 4 s voltage ramp from −100 mV to 10 mV (reduced voltage range is shown) in the absence and presence of 100 μM Ba²⁺. C. Current densities at −60 mV. Ba²⁺-sensitive currents are increased by ∼50%, and Ba²⁺-insensitive currents are not affected by CN treatment (n=11, p<0.05 vs Ctrl, respectively).

Fig. 5. True hypoxia leads to early activation of I_K1

A. Representative Ba²⁺-sensitive current traces recorded in a WT myocyte before and after a 4 min application of nominally O₂-free solution (N₂) at 28 °C. The current activated by hypoxia displays strong inward rectification, confirming it is I_K1. B. At −60 mV the Ba²⁺-sensitive current is increased by 60% when compared to control (n=4, p<0.05).

Fig. 6. K_ATP channels in WT and AAA-TG myocytes

K⁺ currents were measured at +50 mV in high symmetrical K⁺ using excised membrane patches, and K_ATP current amplitudes calculated as a difference between currents in 0 and 1 mM ATP. A. In AAA-TG myocytes, K_ATP current density (per patch) is reduced by ∼25% compared to that in WT cells (p<0.01). B. A representative example of an ATP dose-response relationship in WT cells obtained under the same conditions as above. C. Normalized IK_ATP (I_REL) from individual patches were fit with a Hill equation (insert) to estimate the slope (h) and concentration of half maximum block (K_1/2). The data at each concentration show only a minor variation, thus standard error (SE) bars are too small compared to symbols. Neither the Hill coefficient (h) nor the sensitivity to ATP (K_1/2) are affected in any significant way in AAA-TG myocytes, therefore, for visualization purpose, the averaged data were fit again with the Hill equation and the fit is plotted as indicated (continuous line). n=91 and n=57 for WT and AAA-TG cells, respectively, in all experiments.

Fig. 7. I_Ca and I_NCX in WT and AAA-TG myocytes

A. (1) Representative Ca²⁺ currents in WT myocytes in response to a voltage protocol shown in (2). (3) Current-voltage relationships for peak I_Ca in WT and AAA-TG myocytes. There is no significant difference between the two groups (n=11, n=10, respectively). B. (1) Representative background currents in WT myocytes in response to voltage steps to potentials between −80 mV and +80 mV. (2) and (3) Current-voltage relationships for total, Ni²⁺-insensitive and Ni²⁺-sensitive background currents in WT and AAA-TG myocytes measured at the end of voltage steps. There is no significant difference between any corresponding currents in the two groups (n=14, n=9, respectively). See Methods for solution composition.

Fig. 8. SR Ca²⁺ release is involved in the activation of I_K1 during hypoxia

A. Strong intracellular Ca²⁺ buffering abolishes activation of I_K1 by CN. Current densities at −60 mV were measured before and after application of 1 mM CN (∼3 min) using different concentrations of Ca²⁺ and types of Ca²⁺ buffer in the pipette solution (n=4, 11 and 6 from left to right). pCa_i values were calculated using the following concentrations of Ca²⁺-binding components and Ca²⁺. pCa²⁺_i ∼5 − 5 mM ATP, 2 mM EGTA 190 μM Ca²⁺; pCa²⁺_i ∼8 − 5 mM ATP, 170 μM Ca²⁺; pCa²⁺_i >12 − 5 mM ATP, 20 mM BAPTA, 0.1 μM Ca²⁺; pH 7.3, T=25 °C. Calculations were performed using WinMaxc programs, http://www.stanford.edu/∼cpatton/maxc.html. B. Removal of extracellular Ca²⁺_o does not affect the activation of I_K1 by CN (n=6). In contrast, depletion of Ca²⁺ stores by extracellular application of 10 mM caffeine (Caff) plus 10 μM ryanodine (Rya) or the inhibition of Ca²⁺ release by 10 μM ruthenium red (RR; included into pipette solution) abolishes I_K1 activation (n=7 and 5, respectively).