The role of activation of two different sGC binding sites by NO-dependent and NO-independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes

Abstract

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch-activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC's regulation along NO-dependent and NO-independent pathways, as well as the possibility of S-nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch-clamp method in whole-cell configuration, inward nonselective stretch-activated cation current I_SAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1-subunit of sGC activator BAY41-2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br-cGMP, and S-nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd³⁺ -sensitive nonselective cation current, an analog of I_SAC , while in a stretched cell it eliminates I_SAC . The NO-independent pathway of sGC activation of α subunit, triggered by BAY41-2272, is also important for the regulation of SACs. Since S-nitrosylation inhibitor completely abolishes I_SAC , this mechanism occurs. The application of BAY41-2272 cannot induce I_SAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S-nitrosylation. ODQ eliminates I_SAC , but SNAP added on the background of stretch increases I_SAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in I_SAC . 8Br-cGMP reduces I_SAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S-nitrosylation to the regulation of SACs.

Keywords: 8Br-cGMP; BAY41-2272; KT5823; L-Arginine; ODQ; ascorbic acid; nitric oxide; nitric oxide synthase; patch-clamp, SNAP; soluble guanylyl cyclase; stretch-activated channels; ventricular cardiomyocytes.

FIGURE 1

Induction of net inward currents by a local stretch. (a) Online records (time course) of membrane current, K⁺ currents not suppressed. V _m clamped to a holding potential of −45 mV. The amplitude of stretch (4 µm) and amount of stretch‐induced inward current at −45 mV indicated. (b) Gadolinium completely blocks the stretch‐induced inward current. (c, d, e) Graduation by the extent of stretch. The amplitude of negative current (at −80 mV), reduction of the positive hump of the I/V curve (at −55 to −60 mV), and value of depolarization (change of the zero current potential E ₀, i.e., intercept of I/V curve with current axis) increase with the value of stretch; I/V‐curves before (circles) and during the stretch (triangles) of 4‐µm (c), 6‐µm (d) and 8‐µm (e)

FIGURE 2

Local stretch activates I _SAC, (the current through nonselective cation channels). (a) Online records of I _SAC, K⁺ currents suppressed. V _m clamped to a holding potential of −45 mV. Graduation by the extent of stretch. As an example, the values of stretching 4, 6, and 10 µm are shown. (b) Graduation by the extent of stretch. The amplitude of I _SAC (at −45 mV and −80 mV), and value of depolarization (change of the zero current potential V ₀) increase with the value of stretch; I/V‐curves before (circles) and during the stretch of 6‐µm (triangles), 8‐µm (squares), 10‐µm (rhombuses), 12‐µm (inverse triangles). (c) Online records of I _SAC, gadolinium completely blocks I _SAC. Stretch by 10 µm as an example

FIGURE 3

SNAP shifted net currents to more negative values. Online records (time course) of membrane current, V _m clamped to a holding potential of −45 mV. (a) Different concentrations of SNAP (50, 100, 200, 300, 400 µmol/L) cause the appearance of the maximum peak current (ΔI _max), the highest at 200 µmol/L, with its subsequent decrease. K⁺ currents not suppressed. ${\mathrm{K}}_{\text{in}}^{+}$/${\mathrm{K}}_{\text{out}}^{+}$ environment. (b) In ${\text{Cs}}_{\text{in}}^{+}$/${\text{Cs}}_{\text{out}}^{+}$ medium (K⁺ currents suppressed) SNAP at a concentration of 200 µmol/L cause the appearance of ΔI _max, but the effect develops about twice as long as in ${\mathrm{K}}_{\text{in}}^{+}$/${\mathrm{K}}_{\text{out}}^{+}$ medium at of the same NO donor concentration

FIGURE 4

SNAP changes the voltage dependence of I _L in a ${\mathrm{K}}_{\text{in}}^{+}$/${\mathrm{K}}_{\text{out}}^{+}$ environment. (a, b, c) At a concentration of 100, 200, 300 µmol/L in the first 5 min, a reduction of the positive hump of the I/V curve (at −55 to −60 mV) is noted, an increase in the amplitude of negative current (at −80 mV) and value of depolarization (change of the zero current potential V ₀, ie intercept of I/V curve with current axis). After 15 min, the positive hump of the I/V curve approaches the initial value, the membrane hyperpolarizes, and the emerging negative current (at −80 mV) was inhibited. (d) At a concentration of 400 µmol/L in the first 5 min, the negative current (at −80 mV) was inhibited and a shift of E ₀ to the negative region was observed. Legend: (a) control: circles, perfusion of SNAP 5 min: triangles, 10 min: squares, 15 min: rhombus. (b), (c), (d) control: circles, perfusion of SNAP 5 min: triangles, 15 min: squares

FIGURE 5

Gadolinium reduces SNAP‐induced net currents. SNAP at the concentration of 200 µmol/L. K⁺ currents were not suppressed in ${\mathrm{K}}_{\text{in}}^{+}$/${\mathrm{K}}_{\text{out}}^{+}$ environment. (a) Online records (time course) of membrane current, V _m clamped to a holding potential of −45 mV. Gd³⁺ (5 µmol/L) in the presence of 200 µmol/L SNAP reduces SNAP‐induced net currents and maximum peak current (ΔI _max). (b) Gd³⁺ blocks I _SNAP. Legend ‐ control: circles, perfusion of SNAP 5 min: triangles, perfusion of SNAP 10 min, and Gd³⁺ 5 min: squares. (c) I _SNAP does not develop on the background of the preliminary administration of Gd³⁺. Legend ‐ control: Circles, perfusion of Gd³⁺ 5 min: triangles, perfusion of Gd³⁺ 10 min and SNAP 5 min: squares

FIGURE 6

Gadolinium reduces SNAP‐induced current (I _ns). SNAP at the concentration of 200 µmol/L. K⁺ currents suppressed. ${\text{Cs}}_{\text{in}}^{+}$/${\text{Cs}}_{\text{out}}^{+}$ environment. (a) SNAP changes voltage dependence I _L. In the first 5 min, there was an increase in the value of negative current (I _ns) at −45 and −80 mV (triangles vs circles in control) and value of depolarization (change of the zero current potential V ₀, ie intercept of I/V curve with current axis). After 25 min, the I _ns value decreases (squares vs circles in control). (b) I _SNAP does not develop on the background of preliminary administration of 5 µmol/L Gd³⁺. Legend ‐ control: circles, perfusion of Gd³⁺ 5 min: triangles, perfusion of Gd³⁺ 10 min and SNAP 5 min: squares. (c) Online records (time course) of membrane current, V _m clamped to a holding potential of −45 mV. Gd³⁺ (5 µmol/L) in the presence of 200 µmol/L SNAP reduces SNAP‐induced net currents and maximum peak current (ΔI _max)

FIGURE 7

SNAP at the concentration of 200 µmol/L abolishes stretch‐induced net inwards currents in ${\mathrm{K}}_{\text{in}}^{+}$/${\mathrm{K}}_{\text{out}}^{+}$ environment: Time‐course and voltage dependence. V _m clamped to a holding potential of −45 mV. (a) Online records (time course) of membrane current. Stretch was applied at the level of ΔI _max during perfusion with SNAP. Value of stretch (8 µm) and amount of stretch‐induced inward current at −45 mV, indicated. (b) The same as in A, but the stretch was applied at ΔI _s‐s. (c) Voltage dependence of I _L in control (circles), after 10 min perfusion of SNAP (triangles), after 10 min perfusion of SNAP on the background of stretch by 6 µm (squares), after another 5 min of continued stretching (rhombus). (d) Time course of membrane current. SNAP was applied after stretching the cell by 6 µm. (e) Voltage dependence of I _L in control (circles), I _L after stretching by 8 µm (triangles), after 5 min of a continued stretch at the level of perfusion of SNAP (squares)

FIGURE 8

SNAP at the concentration of 200 µmol/L abolishes stretch induces I _SAC in ${\text{Cs}}_{\text{in}}^{+}$/${\text{Cs}}_{\text{out}}^{+}$ environment. K⁺ currents suppressed. (a) Online record of membrane current, V _m clamped to a holding potential of −45 mV. Stretch was applied at the level near ΔI _max during perfusion with SNAP. The value of stretch (8 µm) and the amount of stretch‐induced inward current at −45 mV are indicated. (b) Time course of membrane current. SNAP was applied after stretching the cell by 6 µm

FIGURE 9

BAY41‐2272 changes the time course and voltage dependence of I _L in a ${\mathrm{K}}_{\text{in}}^{+}$/${\mathrm{K}}_{\text{out}}^{+}$ environment. (a) Online records (time course) of membrane current, V _m clamped to a holding potential of −45 mV. BAY41‐2272 (5 µmol/L) shifted the net currents to more negative values (Curve ‐ a). The additional introduction of SNAP (200 µmol/l) does not lead to a further increase in the net current, but on the contrary, will decrease it with the dynamics characteristic of pure SNAP. (b) At a concentration BAY41‐2272 of 5 µmol/L in the first 5 min, a reduction of the positive hump of the I/V curve (at −55 to −60 mV) is noted, a decrease in the amplitude of negative current (at −90 mV) and an increase V₀ (change of the zero current potential V ₀, i.e., intercept of I/V curve with current axis). Legend ‐ control: circles, perfusion of BAY41‐2272 5 min: triangles, perfusion of BAY41‐2272 10 min: squares, perfusion of BAY41‐2272 15 min: rhombuses. (c) At a concentration BAY41‐2272 of 10 µmol/L in the first 3 min, a reduction of the positive hump of the I/V curve is noted, with a decrease in the amplitude of negative current (at −80 and −90 mV) and an increase V₀ . After 6 min, there are no fundamental changes compared to 3 min. Legend ‐ control: circles, perfusion of BAY41‐2272 3 min: triangles, perfusion of BAY41‐2272 6 min: squares. (d) Changes in I _L circles in control, 6 min after application of 10 µmol/L of BAY41‐2272 (triangles), after 3 (squares), 6 (inverted triangles), and 9 (rhombuses) min after additional application 200 µmol/L of SNAP

FIGURE 10

Changes in I _SAC in a stretched cell under the action of BAY41‐2272. (a) demonstrates the appearance of the I _SAC(−45) with a value of −0.150 nA (−0.195 ± 0.009 nA in control, n = 36) when the cell is stretched by 6 µm (3 min) in ${\mathrm{K}}_{\text{in}}^{+}$/${\mathrm{K}}_{\text{out}}^{+}$ medium with 5 µmol/L of BAY41‐2272. (b) shows the I _SAC(−45) with a value of −0.065 nA (−0.082 ± 0.011 nA in control, n = 5) generated by a stretch of 6 µm (1.5 min) in ${\text{Cs}}_{\text{in}}^{+}$/${\text{Cs}}_{\text{out}}^{+}$ medium with 5 µmol/L of BAY41‐2272. (c) Voltage dependence of I _L in control (circles), after cell stretch by 6 µm (triangles), after 3 (squares), 6 (inverted triangles), and 9 (rhombuses) min of perfusion of BAY41‐2272 (10 µmol/L). (d) Voltage dependence of I _L in control (circles), after cell stretch by 6 µm (triangles), after 6 (squares) min of perfusion of BAY41‐2272 (10 µmol/L), and after 3 (inverted triangles) and 6 (rhombuses) min of perfusion after additional application of SNAP (200 µmol/L)

FIGURE 11

Effect of ODQ (5 μmol/L) and its combination with SNAP (200 μmol/L) on the I/V curve of late current (I _L) and stretch‐activated cation nonselective current (I _L,ns). (a) Changes in I _L in an intact cell against the background of constant ODQ perfusion in the control (circles), after 3 min (triangles), and after 6 min (squares). (b) changes in I _L under the action of ODQ followed by the addition of SNAP to the solution. Circles ‐ control, triangles −6 min of cell perfusion with ODQ, squares ‐ 3 min of perfusion after addition to the SNAP solution, inverted triangles ‐ 6 min with SNAP. (c), I _SAC changes in the stretched control cell (circles), after stretching by 6 µm (triangles), after 3 min (squares), and after 6 min (inverted triangles) of constant ODQ perfusion on the background of stretching. (d) Changes in I _SAC in the stretched control cell (circles), after stretching by 6 µm (triangles), after 6 min of continuous ODQ perfusion (squares), and after 6 min of perfusion after adding to the SNAP solution on the background of continuing stretch (inverted triangles)

FIGURE 12

Effect of KT5823 (1 μmol/L) and its combination with SNAP (200 μmol/L) on late current (I _L) I/V curve and stretch‐activated cation nonselective current (I _L,ns). (a) Changes in I _L in an intact cell against the background of constant perfusion of KT5823 in the control (circles), after 3 min (triangles), and after 6 min (squares). (b) changes in I _L under the action of KT5823 with the subsequent addition of SNAP to the solution. Circles ‐ control, triangles ‐ 6 min perfusion of cells with KT5823, squares ‐ 3 min perfusion after addition to SNAP solution, inverted triangles ‐ 6 min with SNAP. (c) I _SAC changes in the stretched control cell (circles), after stretching by 6 µm (triangles), after 3 min (squares), and after 6 min (inverted triangles) of constant perfusion of KT5823 on the background of stretching. (d) I _SAC changes in the stretched control cell (circles), after stretching by 6 µm (triangles), after 6 min of constant perfusion of KT5823 (squares) and after 3 min (inverted triangles) and 6 min (rhombuses) of perfusion after addition to the solution SNAP against the backdrop of ongoing stretching

FIGURE 13

Effect of 8Br‐cGMP (200 μmol/L) and its combination with SNAP (200 μmol/L) on late current (I _L) I/V curve and stretch‐activated cation nonselective current (I _L,ns). (a) Changes in I _L in an intact cell against the background of its constant perfusion with 8Br‐cGMP in the control (circles), after 3 min (triangles), and after 6 min (squares). (b) changes in I _L under the action of 8Br‐cGMP with the subsequent addition of SNAP to the solution. Circles ‐ control, triangles ‐ 6 min cell perfusion with 8Br‐cGMP, squares ‐ 3 min perfusion after addition to SNAP solution, inverted triangles ‐ 6 min with SNAP. (c) I _SAC changes in the stretched control cell (circles), after stretching by 6 µm (triangles), after 3 min (squares), and after 6 min (inverted triangles) of continuous 8Br‐cGMP perfusion on the background of stretching. (d) I _SAC changes in the stretched control cell (circles), after stretching by 6 µm (triangles), after 6 min of constant perfusion with 8Br‐cGMP (squares), and after 3 min (inverted triangles) and 6 min (rhombuses) of perfusion after addition into the SNAP solution against the background of ongoing stretching

FIGURE 14

Effect of ascorbic acid (10 μmol/L) and its combination with SNAP (200 μmol/L) on I _L. (a) changes in I _L in an intact cell on the background of constant perfusion of ascorbic acid in the control (circles), after 3 min (triangles), and after 6 min (squares). (b) changes in I _L under the action of ascorbic acid with subsequent addition to the SNAP solution. Circles ‐ control, triangles ‐ 6 min of cell perfusion with ascorbic acid, squares ‐ 3 min of perfusion after addition to SNAP solution, inverted triangles ‐ 6 min with SNAP. (c) Changes in I _SAC in the stretched control cell (circles), after stretching by 6 µm (triangles), after 3 min (squares), and after 6 min (inverted triangles) of constant perfusion of ascorbic acid on the background of stretching. (d) Changes in I _SAC in the stretched control cell (circles), after stretching by 6 µm (triangles), after 6 min of constant perfusion of ascorbic acid (squares), and after 6 min (rhombuses) of perfusion after adding to the SNAP solution on the background of continuous stretch

FIGURE 15

Effect of L‐arginine and its combination with SNAP on the I/V curve of I _L. (a) Changes in I _L in an intact cell on the background of its constant perfusion of L‐Arginine at a concentration of 50 μmol/L in the control (circles), after 3 min (triangles), and after 6 min (squares). (b) Changes in I _L on the background of perfusion with L‐arginine at a concentration of 100 μmol/L in the control (circles), after 3 min (triangles), and after 6 min (squares). (c) changes in I _L under the action of L‐arginine followed by the addition of 200 μmol/L SNAP to the solution. Circles ‐ control, triangles ‐ 6 min cell perfusion with L‐arginine, squares ‐ 3 min perfusion after addition to SNAP solution, inverted triangles ‐ 6 min with SNAP