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

Figures

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  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)

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  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

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  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. Kin+/Kout+ environment. (b) In Csin+/Csout+ 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 Kin+/Kout+ medium at of the same NO donor concentration

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  FIGURE 4. SNAP changes the voltage dependence of I L in a Kin+/Kout+ 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

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  FIGURE 5. Gadolinium reduces SNAP‐induced net currents. SNAP at the concentration of 200 µmol/L. K+ currents were not suppressed in Kin+/Kout+ 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

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  FIGURE 6. Gadolinium reduces SNAP‐induced current (I ns). SNAP at the concentration of 200 µmol/L. K+ currents suppressed. Csin+/Csout+ 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)

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  FIGURE 7. SNAP at the concentration of 200 µmol/L abolishes stretch‐induced net inwards currents in Kin+/Kout+ 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)

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  FIGURE 8. SNAP at the concentration of 200 µmol/L abolishes stretch induces I SAC in Csin+/Csout+ 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

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  FIGURE 9. BAY41‐2272 changes the time course and voltage dependence of I L in a Kin+/Kout+ 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…

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  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 Kin+/Kout+ 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 Csin+/Csout+ 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)

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  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)

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  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