Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes

Abstract

1. Ionic currents and unloaded cell shortening were recorded from guinea-pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 C. Patch pipettes (1-3 MOmega) were used to provide intracellular dialysis with pipette solutions. 2. Na+ currents were blocked with 200 microM lidocaine. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and Ca2+-induced Ca2+ release (CICR) in response to L-type Ca2+ current (ICa,L) were separated with voltage clamp protocols. 3. Without 8-bromo cyclic adenosine 3',5'-monophosphate (8-Br-cAMP) in the pipette, small VSRM-induced contractions occurred transiently in only 13% of myocytes. In contrast, large ICa,L-induced contractions were demonstrable in 100% of cells. 4. Addition of 10 or 50 microM 8-Br-cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93%, respectively. With 50 microM 8-Br-cAMP, contractions initiated by the VSRM and ICa,L were not significantly different in amplitude. 5. 8-Br-cAMP-supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd2+ (100 microM) blocked ICa,L and ICa,L contractions but not VSRM contractions. 8-Br-cAMP-supported contractions exhibited steady-state inactivation with parameters characteristic of the VSRM, as well as sigmoidal contraction-voltage relations. 6. Without 8-Br-cAMP in the pipette, contraction-voltage relations determined with steps from a post-conditioning potential (Vpc) of either -40 or -65 mV were bell shaped, with a threshold near -35 mV. With 50 microM 8-Br-cAMP in the pipette, contraction-voltage relations from a Vpc of -65 mV were sigmoidal and the threshold shifted to near -55 mV. Contraction-voltage relations remained bell shaped in the presence of 8-Br-cAMP when the Vpc was -40 mV. 7. H-89, which inhibits cAMP-dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84% with 50 microM 8-Br-cAMP in the pipette. H-89 also significantly reduced the amplitudes of peak ICa, L and ICa,L contractions, although to a lesser extent. 8. We conclude that intracellular dialysis with patch pipettes disrupts the adenylyl cyclase-PKA phosphorylation cascade, and that the VSRM requires intracellular phosphorylation to be available for activation. Intracellular dialysis with solutions that do not maintain phosphorylation levels inhibits a major mechanism in cardiac excitation- contraction coupling.

Figure 1. Effects of 8-Br-cAMP added to the pipette solution on currents and contractions elicited by sequential voltage clamp steps to −40 and 0 mV

The voltage clamp protocol is shown at the top. Ten conditioning steps to 0 mV were followed by a post-conditioning potential (V_pc) of −65 mV, and then sequential 250 ms long test steps to −40 and 0 mV. The holding potential was −80 mV. Each panel shows representative recordings of transmembrane currents (top) and cell shortening (bottom). Each panel was recorded from a different myocyte. A, when the pipette solution contained 0 μm 8-Br-cAMP (8-b-cAMP), the test step to −40 mV elicited only a very small inward deflection in the current record and no contraction. The step to 0 mV elicited a larger inward current and an I_Ca,L contraction. B, when the pipette solution contained 10 μm 8-Br-cAMP, the step to −40 mV still elicited only a small inward current, but now elicited a phasic VSRM contraction. The step to 0 mV activated a larger I_Ca,L than in A, as well as a phasic I_Ca,L contraction. C, the inward current initiated by the step to −40 mV remained small when 50 μm 8-Br-cAMP was included in the pipette solution. However, I_Ca,L elicited by the step to 0 mV became very large. In the presence of 50 μm 8-Br-cAMP, both steps elicited large rapid phasic contractions. The current scale for B is the same as in A. The contraction scale is the same in all 3 panels.

Figure 2. Effects of different concentrations of 8-Br-cAMP on the incidence of VSRM and I_Ca,L contractions

VSRM contractions were elicited by steps to −40 mV and I_Ca,L contractions by steps to 0 mV with the same voltage clamp protocol as that shown in Fig. 1. Conditioning pulses were to 0 mV. When the concentration of 8-Br-cAMP in the pipette was 0 μm 8-Br-cAMP (n = 30), only 13 % of myocytes exhibited a VSRM contraction. When 10 or 50 μm 8-Br-cAMP was added to the pipette solution (n = 31 and 76), VSRM contractions were observed in 68 and 93 % of myocytes, respectively. The increases in incidence with both 10 and 50 μm 8-Br-cAMP were statistically significant (**P < 0.01). The incidence of I_Ca,L contractions was 100 % for all myocytes and thus was not affected by the concentration of 8-Br-cAMP. *P < 0.05.

Figure 3. Effects of the concentration of 8-Br-cAMP in the pipette on the mean magnitudes of contractions and currents elicited by test steps to −40 and 0 mV

The voltage clamp protocol was the same as in Fig. 1. A, effects of 8-Br-cAMP on amplitudes of VSRM and I_Ca,L contractions. The amplitude of VSRM contractions (step to −40 mV) was very small in the absence of 8-Br-cAMP in the pipette (n = 30 cells), but increased significantly with 10 and 50 μm 8-Br-cAMP (n = 31 and 76 cells, respectively). The amplitudes of I_Ca,L contractions were larger than VSRM contractions in the absence of 8-Br-cAMP (P < 0.01) and increased less with addition of 8-Br-cAMP. In the presence of 50 μm 8-Br-cAMP, the amplitudes of VSRM and I_Ca,L contractions were no longer significantly different. B, effects of 8-Br-cAMP on peak inward currents. Peak inward currents elicited by steps to −40 mV were very small and were unaffected by the concentration of 8-Br-cAMP in the patch pipette. Mean peak inward current elicited by the test step to 0 mV (I_Ca,L) was much larger than the inward current elicited by the step to −40 mV, and was increased significantly by 10 and 50 μm 8-Br-cAMP. ns, not significant; *P < 0.05; **P < 0.01.

Figure 4. Effects of 8-Br-cAMP on the time course of cell shortening and relaxation during VSRM and I_Ca,L contractions

A, representative recordings in the presence of 50 μm 8-Br-cAMP showing measurements of time to peak and time to half-relaxation for an I_Ca,L contraction. Time to peak was measured from the beginning of the activation step to peak shortening as indicated. The amplitude of contraction was measured from a point immediately before onset of shortening to peak shortening. Time to half-relaxation was measured from peak shortening to the time at which the myocyte had relaxed to half of peak shortening, as indicated. B, addition of 50 μm 8-Br-cAMP to the pipette solution significantly shortened the time to peak for both VSRM and I_Ca,L contractions. There was no significant difference between the times to peak for VSRM and I_Ca,L contractions in the absence of 8-Br-cAMP, or in the presence of 8-Br-cAMP. C, addition of 50 μm 8-Br-cAMP to the pipette solution also significantly decreased the times to half-relaxation for both VSRM and I_Ca,L contractions. **P < 0.01 with respect to 0 μm 8-Br-cAMP; n = 4–28 cells for VSRM and I_Ca,L contractions without 8-Br-cAMP, and 56–66 cells with 8-Br-cAMP. The number of replicates for VSRM contractions in the absence of 8-Br-cAMP was low because few myocytes exhibited VSRM contractions in the absence of 8-Br-cAMP.

Figure 5. Contractions supported by cAMP are unaffected by blockade of I_Ca,L by Cd²⁺

A schematic protocol of the activation steps is shown at the top of the figure. A, when 50 μm 8-Br-cAMP was included in the pipette, a step to −40 mV elicited a VSRM contraction, and a second step to 0 mV elicited I_Ca,L and an I_Ca,L contraction. B, rapid application of 100 μm Cd²⁺ 3 s in advance of the activation steps strongly inhibited I_Ca,L and the I_Ca,L-induced contraction, but had no effect on the VSRM contraction triggered by the step to −40 mV. Both A and B were recorded in the presence of 200 μm lidocaine plus 50 μm TTX to inhibit sodium current. Cd²⁺ exerted similar differential effects on VSRM and I_Ca,L contractions in 12 of 12 cells.

Figure 6. Selective blockade of I_Ca,L contractions by Cd²⁺ is independent of activation sequence

Voltage clamp protocols used to activate VSRM (left) and I_Ca,L contractions (right) individually are shown at the top. A shows recordings of VSRM contraction and current in response to a voltage step from −70 to −40 mV in the absence of Cd²⁺. B shows that a rapid switch to 100 μm Cd²⁺ 3 s before the activation step had little effect on the VSRM contraction. C shows recordings of I_Ca,L contraction and current in response to a voltage step from −40 to 0 mV in the absence of Cd²⁺. D, application of 100 μm Cd²⁺ blocked I_Ca,L and strongly inhibited I_Ca,L contraction. Similar observations were made in 6 of 6 cells.

Figure 7. Contractions supported by 8-Br-cAMP exhibit steady-state inactivation characteristic of the VSRM

A shows a schematic protocol of the voltage clamp used for steady-state inactivation plus representative recordings of VSRM contractions elicited by a test step to −40 mV. The test step was preceded by a 3 s long V_pc to different potentials. When the V_pc was −40 mV, the VSRM was inactivated and contraction was absent. With progressively more negative V_pc, VRSM contractions of increasing amplitude were observed. B shows mean data for 7 experiments. Data points were fitted to a Boltzmann function as described in the text. Mean values for V_h and k were −54.1 ± 1.2 and 3.8 ± 0.3 mV, respectively. Experiments were conducted in the presence of 200 μm lidocaine plus 50 μm TTX.

Figure 8. Effects of changing V_pc on contraction-voltage and I-V relations in the absence of 8-Br-cAMP in the patch pipette solution

A, representative recordings of currents and contractions elicited by test steps from a V_pc of −40 mV. A test step to 0 mV from a V_pc of −40 mV elicited a large inward current and a phasic contraction. When the test step was increased to +80 mV, both inward current and contraction were minimal. B, representative recordings of currents and contractions elicited by test steps from a V_pc of −65 mV. A test step from −65 to −45 mV elicited no measurable inward current or contraction. A test step from −65 to −5 mV elicited a large inward current and a large phasic contraction. As in A, when the test step was very positive (+85 mV) both inward current and contraction were again minimal. C, mean contraction-voltage relations determined from a V_pc of −40 or −65 mV were bell shaped and similar in amplitude in the absence of 8-Br-cAMP. With both V_pc values, contractions first appeared with test steps to between −40 and −30 mV. Contractions reached a maximum amplitude near 0 mV and declined markedly as test steps became very positive. D, current-voltage relations determined with test steps from a V_pc of −40 or −65 mV. When test steps were made from a V_pc of −40 mV, inward current was first observed near −30 mV, increased to a maximum near 0 mV, and declined with steps to more positive voltages. When the V_pc was changed to −65 mV, inward current first appeared closer to −40 mV but the I-V relation remained bell shaped with a maximum near 0 mV.

Figure 9. Effects of changing V_pc on contraction-voltage and I-V relations with 50 μm 8-Br-cAMP in the patch pipette solution

A, representative recordings of membrane currents and contractions when the V_pc was −40 mV. A test step from −40 to 0 mV activated a large inward current which was accompanied by a large phasic contraction. When the test step was to +80 mV, inward current was abolished and only a very small contraction was observed. B, representative recordings of membrane currents and contractions when the V_pc was −65 mV. A test step from −65 to −45 mV elicited very little inward current but a substantial phasic contraction. When the test step was to −5 mV, a large inward current and large phasic contraction were elicited. When the test step was changed to +85 mV, inward current was abolished but a maximal phasic contraction still occurred. C, effect of V_pc on mean contraction-voltage relationships in the presence of 50 μm 8-Br-cAMP. When the V_pc was −40 mV, contractions first appeared with test steps to −30 mV. The contraction-voltage relationship was bell shaped, and contractions became minimal with voltage steps near +80 mV. When the V_pc was changed to −65 mV, the contraction-voltage relation became sigmoidal rather than bell shaped. Contractions first appeared near −55 mV, became maximal near −20 mV and remained near maximal even at very positive test steps. D, effects of changing V_pc on I–V relations in the presence of 50 μm 8-Br-cAMP. I-V relations were bell shaped for both V_pc values tested.

Figure 10. Direct comparison of contraction-voltage relations in the absence and presence of 50 μm 8-Br-cAMP in the pipette

Data are from experiments illustrated in Figs 8 and 9. A, 8-Br-cAMP had very little effect on the bell-shaped contraction-voltage relations determined with test steps from a V_pc of −40 mV. B, contraction- voltage relations determined with test steps from a V_pc of −65 mV were bell shaped in the absence of 8-Br-cAMP in the pipette and sigmoidal in the presence of 50 μm 8-Br-cAMP. Contractions were significantly larger in amplitude in the presence of 50 μm 8-Br-cAMP compared with 0 μm 8-Br-cAMP, for test steps positive to −50 mV (**P < 0.01). In addition, there was a shift of approximately −20 mV in the threshold voltage for initiation of contraction in the presence of 8-Br-cAMP.

Figure 11. Recordings of currents and contractions with 50 μm 8-Br-cAMP in the pipette, from the same myocyte before and after exposure to H-89, an inhibitor of PKA

A, currents and contractions were elicited by the same voltage clamp protocol as shown in Fig. 1. The voltage step to −40 mV activated a very small inward current and a large phasic contraction. The step to 0 mV activated a large inward current and a second large phasic contraction. B, current and contractions elicited by the same voltage clamp protocol, but after superfusion with 5 μm H-89 for 10 min. The small inward current observed with the step to −40 mV was unaffected, but the phasic contraction was abolished. Both the inward current and the phasic contraction elicited by the step to 0 mV were reduced in amplitude but not abolished.

Figure 12. Mean data illustrating the effects of H-89 on the amplitudes of contractions and currents elicited by sequential steps to −40 and 0 mV with 50 μm 8-Br-cAMP in the pipette solution

A, effects of H-89 on amplitudes of VSRM and I_Ca,L contractions. H-89 markedly and significantly decreased the amplitudes of VSRM contractions. The amplitudes of I_Ca,L contractions were reduced less, but this effect was also significant. B, effects of H-89 on amplitudes of peak inward current. H-89 had no effect on the small inward current activated by the step to −40 mV, but significantly reduced I_Ca,L elicited by the step to 0 mV by approximately 46 % (n = 4). *P < 0.05 and **P < 0.01.

Figure 13. Addition of 8-Br-cAMP to the patch pipette does not increase gain of CICR

Gain of CICR is shown by the relationship between the magnitude of contraction and the magnitude of current. The same relationship between contraction and current was observed with steps from V_pc values of −40 (□) or −65 mV (○) in the absence of 8-Br-cAMP, and from a V_pc of −40 mV (▵) with 50 μm 8-Br-cAMP in the pipette. A single curve was fitted through the data for all 3 conditions (second-order regression) to illustrate the similarity of the relationships. When the V_pc was −65 mV and 8-Br-cAMP was included in the pipette, the VSRM was available and contraction showed no simple relationship to the magnitude of current (•). Data are from the experiments shown in Figs 8 and 9.