L-Type Ca2+ Currents Overlapping Threshold Na+ Currents
Could They Be Responsible for the “Slip-Mode” Phenomenon in Cardiac Myocytes?
Phosphorylation of Na channels has been suggested to increase their Ca permeability. Termed “slip-mode conductance” (SMC), this hypothesis predicts that Ca influx via protein kinase A (PKA)-modified Na channels can induce sarcoplasmic reticulum (SR) Ca release. We tested this hypothesis by determining if SR Ca release is graded with INa in the presence of activated PKA (with Isoproterenol, ISO). Vm, Im, and [Ca]i were measured in feline (n=26) and failing human (n=19) ventricular myocytes. Voltage steps from −70 through −40 mV were used to grade INa. Na channel antagonists (tetrodotoxin), L-type Ca channel (ICa,L) antagonists (nifedipine, cadmium, verapamil), and agonists (Bay K 8644, FPL 64176) were used to separate SMC from ICa,L. In the absence of ISO, INa was associated with SR Ca release in human but not feline myocytes. After ISO, graded INa was associated with small amounts of SR Ca release in feline myocytes and the magnitude of release increased in human myocytes. INa-related SR Ca release was insensitive to tetrodotoxin (n=10) but was blocked by nifedipine (n=10) and cadmium (n=3). SR Ca release was induced over the same voltage range in the absence of ISO with Bay K 8644 and FPL 64176 (n=9). Positive voltage steps (to 0 mV) to fully activate Na channels (SMC) in the presence of ISO and Verapamil only caused SR Ca release when block of ICa,L was incomplete. We conclude that PKA-mediated increases in ICa,L and SR Ca loading can reproduce many of the experimental features of SMC.
A small amount of Ca influx during the beginning of the cardiac action potential induces (“triggers”) the release of larger amounts of Ca from the sarcoplasmic reticulum (SR) of ventricular myocytes.1 This trigger Ca is thought to elevate the subsarcolemmal [Ca] in small, diffusion-limiting spaces between the T-tubular membrane and the junctional face of the SR,2 which promotes Ca binding to and opening of the Ca release channel (ryanodine receptor).
The respective triggering roles of Ca entry through L- and T-type Ca channels and via reverse-mode Na-Ca exchange (NCX) have been well characterized.1,3⇓ Of these pathways the primary trigger of SR Ca release is Ca influx through the L-type Ca channel,4,5,6⇓⇓ with Ca influx through T-type Ca channels and reverse-mode NCX being either weak or ineffective.7,8⇓ Recently, Ca influx through Na channels, termed “slip-mode conductance” (SMC),9 has been proposed as a significant trigger of SR Ca release.
The SMC hypothesis is that the selectivity of Na channels (which normally allow little or no Ca permeation) for Ca is increased by phosphorylation (via activation of protein kinase A, PKA) and cardiac glycosides and that this Ca influx is a potent trigger for SR Ca release.9 Since the original report,9 follow up studies using either heterologously expressed Na channels10,11⇓ or cardiac myocytes12 have not consistently supported this hypothesis. The purpose of the present study was to further test the idea that Ca influx via SMC can induce SR Ca release.
One of the fundamental predictions of the SMC hypothesis is that SR Ca release should be graded with the size of the slip-mode Ca current via the Ca-induced Ca-release (CICR) mechanism. In the present study, we determined if SR Ca release could be graded by Ca entry through the Na channel in normal feline and failing human ventricular myocytes. Failing human myocytes were studied because activation of SMC has been proposed as a novel target for inotropic therapy in human heart failure.13 Our results show that there is SR Ca release associated with the Na current in both feline and human myocytes. However, this SR Ca release was blocked and accentuated by L-type Ca channel antagonists and agonists, respectively, but was not blocked by Na channel antagonists. This raises the possibility that SMC is really L-type Ca current rather than Ca entry through Na channels. If this is true, then SMC is not a new inotropic target for human heart failure.
Materials and Methods
Myocyte isolation, voltage clamp, and statistical techniques have been described in detail in previous publications.14,15⇓ Myocytes were isolated from adult cats (n=26 myocytes, 7 hearts) and failing human hearts (n=19 myocytes, 8 hearts). Adult cats (Liberty Research, Waverly, NY) were cared for and used according to the 1996 ILAR Guide for the Care and Use of Laboratory Animals, and failing human hearts were procured at the time of cardiac transplantation. [Ca]i was measured with Fluo-3K5 (100 μmol/L) via pipette loading. The fluorescence changes during the Ca transient (F) were normalized by the resting fluorescence (Fo) and expressed as F/Fo.
Myocytes were held at −70 mV, and 5 conditioning steps to +10 or +80 mV were used to maintain SR Ca loading. Test voltage steps began from −70 mV to −60 mV and were then incremented by 0.5 or 1 mV to grade the Na currents at their activation threshold. This allowed for study of the SMC hypothesis without substantial concerns related to spurious activation of the L-type Ca current.12 Large (>25 pA/pF) Na currents that caused substantial loss of voltage control (>5 mV) and were associated with SR Ca release were not used. After control measurements, cells were exposed to isoproterenol (ISO, 5×10−8 mol/L feline, 1×10−6 mol/L human) to activate the putative slip-mode conductance mechanism. Some cells were then exposed to Na channel (tetrodotoxin [TTX], 10×10−6 mol/L) or L-type Ca channel (nifedipine [NIF], 25 to 50×10−6 mol/L, or verapamil [VEP], 20×10−6 mol/L) antagonists or to L-type Ca channel agonists (Bay K 8644 [0.5 to 1×10−6 mol/L] and FPL 64176 [0.5×10−6 mol/L]).
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Effects of ISO on Na Current and SR Ca Release
Voltage steps from −70 mV to potentials between −60 to −45 mV were used to progressively increase the activation of the Na conductance and grade the size of Na current (INa) in both feline (n=15) and human (n=10) myocytes. In feline myocytes (Figure 1A) under control conditions, there was no detectable SR Ca release associated with INa unless the current was of sufficient size to induce loss of voltage control (LOVC). In human myocytes under control conditions, there were small amounts of SR Ca release associated with INa between −60 and −45 mV (Figure 1B).
ISO (5×10−8 mol/L feline, 1×10−6 mol/L human) caused a leftward shift in the voltage-dependence of INa activation in both feline and human myocytes (Figures 1C and 1E), consistent with the original SMC report9 and other studies.16,17⇓ In feline myocytes, ISO caused small Ca transients to be seen over the same general voltage range as INa activation (Figures 1A and 1D). These Ca transients were abolished by thapsigargin (data not shown): evidence that they represent SR Ca release. In human myocytes, ISO caused Ca transients induced between −60 and −45 mV to increase in size (Figures 1B and 1F). Although the Ca transients associated with INa in these control experiments are smaller than those in the original SMC report,9 they are nonetheless consistent with the SMC hypothesis. Importantly, all data were obtained with adequate voltage control to prevent uncontrolled depolarization (due to the activation of INa) and the associated spurious activation of the L-type Ca channel.12
Effects of Tetrodotoxin on Na Current and SR Ca Release
TTX (that blocks INa, SMC and the associated SR Ca release9) was tested in feline (n=6) and human (n=4) myocytes. In every myocyte, TTX (10×10−6 mol/L, in the presence of ISO) eliminated most of the INa activated with steps between −60 and −45 mV, but SR Ca release was not significantly reduced (Figure 2). These results suggest that the SR Ca release observed with voltage steps between −60 and −45 mV does not involve Ca influx through TTX-sensitive Na channels. Similar results were obtained when a holding potential of −90 mV was used (data not shown).
Effects of Nifedipine on Na Current and SR Ca Release
The effects of the L-type Ca channel antagonist NIF (25 to 50×10−6 mol/L) on the SR Ca release induced by voltage steps between −60 and −45 mV were examined in feline (n=7) and human (n=3) myocytes. In every myocyte studied, NIF (in the presence of ISO) reduced or abolished this SR Ca release and also caused a small reduction in INa (Figure 3). The effect of NIF on INa is consistent with previous observations.18 Note that after NIF at −50 mV (Figure 3), the peak INa still exceeded that at −51.5 mV before NIF, but there was no Ca transient.
The SR Ca load was maintained in NIF with conditioning pulses to +80 mV, to cause Ca influx via reverse mode NCX,14 as evidenced by similar sized Ca transients during conditioning steps under all conditions. In the presence of NIF these Ca transients were presumably triggered by Ca influx through unblocked Ca channels. This is a likely possibility because the efficacy of Ca channel blockade by NIF is significantly decreased at the negative holding potentials used.1 These results strongly support the idea that Ca influx through the L-type Ca channel is an alternative explanation to SMC for the SR Ca release observed between −60 and −45 mV. Similar results were obtained when cadmium was used to block the L-type Ca current (data not shown).
Effects of Bay K 8644 on SR Ca Release at Negative Potentials
To explore the idea that activation of a small number of the L-type Ca channels at negative potentials (−60 to −45 mV) can induce SR Ca release, feline (n=3) and human (n=3) myocytes were exposed to the Ca channel agonist Bay K 8644 (BayK, 0.5 to 1×10−6 mol/L, Figure 4). 20 In every myocyte, BayK (in the absence of ISO) caused the appearance of SR Ca release with voltage steps to potentials between −60 and −45 mV. BayK did not significantly affect INa. TTX (in the presence of BayK) blocked INa but not SR Ca release. Identical results were obtained with the Ca channel agonist FPL 64176 (FPL, 0.5×10−6 mol/L, n=3, data not shown).20 These results support the idea that a small number of L-type Ca channels are activated during well-controlled voltage steps between −60 and −45 mV and that the resulting Ca influx can induce SR Ca release.
Effects of Verapamil on SR Ca Release in the Presence of ISO at Positive Membrane Potentials
Voltage steps from −90 to 0 mV were used to fully activate the Na conductance, and ISO was employed to activate SMC. Na (10 mmol/L) was used in the bath and pipette so that INa would be small or absent at 0 mV (the reversal potential for INa) so that completeness of ICa,L block could be more clearly observed. Five conditioning steps from −40 (to inactivate INa) to +10 mV were used before each test step to promote use-dependent VEP block of ICa,L.21 Cells in which test steps (from −90 to 0 mV) induced SR Ca release in the presence of VEP and ISO were then exposed to TTX. It is noteworthy that these low-bath Na conditions caused very large SR Ca loads as evidenced by the occasional spontaneous SR Ca release observed in every cell.
Eight feline myocytes were studied. In 4 myocytes, there was no SR Ca release during either the conditioning or test steps in the presence of ISO and VEP (data not shown). In the other 4 myocytes, SR Ca release was observed during both the conditioning and test steps (Figure 5). The Ca release during conditioning steps (when Na channels are inactivated and SMC is not present) shows that ICa,L was not fully blocked by VEP. When these myocytes were subsequently exposed to TTX, SR Ca release during both the conditioning (Na channels inactivated) and test steps (Na channels activated) was abolished. These effects are not explained by TTX block of SMC (see Discussion).
The objective of this study was to test the SMC hypothesis that Ca influx through PKA-modified Na channels can induce SR Ca release. Because Ca release from the SR can be graded by the size of the trigger Ca,1 we examined the relationship between the size of the slip-mode current and the associated SR Ca release. The Na current (INa) was graded to 25 pA/pF with no loss of voltage control by making small voltage steps (0.5 or 1 mV) in the activating voltage range from −60 to −45 mV. The major findings of the study are as follows: (1) in the absence of ISO (PKA activator), there was no detectable SR Ca release associated with INa in feline myocytes, whereas in failing human myocytes small Ca transients were observed; (2) ISO caused a leftward shift in the voltage-dependence of INa activation, and Ca transients were seen concomitant with INa in both feline and human myocytes; (3) TTX (Na channel antagonist) blocked INa (in the activation voltage range studied) but had no significant effect on the associated SR Ca release; (4) NIF (Ca channel antagonist) abolished most of the Ca release at these negative potentials; (5) BayK (Ca channel agonist) induced (feline) or increased (human) SR Ca release at negative test potentials, and these Ca transients were not blocked by TTX; and (6) positive voltage steps (to 0 mV) in the presence of VEP (Ca channel antagonist) and ISO only induced SR Ca release in those myocytes in which there was evidence for incomplete block of ICa,L. The abolition of verapamil-insensitive SR Ca release by TTX is not well explained by SMC and will be discussed later. These results suggest that Ca influx through L-type Ca channels rather than via modified Na channels (SMC) is the trigger for SR Ca release in both normal feline and failing human ventricular myocytes under our conditions. This also suggests that there is sufficient Ca influx through the L-type Ca channel at potentials negative to −50 mV to induce SR Ca release.
Alternative Explanations for SMC-Induced SR Ca Release
Two general lines of evidence support SMC-induced SR Ca release.9 The first is that SR Ca release is associated with INa at negative test potential (without Ca channel blockers). The second is that NIF- and Cd-insensitive SR Ca release produced by depolarizations to more positive potentials are abolished by TTX.9 Our results suggest that Ca influx via the L-type Ca channel rather than SMC may be an alternative explanation for these observations.
Activators of the putative SMC mechanism (PKA activators and cardiotonic steroids) also increase ICa,L14 and the amount of Ca stored in the SR.23 These effects cause the “gain” of excitation-contraction (EC) coupling to increase so that a small quantity of Ca influx via the L-type Ca channel can induce SR Ca release.14,24⇓ Therefore, to prove that Ca influx via a parallel system such as SMC can independently induce SR Ca release requires that Ca influx via ICa,L is completely eliminated. This is difficult to prove because small quantities of ICa,L are not easily detected, especially when overlapping currents are not blocked.14,19⇓
Does “Voltage Escape” Explain SMC?
Spurious activation of ICa,L can occur at the negative membrane potentials (below −50 mV) used to selectively activate INa if the membrane potential is not well controlled. This could be an explanation for the SMC mechanism.12 A useful criterion for LOVC is sudden acceleration of the rate of decay of INa (see inset in Figure 1). 25 We examined this in the original SMC report9 and conclude that LOVC is insufficient to explain these results. A primary purpose of the present study was to define a range of Na currents that could be well controlled before and after exposure to ISO so that we could study SMC in the absence of voltage escape. We used this approach because ISO induces a leftward shift in the voltage-dependence of INa activiation,9,16,17⇓⇓ and if test steps are only performed at a single potential,9 INa will be greater after ISO, increasing the likelihood of voltage escape-induced SR Ca release. By grading INa, we were able to show that the SR Ca release associated with INa in ISO-treated myocytes is not caused by voltage escape-induced activation of ICa,L.12 However, because this SR Ca release was TTX-insensitive, it is not caused by SMC.
Are a Few L-Type Ca Channels Normally Activated at Negative Test Potentials, and Do They Cause the SR Ca Release Attributed to SMC?
It is usually assumed that INa is activated at more negative membrane potentials than ICa,L,1 but our results suggest that this is incorrect. Although TTX blocks the Na channel and SMC,9 we found that it did not block the SR Ca release induced by depolarizations to potentials between −60 to −45 mV (Figures 2 and 4⇑). Because there is no T-type Ca current in normal feline26 or failing human ventricular myocytes,27 we examined whether Ca entry via the L-type Ca channel provided the trigger Ca for this release. The fact that the L-type Ca channel antagonist nifedipine abolished most of the SR Ca release in this voltage range (in the presence of ISO) and that the Ca channel agonist BayK (and FPL) induced SR Ca release (in the absence of ISO) strongly supports a role for ICa,L. These findings support the idea that a small number of L-type Ca channels are activated with depolarizations from the resting potential to the negative potentials (−60 to −45 mV) that are often thought of as subthreshold for activation of ICa,L. This idea is supported by results from other laboratories.1,14,22,28,29⇓⇓⇓⇓ Our experiments show that voltage-dependent activation of L-type Ca channels is responsible for the SR Ca release we observed in association with INa in feline and human myocytes. These data further suggest that Ca influx via ICa,L is an alternative explanation for the triggering of SR Ca release at negative membrane potentials that has previously been attributed to SMC. We also suggest that these small L-type Ca currents are sufficient to induce SR release because of the high SR Ca loads caused by the activators (ISO or cardiotonic steroids) of the putative SMC.14,23⇓
Previous studies of SMC-induced SR Ca release were performed in small animals (rats and mice) in which Ca transients are more dependent on SR Ca release1 and in which SR Ca loading is greater than in myocytes from larger mammals. These physiological differences could cause the EC coupling gain to be greater in small versus large mammals (including humans). Therefore, a small amount of Ca entry via SMC30 in rat or mouse myocytes (with very high SR Ca loads due to the species used and the presence of PKA activation or cardiotonic steroids) might cause SR Ca release.31
Why Does TTX Block SR Ca Release in the Presence of Ca Channel Blockers at Positive Test Potentials in the Presence of ISO?
A significant experimental result favoring the SMC hypothesis is the fact that depolarizations to positive potentials continue to induce SR Ca release in PKA-activated myocytes studied in the presence of ICa,L antagonists and TTX can block this SR Ca release. Previously,14 we showed that when SR loads are maintained in the presence of either 200 or 400 μmol/L Cd (100 μmol/L Cd was used in the original SMC study9), there is sufficient unblocked ICa,L to induce SR Ca release. Therefore, it is likely that there was some unblocked ICa,L in the previous studies of SMC.9 We employed a different ICa,L antagonist, verapamil, in an effort to achieve more complete block. An essential aspect of these experiments was that conditioning steps from −40 to 0 mV were used to evaluate the completeness of VEP block of ICa,L-induced SR Ca release. Na channels and SMC are not activated during these conditioning steps because of steady state inactivation of Na channels at −40 mV. This allowed us to test the idea that subsequent test steps (which included Na current) from −90 to 0 mV involved unblocked ICa,L. We found that when VEP (in the presence of ISO) abolished SR Ca release during the conditioning steps, there was also no Ca release during the test step. Conversely, when VEP did not abolish SR Ca release during the conditioning steps, there was also Ca release during the test steps. These data suggest that unblocked ICa,L rather than SMC induces the SR Ca release we observed at positive test potentials (Figure 5) and may also be an alternative explanation for a related finding in the original SMC report.9
We are left to explain how TTX can block the ICa,L antagonist-insensitive component of SR Ca release, and here we can make only speculations about the mechanism. TTX block of the Na current will eliminate the associated subsarcolemmal Na accumulation32 and will eventually reduce bulk cytosolic Na, thereby shifting the energetics of Ca transport toward forward mode NCX. This will rapidly lead to a reduction in SR Ca stores. Our hypothesis is that in the presence of ICa,L antagonists, EC coupling is on the brink of failure and only occurs when there is sufficient Ca entry through unblocked Ca channels to cause Ca release from a heavily Ca-loaded SR (during PKA activation or with cardiotonic steroids). We suggest that EC coupling can readily fail (block) when it is reliant on a small quantity of unblocked ICa,L if there are small changes in either Ca entry14 or SR Ca loading. TTX block of INa under these conditions might abolish SR Ca release (independent of SMC) by reducing SR Ca loading and/or Ca entry via reverse mode NCX. Ca entry via NCX could be involved if it modulates the effectiveness of ICa,L to trigger SR Ca release.33 For this mechanism to be responsible for the TTX effect we observed, it would have to be very sensitive to small changes in subsarcolemmal Na because there should have been little INa under the conditions we used in experiments to positive test potentials (Figure 5). These ideas are consistent with recent results that support a role for reverse mode NCX in ISO-induced positive inotropic effects in rat ventricular myocytes.34 It is also noteworthy that the elimination of SR Ca release by VEP and TTX confirms our observation14 that the putative voltage-dependent release mechanism35 is not present in feline myocytes.
Differences in experimental conditions between our study and the original SMC report9 do not appear to underlie our failure to observe SMC. We did employ pipette solutions with a lower free [Mg] than in the original SMC study.9 However, it has been known for some time that the Ca sensitivity of Ca-induced SR Ca release is increased at low [Mg],36,37⇓ and this should increase the ability of small amounts of Ca influx to induce SR Ca release. This should have increased our ability to observe SMC-induced SR Ca release and indeed may have allowed us to observe SR Ca release caused by small quantities of ICa,L at negative membrane potentials. Importantly, nothing resembling SMC was ever observed at higher pipette [Mg] (data not shown).
Why Is There SR Ca Release at Negative Potentials in Failing Human Ventricular Myocytes Under Control Conditions?
ICa,L-induced SR Ca release was observed in human myocytes at negative test potentials in the absence of added PKA activators. This is surprising because SR Ca loading is substantially reduced in failing human myocytes.38 Although this observation could represent a fundamental difference in Ca channel behavior in human and feline myocytes, it might also be the result of an increased level of basal L-type Ca channel39 and or RyR40 channel phosphorylation in failing human ventricular myocytes that modify EC coupling. Along these lines, PKA-mediated phosphorylation of Na channels causes a leftward shift in the voltage-dependence of activation.16,17⇓ We found that ISO had substantially smaller effects on the voltage-dependence of Na channel activation in failing human myocytes than in feline cells (Figure 1), consistent with the effects of increased basal phosphorylation.
Summary and Conclusion
The present results provide an alternative explanation for SMC-induced SR Ca release. We provide evidence that Ca influx through the L-type Ca channel rather than the Na channel can account for what has been called slip-mode conductance. If this is true, then SMC is not a useful therapeutic target for human heart failure.
This research was supported by grants from the NIH (HL33921 and HL61495 to S.R.H.) and the American Heart Association (to V.P.). We thank W.J. Lederer, L.F. Santana, and K.S. Ginsburg for helpful discussions and the members of the Cardiovascular Research Laboratories and the Temple University Hospital Cardiac Transplant Team for their assistance.
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received August 16, 2001; resubmission received October 24, 2001; revised resubmission received January 14, 2002; accepted January 14, 2002.
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