This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 90/4/435    most recent hh0402.105666v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Piacentino, V. Articles by Houser, S. R. Search for Related Content PubMed PubMed Citation Articles by Piacentino, V., III Articles by Houser, S. R. Related Collections Contractile function Calcium cycling/excitation-contraction coupling Ion channels/membrane transport

(Circulation Research. 2002;90:435.)
© 2002 American Heart Association, Inc.

Cellular Biology

L-Type Ca²⁺ Currents Overlapping Threshold Na⁺ Currents

Could They Be Responsible for the "Slip-Mode" Phenomenon in Cardiac Myocytes?

Valentino Piacentino, III, John P. Gaughan, Steven R. Houser

From the Cardiovascular Research Group, Molecular and Cellular Cardiology Laboratories, Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Dr Houser, Cardiovascular Research Group, Molecular and Cellular Cardiology Laboratories, Temple University School of Medicine, 3400 North Broad St, Philadelphia, PA 19140. E-mail srhouser{at}unix.temple.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 I_Na in the presence of activated PKA (with Isoproterenol, ISO). V_m, I_m, 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 I_Na. Na channel antagonists (tetrodotoxin), L-type Ca channel (I_Ca,L) antagonists (nifedipine, cadmium, verapamil), and agonists (Bay K 8644, FPL 64176) were used to separate SMC from I_Ca,L. In the absence of ISO, I_Na was associated with SR Ca release in human but not feline myocytes. After ISO, graded I_Na was associated with small amounts of SR Ca release in feline myocytes and the magnitude of release increased in human myocytes. I_Na-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 I_Ca,L was incomplete. We conclude that PKA-mediated increases in I_Ca,L and SR Ca loading can reproduce many of the experimental features of SMC.

Key Words: calcium current • excitation-contraction coupling • sarcoplasmic reticulum • sodium current

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 channels^10,11 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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-3_K5 (100 µmol/L) via pipette loading. The fluorescence changes during the Ca transient (F) were normalized by the resting fluorescence (F_o) and expressed as F/F_o.

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, 5x10^-8 mol/L feline, 1x10^-6 mol/L human) to activate the putative slip-mode conductance mechanism. Some cells were then exposed to Na channel (tetrodotoxin [TTX], 10x10^-6 mol/L) or L-type Ca channel (nifedipine [NIF], 25 to 50x10^-6 mol/L, or verapamil [VEP], 20x10^-6 mol/L) antagonists or to L-type Ca channel agonists (Bay K 8644 [0.5 to 1x10^-6 mol/L] and FPL 64176 [0.5x10^-6 mol/L]).

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (I_Na) 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 I_Na 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 I_Na between -60 and -45 mV (Figure 1B).

View larger version (32K): [in this window] [in a new window]   Figure 1. Effect of isoproterenol on INa and SR Ca release in feline and human myocytes. Voltage-dependent grading of INa in representative feline (A) and human (B) myocytes before (top panels) and after (bottom panels) ISO (0.050 µmol/L feline, 1 µmol/L human) is shown. Conditioning pulses have been eliminated for clarity. In normal bath conditions, INa was well controlled up to -47 mV in the feline myocyte, and there was little or no SR Ca release associated with these currents. The increase in the Ca transient (F/Fo) during conditioning steps to +10 mV during application of ISO is shown in the left inset. After ISO, INa was activated at more negative potentials and was associated with Ca transients. These Ca transients were significantly smaller than during large voltage steps to +10 mV or when there was LOVC (right inset). Arrows indicate test potential(s) at which LOVC was observed. Note that Ca transients associated with LOVC were larger after ISO. The differences in the decay kinetics of the Na current (ISO) during a well-controlled voltage step (to -50 mV) and during loss of voltage control (-49 mV) is shown in the lower right inset. Voltage traces at the top of this and subsequent figures illustrate the voltage measured under the conditions described (control or ISO, respectively). In the failing human myocyte (B), small amounts of SR Ca release were associated with INa under control conditions, and this Ca release was larger after ISO. ISO caused a significant (*P<0.05) leftward shift in the voltage-dependence of INa activation (C and E) and a significant increase in Ca transient (D and F) in both feline (n=15) and human (n=10) myocytes.

ISO (5x10^-8 mol/L feline, 1x10^-6 mol/L human) caused a leftward shift in the voltage-dependence of I_Na 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 I_Na 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 I_Na 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 I_Na) 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 I_Na, SMC and the associated SR Ca release⁹) was tested in feline (n=6) and human (n=4) myocytes. In every myocyte, TTX (10x10^-6 mol/L, in the presence of ISO) eliminated most of the I_Na 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).

View larger version (30K): [in this window] [in a new window]   Figure 2. Effect of tetrodotoxin on INa-associated SR Ca release in feline and human myocytes. The effects of TTX (10 µmol/L) (in the presence of ISO, 0.050 µmol/L feline, 1 µmol/L human) on INa and associated SR Ca release in representative feline (A) and human (B) myocytes are shown. TTX eliminated most of the INa at these potentials in all myocytes, but SR Ca release was not significantly altered. A small amount of SR Ca release was observed under control conditions in the human myocyte. Arrows point to Ca transients induced when there was LOVC. TTX caused a significant decrease in INa (C and E) in both feline (n=6) and human (n=4) myocytes; however, SR Ca release (D and F) was not significantly reduced.

Effects of Nifedipine on Na Current and SR Ca Release
The effects of the L-type Ca channel antagonist NIF (25 to 50x10^-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 I_Na (Figure 3). The effect of NIF on I_Na is consistent with previous observations.¹⁸ Note that after NIF at -50 mV (Figure 3), the peak I_Na still exceeded that at -51.5 mV before NIF, but there was no Ca transient.

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of nifedipine on INa-associated SR Ca release in feline and human myocytes. The effects of NIF (25 to 50 µmol/L) (in the presence of ISO, 0.050 µmol/L feline, 1 µmol/L human) on INa and SR Ca release on representative feline (A) and human (B) myocytes are shown. NIF almost completely abolished the Ca release associated with INa and caused a small decrease in INa. NIF reduced but did not fully block SR Ca release during LOVC, demonstrating that conditioning steps to +80 mV maintained SR Ca loading and that ICa,L was not fully blocked. NIF caused a small reduction in INa (C and E) and significantly reduced SR Ca release (D and F) at these negative test potentials in both feline (n=7) and human (n=3) myocytes.

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 1x10^-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 I_Na. TTX (in the presence of BayK) blocked I_Na but not SR Ca release. Identical results were obtained with the Ca channel agonist FPL 64176 (FPL, 0.5x10^-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.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of Ca channel agonists (Bay K 8644, BayK) on INa-associated SR Ca release in feline and human myocytes. The effects of BayK (0.5 to 1 µmol/L) (no ISO present) on the SR Ca release associated with graded INa in representative feline (A) and human (B) myocytes are shown. BayK increased the magnitude of Ca transients during conditioning steps and LOVC (see arrows, voltage steps to right of dotted line indicate those potentials in which LOVC first occurred) and induced SR Ca release at negative test steps (middle panels) that were similar to those observed in the presence of ISO. BayK had no effect on INa. TTX (in the presence of BayK) eliminated INa, but SR Ca release at negative potentials was still observed (lower panels). BayK caused a significant increase in the F/Fo-voltage relationship (D and F) but no change in INa (C and E) in both feline (n=3) and human (n=3) myocytes

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 I_Na would be small or absent at 0 mV (the reversal potential for I_Na) so that completeness of I_Ca,L block could be more clearly observed. Five conditioning steps from -40 (to inactivate I_Na) to +10 mV were used before each test step to promote use-dependent VEP block of I_Ca,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 I_Ca,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).

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of verapamil and tetrodotoxin on INa-associated SR Ca release in feline myocytes at positive test potentials. Representative experiment to test for SMC-related SR Ca release at positive test potentials in the presence of ISO (0.05 µmol/L) and VEP (20 µmol/L). A, There was no measurable ICa,L during conditioning pulses to +10 mV (where the Na conductance is inactivated because the holding potential was-40 mV, cond) or during test pulses from -90 mV (where Na conductance is activated, test). Ca transients were induced during both the conditioning and test steps, consistent with the idea that unblocked Ca current is the trigger for SR Ca release. Ca transients were abolished when this myocyte was exposed to TTX (B). Similar results were seen in 3 additional cells (see text for discussion). C, Voltage steps to -20 mV (under conditions described in A) elicited TTX-sensitive Na current, which became smaller as the voltage steps were depolarized toward ENa. These results show that Na channels are activated during the test steps shown in A and B.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (I_Na) 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 I_Na in feline myocytes, whereas in failing human myocytes small Ca transients were observed; (2) ISO caused a leftward shift in the voltage-dependence of I_Na activation, and Ca transients were seen concomitant with I_Na in both feline and human myocytes; (3) TTX (Na channel antagonist) blocked I_Na (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 I_Ca,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 I_Na 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 I_Ca,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 I_Ca,L is completely eliminated. This is difficult to prove because small quantities of I_Ca,L are not easily detected, especially when overlapping currents are not blocked.^14,19

Does "Voltage Escape" Explain SMC?
Spurious activation of I_Ca,L can occur at the negative membrane potentials (below -50 mV) used to selectively activate I_Na 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 I_Na (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 I_Na activiation,^9,16,17 and if test steps are only performed at a single potential,⁹ I_Na will be greater after ISO, increasing the likelihood of voltage escape-induced SR Ca release. By grading I_Na, we were able to show that the SR Ca release associated with I_Na in ISO-treated myocytes is not caused by voltage escape-induced activation of I_Ca,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 I_Na is activated at more negative membrane potentials than I_Ca,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 I_Ca,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 I_Ca,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 I_Na in feline and human myocytes. These data further suggest that Ca influx via I_Ca,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 I_Ca,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 I_Ca,L to induce SR Ca release. Therefore, it is likely that there was some unblocked I_Ca,L in the previous studies of SMC.⁹ We employed a different I_Ca,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 I_Ca,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 I_Ca,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 I_Ca,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 I_Ca,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 I_Ca,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 I_Ca,L if there are small changes in either Ca entry¹⁴ or SR Ca loading. TTX block of I_Na 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 I_Ca,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 I_Na 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 I_Ca,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?
I_Ca,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.

	Acknowledgments

 
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.

	Footnotes

 
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received August 16, 2001; revision received January 14, 2002; accepted January 14, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Bers DM. Excitation-Contraction Coupling, and Cardiac Contractile Force. Dordrecht, the Netherlands: Kluwer Academic Publishers; 2001.
2. Barry WH, Bridge JH. Intracellular calcium homeostasis in cardiac myocytes. Circulation. 1993; 87: 1806–15.
3. Litwin SE, Li J, Bridge JH. Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes. Biophys J. 1998; 75: 359–371.
4. London B, Krueger JW. Contraction in voltage-clamped, internally perfused single heart cells. J Gen Physiol. 1986; 88: 475–505.
5. Näbauer M, Callewaert G, Cleemann L, Morad M. Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. Science. 1989; 244: 800–803.
6. Beuckelmann DJ, Wier WG. Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. J Physiol. 1988; 405: 233–255.
7. Sipido KR, Maes M, Van de Werf F. Low efficiency of Ca ²⁺ entry through the Na⁺-Ca²⁺ exchanger as trigger for Ca²⁺ release from the sarcoplasmic reticulum: a comparison between L-type Ca²⁺ current and reverse-mode Na⁺-Ca²⁺ exchange. Circ Res. 1997; 81: 1034–1044.
8. Sipido KR, Carmeliet E, Van de Werf F. T-type Ca²⁺ current as a trigger for Ca²⁺ release from the sarcoplasmic reticulum in guinea-pig ventricular myocytes. J Physiol. 1998; 508: 439–451.
9. Santana LF, Gomez AM, Lederer WJ. Ca²⁺ flux through promiscuous cardiac Na⁺ channels: slip-mode conductance. Science. 1998; 279: 1027–1033.
10. Nuss HB, Marbán E, Balke CW, Goldman L, Aggarwal , Shorofsky SR, Santos Cruz J, Santana LF, Frederick CA, Isom LL, Malhotra JD, Mattei LN, Kass RS, Xia J, An RH, Lederer WJ. Whether "Slip-Mode Conductance" occurs. Science. 1999; 284: A711.
11. Chandra R, Chauhan VS, Starmer CF, Grant AO. ß-Adrenergic action on wild-type and KPQ mutant human cardiac Na⁺ channels: shift in gating but no change in Ca²⁺:Na⁺ selectivity. Cardiovasc Res. 1999; 42: 490–502.
12. DelPrincipe F, Egger M, Niggli E. L-type Ca²⁺ current as the predominant pathway of Ca²⁺ entry during I_Na activation in ß-stimulated cardiac myocytes. J Physiol. 2000; 527: 455–466.
13. Wessely R, Klingel K, Santana LF, Dalton N, Hongo M, Lederer WJ, Kandolf R, Knowlton KU. Transgenic expression of replication-restricted enteroviral genomes in heart muscle induces defective excitation-contraction coupling and dilated cardiomyopathy. J Clin Invest. 1998; 102: 1444–1453.
14. Piacentino VIII, Dipla K, Gaughan JP, Houser SR. Voltage-dependent Ca²⁺ release from the SR of feline ventricular myocytes is explained by Ca²⁺-induced Ca²⁺ release. J Physiol. 2000; 523: 533–548.
15. Dipla K, Mattiello JA, Jeevanandam V, Houser SR, Margulies KB. Myocyte recovery after mechanical circulatory support in humans with end-stage heart failure. Circulation. 1998; 97: 2316–2322.
16. Ono K, Fozzard HA, Hanck DA. Mechanism of cAMP-dependent modulation of cardiac sodium channel current kinetics. Circ Res. 1993; 72: 807–815.
17. Lu T, Lee HC, Kabat JA, Shibata EF. Modulation of rat cardiac sodium channel by the stimulatory G protein alpha subunit. J Physiol. 1999; 518: 371–384.
18. Yatani A, Brown AM. The calcium channel blocker nitrendipine blocks sodium channels in neonatal rat cardiac myocytes. Circ Res. 1985; 56: 868–875.
19. Wier WG, Balke CW. Ca²⁺ release mechanisms, Ca²⁺ sparks, and local control of excitation-contraction coupling in normal heart muscle. Circ Res. 1999; 85: 770–776.
20. Fan JS, Yuan Y, Palade P. Kinetic effects of FPL 64176 on L-type Ca²⁺ channels in cardiac myocytes. Naunyn Schmiedebergs Arch Pharmacol. 2000; 361: 465–476.
21. Motoike HK, Bodi I, Nakayama H, Schwartz A, Varadi G. A region in IVS5 of the human cardiac L-type calcium channel is required for the use-dependent block by phenylalkylamines and benzothiazepines. J Biol Chem. 1999; 274: 9409–9420.
22. Talo A, Stern MD, Spurgeon HA, Isenberg G, Lakatta EG. Sustained subthreshold-for-twitch depolarization in rat single ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release. J Gen Physiol. 1990; 96: 1085–1103.
23. Hussain M, Orchard CH. Sarcoplasmic reticulum Ca²⁺ content, L-type Ca²⁺ current and the Ca²⁺ transient in rat myocytes during ß-adrenergic stimulation. J Physiol. 1997; 505: 385–402.
24. Jing-Song Fan, Palade P. One calcium ion may suffice to open the tetrameric cardiac ryanodine receptor in rat ventricular myocytes. J Physiol. 1999; 516: 769–780.
25. Evans AM, Cannell MB. The role of L-type Ca²⁺ current and Na⁺ current-stimulated Na/Ca exchange in triggering SR calcium release in guinea-pig cardiac ventricular myocytes. Cardiovasc Res. 1997; 35: 294–302.
26. Nuss HB, Houser SR. T-type Ca²⁺ current is expressed in hypertrophied adult feline left ventricular myocytes. Circ Res. 1993; 73: 777–782.
27. Beuckelmann DJ, Nabauer M, Erdmann E. Characteristics of calcium-current in isolated human ventricular myocytes from patients with terminal heart failure. J Mol Cell Card. 1999; 23: 929–937.
28. Sipido KR, Volders PG, Antoons G, Vos M. L-Type Ca current at negative potentials during ß-adrenergic stimulation. Biophys J. 2000; 78: 371A.Abstract.
29. Cannell MB, Cheng H, Lederer WJ. The control of calcium release in heart muscle. Science. 1995; 268: 1045–1049.
30. Guatimosim S, Sobie EA, dos Santos Cruz J, Martin LA, Lederer WJ. Molecular identification of a TTX-sensitive Ca²⁺ current. Am J Physiol. 2001; 280: C1327–C1339.
31. Bassani JW, Yuan W, Bers DM. Fractional SR Ca²⁺ release is regulated by trigger Ca²⁺ and SR Ca²⁺ content in cardiac myocytes. Am J Physiol. 1995; 268: C1313–C1319.
32. Su Z, Sugishita K, Ritter M, Li F, Spitzer KW, Barry WH. The sodium pump modulates the influence of I_Na on [Ca²⁺]_i transients in mouse ventricular myocytes. Biophy J. 2001; 80: 1230–1237.
33. Cordeiro JM, Cannell MB, Bridge JHB. The Na-Ca exchange potentiates I_Ca triggered release in rabbit ventricular myocytes. Biophys J. 2001; 80: 591A.Abstract.
34. Viatchenko-Karpinski S, Gyorke S. Modulation of the Ca²⁺-induced Ca²⁺ release cascade by ß-adrenergic stimulation in rat ventricular myocytes. J Physiol. 2001; 533: 837–848.
35. Ferrier GR, Howlett SE. Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction. Am J Physiol. 2001; 280: H1928–H1944.
36. Ford LE, Podolsky RJ. Regenerative calcium release within muscle cells. Science. 1970; 167: 58–59.
37. Ford LE, Podolsky RJ. Intracellular calcium movements in skinned muscle fibres. J Physiol. 1972; 223: 21–33.
38. Lindner M, Erdmann E, Beuckelmann DJ. Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure. J Mol Cell Card. 1998; 30: 743–749.
39. Schroder F, Handrock R, Beuckelmann DJ, Hirt S, Hullin R, Priebe L, Schwinger RH, Weil J, Herzig S. Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle. Circulation. 1998; 98: 969–976.
40. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365–376.

This article has been cited by other articles:

				H. K. Saini and N. S. Dhalla Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H169 - H181. [Abstract] [Full Text] [PDF]

				J. Altamirano, Y. Li, J. DeSantiago, V. Piacentino 3rd, S. R. Houser, and D. M. Bers The inotropic effect of cardioactive glycosides in ventricular myocytes requires Na+-Ca2+ exchanger function J. Physiol., September 15, 2006; 575(3): 845 - 854. [Abstract] [Full Text] [PDF]

				A. S. Jung, R. Harrison, K. H. Lee, J. Genut, D. Nyhan, E. M. Brooks-Asplund, A. A. Shoukas, J. M. Hare, and D. E. Berkowitz Simulated microgravity produces attenuated baroreflex-mediated pressor, chronotropic, and inotropic responses in mice Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H600 - H607. [Abstract] [Full Text] [PDF]

				A. S. Jung, M. P. Quaile, G. D. Mills, D. P. Bednarik, S. R. Houser, and K. B. Margulies Pharmacological Effects of ATI22-107 [2-(2-{2-[2-Chloro-4-(6-oxo-1,4,5,6-tetrahydro-pyridazin-3-yl)-phenoxy]-acetylamino}-ethoxymethyl)-4-(2-chloro-phenyl)-6-methyl-1,4-dihydro-pyridine-3,5-dicarboxylic Acid Dimethyl Ester)], a Novel Dual Pharmacophore, on Myocyte Calcium Cycling and Contractility J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 517 - 524. [Abstract] [Full Text] [PDF]

				J. L Alvarez, E. Salinas-Stefanon, G. Orta, T. Ferrer, K. Talavera, L. Galan, and G. Vassort Occurrence of a tetrodotoxin-sensitive calcium current in rat ventricular myocytes after long-term myocardial infarction Cardiovasc Res, September 1, 2004; 63(4): 653 - 661. [Abstract] [Full Text] [PDF]

				V. Piacentino III, C. R. Weber, X. Chen, J. Weisser-Thomas, K. B. Margulies, D. M. Bers, and S. R. Houser Cellular Basis of Abnormal Calcium Transients of Failing Human Ventricular Myocytes Circ. Res., April 4, 2003; 92(6): 651 - 658. [Abstract] [Full Text] [PDF]

				R. H.G Schwinger, H. Bundgaard, J. Muller-Ehmsen, and K. Kjeldsen The Na, K-ATPase in the failing human heart Cardiovasc Res, March 15, 2003; 57(4): 913 - 920. [Abstract] [Full Text] [PDF]

				D. W. Hilgemann From a pump to a pore: How palytoxin opens the gates PNAS, January 21, 2003; 100(2): 386 - 388. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 90/4/435    most recent hh0402.105666v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Piacentino, V. Articles by Houser, S. R. Search for Related Content PubMed PubMed Citation Articles by Piacentino, V., III Articles by Houser, S. R. Related Collections Contractile function Calcium cycling/excitation-contraction coupling Ion channels/membrane transport