L-Type Ca2+ Channels Gaining Respect in Heart Failure
L-type Ca2+ channels are essential for the initiation and regulation of excitation-contraction (EC) coupling in adult cardiac muscle.1 The rapid influx of Ca2+ through these channels triggers release of intracellular Ca2+ from the sarcoplasmic reticulum (SR) stores, and the resulting Ca2+ transient activates the myofilaments and thus contraction. Given that failing ventricular myocytes exhibit impaired contractility and abnormal Ca2+ transients, the L-type Ca2+ channel makes for a good suspect as a contributor to the derangement of EC coupling. However, most initial studies of whole-cell currents through L-type Ca2+ channels (ICa) in both human heart failure and animal models did not show a significant difference between nonfailing and failing myocytes.2 In the meantime, important changes in the abundance and/or function of other Ca2+ cycling proteins including the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), Na+-Ca2+ exchanger (NCX), and ryanodine receptors (RyRs) took the spotlight as the bad actors in heart failure.3 The L-type Ca2+ channel was left behind as a boring, obligatory participant in EC coupling.
Times may be changing for the status of the L-type Ca2+ channel in the world of failing hearts. The article by Chen et al,4 in this issue of Circulation Research, unmasks important changes in the density as well as regulation of L-type Ca2+ channels in failing human ventricular myocytes. And, perhaps most encouragingly, the authors show that these changes can be partly reversed in patients with left ventricular assist devices (LVADs). This article adds more evidence to recent publications suggesting significant changes in L-type Ca2+ channel abundance and function in heart failure.5,6⇓ To appreciate the story, one needs to step back from the macroscopic or whole-cell currents through L-type Ca2+ channels and look behind the scenes. Whole-cell currents are determined by the number of functional channels (N), the probability of a channel being open (po), and the current through a single open channel (i) as described by the following simple relationship: ICa,L=N*po*i. Thus, the finding that peak ICa is comparable in failing and nonfailing myocytes when measured using standard voltage-clamp techniques may in fact be hiding counterbalancing changes in channel density, gating, and single-channel current. It is also important to realize that the altered conformation of the action potential in failing myocytes can have a large effect on the influx of Ca2+ through L-type Ca2+ channels even if all of the properties of the channels are unchanged.7,8⇓
Tracking the Density and Gating of L-Type Ca2+ Channels
A number of techniques exist that provide detailed information on the number and gating of L-type Ca2+ channels. Biochemical techniques have generally suggested no change or a decrease in protein abundance of channel subunits or binding sites for Ca2+ antagonists in failing hearts2; however, it is difficult to extrapolate these results to functional channels at the surface membrane. A single-channel electrophysiological study provided the first clue that the story on L-type Ca2+ channels may be more complicated than originally suspected. It was demonstrated that the number of functional channels and the open probability of the channels were markedly increased in failing human ventricular myocytes without changes in single-channel current, just as if the channels had been stimulated by protein kinase A (PKA)-dependent phosphorylation.6 Given that the whole-cell currents were unchanged, the authors speculated that the density of channels is decreased in failing cells with a compensatory increase in the availability and open probability of the remaining channels. The limitation of that study was that by necessity the single-channel recordings were made from channels on the surface sarcolemma, whereas most of Ca2+ channels are concentrated in the t-tubules in ventricular myocytes where the majority of EC coupling action occurs.
An alternative approach measuring intramembrane charge movement associated with L-type Ca2+ channels was taken in the tachycardia-pacing canine heart failure model.5 With appropriate voltage protocols, maximal intramembrane charge movement should be directly proportional to L-type Ca2+ channel number. Despite finding a comparable density of ICa in control and failing myocytes as had others,9 the study showed more than a 50% decrease in maximal amount of intramembrane charge movement again suggesting that the overall density of L-type Ca2+ channels is significantly decreased. The limitation of the charge movement study is that it is difficult to isolate charge movement due to L-type Ca2+ channels in myocytes that have many different voltage-dependent ion channels.
Given this background, Chen and colleagues4 decided to take a closer look at the whole-cell ICa in failing human ventricular myocytes. They similarly found that the peak density of ICa was not changed in a major way; however, unlike previous studies, they did detect a hyperpolarizing shift in the voltage dependence of activation in failing cells. These investigators reasoned that if the basal gating activity of the channels was increased in the presence of reduced channel density, then maximal stimulation of channels by the cAMP/PKA pathway or by dihydropyridine Ca2+ channel activators should unmask a difference in maximally stimulated ICa. Perhaps it was not surprising that maximally stimulated ICa by isoproterenol was significantly less in failing myocytes, given the well-documented blunted response of failing hearts to β-adrenergic agonists.10 Although this may be in part a result of multiple alterations along the β-adrenergic signaling cascade in failing hearts, Chen and colleagues suggested that additional contribution was due to a reduced density of L-type Ca2+ channels in the failing myocytes. To support this contention, the authors bypassed the β-adrenergic receptor through adenylate cyclase by directly testing nonhydrolyzable dibutyryl-cAMP. Although dibutyryl-cAMP was more effective than isoproterenol in stimulating ICa, there was still a 43% smaller maximally stimulated current level in failing myocytes. Therefore, the authors suggest that a major underlying cause for blunted stimulated current levels was the reduction in channel number rather than abnormalities in the β-adrenergic receptor cascade. This interpretation of the results with dibutyryl-cAMP requires that the signaling cascade downstream of cAMP is unaltered, which in fact may not be the case. For example, changes in protein phosphatase activity counterbalancing PKA effects could be important as the authors point out. Alternatively, localization of the PKA to its substrate with the necessary anchoring protein could be impaired in the failing heart, blunting the ability of this pathway to stimulate ICa. Lastly, the substrate for PKA, presumably one or more subunits of the L-type Ca2+ channel, could be altered. To avoid these possible complexities of the PKA signaling cascade, the effect of maximally activating channels via a distinct mechanism using the dihydropyridine BayK 8644 was tested, which acts directly on the channel. Again, in failing myocytes, the maximally stimulated ICa density was reduced compared with nonfailing myocytes, although the difference was smaller than for the experiments with dibutyryl-cAMP. This result is consistent with a lower density of L-type Ca2+ channels in failing myocytes, but the interpretation rests on the assumption that L-type Ca2+ channels in control and failing myocytes respond similarly to dihydropyridines. Thus, a number of recent electrophysiological studies have provided complementary evidence that even if ICa is unchanged in failing myocytes, important differences in channel density and function can be present.
Subcellular Localization of L-Type Ca2+ Channels
Why worry about the number of and gating of L-type Ca2+ channels if the final currency of these channels, ICa, is unchanged in heart failure? One answer lies in the fact that localized, microscopic currents through single L-type Ca2+ channel play an important role in determining the functional effect of these channels. In the case of EC coupling, it is the highly localized influx of Ca2+ through sarcolemmal L-type Ca2+ channels in the restricted space opposite junctional sarcoplasmic reticulum that precisely regulates release of Ca2+ from SR.11 L-type Ca2+ channels outside of these junctional domains have a markedly reduced effect on EC coupling. In addition, subcellular localization of L-type Ca2+ channels in other subdomains such as in complexes with β2-adrenergic receptors may be important in regulating cellular signaling.12,13⇓ Therefore, the present study of Chen et al4 raises the following questions: where are those channels lost and what subpopulation(s) of channels show the counterbalancing increase in channel gating?
In normal adult ventricular myocytes, most L-type Ca2+ channels are concentrated in the t-tubule network and contribute to the synchronized initiation of EC coupling throughout the myocytes.14 Recent studies in the canine tachycardia-dilated cardiomyopathy model and a doxorubicin-induced cardiomyopathy model have suggested that failing myocytes can have dramatic cellular remodeling with loss of t-tubule membranes.5,15⇓ Thus, it is possible that the localization of L-type Ca2+ channels may be quite different in failing myocytes than control myocytes. Defining the subcellular localization, molecular composition, and function of subpopulations of L-type Ca2+ channels represents critical future challenges.
Basal Regulation of L-Type Ca2+ Channels
Chen et al4 hypothesize that there is increased basal phosphorylation of the channel in failing myocytes by cAMP/PKA-mediated pathways based on the shift in the voltage dependence of activation of the channels in failing cells, the effect of PP2a specifically on failing basal ICa, and the blunted response to isoproterenol and dibutyryl-cAMP. In addition, the previous single-channel study in failing human myocytes suggested a gating pattern typical of PKA-stimulated channels.6 They suggest that hyperphosphorylated channels are present in failing hearts, analogous to studies of the RyRs.16 This is an appealing hypothesis, but at this stage no direct evidence has been provided for actual changes in phosphorylation status of the channel measured biochemically. This remains a problem area for L-type Ca2+ channels as the molecular details of PKA regulation of the channel in intact cardiac myocytes have remained relatively refractory to biochemical approaches.17 Furthermore, some of the data by Chen et al4 are difficult to reconcile with this hypothesis in that H89 and Rp-cAMP failed to alter basal currents in both failing and control myocytes. The recovery of regulation by β-adrenergic stimulation of ICa in LVAD patients, despite the continued hyperpolarizing shift in the activation of the currents, suggests multiple points of modulation of channel gating. Another critical factor is that other Ser/Thr kinases can regulate L-type Ca2+ channels including protein kinase C and Ca2+/CaM kinase and potentially more.18 One does not even need to invoke differences in the basal phosphorylation state of the channel to explain increased basal open probability. Changes in channel subunit isoforms can also contribute to this behavior. Alternative isoforms of the pore-forming Cav1.2 channel have been demonstrated in failing hearts,19 and additionally, auxiliary subunits may have important modulatory roles. For example, overexpression of the β2a subunit in rat ventricular myocytes can clearly increase current levels.20 Unraveling the mechanism for upregulated basal activity of L-type Ca2+ channels in failing myocytes will require future studies.
The L-type Ca2+ channel is gaining respect as a contributor to the pathophysiological changes in Ca2+ homeostasis in failing myocytes. Many studies using standard voltage-clamp pulses have not detected significant changes in basal ICa, but the work by Chen and colleagues4 highlights significant decreases in density and altered β-adrenergic regulation of L-type Ca2+ channels in failing human hearts. Fortunately, these changes are at least partially reversible as demonstrated in LVAD patients. The underlying molecular mechanisms of altered channel density and gating as well as the functional impact of these changes on EC coupling are important topics for future research.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Hasenfuss G. Alterations of calcium-regulatory proteins in heart failure. Cardiovasc Res. 1998; 37: 279–289.
- ↵Chen X, Piacentino V, Furukawa S, Goldman B, Margulies KB, Houser SR. L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices. Circ Res. 2002; 91: 517–524.
- ↵He J-Q, Conklin MW, Foell JD, Wolff MR, Haworth RA, Coronado R, Kamp TJ. Reduction in density of transverse tubules and L-type Ca2+ channels in canine tachycardia-induced heart failure. Cardiovasc Res. 2001; 49: 298–307.
- ↵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. Circ Res. 1998; 98: 969–976.
- ↵Sah R, Ramirez RJ, Backx PH. Modulation of Ca2+ release in cardiac myocytes by changes in repolarization rate: role of phase-1 action potential repolarization in excitation-contraction coupling. Circ Res. 2002; 90: 165–173.
- ↵Winslow RL, Rice J, Jafri S, Marbán E, O’Rourke B. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, II: model studies. Circ Res. 1999; 84: 571–586.
- ↵Kaab S, Nuss B, Chiamvimonvat N, O’Rourke B, Pak PH, Kass DA, Marban E, Tomaselli GF. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ Res. 1996; 78: 262–273.
- ↵Davare MA, Avdonin V, Hall DD, Peden EM, Burette A, Weinberg RJ, Horne MC, Hoshi T, Hell JW. A β2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2. Science. 2001; 293: 98–101.
- ↵Xiao R-P, Cheng H, Zhou Y-Y, Kuschel M, Lakatta EG. Recent advances in cardiac β2-adrenergic signal transduction. Circ Res. 1999; 85: 1092–1100.
- ↵Kamp TJ, Hell JW. Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res. 2000; 87: 1095–1102.
- ↵Wei SK, Colecraft HM, DeMaria CD, Peterson BZ, Shang R, Kohout TA, Rogers TB, Yue DT. Ca2+ channel modulation by recombinant auxiliary β subunits expressed in young adult heart cells. Circ Res. 2000; 86: 175–184.