Synergistic interactions between Ca2+ entries through L-type Ca2+ channels and Na+–Ca2+ exchanger in normal and failing rat heart
1 Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, Ohio State University Medical Center, 473 West 12th Avenue, Columbus, OH 43210-1252, USA
2 Division of Cardiology, Texas Tech University Health Science Center, 3601 4th Street, Lubbock, TX 79430, USA
3 Department of Physiology, Texas Tech University Health Science Center, 3601 4th Street, Lubbock, TX 79430, USA
, http://www.100md.com Abstract
We used confocal Ca2+ imaging and the patch-clamp technique to investigate the interplay between Ca2+ entries through L-type Ca2+ channels (LCCs) and reverse-mode Na+–Ca2+ exchange (NCX) in activating Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac myocytes from normal and failing rat hearts. In normal myocytes exposed to N(6),2'-O-dibutyryl adenosine-3',5'-cyclic monophosphate (db-cAMP, membrane-permeable form of cAMP), the bell-shaped voltage dependence of cytosolic Ca2+ transients was dramatically broadened due to activation of SR Ca2+ release at high membrane potentials (30–120 mV). This broadening of Ca2+-transient voltage dependence could be prevented by KB-R7943, an inhibitor of the reverse-mode NCX. Trans-sarcolemmal Ca2+ entries were measured fluorometrically in myocytes during depolarizing steps to high membrane potentials. The total Ca2+ entry (FTot) was separated into two Ca2+ entry components, LCC-mediated (FLCC) and NCX-mediated (FNCX), by exposing the cells to the specific inhibitors of LCCs and reverse-mode NCX, nifedipine and KB-R7943, respectively. In the absence of protein kinase A (PKA) stimulation the amplitude of the Ca2+-inflow signal (FTot) corresponded to the arithmetic sum of the amplitudes of the KB-R7943- and nifedipine-resistant components (FTot=FLCC+FNCX). PKA activation resulted in significant increases in FTot and FLCC. Paradoxically, FTot became threefold larger than the sum of the FNCX and FLCC components. In myocytes from failing hearts, stimulation of PKA failed to induce a shift in Ca2+ release voltage dependence toward more positive membrane potentials. Although the total and NCX-mediated Ca2+ entries were increased again, FTot did not significantly exceed the sum of FLCC and FNCX. We conclude that the LCC and NCX Ca2+-entry pathways interact synergistically to trigger SR Ca2+ release on depolarization to positive membrane potentials in PKA-stimulated cardiac muscle. In heart failure, this new form of Ca2+ release is diminished and may potentially account for the compromised contractile performance and reduced functional reserve in failing hearts.
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Introduction
In cardiac muscle, the process of excitation–contraction (EC) coupling is mediated by Ca2+ influx from the extracellular space activating Ca2+-sensitive Ca2+-release channels (ryanodine receptors, RyR) in the sarcoplasmic reticulum (SR). Elevation of cytosolic Ca2+ concentration resulting from this Ca2+-induced Ca2+ release (CICR) process leads to activation of the contractile filaments and myocyte shortening. A consensus exists that Ca2+ entry via L-type Ca2+ channels (LCCs) represents the main signal for triggering CICR during normal EC coupling (Bers, 2001). This is consistent with the spatial localization of the LCCs just above the RyRs across the junctional cleft (Franzini-Armstrong et al. 1999). However, Ca2+ influx through other entry pathways, including T-type Ca2+ channels and the reverse mode of Na+–Ca2+ exchange (NCX), have been also implicated in activation of SR Ca2+ release under various experimental conditions (Leblanc & Hume, 1990; Lipp & Niggli, 1994; Kohomoto et al. 1994; Sipido, 1998). Additionally, there may be interplay between different Ca2+-entry pathways in initiation of SR Ca2+ release. For example, it has been proposed that Ca2+ entry through LCCs and reverse-mode NCX interact synergistically to activate RyR channels in the SR (Haworth et al. 1991; Litwin et al. 1998; Viatchenko-Karpinski & Gyrke, 2001). Integration of Ca2+-input signals from different entry pathways could provide an important mechanism for regulation of cardiac EC coupling and contractility. However, whether and how such integration of triggers for CICR occurs in cardiac muscle remain to be determined.
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Stimulation through the -adrenergic pathway is a classical regulatory mechanism for controlling the contractile state of the myocardium. -adrenergic agonists increase the amount of Ca2+ released from the SR, resulting in the development of increased contractile force in cardiac muscle. The biochemical signalling mechanisms underlying -adrenergic stimulation involve cAMP-dependent phosphorylation by protein kinase A (PKA) of certain target proteins including the LCC, RyR and phospholamban (Kranias et al. 1985; Bers, 2001; Shannon et al. 2001). In heart failure (HF), the Ca2+-releasing function of the SR appears to be compromised, resulting in depressed intracellular Ca2+ transients and impaired contraction (Houser et al. 2000; Bers, 2001; Eisner et al. 2003). Additionally the Ca2+-release machinery loses its ability to respond to adrenergic stimulation in myocytes from failing hearts, contributing to reduced contractile reserve in HF (Gomez et al. 1997; Bers, 2001; Pogwizd et al. 2001). The precise reasons for these HF-related changes in Ca2+ transients and responsiveness to adrenergic stimulation are not known, and are currently the subject of intense debate.
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In the present study, we investigated the interplay between Ca2+ entries via L-type Ca2+ channels and reverse-mode NCX in activation of SR Ca2+ release, focusing on the influence of adrenergic stimulation and changes occurring after induction of HF. Significantly, our results show that LCCs and NCX interact in a synergistic manner resulting in a larger total Ca2+ entry than the arithmetic sum of entries mediated by each of these transport systems alone. This synergism between the two Ca2+-entry pathways was dependent on stimulation of PKA. The nonlinear summation of Ca2+ triggers and the associated Ca2+-release component was abolished in two experimental models of HF. Our results show for the first time that Ca2+ entries through LCCs and reverse NCX can interact synergistically providing a new Ca2+-release activation mechanism that is turned on by adrenergic stimulation. In HF, this mechanism is compromised, potentially accounting for the reduced SR Ca2+ release and reduced functional reserve in failing hearts.
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Methods
Confocal Ca2+ imaging and patch-clamp measurements
Single ventricular myocytes were isolated by enzymatic dissociation from hearts from male Sprague–Dawley rats (Charles River Laboratories, Raleigh, NC, USA). Adult rats (200–300 g) were killed with an overdose of sodium pentobarbital in accordance with the guidelines of the Institutional Animal Care and Use Committee. Whole-cell transmembrane ionic currents and changes in membrane potential were recorded with an Axopatch 200B amplifier (Axon Instruments, USA) and pClamp (6.03) software. The bathing solution contained (mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 0.5 MgCl2, 10 Hepes and 5.6 glucose; pH 7.3. The pipette solution contained (mM): 70 caesium aspartate, 50 CsCl, 3 ATP(Mg2+), 4 NaCl, 5 Hepes, 0.05 fluo-3 potassium salt; pH 7.3. Voltage pulses of 150 ms duration were applied from a holding potential of –50 mV at 1 min intervals. Signals were sampled at 5 kHz and filtered at 2 kHz. Cytosolic [Ca2+] changes in patch-clamped myocytes were measured with an Olympus Laser Scanning Confocal System (LSM-GB200, Olympus Optical Co. Ltd, Tokyo, Japan), equipped with an Olympus x60 oil, 1.4 NA objective. Fluo-3 was excited at the 488-nm line of an argon laser, with emission collected through a 515-nm long-pass filter. Fluorescence images were recorded in the line-scan mode at the rate of 2 ms per line. In experiments involving measurements of trans-sarcolemmal Ca2+ entries, the pipette solution contained 0.1 mM fluo-3 potassium salt. In certain experiments, the SR Ca2+ content was increased by applying a loading protocol consisting of a train of 50 ms pulses from –50 mV holding potential to 0 mV at 2 Hz. All measurements were performed at 23°C. KB-R7943 (2-(2-(4(4-nitrobenzyloxy) phenyl)ethyl)isothiourea mesylate) was obtained from Tocris (USA). All other reagents were from Sigma-Aldrich (USA).
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Aortocaval shunt-induced HF
The aortocaval shunt (ACS) was produced according to the method described by Garcia & Diebold (1990). Briefly, after the animal was anaesthetized with pentobarbital, the inferior vena cava and abdominal aorta were exposed by laparotomy. An 18 gauge hypodermic needle was advanced into the aorta distal to the left renal artery and through the opposite wall into the inferior vena cava in order to establish a communication between the two vessels. Bulldog vascular clamps were placed across the aorta above and below the entry point, the needle was withdrawn, and the aortic puncture point was sealed with a drop of cyanoacrylate glue. The clamps were removed 30 s later. The presence of a patent shunt was confirmed visually after release of the clamps by the occurrence of swelling of the vena cava and admixture of arterial and venous blood. The laparotomy was then closed. Sham-operated animals (n= 6) serving as controls were subjected to the same surgical procedures except for creation of the shunt. Compensated hypertrophy occurs after 5–6 week of ACS, with progression to HF after 8–12 week (Garcia & Diebold, 1990; Pieruzzi et al. 1995). In vivo cardiac haemodynamic measurements and confirmation of the shunt were done using the Hewlett Packard Sonos 5500 Ultrasound system (12 MHz transducer). M-mode echocardiograms were recorded at the left ventricular (LV) papillary muscle level to measure end-diastolic and end-systolic dimension, fractional shortening, and end-diastolic posterior wall thickness. ACS rats without LV dilatation and decreased shortening after 8–12 weeks of ACS were excluded from the study.
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Isoproterenol-induced cardiomyopathy
Chronic administration of isoproterenol (ISO) was performed either by daily subcutaneous injections or by means of surgically implanted osmotic minipumps for a period of 28 days. Rats were injected daily with either ISO (Sigma-Aldrich, USA; 0.3 mg kg–1n= 6) or 0.9% saline (control, n= 4). An osmotic minipump was surgically implanted subcutaneously into each rat previously anaesthetized with pentobarbital. In the ISO group (n= 10), ISO was delivered at a rate of 4.2 mg kg–1 day–1. In the control group (n= 10), the pumps were filled with 0.9% saline. Cardiomyopathy, manifested by cardiac hypertrophy and reduced functional performance, develops within 28 days of ISO administration (Deshaies et al. 1981). LV hypertrophy and decreased shortening in ISO rats were confirmed by echocardiography as previously described.
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Statistics
The results are presented as means ±S.E.M. Statistical differences were tested using ANOVA (P < 0.05).
Results
Effects of db-cAMP on voltage-dependent Ca2+ release in normal myocytes
It has been shown previously that -adrenergic stimulation results in dramatic alterations in the voltage dependency of SR Ca2+ release in rat ventricular myocytes (Piacentino et al. 2000; Viatchenko-Karpinski & Gyrke, 2001). In the presence of -adrenergic agonists, depolarization triggers SR Ca2+ release at membrane potentials as high as 120 mV, where normally no Ca2+ release occurs. This results in the widening and flattening of the bell-shaped, voltage-dependency curve of the amplitude of Ca2+ transients (Piacentino et al. 2000; Viatchenko-Karpinski & Gyrke, 2001). These effects were reported to take place at a constant SR Ca2+ content and, importantly, require the presence of Na+ in the extracellular solution. This suggested the potential involvement of NCX exchange (Piacentino et al. 2000; Viatchenko-Karpinski & Gyrke, 2001). In accordance with these previous findings, Ca2+-transient voltage dependence became widened on application of the membrane-permeable cAMP analogue, db-cAMP (Fig. 1A and B). Also consistent with our previous report, we were able to obtain these effects upon SR Ca2+ release at relatively low levels of PKA stimulation (300 μM db-cAMP), which resulted only in modest increases in Ca2+current (ICa) (<20%; Fig. 1B). Following the addition of a specific inhibitor of the reverse-mode NCX, KB-R7943, the voltage-dependence relationship of Ca2+ release assumed its normal bell shape. We note that KB-R7943 at the concentration used did not affect ICa. Again, these effects of PKA stimulation were not accompanied by any changes in the SR Ca2+ content, as documented by the observation that application of caffeine induced similar-sized Ca2+ transients or NCX currents before and after treatment of myocytes with db-cAMP (Fig. 1C). As shown previously, the lack of changes in SR Ca2+ content in quiescent myocytes exposed to cAMP may be attributable to the fact that the increase in SR Ca2+ uptake is paralleled by an increase in SR Ca2+ leak, so that no net gain in the SR Ca2+ content is attained (Viatchenko-Karpinski & Gyrke, 2001; Szentesi et al. 2004). The elevation of SR Ca2+ content commonly observed following -adrenergic stimulation in myocytes undergoing periodic pacing is apparently due to the enhanced Ca2+ entry via LCCs (Szentesi et al. 2004). Since the SR Ca2+ release at high membrane potentials seemed to rely for its initiation on Ca2+ entries through both LCCs and the reverse-mode NCX instead of just one of the Ca2+-entry pathways, we refer to it as dual trigger-activated Ca2+ release. The experiments below were designed to explore the mechanisms of this newly identified form of release, and how it is influenced by pathological states such as HF.
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A, representative recordings of fluorescence intensity (F/F0) changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mM db-cAMP (middle panel), and after application of 10 μM KB-R7943 (bottom panel). B, voltage dependence of Ca2+ current (ICa) and intracellular Ca2+ transients in myocytes. Data points are means ±S.E.M. (n= 6–17). C, representative recordings of caffeine-induced intracellular Ca2+ transients (upper traces) and Na+–Ca2+ exchange current (INCX) (lower traces) in myocytes under reference conditions, after addition db-cAMP, and in the presence of both db-cAMP and KB-R7943.
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LCC- and NCX-mediated Ca2+ entries in normal myocytes
To directly quantify trans-sarcolemmal Ca2+ entries mediated by LCC and NCX in control and db-cAMP-treated myocytes, we carried out measurements of cytosolic [Ca2+] using confocal fluorescent microscopy and fluo-3. DelPrincipe et al. (2000) demonstrated that fluorescence measured in the presence of 0.1 mM fluo-3 is a linear function of the amount of Ca2+ entry determined by integration of ICa in rat ventricular myocytes. Since fluo-3 fluorescence is proportional to the number of indicator molecules that bind Ca2+, it can be used to measure Ca2+ influx (Zhou & Bers, 2000; DelPrincipe et al. 2000). In these experiments, myocytes were treated with ryanodine and thapsigargin to disable the SR as a Ca2+-storage site. As shown in Fig. 2A, depolarization to membrane potentials ranging from –30 to 150 mV induced a measurable Ca2+ entry into the cytosol under reference conditions. The voltage dependency of the peak amplitude of the [Ca2+] inflow signal (Fig. 2B, squares) followed that of ICa and SR Ca2+ release in myocytes with intact SR. Application of db-cAMP resulted in dramatic increases in [Ca2+] entry at all membrane potentials, and especially at highly positive membrane potentials (>60 mV). In contrast, only a relatively small amount of Ca2+ entry was detected in the absence of db-cAMP (Fig. 2B, circles). Thus, the voltage dependency of Ca2+ entry paralleled the voltage dependency of SR Ca2+ release in cAMP-stimulated myocytes. These results support the hypothesis that the effects of cAMP on SR Ca2+ release could be due to changes in the properties of the Ca2+ trigger signal rather than the result of possible changes in the properties of the mechanism for Ca2+ release from the SR.
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A, representative line-scan images and time-dependent profiles of Ca2+ changes recorded before (upper panels) and after exposure of the myocyte to db-cAMP (lower panels) during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV. B, voltage dependence of the amplitude of the Ca2+-entry signals measured under reference conditions and in the presence of db-cAMP at the end of the depolarization steps. Data points are means ±S.E.M. (n= 6–9).
To further explore interactions between LCCs and NCX in cAMP-stimulated myocytes, we measured separately the Ca2+ entries mediated by these Ca2+-transport mechanisms. The LCC- and NCX-mediated components of the Ca2+ entry were separated pharmacologically by using the specific inhibitors of LCCs and reverse-mode NCX, nifedipine and KB-R7943, respectively (Fig. 3). KB-R7943 did not affect ICa under the conditions of our experiments (Fig. 1). Nifedipine is a selective blocker of LCCs, with no reported side-effects on NCX at concentrations of the drug used in our study (Shen et al. 2000). Thus the effects of these agents upon NCX and LCCs could be considered indeed specific. Under reference conditions, depolarization to 90 mV induced a relatively large total Ca2+-inflow signal (FTot), and smaller signals in the presence of KB-R7943 or nifedipine (FLCC and FNCX, respectively; Fig. 3A, left panel). Simultaneous application of both compounds eliminated entirely the Ca2+-inflow signal, suggesting that the FTot was entirely due to Ca2+ passing through LCCs and NCX. The averaged peak fluorescence signals for different experimental conditions are plotted in Fig. 3B (grey bars). The sum (FTot) of the KB-R7943- and nifedipine-insensitive components, FLCC and FNCX, respectively (black bar inside grey bar), corresponded to the total Ca2+ entry, suggesting that these two transport systems operate independently of each other in the absence of adrenergic stimulation. Following application of cAMP, FTot increased threefold (Fig. 3A, right panel, and Fig. 3B, hatched bars). At the same time, the KB-R7943-insensitive entry increased only by 50%, and no significant changes were detected in the amplitude of the nifedipine-insensitive Ca2+ entry. Consequently, the magnitude of the total Ca2+ signal was threefold higher than the arithmetic sum of the NCX- and LCC-mediated components (Fig. 3B, black bar inside hatched bar). Thus, Ca2+ entries via LCCs and NCX can sum in a nonlinear fashion implying that a synergistic interaction between the two Ca2+-entry systems takes place in myocytes treated with db-cAMP.
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A, representative recordings of intracellular Ca2+ changes during depolarizing steps to 90 mV from a holding potential of –50 mV under different experimental conditions. From top to bottom: reference (corresponding to FTot), in the presence of 10 μM KB-R7943 (corresponding to FLCC), in the presence of 5 μM nifedipine (corresponding to FNCX), and in the presence of 10 μM KB-R7943 + 5 μM nifedipine (both FNCX and FLCC are blocked). B, bar plot summarizing data. Data points are means ±S.E.M. (n= 5–7). Black bars inside the hatched and grey bars for reference conditions denote the arithmetic sum of the peak amplitudes of KB-R7943- and nifedipine-insensitive components in the presence or absence of db-cAMP, respectively.
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ISO-induced model of HF
HF is characterized by impaired intracellular Ca2+ signalling. In order to study whether the synergistic interactions between LCCs and NCX are altered in HF, we performed experiments using two different models of this disease state: HF induced by chronic administration of ISO, and HF induced by chronic volume overload. In myocytes from ISO-treated rats, the amplitudes of Ca2+ transient were reduced, and decay rates slowed under reference conditions (absence of cAMP) (Fig. 4A and B; Table 1). Additionally, the SR Ca2+ content was reduced to 80% of control (Fig. 4C, Table 1). Such alterations in Ca2+ transients are typical of HF (Yao et al. 1998; Houser et al. 2000; Piacentino et al. 2000). Application of db-cAMP to HF myocytes did not lead to the broadened Ca2+-voltage dependency observed in normal myocytes (Fig. 4B, circles). Since SR Ca2+ release is highly sensitive to the SR Ca2+ content, we considered the possibility that the absence of release at positive membrane potentials was due to the reduced SR Ca2+ content in HF myocytes. Therefore, we increased the SR Ca2+ content in HF myocytes by using a loading protocol (see Methods). This protocol allowed us to increase the SR Ca2+ content of HF myocytes to a level similar to that in normal myocytes (Fig. 4C). However, as shown in Fig. 4B (triangles), increasing the SR Ca2+ content had only a minimal effect on the voltage dependence of SR Ca2+ release. Thus, the loss of Ca2+ release induced by a combination of Ca2+ triggers in db-cAMP stimulated HF myocytes was not simply due to changes in the SR Ca2+ content. To further explore the effects of HF on LCC–NCX interactions, we performed fluorometric Ca2+-entry measurements similar to those carried out in myocytes from normal hearts. In HF myocytes, the LCC-mediated Ca2+ signal remained unchanged, but the total, and especially the NCX-mediated, Ca2+ entries were reduced when compared with normal myocytes (Fig. 5). As in normal myocytes, stimulation by db-cAMP increased the LCC-mediated Ca2+ entry, but had no significant effect on the NCX-mediated Ca2+ signal. However, unlike in normal myocytes, the total Ca2+ influx did not change significantly on addition of db-cAMP, and it continued to correspond to the sum of the NCA and LCC components (black bars). Thus, nonlinear summation of Ca2+ entries mediated by LCC and NCX appears to be lost in the ISO model of HF. This could be associated with the reduced NCX activity in myocytes from ISO-treated rats.
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A, representative recordings of F/F0 changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mM db-cAMP (middle panel), and after application of 10 μM KB-R7943 (bottom panel). B, voltage dependence of ICa and intracellular Ca2+ transients in myocytes. Data points are means ±S.E.M. (n= 4–7). C, representative recordings of caffeine-induced intracellular Ca2+ transients (upper traces) and INCX (lower traces) in myocytes before (reference) and after addition db-cAMP, and following execution of a loading protocol to increase SR Ca2+ content.
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A, representative recordings of intracellular Ca2+ changes during depolarizing steps to 90 mV from a holding potential of –50 mV under different experimental conditions. From top to bottom: reference (corresponding to FTot), in the presence of 10 μM KB-R7943 (corresponding to FLCC), in the presence of 5 μM nifedipine (corresponding to FNCX), and in the presence of 10 μM KB-R7943 + 5 μM nifedipine (both FNCX and FLCC are blocked). B, bar plot summarizing the data. Data points are means ±S.E.M. (n= 4–6). Black bars inside the hatched and grey bars for reference conditions denote the arithmetic sum of the peak amplitudes of KB-R7943- and nifedipine-insensitive components in the presence or absence of db-cAMP, respectively.
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ACS model
The results obtained in myocytes isolated from hearts of animals with overload-induced HF are shown in Fig. 6. Similar to the results obtained with the ISO model (Fig. 5; Table 1), the amplitude of Ca2+ transients was reduced and their decay rate slowed in overload-induced HF myocytes. Also, similar to the ISO model, db-cAMP failed to induce Ca2+ release at positive membrane potentials. The normal bell-shaped relationship between membrane potential and the amplitude of Ca2+ transients was unchanged, even when the SR content was increased by using a prepulse protocol. Interestingly, as indexed by the amplitude of the NCX current, NCX activity was increased in overload-induced HF myocytes, rather than decreased as was observed in the ISO-induced HF myocytes (Fig. 6C, Table 1). This suggests that the loss of synergistic interactions occurs regardless of changes in overall NCX activity in HF.
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A, representative recordings of F/F0 changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mM db-cAMP (middle panel), and after application of 10 μM KB-R7943 (bottom panel). B, voltage dependence of ICa and intracellular Ca2+ transients in myocytes. Data points are means ±S.E.M. (n= 4–6). C, representative recordings of caffeine-induced intracellular Ca2+ transients (upper traces) and INCX (lower traces) in myocytes before (reference) and after addition db-cAMP, and following execution of a loading protocol to increase SR Ca2+ content.
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Discussion
In this study, we investigated the respective roles of LCCs and reverse-mode NCX in promoting Ca2+ entry to trigger SR Ca2+ release in myocytes from normal and diseased hearts. Our principal finding is that Ca2+ entries via LCCs and NCX interact in a synergistic manner resulting in a larger total Ca2+ inflow than the sum of entries mediated by each of these Ca2+ transport mechanisms individually. This synergism between the two Ca2+ entry pathways was dependent on stimulation of PKA, and it led to the activation of SR Ca2+ release at highly positive membrane potentials where no or only minimal Ca2+ release occurred in the absence of PKA stimulation. The nonlinear summation of Ca2+ triggers and the associated Ca2+-release component was abolished in the two experimental models of HF (ISO-induced and overload-induced). Our results provide new insights into the mechanisms involved and the modulation of cardiac EC coupling in normal and failing hearts.
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Mechanism of nonlinear summation of Ca2+ entries via NCX and LCCs
The precise molecular mechanism of synergism between Ca2+ entries through LCCs and NCX is not known. Both transporters are localized in the T-tubular membrane of cardiac myocytes (Frank et al. 1992; Kieval et al. 1992; Carl et al. 1995; Sun et al. 1995). While LCCs are restricted mainly to the dyads, the precise localization of NCXs is less certain. Thus, Scriven et al. (2000), using immunofluorescence and wide-field microscopy, have concluded that NCXs, although located in T-tubules, are excluded from the dyads. However, more recently, Thomas et al. (2003), using a new antibody to NCX in combination with confocal imaging and electron microscopy, found that RyRs, LCCs and NCX are closely associated in the dyadic region. Such colocalization of LCC and NCX would make it possible for communication between these Ca2+ transporters. One possibility is that the crosstalk between LCCs and NCX is mediated by Ca2+. Haworth et al. (1991) and Matsuoka et al. (1995) have shown that NCX can be activated by Ca2+ entry through LCCs, presumably by binding to the cytosolic, catalytic Ca2+-activation site on the exchanger. According to several recent studies, the affinity of the cytosolic Ca2+ activation sites is in the range of 300–600 nM (Levitsky et al. 1994; Reeves & Condrescu, 2003) (although some studies reported lower values, e.g. Miura & Kimura, 1989; Weber et al. 2001). This would imply that NCX is only partially activated in resting myocytes. Ca2+ entry through the LCCs on depolarization could enhance Ca2+ binding to the NCX regulatory sites to increase the Ca2+ inflow via the exchanger and, hence, result in stronger Ca2+-release activation. Another possibility is that the synergism between LCCs and NCX involves a Ca2+-dependent signalling pathway, such as protein phosphorylation by CAMKII. It has been reported that NCX is positively modulated by CAMKII (Isosaki et al. 1994). Ca2+ entry through L-type channels could enhance NCX-mediated Ca2+ entry by promoting CAMKII-dependent phosphorylation of NCX, again resulting in an increased net Ca2+ entry into the cell. In both of these scenarios, activation of NCX by Ca2+ would be expected to be greater after stimulation of ICa by cAMP, potentially accounting for the role of PKA stimulation in the observed effects. Clearly, more studies are needed to determine the mechanisms responsible for the synergism between Ca2+ entry through LCCs and NCX.
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Activation of SR Ca2+ release
While our study demonstrated that synergistic interactions between LCCs and NCX resulted in an increased total Ca2+ entry, it is not clear whether this increased Ca2+ entry by itself was solely responsible for SR Ca2+ release activation at positive membrane potentials. Indeed, several additional processes in the junctional SR could contribute to activation of SR Ca2+ release. For example, pre-elevation of Ca2+ at the cytosolic face of the RyR has been shown to increase RyR activation probability in response to a subsequent Ca2+ stimulus by displacing Mg2+ from the RyR activation sites (Zahradnikova et al. 2004). Recordings of single RyR channel activity in response to isolated and paired photolytically induced Ca2+ stimuli revealed that RyR opens only when all four of its Ca2+-activation binding sites are occupied by Ca2+, and blocking any of these sites by Mg2+ prevents channel opening. Thus, an initial Ca2+ entry to the dyadic space via the L-type channels could sensitize RyRs for a subsequent activation by NCX-mediated Ca2+ entry by replacing Mg2+ at the activation sites. PKA phosphorylation of RyR and/or other proteins of the junctional complex could also contribute to dual activation of Ca2+ release by LCCs and NCX by increasing the functional activity of RyRs in cAMP-treated myocytes (Marx et al. 2000; Viatchenko-Karpinski & Gyorke, 2001; Lindegger & Niggli, 2005). However, the issue of effects of phosphorylation and dephosphorylation on RyR function remains controversial (Bers, Eisner & Valdivia, 2003; Terentyev et al. 2003).
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Implication for EC coupling
The dual activation of release by LCCs and NCX occurred at positive membrane potentials of 30 mV and above. Therefore, it would be expected to operate at the peak of the action potential (40–50 mV) in cardiac myocytes. In our experiments, the synergistic interactions between LCCs and NCX required at least some degree of activation of the PKA pathway. Previous studies, however, provided evidence for synergism between NCX and LCCs in activation of Ca2+ release, even in the absence of PKA stimulation, but usually under conditions that stimulate reverse-mode NCX, such as elevated cytosolic [Na+] (Litwin et al. 1998; Vila Petroff et al. 2003). Thus, although PKA stimulation may not be essential to the observed interplay between LCCs and NCX, it can clearly facilitate this process and the resultant Ca2+ release. It is unclear whether endogenous cytosolic cAMP levels in vivo would be sufficient for dual activation of Ca2+ release by LCCs and NCX to the extent observed in this study in myocytes treated with db-cAMP. It is tempting to speculate that this form of release acts in cardiac muscle as a reserve mechanism that is engaged during adrenergic stimulation. In any event, our study, together with those referenced, suggest that the trigger for SR Ca2+ release in cardiac muscle may be more complex than commonly envisioned, and elucidation of interactions between LCCs and NCX is needed for complete understanding of cardiac EC coupling.
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Implication for HF
One of the most important results of our study is that the nonlinear summation of Ca2+ entries and the resulting Ca2+ release was absent in myocytes from hearts from two different HF models. In the case of ISO-induced HF, this result was not surprising considering the reduction in NCX activity found with this model. However, in the case of overload-induced HF, NCX activity was markedly increased. The loss of dual activation of release by LCCs and NCX that occurred despite the enhanced overall NCX Ca2+-transport activity observed in this model appears paradoxical. Several types of explanations can be proposed for this unexpected result. One possibility is that degradation of the T-tubular system in HF myocytes disrupts the crosstalk between NCX, LCC and RyR. HF is generally accompanied by derangement of the ultrastructural architecture of the myocyte membrane system, including reduction of the T-tubular system (Balijepalli et al. 2003). Thus, despite the increased overall number of NCXs, a potential change in distribution of this protein in the sarcolemma could disrupt the crosstalk among NCX, LCC and RyR in the dyadic region required for Ca2+-release initiation. Another possibility is that the cytosolic Ca2+ regulatory site of NCX involved in the summation phenomenon becomes altered in HF as a result of either altered interactions of the exchanger with other proteins or its post-translational modification. Finally, it is conceivable that downregulation of the -adrenergic signalling pathway, seemingly required for nonlinear summation of triggers in control myocytes, contributes to the loss of this phenomenon in HF myocytes. It is to be noted, however, that, in our experiments, we activated PKA directly with a membrane-permeable form of cAMP. Thus alterations in the cascade would have to be downstream of cAMP. Even though we do not as yet know why dual trigger-activated Ca2+ release does not operate in myocytes from failing hearts, it is reasonable to hypothesize that the loss of this process could account for, or contribute to, the reduced contractile performance and diminished contractile reserve in HF.
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Summary
Our results demonstrate that in cAMP-stimulated cardiomyocytes, Ca2+ entries via LCCs and reverse-mode NCX interact in a synergistic manner resulting in a larger total Ca2+ entry than the sum of entries mediated by each of these transport systems individually. This compound Ca2+-entry signal is at least partially responsible for the induction of SR Ca2+ release at highly positive membrane potentials where no Ca2+ release occurs in the absence of PKA activation. The exact linkage between the two Ca2+-entry pathways and its dependence on PKA activation remains to be determined. The demonstration of the interactive relationship between the LCCs and NCX in cAMP-stimulated myocytes provides new insight into the underlying mechanisms of the positive ionotropic effects of catecholamines in the heart. Further, the finding that this interactive mechanism is nonoperational in myocytes from animals in HF suggests at least a partial explanation for the decreased contractile force as well as the inability of increased adrenergic stimulation to produce the necessary compensatory increase in the force of contraction in the failure state.
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Zhou Z & Bers DM (2000). Ca2+ influx via the L-type Ca2+ channel during tail current and above current reversal potential in ferret ventricular myocytes. J Physiol 523, 57–66., 百拇医药(Serge Viatchenko-Karpinsk)
2 Division of Cardiology, Texas Tech University Health Science Center, 3601 4th Street, Lubbock, TX 79430, USA
3 Department of Physiology, Texas Tech University Health Science Center, 3601 4th Street, Lubbock, TX 79430, USA
, http://www.100md.com Abstract
We used confocal Ca2+ imaging and the patch-clamp technique to investigate the interplay between Ca2+ entries through L-type Ca2+ channels (LCCs) and reverse-mode Na+–Ca2+ exchange (NCX) in activating Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac myocytes from normal and failing rat hearts. In normal myocytes exposed to N(6),2'-O-dibutyryl adenosine-3',5'-cyclic monophosphate (db-cAMP, membrane-permeable form of cAMP), the bell-shaped voltage dependence of cytosolic Ca2+ transients was dramatically broadened due to activation of SR Ca2+ release at high membrane potentials (30–120 mV). This broadening of Ca2+-transient voltage dependence could be prevented by KB-R7943, an inhibitor of the reverse-mode NCX. Trans-sarcolemmal Ca2+ entries were measured fluorometrically in myocytes during depolarizing steps to high membrane potentials. The total Ca2+ entry (FTot) was separated into two Ca2+ entry components, LCC-mediated (FLCC) and NCX-mediated (FNCX), by exposing the cells to the specific inhibitors of LCCs and reverse-mode NCX, nifedipine and KB-R7943, respectively. In the absence of protein kinase A (PKA) stimulation the amplitude of the Ca2+-inflow signal (FTot) corresponded to the arithmetic sum of the amplitudes of the KB-R7943- and nifedipine-resistant components (FTot=FLCC+FNCX). PKA activation resulted in significant increases in FTot and FLCC. Paradoxically, FTot became threefold larger than the sum of the FNCX and FLCC components. In myocytes from failing hearts, stimulation of PKA failed to induce a shift in Ca2+ release voltage dependence toward more positive membrane potentials. Although the total and NCX-mediated Ca2+ entries were increased again, FTot did not significantly exceed the sum of FLCC and FNCX. We conclude that the LCC and NCX Ca2+-entry pathways interact synergistically to trigger SR Ca2+ release on depolarization to positive membrane potentials in PKA-stimulated cardiac muscle. In heart failure, this new form of Ca2+ release is diminished and may potentially account for the compromised contractile performance and reduced functional reserve in failing hearts.
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Introduction
In cardiac muscle, the process of excitation–contraction (EC) coupling is mediated by Ca2+ influx from the extracellular space activating Ca2+-sensitive Ca2+-release channels (ryanodine receptors, RyR) in the sarcoplasmic reticulum (SR). Elevation of cytosolic Ca2+ concentration resulting from this Ca2+-induced Ca2+ release (CICR) process leads to activation of the contractile filaments and myocyte shortening. A consensus exists that Ca2+ entry via L-type Ca2+ channels (LCCs) represents the main signal for triggering CICR during normal EC coupling (Bers, 2001). This is consistent with the spatial localization of the LCCs just above the RyRs across the junctional cleft (Franzini-Armstrong et al. 1999). However, Ca2+ influx through other entry pathways, including T-type Ca2+ channels and the reverse mode of Na+–Ca2+ exchange (NCX), have been also implicated in activation of SR Ca2+ release under various experimental conditions (Leblanc & Hume, 1990; Lipp & Niggli, 1994; Kohomoto et al. 1994; Sipido, 1998). Additionally, there may be interplay between different Ca2+-entry pathways in initiation of SR Ca2+ release. For example, it has been proposed that Ca2+ entry through LCCs and reverse-mode NCX interact synergistically to activate RyR channels in the SR (Haworth et al. 1991; Litwin et al. 1998; Viatchenko-Karpinski & Gyrke, 2001). Integration of Ca2+-input signals from different entry pathways could provide an important mechanism for regulation of cardiac EC coupling and contractility. However, whether and how such integration of triggers for CICR occurs in cardiac muscle remain to be determined.
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Stimulation through the -adrenergic pathway is a classical regulatory mechanism for controlling the contractile state of the myocardium. -adrenergic agonists increase the amount of Ca2+ released from the SR, resulting in the development of increased contractile force in cardiac muscle. The biochemical signalling mechanisms underlying -adrenergic stimulation involve cAMP-dependent phosphorylation by protein kinase A (PKA) of certain target proteins including the LCC, RyR and phospholamban (Kranias et al. 1985; Bers, 2001; Shannon et al. 2001). In heart failure (HF), the Ca2+-releasing function of the SR appears to be compromised, resulting in depressed intracellular Ca2+ transients and impaired contraction (Houser et al. 2000; Bers, 2001; Eisner et al. 2003). Additionally the Ca2+-release machinery loses its ability to respond to adrenergic stimulation in myocytes from failing hearts, contributing to reduced contractile reserve in HF (Gomez et al. 1997; Bers, 2001; Pogwizd et al. 2001). The precise reasons for these HF-related changes in Ca2+ transients and responsiveness to adrenergic stimulation are not known, and are currently the subject of intense debate.
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In the present study, we investigated the interplay between Ca2+ entries via L-type Ca2+ channels and reverse-mode NCX in activation of SR Ca2+ release, focusing on the influence of adrenergic stimulation and changes occurring after induction of HF. Significantly, our results show that LCCs and NCX interact in a synergistic manner resulting in a larger total Ca2+ entry than the arithmetic sum of entries mediated by each of these transport systems alone. This synergism between the two Ca2+-entry pathways was dependent on stimulation of PKA. The nonlinear summation of Ca2+ triggers and the associated Ca2+-release component was abolished in two experimental models of HF. Our results show for the first time that Ca2+ entries through LCCs and reverse NCX can interact synergistically providing a new Ca2+-release activation mechanism that is turned on by adrenergic stimulation. In HF, this mechanism is compromised, potentially accounting for the reduced SR Ca2+ release and reduced functional reserve in failing hearts.
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Methods
Confocal Ca2+ imaging and patch-clamp measurements
Single ventricular myocytes were isolated by enzymatic dissociation from hearts from male Sprague–Dawley rats (Charles River Laboratories, Raleigh, NC, USA). Adult rats (200–300 g) were killed with an overdose of sodium pentobarbital in accordance with the guidelines of the Institutional Animal Care and Use Committee. Whole-cell transmembrane ionic currents and changes in membrane potential were recorded with an Axopatch 200B amplifier (Axon Instruments, USA) and pClamp (6.03) software. The bathing solution contained (mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 0.5 MgCl2, 10 Hepes and 5.6 glucose; pH 7.3. The pipette solution contained (mM): 70 caesium aspartate, 50 CsCl, 3 ATP(Mg2+), 4 NaCl, 5 Hepes, 0.05 fluo-3 potassium salt; pH 7.3. Voltage pulses of 150 ms duration were applied from a holding potential of –50 mV at 1 min intervals. Signals were sampled at 5 kHz and filtered at 2 kHz. Cytosolic [Ca2+] changes in patch-clamped myocytes were measured with an Olympus Laser Scanning Confocal System (LSM-GB200, Olympus Optical Co. Ltd, Tokyo, Japan), equipped with an Olympus x60 oil, 1.4 NA objective. Fluo-3 was excited at the 488-nm line of an argon laser, with emission collected through a 515-nm long-pass filter. Fluorescence images were recorded in the line-scan mode at the rate of 2 ms per line. In experiments involving measurements of trans-sarcolemmal Ca2+ entries, the pipette solution contained 0.1 mM fluo-3 potassium salt. In certain experiments, the SR Ca2+ content was increased by applying a loading protocol consisting of a train of 50 ms pulses from –50 mV holding potential to 0 mV at 2 Hz. All measurements were performed at 23°C. KB-R7943 (2-(2-(4(4-nitrobenzyloxy) phenyl)ethyl)isothiourea mesylate) was obtained from Tocris (USA). All other reagents were from Sigma-Aldrich (USA).
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Aortocaval shunt-induced HF
The aortocaval shunt (ACS) was produced according to the method described by Garcia & Diebold (1990). Briefly, after the animal was anaesthetized with pentobarbital, the inferior vena cava and abdominal aorta were exposed by laparotomy. An 18 gauge hypodermic needle was advanced into the aorta distal to the left renal artery and through the opposite wall into the inferior vena cava in order to establish a communication between the two vessels. Bulldog vascular clamps were placed across the aorta above and below the entry point, the needle was withdrawn, and the aortic puncture point was sealed with a drop of cyanoacrylate glue. The clamps were removed 30 s later. The presence of a patent shunt was confirmed visually after release of the clamps by the occurrence of swelling of the vena cava and admixture of arterial and venous blood. The laparotomy was then closed. Sham-operated animals (n= 6) serving as controls were subjected to the same surgical procedures except for creation of the shunt. Compensated hypertrophy occurs after 5–6 week of ACS, with progression to HF after 8–12 week (Garcia & Diebold, 1990; Pieruzzi et al. 1995). In vivo cardiac haemodynamic measurements and confirmation of the shunt were done using the Hewlett Packard Sonos 5500 Ultrasound system (12 MHz transducer). M-mode echocardiograms were recorded at the left ventricular (LV) papillary muscle level to measure end-diastolic and end-systolic dimension, fractional shortening, and end-diastolic posterior wall thickness. ACS rats without LV dilatation and decreased shortening after 8–12 weeks of ACS were excluded from the study.
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Isoproterenol-induced cardiomyopathy
Chronic administration of isoproterenol (ISO) was performed either by daily subcutaneous injections or by means of surgically implanted osmotic minipumps for a period of 28 days. Rats were injected daily with either ISO (Sigma-Aldrich, USA; 0.3 mg kg–1n= 6) or 0.9% saline (control, n= 4). An osmotic minipump was surgically implanted subcutaneously into each rat previously anaesthetized with pentobarbital. In the ISO group (n= 10), ISO was delivered at a rate of 4.2 mg kg–1 day–1. In the control group (n= 10), the pumps were filled with 0.9% saline. Cardiomyopathy, manifested by cardiac hypertrophy and reduced functional performance, develops within 28 days of ISO administration (Deshaies et al. 1981). LV hypertrophy and decreased shortening in ISO rats were confirmed by echocardiography as previously described.
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Statistics
The results are presented as means ±S.E.M. Statistical differences were tested using ANOVA (P < 0.05).
Results
Effects of db-cAMP on voltage-dependent Ca2+ release in normal myocytes
It has been shown previously that -adrenergic stimulation results in dramatic alterations in the voltage dependency of SR Ca2+ release in rat ventricular myocytes (Piacentino et al. 2000; Viatchenko-Karpinski & Gyrke, 2001). In the presence of -adrenergic agonists, depolarization triggers SR Ca2+ release at membrane potentials as high as 120 mV, where normally no Ca2+ release occurs. This results in the widening and flattening of the bell-shaped, voltage-dependency curve of the amplitude of Ca2+ transients (Piacentino et al. 2000; Viatchenko-Karpinski & Gyrke, 2001). These effects were reported to take place at a constant SR Ca2+ content and, importantly, require the presence of Na+ in the extracellular solution. This suggested the potential involvement of NCX exchange (Piacentino et al. 2000; Viatchenko-Karpinski & Gyrke, 2001). In accordance with these previous findings, Ca2+-transient voltage dependence became widened on application of the membrane-permeable cAMP analogue, db-cAMP (Fig. 1A and B). Also consistent with our previous report, we were able to obtain these effects upon SR Ca2+ release at relatively low levels of PKA stimulation (300 μM db-cAMP), which resulted only in modest increases in Ca2+current (ICa) (<20%; Fig. 1B). Following the addition of a specific inhibitor of the reverse-mode NCX, KB-R7943, the voltage-dependence relationship of Ca2+ release assumed its normal bell shape. We note that KB-R7943 at the concentration used did not affect ICa. Again, these effects of PKA stimulation were not accompanied by any changes in the SR Ca2+ content, as documented by the observation that application of caffeine induced similar-sized Ca2+ transients or NCX currents before and after treatment of myocytes with db-cAMP (Fig. 1C). As shown previously, the lack of changes in SR Ca2+ content in quiescent myocytes exposed to cAMP may be attributable to the fact that the increase in SR Ca2+ uptake is paralleled by an increase in SR Ca2+ leak, so that no net gain in the SR Ca2+ content is attained (Viatchenko-Karpinski & Gyrke, 2001; Szentesi et al. 2004). The elevation of SR Ca2+ content commonly observed following -adrenergic stimulation in myocytes undergoing periodic pacing is apparently due to the enhanced Ca2+ entry via LCCs (Szentesi et al. 2004). Since the SR Ca2+ release at high membrane potentials seemed to rely for its initiation on Ca2+ entries through both LCCs and the reverse-mode NCX instead of just one of the Ca2+-entry pathways, we refer to it as dual trigger-activated Ca2+ release. The experiments below were designed to explore the mechanisms of this newly identified form of release, and how it is influenced by pathological states such as HF.
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A, representative recordings of fluorescence intensity (F/F0) changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mM db-cAMP (middle panel), and after application of 10 μM KB-R7943 (bottom panel). B, voltage dependence of Ca2+ current (ICa) and intracellular Ca2+ transients in myocytes. Data points are means ±S.E.M. (n= 6–17). C, representative recordings of caffeine-induced intracellular Ca2+ transients (upper traces) and Na+–Ca2+ exchange current (INCX) (lower traces) in myocytes under reference conditions, after addition db-cAMP, and in the presence of both db-cAMP and KB-R7943.
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LCC- and NCX-mediated Ca2+ entries in normal myocytes
To directly quantify trans-sarcolemmal Ca2+ entries mediated by LCC and NCX in control and db-cAMP-treated myocytes, we carried out measurements of cytosolic [Ca2+] using confocal fluorescent microscopy and fluo-3. DelPrincipe et al. (2000) demonstrated that fluorescence measured in the presence of 0.1 mM fluo-3 is a linear function of the amount of Ca2+ entry determined by integration of ICa in rat ventricular myocytes. Since fluo-3 fluorescence is proportional to the number of indicator molecules that bind Ca2+, it can be used to measure Ca2+ influx (Zhou & Bers, 2000; DelPrincipe et al. 2000). In these experiments, myocytes were treated with ryanodine and thapsigargin to disable the SR as a Ca2+-storage site. As shown in Fig. 2A, depolarization to membrane potentials ranging from –30 to 150 mV induced a measurable Ca2+ entry into the cytosol under reference conditions. The voltage dependency of the peak amplitude of the [Ca2+] inflow signal (Fig. 2B, squares) followed that of ICa and SR Ca2+ release in myocytes with intact SR. Application of db-cAMP resulted in dramatic increases in [Ca2+] entry at all membrane potentials, and especially at highly positive membrane potentials (>60 mV). In contrast, only a relatively small amount of Ca2+ entry was detected in the absence of db-cAMP (Fig. 2B, circles). Thus, the voltage dependency of Ca2+ entry paralleled the voltage dependency of SR Ca2+ release in cAMP-stimulated myocytes. These results support the hypothesis that the effects of cAMP on SR Ca2+ release could be due to changes in the properties of the Ca2+ trigger signal rather than the result of possible changes in the properties of the mechanism for Ca2+ release from the SR.
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A, representative line-scan images and time-dependent profiles of Ca2+ changes recorded before (upper panels) and after exposure of the myocyte to db-cAMP (lower panels) during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV. B, voltage dependence of the amplitude of the Ca2+-entry signals measured under reference conditions and in the presence of db-cAMP at the end of the depolarization steps. Data points are means ±S.E.M. (n= 6–9).
To further explore interactions between LCCs and NCX in cAMP-stimulated myocytes, we measured separately the Ca2+ entries mediated by these Ca2+-transport mechanisms. The LCC- and NCX-mediated components of the Ca2+ entry were separated pharmacologically by using the specific inhibitors of LCCs and reverse-mode NCX, nifedipine and KB-R7943, respectively (Fig. 3). KB-R7943 did not affect ICa under the conditions of our experiments (Fig. 1). Nifedipine is a selective blocker of LCCs, with no reported side-effects on NCX at concentrations of the drug used in our study (Shen et al. 2000). Thus the effects of these agents upon NCX and LCCs could be considered indeed specific. Under reference conditions, depolarization to 90 mV induced a relatively large total Ca2+-inflow signal (FTot), and smaller signals in the presence of KB-R7943 or nifedipine (FLCC and FNCX, respectively; Fig. 3A, left panel). Simultaneous application of both compounds eliminated entirely the Ca2+-inflow signal, suggesting that the FTot was entirely due to Ca2+ passing through LCCs and NCX. The averaged peak fluorescence signals for different experimental conditions are plotted in Fig. 3B (grey bars). The sum (FTot) of the KB-R7943- and nifedipine-insensitive components, FLCC and FNCX, respectively (black bar inside grey bar), corresponded to the total Ca2+ entry, suggesting that these two transport systems operate independently of each other in the absence of adrenergic stimulation. Following application of cAMP, FTot increased threefold (Fig. 3A, right panel, and Fig. 3B, hatched bars). At the same time, the KB-R7943-insensitive entry increased only by 50%, and no significant changes were detected in the amplitude of the nifedipine-insensitive Ca2+ entry. Consequently, the magnitude of the total Ca2+ signal was threefold higher than the arithmetic sum of the NCX- and LCC-mediated components (Fig. 3B, black bar inside hatched bar). Thus, Ca2+ entries via LCCs and NCX can sum in a nonlinear fashion implying that a synergistic interaction between the two Ca2+-entry systems takes place in myocytes treated with db-cAMP.
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A, representative recordings of intracellular Ca2+ changes during depolarizing steps to 90 mV from a holding potential of –50 mV under different experimental conditions. From top to bottom: reference (corresponding to FTot), in the presence of 10 μM KB-R7943 (corresponding to FLCC), in the presence of 5 μM nifedipine (corresponding to FNCX), and in the presence of 10 μM KB-R7943 + 5 μM nifedipine (both FNCX and FLCC are blocked). B, bar plot summarizing data. Data points are means ±S.E.M. (n= 5–7). Black bars inside the hatched and grey bars for reference conditions denote the arithmetic sum of the peak amplitudes of KB-R7943- and nifedipine-insensitive components in the presence or absence of db-cAMP, respectively.
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ISO-induced model of HF
HF is characterized by impaired intracellular Ca2+ signalling. In order to study whether the synergistic interactions between LCCs and NCX are altered in HF, we performed experiments using two different models of this disease state: HF induced by chronic administration of ISO, and HF induced by chronic volume overload. In myocytes from ISO-treated rats, the amplitudes of Ca2+ transient were reduced, and decay rates slowed under reference conditions (absence of cAMP) (Fig. 4A and B; Table 1). Additionally, the SR Ca2+ content was reduced to 80% of control (Fig. 4C, Table 1). Such alterations in Ca2+ transients are typical of HF (Yao et al. 1998; Houser et al. 2000; Piacentino et al. 2000). Application of db-cAMP to HF myocytes did not lead to the broadened Ca2+-voltage dependency observed in normal myocytes (Fig. 4B, circles). Since SR Ca2+ release is highly sensitive to the SR Ca2+ content, we considered the possibility that the absence of release at positive membrane potentials was due to the reduced SR Ca2+ content in HF myocytes. Therefore, we increased the SR Ca2+ content in HF myocytes by using a loading protocol (see Methods). This protocol allowed us to increase the SR Ca2+ content of HF myocytes to a level similar to that in normal myocytes (Fig. 4C). However, as shown in Fig. 4B (triangles), increasing the SR Ca2+ content had only a minimal effect on the voltage dependence of SR Ca2+ release. Thus, the loss of Ca2+ release induced by a combination of Ca2+ triggers in db-cAMP stimulated HF myocytes was not simply due to changes in the SR Ca2+ content. To further explore the effects of HF on LCC–NCX interactions, we performed fluorometric Ca2+-entry measurements similar to those carried out in myocytes from normal hearts. In HF myocytes, the LCC-mediated Ca2+ signal remained unchanged, but the total, and especially the NCX-mediated, Ca2+ entries were reduced when compared with normal myocytes (Fig. 5). As in normal myocytes, stimulation by db-cAMP increased the LCC-mediated Ca2+ entry, but had no significant effect on the NCX-mediated Ca2+ signal. However, unlike in normal myocytes, the total Ca2+ influx did not change significantly on addition of db-cAMP, and it continued to correspond to the sum of the NCA and LCC components (black bars). Thus, nonlinear summation of Ca2+ entries mediated by LCC and NCX appears to be lost in the ISO model of HF. This could be associated with the reduced NCX activity in myocytes from ISO-treated rats.
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A, representative recordings of F/F0 changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mM db-cAMP (middle panel), and after application of 10 μM KB-R7943 (bottom panel). B, voltage dependence of ICa and intracellular Ca2+ transients in myocytes. Data points are means ±S.E.M. (n= 4–7). C, representative recordings of caffeine-induced intracellular Ca2+ transients (upper traces) and INCX (lower traces) in myocytes before (reference) and after addition db-cAMP, and following execution of a loading protocol to increase SR Ca2+ content.
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A, representative recordings of intracellular Ca2+ changes during depolarizing steps to 90 mV from a holding potential of –50 mV under different experimental conditions. From top to bottom: reference (corresponding to FTot), in the presence of 10 μM KB-R7943 (corresponding to FLCC), in the presence of 5 μM nifedipine (corresponding to FNCX), and in the presence of 10 μM KB-R7943 + 5 μM nifedipine (both FNCX and FLCC are blocked). B, bar plot summarizing the data. Data points are means ±S.E.M. (n= 4–6). Black bars inside the hatched and grey bars for reference conditions denote the arithmetic sum of the peak amplitudes of KB-R7943- and nifedipine-insensitive components in the presence or absence of db-cAMP, respectively.
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ACS model
The results obtained in myocytes isolated from hearts of animals with overload-induced HF are shown in Fig. 6. Similar to the results obtained with the ISO model (Fig. 5; Table 1), the amplitude of Ca2+ transients was reduced and their decay rate slowed in overload-induced HF myocytes. Also, similar to the ISO model, db-cAMP failed to induce Ca2+ release at positive membrane potentials. The normal bell-shaped relationship between membrane potential and the amplitude of Ca2+ transients was unchanged, even when the SR content was increased by using a prepulse protocol. Interestingly, as indexed by the amplitude of the NCX current, NCX activity was increased in overload-induced HF myocytes, rather than decreased as was observed in the ISO-induced HF myocytes (Fig. 6C, Table 1). This suggests that the loss of synergistic interactions occurs regardless of changes in overall NCX activity in HF.
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A, representative recordings of F/F0 changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of –50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mM db-cAMP (middle panel), and after application of 10 μM KB-R7943 (bottom panel). B, voltage dependence of ICa and intracellular Ca2+ transients in myocytes. Data points are means ±S.E.M. (n= 4–6). C, representative recordings of caffeine-induced intracellular Ca2+ transients (upper traces) and INCX (lower traces) in myocytes before (reference) and after addition db-cAMP, and following execution of a loading protocol to increase SR Ca2+ content.
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Discussion
In this study, we investigated the respective roles of LCCs and reverse-mode NCX in promoting Ca2+ entry to trigger SR Ca2+ release in myocytes from normal and diseased hearts. Our principal finding is that Ca2+ entries via LCCs and NCX interact in a synergistic manner resulting in a larger total Ca2+ inflow than the sum of entries mediated by each of these Ca2+ transport mechanisms individually. This synergism between the two Ca2+ entry pathways was dependent on stimulation of PKA, and it led to the activation of SR Ca2+ release at highly positive membrane potentials where no or only minimal Ca2+ release occurred in the absence of PKA stimulation. The nonlinear summation of Ca2+ triggers and the associated Ca2+-release component was abolished in the two experimental models of HF (ISO-induced and overload-induced). Our results provide new insights into the mechanisms involved and the modulation of cardiac EC coupling in normal and failing hearts.
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Mechanism of nonlinear summation of Ca2+ entries via NCX and LCCs
The precise molecular mechanism of synergism between Ca2+ entries through LCCs and NCX is not known. Both transporters are localized in the T-tubular membrane of cardiac myocytes (Frank et al. 1992; Kieval et al. 1992; Carl et al. 1995; Sun et al. 1995). While LCCs are restricted mainly to the dyads, the precise localization of NCXs is less certain. Thus, Scriven et al. (2000), using immunofluorescence and wide-field microscopy, have concluded that NCXs, although located in T-tubules, are excluded from the dyads. However, more recently, Thomas et al. (2003), using a new antibody to NCX in combination with confocal imaging and electron microscopy, found that RyRs, LCCs and NCX are closely associated in the dyadic region. Such colocalization of LCC and NCX would make it possible for communication between these Ca2+ transporters. One possibility is that the crosstalk between LCCs and NCX is mediated by Ca2+. Haworth et al. (1991) and Matsuoka et al. (1995) have shown that NCX can be activated by Ca2+ entry through LCCs, presumably by binding to the cytosolic, catalytic Ca2+-activation site on the exchanger. According to several recent studies, the affinity of the cytosolic Ca2+ activation sites is in the range of 300–600 nM (Levitsky et al. 1994; Reeves & Condrescu, 2003) (although some studies reported lower values, e.g. Miura & Kimura, 1989; Weber et al. 2001). This would imply that NCX is only partially activated in resting myocytes. Ca2+ entry through the LCCs on depolarization could enhance Ca2+ binding to the NCX regulatory sites to increase the Ca2+ inflow via the exchanger and, hence, result in stronger Ca2+-release activation. Another possibility is that the synergism between LCCs and NCX involves a Ca2+-dependent signalling pathway, such as protein phosphorylation by CAMKII. It has been reported that NCX is positively modulated by CAMKII (Isosaki et al. 1994). Ca2+ entry through L-type channels could enhance NCX-mediated Ca2+ entry by promoting CAMKII-dependent phosphorylation of NCX, again resulting in an increased net Ca2+ entry into the cell. In both of these scenarios, activation of NCX by Ca2+ would be expected to be greater after stimulation of ICa by cAMP, potentially accounting for the role of PKA stimulation in the observed effects. Clearly, more studies are needed to determine the mechanisms responsible for the synergism between Ca2+ entry through LCCs and NCX.
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Activation of SR Ca2+ release
While our study demonstrated that synergistic interactions between LCCs and NCX resulted in an increased total Ca2+ entry, it is not clear whether this increased Ca2+ entry by itself was solely responsible for SR Ca2+ release activation at positive membrane potentials. Indeed, several additional processes in the junctional SR could contribute to activation of SR Ca2+ release. For example, pre-elevation of Ca2+ at the cytosolic face of the RyR has been shown to increase RyR activation probability in response to a subsequent Ca2+ stimulus by displacing Mg2+ from the RyR activation sites (Zahradnikova et al. 2004). Recordings of single RyR channel activity in response to isolated and paired photolytically induced Ca2+ stimuli revealed that RyR opens only when all four of its Ca2+-activation binding sites are occupied by Ca2+, and blocking any of these sites by Mg2+ prevents channel opening. Thus, an initial Ca2+ entry to the dyadic space via the L-type channels could sensitize RyRs for a subsequent activation by NCX-mediated Ca2+ entry by replacing Mg2+ at the activation sites. PKA phosphorylation of RyR and/or other proteins of the junctional complex could also contribute to dual activation of Ca2+ release by LCCs and NCX by increasing the functional activity of RyRs in cAMP-treated myocytes (Marx et al. 2000; Viatchenko-Karpinski & Gyorke, 2001; Lindegger & Niggli, 2005). However, the issue of effects of phosphorylation and dephosphorylation on RyR function remains controversial (Bers, Eisner & Valdivia, 2003; Terentyev et al. 2003).
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Implication for EC coupling
The dual activation of release by LCCs and NCX occurred at positive membrane potentials of 30 mV and above. Therefore, it would be expected to operate at the peak of the action potential (40–50 mV) in cardiac myocytes. In our experiments, the synergistic interactions between LCCs and NCX required at least some degree of activation of the PKA pathway. Previous studies, however, provided evidence for synergism between NCX and LCCs in activation of Ca2+ release, even in the absence of PKA stimulation, but usually under conditions that stimulate reverse-mode NCX, such as elevated cytosolic [Na+] (Litwin et al. 1998; Vila Petroff et al. 2003). Thus, although PKA stimulation may not be essential to the observed interplay between LCCs and NCX, it can clearly facilitate this process and the resultant Ca2+ release. It is unclear whether endogenous cytosolic cAMP levels in vivo would be sufficient for dual activation of Ca2+ release by LCCs and NCX to the extent observed in this study in myocytes treated with db-cAMP. It is tempting to speculate that this form of release acts in cardiac muscle as a reserve mechanism that is engaged during adrenergic stimulation. In any event, our study, together with those referenced, suggest that the trigger for SR Ca2+ release in cardiac muscle may be more complex than commonly envisioned, and elucidation of interactions between LCCs and NCX is needed for complete understanding of cardiac EC coupling.
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Implication for HF
One of the most important results of our study is that the nonlinear summation of Ca2+ entries and the resulting Ca2+ release was absent in myocytes from hearts from two different HF models. In the case of ISO-induced HF, this result was not surprising considering the reduction in NCX activity found with this model. However, in the case of overload-induced HF, NCX activity was markedly increased. The loss of dual activation of release by LCCs and NCX that occurred despite the enhanced overall NCX Ca2+-transport activity observed in this model appears paradoxical. Several types of explanations can be proposed for this unexpected result. One possibility is that degradation of the T-tubular system in HF myocytes disrupts the crosstalk between NCX, LCC and RyR. HF is generally accompanied by derangement of the ultrastructural architecture of the myocyte membrane system, including reduction of the T-tubular system (Balijepalli et al. 2003). Thus, despite the increased overall number of NCXs, a potential change in distribution of this protein in the sarcolemma could disrupt the crosstalk among NCX, LCC and RyR in the dyadic region required for Ca2+-release initiation. Another possibility is that the cytosolic Ca2+ regulatory site of NCX involved in the summation phenomenon becomes altered in HF as a result of either altered interactions of the exchanger with other proteins or its post-translational modification. Finally, it is conceivable that downregulation of the -adrenergic signalling pathway, seemingly required for nonlinear summation of triggers in control myocytes, contributes to the loss of this phenomenon in HF myocytes. It is to be noted, however, that, in our experiments, we activated PKA directly with a membrane-permeable form of cAMP. Thus alterations in the cascade would have to be downstream of cAMP. Even though we do not as yet know why dual trigger-activated Ca2+ release does not operate in myocytes from failing hearts, it is reasonable to hypothesize that the loss of this process could account for, or contribute to, the reduced contractile performance and diminished contractile reserve in HF.
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Summary
Our results demonstrate that in cAMP-stimulated cardiomyocytes, Ca2+ entries via LCCs and reverse-mode NCX interact in a synergistic manner resulting in a larger total Ca2+ entry than the sum of entries mediated by each of these transport systems individually. This compound Ca2+-entry signal is at least partially responsible for the induction of SR Ca2+ release at highly positive membrane potentials where no Ca2+ release occurs in the absence of PKA activation. The exact linkage between the two Ca2+-entry pathways and its dependence on PKA activation remains to be determined. The demonstration of the interactive relationship between the LCCs and NCX in cAMP-stimulated myocytes provides new insight into the underlying mechanisms of the positive ionotropic effects of catecholamines in the heart. Further, the finding that this interactive mechanism is nonoperational in myocytes from animals in HF suggests at least a partial explanation for the decreased contractile force as well as the inability of increased adrenergic stimulation to produce the necessary compensatory increase in the force of contraction in the failure state.
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