HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 291/4/H1623    most recent 01245.2005v1 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 ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Shutt, R. H. Articles by Howlett, S. E. Search for Related Content PubMed PubMed Citation Articles by Shutt, R. H. Articles by Howlett, S. E.

Increases in diastolic [Ca²⁺] can contribute to positive inotropy in guinea pig ventricular myocytes in the absence of changes in amplitudes of Ca²⁺ transients

Robin H. Shutt, Gregory R. Ferrier, and Susan E. Howlett

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada

Submitted 25 November 2005 ; accepted in final form 9 May 2006

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Increases in contraction amplitude following rest or in elevated extracellular Ca²⁺ concentration ([Ca²⁺]) have been attributed to increased sarcoplasmic reticulum (SR) Ca²⁺ stores and/or increased trigger Ca²⁺. However, either manipulation also may elevate diastolic [Ca²⁺]. The objective of this study was to determine whether elevation of diastolic [Ca²⁺] could contribute to positive inotropy in isolated ventricular myocytes. Voltage-clamp experiments were conducted with high-resistance microelectrodes in isolated myocytes at 37°C. Intracellular free [Ca²⁺] was measured with fura-2, and cell shortening was measured with an edge detector. SR Ca²⁺ stores were assessed with 10 mM caffeine (0 mM Na⁺, 0 mM Ca²⁺). Following a period of rest, cells were activated with trains of pulses, which generated contractions of increasing amplitude, called positive staircases. Positive staircases were accompanied by increasing diastolic [Ca²⁺] but no change in Ca²⁺ transient amplitudes. When extracellular [Ca²⁺] was elevated from 2.0 to 5.0 mM, resting intracellular [Ca²⁺] increased and resting cell length decreased. Amplitudes of contractions and L-type Ca²⁺ current increased in elevated extracellular [Ca²⁺], although SR Ca²⁺ stores, assessed by rapid application of caffeine, did not increase. Although Ca²⁺ transient amplitude did not increase in 5.0 mM extracellular [Ca²⁺], diastolic [Ca²⁺] continued to increase with increasing extracellular [Ca²⁺]. These data suggest that increased diastolic [Ca²⁺] contributes to positive inotropy following rest or with increasing extracellular [Ca²⁺] in guinea pig ventricular myocytes.

excitation-contraction coupling; fura-2 fluorescence; extracellular Ca²⁺ concentration; positive staircases

CICR is not all or none; rather, the amplitudes of Ca²⁺ transients are graded by the amplitude of I_Ca-L. Gradation of CICR is believed to occur by way of local control of Ca²⁺ release by L-type Ca²⁺ channels (3, 32). SR Ca²⁺ is released in units called Ca²⁺ sparks (8). Ca²⁺ sparks represent the coordinated opening of a number of RyRs, which function as a Ca²⁺ release unit (8). A Ca²⁺ release unit can be activated by Ca²⁺ entry via an adjacent L-type Ca²⁺ channel. However, these Ca²⁺ release units are not normally activated by Ca²⁺ released by neighboring groups of RyRs (8, 3, 32). Therefore, the number of L-type Ca²⁺ channels that open determines the number of local Ca²⁺ release units activated (3, 32). The cardiac action potential is believed to activate a large number of L-type Ca²⁺ channels simultaneously and therefore activate a large number of Ca²⁺ release units to produce a large Ca²⁺ transient. In contrast, if the depolarization is weak and few L-type Ca²⁺ channels are activated, then fewer release units will open and the transient will be smaller (5).

Studies have shown that increases in SR Ca²⁺ load also can increase the amplitude of Ca²⁺ transients (22). Increased SR Ca²⁺ load is thought to increase the amount of Ca²⁺ released in each Ca²⁺ spark by increasing the driving force for SR Ca²⁺ release (3, 41). In addition, increased SR Ca²⁺ content also can increase RyR sensitivity to single L-type channel currents, thereby increasing the probability of activation of a Ca²⁺ release unit by any given Ca²⁺ current (3, 8). Thus increased SR Ca²⁺ load can increase the amplitude of Ca²⁺ transients through an increase in the amount of SR Ca²⁺ released through each cluster of RyRs, as well as by increasing the number of Ca²⁺ release units activated (3, 8, 41).

Although I_Ca-L and SR Ca²⁺ load are important mediators of inotropic effects in the heart, several lines of evidence suggest that diastolic Ca²⁺ levels also may play a role in regulation of the amplitude of cardiac contraction (3). Frampton et al. (19) reported that, when extracellular [Ca²⁺] was increased, or when stimulation rate was increased, diastolic [Ca²⁺] increased. The authors suggested that this increase in diastolic Ca²⁺ contributed to positive inotropic effects by increasing SR stores (19). Interestingly, Hattori et al. (20) showed that increases in diastolic Ca²⁺ might play a direct role in mediating positive staircases, which were generated by stimulating cardiac myocytes following a period of rest. In this study, positive staircases were directly related to progressive increases in diastolic [Ca²⁺] and were preserved when SR Ca²⁺ release was blocked by ryanodine (20). Ryanodine should remove the contribution of SR Ca²⁺ release to staircases, and therefore this observation suggests a role for changes in diastolic Ca²⁺ in generation of staircases (20).

Despite evidence demonstrating a role for diastolic Ca²⁺ in inotropy, the contribution of diastolic [Ca²⁺] to regulation of contraction amplitude is disputed. duBell and Houser (11) showed that Ca²⁺ transient amplitude and SR Ca²⁺ content are the primary contributors to positive staircases of contraction. Also, Suda and Kokubun (34) did not detect significant changes in diastolic Ca²⁺ when they elevated extracellular [Ca²⁺] to produce a positive inotropic effect. Although some studies show that positive staircases and inotropic responses to elevation of extracellular [Ca²⁺] occur in the absence of changes in diastolic Ca²⁺, they do not eliminate the possibility that diastolic Ca²⁺ could contribute to positive inotropy. Thus the role of diastolic Ca²⁺ in positive inotropy in the heart remains controversial.

In the present study, we examined inotropic effects of diastolic Ca²⁺ during positive staircases initiated by electrical stimulation following rest and in response to elevated extracellular [Ca²⁺] in isolated guinea pig ventricular myocytes at physiological temperatures. As elevation of extracellular [Ca²⁺] increases the amplitudes of contractions in intact cardiac muscle (7) and amplitudes of I_Ca-L and contraction in isolated myocyte models (19, 22, 4), we used elevation of extracellular [Ca²⁺] as an experimental manipulation to produce positive inotropy. Positive inotropy was measured as an increase in the degree of unloaded cell shortening. Although cell shortening does not definitively describe whole heart changes in force of contraction, inotropic responses recorded from unloaded cells parallel responses measured from loaded cells and intact tissue (6, 7). Increased extracellular [Ca²⁺] can modulate EC coupling through several factors that affect CICR, including peak amplitude of I_Ca-L, SR Ca²⁺ load, and/or diastolic [Ca²⁺]. The specific objectives of this study are 1) to determine whether diastolic Ca²⁺ plays a role in positive staircases and 2) to determine whether diastolic Ca²⁺ contributes to the inotropic effects caused by experimental increases in extracellular [Ca²⁺] in myocytes at physiological temperatures.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Myocyte isolation. Experiments were approved by the Dalhousie University Committee on Laboratory Animal Care, in accordance with Canadian Council on Animal Care guidelines. Guinea pig ventricular myocytes were prepared as described previously (38). Briefly, guinea pigs were anesthetized with pentobarbital sodium (Somnatol; 120 mg/kg ip), coinjected with heparin (3,300 IU/kg). Following induction of anesthesia, the chest was opened, and the heart was cannulated in situ. The heart was perfused with a nominally Ca²⁺-free isolation buffer (in mM: 120.5 NaCl, 4 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 10 HEPES, 12 glucose, pH to 7.4 with NaOH). When contractions ceased and blood had been washed from the heart, the isolation buffer was replaced with a buffer of the same composition but with the addition of collagenase (Worthington Type II, 16–23 mg/50 ml) and protease (Sigma, 4 mg/50 ml). Following digestion, the ventricles were excised and placed in a petri dish containing a high K⁺ buffer (in mM: 30 KCl, 3 MgSO₄, 50 L-glutamic acid, 30 KH₂PO₄, 20 taurine, 0.5 EGTA, 10 HEPES, 10 glucose, pH to 7.4 with KOH) and then minced. In some experiments, EGTA was excluded from the high K⁺ buffer. Cells were dissociated from the tissue by gentle agitation and were filtered through a 225-µm filter (Spectra/Mesh). Isolated cells, suspended in 1.0–1.5 ml of high-K⁺ buffer, were incubated for 15–20 min, at room temperature in the dark, with 5 µM fura-2 AM (fura-2 AM stock solution in anhydrous DMSO). Myocytes were then transferred to an experimental chamber installed on the stage of an inverted microscope (Nikon TE200) and were allowed to settle on the glass coverslip bottom of the chamber. Myocytes were superfused with a buffer of the following composition (mM): 45 NaCl, 100 choline chloride, 10 HEPES, 10 glucose, 4 KCl, 1 MgCl₂, 2 CaCl₂, 0.3 lidocaine (37°C). Superfusion of myocytes and isolated cardiac muscle with buffers at hypothermic temperature (22°C) has been shown to produce dramatic changes in EC coupling. Hypothermia inhibits Na⁺ pump function (12), elevates SR Ca²⁺ load, alters myofilament Ca²⁺ sensitivity, and changes I_Ca-L amplitude and inactivation (4). The superfusion buffer, therefore, was warmed to 37°C to approximate guinea pig core temperature (39°C; Ref. 30).

Voltage clamp. Discontinuous single-electrode voltage clamp at a sampling rate of 7–10 kHz was conducted with an Axoclamp 2B current and voltage-clamp amplifier (Axon Instruments, Foster City, CA) and ClampEx software (version 8.1, Axon Instruments). In most experiments, cells were impaled with high-resistance microelectrodes (15–25 M) filled with 2.7 M KCl. In some experiments, myocytes were voltage clamped with patch pipettes (1–2 M). Patch pipettes were filled with a nominally Na⁺-free intracellular solution of the following composition (in mM): 70 KCl, 70 potassium aspartate, 1 MgCl₂, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 2.5 KH₂PO₄, 0.12 CaCl₂ and 0.05 8-bromo cAMP, pH adjusted to 7.2 (16). All test steps were preceded by trains of ten 200-ms-long conditioning pulses from –80 to 0 mV, delivered at a rate of 2 Hz. Na⁺ current was inhibited with 300 µM lidocaine. Cells were voltage clamped at a holding potential of –80 mV, except following conditioning pulse trains when a holding potential of either –60 or –50 mV preceded the test step to facilitate inhibition of Na⁺ current by lidocaine. At concentrations <0.5 mM, lidocaine has been reported to have little effect on I_Ca-L (26) and no effect on the arrhythmogenic transient inward current (26) or on the T-type Ca²⁺ current (36).

[Ca²⁺] in the control solution was 2.0 mM, and this solution was used to superfuse the cells throughout the preceding trains of 10 conditioning pulses. Extracellular [Ca²⁺] was rapidly changed from 2.0 mM to 0.1, 0.5, or 5.0 mM by bathing the cell in buffers of different [Ca²⁺]. A computer-controlled rapid solution changer was used to rapidly expose the cell to solutions with different [Ca²⁺] for 3 s following the conditioning pulse train and throughout the test step (17). In some experiments, the extracellular Mg²⁺ concentration also was elevated from 1 to 4 mM. The elevated Mg²⁺ was applied with the rapid solution changer. The rapid solution changer also was used in other experiments where 0.1 mM Cd²⁺ was added to a 5.0 mM extracellular [Ca²⁺] test solution. Finally, the rapid solution changer was used to rapidly expose the cell to a buffer containing 10 mM caffeine (1.0 s application). Caffeine was used to release SR Ca²⁺ and thereby estimate SR Ca²⁺ load. To inhibit extrusion of Ca²⁺ from the cytosol by the Na⁺/Ca²⁺ exchanger (NCX), caffeine was applied in a nominally Ca²⁺- and Na⁺-free solution (23) of the following composition (in mM): 140 LiCl, 4 KCl, 10 glucose, 5 HEPES, 4 MgCl₂, and 10 caffeine. In a few experiments, caffeine was applied in a buffer of the following composition (in mM): 145 NaCl, 10 HEPES, 10 glucose, 4 KCl, 1 MgCl₂, 2 CaCl₂, 0.3 lidocaine, and 10 caffeine.

Measurement and analysis. Fura-2 fluorescence was recorded and measured as previously described (18). Briefly, an aperture in front of the photomultiplier tube was used to isolate the fura-2-loaded cell in a sampling window. Ca²⁺ bound and unbound fura-2 were excited by UV light at 340 and 380 nm, respectively, by a DeltaRam fluorescence system (Photon Technologies International). Fluorescence emission at 510 nm was measured, at 100 samples/s for each of the 340- and 380-nm excitation wavelengths. Following fluorescence recordings from cells, background fluorescence was determined. To determine intracellular [Ca²⁺], the background values for each excitation wavelength were subtracted from the recordings made during the experimental protocol. The ratio of fluorescence emission recorded during excitation at 340 and 380 nm was determined. Emission ratios were converted to [Ca²⁺] with an in vitro calibration curve determined from known concentrations of Ca²⁺. Simultaneous recordings of contractions and fluorescence were made by splitting the light from the microscope with a dichroic cube. Red light was sent to the closed-circuit camera and video edge detection system, while the remaining light was delivered to the photomultiplier tube for fluorescence measurement. Contraction amplitudes were recorded by tracking one edge of the cell with a Crescent Electronics (Sandy, UT) video edge detector at 120 Hz. In some experiments, resting cell length was determined by tracking both ends of the cell. To determine resting cell length and resting intracellular [Ca²⁺], cells were activated with a short activation protocol to show that they responded to electrical stimuli with contractions and Ca²⁺ transients. Cells were then voltage clamped at a resting potential of –80 mV and exposed to different extracellular [Ca²⁺]. Cells that generated spontaneous Ca²⁺ waves were excluded.

Contraction amplitudes, cell length, and whole cell currents were digitized by a Digidata 1322A analog-to-digital interface (Axon Instruments). Digitization rates varied from 0.7 to 4.7 kHz, depending on the duration of the specific voltage clamp protocol. Contraction amplitudes, cell length, and currents were recorded with ClampEx 8.1 (Axon Instruments) software and analyzed with ClampFit 8.1 (Axon Instruments) software. Sigmaplot 2001 (Jandel Scientific, SPSS) was used to construct graphs. Statistical analyses were performed with either Sigmaplot 2001 or Sigmastat, version 2.03 (Jandel Scientific, SPSS). Mean data were expressed ± SE; * denotes P < 0.05.

Following steps from –50 to 0 mV, peak amplitudes of I_Ca-L were measured as the peak inward current with respect to net current at the end of the 250-ms test step. Following steps from –60 to 0 mV, peak amplitudes of I_Ca-L were measured as the peak current with respect to net current at the time point where I_Ca-L was 90% inactivated on a step to 0 mV. In some experiments, identity of I_Ca-L was confirmed, and measurement techniques validated, with application of Cd²⁺ and determination of Cd²⁺-sensitive current amplitude. These experiments established that measurement of peak inward current, as described above, resulted in minimal underestimation of I_Ca-L. Contraction amplitudes were measured as the difference between peak contraction and the diastolic value immediately preceding the onset of contraction. Following conversion of the fura-2 fluorescence ratio to [Ca²⁺], diastolic [Ca²⁺] (the concentration immediately preceding the test step), peak systolic [Ca²⁺], and Ca²⁺ transient amplitude (difference between systolic and diastolic) were measured.

Sources of chemicals. Lidocaine, choline chloride, HEPES buffer, MgCl₂, anhydrous DMSO, and caffeine were purchased from Sigma Aldrich Canada (Oakville, ON). Invitrogen (Burlington, ON) was the supplier for fura-2 AM. All other chemicals were purchased from BDH (Toronto, ON).

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Role of diastolic [Ca²⁺] in increasing contraction amplitude during positive staircases. In our first set of experiments, we simultaneously recorded contractions and Ca²⁺ transients from voltage-clamped guinea pig ventricular myocytes during positive staircases initiated by regular depolarization following a rest period. Myocytes were activated with trains of ten 200-ms rectangular pulses from –80 to 0 mV, following a 4-s rest period. Pulses were delivered at a rate of 2 Hz. Figure 1 shows a schematic of the activation train (A), as well as representative recordings of contractions and intracellular Ca²⁺ transients (B and C, respectively). The recording of contractions in Fig. 1B shows that the first contraction initiated after a period of rest was small. However, the amplitude of contractions increased stepwise with each sequential activation during the first part of the train. Therefore, these contractions exhibit positive staircases. In contrast, Ca²⁺ transient amplitudes appeared to remain constant throughout the train (Fig. 1C). Mean amplitudes of contraction and intracellular [Ca²⁺] during positive staircases are shown in Fig. 1, D–F. Only data from responses to activation pulses 2–10 were fitted, as the first response represents a rest response which followed a long period of quiescence. All responses were normalized to the mean magnitude of the 9th and 10th responses to compensate for cell-to-cell variability. Contraction amplitudes increased significantly during the pulse train (Fig. 1D). The solid regression line shows that amplitude of contraction increased over the first half of the train and then remained constant. Figure 1E illustrates the relationship between Ca²⁺ transient amplitudes and beat number for the 10 pulses. Amplitudes of Ca²⁺ transients only increased slightly at the end of the train, but this was not significant. There was no clear relationship between Ca²⁺ transient amplitude and contraction. However, as shown in Fig. 1F, diastolic intracellular [Ca²⁺] increased significantly over the series of pulses. The regression line for diastolic [Ca²⁺] appeared to increase in parallel with the regression for mean amplitudes of contractions. Both peak systolic Ca²⁺ and diastolic Ca²⁺ increased over the 10-pulse train.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Positive staircases of contraction occur in parallel with increasing diastolic Ca2+ concentration ([Ca2+]), not increasing Ca2+ transient amplitude. A: schematic representation of voltage-clamp protocol used to activate the cells. Contractions and Ca2+ transients were elicited by a series of ten 200-ms square pulses from –80 to 0 mV at 2 Hz. Representative examples of contractions (B) and Ca2+ transients (C) were recorded simultaneously. Mean data are expressed in panels D, E, and F. Mean data were normalized to the means of the 9th and 10th responses. The first response was omitted. D: mean contraction amplitudes increased with increasing pulse number. E: mean Ca2+ transient amplitudes do not differ significantly during the train of pulses. F: mean diastolic [Ca2+] increased significantly over the train of 10 pulses. n = 9 Cells. All data are paired. *P < 0.05 as tested by one-way repeated-measures ANOVA.

View larger version (9K): [in this window] [in a new window]   Fig. 2. During positive staircases, increasing contraction amplitudes show a direct relationship to the increase in diastolic [Ca2+]. A: contraction-Ca2+ transient amplitude relationship from the data shown in Fig. 2. Ca2+ transient amplitude shows no relationship to increasing contraction amplitude. B: contraction-diastolic [Ca2+] relationship determined from the data presented in Fig. 2. Contraction amplitude is directly related to diastolic Ca2+. n = 9 Cells. All data are paired.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Increased [Ca2+] causes an elevation in diastolic [Ca2+] and a decrease in cell length. Recordings are from a resting myocyte voltage clamped at –80 mV. The bar indicates the approximate point at which extracellular was elevated from 2.0 to 5.0 mM Ca2+. A: representative recording of diastolic [Ca2+] and cell length during a rapid increase in extracellular [Ca2+] from 2.0 to 5.0 mM. B: when extracellular [Ca2+] was increased, diastolic [Ca2+] increased and resting cell length decreased significantly. n = 8 Cells. All data are paired. *P < 0.05 as tested by paired Student's t-test.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Contraction amplitudes, inward L-type Ca2+ current (ICa-L), and Ca2+ transient amplitudes increase with increasing extracellular [Ca2+]. A: different extracellular [Ca2+] (0.1, 0.5, 2.0, and 5.0 mM) were applied with a computer-controlled rapid solution changer for 3 s before and throughout a step from –50 to 0 mV. The solution application and test step were preceded by 10 pulses at a rate of 2 Hz. Representative transmembrane currents (B), Ca2+ transients (C), and contractions (D) recorded in 0.1 (top), 0.5, 2.0, and 5.0 mM (bottom) extracellular [Ca2+] are shown. Concentration-response curves for ICa-L (E), Ca2+ transient (F), and contraction (G) amplitudes are shown. Dotted lines represent regressions to the mean data. ICa-L (E) and contractions (G) increased in parallel with increasing extracellular [Ca2+]. Ca2+ transient amplitude (F) increased with increasing extracellular [Ca2+]; however, it appeared to saturate and did not increase in 5.0 mM extracellular [Ca2+]. n = 6–14 Cells. All data are paired.

As we were interested in the roles of diastolic Ca²⁺, I_Ca-L, and Ca²⁺ transient amplitude in regulation of the amplitude of contraction, we compared mean amplitudes of contractions, I_Ca-L, and Ca²⁺ transients when extracellular [Ca²⁺] was elevated from 2.0 to 5.0 mM in Fig. 5. Figure 5A shows that contraction amplitude increased significantly in 5.0 mM extracellular [Ca²⁺] compared with 2.0 mM [Ca²⁺]. Elevation of extracellular [Ca²⁺] from 2.0 to 5.0 mM also increased the amplitude of I_Ca-L significantly (Fig. 5B). As shown in Fig. 5C, diastolic and peak systolic intracellular [Ca²⁺] also both increased with increasing extracellular [Ca²⁺]. In contrast, elevation of the extracellular [Ca²⁺] from 2.0 to 5.0 mM did not significantly affect the amplitudes of Ca²⁺ transients. Ca²⁺ transient amplitude appeared to have saturated in 5.0 mM extracellular Ca²⁺. Interestingly, increasing extracellular [Ca²⁺] from 2.0 to 5.0 mM led to positive inotropy under our experimental conditions. Although both the trigger for EC coupling and the final output, contraction, were increased, the positive inotropic effect occurred, despite saturation of Ca²⁺ transient amplitude.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Ca2+ transient amplitude saturates in 5.0 mM Ca2+, despite increases in mean contraction and ICa-L amplitudes as well as diastolic [Ca2+]. Activation protocol is as shown in Fig. 4A. Extracellular [Ca2+] was increased from 2.0 to 5.0 mM for 3 s following conditioning pulse train and throughout a test step from –50 to 0 mV. A and B: mean amplitudes of ICa-L and contractions increased significantly in 5.0 mM extracellular [Ca2+]. C: diastolic and peak systolic intracellular [Ca2+] increased significantly when extracellular [Ca2+] was elevated. Ca2+ transient amplitude did not increase when extracellular [Ca2+] was elevated from 2.0 to 5.0 mM. n = 14 Cells. All data are paired.

Voltage dependence of amplitudes of I_Ca-L, contractions, and Ca²⁺ transients in elevated extracellular [Ca²⁺]. As amplitudes of contractions, I_Ca-L, and Ca²⁺ transients vary with the voltage of test steps to different membrane potentials, it was important to examine responses to a wide range of test step potentials. It was our goal to determine whether the relationship between these variables remained similar to that seen with test steps to only one potential. To that end, test steps were made from a holding potential of –60 mV, following conditioning pulse trains. This activation protocol, shown as a schematic in Fig. 6A, was repeated eight times. The test step potential was increased by 20 mV following each repetition of the protocol to initiate responses by test steps from –60 to +80 mV. This whole sequence was then repeated with the extracellular [Ca²⁺] increased to 5.0 mM for 3 s before and throughout the test step. Contraction, current, and Ca²⁺ transient amplitudes were plotted as functions of test step amplitudes to generate contraction-voltage, current-voltage, and Ca²⁺ transient-voltage relationships as shown in Fig. 6, B–D. Figure 6B shows that elevated extracellular [Ca²⁺] caused a significant increase in the amplitude of contraction and that this increase was observed over a wide range of membrane potentials.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Amplitudes of contraction-voltage, current-voltage, and Ca2+ transient-voltage relationships increase in 5.0 mM extracellular [Ca2+]. A: representative activation protocol. Extracellular [Ca2+] was increased for 3 s before and throughout the test pulse. Amplitudes of mean contraction-voltage (B) and current-voltage (C) relationships are significantly increased by 5.0 mM extracellular [Ca2+]. D: transient amplitude did not increase over the range of voltages tested. E: diastolic [Ca2+] before each of the test steps increased significantly when extracellular [Ca2+] was elevated. n = 14. All data are paired. *P < 0.05, tested with two-way repeated-measures ANOVA.

Surface charge screening caused by high extracellular divalent (21) concentrations might have influenced Ca²⁺ transient amplitude in 5.0 mM extracellular Ca²⁺. To further investigate this possibility, intracellular Ca²⁺ also was measured in myocytes exposed to 5.0 mM extracellular Ca²⁺ and 1.0 mM extracellular Mg²⁺ and compared with intracellular Ca²⁺ measurements from myocytes exposed to 2.0 mM extracellular Ca²⁺ and 4.0 mM Mg²⁺. Responses were elicited by the voltage protocol shown in Fig. 4A. Extracellular solution containing 2.0 mM Ca²⁺ and 4.0 mM Mg²⁺ has the same divalent concentration as 5.0 mM Ca²⁺ and 1.0 mM Mg²⁺. When the mean responses of myocytes to 2.0 and 5.0 mM extracellular Ca²⁺ were compared at the same extracellular divalent concentration, diastolic and peak systolic Ca²⁺ increased significantly with increased extracellular Ca²⁺ (diastolic 167 ± 24 vs. 201 ± 28 nM and systolic 229 ± 29 vs. 276 ± 33 nM, n = 7, P < 0.05). Ca²⁺ transient amplitude, however, did not increase in 5.0 mM extracellular Ca²⁺ (62 ± 8 vs. 75 ± 13 nM, n = 7). Therefore, surface charge effects of elevated extracellular [Ca²⁺] did not alter Ca²⁺ transient amplitude.

Effect of changing extracellular [Ca²⁺] on SR load. In the next series of experiments, we determined whether elevation of the extracellular [Ca²⁺] and the subsequent elevation in diastolic Ca²⁺ levels had an effect on SR Ca²⁺ content. To that end, SR Ca²⁺ content was estimated by rapid application of caffeine. As in previous protocols, extracellular [Ca²⁺] was increased for 3 s following the conditioning pulse train. However, rather than a test step, a 1-s rapid application of 10 mM caffeine solution was used to induce a Ca²⁺ transient. A schematic representation of this protocol is shown in Fig. 7A. Figure 7B shows representative caffeine-induced Ca²⁺ transients. The amplitude of the caffeine-induced Ca²⁺ transient was not elevated when extracellular [Ca²⁺] was increased in this example. The mean data shown in Fig. 7C indicate that SR Ca²⁺ load was not significantly increased by elevation of extracellular [Ca²⁺] from 2.0 to 5.0 mM.

View larger version (15K): [in this window] [in a new window]   Fig. 7. When extracellular [Ca2+] is elevated, sarcoplasmic reticulum (SR) load remains constant. A: activation protocol. Extracellular [Ca2+] was increased for 3 s following a train of 10 pulses from –80 to 0 mV at a rate of 2 Hz. The extracellular bathing solution was then rapidly changed for 1.0 s to a nominally Na+- and Ca2+-free buffer containing 10 mM caffeine to assess SR stores. B: representative caffeine transients elicited following application of either 2.0 (top) or 5.0 mM (bottom) extracellular [Ca2+] for 3 s following the conditioning pulse train. C: mean data show that caffeine-releasable SR stores were similar in 5.0 mM extracellular [Ca2+].

Effect of 100 µM Cd²⁺ on the increase in diastolic Ca²⁺ in myocytes at –50 mV. Myocytes were exposed to buffer containing 100 µM CdCl₂ to inhibit I_Ca-L window current, which could be activated when myocytes are held at –50 mV for 3 s before the test step, as shown in the protocol in Fig. 8A. Figure 8B shows the mean intracellular Ca²⁺ responses of myocytes exposed to 2.0, 5.0, or 5.0 mM extracellular Ca²⁺ plus 100 µM Cd²⁺. Cd²⁺ (100 µM) did not abolish the increases in diastolic and peak systolic intracellular Ca²⁺ associated with the application of 5.0 mM extracellular Ca²⁺. Therefore, in 5.0 mM extracellular Ca²⁺, diastolic Ca²⁺ does not increase due to prolonged activation of I_Ca-L window currents during the 3 s before the test step.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Diastolic and peak systolic Ca2+ increase with increasing extracellular Ca2+ when ICa-L window currents are inhibited. A: activation protocol. Extracellular Ca2+ was increased from 2.0 to 5.0 mM for 3 s before and throughout the test step, either in the absence or the presence of 0.1 mM Cd2+. B: diastolic Ca2+ increased significantly when extracellular Ca2+ was elevated, and this increase was not diminished in the presence of 0.1 mM Cd2+. Peak systolic Ca2+ also increased in 5.0 mM extracellular Ca2+, both in the absence and presence of 0.1 mM Cd2+. n = 7. All data are paired.

View larger version (13K): [in this window] [in a new window]   Fig. 9. Diastolic and peak systolic Ca2+ increase with increasing extracellular Ca2+ when reverse mode Na+/Ca2+ exchanger is inhibited. A: representative activation protocol. Myocytes were voltage clamped with patch pipettes containing a nominally Na+-free intracellular solution. Myocytes then were stimulated with a series of 10 conditioning pulses from –80 to 0 mV at a rate of 2 Hz. Extracellular [Ca2+] was elevated from 2.0 to 5.0 mM for 3 s before and throughout a test step from –50 to 0 mV. B: representative intracellular Ca2+ recordings from a single myocyte. Diastolic and peak systolic Ca2+ appeared to increase with increasing extracellular [Ca2+]. C: mean intracellular [Ca2+] recorded during the step from –50 to 0 mV. Diastolic and peak systolic Ca2+ were significantly elevated in 5.0 mM extracellular [Ca2+]. Ca2+ transient amplitude was not significantly increased in 5.0 mM extracellular [Ca2+] compared with 2.0 mM. n = 5. All data are paired.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

An additional objective of this study was to determine whether diastolic Ca²⁺ contributes to positive inotropy arising from experimental manipulation of extracellular Ca²⁺. When extracellular Ca²⁺ was elevated, we found that resting intracellular [Ca²⁺] levels increased in guinea pig myocytes voltage clamped at –80 mV. These observations agree with the findings of Frampton et al. (19), who showed that diastolic [Ca²⁺] increased in response to elevated extracellular [Ca²⁺] in field-stimulated rat myocytes. In contrast, Janczewski et al. (22) did not report changes in diastolic [Ca²⁺] in response to elevated extracellular [Ca²⁺] in rat myocytes. Our experiments also showed that elevated resting intracellular [Ca²⁺] produced a decrease in resting cell length. These data support the observation that cardiac myocyte resting tension is active and generated by actin-myosin interactions (31). Therefore, experimental elevation of extracellular Ca²⁺ can influence diastolic [Ca²⁺] and thereby influence myofilament activation.

We also found that I_Ca-L, contraction amplitude, diastolic [Ca²⁺], and systolic [Ca²⁺] increased in 5.0 mM extracellular [Ca²⁺]. However, Ca²⁺ transient amplitude did not increase significantly when extracellular [Ca²⁺] was elevated and appeared to be saturated between 2.0 and 5.0 mM extracellular [Ca²⁺]. The increase in amplitudes of contractions, despite saturation of the amplitudes of Ca²⁺ transients, was unanticipated. One possible explanation for our observation is that the amplitude of contractions increased because diastolic [Ca²⁺] increased when extracellular [Ca²⁺] was elevated. Bers (3, 4) has hypothesized that elevated diastolic [Ca²⁺] should be inherently positively inotropic, because it necessarily increases the peak systolic [Ca²⁺] and decreases the intracellular Ca²⁺ buffering capacity. The data presented in the present study support this hypothesis and demonstrate that positive inotropy can arise from elevation of diastolic [Ca²⁺]. The elevation in diastolic [Ca²⁺], which occurred in response to the increase in extracellular [Ca²⁺], led to a corresponding increase in peak systolic [Ca²⁺]. Thus positive inotropy occurred in the absence of increasing Ca²⁺ transient amplitude.

Figure 10A shows the hypothetical cell shortening-intracellular [Ca²⁺] relationship (3, 4). When a Ca²⁺ transient of a given amplitude occurs, the intracellular [Ca²⁺] increases from the diastolic [Ca²⁺] to a peak systolic [Ca²⁺], and cell shortening occurs. When the diastolic [Ca²⁺] is elevated, as in Fig. 10B, a rightward shift in the starting point on the cell shortening-intracellular [Ca²⁺] relationship occurs. This rightward shift leads to a larger contraction for a transient of the same amplitude as in Fig. 10A. This conclusion is supported by our contraction amplitude data, which show that contraction amplitude continues to increase for a constant Ca²⁺ transient amplitude where diastolic [Ca²⁺] is increasing. Furthermore, we have shown that increases in diastolic [Ca²⁺] caused by increasing extracellular [Ca²⁺] in resting myocytes cause cell shortening. This indicates that we are working in a range of diastolic [Ca²⁺] where active resting tension can be produced.

View larger version (9K): [in this window] [in a new window]   Fig. 10. Cell shortening-[Ca2+] relationship. The effect of increasing diastolic [Ca2+] on cell length can be described by a curve similar to that for the relationship between contractile force and pCa2+. A: the Ca2+ transient (the difference between systolic and diastolic [Ca2+]) will increase intracellular [Ca2+], which in turn increases peak systolic [Ca2+] and leads to cell shortening. B: based on this model, when a higher initial diastolic [Ca2+] is combined with a Ca2+ transient of the same amplitude, the result will be a higher peak systolic [Ca2+], which should lead to further cell shortening. The greater distance between the lines labeled as peak systolic and diastolic where they meet the y-axis (compared with A) indicates a greater degree of cell shortening.

The mechanism responsible for the elevation in diastolic Ca²⁺ following an increase in extracellular Ca²⁺ was not clear. It is possible that Ca²⁺ influx through I_Ca-L window currents is responsible for the increase in diastolic Ca²⁺. Previous studies have shown that 100 µM Cd²⁺ blocks I_Ca-L window current (35). Interestingly, when we blocked I_Ca-L window currents with 100 µM Cd²⁺, we found that diastolic Ca²⁺ levels still increased in 5.0 mM extracellular [Ca²⁺]. Thus the increase in diastolic Ca²⁺ associated with elevated extracellular Ca²⁺ cannot be attributed to I_Ca-L window currents.

Another mechanism that could account for the increase in diastolic Ca²⁺ is reverse mode NCX. However, when we dialyzed myocytes with patch pipettes containing a nominally Na⁺-free intracellular solution to exclude the contribution of reverse mode NCX (16), diastolic Ca²⁺ increased significantly in 5.0 mM extracellular [Ca²⁺]. Thus intracellular Ca²⁺ increased in response to increased extracellular [Ca²⁺] when reverse-mode NCX was inhibited by the absence of intracellular Na⁺. Therefore, although NCX-induced Ca²⁺ entry can contribute to changes in intracellular Ca²⁺ in some situations (4, 24, 37), NCX-induced Ca²⁺ entry is not responsible for increases in diastolic Ca²⁺ in voltage-clamped myocytes held at –50 mV.

Therefore, the increased diastolic Ca²⁺, which occurs in response to elevated extracellular [Ca²⁺], must arise through a mechanism other than I_Ca-L window current or reverse-mode NCX. Other possible mechanisms that might lead to the elevation of diastolic Ca²⁺ must, therefore, be responsible for the increase in diastolic Ca²⁺ associated with elevated extracellular [Ca²⁺]. Enhanced SR Ca²⁺ leak or a decrease in the extrusion of the Ca²⁺ that has leaked from the SR could contribute to the increase in diastolic Ca²⁺ in myocytes voltage clamped at –50 mV.

Our results showed that Ca²⁺-transient amplitudes did not increase in 5.0 mM extracellular [Ca²⁺], despite increased I_Ca-L amplitude. This remained true when extracellular [Ca²⁺] was elevated in the presence of 0.1 mM Cd²⁺, when myocytes were voltage clamped with patch pipettes containing a nominally Na⁺-free intracellular solution, throughout the entire transient-voltage relationship, and when extracellular divalent was balanced in 2.0 mM Ca²⁺ by increasing extracellular [Mg²⁺]. Thus, despite many manipulations, we could not produce a statistically significantly larger Ca²⁺ transient, despite increased I_Ca-L amplitude, in 5.0 mM extracellular Ca²⁺.

There are several possible explanations that could account for our finding that Ca²⁺ transient amplitudes did not increase when I_Ca-L amplitude continued to increase. First, it is possible that saturation of Ca²⁺ transient amplitude occurs in response to inhibition of CICR by elevated diastolic [Ca²⁺]. Xu et al. (39) have shown that the RyR can be inhibited by high diastolic [Ca²⁺], although the [Ca²⁺] in their experiments were substantially higher than those seen in our experiments. Second, elevated intracellular [Ca²⁺] may increase spark frequency (18), which would lead to an increase in the number of refractory spark units at any one time (33), and Ca²⁺ transient amplitude could saturate due to inactivation of that subset of refractory RyRs (33). Also, Ca²⁺ transient amplitude may saturate due to saturation of local control of CICR at the level of the RyR. A finite number of Ca²⁺ release units are coupled to L-type Ca²⁺ channels (32, 33); therefore, as the amplitude of I_Ca-L increases, the number of Ca²⁺ release units available for activation must approach a maximum. Once this maximum is reached, no further increase in Ca²⁺ transient amplitude could occur. Finally, it is possible that the saturation of Ca²⁺ transient amplitudes arises from a saturation of the amount of SR Ca²⁺ available for release in response to the trigger Ca²⁺ current. In support of this view, Janczewski et al. (22) have shown that, when extracellular Ca²⁺ is elevated, Ca²⁺ transient amplitude does not increase, despite increases in the amplitude of I_Ca-L, unless SR Ca²⁺ load also is elevated. When the findings of Janczewski et al. are considered in conjunction with the findings of the present study, the data suggest strongly that saturation of the amount of Ca²⁺ available for release from the SR is responsible for the saturation of the Ca²⁺ transient, which we report in this study.

The results of this study may be relevant to disease states where diastolic Ca²⁺ is elevated. Following myocardial ischemia, in late reperfusion, Ca²⁺ transient amplitudes are depressed; however, diastolic [Ca²⁺] is elevated (25). Elevated diastolic Ca²⁺ in this situation could contribute to larger contraction amplitudes, abrogating the contractile depression associated with myocardial stunning. However, in general, prolonged periods of elevated intracellular [Ca²⁺] are damaging to the heart. Studies have shown that intracellular [Ca²⁺] is increased in heart disease (40). In most disease states, such as heart failure, the increased intracellular [Ca²⁺] levels do not improve contractile function (40). Rather, prolonged exposure to increased intracellular [Ca²⁺] can activate pathways in which Ca²⁺ acts as a second messenger to produce deleterious genomic effects (1, 2, 10). Ca²⁺-activated second-messenger signaling cascades, such as the Ca²⁺-calcineurin/NFAT cascade, lead to gene expression changes seen in cardiac myocytes undergoing pathological hypertrophy (27, 28). Thus the increased diastolic [Ca²⁺] level seen in heart failure is associated with deficits in contractile force generation, rather than improvements.

In summary, the results of this study demonstrate that diastolic [Ca²⁺] can contribute to the positive inotropic effects that occur following rest or when extracellular [Ca²⁺] is elevated. Therefore, although amplitudes of Ca²⁺ transients can be an important determinant of amplitudes of contractions, the amplitude of contraction can be further modulated by the contribution of diastolic [Ca²⁺], which elevates the peak systolic [Ca²⁺]. By causing a rightward shift in the cell shortening-intracellular [Ca²⁺] relationship start point, increases in diastolic [Ca²⁺] contribute significantly to the positive inotropy associated with the experimental manipulation of extracellular [Ca²⁺] and during positive staircases. In the situations examined throughout the course of this study, diastolic [Ca²⁺] was the primary contributor to the positive inotropic effects we observed.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported in part by grants from the Heart and Stroke Foundation of Nova Scotia and from The Canadian Institutes of Health Research. During this study R. Shutt received studentship support from the Heart and Stroke Foundation of Canada.

	ACKNOWLEDGMENTS

 
The authors thank Peter Nicholl, Steven Foster, Cindy Mapplebeck, and Dr. Jiequan Zhu for excellent laboratory and technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. E. Howlett, Dept. of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie Univ., 5850 College St., Halifax, NS, Canada B3H 1X5 (e-mail: susan.howlett{at}dal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Deceased 30 August 2005.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Berridge MJ. The AM and FM of calcium signaling. Nature 386: 759–760, 1997.[CrossRef][Medline]
2. Berridge MJ, Lipp P, and Bootman MD. The versatility and universality of calcium signaling. Nat Rev Mol Cell Biol 1: 11–21, 2000.[CrossRef][ISI][Medline]
3. Bers DM. Mechanisms contributing to the cardiac inotropic effect of Na pump inhibition and reduction of extracellular Na. J Gen Physiol 90: 479–504, 1987.[Abstract/Free Full Text]
4. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force (2nd Ed.). Boston, MA: Kluwer Academic, 2001.
5. Beuckelmann DJ and Wier WG. Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. J Physiol 405: 233–255, 1988.[Abstract/Free Full Text]
6. Brady AJ. Mechanical properties of isolated cardiac myocytes. Physiol Rev 71: 413–428, 1990.
7. Capogrossi MC, Kort AA, Spurgeon HA, and Lakatta EG. Single adult rabbit and rat cardiac myocytes retain the Ca²⁺- and species-dependent systolic and diastolic contractile properties of intact muscle. J Gen Physiol 88: 589–613, 1986.[Abstract/Free Full Text]
8. Cheng H, Lederer MR, Lederer WJ, and Cannell MB. Calcium sparks and [Ca²⁺]_i waves in cardiac myocytes. Am J Physiol Cell Physiol 270: C148–C159, 1996.[Abstract/Free Full Text]
9. Dirksen RT and Beam KG. Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling. J Gen Physiol 114: 393–403, 1999.[Abstract/Free Full Text]
10. Dolmetsch RE, Lewis RS, Goodnow CC, and Healy JI. Differential activation of transcription factors induced by Ca response amplitude and duration. Nature 386: 855–858, 1997.[CrossRef][Medline]
11. DuBell WH and Houser SR. Voltage and beat dependence of Ca²⁺ transient in feline ventricular myocytes. Am J Physiol Heart Circ Physiol 257: H746–H759, 1989.[Abstract/Free Full Text]
12. Eisner DA and Lederer WJ. The relationship between sodium pump activity and twitch tension in cardiac Purkinje fibres. J Physiol 303: 475–494, 1980.[Abstract/Free Full Text]
13. Fabiato A. Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell. J Gen Physiol 85: 189–246, 1985.[Abstract/Free Full Text]
14. Fabiato A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol 85: 247–289, 1985.[Abstract/Free Full Text]
15. Fabiato A. Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol 85: 291–320, 1985.[Abstract/Free Full Text]
16. Ferrier GR and Howlett SE. Differential effects of phosphodiesterase-sensitive and -resistant analogs of cAMP on initiation of contraction in cardiac ventricular myocytes. J Pharmacol Exp Ther 306: 166–178, 2003.[Abstract/Free Full Text]
17. Ferrier GR, Redondo IM, Mason CA, Mapplebeck C, and Howlett SE. Regulation of contraction and relaxation by membrane potential in cardiac ventricular myocytes. Am J Physiol Heart Circ Physiol 278: H1618–H1626, 2000.[Abstract/Free Full Text]
18. Ferrier GR, Smith RH, and Howlett SE. Calcium sparks in mouse ventricular myocytes at physiological temperature. Am J Physiol Heart Circ Physiol 285: H1495–H1505, 2003.[Abstract/Free Full Text]
19. Frampton JE, Orchard CH, and Boyett MR. Diastolic, systolic and sarcoplasmic reticulum [Ca²⁺] during inotropic interventions in isolated rat myocytes. J Physiol 437: 351–375, 1991.[Abstract/Free Full Text]
20. Hattori Y, Toyama J, and Kodama I. Cytosolic calcium staircase in ventricular myocytes isolated from guinea pigs and rats. Cardiovasc Res 25: 622–629, 1991.[ISI][Medline]
21. Hille B. Ion Channels of Excitable Membranes (3rd Ed.). Sunderland, MA: Sinauer Associates, 2001.
22. Janczewski AM, Lakatta EG, and Stern MD. Voltage-independent changes in L-type Ca²⁺ current uncoupled from SR Ca²⁺ release in cardiac myocytes. Am J Physiol Heart Circ Physiol 279: H2024–H2031, 2000.[Abstract/Free Full Text]
23. Katoh H, Schlotthauer K, and Bers DM. Transmission of information from cardiac dihydropyridine receptor to ryanodine receptor: evidence from BayK 8644 effects on resting Ca²⁺ sparks. Circ Res 87: 106–111, 2000.[Abstract/Free Full Text]
24. Levesque PC, Leblanc N, and Hume JR. Release of calcium from guinea pig cardiac sarcoplasmic reticulum induced by sodium-calcium exchange. Cardiovasc Res 28: 370–378, 1994.[Abstract/Free Full Text]
25. Louch WE, Ferrier GR, and Howlett SE. Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning. Am J Physiol Heart Circ Physiol 283: H800–H810, 2002.[Abstract/Free Full Text]
26. Mitchell MR and Plant S. Effect of lidocaine on action potentials, currents and contractions in the absence and presence of ouabain in guinea-pig ventricular cells. Q J Exp Physiol 73: 379–390, 1988.[Abstract/Free Full Text]
27. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, and Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215–228, 1998.[CrossRef][ISI][Medline]
28. Nadal-Ginard B and Mahdavi V. Molecular basis of cardiac performance. J Clin Invest 84: 1693–1700, 1989.[ISI][Medline]
29. Periyasamy SM, Chen J, Cooney D, Carter P, Omran E, Tian J, Priyadarshi S, Bagrov A, Fedorova O, Malhotra D, Xie Z, and Shapiro JI. Effects of uremic serum on isolated cardiac myocyte calcium cycling and contractile function. Kidney Int 60: 2367–2376, 2001.[CrossRef][ISI][Medline]
30. Shido O, Romanovsky AA, Ungar AL, and Blatteis CM. Role of intrapreoptic norepinephrine in endotoxin-induced fever in guinea pigs. Am J Physiol Regul Integr Comp Physiol 265: R1369–R1375, 1993.[Abstract/Free Full Text]
31. Sollott SJ, Ziman BD, Warshaw DM, Spurgeon HA, and Lakatta EG. Actomyosin interaction modulates resting length of unstimulated cardiac ventricular cells. Am J Physiol Heart Circ Physiol 271: H896–H905, 1996.[Abstract/Free Full Text]
32. Stern MD. Theory of excitation-contraction coupling in cardiac muscle. Biophys J 63: 497–517, 1992.[Abstract/Free Full Text]
33. Stern MD and Cheng H. Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle? Cell Calcium 35: 591–601, 2004.[CrossRef][ISI][Medline]
34. Suda N and Kokubun S. The effect of extracellular Ca²⁺ concentration on the negative staircase of Ca²⁺ transient in field-stimulated rat ventricular cells. Pflügers Arch 429: 7–13, 1994.[ISI][Medline]
35. Talo A, Stern MD, Spurgeon HA, Isenberg G, and 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 96: 1085–1103, 1990.[Abstract/Free Full Text]
36. Tytgat J, Nilius B, and Carmeliet E. Modulation of the T-type cardiac Ca channel by changes in proton concentration. J Gen Physiol 96: 973–990, 1990.