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Cardiovascular Research 2004 62(3):529-537; doi:10.1016/j.cardiores.2004.02.008
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Functional consequences of detubulation of isolated rat ventricular myocytes

Mark R Fowler, Rebekah S Dobson, Clive H Orchard and Simon M Harrison*

School of Biomedical Sciences, University of Leeds, Worsley Building, Leeds, West Yorkshire, LS2 9NQ, UK

* Corresponding author. Tel.: +44-113-343-4262; fax: +44-113-343-4262. Email address: s.m.harrison{at}leeds.ac.uk

Received 1 July 2003; revised 22 January 2004; accepted 13 February 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Recent work has suggested that Na+/Ca2+ exchange (NCX) and L-type Ca2+ current (ICa) are located predominantly in the t-tubules of cardiac ventricular myocytes, which therefore represent a microdomain for the regulation of intracellular Na+ (Nai) and Ca2+ (Cai). The aim of this study was to investigate the role of the t-tubules in the response of Cai and contraction to interventions that alter the transsarcolemmal Na+gradient. Methods: Enzymatically isolated and detubulated Wistar rat ventricular myocytes were investigated using fluorescence microscopy and optical detection of cell length. Results: In unstimulated cells, spontaneous contractile activity increased when extracellular [Na+] was decreased or strophanthidin (100 µM) was added to the bathing solution, but the increase was significantly smaller in detubulated cells than in control cells. In electrically stimulated cells, strophanthidin increased Nai to a similar extent in normal and detubulated cells, although the associated increase in Ca2+ transient amplitude and contraction were significantly smaller in detubulated cells. Similarly, tetrodotoxin (TTX, 10 µM) attenuated the Ca2+ transient and contraction less in detubulated than in control cells. Increasing stimulation rate (0.05–1 Hz) caused little change or a small increase in contraction amplitude in control cells, but a significant decrease in contraction amplitude in detubulated cells, although the change of Nai caused by increasing stimulation rate from 0 to 1 Hz was not significantly different in the two cells types. Conclusion: It is concluded that although some Na/K ATPase, NCX and Na+channel activity is present on the surface membrane, the t-tubules play a major role in the modulation of contraction via NCX, allowing changes of the transsarcolemmal Na+gradient to be translated into changes of Cai.

KEYWORDS Sodium; Calcium; Cardiac muscle; t-tubules; Na/Ca exchanger


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Contraction of cardiac ventricular myocytes is initiated by Ca2+ influx across the cell membrane, predominantly via L-type Ca2+ channels [6] although Ca2+ entry via Na/Ca exchange (NCX) may also play a role (e.g., Refs. [15,21,22,27]). This relatively small Ca2+ influx triggers the release of more Ca2+ from the sarcoplasmic reticulum (SR) [6,12,13], inducing a rapid increase in cytosolic Ca2+ (the Ca2+ transient) that activates the myofilaments to cause contraction. Relaxation is brought about by removal of Ca2+ from the cell cytoplasm, predominantly by the SR Ca2+ ATPase and sarcolemmal NCX [4].

In addition to triggering contraction [9], the L-type Ca2+ current (ICa) and NCX can alter the strength of contraction. This can occur either by changing the magnitude of the (trigger) Ca2+ influx, which produces transient changes in Ca2+ transient amplitude [11], or by changing the Ca2+ load of the cell—in particular SR Ca2+ content—which can produce sustained changes of Ca2+ transient amplitude. For example, application of cardiac glycosides (which block the Na/K ATPase), or increasing stimulation rate, increase intracellular [Na+] (Nai). This decreases the transsarcolemmal Na+gradient, and hence the driving force for Ca2+ extrusion via NCX [18], thereby increasing the Ca2+ load of the cell, Ca2+ transient amplitude and the accompanying contraction.

ICa, NCX and the Na/K ATPase appear to be located predominantly within the t-tubules of rat cardiac ventricular myocytes [10,26,29], which therefore represent a specialized microdomain for Ca2+ and Na+ regulation. It has recently been shown that t-tubule density is decreased in ventricular myocytes from failing hearts [2]; however, the functional role of the t-tubules in modulating contraction, and hence the possible impact of such loss, is not clear. The aim of the present study was to investigate the role of the t-tubules in modulating cytosolic [Ca2+] (Cai) and contraction in ventricular myocytes during interventions that change the transsarcolemmal Na+ gradient, and hence NCX activity.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Isolation of rat ventricular myocytes
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats (250 g, Harlan, UK) were killed using a Schedule 1 procedure sanctioned by the UK Home Office Animals (Scientific Procedures) Act of 1986. The heart was removed and perfused retrogradely with ‘isolation solution’ (mmol/l): NaCl, 130; KCl, 5.4; NaH2PO4, 0.4; MgCl2.6H2O, 1.4; CaCl2, 0.75; HEPES, 10; Glucose, 10; Taurine, 20; Creatine, 10; pH 7.3 with NaOH, to clear the coronary circulation of blood. The perfusate was then changed to a Ca2+-free solution (isolation solution with 0.1 mmol/l EGTA replacing CaCl2) for 4 min, followed by 3–5-min perfusion with isolation solution containing 0.05 mmol/l Ca2+, 0.8 mg/ml collagenase (Type 1, Worthington Biochemical, Lakewood, NJ) and 0.08 mg/ml protease (Type XIV, Sigma, St. Louis, MO). The ventricles were then removed, cut into small pieces and agitated gently at 37 °C in recycled collagenase/protease-containing solution (above) supplemented with 1% BSA. Dispersed myocytes were resuspended in isolation solution and stored at room temperature until use. Unless otherwise stated, all chemicals were from Sigma.

2.2. Preparation of detubulated myocytes
An aliquot of isolated ventricular myocytes was centrifuged (40 x g for 45 s) and resuspended in 0.94 ml of ‘detubulation solution’ [20] (mmol/l): KCl, 100; MgCl2, 5.72; ATP, 5; creatine phosphate, 10; HEPES, 25; EGTA, 0.1; pH 7.1 with KOH. Formamide (0.06 ml) was added to give a final concentration of 1.5 M. Cells were agitated gently for 15–20 min at room temperature before resuspension in detubulation solution without formamide for a further 15 min. Cells were resuspended in isolation solution and stored at room temperature until use.

2.3. Experimental solutions
The control Tyrode solution contained (mmol/l): NaCl, 140; KCl, 5.4; MgCl2, 1.2; CaCl2, 1; NaH2PO4, 0.4; HEPES, 5; Glucose, 10; pH 7.4 with NaOH. For experiments in which spontaneous contractile activity was monitored, a low [Na+] Tyrode solution was made by complete equimolar substitution of NaCl with LiCl; bathing [Na+] was reduced by adding the appropriate amount of the low Na+ solution to control Tyrode. Tetrodotoxin (TTX-citrate, Tocris, Bristol, UK) and strophanthidin were diluted in Tyrode, to a final concentration of 10 and 100 µM, respectively. NiCl2, ryanodine and nifedepine were diluted in Tyrode to final concentrations of 5 mM, 1 and 20 µM, respectively. For all experiments except those measuring spontaneous contractile activity, solutions were gravity fed to the experimental chamber (volume 0.1 ml) at a rate of 1–2 ml/min and were switched at the point of entry to the chamber. All experiments were performed at room temperature (22–24 °C).

2.4. Measurement of spontaneous contractile activity
For these experiments (Fig. 1), a small petri dish was used as the experimental chamber. A 0.5 ml aliquot of cells in Tyrode solution was placed in the dish and the appropriate volume of low Na+ solution added within 2–3 s to obtain the required final [Na+]. A new batch of cells was used for each test [Na+] and for each condition (e.g., the absence or presence of pharmacological agents), so that each measurement was independent; 25 striated rod shaped cells chosen at random from those within the dish were observed within 1–4 min of reducing [Na+]o from 140 mmol/l, and were classified as spontaneous if such activity occurred within 5 s of observation. Data are shown from this protocol performed in control cells [n=200 cells at each Nao, comprising 50 cells (two dishes) from each of four hearts], detubulated cells [n=100: 25 cells (one dish) from each of four hearts], control cells in the presence of 5mM Ni2+ (to inhibit NCX; n=100), detubulated cells in the presence of Ni2+ (n=50), control cells in the presence of 100 µM strophanthidin (to inhibit the Na/K ATPase; n=50) and detubulated cells in the presence of strophanthidin (n=50); the data obtained in the presence of pharmacological agents were obtained from cells from two hearts.


Figure 1
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  		Fig. 1 The effect of reducing Nao on spontaneous contractile activity in control and detubulated cells: (A) in the absence and presence of Ni2+; (B) in the absence and presence of 100 µM strophanthidin (the data for control and detubulated cells in the absence of strophanthidin are the same as in panel A and are shown for comparison). Reduction of Nao in control cells significantly increased spontaneous activity at all concentrations below 140 mmol/l as indicated by the open bar above panel A. Detubulation significantly decreased spontaneous contractile activity over the range of Nao concentrations indicated by the filled bar above panel A (all P<0.001 except at 35 mM Nao where P=0.049; chi-squared test). Ni2+ significantly inhibited spontaneous contractile activity in control and detubulated myocytes over the range of Nao concentrations indicated by the open bar above panel A (all P<0.001 except in control cells at 70 mM Nao, where P=0.001, and in detubulated cells at 70 and 46 mM Nao where P=0.035 and 0.002, respectively). Strophanthidin significantly increased spontaneous contractile activity in control cells over the range of Nao indicated by the filled bar above panel B (all P<0.001, except at 70 and 23 mM Nao, where P=0.03 and 0.005, respectively) and in detubulated cells over the range of Nao indicated by the open bar above panel B (all P<0.001 except 35 mM Nao where P=0.021). The number of cells for each data point are as follows. Control: 200; detubulated: 100; control+Ni: 100; detubulated+Ni: 50; control+strophanthidin: 50; detubulated+strophanthidin: 50 (see Materials and methods for more detail). Error bars show S.E.M. between samples (i.e., between dishes; see Materials and methods).

 
2.5 Measurement of Cai
Cells were incubated with 3 µmol/l fura-2-AM (Molecular Probes, Eugene, OR) in isolation solution for 10 min in the dark at room temperature before being resuspended in Tyrode solution and stored in the dark until use. An aliquot of cells was then placed in an experimental chamber mounted on the stage of an inverted microscope (Nikon Diaphot, Nikon, Japan). Cells were viewed using a x 40 oil immersion objective (NA 1.3) and stimulated electrically via platinum electrodes on each side of the chamber. Fura-2 was alternately excited at 340 and 380 nm every 2 ms using a spectrophotometer (Cairn, Kent UK) and emitted fluorescence collected at 510±40 nm. The signal during 340 and 380 nm excitation and the 340/380 ratio (a function of Cai) were digitised at 1 kHz and stored on a PC using an AD converter (CED 1401 plus) and displayed using ‘Signal’ software (CED, Cambridge, UK), which was also used for off-line analysis.

Calibration of fura-2 fluorescence was used to ensure that differences in the relationship between fluorescence and Cai in control and detubulated cells could not account for observed differences in the Cai transient. Fura-2 loaded myocytes were perfused with Tyrode solution (above) and impaled with a patch pipette [3] containing the same solution; the plateau of the fluorescence signal thus obtained was taken as Rmax. Rmin was obtained in the same way, except that 10 mM EGTA was substituted for Ca2+ and Mg2+was increased to compensate for binding by EGTA (React 2 software). Ca2+ was calculated using the Kd [19] and equation [16] given previously. The calibrated fluorescence signals showed similar changes to the fluorescence signals; however, given the uncertainties of calibrating fura-2 signals in vivo (particularly in AM-loaded cells [5]), the data are presented as the original fluorescence signals.

2.6 Measurement of Nai
Cells were incubated with 11 µmol/l SBFI-AM (Molecular Probes), for 2 h in the dark at room temperature before being resuspended in Tyrode solution and stored in the dark until use. SBFI loaded cells were excited, and emitted fluorescence collected, at the same wavelengths as for fura-2. However excitation occurred every 8 ms, a 50% transmission neutral density filter was placed in the excitation light path to reduce photobleaching and the ratio signal was filtered with a {tau} of 0.66 s before sampling at 20 Hz.

To calibrate Nai, SBFI-loaded myocytes were superfused with normal Tyrode solution before being exposed to a calibration solution (mmol/l): EGTA, 10; HEPES, 5; strophanthidin, 0.1; gramicidin D, 1 mg/l; monensin, 0.04; Na+, 5 or 15; KCl, 150 minus [NaCl]. In early experiments a third [Na+] was also used to ensure that the relationship between fluorescence ratio and [Na+] concentration was linear [18]. This relationship had a slope of 0.12±0.009 mM per 10 mmol/l change in Na+ (n=17), which was used to convert changes in SBFI fluorescence ratio to changes in Nai.

2.7. Measurement of cell length
In addition to 340 and 380 nm light, each myocyte was bright-field illuminated with red light ({lambda}>600 nm). The cell image created by this light was separated from the fluorescence signal using a dichroic mirror; the cell image was focused onto a 1024 element photodiode array, the output of which was proportional to cell length [7].

2.8. Statistical analysis
Data were analysed using SigmaStat (Jandel, USA) using ANOVA, paired or unpaired t-tests or Chi-squared tests as appropriate. Significance was taken at the 5% level. Data are expressed as mean±S.E.M. for n cells.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Effect of changing the transsarcolemmal Na+ gradient on spontaneous Ca2+ release
Reducing extracellular [Na+] (Nao) or increasing Nai decreases the electrochemical gradient for Na+ influx, and hence Ca2+ extrusion via NCX. The consequent increase in Cai causes spontaneous SR Ca2+ release, and hence contraction [1]. The first series of experiments was designed to investigate this response in detubulated cells.

Spontaneous contractile activity was monitored visually using bright-field microscopy in control and detubulated cells and was recorded as either present or absent depending on whether it occurred within 5 s of observation of the cell (see Materials and methods). This protocol was performed in normal (140 mM) Nao and between 1 and 4 min after reducing Nao to a series of values between 140 and 15.5 mmol/l (see Materials and methods). Reducing Nao to 70 mM or lower significantly increased spontaneous contractile activity (P<0.001 at each Nao, compared to 140 mM Nao). Fig. 1 shows that this increase in activity was significantly lower in detubulated cells; it only increased significantly from that in 140 mM Nao when Nao was reduced to 35 mM (P=0.049) or below (P<0.001). Fig. 1 also shows that the increase in spontaneous activity induced by reduction of Nao was significantly inhibited in control and detubulated cells by 5 mM Ni2+, an inhibitor of NCX. The Ca2+ channel blocker nifedipine (20 µM) had little effect on the increase in spontaneous activity observed in either cell type, but this activity was abolished by the SR inhibitor ryanodine (1 µM; not shown) in both control and detubulated cells. Thus, the increase in spontaneous activity was not the result of Ca2+ influx via L-type Ca2+ channels in either cell type, but was Ni2+ sensitive and SR dependent.

Fig. 1B shows that inhibition of the Na/K ATPase by strophanthidin significantly increased spontaneous contractile activity in both cell types, and that in the presence of strophanthidin the spontaneous contractile activity of detubulated cells (open triangles) remained lower than that of control cells (filled triangles) at all Nao concentrations tested. These increases in spontaneous contractile activity were also inhibited by Ni2+(not shown).

These data suggest that the observed increases in spontaneous activity are the result of enhanced SR Ca2+ loading predominately via NCX, and that this is significantly lower, but still present, in detubulated cells. It is also worth noting that previous work from our laboratory has shown that ~13% of cells are not detubulated by treatment with formamide [20]. Other laboratories have provided lower estimates: 10% (assessed using cell capacitance:volume ratio [10]) and 0% (using Di-8-ANNEPS staining [28]). Thus, an increase of spontaneous activity in a maximum of ~13% of formamide-treated cells is likely to be due to the presence of nondetubulated cells.

3.2 Effect of changing the transsarcolemmal Na+ gradient on electrically stimulated Ca2+ release
The effect of strophanthidin and TTX on Nai, Cai and contraction was investigated further in electrically stimulated control and detubulated cells. Fig. 2 shows representative Ca2+ transients (top) and accompanying contractions (bottom) recorded from a normal (A) and a detubulated (B) cell following stimulation at 1 Hz before and during the addition of 100 µM strophanthidin to the bathing solution. In the absence of strophanthidin, the Ca2+ transient and contraction were significantly smaller (P=0.032 and 0.001, respectively) in detubulated myocytes (note different scale bars in A and B). In addition, the duration of the Ca2+ transient was significantly prolonged: that is, the time taken for 50% decay of the Ca2+ transient at 1 Hz was 158±26 ms in control cells and 232±13 ms in detubulated cells (n=6, P=0.015), consistent with loss of Ca2+ extrusion mechanisms, such as Na/Ca exchange, following detubulation (see Discussion).


Figure 2
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  		Fig. 2 Representative Cai transients (top) and contractions (bottom) in control (A) and detubulated (B) cells in the absence and presence of 100 µM strophanthindin during 1 Hz stimulation. Traces are the average of 10–20 consecutive records. (C) Mean (±S.E.M.) Ca2+ transient and (D) contraction amplitude in control (n=7) and detubulated (n=7) cells in the presence and absence of strophanthidin during 1 Hz stimulation. Data within a cell type (control or detubulated) are paired; control vs. detubulated is unpaired. *P<0.05, paired t-test.

 
Strophanthidin significantly increased Ca2+ transient amplitude in control cells during stimulation at 0.1, 0.5 and 1 Hz (e.g., from 0.7±0.08 to 0.85±0.07 ratio units at 1 Hz; P=0.0011). In detubulated cells, strophanthidin had no significant effect on Ca2+ transient amplitude at 0.1 and 0.5 Hz, but caused a significant increase at 1 Hz (from 0.2±0.02 to 0.29±0.02 ratio units, P=0.016; Fig. 2C). There was no significant difference in diastolic Ca2+ between control and detubulated cells at any stimulation rate investigated, either in the absence or presence of strophanthidin. Contraction amplitude showed similar changes to Ca2+ transient amplitude (Fig. 2D).

Nai was monitored in control and detubulated cells to investigate whether the reduced response of Cai to strophanthidin in detubulated cells was secondary to smaller changes in Nai. Fig. 3 shows that the increase in Nai induced by increasing stimulation rate from 0 to 1 Hz (encompassing the range over which Cai was investigated) was not significantly different in normal and detubulated cells: that is, Nai increased by 3.5±1.2 mmol/l (n=8) in control cells (from a resting value of 10.4±1.3 mmol/l) and by 4.7±1.6 mmol/l (n=7) in detubulated cells (from a resting value of 8.5±2.3 mmol/l). The resting values and the increase of Nai were not significantly different in control and detubulated cells. In the presence of strophanthidin, increasing stimulation rate over the same range resulted in a significantly greater increase of Nai in normal (10.6±1.3 mmol/l) and detubulated (9.1±1.2 mmol/l) cells (Fig. 3), which was not significantly different in the two cell types. Thus, it appears unlikely that the reduced effect of strophanthidin on the amplitude of the Cai transient in detubulated cells is due to a smaller increase of Nai.


Figure 3
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  		Fig. 3 Mean (±S.E.M.) increase of Nai in control (n=8) and detubulated (n=7) cells in the absence and presence of 100 µM strophanthidin when stimulation rate was increased from 0 to 1 Hz. Data within a cell type (control or detubulated) are paired; control vs. detubulated is unpaired. *P<0.05, paired t-test.

 
Fig. 4 shows the effect of the Na+ channel blocker TTX (10 µM) on the Ca2+ transient and contraction. TTX has previously been shown to decrease the strength of contraction by decreasing Nai thus altering NCX activity [17]. TTX decreased Ca2+ transient amplitude (top) and contraction (bottom) in control (A) and detubulated (B) cells, consistent with the suggestion that INa is present in the surface sarcolemma as well as the t-tubule [29]. Fig. 4 also shows mean data summarising the effect of TTX on the Ca2+ transient (C) and contraction (D) amplitude of normal and detubulated cells. TTX significantly decreased Ca2+ transient and contraction amplitude in control (P=0.031 and 0.005, respectively) and detubulated (P=0.002 and 0.019, respectively) cells, although the decrease in Ca2+ transient and contraction amplitude was much larger in control (to 31±11% and 18±7% of control, respectively) than detubulated (to 61±5% and 45±8% of control, respectively) cells so that the Ca2+ transient and contraction amplitudes were not significantly different between the two cell types in the presence of TTX (P=0.662 and 0.792, respectively).


Figure 4
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  		Fig. 4 Representative Ca2+ transients (top) and contractions (bottom) in control (A) and detubulated (B) cells in the absence and presence of 10 µM TTX. Traces are an average of 10–20 consecutive records. (C) Mean (±S.E.M.) Ca2+ transient amplitude in control (n=6) and detubulated (n=5) cells in the presence and absence of TTX. (D) Mean (±S.E.M.) cell shortening (contraction) in control (n=6) and detubulated (n=5) cells in the presence and absence of TTX. Data within a cell type (control or detubulated) are paired; control vs. detubulated is unpaired. *P<0.05, paired t-test.

 
3.3. Contraction–frequency relationship of normal and detubulated ventricular myocytes
An increase in stimulation frequency of cardiac myocytes normally increases the amplitude of the systolic Ca2+ transient and accompanying contraction, due predominantly to a rise of Nai leading to an increase in Cai via NCX [14,17]. We therefore investigated whether detubulation alters the contraction–frequency relationship.

Fig. 5A illustrates the cytosolic Ca2+ transient (top) and accompanying contraction (bottom) from a representative control cell at stimulation rates of 0.05 and 1 Hz, showing that as stimulation rate is increased there is an increase in Ca2+ transient amplitude and contraction [14]. Fig. 5B shows similar data from a detubulated cell, showing a decrease in Ca2+ transient amplitude and contraction as stimulation frequency is increased. Fig. 5 also shows mean data for the Ca2+ transient (C) and contraction (D) amplitude at different stimulation rates, showing that control cells showed a flat or slightly positive contraction–frequency relationship, whereas detubulated cells showed a clear negative contraction–frequency relationship. Diastolic Ca2+ was not significantly different between the cell types at any stimulation frequency.


Figure 5
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  		Fig. 5 Representative Ca2+ transients (top) and contractions (bottom) in control (A) and detubulated (B) cells at 0.05 and 1 Hz. Traces are an average of 10–20 consecutive records. (C) The effect of stimulation frequency on mean (±S.E.M.) Ca2+ transient amplitude in control (n=6) and detubulated (n=10) ventricular myocytes. (D) The effect of stimulation frequency on mean (±S.E.M.) contraction amplitude in control (n=6) and detubulated (n=10) ventricular myocytes. *P<0.05, unpaired t-test between cell types at each stimulation frequency.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The detubulation method used has been described and validated previously [8,20]. Following formamide treatment, the t-tubules appear to detach from the surface membrane and reseal within the cell with an accompanying decrease in cell capacitance [10]. The release of Ca2+, which is normally temporally and spatially synchronous, occurs initially at the cell surface and then propagates into the centre of the cell. As a result of this nonsynchronous Ca2+ release and loss of Ca2+ extrusion mechanisms, the Ca2+ transient is smaller and prolonged compared to control cells, although interestingly the SR Ca2+ content appears normal [29].

Previous studies using detubulated cells have shown that many proteins associated with excitation–contraction coupling, in particular ICa and NCX activity, appear to be concentrated in the t-tubules, whereas INa appears to be more uniformly distributed between the surface sarcolemma and t-tubule membrane [20,29].

4.1 The effect of changing the transsarcolemmal electrochemical gradient for Na+
The present study showed that decreasing Nao, or increasing Nai using strophanthidin resulted in an increase in spontaneous contractile activity in control cells. The inhibition of the increase by Ni2+and ryanodine (but not nifedipine) suggests that it was due to decreased Ca2+ efflux or increased influx on NCX, leading to Ca2+ overload and hence spontaneous SR Ca2+ release. Calculation of ENaCa in unstimulated cells (assuming Nai=10 mM and Cai=100 nM) shows that as Nao is reduced from 140 to 70 mM, ENaCa decreases from –33 mV at 140 Nao to –86 mV at 70 mM Nao, which is approximately equal to resting membrane potential, so that there would be no driving force for Ca2+ efflux via NCX at this Nao. As Nao is reduced further, ENaCa would become more negative (e.g., at 46.7 mM, ENaCa=–117 mV; at 35 mM, ENaCa=–140 mV), favouring Ca2+ influx via reverse NCX, and over this range of Nao the increase in spontaneous contractile activity as Nao was reduced became more marked. These data suggest that a reduction in the driving force for Ca2+ efflux is a less potent mechanism for enhancing SR Ca2+ content in unstimulated cells than increasing the driving force for reverse NCX.

The increase in spontaneous activity was significantly reduced in detubulated cells at 35 mM Nao and below (Fig. 1) and some of the remaining increase is likely to have been due to nondetubulated cells (see Results). These data suggest that NCX activity is markedly reduced following detubulation, so that changes of Nao or Nai have less effect on Cai.

In stimulated detubulated cells, strophanthidin increased Nai, suggesting that at least some Na/K ATPase activity is located on the surface sarcolemma [10]. This increase in Nai was associated with an increase of Cai and contraction during 1 Hz stimulation, suggesting that some NCX activity also remains in detubulated cells. However, although increasing stimulation rate in the presence of strophanthidin caused a similar increase in Nai in control and detubulated cells (presumably because approximately equal proportions of influx and efflux pathways are lost on detubulation), the response of Cai was different: that is, Cai was not significantly increased by strophanthidin at 0.1 and 0.5 Hz in detubulated cells, only at 1 Hz, whereas in control cells strophanthidin increased Cai significantly at all frequencies. Similarly, TTX caused a smaller decrease of Cai transient and contraction amplitude in detubulated cells than in control cells (Fig. 4). These data suggest that, as in the spontaneously contracting cells, a given change in transsarcolemmal Na+ gradient causes a smaller change in Cai in detubulated cells, consistent with marked loss of NCX.

It is also worth noting that changes in contraction amplitude reflect the changes in Cai transient amplitude caused by strophanthidin (Fig. 3), TTX (Fig. 4) and changes of stimulation rate (Fig. 5; below). This is consistent with Cai transient amplitude being the main determinant of contraction amplitude under these conditions, and provides support for the changes in the Ca2+ transient reported by fura-2 fluorescence (see Materials and methods).

4.2. The effect of changing stimulation rate
Increasing stimulation rate results in a number of changes that alter Ca2+ transient amplitude. These include:

(i) abbreviation of the action potential;
(ii) decreased ICa amplitude but,
(iii) increased time-averaged Ca2+ influx via ICa as a result of more action potentials per unit time;
(iv) increased Nai, due to an increase in time-averaged Na+ influx via INa;
(v) increased SR Ca2+ content as a direct consequence of (iii) and an indirect consequence (via NCX) of (iv);
(vi) decreased diastolic period, allowing less time for Ca2+ efflux, which will also increase the cell's Ca2+ load;
(vii) decreased SR restitution time.

Some of these changes (e.g., [(i),(ii) and (vii)]) will decrease Ca2+ transient amplitude. The others, which will increase Ca2+ transient amplitude, depend mainly on ICa and NCX, which are thought to be concentrated in the t-tubules [10,20,29]. Previous work has shown that the increase in the amplitude of the Ca2+ transient that occurs on increasing stimulation rate depends principally on the increase of Nai that, via NCX, increases Cai [17]. Thus, the observation in the present study that the normal positive contraction–frequency relationship observed in control cells is transformed into a negative contraction–frequency relationship following detubulation, although the change of Nai on increasing stimulation rate over the range investigated is little altered (Fig. 3), is compatible with the idea that NCX activity has been lost during detubulation. A reduction in NCX will mean that the increase in Nai is not translated into an increase in Cai in detubulated cells and as a consequence the rise in Ca2+ with higher stimulation rate is not sufficient to overcome the changes (above) that decrease Ca2+ transient amplitude [14].

In conclusion, these data suggest that the t-tubules of cardiac ventricular myocytes, as well as causing synchronous Ca2+ release throughout the cell, also play an important role in modulating the amplitude of the Ca2+ transient, and hence contraction. In particular it appears that NCX in the t-tubules is important in translating changes of transsarcolemmal Na+ gradient into changes of Cai, and hence in determining the contractile response to cardiac glycosides and changes of stimulation rate. Thus, it is possible that the reduction in t-tubule density that occurs during heart failure [2] may contribute to the inhibition and prolongation of contraction observed in cells from failing hearts [23,24] and to the altered response of such cells to changes of heart rate [25]. However, the increase in NCX expression that occurs during heart failure might help compensate for the loss of t-tubules, although its distribution between the t-tubule and surface membranes may be different as a result of the loss of t-tubules.


   Acknowledgements
 
This work was supported by the British Heart Foundation and Wellcome Trust.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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