Copyright © 2006, European Society of Cardiology
A novel Z-groove index characterizing myocardial surface structure
aNational Heart and Lung Institute, Imperial College, London SW3 6LY, UK
bDivision of Medicine, Imperial College, London SW3 6LY, UK
* Corresponding author. Cardiac Medicine, NHLI, Imperial College London, Dovehouse St., London SW3 6LY, UK. Tel.: +44 20 7351 8146; fax: +44 20 7823 3392. Email address: sian.harding{at}imperial.ac.uk
Received 2 August 2006; revised 12 September 2006; accepted 13 September 2006
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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Objective: The role of t-tubule structures in excitation–contraction coupling of ventricular myocytes has been investigated by disruption using prolonged culture, or osmotic shock with formamide. We have used a new method, the Scanning Ion Conductance Microscope (SICM), to investigate in more detail the changes in surface structure of live myocytes during these interventions and to relate them to contractile effects.
Methods: Freshly isolated ventricular myocytes from adult rat hearts were either incubated with formamide, then washed to produce osmotic shock, or put into culture for 2, 4 and 7 days. Contractile characteristics of single myocytes were then measured using the IonOptix system, and in parallel imaged using the SICM which produces a 3-dimensional topographical representation of the cell surface. Loss of t-tubules was quantitated with confocal microscopy after staining with the membrane dye di-8-ANNEPS, and sarcomere structure revealed by immunocytochemical detection of 
-actinin.
Results: Detubulation was produced by either method, with formamide equivalent to 4 day culture in quantitative measures of ANNEPS t-tubule/membrane ratio. SICM images confirmed the loss of t-tubule indentations. Disruption of the Z-groove structure and flattening of the surface were also noted with formamide and, to a lesser extent, culture. A novel Z-groove index was introduced to describe this effect more quantitatively. Contraction and relaxation were impaired by the detubulation methods, but formamide had a markedly greater depressant effect on contraction amplitude than equivalent detubulation by culture.
Conclusion: Changes in contraction amplitude after detubulation with formamide were more closely related to the alteration in Z-groove structure than loss of t-tubules alone. As well as disrupting t-tubule-induced excitation and calcium movements, formamide may alter the transmission of contraction in the myocyte by interference with sarcomere attachment at the Z-line.
KEYWORDS Cardiomyocyte; T-tubule; Contraction; Topography
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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Efficiency of excitation–contraction in the adult ventricular myocyte is enhanced by the structural features of this highly organised cell, with its scalloped surface formed by periodic anchorage points at the Z-line. T-tubules, deep invaginations of the sarcolemma at the Z-line, bring the action potential into the centre of the cell, so that calcium entry is close to the effective site for contraction. The functional significance of the t-tubule has been closely studied, because immunohistochemical studies have shown a concentration of ion channels and transporters there [1,2]. We have previously added evidence obtained using a novel method, the "smart patch" technique, which is based on the combination of the Scanning Ion Conductance Microscope (SICM) with conventional patch clamp, to show channel distribution in living myocytes [3]. The majority of L-type Ca2+ channels were found in the t-tubule-associated patch, rather than in patches between the Z-lines [4]. Structures (dyads) within the t-tubule maintain Ca2+ entry channels (L-type Ca2+ channels) in apposition with the Ca2+ release channels for the intracellular Ca2+ store, the sarcoplasmic reticulum (SR) [5]. Ca2+-induced Ca2+ release from the SR amplifies the small Ca2+ entry signal to control the strength of contraction [6].
Disruption of t-tubules by various methods has produced additional evidence for the localisation of ion channels and transporters. Importantly, it has also given information about the functional contribution of these structures to contraction and Ca2+ movements. For example, it has been shown that synchronisation of the Ca2+ sparks to form the final Ca2+ transient is markedly reduced after experimental detubulation [7,8], but can be normalised by β-adrenoceptor stimulation [7]. Investigation of the role of t-tubules is pertinent to the study of heart failure: myocytes from failing hearts have areas of detubulation, but the functional relevance of this observation has not been clear [8,11].
The two main detubulation methods are prolonged culture of the adult myocytes and osmotic shock using formamide [9,10]. In the present study, we have used the SICM to compare the changes in the morphology of t-tubule openings on the surface of living cardiomyocytes after both of these treatments. During SICM operation, the cell is scanned by a micropipette probe which is maintained at a constant distance from the surface by feedback control. This is similar in principle to the atomic force microscope but does not involve contact with the sample [12]. The SICM scan produces 3-dimensional topographical image of the myocyte surface in which the t-tubule openings and Z-grooves are clearly visible. While investigating the effect of detubulation using the SICM, we also observed other qualitative differences in 3-dimensional surface structure when comparing the two detubulation methods. In order to approach a quantitation of these topographical alterations we have introduced a novel parameter, the Z-groove index, which gives information about the dome and groove structure of the myocyte surface. We then compared Z-groove index and t-tubule ratio during detubulation by formamide or culture, to determine which is most closely related to the alterations in contraction parameters observed.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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2.1. Isolation of single rat ventricular myocytes
The investigation conforms with 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). Cardiac myocytes from adult rats were isolated by digestion of intact perfused ventricle as previously described [13]. Male Sprague–Dawley rats were heparinised, killed by cervical dislocation and the heart was rapidly excised and placed in ice-cold Krebs–Henseleit (KH) solution of composition (mM): NaCl 119, KCl 4.7, MgSO4 0.94, KH2PO4 1.2, NaHCO3 25, glucose 11.5, CaCl2 1 and equilibrated to pH 7.4 with 95% O2/5% CO2. A Langendorff perfusion method was used and the heart stabilised for 5 min with KH solution. A further 5 min perfusion was carried out with a low calcium solution of composition (mM): NaCl 120, KCl 5.4, MgSO4 5, pyruvate 5, glucose 20, taurine 20, HEPES 10 and nitrilotriacetic acid (NTA) 5, with a pH of 6.95 and calcium added to give a final concentration of 12–14 mmol/L as measured with a calcium electrode. This solution was equilibrated with 100% O2. A solution similar to the low calcium one, but with no NTA, and with added Ca2+ (200 µM), collagenase (1 mg/ml) and hyaluronidase (0.6 mg/ml), was perfused through the heart for 10 min. The two ventricles were then separated, keeping the interventricular septum with the left ventricle, and the right ventricle discarded. After chopping with scissors, the pieces were shaken at 35 °C under 100% O2 for 5 min with the same enzyme-containing solution, strained through gauze of mesh size 300 µm and incubated for a further 5 min with enzyme. The supernatant was centrifuged at 400 g for 1 min at room temperature. Cells were washed and resuspended in the low calcium solution, with no NTA and Ca2+ at a concentration of 200 µmol/L (ES).
2.2. Disruption of t-tubule structures by prolonged culture or treatment with formamide
Cells were cultured in medium M199 (Invitrogen, UK) plus 5 mM creatine, 5 mM taurine, 2 mM carnitine, 0.1 M insulin, 100 mM ascorbate and penicillin/streptomycin for 2, 4 and 7 days in this study to induce loss of t-tubule structures. Disruption of t-tubules was achieved in other preparations by incubating myocytes with 1.5 M formamide for 15 min at room temperature, as previously described [14]. The cells were then washed twice with ES before being labelled with ANNEPS or used for contraction/scanning studies.
2.3. T-tubule labelling
To label the t-tubules, ventricular myocytes were incubated with 10 nM Di-8-ANNEPS (Molecular Probes, Oregon, USA) for 15 min and then left in ES for 30 min before being observed under the confocal microscope [14]. AfterDi-8-ANEPPS labelling the density of t-tubules was quantified by the ratio of t-tubule fluorescence (t-tubule membrane) to total plasma membrane fluorescence (total membrane) in the same confocal slice, with excitation at 488 nm and emission detected at 520 nm, as described previously [8]. The comparison was made among fresh, 2-day, 4-day, 7-day cultured cells, and formamide-treated cells.
2.4. Immunocytochemical detection of -actinin
Cells were fixed with 4% formalin solution in PBS, permeabilised with 0.1% Triton-X 100 in PBS for 20 min and then blocking solution of 0.2% normal goat serum was applied for 20 min. The cells were incubated overnight at 4 °C with monoclonal antibodies to sarcomeric -actinin, clone EA-53 (Sigma-Aldrich, UK) in 1:200 dilution. The antigen was detected using Alexa 488 conjugated secondary antibodies (1:500) against mouse IgG (Invitrogen, UK). Slides were observed under a Nikon TU 2000 microscope.
2.5. Measurement of contraction of adult myocytes
Myocytes in suspension were placed into a perspex bath with a glass floor on the stage of an inverted microscope and superfused with KH solution (1 mM Ca2+) equilibrated with 95% O2/5% CO2 at 37 °C. Cells were electrically stimulated at 0.5 Hz and cell shortening with each beat followed using the IonOptix system, scanning at 120 Hz. Contraction amplitude (% shortening), time-to-peak contraction (TTP) and time-to-50% relaxation (R50) were analysed offline.
2.6. Scanning Ion Conductance Microscopy (SICM)
The basic arrangement of the SICM for topographical imaging of living cells has previously been described [12,15]. Briefly, the sensitive probe of the SICM is a glass micropipette filled with electrolyte which is connected to a high impedance head stage current amplifier, and mounted on a piezo-driven three-axis translation stage. The sample on a coverslip (thickness: No. 0) was also mounted on another piezo translation stage. To facilitate attachment of the cardiac myocytes, the coverslips were coated with laminin (25 µg/ml, Sigma-Aldrich) for 1 h before plating cells.
The control electronics drive the translation stage to scan the specimen under the micropipette probe. Control/data acquisition hardware and software are produced by East Coast Scientific (Cambridge, UK). The electronics comprise a decoder, four digital-to-analog converters and two analog-to-digital converters. The digital signal processor card (DSP32C PC, Loughborough Sound Images PLC, Loughborough, UK) of a PC functions as a "front-end" controller and provides digital feedback and scanning control. White light illumination and a camera on one port of the microscope are used to approach the pipette to the sample. To perform distance modulation, an AC voltage was applied to the piezo on which either the sample or micropipette was mounted. This led to a modulation of the distance between the sample and pipette. The frequency of modulation was from 100 to 1000 Hz depending on the piezo loading. Piezo loading lowers the resonance frequency of the piezo and, hence, lowers the maximum modulation frequency possible. We operated close to this limit but also selected modulation frequencies away from the noise in our system, such as harmonics of main frequency. The modulated ion current is fed into a lock-in amplifier (SR830 DSP, Stanford Research Systems, Sunnyvale, CA), which provides a signal for the feedback loop to keep the separation between the sample and micropipette. The micropipettes were made from 1.00 mm outer diameter, 0.58 mm inner diameter glass microcapillaries (Intracel, Herts, UK) on a laser-based Brown-Flaming puller (model P-2000, Sutter Instrument Company, San Rafael, CA). In all experiments, micropipettes and the bath solution contained the same physiological L-15 medium (Gibco, Parsley, UK), so that salt concentration gradient potentials and liquid junction potentials were not generated. The measured micropipette resistance was usually 100 M
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2.7. Statistics
Results are expressed as mean and standard deviation (SD) or standard error of the mean (SEM). The data were checked for normal distribution prior to parametric analyses. Comparisons were made using the unpaired Student t-test, or one-way ANOVA with Tukey's post-hoc test (parametric) or Dunn's (non-parametric). P<0.05 was considered to be statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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3.1. Z-groove ratio
SICM images show clearly the surface topography of the cardiomyocyte (Fig. 1). As well as the t-tubule openings, the domed crest between the Z-grooves was very evident in rat myocytes. Profile measurements showed that spacing between Z-grooves was approximately 2 µm, corresponding to the predicted sarcomere length for quiescent ventricular myocytes. To quantitate the data obtained during scanning we introduced an index of the completeness of the Z-grooves on the surface of cardiomyocytes (Z-groove ratio, or Z). To calculate Z-groove ratio we divided the length of Z-grooves seen on a single image (represented by dashed lines on Fig. 1C, right image) to the total estimated Z-groove length, as if they all were present on the surface (represented by solid lines on Fig. 1C, right image).
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3.2. SICM imaging of cultured and formamide-treated myocytes
In the surface scans of control cells (Figs. 1C and 2A
) t-tubules were clearly identified in addition to the Z-grooves. Quantitative information from the SICM can be obtained along a line of the image: these depth profiles are shown at the bottom of SICM images in Fig. 2A, B and C. The height of this profile represents the vertical distance between the crest and the depth to which the pipette can penetrate the t-tubule opening, and the distance between crests shows the 
2 µm spacing in the fresh myocyte (Fig. 2A). T-tubule openings were also visible on the confocal fluorescent images of control cells stained with Di-8-ANNEPS (Figs. 1B and 2D), and could be quantitated relative to the total membrane surface (see below). Cells after formamide treatment had severe changes to membrane topography (Fig. 2B). T-tubules and Z-grooves were absent over large areas, and the domed crest was markedly reduced. Some scans showed areas of complete flattening (Fig. 2B), while in others a ruffled appearance was observed (not shown). Depth profiling after formamide treatment showed small irregularities separated at 0.5 µm to 1 µm intervals. The corresponding fluorescent image shows an example of a partially detubulated formamide-treated myocyte (Fig. 2E). Cells after 2 days of culture showed some loss of structure, but still had many well-preserved areas with visible t-tubule opening areas and Z-grooves (Fig. 1A Supplementary materials). Profiles through the surfaces showed well-defined regular striations with intervals of around 2 µm. The fluorescent image in Fig. 1B, Supplementary materials shows a cardiomyocyte at 2 days of culture with t-tubules well preserved. However, cells after 4 days of culture had lost these regular structures to a greater extent (Fig. 2C). The profile shows irregular structures distributed at variable intervals from 0.5 µm to 3.5 µm. The fluorescent image of a cell from 4 day-long culture also reveals a partially detubulated cardiomyocyte (Fig. 2F). Cardiomyocytes after 7 days of cultivation did not have any regular structures on the surface, only large blebs were seen (Fig. 2A, Supplementary materials). Profiling of the surface of these cells confirmed the absence of any striation-like structures (Fig. 2A, Supplementary materials). A fully detubulated cardiomyocyte after 7 days in culture is also shown in the corresponding fluorescent image (Fig. 2B, Supplementary materials).
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3.3. Immunohistochemistry of sarcomere structure of cultured and formamide-treated myocytes
Underlying sarcomeric structures were revealed using
-actinin staining for each treatment group (Figs. 2G–J, and 3, Supplementary materials). These were preserved to a much greater extent than the surface structures after culturing, as compared with control cells (Fig. 2G). Cells after 2 days of culture (Fig. 3A, Supplementary materials) and cells after 4 days of culture (Fig. 2J) had regular striations, whereas cells after 7 days in culture had lost this regularity of staining (Fig. 3B, Supplementary materials). After formamide treatment, regular staining was preserved only in 40% of myocytes (Fig. 2H, I).
3.4. Di-8-ANNEPS labelling of cultured and formamide-treated myocytes
To demonstrate the amount of t-tubules remaining at the cell membranes after cultivation and detubulation with formamide, we calculated the ratio of fluorescence volume of t-tubule membrane to total cell membrane after labelling with Di-8-ANNEPS (Fig. 3A). The average fluorescence volume ratio of freshly isolated rat ventricular myocytes was 0.728±0.079 (mean±SD, n=25). The number of t-tubules decreased with time in rat ventricular myocytes cultured for 2 days, 4 days and 7 days, with their average fluorescence volume ratio reduced to 0.547±0.117 (n=50), 0.466±0.165 (n=53) and 0.378±0.135 (n=18), respectively. Few myocytes survived as rod-shaped for 7 days, hence the small numbers in this group. Formamide induced a decrease in average fluorescence volume ratio to 0.432±0.146 (n=76), similar to that after 4 days in culture. Histograms of t-tubule volumes for 4-day cultured and formamide-treated myocytes are shown in Fig. 3C, and demonstrate that detubulation was similar for the two treatments.
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3.5. Z-groove ratio calculation of cultured and formamide-treated myocytes
Next, to demonstrate the changes of surface structure of the cell membranes after cultivation and detubulation with formamide we calculated the ratio of Z-groove to the total estimated Z-groove length, as if they all were present on the surface (Fig. 3B). The average Z-groove ratio of freshly isolated rat ventricular myocytes was 0.835±0.016 (mean±SEM, n=12). The ratio of Z-grooves decreased significantly with time in rat ventricular myocytes cultured for 2 days, 4 days and 7 days, with their average ratio reduced to 0.743±0.029 (n=11), 0.431±0.091 (n=7) and 0.095±0.05 (n=4), respectively. Formamide induced a decrease in average Z-grooves ratio to 0.056±0.006 (n=12), similar to that at 7 days of culture.
3.6. Contraction characteristics of cultured and formamide-treated myocytes
Freshly isolated, formamide-treated and 2- or 4-day cultured rat ventricular myocytes were compared in terms of their contractility. Seven-day cultured myocytes were not included, as they represented such a sparse and varied population. Mean contraction amplitudes (percentage of shortening) and example beats are shown in Fig. 4. Time-to-peak contraction (TTP) and time-to-50% relaxation (R50) are presented in Table 1. The amplitude of cardiomyocyte contraction was reduced by 50% after detubulation by formamide (Fig. 4A), but there were no significant differences after the detubulation induced by culturing (Fig. 4B). Average TTP was significantly prolonged in ventricular myocytes detubulated by culturing, whereas this was not the case in myocytes detubulated by formamide (Table 1). The R50 showed similar modest increases (15–16%) at 2 and 4 days of culture, but there was no significant increase with formamide.
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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Cell surface scanning using SICM confirmed the loss of t-tubule openings in both cultured and formamide-treated ventricular myocytes. In control cells, t-tubule openings were clearly distinguishable as deep indentations at the Z-lines, which were spaced regularly at
2 µm intervals, but profiles through the scan show the increasing disorganisation and loss of this pattern gradually during culture, or immediately following formamide treatment. Quantitation of this effect was obtained by conventional methods after ANNEPS staining. In addition cultured and, particularly, formamide-treated myocytes lost the crest and groove appearance defined by the spacing of the Z-line. We have introduced a new parameter, the Z-groove ratio, which gives more information about surface structure changes during detubulation process. Comparing the two measurements shows that changes in Z-groove and t-tubule occur in parallel in cultured myocytes, but that formamide treatment has more effect on Z-groove than t-tubule ratio. The difference was significant between Z-groove indices of 4 days old cardiac myocytes and formamide-treated myocytes, whereas the difference in t-tubule indices was not.
Electron microscopical sections of rat ventricular myocytes show that the Z-groove represents an area of sarcolemmal attachment to the sarcomere, and that the domed appearance can correspond to mitochondria or other structures lying between the myofilaments and the sarcolemma in between the sarcomeric attachment points [16]. We have noticed that this differs between species with rabbit, for example, showing much less pronounced crest and groove morphology (unpublished observations). Surface disruption was particularly evident after formamide treatment, when areas of almost complete flattening were observed. This suggests that structural changes in addition to simple pinching off of the t-tubules had occurred. Loss of regularly-spaced -actinin staining in 60% of the formamide-treated myocytes points to sarcomeric disturbance.
Quantitation of t-tubule loss with di-8-ANNEPS produced similar results to those previously reported for either cultured or formamide-treated myocytes, although species difference and slight methodological variations preclude exact numerical comparison. For formamide treatment, 76% of myocytes in the present study had t-tubule/membrane ratios lower than any of the control cells (Fig. 1B): this compares to a previous report of 609 of 700 (87%) myocytes detubulated [13]. For culture, we showed a decrease to 64% of control at 4 days, compared to a reduction in t-tubule density to 46.5% of control in rabbit myocytes over the same period [17]. Qualitative changes in contraction characteristics were also in accordance with previous reports, where t-tubule loss correlated to reduction in contraction amplitude and slowing of contraction and relaxation [7,18]. Similar alterations have been seen in the underlying Ca2+ transient [18]. Extensive investigation has shown that this is related to loss of the L-type Ca2+ channel (which is preferentially located at the t-tubule), leading to reduced Ca2+ entry and decreased sychronisation of Ca2+ release [19]. In line-scan real-time confocal images of the myocyte, areas of delayed Ca2+ release are seen which correspond to the areas of t-tubule depletion [7,8]. Changes in action potential duration may also contribute to the contractile depression [8]. Sarcoplasmic reticulum (SR) Ca2+ stores appear to be preserved while recovery after SR Ca2+ depletion is slower: this can be explained by decreased Ca2+ entry [20].
Although the general features of the contractile alterations are similar between cultured and formamide-treated myocytes, quantitative discrepancies are revealed by a direct comparison. Formamide-treated and 4 day cultured myocytes, for example, have approximately the same degree of detubulation, shown both in the mean value and the distribution (Fig. 3C). However, in the formamide-treated cells the Z-groove ratio is significantly low than after 4 days in culture. The scale of contractile impairment is also markedly different between the two conditions. After 4 days in culture, the reduction in contraction amplitude was not significant and there was only a modest increase in either TTP or R50, while formamide treatment reduced amplitude by about 50%. Either changes in expression of ion channels and calcium handling proteins in the cultured myocytes have opposed the effects of detubulation, or formamide treatment has had functional effects beyond those related to removal of t-tubules. We hypothesise that the acute mechanical strain induced during rapid relengthening after removal of formamide has severed at some points the membrane attachment, and disrupted the transmission of force from the myofilaments to the membrane. Direct comparison has shown a more pronounced effect of formamide treatment on cell shortening than on the Ca2+ transients in the same myocytes [18]. This supports the suggestion of problems with mechanical transduction of the Ca2+ signal, which would also be consistent with the Z-line detachment and disrupted -actinin pattern seen in the formamide-treated cohort. Future studies to perform detailed direct comparisons of the change in the calcium transient with the alteration in contraction in individual cells will be necessary. One implication of these results is that, while the use of formamide to investigate the effects of detubulation on calcium movements is both valid and useful, more caution should be used in interpreting the effects on contraction, since there may be additional changes in transmission of force produced by formamide treatment.
In conclusion, SICM images of the live myocytes, combined with ANNEPS staining, have confirmed that both prolonged culturing and formamide treatment are efficient methods for disrupting t-tubules. Quantitative comparison of the two methods suggests that formamide may have additional effects on the structure of the myocyte which contributes to the marked contractile abnormalities in treated cells. It will be of interest in the future to quantitate the Z-groove ratio in addition to the t-tubule structure in myocytes from failing heart.
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Appendix A. Supplementary data |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.09.009.
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Acknowledgments |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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We thank Peter O'Gara for the preparation of adult rat ventricular myocytes, and Prof Clive Orchard for helpful discussion and comments on the manuscript. Lin Yang was supported by an NHLI Foundation award, Julia Gorelik is the Daphne Lott-Taylor Lecturer.
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Notes |
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Time for primary review 26 days
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataAcknowledgmentsReferences |
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