mechanisms of calcium release and propagation in cardiac cells. do studies with confocal microscopy...

Cd Chk8-n (1993) 14, 724-735 Q Longman Group UK Ltd lW3

Mechanisms of calcium release and propagation in cardiac cells. Do studies with confocal microscopy add to our understanding?

D.A. WILLIAMS

Depadment of Physiology, The University of Melbourne, Parkville, Victoria, Australia

Abstract - Laser-scanning confocal microscopy (LSCM) has a number of recognised advantages over other techniques of light microscopy for the study of cell and tissue structure. These include increased image spatial resolution, and even more importantly, removal of out-of-focus information from Pdlmensional images of 3dimensional structures. Moreover, these features have also recently proved to be of immense benefit when coupled with ion-sensitive fluorescent probes, in the study of second messenger systems in relation to cell function. This review summarises the contribution that recent studies with LSCM have made to our understanding of the important patho-physiological state, spontaneous Ca2+-release (SCR) in isolated cardiac myocytes, and the relationship of this phenomenon to the induction of abnormal cell automaticity or cardiac arrhythmia. In some components of SCR and propagation, our existing knowledge has only been confirmed by recent results, while in others facets of this complex process, our understanding is being greatly enhanced by LSCM.

As in most mammalian cell systems the efficient regulation of cytosolic Ca2+ concentrations ([Ca2+]i) in cardiac myocytes is important in controlling normal cell function. In non-contracting cardiac cells [Ca2+]i is maintained at approximately 100-200 nM [l-3], by the balance of activity of sarcolemmal and sarcoplasmic reticular Ca-ATPase pumps, Ca2’ channels and Nat/Ca2’ exchangers. The contribution that each individual component makes to the regulation process will vary Iiom species to species, and this is perhaps most evident in the diversity of development of the cardiac samoplasmic reticulum (SR) between different species. Although in cardiac muscle the SR is much less abundant than in skele-

tal muscle, cells from the rat ventricle appear to have only low dependence for contraction, on calcium influx from the extracellular space [4]. Not surprisingly, it is isolated cells from this species that show the greatest propensity to undergo spontaneous Ca2+ release 151.

Cardiac myocytes have often been reported to exhibit oscillations in cytosolic Ca2+, with the earliest measurements in intact cells made over a de- cade ago [6,7]. These oscillations have been observed in cells in isolation, within tissues and in the intact myocardium, and are thought to originate from spontaneous calcium release (SCR) from Ca2+ pools within the sarcoplasmic reticulum (for review

724

CONFOCAL h4ICROSCOPY OF CALCIUM RELEASE IN CARDIAC CELLS 125

see [5]). SCR in the myocardium has recently been defined as ‘the release of calcium from the SR that is not elicited by an action potential’ 181.

The wide spread nature of these occurrences of SCR strongly suggests that the process has physiological significance, and this has prompted many previous studies of this process in cardiac preparations. However, it has not been an easily studied phenomenon because of both the speed of Ca2+ changes involved, and the intracellular and intercel- lular heterogeneity of the response. Ca2+ oscillations in spontaneously contracting cardiac cells and tissues have previously been directly recorded by several groups [3,9,101, or have been inferred from aua- lysis of the contractile behaviour itself 111-131, with most previous observations of this phenomenon restricted by the temporal or spatial limitations of the imaging methodologies employed.

In particular, the results of signal averaging techniques, have been difficult to interpret due to the considerable variability in Ca2t-transients observed from individual cells within a multicellular tissue sample 161. As a result most recent studies of SCR in cardiac cells have utilised techniques of digital imaging microscopy, coupled with specific Ca2+- sensitive fluorophoms (for review see [14]), but even here limitations exist. Generation of ratio images of Fura- fluorescence has been temporally dependant upon conventional video-frame rates (25-33 ms/frame), and the requirement to frame average at each component wavelength to achieve statistical significance in the pixel intensities of cellular regions within the ratio image (see [15]). Where ra- tiomettic measurement was overlooked, and a single wavelength approach was used with Fura- to increase data acquisition speed, many important observations about spontaneous cardiac contractile behaviour were made 191. However, it is always necessary to temper quantitative interpretation in these cases, because of the possibility of fluorescence changes dye to cell movement, leakage, redistribu- tion and photobleaching of fluorophore, and other potential experimental artefacts. As a result it is challenging to obtain long sequences of image data from spontaneously contracting cardiac cells, mak- ing it extremely difficult to determine the sources and sinks for cytosolic Ca2’ , calculate the rates of Ca2’ propagation and Ca2’ levels, or to analyse the

interactions of Ca2+ where more than one site of Ca2+ elevation occurs within a cell. The future ad- vent of new Ca2+-sensitive fluorophores is not likely to alleviate these deficiencies since it is mainly a methodological limitation. Takamatsu and Wier [16], described a video-based, dual emission system consisting of well matched micro-channel intensi- fiers, optically coupled to CCD-sensors, and were able to provide some of the first artefact-free, quantitative images of Ca2’ bands (waves) in cardiac cells (see also [8]). However, laser scanning confo- caI microscopy (LSCM) has the potential to add even further to mechanistic studies of SCR and propagation in cardiac cells as shall be described in this review.

Laser scanning confocal microscopy

LSCM is finding increased use in physiological research because of the improvements in image spatial resolution (x-y and z-depth), but perhaps most importantly, because of the removal of most of the out- of-focus information from 2-D images (for review see [17]). This feature enables, in essence, produc- tion of optical sections of living cells in isolation or within the 3-D tissue syncitium (see for example 118-211). This is particularly important in the study of cellular physiology, anatomy and pathology. For example, in motile cell systems, such as isolated muscle cells, the confounding effects of cell movement (contraction), and out-of-focus information, on measured fluorescence levels can be minim&d through the ability to confine data acquisition to well defined volumes within the cell. These volumes may be represented by complete 2-D slices of the cell, or by specific sub-cellular areas-of-interest, both of which have been demonstrated in the studies to date. The combination of LSCM and ion-sensitive fluorescent probes is relatively new and the present review deals with some features of this combination of specific interest to the study of SCR and Ca2’ propagation in isolated cardiac cells.

One of the first studies of this type with cardiac cells appeared only recently [22]. Although a brief report, dealing mainly with optical and hardware considerations of the combination, it did serve to illustrate some of the potential of the technique in physiological studies. A methodological description

726 CELL CALCIUM

was also presented by Takamatsu et al., [23], with observations of Ca2’ waves measured in rat cardiac cells loaded with Fluo-3, in a companion publication 1241. During this same period in this laboratory we have also presented extensive observations on Ca2+ kinetics in spontaneously active rat ventricular myocytes, with a similar confocal microscopic approach [10,25,26].

Since most conventional confocal microscopes utilise monochromatic laser illumination as a coher- ent excitation source, a number of different spe- cimen-scanning modalities are possible through con- trol of the laser scanning pattern. This feature has added great flexibility to the ways in which confocal images of cardiac myocytes can be collected. For example, in our recent studies with LSCM, non- weighted averaged images of 8-16 frames (desig- nated ‘slow-scan’ images; 768 pixel x 512 lines resolution in a BioRad MRC-500) were collected, where high temporal resolution was not required. However, time resolution could be increased at the expense of some spatial resolution by collecting images which were either: (i) single frames (e.g. 768 pixels x 128 lines) repeated every 250 ms (desig- nated ‘fast-scan’ images); or (ii) ‘line-scan’ images which represented multiple versions (up to 512) of a single scan line (oriented along the major cell axis). This latter option in particular provides important information about propagated SCR by directly generating a plot of cell length and fluorescence intensity (and hence [Ca2+]i), as a function of time (2- 6 mskne), and has already been used to effect by several research groups [10,22.24,27,281.

What do we know about spontaneous cardiac contractility?

The mechanism for the initiation of SCR has not been fully resolved, although it has been extensively reviewed in the last 5 years [5,8]. It has been proposed that a critical level of SR Ca2’ loading, is a prerequisite for initiation of SCR. The overloading of the SR with Ca2+ usually takes place in local&d cell areas, which then become the focal points for the sudden release of excess Ca2’. Based on the demonstration of Ca2+-induced Ca2+-release (CICR) by the SR of shinned cardiac myocytes, and the ob-

servation that the species dependence on the extracellular Ca2’ level required to generate contractile waves, parallels the species sensitivity of CICR in skinned fibres [29], it has been proposed that sub- sequent SCR is triggered, and then propagated by the process of CICR. However, the method for Ca2+ wave propagation is by no means unequivocal, and several alternatives ate possible (e.g. see [8]). A number of the steps in this release/propagation process have become clearer, and some possibilities for potential mechanisms can be dismissed, as the result of studies with LSCM, while in some features of this sequence LSCM has added little additional functional information, merely serving as further contirmation for existing knowledge. Some of these observations are discussed in the following sections.

Contractile ban& in spontaneously active cardiac cells resultj-om waves of elevated Ca2+

This is an inference that has been often made in studies of cardiac SCR, and is based mainly on de- tection of Ca2+ transients in spontaneously-active cells with Indo-l [5], or Fura- [3]. However, it is only recently that the direct temporal and spatial correlation of the contractile band and the under- lying Ca2’ waves has been made [8-10,16,24,25]. All of these studies have clearly shown distinct, lo- calised areas of Ca2+ elevation (or Ca2+ waves), which are coincident with (or indeterminately preceding), the bands of sarcomere constriction evident in spontaneously contracting cardiac myocytes 1301.

An example of the propagating Ca2+ waves commonly seen in spontaneously contracting cells in shown in the LSCM fast-scan images presented in Figure 1. In this image series the SCR was initiated near the lower right edge of the cell, and propagated towards, and terminated at, both ends of the cell. In particular, line-scan images have the capacity to concurrently show the timecourse of shortening of cells which are propagating these Ca2+ waves. A line-scan image for this cell is displayed in Figure 2. The narrowing of this fluorescence profile (cell length) can be seen to closely follow the onset of SCR. This type of image provides re2yly available quantitative data for anaIl$is of Ca propagation rate, local change in Ca level, and extent and

CONFOCAL MICROSCOPY OF CALCIUM RELEASE IN CARDIAC CELLS 127

Ng. 1 Fast scan image series of a spontaneously contmcting cell containing Flue-3. Images were collected at 250 ms intervals. Initial

cell length 143 jun.

timecourse of cell shortening, as has recently been described (see [ 10,251).

The sarcoplasmic reticulum is the primary source of Ca2+ for spontaneous calcium release

Direct observations of Ca2’-cycling of the SR of mechanically skinned cardiac cells [31], coupled with results of some previous studies of SCR where agents such as ryanodine and caffeine have marked-

Ng. 2 Line-scan image of cell (shown in Fig. 2) showing centml

initiation site of SCR. The rate of Ca’+ wave propagation rate was

70 pm/s towards both cell ends. Initial cell length 143 w.

ly impaired the contractility of spontaneously active cardiac cells 29,321, strongly support the idea of the SR as the 1 Ca ’ store of importance in SCR and propagation. This contention is also convincingly confirmed by descriptions of the annihilation of Ca2+ waves following their intersection in individual cells, data which can be derived from the processing of standard (wide-field) fluorescence images [9], but which are readily available with LSCM with no image processing. An example of this type of Ca2+ wave interaction is shown in the line-scan image displayed in Figure 3. Propagation of Ca2’ from a site of SCR near the left cell margin is terminated soon after initiation by a second, and separate Ca2’ wave which emanated from near the right cell end. The abolition of Ca2’ waves in this fashion is convincing evidence that a limited, intracellular store acts as the primary supply for cycling Ca2’. More- over, wave cancellation shows that SR Ca2’ release is followed by a refractory period during which further release to a similar stimulus is restricted. This is not a surprising observation as the transition to au inactive state is a common step in functional modelling schemes for the actions of most ion channels of cell membranes. This may also be similar to the refractoriness of Ca2+ release previously described

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lease. Further quantitative analysis of these occurrences, which will be important in defining the tbre- shold Ca2+ levels for propagation, is the subject of a separate investigation (D.A. Williams and S.H. Cody, unpublished observations).

It is unlikely that the Ca2+-release sites in these image sequences are all the result of numerous damage sites, as in some cells the number of SCR initiation sites were seen to increase and vary during the recording period. These observations are more consistent with the idea that initiation sites represent cell areas where the SR shows progressive, and highly local&d, overloading with Ca2+. Maybe cells which display such sites of SR Ca-overload

Fig. 3 Line-scan image of cell showing the intelsection of could conceivably be considered to be functionally multiple Ca2’ waves. Also highlighted (arrows) are focal point8 of damaged. However, it is important to note that some SCR which do not successfully propagate within the cell. Initial isolated cardiac cells, which are functionally normal ccl1 length 153 pm, propagation rate 100 pm/s in both directions. with low resting [Ca2+]i and typical contractile re-

sponses to agonists and depolarisation, can be induced to generate Ca2+ the extracellular [Ca2+l.

waves simply by elevating

in skinned cardiac preparations by Fabiato [33]. In the present case the SR Ca2+-release channel may show a transient insensitivity to the trigger for Ca2+ release. However, this refractoriness may also be the result of the releasable pool of SR Ca2’ being below a release threshold concentration, as may occur when Ca2+ is resequestemd into a nonreleasable, up- take compartment prior to transfer to the release compartment.

The site of SCR initiation is a site of local cell damage, usually at a cell end

The ability to collect long sequences of images in individual active cells is a decided advantage of confocal microscopy. In many of these sequences, initiation of Ca2+ release cau be seen to constantly change between many focal sites, apparently any- where within a given cell. For example, in the line- scan image shown in Figure 3 two main sites of SCR were apparent, as previously described. How- ever, a number of other smaller hot-spots of SCR were also conspicuous (arrows). These areas may represent focal sites where the Ca2+ that is released is sufficient to be detected by the fluorophom, but insufficient to trigger the propagation of further re-

CICR is the mechanism for propagation of Ca2+ waves

The most likely mechanisms responsible for the propagation of spontaneously released Ca2+ have been recently reviewed by Wier and Blatter [S]. The most strongly supported of these possibilities am: (i) Ca2’ diffusion from a release point to act at an external site on the SR to trigger the release of SR Ca2’; and (ii) Ca2’ sequestered by the SR from the cytosol ac- tings on an internal site of the SR to trigger Ca2’ release. The former scheme, relying on calcium-induced calcium-release (CICR) and diffusion, has been developed into a convincing mathematica.l scheme which accurately explains many observations of the Ca2+ wave propagation process in cardiac cells [34]. The validity of this scheme rests heavily on the requirement for the rate of increase of [Ca2+]i in the cell to be in the range reviously

eL+ determined as necessary to trigger SR Ca release by CICR [33]. A number of recent measurements show that this is the case. Wier and Blatter [8], reported an average rate of change of [Ca2+]i of 2.3 pM/s in cardiac cells treated with BDM to limit cell movement artefacts, while in our recent measurements with LSCM values of up to 20 J.&Us can be

CONFOCAL MICROSCOPY OF CALCIUM RELEASE IN CARDIAC CELti 129

derived (e.g. see Fig. 11 of [251). A scheme incorporating an internal trigger for

release of SR Ca2+ has been described in detail for cells types other than cardiac cells 1351. A possible implication of this type of mechanism in cardiac cells would be the likelihood of measurement of higher Ca2+ levels occurring during Ca2’ waves than in the Ca2+ transients of E-C coupling (‘normal release’), due to the requirement for an overloading of the SR (as mentioned in 181). As described later in this review, measurements of both processes with LSCM in individual cells, show clearly that this is not usually the case for cardiac cells. This may suggest that the internal trigger mechanism for release of SR Ca2’ is unlikely. Alter- natively, the [Ca2+]i resulting from release from a greatly overloaded SR may be restricted by other factors, such as time- or Ca2’-dependent closure of the SR Ca2+-release channels [33], which would limit Ca2+ levels to values similar to those resulting from ‘normal release’ processes. Although the mechanism of CICR is more readily acceptable, the existing possibilities cannot be unequivocally distin- guished from present results.

The velocity of Ca2+ wave propagation is between SO-150 p/s

The propagation rate for the contractile band in spontaneously active cardiac cells has been reported in numerous previous studies to vary from 5&150 @s with often quoted average values of about 100 pm/s 15,12,36,37]. These figures have not been modified by studies which have directly measured the propagation rate for the Ca” wave with fluoms- cence microscopy or LSCM [9,10]. However, these recent studies have also clearly shown that the propagating Ca2+ waves display remarkably constant velocity through the cell, similar Ca2” level and almost identical timecourse and duration in different cells areas. Such consistency must be the result of the constant level and rate of the Ca2+-release (SR and/or channel influx), resequestration (SR and plas- malemmal Ca2+-ATPase, Nat/Ca2+ exchange, cytosolic buffering) processes, and trigger sensitivity for the release process during wave propagation along the cell length.

A number of studies modelling these compo-

Ng. 4 Spontaneously contracting cardiac myocyte showing a

circular wave front for the propagation of Ca2+ (consecutive

fast-scan images). Cell length 112 pm.

nents in a variety of cell types have appeared (e.g. [34,37]), and some of these have recently been reviewed in the formulation of a hypotbesis to unify the observations of cytosolic Ca2’ oscillations made in a variety of cell types [38]. Recent studies with fluorescent Ca2’-indicators [16.25,39], have provided alternative estimates for the Ca2+ levels reached during spontaneous activity, to those derived from investigations with Ca2+-photopro- teins, and used in these models until now. These lower peak values measured in propagated SCR necessitate revision of the present estimates for the threshold [Ca2+]l for CICR (21.2 p.M). Importantly, these lower estimates of Ca + levels suggest that Ca2+-dependent inactivation of CICR ]33] would not be expected to prohibit the involvement of CICR in the process of propagated SCR.

A feature of propagated SCR which has not been rigourously addressed in these studies is the ques- tion of whether the propagation rates am equivalent in all spatial dimensions (3-D). A Ca2+ wave propagating from a highly defined initiation site in the

730 CELL CALCIUM

cell is likely to traverse a number of different intracellular paths with respect to the arrangement of intracellular organelles encountered in cardiac cells. These cells are polarised, possess distinct longitudi- nal bands of mitochondria, and are mostly bi-nu- cleated with distinct peripolar regions lacking myoflbrils. However, both x-z images [24], and the x-y fast-scan images shown in Fi

JY re 4, clearly in-

dicate that the propagating Ca wave front is spherical, a reflection of Ca +-release and propagation processes which are equivalent in all directions, and generally not affected by the different intracellular structures encountered along and across the cell. The few exce tions to this generalisation seem

R to occur when Ca waves are in the vicinity of a cardiac cell nucleus [26].

Rarely, a cell is also encountered which shows a completely different pattern of Ca2+ wave propagation, Fast spiral bands of Ca2’. similar to the spiral waves recently reported in Xen0pu.s oocytes PO], were evident in some cells (D.A. Williams and S.H Cody, unpublished results). These sequences are difficult to analyse, almost impossible to display effec- tively for publication, and present a nightmare for the mathematician to model.

The magnitude of the Ca2+ change in SCR is the same as that produced during normal synchrom’sed contraction

The equivalence of Ca2’ levels in triggered and spontaneous Ca2+ release is a contention which has been inferred by the earliest observations of Ca2’ fluctuations in mammalian cardiac muscle [6,7]. Nevertheless, it has not been straightforward to experimentally confirm or illustrate this point, even with fluorescence imaging, because of the inhomo- geneity of SCR [5], and the hardware constraints discussed previously. One very important advantage brought to these studies by LSCM is the ability to measure Ca2’ changes directly at fixed voxels (volume elements) within cells. This feature allows a direct comparison of Ca2+ levels occurring in identical, specific, cell volumes during spontaneous Ca2’ release (SCR), and the synchronous Ca2’ elevation that occurs with co-ordinated cell activation (either induced by the spontaneous activity, or from direct electrical cell depolarisation). It is clear from these

recent quantitative results [25], that Ca2+ transients accompanying SCR and the synchronised contraction of the same cell, are almost equivalent in magnitude, although significantly variant in time-course. In addition, the extent of samomere shortening was almost identical in both types of cell response (approximately 0.29 pm/activated sarcomere). The ob servation of a difference in time-course for each of the Ca2+-release processes was important and had not been previous1

It shown. It indicated that the

same supply of Ca was likely to be involved in both responses, but that the trigger for Ca2+-release was different for each response. The most likely release schemes for these processes, CICR and charge-coupled release, have been recently reviewed by Fabiato [41 I.

These observations also confirm that the SR must contribute nearly all of the Ca2’ required for activation of the contractile apparatus during the twitch of rat cardiac cells. This observation is consistent with the earlier observations in shinned and intact rat cardiomyocytes of Fabiato [31], and with the report of a large SR volume and low external Ca2+-dependence for normal E-C coupling of these cells 141.

SCR leads to small changes in membrane potential (Vm) which may be linked to cardiac arrhythmia

A direct correlation between spontaneous Ca2’ waves and the induction of a transient inward current has previously been reported by several groups. Direct measurements with intracellular electrodes in single myocytes undergoing spontaneous Ca2’ release have clearly shown that each spontaneous contraction is accompanied by a small depolarisation, rather than triggered by an action potential [36,42]. In fact, under conditions where there are multiple sites of elevated Ca2+ within a cell at a given time (multifocal spontaneous SR Ca2’ release), summa- tion of these small depolarisations may be sufficient to exceed threshold to induce an action potential [42], leading to a type of ‘abnormal automalicity’, with an obvious relationship to cardiac arrhythmia 1431.

Observations with LSCM make. it necessary to re-classify the term multi-focal SCR [42]. This term itself is best reserved to describe cells which con-


comitantly exhibit more than one site acting as initiation points for Ca2’ release (e.g. Fig. 3). This oc-

Fig. 5 Line scan images of spontaneously active myocytes. (a)

High frequency unifocal wave, propagating at 121 pm/s, initial

cell length 132 p. (b) High frequency multifocal Ca’+ waves

showing collisions and interactions, average propagation rate 92

@s. Initial ccl1 length 143 pm. (c) Global Ca2” release (within 6

ms).

cunence is distinct from that whereby a cell generates Ca2+ waves from a single site, either at a cen- tral cell position that allows Ca2+ waves to propagate in both directions (bidirectional, unifocal SCR; e.g. Fig. 2). or with high enough frequency to allow multiple waves to coexist (high-frequency uni-focal SCR). These later phenomena, which we previously shown to lead to rapid, global elevations of cell Ca2’ [25], are depicted in the line-scan images in Figure 5.

All three of these situations result in multiple, coincident sites of Ca2+ elevation within a cell with a high likelihood of the accompanying cell depolarisations triggering an action potential, and synchronised cell contraction. However, unless the release sites are many, or act frequently, true multifocal SCR may be of least importance in generating arrhythmia, because of the potential for wave interaction and annihilation in this case (e.g. Fig. 5b). It has not previously been possible to distinguish these different situations, because in a single standard video image frame these different behavioural processes would appear identical. These distinctions may seem trivial but it is important to separate the terminology related to the initiation process for SCR, from that of the likely wave propagation mechanism (CICR).

Our attempts with LSCM of voltage-sensitive dyes such as RH160 and R649 (D.A. Williams, L.M. Delbridge and S.H. Cody, unpublished results). proved to be abortive as we were not able to plainly resolve the small changes in membrane potential, clearly shown with basic electrophysiological techniques, to accompany SCR. This made it difficult to directly couple optical measurement of both Ca2’ and membrane potential changes in these cells. However, LSCM did produce important image sequences and accompanying line scan images, which clearly illustrated that high-frequency uni-focal Ca2’ waves could lead directly to synchronised cellular elevations in Ca2+ , with a timecourse consistent with an depolarisation-linked event (see Fig. 9 of Em.

Interestingly, cells which showed global synchronised elevations in Ca2+ following wave propa- gations, maz+have provided evidence for rapid recy- cling of Ca by the SR. Analysis of Ca2+ levels at distinct cell locations indicated that two distinct

732 CELL CALCIUM

Ca2’ peaks could be resolved provided that a period of at least 300 ms separated the two Ca2’ changes, while at shorter intervals a single prolonged Ca2+ transient was evident. These observations suggest that the global release of Cazt that follows a Ca2’ wave may involve a different trigger for release. If E-C coupling involves the charge-coupled release of Ca2+ from the SR (reviewed in [41]), the apparent shorter refractory period seen with synchronised Ca2+ increases may reflect the efficiency of this stimulus in releasing Ca2’ following shorter delays than for CICR. Alternatively, this stimulus may have access to another Ca2’ pool which was not de- pleted by the prior wave of CICR.

Ca2+-sensitive fluorophores for use with confocal microscopy

Single excitation wavelength Ca2+ indicators

At this stage confocal microscopy has mainly employed a single wavelength mode of fluorescence excitation, with Flu*3 the most commonly used Ca2’-sensitive fluorophore for analysis of Ca2’ dis- tribution and kinetics in cells and tissues 1441. Other probes that have been used include Rhod-2, calcium-green(s) and Fur&Red, although the latter fluorophore could be used ratiometrically with the right combination of laser sources. Accurate calibration of the fluctuations in fluorescence intensity of inter- naked indicators in terms of ionised Ca2’ levels presents a number of challenges that were common in the use of a fluorophore that does not undergo a significant Ca2+-dependant change in spectral characteristics. This was previously the case with another Ca2’ fluorophom Quin-2 [45], which was widely used in the early 1980s. Variation of the ab- solute concentration of intracellular fluorophore within cells or organelles, or as a function of time, may produce intensity variations which could be er- roneously interpreted as differences in ion distribu- tions. This is particularly evident in cells which exhibit large regional variations in volume (e.g. flat- tened cells which taper at the edges), cells with large volumes occupied by intracellular organelles or vacuoles, or cells which change shape during the activity of interest (e.g. contractile cells).

Ng. 6 Slow-scan images (8 fmmes averaged) of a single cardiac

myocyte immediately before (a) and 4 B afk (b) application of

the detergent saponin (2% v/v) to the bathing solution. Average

cellular f’hmescence intensity was decreased by 90% by this

treatment Initial cell length 121 pm.

Importantly, the majority of Flue-3 in these cells is located in the cell cytosol, as 8hOWII in Figure 6, since it is rapidly released (within seconds) from cells treated with the detergent saponin. This is important fact to establish because of the possibility of intemalised fluorophom localising in the intracellular organelles when using esterified derivatives for cell loading.

Calibration of Flue-3 fluorescence intensity in cardiac myocytes has been performed in several ways. The simplest calibration resulted from the determination of the ratio of maximum (Fmm) and minimum (Fti) fluorescence intensity levels for the fluorophore within individual cells. Experimental fluorescence levels were scaled relative to these fluorescence limits to allow for the determination of fCa2’] at any point in time as has previously been described in detail for Quin-2 [45], and Flue-3 1251.

Alternatively, cells have been loaded with both FM-3 and a second Ca2+-sensitive fluorophore Fura- to allow for implementation of a cross-calibration technique recently described by Williams [lo]. The direct Ca2+-calibration of Fura- ratio images, as has been described in detail elsewhere 1461. provided a baseline average [Ca2+] for individual non-stimulated cells, upon which changes in Flue-3

CONFOCAL MICROSCOPY OF CALCIUM RELEASE IN CARDIAC CELLS

Ng. 7 Simultaneous dual channel (fast-scan) images of cardiac

myocytes co-loaded with the Ca*+-sensitive fluorophores Flu+3

(a) and Fura-Red @). A Ca*’ wave can be seen to propagate from

the top edge of the lower cell of both images (arrows).

fluorescence intensity could be expressed as [Ca2’] changes. This technique also required knowledge of the fluorescence enhancement of Fluo-3 upon Ca2+ binding (Fn&Fb), for the experimental system in use. These procedures have increased the certainty with which quantitative information can be derived from single wavelength indicators (Flue-3, Rhod-2 and calcium green), and other indicators (e.g. Fura- Red), which may be confined to a single wavelength mode of data acquisition by the constraints of the standard hardware configurations (e.g. argon ion la- sers with confocal microscopes).

Dual wavelength Ca2’ indicators

Although a number of dual wavelength indicators presently exist for the measurement of intracellular Ca2’ there are few reports outlining their use with confocal microscopy. Most interest in this area has focus& on the recent availability of confocal microscopes with the capacity for UV-excitation of fluorophores, thereby allowing dualemission fluorophores, such as Indo-l [47], to be of value with confocal microscopy [48]. A novel variation in the use of dual wavelength indicators results from the intracellular combination of two visible wavelength indicators, Fluo-3 and Fura-Red [28]. Fura-Red re-

flects an increase in Ca” levels by exhibiting a significant decrease in fluorescence intensity, as previously shown in spontaneously contracting cardiac cells 1491. The combination of Fura-Red with Flue- 3, both of which can be excited by the 488 nm band of the argon ion laser, produces emission image pairs similar to those expected of true ratiometric Ca2+-indicators (see Fig. 7). The passage of a propagated Ca2’ wave is indicated by increased fluorescence intensity in one image (Fig. 7a, Flue-3), and decreased intensity in the other (Fig. 7b, Fura-Red). These features, coupled with initial studies which in- dicate that the two fluorophores co-localise and exhibit similar Ca2t-binding kinetics 1281, make this combination of possible benefit in future quantitative study of SCR and propagation with LSCM.

Concluding remarks

Ca2+ oscillations occur in unstimulated cardiac cells and tissues, and in the intact myocardium from many species, in the presence of physiological Ca2’ levels in the bathing solutions, and with minimal experimental perturbations of the Ca2+ loading state of the cell. Such a powerful and fundamental phenomenon occurring under relatively physiological conditions is clearly relevant to an understanding of the generation of cardiac arrhythmias. The advantages of laser scanning confocal microscopy, increased image spatial resolution, and even more importantly, removal of out-of-focus information from 2-D images of 3-D structures, make it an invaluable technique for use at all levels of structural organisa- tion. The coupling of laser scanning confocal microscopy with ion-sensitive fluorescent indicators ap- pears to present the best suited approach to carry 02; the necessary further investigation of SCR, Ca propagation and cardiac arrhythmia.

Acknowledgements

I am grateful for the involvement of Stephen H. Cody in the majority of these studies and for the support of the National Heart Foundation of Australia.

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Please send reprint requests to : Dr David A. Williams, Department

of Physiology, The University of Melbourne, Parkville, Victoria 3052. Australia.

Received : 28 July 1993

Aaepted : 28 July 1993

mechanisms of calcium release and propagation in cardiac cells. do studies with confocal microscopy...

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