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Cardiovascular Research 1998 39(1):136-147; doi:10.1016/S0008-6363(98)00093-5
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Physiopharmacological evaluation of myocardial performance

how to study modulation by cardiac endothelium and related humoral factors?

Stanislas U Sys*, Gilles W De Keulenaer and Dirk L Brutsaert

Department of Physiology and Medicine, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium

* Corresponding address: Tel.: +32 3 218 02 77; Fax: +32 3 218 02 76; E-mail: stsys@ruca.ua.ac.be

Received 5 January 1998; accepted 11 March 1998


   1 Introduction — the reductionist approach: how far?
Top
 1 Introduction -- the...
2 Endothelial modulation of...
 3 Minimally required information...
 4 Specific properties of...
 5 Specific properties of...
 6 Conclusion
 References
 
When conducting human or animal experiments in a search for an understanding of the regulation and dysfunction of the cardiovascular system, clinicians and scientists have been confronted with the complexity of this system. Although complexity could be studied at any magnification, it has, in general, followed a reductionist approach [1, 2]. In this way, by bypassing or eliminating as many interacting processes as possible, our view on cardiac performance has become ever narrower in scope. For example, excluding neurohumoral control and uncoupling the heart from the peripheral circulation reduced our experimental approach from in-situ animal experiments to isolated experimental conditions, as e.g. the Langendorff-perfused heart and various, more recent, modified versions of it, where the heart is no longer necessarily considered as an input–output pump with ventricular filling (end-diastolic pressure and volume) and cardiac output (systolic pressure and stroke volume) as major features. With the heart as a muscular pump and, more specifically, in view of the strongly time-dependent activation and inactivation processes, time became inherent in any consideration concerning the performance, regardless of the hierarchic level at which it is being studied. Next, attempts to eliminate the complex dynamic architecture of the intact heart have led to the usage of isolated papillary muscles. This allowed cardiac physiologists to apply technology and principles from skeletal muscle physiology, with the addition of the dimension of time through close analysis of twitches, instead of tetani. Isolated single cardiomyocytes were introduced to bypass multicellularity and nonuniform behavior (damaged ends); nonuniformity being evidenced, in multicellular cardiac muscle preparations, by laser diffraction methods [3]or by segmental analysis [4, 5]. In single cardiomyocytes, techniques were developed to bypass the natural membrane-linked activation processes, e.g. by (hyper)permeabilizing the cardiomyocytes [6, 7], or to eliminate cytoplasmic activation control, e.g. by skinning the cardiomyocytes [8, 9]; such procedures further magnified our observations to sarcomeric behavior. More recently still, technological advances have allowed us to focus on the interaction between single molecules of myosin and actin.

From a clinical point of view, the key question with respect to such a magnifying, or reductionist, approach is how far "down" the hierarchic scale of performance one is allowed to descend without losing or obscuring some essential features of organ function as a whole. In other words, how far can the system be reduced, or how far can the subsystems be magnified, and still reflect the dynamic and kinetic specificity of in vivo myocardial performance? Ideal reductionism should clearly stop at the smallest possible integrative functional unit below which it starts to lose some of its properties, which impedes, when multiplied, that organ function re-emerges.


   2 Endothelial modulation of myocardial performance — the need for cardiomyocyte cell membrane and cytoplasm
Top
 1 Introduction -- the...
 2 Endothelial modulation of...
3 Minimally required information...
 4 Specific properties of...
 5 Specific properties of...
 6 Conclusion
 References
 
The study of the interaction of cardiomyocytes with cardiac endothelial cells and with related humoral factors illustrates the above limitation to reductionism. Modulation of myocardial performance by cardiac endothelium was first observed for the endocardial endothelium in the multicellular isolated papillary muscle [10–13]. Subsequently, these observations were extended to the vascular endothelium of myocardial capillaries [14]and to many different levels of integration within the hierarchic scale of myocardial performance, i.e. myocardial performance was studied in isolated single cardiomyocytes that were either superfused with endothelium-conditioned medium [15]or examined in coculture with endothelial cells [16, 17], up the hierarchic scale to the multicellular isolated papillary muscle from various animal species [18–20], to Langendorff-perfused and ejecting isolated hearts, and to the in vivo heart in animal and man [14, 21–23]. In analyzing and evaluating data from these studies performed at different hierarchic levels of performance, one must give careful consideration to the strengths and limitations of each type of preparation.

Because most mechanisms of modulation of the cardiomyocyte by cardiac endothelium and by related humoral factors are closely linked to membrane receptors and receptor-linked intracellular messenger systems, the presence of intact cell membranes and of a preserved cytoplasm in the cardiomyocytes are prerequisites to the evaluation of their effect on myocardial performance. Hence, in order to study cardiac endothelial–myocardial interaction, the reductionist approach cannot descend the hierarchic scale of performance to below the level of the isolated intact cardiomyocyte. This excludes the usage of skinned and (hyper)permeabilized preparations, as well as all other subcellular preparations further down the hierarchic scale. From a physiological point of view, one may wonder if, among the above experimental levels, there is perhaps a more ideal or more appropriate one, with a minimal complexity, that still allows extrapolation in terms of organ function, i.e. myocardial muscle-pump function.


   3 Minimally required information — instantaneous force and triple control of it
Top
 1 Introduction -- the...
 2 Endothelial modulation of...
 3 Minimally required information...
4 Specific properties of...
 5 Specific properties of...
 6 Conclusion
 References
 
Regardless of the level of analysis, it must be emphasized that any study of myocardial performance, to be physiologically relevant in terms of ventricular performance in vivo, must include components of the time course of pressure and volume (including flow) in the ventricle during a cardiac cycle. The idea in using single cardiomyocytes or papillary muscles is that these preparations display transient activation, during which, the above ventricular events are reflected in the time course of force and length (including velocity) during each contraction–relaxation cycle or twitch. We will now see how the determinants of instantaneous force developed by isolated myocardium during transient activation unmask fundamental information about in vivo myocardial contraction–relaxation performance (Fig. 1).


Figure 1
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  		Fig. 1 Determinants of instantaneous force. Schematic diagram of the determinants of instantaneous force in an individual cardiomyocyte (delineated by the hatched cell membrane). Passive force reflects contributions of both intracellular and extracellular load. Active force is determined by the number of crossbridges (CB) and their average force, reflecting the properties of the contractile proteins (CP, myosin and actin and the respective regulatory systems) and the availability of activating calcium (role of sarcolemma, SL, and sarcoplasmic reticulum, SR). Instantaneous force is balanced against the external mechanical factors, i.e. cellular load and length, or indirectly, against ventricular pressure and volume. Neurohumoral and cardiac endothelial control can further modify instantaneous force through any of the above determinants. Nonuniformities in space and in time do occur at each experimental level of the hierarchic scale of performance. It may be important to remember that this schematic diagram incorporates the three components of the previously proposed "triple control" of myocardial performance, i.e. (i) the interaction of the contractile proteins and the activating [Ca2+]i, which constitute the two major determinants of the intracellular activation and inactivation processes during a twitch, (ii) intra- and extracellular loading, and (iii) nonuniformity of (in)activation and loading in space and in time [1, 2].

 
3.1 Load and (in)activation
For any single cardiomyocyte in the myocardium, instantaneous force is composed of a passive and an active component. Passive force reflects the contribution of different extracellular (e.g. collagen) [24, 25], cytoskeletal (e.g. microtubules and intermediate filaments) [26, 27]and sarcomeric (e.g. titin) [27, 28]structures at different muscle lengths (see also review by Brady [29]). Active force reflects the intracellular interaction between myosin and actin; it integrates, through the number of crossbridges and their average force, the properties of the contractile proteins and the available activating calcium. Instantaneous force is then balanced against the actual cellular load and length, acting as external mechanical forces, in the same way as pressure and volume changes in the ventricle deal with the continuously changing, auxotonic loading. The continuous interplay between the former (in)activation processes (contractile proteins and activating calcium) and the latter loading conditions (load and length/pressure and volume) controls both the contraction and the relaxation phases of the cardiac cycle. This interplay has been illustrated by numerous concepts, as e.g. length-dependent activation [30]and inactivation [31], cooperative activity of crossbridges [32, 33]and load dependence of relaxation [34].

3.2 (Non)uniformity
Along with load and (in)activation throughout the cardiac cycle, the degree of (non)uniformity of the distribution of load and (in)activation in space and in time should be considered as a third important determinant of myocardial performance during both contraction and relaxation, hence, the previously proposed concept of a triple control of myocardial performance [2, 35, 36]. To function optimally at the global organ scale, the heart can tolerate only a limited degree of nonuniformity at the lower scales. Although the heart would thus be expected to function with little nonuniformity, this small degree of nonuniformity may vary during the cardiac cycle, and thereby modulate myocardial performance. As such, a limited but variable degree of nonuniformity at all scales would constitute an essential and fundamental property of the pumping heart and should be regarded as a normal physiological modulator of performance [1].

3.3 Extracardiac modulators
In the intact heart, extracardiac modulators, such as neurohumoral and cardiac endothelial control, can modify instantaneous force through any of the above factors. Whereas preservation of an intact innervation and circulation are experimental prerequisites to examine the former (neurohumoral) control of the heart, the latter (cardiac endothelial) control of myocardial performance can be conveniently studied in isolated cardiac muscle preparations, such as the papillary muscle; here, endothelial cells and cardiomyocytes coexist and interact to sustain optimal performance.

Given the above restrictions, we will now critically explore the appropriateness of experimenting with two hierarchic levels of the cardiac muscle-pump for the evaluation of myocardial performance, (i) isolated intact single cardiomyocytes (i.e. surrounded by a presumably intact cell membrane) and (ii) multicellular cardiac muscle preparations, such as the isolated papillary muscle. Both have been used by numerous investigators to distinguish normal from abnormal features of performance of the cardiac organ as a whole. Particular attention will be given to the soundness of information that can be gained from these two different experimental preparations about the effects of cardiac endothelium and related humoral factors.


   4 Specific properties of the isolated intact cardiomyocyte — the need for force measurement at physiologic lengths
Top
 1 Introduction -- the...
 2 Endothelial modulation of...
 3 Minimally required information...
 4 Specific properties of...
5 Specific properties of...
 6 Conclusion
 References
 
During the last decade, we have seen a revival of studies on isolated intact cardiomyocytes; at least one of the reasons being the easy access to specimens from human, in particular explanted, hearts, and one of the advantages being that mechanical twitch analysis in these single cardiomyocytes can conveniently be combined with the simultaneous recording of electrophysiological events or of intracellular Ca2+ transients.

The simultaneous measurement of traditional membrane action potentials or of underlying transmembraneous ion currents by voltage clamp techniques has indeed been considered as a major advantage of using single cardiomyocytes to analyze excitation–contraction coupling and to study modulation of myocardial contractile performance [37](Fig. 2). Apart from the fact that an intact glycocalyx appeared to be unnecessary for the normal functioning of transmembraneous calcium channels [39], which was seen as an unexpected advantage, most electrophysiologists continued to warn that receptors, ion channels and gap junctions may be damaged or disrupted by the isolation procedure. A critical evaluation of the effects of various isolation procedures, as well as of the mere disconnection of cardiomyocytes from neighbouring cells, on the electrophysiological membrane properties has, as yet, not been performed. Hence, the connotation "intact" in single isolated cardiomyocytes requires a more strict and critical reassessment.


Figure 2
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  		Fig. 2 Intact myocyte performance. During electrically stimulated twitch shortening from slack length, freeloaded cardiomyocytes shorten by about 5–15% (lower panels; 33°C left and 23°C right). Left panels (guinea pig cardiomyocyte; resting cell length, 131 µm): Impalement of conventional microelectrodes allowed for the simultaneous recording of electrical activity with cardiomyocyte shortening: Action potentials were recorded (upper panel) and membrane currents were monitored through voltage clamp techniques. Right panels (rat cardiomyocyte): Loading of a cardiomyocyte with a calcium-sensitive fluorescent probe, such as Indo-1, allowed for the simultaneous recording of cardiomyocyte shortening and cytosolic [Ca2+]i, estimated from the ratio of Indo-1 emission at two wavelengths (410 and 490 nm). Left panel modified after White et al. [37]; right panel after Spurgeon et al. [38].

 
Another advantage of using intact single cardiomyocytes is that contraction and relaxation can be studied simultaneously with measurements of intracellular calcium transients using various calcium-binding chemiluminescent proteins or fluorescent probes. From these combined measurements, conclusions can be drawn about intracellular calcium availability and the calcium responsiveness of the contractile proteins, i.e. the link between activating calcium and the force-generating crossbridges, which underlie the measured changes in length and force during contraction and relaxation. Recently, some investigators have extended the analysis of combining these two measurements by displaying them in phase–plane plots of length versus [Ca2+]i [38]. However, in agreement with Blinks [40], we have serious doubts about the idea that cardiomyocyte length, in contrast to force, could serve as a quantitative mechanical indicator of activation (see below), particularly at the very low levels of activation just before relaxation is completed. Moreover, interventions that increased the myofilament Ca2+ response also reduced cardiomyocyte resting length, which may be expected to directly affect [Ca2+]i; phase plane length–[Ca2+]i plots, as a way to study myocardial performance, would be further invalidated by the fact that the contribution of the length change to the observed shifts in the plots cannot be estimated.

4.1 Measurements of length
The two above attractive advantages of the single cardiomyocytes are eclipsed by the fact that most investigators in this area continue to analyze length changes as sole measure of mechanical performance; the major obstacle being that conventional attachment methods necessary for force measurement cannot readily be applied to intact cardiomyocytes. Yet, most knowledge on myocardial performance during contraction and relaxation has been derived from the combined analysis, in multicellular preparations, of force and length in strict isometric and pre- or afterloaded isotonic conditions. Furthermore, although most inotropic interventions appear to influence the extent or rate of shortening under lightly loaded conditions in these multicellular preparations in a similar way as they affect isometric force development [40, 41], some interventions related to important intracellular features, such as the energy metabolism or the cAMP second messenger system, do not influence force development and the extent or rate of shortening in a similar manner. For example, in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs at 2 mmol/l MgATP2–, an increase in creatine phosphate concentration from 0 to 10 mmol/l was shown to increase the unloaded isotonic shortening rate by ~40%, while it decreased the developed force by ~35% [42]. These observations are reminiscent of the effects of β-adrenergic stimulation, e.g. by isoproterenol, on force–velocity relations in multicellular preparations, i.e. inconsistent changes in the velocity of unloaded shortening, despite a consistent increase in developed force and the extent of shortening [43, 44]. Numerous other examples from literature could be provided to further endorse this point. Hence, although it cannot be denied that numerous, e.g. inotropic, interventions resulted in parallel patterns of response in single cardiomyocyte shortening and in multicellular preparation force development, length change and force development can clearly not be used interchangeably as measurements of mechanical performance in muscle.

In addition, when single cardiomyocytes are free-floating in the bathing solution or attached to the substrate or its coating, the mode of contraction is neither isotonic nor isometric, but auxotonic. During a twitch, there is a close bidirectional feedback between excitation and contraction [31, 45, 46], in which the changing length and force participate in a reciprocal fashion. Hence, in order to correctly interpret length changes of an isolated cardiomyocyte during a shortening twitch under auxotonic loading conditions, one should at least have a measure of the concomitant instantaneous force, or, have an appreciation of the load which is balanced against the shortening myocyte.

More importantly still, during shortening from slack length to a peak shortening extent of about 5 to 15% below slack length (Fig. 3), intact freeloaded cardiomyocytes contract over a range of lengths that is substantially shorter than ever reached by muscle fibers in an ejecting ventricle [48, 49]. Whenever ventricular fibers shorten below slack length, as isolated freeloaded cardiomyocytes do, internal restoring forces, by promoting longitudinal expansion, will impede the shortening speed, limit the extent of shortening and fasten relaxation. The cytoskeletal microtubular system has recently been suggested as a major source of these internal restoring forces below slack length. Microtubules display longitudinal and transverse orientation in the cardiomyocytes. They consist of the ubiquitous {alpha}-β-tubulin heterodimers, which have phosphorylation and acetylation sites, and are associated with more tissue-specific regulatory microtubule-associated proteins (MAPs) [26]. Microtubules are polymerized by GTP, Mg2+ and taxol. On the other hand, exposure to cold or to increased intracellular calcium, as induced by increased [Ca2+]o, by β-adrenoceptor stimulation or by many other positive inotropic interventions, has been shown to depolymerize, or disrupt, the microtubular system [50](Fig. 4). Polymerization and depolymerization of this microtubular system may occur within a time scale of minutes and would thus be expected to induce a highly variable internal restoring force. In this respect, in a study on contractile dysfunction of hypertrophied cardiomyocytes isolated from the right ventricle after pulmonary artery banding [50](Fig. 5), a stress-induced increase of the microtubule component in the cytoskeleton was shown to be entirely responsible for the decreased speed and extent of shortening of these cardiomyocytes. This contractile deficit could be mimicked in normal cardiomyocytes after microtubule hyperpolymerization through chemical means, e.g. taxol. Taxol-induced microtubule hyperpolymerization indeed proved to increase stiffness and the apparent viscosity in normal cardiomyocytes [52].


Figure 3
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  		Fig. 3 Operating length range of cardiomyocytes in vivo and in vitro. This figure is composed of the lower part of a pressure–volume diagram of the left ventricle (upper trace), a passive force–sarcomere length (sl) relation (middle trace) and a hypothetical force–sl relation (lower trace), extrapolated to lengths below slack length (lslack). The reference lengths are lmax, i.e. the muscle length for maximal active force development during isometric twitch, and lslack, i.e. the muscle length where the passive force becomes zero, corresponding to about 85% of lmax [47]. The range of sarcomere lengths reached by individual muscle fibers in the left ventricle (black and crosshatched area) during a cardiac cycle varies between 1.86 and 1.96 µm at end-ejection volumes (VES) and between 2.2 and 2.3 µm at end-diastolic volumes (VED) [48]. Volume- and sl-axes have been scaled and positioned accordingly. In a manner similar to that of the pump, and because lmax is commonly used as the resting length of a preloaded muscle, in vitro multicellular preparations operate over the same indicated sl range. By contrast, freeloaded single cardiomyocytes have a resting length corresponding to lslack; with inotropic stimulation, the "resting" length may shorten somewhat further through the development of some myocardial resting tone [19, 38]. Reported active shortening of freeloaded single cardiomyocytes amounts to 5–15% below lslack, thus encountering major intracellular viscoelastic loading. As a result, the weak instantaneous force exerted by the cycling crossbridges in shortening freeloaded single cardiomyocytes is balanced both against these intracellular loadings as well as against some extracellular loadings, which are not well defined, such as e.g. adhesion to the supporting surface and viscous loading of the perfusion media. Middle panel after de Tombe and ter Keurs [47].

 

Figure 4
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  		Fig. 4 Microtubule disruption by second messengers. Bar graphs showing the effects of the phosphodiesterase inhibitor, forskolin, and dibutyryl–cAMP (left) and of increasing [Ca2+]o and the calcium-ionophore, A23187 (right), on the microtubular system in neonatal rat cultured cardiomyocytes. The microtubule disruption score was determined as the sum/3 of the semiquantitative grades of disruption (from normal=0 to severely injured=3) in 100 randomly chosen cells: The maximal value was 100, i.e. when the microtubular system was severely damaged in all cells. In a manner similar to that of β-adrenergic stimulation, forskolin and dibutyryl–cAMP increased the microtubule disruption score dose-dependently. Even without β-adrenergic stimulation, increases in [Ca2+]o or treatment with A23187 severely disrupted the microtubular system. The microtubular system can thus be disrupted by the second messenger systems (i.e. directly by an increased calcium influx or indirectly by increased cAMP levels) of many (positive) inotropic interventions, such as β-adrenergic stimulation. (*: p<0.05 versus control). Modified from Hori et al. [50].

 

Figure 5
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  		Fig. 5 Microtubules influence cardiomyocyte performance. The extent of shortening of isolated adult cat cardiomyocytes is plotted as a function of the time after addition of 1 µmol/l colchicine (left) or of 10 µmol/l taxol (right). Left panel: Cardiomyocytes were obtained from the hypertrophied right ventricle (RV) and from the control left ventricle (LV), after a six-month (6 mo) pulmonary artery (PA) banding. Colchicine binds to the β-subunit of {alpha}β-tubulin, prevents assembly of microtubules and, at micromolar concentrations, thereby disrupts the microtubular system in 1 h. In the control LV cardiomyocytes, colchicine caused only a small increase in the extent of shortening. In the hypertrophied RV cardiomyocytes, the extent of shortening was markedly suppressed at baseline as a consequence of an increased microtubule content. By disrupting the microtubular system, colchicine, after 30 min, restored the extent of shortening to a level that could not be distinguished from control LV cardiomyocyte shortening. Right panel: Taxol induced excessive microtubule polymerization. The extent of shortening of normal cardiomyocytes, exposed to taxol, was significantly depressed by 2 h of exposure, compared to cardiomyocytes exposed to vehicle alone. Modified from Tsutsui et al. [51].

 
Hence, minor variations in the variable restoring forces below slack length may, on a short time scale, introduce substantial changes in the extent and rate of shortening of intact freeloaded cardiomyocytes in the absence of any change in the excitation or in the calcium transient or in the properties of the contractile proteins. Indeed, whereas in the in vivo intact heart, intracellular loading has been calculated not to exceed 3 to 5% of total myocardial force development, in the single cardiomyocyte, intracellular loading may not only constitute most of the total loading, but, in addition, be highly variable. In other words, changes in the extent or rate of shortening, below slack length, of intact cardiomyocytes do not necessarily reflect genuine changes in the underlying contractile processes; instead, the observed changes may be the mere manifestation of a highly variable intracellular loading. Similarly, changes in the timing of relaxation in intact cardiomyocytes, i.e. twitch abbreviation and twitch prolongation, as induced by cardiac endothelium and related humoral factors, do not necessarily reflect changes in the contractile processes, but may also be the manifestation of changes in intracellular loading.

4.2 Measurement of force
Only a few investigators have successfully measured force development, instead of shortening, in intact single cardiomyocytes. For example, Brady et al. [53]and Fabiato [54], albeit with very low success rates, have recorded force from intact cardiomyocytes by attachment with suction micropipettes or by impalement of adjacent cells, respectively. Brady [29]also reported that Copelas et al. [55]have had success with fibrin attachment to the stylus of a force transducer, and Shepherd et al. [56]with adhesion to a poly-L-lysine-coated surface. Sollott and Lakatta [57]imposed a given load during the contraction–relaxation cycle by using an elastic substrate to which the cardiomyocytes adhered; White et al. [37]attached the cardiomyocytes to carbon fibers of known compliance, which resulted in auxotonic contractions. But even in these more appropriate experimental conditions, several drawbacks remained. Indeed, "maximal" force development predicted from tonically activated cardiomyocytes always remained appreciably lower than "maximal" twitch force in isolated papillary muscle [1]. Along the same line, data about the "maximal" aerobic capacity of the in vivo heart could not reliably be extrapolated from the "maximal" oxygen consumption of uncoupled mitochondria in isolated single cardiomyocytes [58]. It, therefore, seems as if some basic functional properties vanish when one moves down the hierarchic scale of performance from multicellularity to single cells. Conversely, as we move up the hierarchic scale, it would seem as if some unexpected optimization or major intercellular signalling processes emerge within the system. To what extent interactions in a more than additive fashion of the connected cardiomyocytes in multicellular preparations, as well as interaction with other cell types, such as e.g. endothelial cells and fibroblasts, and with nerve endings, cytokines, etc., may underlie these unexpected emergent features of multicellularity, needs further exploration.

Our conclusion of these paragraphs must be that the isolated intact cardiomyocyte will remain inappropriate as an experimental model for the physiopharmacological evaluation of myocardial performance and of the modulation by cardiac endothelium and related humoral factors, as long as the above shortcomings have not been taken care of experimentally. Does the use of isolated papillary muscles or isolated trabeculae constitute an alternative option?


   5 Specific properties of the isolated papillary muscle — the need for reappraisal
Top
 1 Introduction -- the...
 2 Endothelial modulation of...
 3 Minimally required information...
 4 Specific properties of...
 5 Specific properties of...
6 Conclusion
 References
 
Although the intact cardiomyocyte has in many ways supplanted the traditional isolated papillary muscle and trabecular cardiac preparation in understanding many cellular and subcellular functions, it is timely, as also emphasized by Bers [59], to take reductionist science a step back, towards a more integrated physiological level. In his editorial on the well-characterized multicellular preparation (i.e. the intact rat right ventricular trabecula, developed by ter Keurs and Backx and extended by Gao, Backx and Marban, as used by Wier et al. [60]), Bers emphasized the importance, in this multicellular preparation, of the relatively simple geometry, the undisturbed extracellular matrix and cellular connections, and the energy balance. Similar to the intact cardiomyocyte, this complex preparation may also provide information about [Ca2+]i transients. In view of all of the above, it may be clear that we could not agree more with Bers' view about the need for reappraisal of multicellular preparations. In what follows, we will outline how an appropriate analysis of data obtained from intact isolated papillary muscle could offer an experimentally more simple, more suitable and, at the same time for our present needs, satisfactory multicellular preparation or experimental model to study myocardial performance under various physiopharmacological conditions. It is, however, important to note that these isolated multicellular preparations, in contrast to in vivo conditions, are generally superfused, rather than perfused, with a physiological bathing solution, rather than with blood. Triggered by the wide usage of in vitro cell culture and the search for optimal tissue and organ preservation solutions in view of transplantation in man, Krebs–Ringer–Henseleit solutions have seen numerous modifications aimed at optimization of both myocardial and endothelial preservation in vitro.

The intact isolated papillary muscle is a multicellular preparation, the mechanical behaviour of which has been studied for many decades. In most of these studies, force and length (or velocity of length changes) during contraction and relaxation of isometric and isotonic twitches have been analyzed as functions of time. From measurements at different pre- and afterloads (Table 1), force–velocity, length–force, velocity–length and force–velocity–length relations have been derived and have provided fundamental insights into the energetics and mechanics of myocardial performance. Peak active force has been used as a measure of the number of crossbridges and Vmax as a measure of the crossbridge cycling rate and of myosin ATPase activity. Moreover, the existence of length-dependent activation and an appreciation of global myocardial contractility has been derived. Load dependence of relaxation has provided insight into sarcoplasmic reticular calcium reuptake, a prerequisite for early and fast isotonic relaxation, and into cooperative activity of contractile proteins, which results in prolongation of force development and slow isometric relaxation. Further information has been obtained about intracellular events by applying a wide spectrum of different cardiac frequencies. Manipulation of the interval between beats, as with changing frequency of stimulation and with paired stimulus potentiation, may indeed substantially change myocardial performance through changes in the intracellular calcium homeostasis.


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  		Table 1 Analysis of the mechanical performance of isolated intact multicellular cardiac muscle preparations provides indirect information about most essential intracellular contractile events

 
5.1 Nonuniformity in isolated papillary muscle
The multicellular scale, as in isolated papillary muscles or in trabeculae, demonstrates substantial segmental nonuniformity, even when the damaged ends are taken into account. Hence, before proceeding with this review, the problem of nonuniformity should first be dealt with more critically. Simultaneously with whole-muscle function, we measured regional (segmental) muscle function by optical detection of microelectrodes impaled to delineate adjacent longitudinal segments (Fig. 6). Isometric twitches were selected from three different muscles, because peak isometric force and rate of force development were similar, without any correction or normalization, except for cross-sectional area. Despite the almost superimposable force traces, there was a wide variation in segment length behaviour, not only within a given muscle but also from one muscle to another. The pattern of nonuniformity was characteristic for any given normal papillary muscle. Despite the presence of a wide spectrum of segment length behaviour, contraction and relaxation of the muscle as a whole, as well as responses to changes in length or load, were predictably uniform and qualitatively identical from one normal preparation to another. We have previously [1, 2]suggested that a perfectly tuned "coherence of nonuniformities at the smaller scale leads to uniformity of function at the immediately larger scale", or that "the heart attains uniformity through nonuniformity" (‘order through fluctuations’, [68]). In addition, it has been shown that maximal isometric twitch force of isolated papillary muscles was at least as large as isometric tetanic force, and, as stated above, it also by far exceeded the extrapolated maximal force developed by single cardiomyocytes [1]. The concept of optimization has been outlined at different hierarchic levels of myocardial performance in a previous review [2]. As our plea for a reappraisal of the multicellular papillary muscle relies heavily on this concept of optimization, we would highly recommend the erudite scholar to read this text.


Figure 6
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  		Fig. 6 Nonuniform segmental behaviour during isometric twitch in isolated papillary muscle. Line graphs illustrating nonuniform kinetics of adjacent segments (S1, S2 and S3, as indicated in the lower left panel) of three different muscles (a, b and c) during one isometric twitch in each muscle. The isometric twitches were obtained under identical conditions: Muscle length, 1.05 lmax and [Ca2+]o, 2.5 mmol/l. All values are individual measurements. Notice that differences between the three segments, including the basal segment S3, also occurred between the two central segments S1 and S2. Reproduced with permission from Brutsaert and Sys [1].

 
5.2 Importance of variations in isometric twitch duration
The effect of endocardial endothelium on myocardial performance was first observed in isolated papillary muscle. It revealed important changes in isometric twitch duration and, less so, in isotonic twitch duration; this effect was induced by selectively damaging the endocardial endothelium lining the surface of the muscle. Initial rate of force development or of shortening hardly changed (particularly not at a high [Ca2+]o of 7.5 mmol/l, or at 29°C), and yet, peak force was consistently reduced as a consequence of isometric twitch abbreviation [10, 69]. When endothelium-related factors affected peak twitch force, as e.g. a negative inotropic response to nitric oxide [19, 71, 72]or a positive inotropic response to endothelin [73–77], it was similarly largely mediated by a change in twitch duration. In addition, variability of isometric twitch duration in intact isolated cat papillary muscles (mean±SD=451±47 ms; variation coefficient=11.9%; n=19) was significantly higher than in muscles where the endocardial endothelium was selectively damaged (439±10 ms; variation coefficient=2.3%; n=9, p<0.001) [70]. Although this intriguing feature has as yet not been explained, we would suggest that it reflects an additional advantage in the modulation of myocardial performance, i.e. flexibility: The presence of an intact endocardial endothelium seems to allow a broader range of isometric twitch duration in order to better adapt myocardial performance to changing demands by the environmental conditions.

Variations in isotonic twitch duration have been well studied, in particular, with respect to the relaxation phase under changing load, and hence, as a basis for the concept of load dependence of relaxation (for review, see ref. [2]) (Table 1). Surprisingly however, in most previous studies on myocardial contractile performance, changes in isometric twitch duration were either not taken into account or not taken seriously. In order to answer the question of whether or not changes in isometric twitch duration could help to distinguish normal from abnormal myocardial performance, Rouleau et al. [78]systematically measured the duration of isometric twitches in papillary muscles obtained from various models of cardiac failure in different animals. They observed, as expected, a decrease in the rate of both force development and shortening, but no consistent picture about isometric twitch duration emerged; isometric twitch duration was abbreviated, unchanged or prolonged, depending on the species and on the underlying etio-pathological processes. As previously suggested [2, 10, 79], changes in isometric twitch duration may, at least in the absence of significant changes in action potential duration [45], reflect changes in the properties of the contractile proteins. A critical re-evaluation of myocardial isometric twitch duration became, therefore, mandatory. In order to answer the question of the extent to which isometric twitch duration contributes to myocardial performance, we reinvestigated our traditional way of looking at mechanical performance in isolated multicellular cardiac muscle.

5.3 Isometric twitch duration and endothelial modulation of myocardial performance
Following up our earlier hypothesis that the endocardial endothelium-mediated chain of events may be mediated by changes in the sensitivity of the contractile proteins to Ca2+ [10], Wang and Morgan [80]demonstrated that endocardial endothelial damage caused a marked decrease in myocardial calcium sensitivity and had smaller, but variable, effects on myocardial calcium availability, the latter being dependent upon [Ca2+]o. The analysis of mechanical performance of multicellular cardiac preparations before and after selective damage of the endocardial endothelium (Fig. 7; average values for 21 muscles) can be presented in a novel way, based on peak rate of force development (+dF/dt) and time to half relaxation (tHR; measured from the stimulus to 50% isometric relaxation). The most marked effect of endothelial damage was a shift to the left of the data points along the tHR axis or the curvilinear length axis, in a manner similar to that which would occur if the muscle were operating at a shorter length. According to Allen and Kentish [81]and to Backx and ter Keurs [82], the intracellular calcium transient in the first twitch after a step change in resting muscle length can hardly be distinguished from the calcium transient at the control resting muscle length; any important difference in isometric twitch performance induced by changes in resting muscle length must therefore reflect changes in the calcium sensitivity of the contractile proteins. The effect of endocardial endothelial damage, similar to the effect of a shorter muscle length, therefore also suggested decreased calcium sensitivity as the major mechanism. Near lmax, an additional, slightly downward shift along the +dF/dt axis or the curvilinear calcium axis, suggested a small decrease in calcium availability after endothelial damage, similar to that which would occur if the muscle were operating at a slightly lower [Ca2+]o. Indeed, earlier observations have demonstrated a strong correlation of +dF/dt, rather than of peak twitch force, with peak intracellular calcium during the calcium transient of a twitch [83]. At shorter lengths, there was a tendency towards increased calcium availability after endothelial damage, in particular, at the higher [Ca2+]o.


Figure 7
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  		Fig. 7 Effects of endocardial endothelial damage on the determinants of peak isometric twitch force. To analyze the effects of endocardial endothelial damage, a series of isometric twitches at different muscle lengths and [Ca2+]o were obtained from isolated cat papillary muscle. Mean data for peak rate of force development (+dF/dt) were plotted against mean data for the time to half relaxation (tHR; measured from the stimulus to 50% isometric relaxation), both expressed as % of the control value, i.e. the value obtained at lmax and at [Ca2+]o=7.5 mmol/l, in 21 isolated cat papillary muscles. Closed symbols: Obtained with intact endocardial endothelium: {diamondsuit}, {blacktriangleup}, {blacksquare}, bullet and {blacktriangledown} at 0.625, 1.25, 2.5, 5.0 and 7.5 mmol/l [Ca2+]o, respectively, along the calcium axis arrow (from bottom to top); the data points corresponding to isometric twitches at different resting muscle lengths, from 88 to 100% lmax are indicated by the length axis arrow (from left to right). Open symbols: Data obtained after selective damaging of the endocardial endothelium by brief (1 s) immersion in 0.5% Triton-X-100 [10]. The position of the open symbols, relative to the closed symbols, indicated the hypothetical changes in [Ca2+]o and in resting muscle length that would have mimicked the effects of endothelial damage. To clarify these relative positions, the grid in (A), consisting of broken lines connecting equal [Ca2+]o data points and lines connecting equal muscle length data points, was straightened by interpolation for individual muscle data and summarized again for all 21 muscles in panel B. In panel B, the straight grid thus corresponded to the filled symbols in (A), i.e. with intact endocardial endothelium. The open symbols represented the data after endocardial endothelial damage, obtained from interpolation in (A). (Some points obviously required extrapolation in one or two dimensions and were, therefore, less reliable). Accordingly, the above analysis of the experimental data indicated that endocardial endothelial damage could be mimicked by the simultaneous effects of (i) a marked decrease in resting muscle length (leftward shift of the data points relative to the grid) and (ii) variable small changes in [Ca2+]o (upward or downward shift).

 
This novel approach of isometric twitch analysis in a coordinate system of +dF/dt versus tHR, or, interchangeably, [Ca2+]o versus length, provides an integrated analysis of mechanical performance, including not only parameters of peak performance (peak active force) but also parameters which underlie the time pattern of twitch contraction (+dF/dt) and twitch relaxation (tHR) phases. At the same time, this analysis of isometric twitches from multicellular preparations, recorded at multiple [Ca++]o and various initial muscle lengths, also allows us to separate effects of interventions on calcium availability versus calcium sensitivity.


   6 Conclusion
Top
 1 Introduction -- the...
 2 Endothelial modulation of...
 3 Minimally required information...
 4 Specific properties of...
 5 Specific properties of...
 6 Conclusion
References
 
In conclusion, in order to evaluate myocardial performance and, in particular, modulation by cardiac endothelium and related humoral factors, the isolated papillary muscle has, at present, proven to be superior to the isolated intact cardiomyocyte. This can be ascribed in part to technical problems involved when studying single cardiomyocytes (such as the mere measurement of freeloaded cardiomyocyte length and shortening, and the operating length range exclusively below slack length in the presence of variable restoring forces), but, more importantly, to the fact that some basic functional properties vanish when one moves down the hierarchic scale from multicellularity to single cells. By inference, a similar criticism can probably be formulated against most previous studies investigating physiopharmacological interventions on myocardial mechanical performance using single cardiomyocytes. A large number of the major intra- and extracellular features that are required to describe myocardial performance can be derived from analyzing twitch contraction and relaxation in the multicellular isolated papillary muscle, e.g. energetics and mechanics (peak active force, Po; number of crossbridges; Vmax, crossbridge cycling rate and ATPase activity), global myocardial contractility, intracellular Ca2+ homeostasis (sarcoplasmic reticulum function and sarcolemmal calcium fluxes) and nonuniformity. In addition, the present paper illustrates the possibility of differentiating between activating Ca2+ and Ca2+ sensitivity. Indeed, a grid analysis of isometric twitches from multicellular preparations, recorded at multiple [Ca++]o and various initial muscle lengths, in a coordinate system of +dF/dt versus tHR, or, interchangeably, [Ca2+]o versus length, provides an integrated evaluation of the minimally required information about myocardial performance, allowing, at the same time, one to separate effects of interventions on calcium availability versus calcium sensitivity. The abundance of information about myocardial performance and its modulation by cardiac endothelium that can be derived from the easily accessible multicellular preparation reflects its physiological kinship with the intact ventricle; it contrasts with the artifact-rich information that derived from an over-reductionist approach in isolated freeloaded cardiomyocyte studies.

Time for primary review 24 days.


   References
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 1 Introduction -- the...
 2 Endothelial modulation of...
 3 Minimally required information...
 4 Specific properties of...
 5 Specific properties of...
 6 Conclusion
 References
 

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