In vitro studies have traditionally used multicellular preparations, such as papillary muscle or trabeculae, for the evaluation of cardiac mechanical properties including inotropic, lusitropic as well as basic electrophysiological and pharmacological properties. Actually, these preparations represent one of the oldest techniques used for the assessment of myocardial contractility and many fundamental concepts of muscle mechanics have been based on studies in this preparation, dating back more than 60 years [4]. Multicellular preparation data closely correlate with qualitatively results obtained from intact ventricle studies and thus become an invaluable tool to assess myocardial performance in conditions such as cardiomyopathy or heart failure. In these circumstances, parameters such as pressure and volume, which apply to the whole heart, are replaced by force or tension and length. By definition, force means the absolute amount of contractile strength, measured in newtons (N), whereas tension is force related to cross sectional area, measured in newtons per squared millimetre (N/mm²).

Protocols aimed to assess cardiac function in multicellular preparations typically involve muscle twitches (contraction–relaxation sequences) of either isotonic or afterloaded isometric contractions, but the frequency of contraction is very low compared to the physiological frequencies [22]. An isolated papillary muscle preparation responds to an electrical stimulus of suprathreshold intensity with an isotonic, an isometric or an auxotonic contraction (Fig. 20.2).

During contraction of a muscle in an isotonic setup, the muscle is fixed to the base, while the other end is attached to a loose transducer. Thus, when the muscle is electrically stimulated to contract under isotonic conditions there will be a displacement of the transducer and this movement is registered [23] (Fig. 20.2a).

During an isometric force measurement, force develops while shortening is prevented because the muscle is attached to two fixed ends one of which is a transducer. Upon electrical stimulation, the developed force is recorded under isometric conditions by the transducer. An important parameter to assess in such in vitro preparations is the L_max – the length for which the muscle develops maximal force (Fig. 20.2b).

In these multicellular setups mechanical load, such as preload and afterload are controlled and influence the pattern of contraction of the muscles. Also quick changes in load and length have been used to give information about cardiac muscle mechanics. The preload is responsible for the initial muscle stretch and thus its length. After stimulation, the contractile elements of the muscle begin to shorten, but the length does not change: the muscle fibers shorten at the expense of the interaction of the myofilaments. The stretching of the elastic members results in a progressive increase of the force developed, but no muscle shortening. When the contractile force equals the load (afterload), the muscle shortens without increasing the developed force. The speed and force of contraction are dependent on the concentration of intracellular free Ca²⁺. Muscle force and speed are inversely related, so without load the speed is maximal but the force is residual. In contrast, in isometric contractions muscle does not shorten, its velocity of shortening is zero but develops maximal force [24].

These experiments are usually performed in organ bath heated to physiological temperatures containing oxygenated carbonate buffers (see Sect. 20.2.3.1). At the bottom of the bath there is a small device to oxygenate the solution or instead oxygen can be dissolved in the carbonate buffer that is perfusing the tissue. The pH of this solution should be strictly monitored and kept between 7.37 and 7.42 by altering the 95 % O₂/5 % CO₂ bubbling intensity. The base of the papillary muscle/trabeculae is fixed by a clamp, while the opposite end is attached to a transducer. In the organ bath there is a sensor that, together with a heater, controls the bath temperature [25]. The papillary muscles need to be stimulated electrically. Since the solution contains salts, it is possible that electrolytic processes occurring at the electrodes could lead to polarization and release of metals from the electrodes. This is usually prevented by using non polarizable electrodes such as platinum [25]. The buffer solution(s) chosen for the dissection and maintenance of the tissue is important as it will affect the viability of the preparation and hence the experimental protocol.

The basic procedure to obtain a stable preparation before starting any protocol typically includes the intravenous administration of anesthesia and heparin to the animal, to avoid the deposition of blood clots that potentially damage the endocardial endothelium. A thoracotomy is performed followed by the excision and fast immersion of the heart in the carbonate buffer. The heart is allowed to contract for a few beats to eject all the blood kept inside the ventricles coronary circulation and then is transferred to a similar solution but containing a cardioplegic agent to arrest the heart. Cardioplegia allows a safe dissection of the trabeculae or papillary muscles, which are subsequently transferred to the heated organ bath. The electric stimulation is turned on and the cardioplegic solution is replaced by normal carbonate buffer. During all this procedure the temperature should be kept as close as possible to the physiologic temperature of the animal.

Usually the derived parameters include active tension, maximum velocity of tension rise (dT/dt_max), maximum velocity of tension decline (dT/dt_min), peak shortening, time to peak shortening, maximum velocity of shortening (dL/dt_max), maximum velocity of lengthening (dL/dt_min) and time to half relaxation (Fig. 20.2). However other parameters can be derived from these, such as coefficients R1 and R2. Because changes in the contraction phase induce coordinated changes in the relaxation phase, variations in contraction and relaxation must be considered simultaneously to quantify drug induced changes in lusitropy [26]. Coefficient R1 = dL/dt_max/dL/dt_min evaluates the lusitropy under isotonic conditions where the amplitude of sarcomere shortening is greater than that observed under isometric conditions [27]. Because of the lower sensitivity of myofilament for calcium when cardiac muscle is markedly shortened under low load, relaxation proceeds more rapidly than contraction, apparently due to the rapid uptake of calcium by the sarcoplasmatic reticulum. Thus, R1 tests sarcoplasmic reticulum uptake function [28]. Coefficient R2 = dT/dt_max/dT/dt_min evaluates the lusitropy under a high load. When the muscle contracts isometrically, the sarcomeres shorten very little [27]. Because of the higher sensitivity of myofilament for calcium [29], the time course of relaxation is determined by calcium release from troponin C rather than by calcium sequestration by the sarcoplasmatic reticulum. Thus, R2 indirectly reflects myofilament calcium sensitivity [28].

Some of the advantages of the multicellular preparations include their great usefulness in elucidating the myocardial effects and mechanisms of action of certain drugs and neurohumoral agents while excluding the influence of systemic and other neurohumoral processes that occur in intact animals. Thus, they represent a classical pharmacological tool to assess dose response relationships in contractile tissue which is widely used in preclinical safety studies. Secondly, these preparations allow to measure and control force and length in preparations with relatively uniform fiber orientation, more than in the whole heart but less than in single cardiomyocytes, and to measure isotonic or isometric muscle performance independently of geometric constraints and interaction with blood flow and vascular tone. The results obtained from these organ bath systems are generally more consistent, reproducible and useful for measurement of concentration response curves. Finally, papillary muscles or trabeculae can be excised with minimal mechanical injury and remain stable for hours.

Some limitations of this preparation include the fact that: (1) the papillary muscle preparation has a higher elasticity than the one assumed due to end compliance, related to injured muscle extremities and the compliance of attached recording devices. This often results in significant mid-segmental shortening (up to 12 % L_max) during supposedly isometric contractions [4]; (2) the resultant spatial inhomogeneity of sarcomere length and intracellular Ca²⁺ may invalidate many of the assumptions required for cardiac function assessment; (3) there is no simple relationship between active and passive elements in this preparation; (4) it is not possible to undertake voltage clamp studies, thus greatly diminishing its usefulness in studying excitation-contraction coupling processes [4]; (5) there is a potential inadequacy of oxygenation and substrate supply by the carbonate buffer to the inner parts of the muscles (hypoxic core) and (6) the accumulation of metabolites, such as inorganic phosphate, and concentration gradients in the organ bath [4]. In this regard, an appropriate selection of muscles with relatively low cross-sectional areas (<0.5 mm thick) is critical and may surpass these limitations.

Regarding the influence of endothelial cells on myocardial function, it is important to account for the important functional differences between endocardial and coronary vascular endothelial cells [30]. Moreover, the coronary vascular endothelial cells, unless perfused, are probably non-functional in this preparation [31] in contrast to the endocardial endothelial cells whose interaction with the myocardium has been quite well studied in the papillary muscle [30]. In addition, important and relevant aspects of endothelial function, such as cyclical mechanotransduction mediated by shear stress are largely inoperative.

Despite these limitations, a number of recent technical breakthroughs provide the potential to overcome some of these problems. Among the most important of these is the ability to accurately assess sarcomere length, as well as, to obtain true isosarcometric contractions using laser diffraction to monitor sarcomere length and adaptive control systems to regulate it [32]. Interestingly, this approach demonstrated that many “isometric” conditions assumed in previous studies may not be totally reliable. Laser diffraction analysis coupled with force measurements provides important information about muscle contractile properties. Moreover, the incorporation of objective measurement of variables such as intracellular Ca²⁺ transients, ATPase activity, heat production and O₂ consumption [32, 33] are a major advance over much less precise and speculative interpretative assessments based solely on mechanical correlates. Other progresses include the use of skinned fibres with careful, uniform control of Ca²⁺ levels for studying Ca²⁺-myofilament interaction, and the use of caged compounds [34]. In addition, information about the availability of stored calcium to activate muscle contraction is studied using rapid cooling contracture techniques. Rapid cooling contractures are also useful for assessing sarcoplasmic reticulum Ca²⁺, especially in multicellular preparations where slow caffeine diffusion to all the cells limits the utility of the caffeine approach. Cooling to 0 °C inhibits Ca²⁺ pumping and also causes rapid sarcoplasmic reticulum Ca²⁺ release (presumably due to very long ryanodine receptor openings). Then, one can measure either Δ[Ca²⁺]i or contractile force (which develops slowly at 0 °C). This technique is less quantitative concerning absolute amounts of Ca²⁺ but is useful for measuring changes in sarcoplasmatic reticulum Ca²⁺ content under different conditions.

The ex vivo isolated perfused heart includes two different preparations: the Langendorff preparation, first described by Oskar Langendorff in 1895 [35] who demonstrated that the heart receives its nutrients and oxygen from blood via the coronary arteries and that cardiac mechanical function is reflected by changes in the coronary circulation and the ejecting heart or “working heart” system developed by Howard Morgan and James Neeley in 1967 to investigate the metabolism of the heart and coronary regulation [36].