Taberner AJ, Han J-C, Loiselle DS, Nielsen PM. An innovative work-loop calorimeter for in vitro measurement of the mechanics and energetics of working cardiac trabeculae. J Appl Physiol 111: 1798 -1803. First published September 8, 2011 doi:10.1152/japplphysiol.00752.2011.-We describe a unique work-loop calorimeter with which we can measure, simultaneously, the rate of heat production and force-length work output of isolated cardiac trabeculae. The mechanics of the force-length work-loop contraction mimic those of the pressure-volume work-loops experienced by the heart. Within the measurement chamber of a flowthrough microcalorimeter, a trabecula is electrically stimulated to respond, under software control, in one of three modes: fixed-end, isometric, or isotonic. In each mode, software controls the position of a linear motor, with feedback from muscle force, to adjust muscle length in the desired temporal sequence. In the case of a work-loop contraction, the software achieves seamless transitions between phases of length control (isometric contraction, isometric relaxation, and restoration of resting muscle length) and force control (isotonic shortening). The area enclosed by the resulting force-length loop represents the work done by the trabecula. The change of enthalpy expended by the muscle is given by the sum of the work term and the associated amount of evolved heat. With these simultaneous measurements, we provide the first estimation of suprabasal, net mechanical efficiency (ratio of work to change of enthalpy) of mammalian cardiac trabeculae. The maximum efficiency is at the vicinity of 12%.work; heat; enthalpy; efficiency; microcalorimeter THE ADVANTAGE OF USING ISOLATED tissue preparations for the study of cardiac energetics arises from the ease of administration of ionic or pharmacological interventions. The disadvantage of their use is that it is difficult to achieve realistic approximations of the mechanics of the intact heart. In situ, the heart repetitively undergoes a pressure-volume-time trajectory consisting of sequential periods of isovolumic contraction, emptying, isovolumic relaxation, and passive refilling. Commonly, experiments consist entirely of unloaded, isometric or, more usually, fixed-end contractions. Such artificial mechanical events are rarely, if ever, encountered by the heart. To enhance our understanding of cardiac energetics, it is thus desirable to adopt contraction protocols that more closely resemble the repetitive pressure-volume-time behavior of the heart. Such approximations have been achieved previously using multicellular, isolated, cardiac preparations. Gibbs and colleagues (6, 22) routinely measured the heat and work output of papillary muscles undergoing afterloaded isotonic contractions, thereby generating characteristic enthalpy-load relations and allowing derivation of the load-dependence of mechanical efficiency. Peterson et al. (21) were the first to apply a computer-based "adaptive" approach to the control of muscle segment length to generate isotonic con...

Pulmonary arterial hypertension (PAH) greatly increases the afterload on the right ventricle (RV), triggering RV hypertrophy, which progressively leads to RV failure. In contrast, the disease reduces the passive filling pressure of the left ventricle (LV), resulting in LV atrophy. We investigated whether these distinct structural and functional consequences to the ventricles affect their respective energy efficiencies. We studied trabeculae isolated from both ventricles of Wistar rats with monocrotaline-induced PAH and their respective Control groups. Trabeculae were mounted in a calorimeter at 37°C. While contracting at 5 Hz, they were subjected to stress-length work-loops over a wide range of afterloads. They were subsequently required to undergo a series of isometric contractions at various muscle lengths. In both protocols, stress production, length change and suprabasal heat output were simultaneously measured. We found that RV trabeculae from PAH rats generated higher activation heat, but developed normal active stress. Their peak external work output was lower due to reduced extent and velocity of shortening. Despite lower peak work output, suprabasal enthalpy was unaffected, thereby rendering suprabasal efficiency lower. Crossbridge efficiency, however, was unaffected. In contrast, LV trabeculae from PAH rats maintained normal mechano-energetic performance. Pulmonary arterial hypertension reduces the suprabasal energy efficiency of hypertrophied right ventricular tissues as a consequence of the increased energy cost of Ca cycling.

To study cardiac muscle energetics quantitatively, it is of paramount importance to measure, simultaneously, mechanical and thermal performance. Ideally, this should be achieved under conditions that minimize the risk of tissue anoxia, especially under high rates of energy expenditure. In vitro, this consideration necessitates the use of preparations of small radial dimensions. To that end, we have constructed a unique micromechanocalorimeter, consisting of an open-ended flow-through microcalorimeter, a force transducer, and a pair of muscle-length actuators. The device enables the metabolic and mechanical performance of cardiac trabeculae carneae to be investigated for prolonged periods in a continuously replenished oxygen- and nutrient-rich environment.

The study of cardiac energetics commonly involves the use of isolated muscle preparations (papillary muscles or trabeculae carneae). Their contractile performance has been observed to vary inversely with thickness. This inverse dependence has been attributed, almost without exception, to inadequate diffusion of oxygen into the centers of muscles of large diameter. It is thus commonly hypothesized that the radius-dependent diminution of performance reflects the development of an anoxic core. We tested this hypothesis theoretically by solving a modification of the diffusion equation, in which the rate of oxygen consumption is a sigmoidal function of the partial pressure of oxygen. The model demonstrates that sufficiently thick muscles, operating at sufficiently high rates of oxygen demand or sufficiently low ambient partial pressures of oxygen, will indeed show diminished energetic performance, whether indirectly indexed as stress (force per cross-sectional area) development or as the rate of heat production. However, such simulated behavior requires the adoption of extreme parameter values, often differing by an order of magnitude from their experimental equivalents. We thus conclude that the radius-dependent diminution of muscle performance in vitro cannot be attributed entirely to an insufficient supply of oxygen via diffusion.

Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca pumping by the sarcoplasmic reticular Ca -ATPase and Ca translocation by the Na /Ca exchanger coupled to the Na ,K -ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l ), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat-stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat-force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long-standing disputes in the literature.

The heat liberated upon stress production in isolated cardiac muscle provides insights into the complex thermodynamic processes underlying mechanical contraction. To that end, we simultaneously measured the heat and stress (force per cross-sectional area) production of cardiac trabeculae from rats using a flow-through micromechanocalorimeter. In a flowing stream of O2-equilibrated Tyrode solution (ϳ22°C), the stress and heat production of actively contracting trabeculae were varied by 1) altering stimulus frequency (0.2-4 Hz) at optimal muscle length (Lo), 2) reducing muscle length below Lo at 0.2 and 2 Hz, and 3) changing extracellular Ca 2ϩ concentrations ([Ca 2ϩ ]o; 1 and 2 mM). Linear regression lines were adequate to fit the active heat-stress data. The active heat-stress relationships were independent of stimulus frequency and muscle length but were dependent on [Ca 2ϩ ]o, having greater intercepts at 2 mM [Ca 2ϩ ]o than at 1 mM [Ca 2ϩ ]o (3.5 and 2.0 kJ·m Ϫ3 ·twitch Ϫ1 , respectively). The slopes among the heat-stress relationships did not differ. At the highest experimental stimulus frequency, pronounced elevation of diastolic Ca 2ϩ resulted in incomplete twitch relaxation. The resulting increase of diastolic stress, which occurred with negligible metabolic energy expenditure, subsequently diminished due to the time-dependent loss of myofilament Ca 2ϩ -sensitivity.cardiac thermodynamics; heat-stress relationships; dynamic stiffness; diastolic calcium, myofilament calcium sensitivity WHEN AN ISOLATED CARDIAC MUSCLE, held fixed at both ends, is electrically stimulated, twitch force is produced. A consequence of this mechanical contraction is the simultaneous liberation of heat. Thus, the relationship between heat and stress (force per cross-sectional area) production provides insights into the complex thermomechanical processes occurring within the muscle. In selecting suitable isolated multicellular tissue preparations for in vitro experiments, two important criteria have to be met. First, for an unambiguous interpretation of stress production, the myocytes should be aligned in parallel with the direction of force measurement. Second, to avoid the risk of tissue anoxia, the preparation should be sufficiently small in radial dimension for O 2 to diffuse into the muscle core under high rates of O 2 demand. The first criterion can be achieved using either papillary muscles or cardiac trabeculae. Compared with papillary muscles, cardiac trabeculae have cross-sectional areas an order of magnitude smaller. Thus, to satisfy the second criterion, cardiac trabeculae are preferable, owing to their minute radial dimensions (about that of a human hair), which greatly facilitates the diffusion of O 2 .Using a flow-through micromechanocalorimeter (11), we simultaneously measured the heat and stress production of cardiac trabeculae excised from the right ventricles of rat hearts. Optimized in vitro metabolic conditions were achieved by continuous provision of O 2 and removal of waste products, enabling measureme...

When a muscle shortens against an afterload, the heat that it liberates is greater than that produced by the same muscle contracting isometrically at the same level of force. This excess heat is defined as 'shortening heat', and has been repeatedly demonstrated in skeletal muscle but not in cardiac muscle. Given the micro-structural similarities between these two muscle types, and since we imagine that shortening heat is the thermal accompaniment of cross-bridge cycling, we have re-examined this issue. Using our flow-through microcalorimeter, we measured force and heat generated by isolated rat trabeculae undergoing isometric contractions at different muscle lengths and work-loop (shortening) contractions at different afterloads. We simulated these experimental protocols using a thermodynamically constrained model of cross-bridge cycling and probed the mechanisms underpinning shortening heat. Predictions generated by the model were subsequently validated by a further set of experiments. Both our experimental and modelling results show convincing evidence for the existence of shortening heat in cardiac muscle. Its magnitude is inversely related to the afterload or, equivalently, directly related to the extent of shortening. Computational simulations reveal that the heat of shortening arises from the cycling of cross-bridges, and that the rate of ATP hydrolysis is more sensitive to change of muscle length than to change of afterload. Our results clarify a long-standing uncertainty in the field of cardiac muscle energetics.

Key points• With each beat of the heart, the left and right ventricles must overcome substantially different arterial pressures in order to eject blood.• We have tested whether this difference in mechanical demand is reflected in different metabolic energy expenditure.• Our experimental preparations were trabeculae isolated from both ventricles; our index of metabolic energy expenditure was their heat production in response to electrical stimulation.• We found that the cost of activating contraction (i.e. the production of heat in the absence of force generation) was higher in trabeculae from the left ventricle, thereby conferring a lower mechanical efficiency on that ventricle.• Correction for the activation heat reveals the thermodynamic efficiency of the actomyosin crossbridges; whereas there was no interventricular difference in this fundamental property of cardiac muscle, its dependence on force development is baffling.Abstract We compare the energetics of right ventricular and left ventricular trabeculae carneae isolated from rat hearts. Using our work-loop calorimeter, we subjected trabeculae to stress-length work (W ), designed to mimic the pressure-volume work of the heart. Simultaneous measurement of heat production (Q) allowed calculation of the accompanying change of enthalpy ( H = W + Q). From the mechanical measurements (i.e. stress and change of length), we calculated work, shortening velocity and power. In combination with heat measurements, we calculated activation heat (Q A ), crossbridge heat (Q xb ) and two measures of cardiac efficiency: 'mechanical efficiency' (ε mech = W / H) and 'crossbridge efficiency' (ε xb = W /( H -Q A )). With respect to their left ventricular counterparts, right venticular trabeculae have higher peak shortening velocity, and higher peak mechanical efficiency, but with no difference of stress development, twitch duration, work performance, shortening power or crossbridge efficiency. That is, the 35% greater maximum mechanical efficiency of right venticular than left ventricular trabeculae (13.6 vs. 10.2%) is offset by the greater metabolic cost of activation (Q A ) in the latter. When corrected for this difference, crossbridge efficiency does not differ between the ventricles.