Myocardial twitch duration and the dependence of oxygen consumption on pressure-volume area: experiments and modelling.

We tested the proposition that linear length dependence of twitch duration underlies the well-characterised linear dependence of oxygen consumption (V(O(2)) ) on pressure­volume area (PVA) in the heart. By way of experimental simplification, we reduced the problem from three dimensions to one by substituting cardiac trabeculae for the classically investigated whole-heart. This allowed adoption of stress­length area (SLA) as a surrogate for PVA, and heat as a proxy for V(O(2)) . Heat and stress (force per cross-sectional area), at a range of muscle lengths and at both 1 mM and 2 mM [Ca(2+)](o), were recorded from continuously superfused rat right-ventricular trabeculae undergoing fixed-end contractions. The heat­SLA relations of trabeculae (reported here, for the first time) are linear. Twitch duration increases monotonically (but not strictly linearly) with muscle length. We probed the cellular mechanisms of this phenomenon by determining: (i) the length dependence of the duration of the Ca(2+) transient, (ii) the length dependence of the rate of force redevelopment following a length impulse (an index of Ca(2+) binding to troponin-C), (iii) the effect on the simulated time course of the twitch of progressive deletion of length and Ca(2+)-dependent mechanisms of crossbridge cooperativity, using a detailed mathematical model of the crossbridge cycle, and (iv) the conditions required to achieve these multiple length dependencies, using a greatly simplified model of twitch mechano-energetics. From the results of these four independent investigations, we infer that the linearity of the heat­SLA relation (and, by analogy, the V(O(2))­PVA relation) is remarkably robust in the face of departures from linearity of length-dependent twitch duration.

The Noble cardiac ventricular electrophysiology models in CellML.

We present a review of the cardiac ventricular cell electrophysiology models developed by Prof. Denis Noble and colleagues as an example of how models may be published using a web-based CellML publication framework. The models reviewed have been marked-up in CellML and then used to compute all results presented here. The models are freely available from a website as are the specific numerical experiments discussed in this review and the tools used to perform the simulations.

Modelling the passive and nerve activated response of the rectus femoris muscle to a flexion loading: a finite element framework.

A muscle modelling framework is presented which relates the mechanical response of the rectus femoris muscle (at the organ level) to tissue level properties, with the capability of linking to the cellular level as part of the IUPS Physiome Project. This paper will outline our current approach to muscle modelling incorporating micro-structural passive and active properties including fibre orientations and nerve innervation. The technique is based on finite deformation (using FE analysis) coupled to electrical nerve initiated muscle activation, and we present the influence of active tension through an eccentric contraction at specific flexion angles. Finally we discuss the future goals of incorporating cell mechanics and validating at the organ level to provide a complete diagnostic tool with the ability to relate mechanisms of failure across spatial scales.

The catalytic activity of cytochrome P450cam towards styrene oxidation is increased by site-specific mutagenesis.

The styrene oxidation activity of cytochrome P450cam, has been greatly improved by rational protein engineering. Compared to the wild-type enzyme, the active-site mutants Y96A and Y96F bound styrene more tightly, consumed NADH more rapidly, and were more efficient at utilising reducing equivalents for product formation. Styrene oxide formation rates were enhanced 9-fold in the Y96A mutant relative to wild-type, and 25-fold in the Y96F mutant, thus demonstrating the effectiveness of active-site redesign in improving the activity of a haem monooxygenase towards an unnatural substrate.

Relating components of pressure-volume area in Suga's formulation of cardiac energetics to components of the stress-time integral.

The concept of pressure-volume area (PVA) in whole heart studies is central to the phenomenological description of cardiac energetics proposed by Suga and colleagues (Physiol Rev 70: 247-277, 1990). PVA consists of two components: an approximately rectangular work loop (W) and an approximately triangular region of potential energy (U). In the case of isovolumic contractions, PVA consists entirely of U. The utility of Suga's description of cardiac energetics is the observation that the oxygen consumption of the heart (Vo(2)) is linearly dependent on PVA. By using isolated ventricular trabeculae, we found a basis on which to correlate the components of stress-length area (SLA; i.e., the 1-D equivalent of PVA) with specific regions of the stress-time integral (STI; i.e., the area under the force-time profile of a single twitch). In each case, proportionality obtains and is robust, independent of the type of twitch contraction (isometric or isotonic), and insensitive to changes of preload or afterload. We apply our results by examining retrospectively the interpretations reached in three independent studies published in the literature.

Comparison of the Gibbs and Suga formulations of cardiac energetics: the demise of "isoefficiency".

Two very different sorts of experiments have characterized the field of cardiac energetics over the past three decades. In one of these, Gibbs and colleagues measured the heat production of isolated papillary muscles undergoing isometric contractions and afterloaded isotonic contractions. The former generated roughly linear heat vs. force relationships. The latter generated enthalpy-load relationships, the peak values of which occurred at or near peak isometric force, i.e., at a relative load of unity. Contractile efficiency showed a pronounced dependence on afterload. By contrast, Suga and coworkers measured the oxygen consumption (Vo(2)) while recording the pressure-volume-time work loops of blood-perfused isolated dog hearts. From the associated (linear) end-systolic pressure-volume relations they derived a quantity labeled pressure-volume area (PVA), consisting of the sum of pressure-volume work and unspent elastic energy and showed that this was linearly correlated with Vo(2) over a wide range of conditions. This linear dependence imposed isoefficiency: constant contractile efficiency independent of afterload. Neither these data nor those of Gibbs and colleagues are in dispute. Nevertheless, despite numerous attempts over the years, no demonstration of either compatibility or incompatibility of these disparate characterizations of cardiac energetics has been forthcoming. We demonstrate that compatibility between the two formulations is thwarted by the concept of isoefficiency, the thermodynamic basis of which we show to be untenable.

The dimerization of Pseudomonas putida cytochrome P450cam: practical consequences and engineering of a monomeric enzyme.

Cytochrome P450cam dimerizes via the formation of an intermolecular disulfide bond, complicating the storage and handling of the enzyme, particularly at higher concentrations. The dimeric enzyme is 14% less active than the monomer and forms at a slow but significant rate even at 4 degrees C [k = 1.09 x 10(-3) mM(-1) h(-1)]. To eliminate any ambiguity introduced by dimer formation and to simplify handling and storage of the enzyme, site-directed mutagenesis was used to identify C334 as the single cysteine residue responsible for the formation of the disulfide linkage and to engineer a monomeric enzyme by substituting an alanine in its place. The C334A mutant is identical with the wild-type P450cam monomer in terms of optical spectra, camphor binding and turnover activity, but shows no evidence of dimerization and aggregation even at millimolar concentrations. Preliminary 1H NMR investigations also indicate a significant improvement in the quality of spectra obtained with this mutant. (C334A)P450cam is therefore proposed as an alternative to the wild-type enzyme-a base mutant otherwise identical with the wild-type but with improved handling characteristics.