Denis S. Loiselle

A Thermodynamic Model of the Cardiac Sarcoplasmic/Endoplasmic Ca2+ (SERCA) Pump

We present a biophysically based kinetic model of the cardiac SERCA pump that consolidates a range of experimental data into a consistent and thermodynamically constrained framework. The SERCA model consists of a number of sub-states with partial reactions that are sensitive to Ca(2+) and pH, and to the metabolites MgATP, MgADP, and Pi. Optimization of model parameters to fit experimental data favors a fully cooperative Ca(2+)-binding mechanism and predicts a Ca(2+)/H(+) counter-transport stoichiometry of 2. Moreover, the order of binding of the partial reactions, particularly the binding of MgATP, proves to be a strong determinant of the ability of the model to fit the data. A thermodynamic investigation of the model indicates that the binding of MgATP has a large inhibitory effect on the maximal reverse rate of the pump. The model is suitable for integrating into whole-cell models of cardiac electrophysiology and Ca(2+) dynamics to simulate the effects on the cell of compromised metabolism arising in ischemia and hypoxia.

Reduced contraction strength with increased intracellular [Ca2+] in left ventricular trabeculae from failing rat hearts

Intracellular calcium ([Ca2+]i) and isometric force were measured in left ventricular (LV) trabeculae from spontaneously hypertensive rats (SHR) with failing hearts and normotensive Wistar‐Kyoto (WKY) controls. At a physiological stimulation frequency (5 Hz), and at 37 °C, the peak stress of SHR trabeculae was significantly (P≤; 0.05) reduced compared to WKY (8 ± 1 mN mm−2(n= 8)vs. 21 ± 5 mN mm−2(n= 8), respectively). No differences between strains in either the time‐to‐peak stress, or the time from peak to 50 % relaxation were detected. Measurements using fura‐2 showed that in the SHR both the peak of the Ca2+ transient and the resting [Ca2+]i were increased compared to WKY (peak: 0.69 ± 0.08 vs. 0.51 ± 0.08 μm (P≤ 0.1) and resting: 0.19 ± 0.02 vs. 0.09 ± 0.02 μm (P≤ 0.05), SHR vs. WKY, respectively). The decay of the Ca2+ transient was prolonged in SHR, with time constants of: 0.063 ± 0.002 vs. 0.052 ± 0.003 s (SHR vs. WKY, respectively). Similar results were obtained at 1 Hz stimulation, and for [Ca2+]o between 0.5 and 5 mm. The decay of the caffeine‐evoked Ca2+ transient was slower in SHR (9.8 ± 0.7 s (n= 8)vs. 7.7 ± 0.2 s (n= 8) in WKY), but this difference was removed by use of the SL Ca2+‐ATPase inhibitor carboxyeosin. Histological examination of transverse sections showed that the fractional content of perimysial collagen was increased in SHR compared to WKY (18.0 ± 4.6 % (n= 10)vs. 2.9 ± 0.9 % (n= 11) SHR vs. WKY, respectively). Our results show that differences in the amplitude and the time course of the Ca2+ transient between SHR and WKY do not explain the reduced contractile performance of SHR myocardium per se. Rather, we suggest that, in this animal model of heart failure, contractile function is compromised by increased collagen, and its three‐dimensional organisation, and not by reduced availability of intracellular Ca2+.

A unique micromechanocalorimeter for simultaneous measurement of heat rate and force production of cardiac trabeculae carneae

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.

An innovative work-loop calorimeter for in vitro measurement of the mechanics and energetics of working cardiac trabeculae

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 flow-through 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%.

Trabeculae carneae as models of the ventricular walls: implications for the delivery of oxygen

Trabeculae carneae are the smallest naturally arising collections of linearly arranged myocytes in the heart. They are the preparation of choice for studies of function of intact myocardium in vitro. In vivo, trabeculae are unique in receiving oxygen from two independent sources: the coronary circulation and the surrounding ventricular blood. Because oxygen partial pressure (PO2) in the coronary arterioles is identical in specimens from both ventricles, whereas that of ventricular blood is 2.5-fold higher in the left ventricle than in the right ventricle, trabeculae represent a “natural laboratory” in which to examine the influence of “extravascular” PO2 on the extent of capillarization of myocardial tissue. We exploit this advantage to test four hypotheses. (1) In trabeculae from either ventricle, a peripheral annulus of cells is devoid of capillaries. (2) Hence, sufficiently small trabeculae from either ventricle are totally devoid of capillaries. (3) The capillary-to-myocyte ratios in specimens from either ventricle are identical to those of their respective walls. (4) Capillary-to-myocyte ratios are comparable in specimens from either ventricle, reflecting equivalent energy demands in vivo, driven by identical contractile frequencies and comparable wall stresses. We applied confocal fluorescent imaging to trabeculae in cross section, subsequently using semi-automated segmentation techniques to distinguish capillaries from myocytes. We quantified the capillary-to-myocyte ratios of trabeculae from both ventricles and compared them to those determined for the ventricular free walls and septum. Quantitative interpretation was furthered by mathematical modeling, using both the classical solution to the diffusion equation for elliptical cross sections, and a novel approach applicable to cross sections of arbitrary shape containing arbitrary disposition of capillaries and non-respiring collagen cords.

Mitochondrial inefficiencies and anoxic ATP hydrolysis capacities in diabetic rat heart

As ~80% of diabetic patients die from heart failure, an understanding of diabetic cardiomyopathy is crucial. Mitochondria occupy 35-40% of the mammalian cardiomyocyte volume and supply 95% of the hearts ATP, and diabetic heart mitochondria show impaired structure, arrangement, and function. We predict that bioenergetic inefficiencies are present in diabetic heart mitochondria; therefore, we explored mitochondrial proton and electron handling by linking oxygen flux to steady-state ATP synthesis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (ΔΨ) within rat heart tissues. Sprague-Dawley rats were injected with streptozotocin (STZ, 55 mg/kg) to induce type 1 diabetes or an equivalent volume of saline (control, n = 12) and fed standard rat chow for 8 wk. By coupling high-resolution respirometers with purpose-built fluorometers, we followed Magnesium Green (ATP synthesis), Amplex UltraRed (ROS production), and safranin-O (ΔΨ). Relative to control rats, the mass-specific respiration of STZ-diabetic hearts was depressed in oxidative phosphorylation (OXPHOS) states. Steady-state ATP synthesis capacity was almost one-third lower in STZ-diabetic heart, which, relative to oxygen flux, equates to an estimated 12% depression in OXPHOS efficiency. However, with anoxic transition, STZ-diabetic and control heart tissues showed similar ATP hydrolysis capacities through reversal of the F1F0-ATP synthase. STZ-diabetic cardiac mitochondria also produced more net ROS relative to oxygen flux (ROS/O) in OXPHOS. While ΔΨ did not differ between groups, the time to develop ΔΨ with the onset of OXPHOS was protracted in STZ-diabetic mitochondria. ROS/O is higher in lifelike OXPHOS states, and potential delays in the time to develop ΔΨ may delay ATP synthesis with interbeat fluctuations in ADP concentrations. Whereas diabetic cardiac mitochondria produce less ATP in normoxia, they consume as much ATP in anoxic infarct-like states.

Characterization of a flow-through microcalorimeter for measuring the heat production of cardiac trabeculae

The energy consumption of isolated cardiac trabeculae can be inferred from measurements of their heat production. Once excised from the heart, to remain viable, trabeculae require continuous superfusion with an oxygen- and nutrient-rich solution. Flow-through calorimeters enable trabeculae to be maintained in a stable and controlled environment for many hours at a time. In this paper we describe and characterize a flow-through microcalorimeter, with sensitivity in the 1μW range, for measuring the heat output of 10μg cardiac trabeculae. The device uses infrared-sensitive, thin-film thermopile sensors to provide a noncontact method for measuring temperature differences. The sensors are capable of resolving 5μK temperature differences within the superfusing fluid. The microcalorimeter has a sensitivity of 2.56V∕W at a flow rate of 1μl∕s, with a time constant of approximately 3.5 s. The sensitivity and time constant are strongly dependent upon the flow rate. Predictions of a finite-element model of the calori...

A Metabolite-Sensitive, Thermodynamically Constrained Model of Cardiac Cross-Bridge Cycling: Implications for Force Development during Ischemia

We present a metabolically regulated model of cardiac active force generation with which we investigate the effects of ischemia on maximum force production. Our model, based on a model of cross-bridge kinetics that was developed by others, reproduces many of the observed effects of MgATP, MgADP, Pi, and H(+) on force development while retaining the force/length/Ca(2+) properties of the original model. We introduce three new parameters to account for the competitive binding of H(+) to the Ca(2+) binding site on troponin C and the binding of MgADP within the cross-bridge cycle. These parameters, along with the Pi and H(+) regulatory steps within the cross-bridge cycle, were constrained using data from the literature and validated using a range of metabolic and sinusoidal length perturbation protocols. The placement of the MgADP binding step between two strongly-bound and force-generating states leads to the emergence of an unexpected effect on the force-MgADP curve, where the trend of the relationship (positive or negative) depends on the concentrations of the other metabolites and [H(+)]. The model is used to investigate the sensitivity of maximum force production to changes in metabolite concentrations during the development of ischemia.

Changes of surface and t-tubular membrane excitability during fatigue with repeated tetani in isolated mouse fast- and slow-twitch muscle

We investigated whether impaired sarcolemmal excitability causes severe fatigue during repeated tetani in isolated mouse skeletal muscle. Slow-twitch soleus or fast-twitch extensor digitorum longus (EDL) muscles underwent intensive stimulation (standard protocol: 125 Hz for 500 ms, every second, parallel plate electrodes, 20 V, 0.1-ms pulses). Interventions with altered stimulation characteristics were tested either on the entire fatigue profile or after 90- to 100-s stimulation. d-tubocurarine did not alter the fatigue profile in soleus thereby eliminating impaired neuromuscular transmission. Lower stimulation frequencies partially restored peak force, especially in soleus. The twitch force-stimulation strength relationship shifted towards higher voltages in both muscle types, with a much larger shift in EDL. Augmenting pulse strength restored tetanic force from 29% (4.4 V) to 79% (20 V), or slowed fatigue in soleus. Increasing pulse duration (0.1 to 1.0 ms) restored tetanic force from 8 to 46% in EDL and from 41 to 90% in soleus; 0.25-ms pulses restored tetanic force to 83% in soleus. Switching from transverse wire to parallel plate stimulation increased tetanic force from 34 to 63%, and fatigue was exacerbated with wires compared with plates in soleus. The combined data suggest that impaired excitability (disrupted action potential generation) within trains is the main contributor ( approximately 50% initial force) to severe fatigue in both muscle types, the surface rather than t-tubular membrane is the main site of impairment during wire stimulation, and extreme fatigue in EDL includes an increased action potential threshold leading to inexcitable fibers. Moreover, mathematical modeling discounts anoxia as the major contributor to fatigue during our stimulation regime in isolated muscles.

Double-sigmoid model for fitting fatigue profiles in mouse fast- and slow-twitch muscle.

We present a curve‐fitting approach that permits quantitative comparisons of fatigue profiles obtained with different stimulation protocols in isolated slow‐twitch soleus and fast‐twitch extensor digitorum longus (EDL) muscles of mice. Profiles from our usual stimulation protocol (125 Hz for 500 ms, evoked once every second for 100–300 s) could be fitted by single‐term functions (sigmoids or exponentials) but not by a double exponential. A clearly superior fit, as confirmed by the Akaiki Information Criterion, was achieved using a double‐sigmoid function. Fitting accuracy was exceptional; mean square errors were typically <1% and r2 > 0.9995. The first sigmoid (early fatigue) involved ∼10% decline of isometric force to an intermediate plateau in both muscle types; the second sigmoid (late fatigue) involved a reduction of force to a final plateau, the decline being 83% of initial force in EDL and 63% of initial force in soleus. The maximal slope of each sigmoid was seven‐ to eightfold greater in EDL than in soleus. The general applicability of the model was tested by fitting profiles with a severe force loss arising from repeated tetanic stimulation evoked at different frequencies or rest periods, or with excitation via nerve terminals in soleus. Late fatigue, which was absent at 30 Hz, occurred earlier and to a greater extent at 125 than 50 Hz. The model captured small changes in rate of late fatigue for nerve terminal versus sarcolemmal stimulation. We conclude that a double‐sigmoid expression is a useful and accurate model to characterize fatigue in isolated muscle preparations.