Rapid myocardial relaxation is essential in maintaining cardiac output, and impaired relaxation is an early indicator of diastolic dysfunction. While the biochemical modifiers of relaxation are well known to include calcium handling, thin filament activation, and myosin kinetics, biophysical and biomechanical modifiers can also alter relaxation. We have previously shown that the relaxation rate is increased by an increasing strain rate, not a reduction in afterload. The slope of the relaxation rate to strain rate relationship defines Mechanical Control of Relaxation (MCR). To investigate MCR further, we performed in vitro experiments and computational modeling of preload-adjustment using intact rat cardiac trabeculae. Trabeculae studies are often performed using isometric (fixed-end) muscles at optimal length (Lo, length producing maximal developed force). We determined that reducing muscle length from Lo increased MCR by 20%, meaning that reducing preload could substantially increase the sensitivity of the relaxation rate to the strain rate. We subsequently used computational modeling to predict mechanisms that might underlie this preload-dependence. Computational modeling was not able to fully replicate experimental data, but suggested that thin-filament properties are not sufficient to explain preload-dependence of MCR because the model required the thin-filament to become more activated at reduced preloads. The models suggested that myosin kinetics may underlie the increase in MCR at reduced preload, an effect that can be enhanced by force-dependence. Relaxation can be modified and enhanced by reduced preload. Computational modeling implicates myosin-based targets for treatment of diastolic dysfunction, but further model refinements are needed to fully replicate experimental data.

DCM phenotype are lacking. However, we recently introduced a metric capable of predicting the type and severity of myocardial remodeling in progressive cardiomyopathies (Davis et al. Cell, 2016), which hinges on relating force-time integrals of computationally derived twitches of diseased cardiomyocytes to wild-type. Negative values of this metric (the 'tension index') correlate with eccentric hypertrophy associated with DCM, and the magnitude correlates with the severity of phenotype. Here, we test the hypothesis that an experimental analogue of the tension index can be generated from twitches of intact cardiac trabeculae from genetically engineered murine models of DCM, and that it will predict the degree of myocardial remodeling. Intact trabeculae from a DCM model-a calcium-desensitizing tropomyosin mutation (D230N)-have significantly decreased twitch force compared to wild-type trabeculae (1652 versus 3153 kPa, respectively). The corresponding tension index for D230N trabeculae is -6.62x10 3 (normalized force,ms), predicting significant eccentric hypertrophy. Indeed, our predictions are verified by echocardiographic measurements of ventricular diameters in vivo. In D230N hearts, the left-ventricular systolic and diastolic diameters are significantly greater than wild-type (3.1 and 4.3mm, versus 2.4 and 3.8mm, respectively). Furthermore, intact trabeculae from a double-transgenic (DTG) murine model, (D230N plus the calcium-sensitizing troponin mutation, L48Q), produce twitch forces similar to wild-type (3253 kPa). The tension index for DTG trabeculae is -1.48x10 3 (normalized force,ms), predicting less remodeling than D230N alone. Consistently, systolic and diastolic diameters of DTG hearts are not different from wild-type (2.5 and 3.9mm, respectively). Our work demonstrates the ability of a trabecula-based tension index to predict organ-level morphology in DCM hearts and to use these predictions to develop interventions that prevent pathological remodeling. 1306-PosMolecular Dynamics Studies of Myosin Structure with 2-Deoxy-ADP The mitochondrial inner membrane (IM) has a dynamic and complex structure that plays a significant role in mitochondrial function and energy metabolism.

Impaired relaxation is a prevalent form of diastolic dysfunction, present in nearly all cases of heart failure and in many asymptomatic adults. To date, there are no accepted treatments for impaired relaxation, despite its biochemical control by calcium reuptake, thin filament deactivation, and crossbridge kinetics. Mechanical modification of relaxation was previously theorized to occur through afterload; we, however, have recently shown that relaxation was actually modified by the strain rate of myocardial lengthening. We termed this Mechanical Control of Relaxation, or the sensitivity of the relaxation rate to the strain rate. The mechanisms underlying Mechanical Control of Relaxation are unknown, but computational models and our preliminary data suggest a dependence on myosin detachment. The objective of this study was to evaluate whether myosin was a modifying factor of Mechanical Control of Relaxation. Intact cardiac trabeculae and cardiomyocytes were obtained from rats (Sprague Dawley both treated and untreated with propylthiouracil, Spontaneously Hypertensive, and Wistar Kyoto strains) and underwent load-clamp studies. Mechanical Control of Relaxation could be improved by reducing preload (length) 5%, increasing the sensitivity of the relaxation rate to strain rate by 28±16%. Treatment with 400μM Omecamtiv Mecarbil, a myosin-ATPase specific drug, induced similar increases. Myosin isoform differences were also studied. Collagenase treatment of intact trabeculae increased Mechanical Control by 21±3%; intact myocyte studies show no collagen-dependence. These data provide evidence that the properties of myosin’s response to strain rate are major factors that modify Mechanical Control of myocardial Relaxation.

Impaired cardiac relaxation is present in nearly all cases of heart failure and possibly in up to 25% of the asymptomatic population. Myocardial relaxation is known to be biochemically modified by the calcium reuptake rate, thin filament calcium sensitivity, and crossbridge kinetics. Mechanical regulation of relaxation was thought to be regulated via afterload, but we have recently shown that a lengthening strain was sufficient to modify relaxation. Further, the relaxation rate is actually dependent on the strain rate, a relationship that we termed Mechanical Control of Relaxation. Computational modeling suggests that myosin detachment is a key mechanism underlying Mechanical Control of Regulation, but to date, no experimental evidence for this was available. The objective of this study was to determine if myosin head position changed in response to lengthening strains during relaxation. Intact cardiac trabeculae were mounted within the beamline of the Biophysical Collaborative Access Team (BioCAT) beamline at the Advanced Photon Source at Argonne National Laboratories. The trabeculae were paced and load-clamps were performed during time-resolved imaging of the equatorial axis, which primarily reflects myosin head positioning. Activation (pacing) caused the myosin head localization to shift from the thick filament to near the thin filament (increased I 1,1 /I 1,0 ratio). During stretch, there was a transient decline of the I 1,1 /I 1,0 ratio which recovered until relaxation was complete, when the ratio again reduced indicating myosin returned to the thick filament. These preliminary data suggest that Mechanical Control of Relaxation is caused by perturbations in myosin, but the late-diastolic kinetics suggests that the strain-rate dependent detachment does not lead to immediate deactivation of myosin heads. Modifications of myosin ATPase properties may reveal more specific regulatory targets, which may provide new insight and targets for treating impaired myocardial relaxation.