Cooling intact and demembranated trabeculae from rat heart releases myosin motors from their inhibited conformation.

Myosin filament-based regulation supplements actin filament-based regulation to control the strength and speed of contraction in heart muscle. In diastole, myosin motors form a folded helical array that inhibits actin interaction; during contraction, they are released from that array. A similar structural transition has been observed in mammalian skeletal muscle, in which cooling below physiological temperature has been shown to reproduce some of the structural features of the activation of myosin filaments during active contraction. Here, we used small-angle x-ray diffraction to characterize the structural changes in the myosin filaments associated with cooling of resting and relaxed trabeculae from the right ventricle of rat hearts from 39°C to 7°C. In intact quiescent trabeculae, cooling disrupted the folded helical conformation of the myosin motors and induced extension of the filament backbone, as observed in the transition from diastole to peak systolic force at 27°C. Demembranation of trabeculae in relaxing conditions induced expansion of the filament lattice, but the structure of the myosin filaments was mostly preserved at 39°C. Cooling of relaxed demembranated trabeculae induced changes in motor conformation and filament structure similar to those observed in intact quiescent trabeculae. Osmotic compression of the filament lattice to restore its spacing to that of intact trabeculae at 39°C stabilized the helical folded state against disruption by cooling. The myosin filament structure and motor conformation of intact trabeculae at 39°C were largely preserved in demembranated trabeculae at 27°C or above in the presence of Dextran, allowing the physiological mechanisms of myosin filament-based regulation to be studied in those conditions.

Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing.

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

Right Ventricle Has Normal Myofilament Function But Shows Perturbations in the Expression of Extracellular Matrix Genes in Patients With Tetralogy of Fallot Undergoing Pulmonary Valve Replacement.

Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF-PVR and RV donor samples, as was collagen content and cross-linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine-rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.

Myosin filament-based regulation of the dynamics of contraction in heart muscle.

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone." Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

A model of cardiac contraction based on novel measurements of tension development in human cardiomyocytes.

Experimental data from human cardiac myocytes at body temperature is crucial for a quantitative understanding of clinically relevant cardiac function and development of whole-organ computational models. However, such experimental data is currently very limited. Specifically, important measurements to characterize changes in tension development in human cardiomyocytes that occur with perturbations in cell length are not available. To address this deficiency, in this study we present an experimental data set collected from skinned human cardiac myocytes, including the passive and viscoelastic properties of isolated myocytes, the steady-state force calcium relationship at different sarcomere lengths, and changes in tension following a rapid increase or decrease in length, and after constant velocity shortening. This data set is, to our knowledge, the first characterization of length and velocity-dependence of tension generation in human skinned cardiac myocytes at body temperature. We use this data to develop a computational model of contraction and passive viscoelasticity in human myocytes. Our model includes troponin C kinetics, tropomyosin kinetics, a three-state crossbridge model that accounts for the distortion of crossbridges, and the cellular viscoelastic response. Each component is parametrized using our experimental data collected in human cardiomyocytes at body temperature. Furthermore we are able to confirm that properties of length-dependent activation at 37°C are similar to other species, with a shift in calcium sensitivity and increase in maximum tension. We revise our model of tension generation in the skinned isolated myocyte to replicate reported tension traces generated in intact muscle during isometric tension, to provide a model of human tension generation for multi-scale simulations. This process requires changes to calcium sensitivity, cooperativity, and crossbridge transition rates. We apply this model within multi-scale simulations of biventricular cardiac function and further refine the parametrization within the whole organ context, based on obtaining a healthy ejection fraction. This process reveals that crossbridge cycling rates differ between skinned myocytes and intact myocytes.

Quantification of [11C]GB67 binding to cardiac alpha1-adrenoceptors with positron emission tomography: validation in pigs.

INTRODUCTION: An increase in human cardiac alpha(1)-adrenoceptor (alpha(1)-AR) density is associated with various diseases such as myocardial ischemia, congestive heart failure, hypertrophic cardiomyopathy and hypertension. Positron emission tomography (PET) with an appropriate radioligand offers the possibility of imaging receptor function in the normal and diseased heart. [(11)C]GB67, an analogue of prazosin, has been shown in rats to have potential as a PET ligand with high selectivity to alpha(1)-AR. However, alpha(1)-AR density is up to ten times higher in rat heart compared to that in man. The aim of the present preclinical study was to extend the previous evaluation to a large mammal heart, where the alpha(1)-AR density is comparable to man, and to validate a method for quantification before PET studies in man. METHODS: Seven [(11)C]GB67 PET studies, with weight-adjusted target dose of either 5.29 MBq kg(-1) (pilot, test-retest and baseline-predose studies) or 8.22 MBq kg(-1) (baseline-displacement studies), were performed in four anaesthetised pigs (39.5 +/- 3.9 kg). Total myocardial volume of distribution (V (T)) was estimated under different pharmacological conditions using compartmental analysis with a radiolabelled metabolite-corrected arterial plasma input function. A maximum possible blocking dose of 0.12 mumol kg(-1) of unlabeled GB67 was given 20 min before [(11)C]GB67 administration in the predose study and 45 min after administration of [(11)C]GB67 in the displacement study. In addition, [(15)O]CO (3,000 MBq) and [(15)O]H(2)O, with weight adjusted target dose of 10.57 MBq kg(-1), were also administered for estimation of blood volume recovery (RC) of the left ventricular cavity and myocardial perfusion (MBF), respectively. RESULTS: [(11)C]GB67 V (T) values (in ml cm(-3)) were estimated to be 24.2 +/- 5.5 (range, 17.3-31.3), 10.1 (predose) and 11.6 (displacement). MBF did not differ within each pig, including between baseline and predose conditions. Predose and displacement studies showed that specific binding of [(11)C]GB67 to myocardial alpha(1)-ARs accounts for approximately 50% of V (T). CONCLUSION: The present study offers a methodology for using [(11)C]GB67 as a radioligand to quantify human myocardial alpha(1)-ARs in clinical PET studies.

Long-term precision of 18F-fluoride PET skeletal kinetic studies in the assessment of bone metabolism.

UNLABELLED: (18)F-Fluoride PET allows noninvasive evaluation of regional bone metabolism and has the potential to become a useful tool for assessing patients with metabolic bone disease and evaluating novel drugs being developed for these diseases. The main PET parameter of interest, termed K(i), reflects regional bone metabolism. The aim of this study was to compare the long-term precision of (18)F-fluoride PET with that of biochemical markers of bone turnover assessed over 6 mo. METHODS: Sixteen postmenopausal women with osteoporosis or significant osteopenia and a mean age of 64 y underwent (18)F-fluoride PET of the lumbar spine and measurements of biochemical markers of bone formation (bone-specific alkaline phosphatase and osteocalcin) and bone resorption (urinary deoxypyridinoline) at baseline and 6 mo later. Four different methods for analyzing the (18)F-fluoride PET data were compared: a 4k 3-compartmental model using nonlinear regression analysis (K(i-4k)), a 3k 3-compartmental model using nonlinear regression analysis (K(i-3k)), Patlak analysis (K(i-PAT)), and standardized uptake values. RESULTS: With the exception of a small but significant decrease in K(i-3k) at 6 mo, there were no significant differences between the baseline and 6-mo values for the PET parameters or biochemical markers. The long-term precision, expressed as the coefficient of variation (with 95% confidence interval in parentheses), was 12.2% (9%-19%), 13.8% (10%-22%), 14.4% (11%-22%), and 26.6% (19%-40%) for K(i-3k), K(i-PAT), mean standardized uptake value, and K(i-4k), respectively. For comparison, the precision of the biochemical markers was 10% (7%-15%), 18% (13%-27%), and 14% (10%-21%) for bone-specific alkaline phosphatase, osteocalcin, and urinary deoxypyridinoline, respectively. Intraclass correlation between the baseline and 6-mo values ranged from 0.44 for K(i-4k) to 0.85 for K(i-3k). No significant correlation was found between the repeated mean standardized uptake value measurements. CONCLUSION: The precision and intraclass correlation observed for K(i-3k) and K(i-PAT) was equivalent to that observed for biochemical markers. This study provided initial data on the long-term precision of (18)F-fluoride PET measured at the lumbar spine, which will aid in the accurate interpretation of changes in regional bone metabolism in response to treatment.

Conventional measurements of GFR using 51Cr-EDTA overestimate true renal clearance by 10 percent.

It is widely believed that measurement of the area under the plasma clearance curve (AUC) following a single intravenous injection of chromium-51 labelled ethylene diamine tetra-acetic acid ((51)Cr-EDTA) is a gold standard method for determining glomerular filtration rate (GFR). However, there are reports that (51)Cr-EDTA may have a significant extrarenal clearance. The aim of this study was to identify the non-renal component of (51)Cr-EDTA plasma clearance contributing to the AUC measurement of GFR. Seventy healthy postmenopausal women (mean age 60 years, range 45-79 years) were injected with 3 MBq (51)Cr-EDTA and 0.25 MBq iodine-125 labelled human serum albumin and 11 blood samples taken between 0 and 4 h through an indwelling venous cannula. For the first 21 subjects, two complete urine collections were made 0-2 h and 2-4 h after injection, and for the final 49 patients, four 1-h urine collections were made. The mean (51)Cr-EDTA total plasma clearance was 84 ml/min (range 50-132 ml/min). The mean ratio (SEM) of urine to total clearance determined from the cumulative 1-, 2-, 3- and 4-h data was 0.903 (0.018), 0.891 (0.013), 0.898 (0.011) and 0.899 (0.010) respectively and remained constant despite the mean urine concentration decreasing from 122% to 15%/litre during this period. A least squares fit to data from the 238 individual urine collections was used to determine the fraction of the total plasma clearance attributable to renal clearance, alpha(0), and the residual urine volume, delta V. The results were alpha(0)=0.910 (95% CI: 0.889-0.932) and delta V=14 ml (95% CI: -4 to +34 ml). The overestimation of the true renal clearance of (51)Cr-EDTA by the AUC method is believed to be due to the failure of the plasma clearance curve to reach the true terminal exponential by 2 h after injection as usually assumed. As a result, conventional measurements of GFR using (51)Cr-EDTA overestimate the true renal clearance of tracer by approximately 10%.

Quantitative studies of bone in postmenopausal women using (18)F-fluoride and (99m)Tc-methylene diphosphonate.

UNLABELLED: Quantitative radionuclide studies of bone using the short-lived tracers (18)F-fluoride and (99m)Tc-methylene diphosphonate (MDP) are an alternative method to biochemical markers of bone turnover for investigating the dynamic state of the skeleton. In this study we evaluated their use to quantify bone turnover in women receiving antiresorptive therapy compared with that of untreated control subjects. METHODS: The patients were 69 healthy postmenopausal women. Twenty-six women were receiving hormone replacement therapy (HRT) and 43 were untreated age-matched control subjects. After bolus injection of (18)F-fluoride (1 MBq), (99m)Tc-MDP (1 MBq), (51)Cr-ethylenediaminetetraacetic acid (3 MBq), and (125)I-labeled human serum albumin (0.25 MBq), multiple blood samples and urine collections were taken between 0 and 4 h. The clearance to bone mineral K(bone) was first evaluated using the area under the plasma concentration curve (AUC) on the assumption that the rate constant k(4) for the outflow of tracer from bone was negligibly small. AUC values of K(bone) were then compared with those found using a compartmental model method that allowed k(4) to be fitted as a free parameter. RESULTS: Using the AUC method the mean plus minus SD for K(bone) for the 2 tracers were: (18)F-fluoride, 61.8 plus minus 12.0 mL center dot min(-1) (HRT group) versus 67.2 plus minus 12.6 mL center dot min(-1) (control group) (P = 0.045); and (99m)Tc-MDP, 40.3 plus minus 8.2 mL center dot min(-1) (HRT group) versus 44.2 plus minus 7.6 mL center dot min(-1) (control group) (P = 0.024). Values for the 2 tracers in individual patients were moderately well correlated (r = 0.76; P < 0.001). Using the compartmental model method, k(4) for (18)F-fluoride was shown to lie in the range 0--0.0025 min(-1) with a best-fit value of 0.0018 min(-1). Values of K(bone) determined using k(4) = 0.0018 min(-1) were highly correlated with the AUC values (r = 0.989; SEE = 2.05 mL center dot min(-1)) with numeric values that were larger by a factor of 1.53. Analysis of the (99m)Tc-MDP data was more difficult because of uncertainties in protein binding in the extracellular fluid compartment space. The best fit for k(4) was in the range 0.0010--0.0014 min(-1) with values of K(bone) similar to those found using the AUC method. CONCLUSION: Values of K(bone) determined using the AUC method were able to differentiate between HRT-treated women and postmenopausal women who were not treated and were highly correlated with those determined using a compartmental model method with nonzero values of k(4).