Pediatric and adult-onset HCM mutations in the myosin motor domain have similar properties

Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder that typically involves left ventricular hypertrophy and abnormal cardiac contractility. Mutations in β-MyHC are a major cause of HCM and are typically characterized with cardiac hypercontractility, but the specific mechanistic changes to myosin function that lead to the disease remain incompletely understood. Predicting the severity of any single β-MyHC mutation is hindered by a lack of detailed evaluation at the molecular level. In addition, since the cardiomyopathy can take 20 - 40 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations which result in childhood cardiomyopathies may show a more severe indication of molecular changes in myosin and be therefore easier to identify. In this work, we performed steady-state and transient kinetics analysis of the myosin carrying one of eight miss sense mutations in the motor domain. Five of these have been identified in childhood cardiomyopathies. The derived parameters were used to model the ATP driven cross bridge. Contrary to our hypothesis, the results show no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted A·M·D occupancy for [A] = 3 Kapp along with the closely related Duty Ratio (DR) and the measured ATPases all change in parallel (in both sign and degree of change) compared to the WT values. Six of the eight HCM mutations are clearly distinct from a set of DCM mutations previously characterized

Modeling the Actin.myosin ATPase cross-bridge cycle for skeletal and cardiac muscle myosin isoforms

Modeling the complete actin.myosin ATPase cycle has always been limited by the lack of experimental data concerning key steps of the cycle, because these steps can only be defined at very low ionic strength. Here, using human ?-cardiac myosin-S1, we combine published data from transient and steady-state kinetics to model a minimal eight-state ATPase cycle. The model illustrates the occupancy of each intermediate around the cycle and how the occupancy is altered by changes in actin concentration for [actin] = 1–20Km. The cycle can be used to predict the maximal velocity of contraction (by motility assay or sarcomeric shortening) at different actin concentrations (which is consistent with experimental velocity data) and predict the effect of a 5 pN load on a single motor. The same exercise was repeated for human ?-cardiac myosin S1 and rabbit fast skeletal muscle S1. The data illustrates how the motor domain properties can alter the ATPase cycle and hence the occupancy of the key states in the cycle. These in turn alter the predicted mechanical response of the myosin independent of other factors present in a sarcomere, such as filament stiffness and regulatory proteins. We also explore the potential of this modeling approach for the study of mutations in human ?-cardiac myosin using the hypertrophic myopathy mutation R453C. Our modeling, using the transient kinetic data, predicts mechanical properties of the motor that are compatible with the single-molecule study. The modeling approach may therefore be of wide use for predicting the properties of myosin mutations

Dilated cardiomyopathy myosin mutants have reduced force-generating capacity

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human ?-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M.D complex in the steady-state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force holding capacity due to the reduced occupancy of the force-holding state

Detailed Thermal Characterization of Acrylonitrile Butadiene Styrene and Polylactic Acid Based Carbon Composites Used in Additive Manufacturing

Currently, 3D printing is an affordable technology for industry, healthcare, and individuals. Understanding the mechanical properties and thermoplastic behaviour of the composites is critical for the users. Our results give guidance for certain target groups including professionals in the field of additive manufacturing for biomedical components with in-depth characterisation of the examined commercially available ABS and PLA carbon-based composites. The study aimed to characterize these materials in terms of thermal behaviour and structure. The result of the heating-cooling loops is the thermal hysteresis effect of Ohmic resistance with its accommodation property in the temperature range of 20–84 °C for ESD-ABS and 20–72 °C for ESD-PLA. DSC-TGA measurements showed that the carbon content of the examined ESD samples is ~10–20% (m/m) and there is no significant difference in the thermodynamic behaviour of the basic ABS/PLA samples and their ESD compounds within the temperature range typically used for 3D printing. The results support the detailed design process of 3D-printed electrical components and prove that ABS and PLA carbon composites are suitable for prototyping and the production of biomedical sensors

Evaluation and Comparison of Traditional Plaster and Fiberglass Casts with 3D-Printed PLA and PLA–CaCO3 Composite Splints for Bone-Fracture Management

Bone fractures pose a serious challenge for the healthcare system worldwide. A total of 17.5% of these fractures occur in the distal radius. Traditional cast materials commonly used for treatment have certain disadvantages, including a lack of mechanical and water resistance, poor hygiene, and odors. Three-dimensional printing is a dynamically developing technology which can potentially replace the traditional casts. The aim of the study was to examine and compare the traditional materials (plaster cast and fiberglass cast) with Polylactic Acid (PLA) and PLA–CaCO3 composite materials printed using Fused Filament Fabrication (FFF) technology and to produce a usable cast of each material. The materials were characterized by tensile, flexural, Charpy impact, Shore D hardness, flexural fatigue, and variable load cyclic tests, as well as an absorbed water test. In addition, cost-effectiveness was evaluated and compared. The measured values for tensile strength and flexural strength decreased with the increase in CaCO3 concentration. In the fatigue tests, the plaster cast and the fiberglass cast did not show normal fatigue curves; only the 3D-printed materials did so. Variable load cyclic tests showed that traditional casts cannot hold the same load at the same deflection after a higher load has been used. During these tests, the plaster cast had the biggest relative change (−79.7%), compared with −4.8 % for the 3D-printed materials. The results clearly showed that 3D-printed materials perform better in both static and dynamic mechanical tests; therefore, 3D printing could be a good alternative to customized splints and casts in the near future

Development of a Novel X-ray Compatible 3D-Printed Bone Model to Characterize Different K-Wire Fixation Methods in Support of the Treatment of Pediatric Radius Fractures

Additive manufacturing technologies are essential in biomedical modeling and prototyping. Polymer-based bone models are widely used in simulating surgical interventions and procedures. Distal forearm fractures are the most common pediatric fractures, in which the Kirschner wire fixation is the most widely used operative method. However, there is still lingering controversy throughout the published literature regarding the number of wires and sites of insertion. This study aims to critically compare the biomechanical stability of different K-wire fixation techniques. Different osteosyntheses were reconstructed on 189 novel standardized bone models, which were created using 3D printing and molding techniques, using PLA and polyurethane materials, and it has been characterized in terms of mechanical behavior and structure. X-ray imaging has also been performed. The validation of the model was successful: the relative standard deviations (RSD = 100 × SD × mean−1, where RSD is relative standard deviation, SD is the standard deviation) of the mechanical parameters varied between 1.1% (10° torsion; 6.52 Nm ± 0.07 Nm) and 5.3% (5° torsion; 4.33 Nm ± 0.23 Nm). The simulated fractures were fixed using two K-wires inserted from radial and dorsal directions (crossed wire fixation) or both from the radial direction, in parallel (parallel wire fixation). Single-wire fixations with shifted exit points were also included. Additionally, three-point bending tests with dorsal and radial load and torsion tests were performed. We measured the maximum force required for a 5 mm displacement of the probe under dorsal and radial loads (means for crossed wire fixation: 249.5 N and 355.9 N; parallel wire fixation: 246.4 N and 308.3 N; single wire fixation: 115.9 N and 166.5 N). We also measured the torque required for 5° and 10° torsion (which varied between 0.15 Nm for 5° and 0.36 Nm for 10° torsion). The crossed wire fixation provided the most stability during the three-point bending tests. Against torsion, both the crossed and parallel wire fixation were superior to the single-wire fixations. The 3D printed model is found to be a reliable, cost-effective tool that can be used to characterize the different fixation methods, and it can be used in further pre-clinical investigations

Stereolithography 3D Printing of a Heat Exchanger for Advanced Temperature Control in Wire Myography

We report the additive manufacturing of a heat-exchange device that can be used as a cooling accessory in a wire myograph. Wire myography is used for measuring vasomotor responses in small resistance arteries; however, the commercially available devices are not capable of active cooling. Here, we critically evaluated a transparent resin material, in terms of mechanical, structural, and thermal behavior. Tensile strength tests (67.66 ± 1.31 MPa), Charpy impact strength test (20.70 ± 2.30 kJ/m2), and Shore D hardness measurements (83.0 ± 0.47) underlined the mechanical stability of the material, supported by digital microscopy, which revealed a glass-like structure. Differential scanning calorimetry with thermogravimetry analysis and thermal conductivity measurements showed heat stability until ~250 °C and effective heat insulation. The 3D-printed heat exchanger was tested in thermophysiology experiments measuring the vasomotor responses of rat tail arteries at different temperatures (13, 16, and 36 °C). The heat-exchange device was successfully used as an accessory of the wire myograph system to cool down the experimental chambers and steadily maintain the targeted temperatures. We observed temperature-dependent differences in the vasoconstriction induced by phenylephrine and KCl. In conclusion, the transparent resin material can be used in additive manufacturing of heat-exchange devices for biomedical research, such as wire myography. Our animal experiments underline the importance of temperature-dependent physiological mechanisms, which should be further studied to understand the background of the thermal changes and their consequences