Hyaluronan (HA) is a naturally occurring linear, negatively charged polysaccharide that plays a vital role in the organization and function of pericellular coats and extracellular matrices in vertebrates, and that is becoming increasingly popular in biomedical applications. To gain insight into the physical phenomena that govern the mechanical behavior of HA assemblies, we have studied the response of films of end-grafted HA to compression over a large range of ionic strength. Compression forces were measured as a function of the absolute distance between a colloidal probe and the planar surface on which the HA film was constructed, using a combined atomic force microscopy and reflection interference contrast microscopy setup. The HA films were well-defined in the sense that they are made of chains with a narrow size distribution that are grafted at controlled density to a solid support. Detailed comparison of the experimental data with analytical expressions derived from polymer and polyelectrolyte brush theory reveals that films of end-grafted HA behave as strongly charged polyelectrolyte brushes. To quantitatively reproduce the experimental data, intrinsic excluded volume interactions and chain stiffness of the polymer backbone must be taken into account. At low ionic strength, chains become almost fully stretched. In our experimental system, several micrometer thick films are formed that reach a hydration of up to 99.98%, and the brush thickness decreases by more than 5-fold with increasing ionic strength. More generally, the study provides quantitative theoretical predictions for the film thickness and compressive response as a function of HA length, grafting density and ionic strength. ■ INTRODUCTIONThe polysaccharide hyaluronan (HA) is ubiquitous in the pericellular and extracellular space of vertebrates. It is also becoming increasingly popular in biomedical applications as surface coating 1 and in tissue engineering. 2,3 HA is a linear polymer of disaccharides, made of glucuronic acid and Nacetylglucosamine, that are linked via alternating β-1,4 and β-1,3 glycosidic bonds. Each disaccharide has a length of 1 nm 4 and carries one chargeable group in the form of a carboxylic acid. For polymeric HA, the pK a is approximately 3, 5 and HA is, hence, negatively charged above pH ≈ 3. In vivo, HA is expressed by HA synthases at the cell membrane and extruded into the extracellular space. These HA molecules have a molecular mass of typically a few million Daltons 6 or a contour length of several micrometers. Once produced, they can stay grafted to the synthases and/or bind to other HA receptors at the cell surface, such as CD44 7 and form so-called pericellular coats (PCCs), or they can be released into solution to serve other functions in the extracellular space. Numerous biological functions of PCCs 8−11 and extracellular assemblies of HA in general (e.g., in cartilage) have been related to their mechanical properties. To better understand how the mechanical properties of PCCs are connected to their ...

We describe a method that combines colloidal probe atomic force microscopy (AFM) and reflection interference contrast microscopy (RICM) to characterize the mechanical properties of thin and solvated polymer films. When analyzing polymer films, a fundamental problem in colloidal probe AFM experiments is to determine the distance at closest approach between the probe and the substrate on which the film is deposited. By combining AFM and RICM in situ, forces and absolute distances can be measured simultaneously. Using the combined setup, we quantify the compressive mechanics of films of the polysaccharide hyaluronan that is end-grafted to a supported lipid bilayer. The experimental data, and comparison with polymer theory, show that hyaluronan films are well-described as elastic, very soft and highly solvated polymer brushes. The data on these well-defined films should be a useful reference for the investigation of the more complex hyaluronan-rich coats that surround many living cells.

Movements in animals arise through concerted action of neurons and skeletal muscle. General anaesthetics prevent movement and cause loss of consciousness by blocking neural function. Anaesthetics of the amino amide-class are thought to act by blockade of voltage-gated sodium channels. In fish, the commonly used anaesthetic tricaine methanesulphonate, also known as 3-aminobenzoic acid ethyl ester, metacaine or MS-222, causes loss of consciousness. However, its role in blocking action potentials in distinct excitable cells is unclear, raising the possibility that tricaine could act as a neuromuscular blocking agent directly causing paralysis. Here we use evoked electrical stimulation to show that tricaine efficiently blocks neural action potentials, but does not prevent directly evoked muscle contraction. Nifedipine-sensitive L-type Cav channels affecting movement are also primarily neural, suggesting that muscle Nav channels are relatively insensitive to tricaine. These findings show that tricaine used at standard concentrations in zebrafish larvae does not paralyse muscle, thereby diminishing concern that a direct action on muscle could mask a lack of general anaesthesia.

We assemble aggrecan-containing hyaluronan brushes to study how the supramolecular structure and dynamics relate to material properties in hyaluronan-rich pericellular matrices.

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The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function.

In healthy hearts, myofilaments become more sensitive to Ca2+ as the myocardium is stretched. This effect is known as length-dependent activation and is an important cellular-level component of the Frank–Starling mechanism. Few studies have measured length-dependent activation in the myocardium from failing human hearts. We investigated whether ischemic and non-ischemic heart failure results in different length-dependent activation responses at physiological temperature (37°C). Myocardial strips from the left ventricular free wall were chemically permeabilized and Ca2+-activated at sarcomere lengths (SLs) of 1.9 and 2.3 µm. Data were acquired from 12 hearts that were explanted from patients receiving cardiac transplants; 6 had ischemic heart failure and 6 had non-ischemic heart failure. Another 6 hearts were obtained from organ donors. Maximal Ca2+-activated force increased at longer SL for all groups. Ca2+ sensitivity increased with SL in samples from donors (P < 0.001) and patients with ischemic heart failure (P = 0.003) but did not change with SL in samples from patients with non-ischemic heart failure. Compared with donors, troponin I phosphorylation decreased in ischemic samples and even more so in non-ischemic samples; cardiac myosin binding protein-C (cMyBP-C) phosphorylation also decreased with heart failure. These findings support the idea that troponin I and cMyBP-C phosphorylation promote length-dependent activation and show that length-dependent activation of contraction is blunted, yet extant, in the myocardium from patients with ischemic heart failure and further reduced in the myocardium from patients with non-ischemic heart failure. Patients who have a non-ischemic disease may exhibit a diminished contractile response to increased ventricular filling.

in physiological and pathological processes and thus central for the function of biological systems. We describe a fast and reliable ratiometric Fluorescence Lifetime Imaging Microscopy (rmFLIM) approach to analyze the distribution of protein-ligand complexes in the cellular context. 1 Binding of the fluorescently labeled antagonist naloxone to the G-protein coupled m-opioid receptor is used as an example. To show the broad applicability of the rmFLIM method we extended this approach to investigate the distribution of polymer-based nanocarriers in histological liver sections. References (1) Boreham et al. (2011)