Sarcomere, troponin, and myosin X-ray diffraction signals can be resolved in single cardiomyocytes.

Cardiac function relies on the autonomous molecular contraction mechanisms in the ventricular wall. Contraction is driven by ordered motor proteins acting in parallel to generate a macroscopic force. The averaged structure can be investigated by diffraction from model tissues such as trabecular and papillary cardiac muscle using collimated synchrotron beams, offering high resolution in reciprocal space. In the ventricular wall, however, the muscle tissue is compartmentalized into smaller branched cardiomyocytes, with a higher degree of disorder. We show that X-ray diffraction is now also capable of resolving the structural organization of actomyosin in single isolated cardiomyocytes of the ventricular wall. In addition to the hexagonal arrangement of thick and thin filaments, the diffraction signal of the hydrated and fixated cardiomyocytes was sufficient to reveal the myosin motor repeat (M3), the troponin complex repeat (Tn), and the sarcomere length. The sarcomere length signal comprised up to 13 diffraction orders, which were used to compute the sarcomere density profile based on Fourier synthesis. The Tn and M3 spacings were found in the same range as previously reported for other muscle types. The approach opens up a pathway to record the structural dynamics of living cells during the contraction cycle, toward a more complete understanding of cardiac muscle function.

Lipid vesicle pools studied by passive X-ray microrheology.

Vesicle pools can form by attractive interaction in a solution, mediated by proteins or divalent ions such as calcium. The pools, which are alternatively also denoted as vesicle clusters, form by liquid-liquid phase separation (LLPS) from an initially homogeneous solution. Due to the short range liquid-like order of vesicles in the pool or cluster, the vesicle-rich phase can also be regarded as a condensate, and one would like to better understand not only the structure of these systems, but also their dynamics. The diffusion of vesicles, in particular, is expected to change when vesicles are arrested in a pool. Here we investigate whether passive microrheology based on X-ray photon correlation spectroscopy (XPCS) is a suitable tool to study model systems of artificial lipid vesicles exhibiting LLPS, and more generally also other heterogeneous biomolecular fluids. We show that by adding highly scattering tracer particles to the solution, valuable information on the single vesicle as well as collective dynamics can be inferred. While the correlation functions reveal freely diffusing tracer particles in solutions at low CaCl[Formula: see text] concentrations, the relaxation rate [Formula: see text] shows a nonlinear dependence on [Formula: see text] at a higher concentration of around 8 mM CaCl[Formula: see text], characterised by two linear regimes with a broad cross-over. We explain this finding based on arrested diffusion in percolating vesicle clusters.

Contractility measurements for cardiotoxicity screening with ventricular myocardial slices of pigs.

AIMS: Cardiotoxicity is one major reason why drugs do not enter or are withdrawn from the market. Thus, approaches are required to predict cardiotoxicity with high specificity and sensitivity. Ideally, such methods should be performed within intact cardiac tissue with high relevance for humans and detect acute and chronic side effects on electrophysiological behaviour, contractility, and tissue structure in an unbiased manner. Herein, we evaluate healthy pig myocardial slices and biomimetic cultivation setups (BMCS) as a new cardiotoxicity screening approach. METHODS AND RESULTS: Pig left ventricular samples were cut into slices and spanned into BMCS with continuous electrical pacing and online force recording. Automated stimulation protocols were established to determine the force-frequency relationship (FFR), frequency dependence of contraction duration, effective refractory period (ERP), and pacing threshold. Slices generated 1.3 ± 0.14 mN/mm2 force at 0.5 Hz electrical pacing and showed a positive FFR and a shortening of contraction duration with increasing pacing rates. Approximately 62% of slices were able to contract for at least 6 days while showing stable ERP, contraction duration-frequency relationship, and preserved cardiac structure confirmed by confocal imaging and X-ray diffraction analysis. We used specific blockers of the most important cardiac ion channels to determine which analysis parameters are influenced. To validate our approach, we tested five drug candidates selected from the Comprehensive in vitro Proarrhythmia Assay list as well as acetylsalicylic acid and DMSO as controls in a blinded manner in three independent laboratories. We were able to detect all arrhythmic drugs and their respective mode of action on cardiac tissue including inhibition of Na+, Ca2+, and hERG channels as well as Na+/Ca2+ exchanger. CONCLUSION: We systematically evaluate this approach for cardiotoxicity screening, which is of high relevance for humans and can be upscaled to medium-throughput screening. Thus, our approach will improve the predictive value and efficiency of preclinical cardiotoxicity screening.