#weekendusers The challenges of studying the heartbeat

18-11-2016

​A team from the University of Florence (Italy) and VU university Medical School of Amsterdam (the Netherlands) is working this weekend on beamline ID02 doing a very complex experiment to learn how the heart contraction is regulated with the ultimate aim to better tackle cardiac diseases.

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Diseases affecting the heart muscle affect 8 million people worldwide. These diseases, also known as cardiomyopathies, alter the mechanism of the heart, which pumps blood through the vessels keeping the two circulatory systems, pulmonary and systemic, in balance. This is achieved thanks to a regulatory mechanism intrinsic to the heart, that allows to adapt the force developed in the systole to the filling  of the ventricles at the end of the diastole. ​A team from the University of Florence (Italy) and VU university Medical School of Amsterdam (the Netherlands) is working this weekend on beamline ID02 doing a very complex experiment to learn how the heart contraction is regulated with the ultimate aim to better tackle cardiac diseases.

The sample is a multicellular preparation from the heart ventricle of the rat, and it needs to be continuously perfused with an oxygenated physiological solution containing glucose to keep it alive for the time of the experiment. It is a tough experiment and time is precious. “It is so hectic over here that we don’t have much time for an interview”, says Vincenzo Lombardi, leader of the team. Passionate about his work, and long-term user at the ESRF, his research has advanced by leaps and bounds in recent years studying how skeletal muscle contracts.

Now his team is facing a new challenge: they have swapped skeletal muscle for heart muscle. “It is more complicated to study, because skeletal muscle has straight long gigantic cells, the muscle fibres, that can be isolated and attached via their tendinuous ends to a motor and a force transducer for fine mechanical control, whereas the heart is made by a circular meshwork of very small cells, the myocites. The same mechanical methods can be applied to small pieces of the heart tissue, the trabeculae, elongated multicellular bundles (about 2 mm long and 0.2 mm wide) that connect the tip of the valve to the internal cardiac wall.   

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Part of the team in front of the experimental hutch. From left to right: Gabriella Piazzesi, Marco Camerani, Vincenzo Lombardi and Ger Stiene.

In this way it is possible to mechanically control  the structural unit of the muscle cell, the 2 µm long sarcomere where the molecular motors, made by the protein myosin, generate force and shortening by pulling on the  actin filament. The sarcomeres increase their contractility with the increase of the sarcomere’s length in a still unexplained process, called length-dependent activation (LDA), which is the cellular basis of the mechanism by which the heart is able to adapt the systolic force to the ventricular filling.  The goal is to find, at molecular level, how the LDA works and fails, which is what causes heart failure.

It all takes place thanks to the integration of the function of the various kinds of proteins constituting the sarcomere (contractile, regulatory and cytoskeletal),and therefore must be investigated at the nanometre level, which makes it a challenge for researchers. How can scientists study  the heart mechanism at a nanometre scale on a X-ray Small Angle Scattering beamline? At ID02, the team isolates the trabecula from the ventricle of the rat heart and mounts it between a force transducer and a motor for fast mechanical control. Cycles of contraction-relaxation are elicited by  electrical stimulation.  At ID02 the camera length can be rapidly changed during the same experiment, so that  the nanometre-scale signals from the contractile proteins along the myosin and actin filament can be recorded,  together with the micrometer-scale changes in sarcomere length. According to Lombardi, “ID02 is the best beamline worldwide for these experiments, as it offers an unparalleled combination of high monochromatic flux in a small beam and a camera length adjustable in the range 0.5-30 m”.

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Marco Camerani and the set-up.

With such a complicated experiment on a live preparation, five days of beamtime can go very quickly so organisation is crucial. The team consists of eight people, and they take shifts to do the experiment. A lot of effort is spent in the preparation of the trabeculae. On top of that, their survival to radiation is quite short: a few hundred of milliseconds, with the flux of X-ray necessary to collect good signals from such a small and poorly-diffracting mass as a trabecula. This time must be efficiently distributed in several snapshots of a few tens of milliseconds selected on the basis of the mechanical protocol. “We do a first analysis of the data in real time”, explains Gabriella Piazzesi, scientist in the team, “so we know roughly whether we are doing things the right way and take advantage of the response of the ongoing experiment to optimise the protocol for the next exeriment, she adds. So far, the data looks promising.

This is the first study in which sarcomere mechanics are combined with time-resolved X-ray interference to record the dynamics of the sarcomeres during the contraction process. “Ultimately, we want to understand how the heart works so that we can, in the future, cure heart diseases”, explains Lombardi. 

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The set-up.

Text by Montserrat Capellas Espuny