Scientists visualise the paths controlling heart performance

The heart works using tiny muscle filaments, and two main steps control how they interact: first calcium signals cause changes in the thin filament (actin), allowing it to connect with myosin motors. Then myosin motors on the thick filament switch from "off" (inactive) to "on" (active), so they can pull on actin and create force and shortening. The thick filament can sense how much force is needed and adjust how many motors to turn on, but scientists are still figuring out exactly how this works.

Cardiomyopathies take place when these mechanisms regulating the contraction-relaxation cycle of the heart malfunction. This is due to mutations in the contractile proteins (myosin and actin) but also in accessory proteins (Myosin-binding protein-C and titin). Broadly, MyBP-C and titin ensure together that the heart contracts effectively while maintaining responsiveness to changes in demand.

Theyencheri Narayanan, scientist in charge of ID02, on the beamline. Credits: S. Candé.

A team from the University of Firenze (Italy) used small-angle X-ray diffraction at the ESRF’s ID02 beamline back in 2017 and showed that, during muscle contraction, myosin motors move outward from the thick filament, enabling interaction with actin based on the load, which in the cardiac ventricle is the arterial pressure against which blood must be pumped (Reconditi et al. PNAS 2017). Evidence from other laboratories also suggested that thick filament activation is modulated by regulatory domains in the myosin motor itself and by accessory proteins like MyBP-C and titin. However, their specific function in cardiac performance was not known in detail.

Now the same team has found the role that MyBP-C and titin play in the functioning of the heart, at beamline ID02. “The beamline ID02 is ideal thanks to its camera length, so we can have the detector closer or further to the samples in a range from 0.5 to 31 m. This lets us explore the contractile proteins as well as the sarcomere, the unit cell of muscle, which is crucial for our research”, says Massimo Reconditi, scientist at the University of Firenze and corresponding author of the paper. “With EBS, thanks to its low divergence, we can observe on the same diffraction pattern a high number of reflections together with their fine structure”, he adds. 

Understanding mutations in cardiomyopathy

They found that, as suggested by recent X-ray diffraction experiments on skeletal muscle fibres done at ID02 (Squarci et al.  PNAS 2023), calcium has an effect on titin, which becomes the first activator: it becomes stiffer and may give the first stroke to both motors and MyBP-C. MyBP-C communicates between actin and myosin and, in this way, it starts the process of myosin motor attachment.

The classical mechanosensing hypothesis implies that the motor starts attachment from the periphery of the thick filament. “Now we have evidence that motor attachment happens first in the region where the MyBP-C lays and then it spreads through the thick filaments toward the edges as a function of the load”, explains Reconditi.

From the medical point of view, the finding could help in the treatment of heart disease. “Most of cardiomyopathies are associated with mutations in MyBP-C, so  knowing the role of this protein is the prerequisite for its targeting in the development of therapeutic interventions”, says Reconditi.

The next step for the team is to characterise the development of the complex interactions between the contractile and accessory proteins while the sarcomeres shorten as heart pumps blood against different arterial pressures.

References:
Moroti, I., et al, PNAS, December 4, 2024. 121 (50) e2410893121  https://doi.org/10.1073/pnas.2410893121

Reconditi et al., PNAS, 114, 3240–3245 (2017). https://doi.org/10.1073/pnas.1619484114

Squarci et al.,  PNAS, 120, e2219346120 (2023). https://doi.org/10.1073/pnas.2219346120

Text by Montserrat Capellas Espuny

Top image: Enlarged scale of overlap region showing: i) on the thin filament, the double-stranded helix formed by actin monomers, the regulatory proteins tropomyosin (red) and the troponin complex (TnC, brown, TnT, gray, and TnI, dark gray) and ii) on the thick filament, the two motor domains (S1 fragments, orange) of each myosin molecule lying tilted back on their tail (S2 fragment, blue) in three-stranded helical tracks with 43 nm periodicity. Titin (magenta) and MyBP-C (green, with M domain identified by light green) are also shown. Left panel all motors in the OFF state; Right panel one dimer is switched ON. Credits: ​​​​​​​Moroti, I., et al, PNAS, December 4, 2024. 121 (50) e2410893121  https://doi.org/10.1073/pnas.2410893121