Periodic Reporting for period 2 - Heart Fi-Re (HEART FIne REgulation through mechanosensing in myosin filaments: merging theory and experiments into a multi-scale heart simulator)
Berichtszeitraum: 2023-07-01 bis 2024-06-30
Muscle force is directly proportional to the number of myosin motors going through the so-called cross-bridge cycle, the ATP-driven interaction between myosin proteins, protruding from the thick filament, and actin proteins, forming the thin filament. Classically, the calcium concentrations ([Ca2+]) in the myofibrillar space is known to modulate the activation of the thin filament and, through it, the number of detached but active (ON, pointing toward the thin filament) myosin motors, which generate the cross-bridges.
However, in 2010, two detached states have been identified, biochemically defined on the basis of their rate of ATP consumption: a classical disordered relaxed state (DRX) and an unexpected stable state with an ATPase rate of one order of magnitude lower, the super-relaxed state (SRX). This SRX state has also been proposed to be the biochemical counterpart of the structurally defined detached state where motors lie on the thick filament and are unable to interact with activated actin (OFF state).
Importantly, ground-breaking data in 2015 have proved the existence of an internal mechano-sensing (MS) mechanism that relates the ratio of ON-to-OFF motors to the tension sustained by the thick myosin filament (Figure 1).
Yet the molecular bases of the MS mechanism remain mostly unknown, and this limits the strategies to address cardiac pathologies related to its dysfunction.
The MS mechanism creates a critical cellular feedback mechanism, in which malfunction can be at play in hypertrophic cardiomyopathies (HCM). Accordingly, the pharmacological “stabilization” of the OFF state has been shown to prevent or reduce HCM consequences, a therapeutic option that already reached the clinical stage. Then, the MS mechanism play a fundamental role in our basic understanding of the physiological aspects of the skeletal and cardiac muscle contraction, but also it has implications in the treatment of hypertrophic and dilated cardiomyopathies, allowing the developed of a drug for the treatment of HCM that, in essence, is a stabilizer of the OFF state.
On these grounds, the project wants to shape the theoretical description of this mechanism and usher it into a multiscale model - from the molecule to the organ. Doing so will enable to create a benchmark to drive pharmacological applications, aiming at reducing the failure rate in this drug discovery pipeline.
At its conclusion, the project made a substantial contribution to highlight the differences between the biochemically defined SRX state and structurally defined Off state, and the interconnected mechanism between the thin filament activation and the thick filament activation through a cross-talk mechanism. Moreover, the project shown the need to characterize the diffusion of calcium ions inside the muscle cell, to properly understand the mechanical activation of the thick filament.
As a long-term goal reached by the project, a cluster of researchers with complementary expertise has been created at the University of Padova and integrated, as a crucial node, in an international network of collaborations, both within Europe and outside it. The collaboration will foster new results in the aim of personalized medicine.
However, in 2010, two detached states have been identified, biochemically defined on the basis of their rate of ATP consumption: a classical disordered relaxed state (DRX) and an unexpected stable state with an ATPase rate of one order of magnitude lower, the super-relaxed state (SRX). This SRX state has also been proposed to be the biochemical counterpart of the structurally defined detached state where motors lie on the thick filament and are unable to interact with activated actin (OFF state).
Importantly, ground-breaking data in 2015 have proved the existence of an internal mechano-sensing (MS) mechanism that relates the ratio of ON-to-OFF motors to the tension sustained by the thick myosin filament (Figure 1).
Yet the molecular bases of the MS mechanism remain mostly unknown, and this limits the strategies to address cardiac pathologies related to its dysfunction.
The MS mechanism creates a critical cellular feedback mechanism, in which malfunction can be at play in hypertrophic cardiomyopathies (HCM). Accordingly, the pharmacological “stabilization” of the OFF state has been shown to prevent or reduce HCM consequences, a therapeutic option that already reached the clinical stage. Then, the MS mechanism play a fundamental role in our basic understanding of the physiological aspects of the skeletal and cardiac muscle contraction, but also it has implications in the treatment of hypertrophic and dilated cardiomyopathies, allowing the developed of a drug for the treatment of HCM that, in essence, is a stabilizer of the OFF state.
On these grounds, the project wants to shape the theoretical description of this mechanism and usher it into a multiscale model - from the molecule to the organ. Doing so will enable to create a benchmark to drive pharmacological applications, aiming at reducing the failure rate in this drug discovery pipeline.
At its conclusion, the project made a substantial contribution to highlight the differences between the biochemically defined SRX state and structurally defined Off state, and the interconnected mechanism between the thin filament activation and the thick filament activation through a cross-talk mechanism. Moreover, the project shown the need to characterize the diffusion of calcium ions inside the muscle cell, to properly understand the mechanical activation of the thick filament.
As a long-term goal reached by the project, a cluster of researchers with complementary expertise has been created at the University of Padova and integrated, as a crucial node, in an international network of collaborations, both within Europe and outside it. The collaboration will foster new results in the aim of personalized medicine.
I have reviewed the most recent discoveries about the biochemically defined super-relaxed state (SRX) and its structural counterpart, the interacting heads motif (IHM), organizing, in a structural way from the mechanical point of view, more than a hundred papers published in the last years. The review highlighted the emerging view, not foreseen at the time of the proposal submission, that there is a clear distinction between the SRX and the IHM, which only partially can be overlapped (Figure 2).
Then, I have developed a theoretical quantitative assessment of thick filament activation in physiological situations, through labeling with the fluorescent probes the myosin protein in the skinned single fiber of psoas muscle, a unique technique developed at the institution selected for the secondment. The analysis of the emission of the probe at different [Ca2+], offered a unique possibility to estimate the population of myosin in the ON and OFF states in the muscle fiber through the so-called P2 parameter. Our joint work, as a main scientific achievement and contribution to the state of the art, showed the existence of two positive feedback loops controlling the activation of thin (myosin-sensing) and thick (MS) filaments triggered by calcium (Figure 3).
The existence of these two positive feedback loops controlling muscle activation prompted the project to better explore the effect of the Ca2+ diffusion, also based on the evidence of a sequential activation of the thick filament on cardiac muscle, obtained by the group of the secondment institution. Then I have developed a Ca2+ diffusion model and a technique of Ca2+ signaling analysis, to explore the effect of Ca2+ both in resting state (figure 4) and during activation (Figure 5). Current work aims at exploring the limits of the commonly used technique to characterize the biochemical state of SRX, to complete the comparison between this state and its structural counterpart.
Then, I have developed a theoretical quantitative assessment of thick filament activation in physiological situations, through labeling with the fluorescent probes the myosin protein in the skinned single fiber of psoas muscle, a unique technique developed at the institution selected for the secondment. The analysis of the emission of the probe at different [Ca2+], offered a unique possibility to estimate the population of myosin in the ON and OFF states in the muscle fiber through the so-called P2 parameter. Our joint work, as a main scientific achievement and contribution to the state of the art, showed the existence of two positive feedback loops controlling the activation of thin (myosin-sensing) and thick (MS) filaments triggered by calcium (Figure 3).
The existence of these two positive feedback loops controlling muscle activation prompted the project to better explore the effect of the Ca2+ diffusion, also based on the evidence of a sequential activation of the thick filament on cardiac muscle, obtained by the group of the secondment institution. Then I have developed a Ca2+ diffusion model and a technique of Ca2+ signaling analysis, to explore the effect of Ca2+ both in resting state (figure 4) and during activation (Figure 5). Current work aims at exploring the limits of the commonly used technique to characterize the biochemical state of SRX, to complete the comparison between this state and its structural counterpart.
The results reached so far in the project have pushed the state of the art in understanding the regulation of muscle force generation.
The characterization of the two positive feedback loops controlling muscle activation prompted the project to better explore the effect of the Ca2+ diffusion, with the definition of a new mathematical model of calcium diffusion into a muscle sarcomere. Its application to the experimental data obtained in the Italian hosting institution contributed to the quantitative understanding of how local gradients are generated, not only during contraction but also at rest, and how relevant they are to mitochondrial Ca2+ regulation.
These results supported the holistic view of dual filament regulation where the positive feedback loop drives both the thin and the thick filament activations. All these aspects can be clarified, from the single molecule to the whole muscle, only through a multi-scale and multi-disciplinary approach: in vitro, in situ, in vivo, and in silico, an approach that will be developed in the next collaborations.
The validation of the multi-scale simulator for clinical application will be proposed to drive clinical aspects, such as the dose estimation for each patient, with a wide impact on society.
The characterization of the two positive feedback loops controlling muscle activation prompted the project to better explore the effect of the Ca2+ diffusion, with the definition of a new mathematical model of calcium diffusion into a muscle sarcomere. Its application to the experimental data obtained in the Italian hosting institution contributed to the quantitative understanding of how local gradients are generated, not only during contraction but also at rest, and how relevant they are to mitochondrial Ca2+ regulation.
These results supported the holistic view of dual filament regulation where the positive feedback loop drives both the thin and the thick filament activations. All these aspects can be clarified, from the single molecule to the whole muscle, only through a multi-scale and multi-disciplinary approach: in vitro, in situ, in vivo, and in silico, an approach that will be developed in the next collaborations.
The validation of the multi-scale simulator for clinical application will be proposed to drive clinical aspects, such as the dose estimation for each patient, with a wide impact on society.