Periodic Reporting for period 1 - Heart Fi-Re (HEART FIne REgulation through mechanosensing in myosin filaments: merging theory and experiments into a multi-scale heart simulator)
Période du rapport: 2021-07-01 au 2023-06-30
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, recently, a detached state where motors lie on the thick filament and are unable to interact with activated actin has been identified: the super-relaxed (SRX or OFF) state .
Importantly, ground-breaking data have recently 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. Briefly, at higher active or passive tensions sustained by the thick filament, the MS mechanism generates a higher probability that OFF myosin motors, unable to enter the conventional cycle, are recruited to the ON state (Figure 1, adapted from Marcucci, et al., PLoS Comp Bio, 2016).
Yet the molecular bases of the MS mechanism remain mostly unknown at the molecular level, and this limits the strategies to address cardiac pathologies related to its dysfunction.
The MS mechanism creates a critical cellular feedback mechanism, likely associated with the Frank-Starling law, 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. Since its discovery, the MS mechanism has attracted more and more interest in the scientific community. This is not only because it may play a fundamental role in the physiology of basic understanding of the physiological aspects of the skeletal and cardiac muscle contraction, but also for its implications in the treatment of hypertrophic and dilated cardiomyopathies, as done by a US-based company which developed a drug for the treatment of HCM that, in essence, is a stabilizer of the OFF state. Phase III clinical trials testing the effects of this compound are already made in the EU and USA and represent a unique opportunity for validating the pragmatic implications of the findings emanating from this project.
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.
Finally, the long-term purpose of the present project is to create a cluster of researchers with complementary expertise at the University of Padova and integrate it as a crucial node in an international network of collaborations
However, recently, a detached state where motors lie on the thick filament and are unable to interact with activated actin has been identified: the super-relaxed (SRX or OFF) state .
Importantly, ground-breaking data have recently 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. Briefly, at higher active or passive tensions sustained by the thick filament, the MS mechanism generates a higher probability that OFF myosin motors, unable to enter the conventional cycle, are recruited to the ON state (Figure 1, adapted from Marcucci, et al., PLoS Comp Bio, 2016).
Yet the molecular bases of the MS mechanism remain mostly unknown at the molecular level, and this limits the strategies to address cardiac pathologies related to its dysfunction.
The MS mechanism creates a critical cellular feedback mechanism, likely associated with the Frank-Starling law, 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. Since its discovery, the MS mechanism has attracted more and more interest in the scientific community. This is not only because it may play a fundamental role in the physiology of basic understanding of the physiological aspects of the skeletal and cardiac muscle contraction, but also for its implications in the treatment of hypertrophic and dilated cardiomyopathies, as done by a US-based company which developed a drug for the treatment of HCM that, in essence, is a stabilizer of the OFF state. Phase III clinical trials testing the effects of this compound are already made in the EU and USA and represent a unique opportunity for validating the pragmatic implications of the findings emanating from this project.
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.
Finally, the long-term purpose of the present project is to create a cluster of researchers with complementary expertise at the University of Padova and integrate it as a crucial node in an international network of collaborations
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, from Marcucci, L., IJMS, 2023).
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 adapted from Brunello, E.,L. Marcucci, M. Irving and L. Fusi PNAS, 2023).
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. I have developed a Ca2+ diffusion model and a technique of Ca2+ signaling analysis, which led to an in-silico-based evidence of local Ca2+ microdomains in resting cells due to the leakage in the sarcoplasmic reticulum, and an extensive analysis of the Ca2+ diffusion in the presence and in the absence of the major Ca2+ buffer in the cytosol (figure 4, adapted from Marcucci, L. et al. Biophysical Reports 2023).
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 adapted from Brunello, E.,L. Marcucci, M. Irving and L. Fusi PNAS, 2023).
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. I have developed a Ca2+ diffusion model and a technique of Ca2+ signaling analysis, which led to an in-silico-based evidence of local Ca2+ microdomains in resting cells due to the leakage in the sarcoplasmic reticulum, and an extensive analysis of the Ca2+ diffusion in the presence and in the absence of the major Ca2+ buffer in the cytosol (figure 4, adapted from Marcucci, L. et al. Biophysical Reports 2023).
The results reached so far in the project have pushed the state of the art in understanding the regulation of muscle force generation. I have contributed to showing that the MS in the thick filament and myosin-sensing in the thin filament constitutes two positive feedback loops in the muscle activation, where both the steady-state and dynamic mechanisms of force generation depend on a dual-filament paradigm of muscle regulation.
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.
In particular, in the absence of the major Ca2+ buffer in skeletal muscle, the experimental data and the model results confirmed the hypothesis that mitochondria contribute to the removal of cytosolic Ca2+, likely accompanied by increased Ca2+ exchange with the extracellular space. This conclusion is important within a wider debate on the modulation of cytosolic Ca2+ transients by mitochondria and highlights the importance of including the mitochondrial compartment in calcium diffusion mathematical models.
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 last year of the project.
The validation of the multi-scale simulator for clinical application in a patient-specific way by comparing healthy data with pre-and post-treatment pathological data derived by the pharmacological compound currently in Phase III clinical trials, in a fixed, patient-specific, heart geometry. If the validation process is successful, the model 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.
In particular, in the absence of the major Ca2+ buffer in skeletal muscle, the experimental data and the model results confirmed the hypothesis that mitochondria contribute to the removal of cytosolic Ca2+, likely accompanied by increased Ca2+ exchange with the extracellular space. This conclusion is important within a wider debate on the modulation of cytosolic Ca2+ transients by mitochondria and highlights the importance of including the mitochondrial compartment in calcium diffusion mathematical models.
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 last year of the project.
The validation of the multi-scale simulator for clinical application in a patient-specific way by comparing healthy data with pre-and post-treatment pathological data derived by the pharmacological compound currently in Phase III clinical trials, in a fixed, patient-specific, heart geometry. If the validation process is successful, the model will be proposed to drive clinical aspects, such as the dose estimation for each patient, with a wide impact on society.