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Malcolm Irving - One of the best experts on this subject based on the ideXlab platform.
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Myosin Filament based regulation of the dynamics of contraction in heart muscle
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Elisabetta Brunello, Luca Fusi, Theyencheri Narayanan, Andrea Ghisleni, Sojin Parkholohan, Jesus G Ovejero, Malcolm IrvingAbstract:Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the Myosin Filament during contraction of heart muscle. We show that, although Myosin motors throughout the Filament contribute to force development, only about 10% of the motors in each Filament bear the peak force, and these are confined to the Filament domain containing Myosin binding protein-C, the "C-zone." Myosin motors in domains further from the Filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated Myosin motors are folded against the Filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin Filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of Myosin Filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.
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omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the Myosin Filament
The Journal of Physiology, 2018Co-Authors: Thomas Kampourakis, Xuemeng Zhang, Yinbiao Sun, Malcolm IrvingAbstract:Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick Filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick Filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-Myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the thick Filament OFF state and inhibiting acto-Myosin ATPase. Thick Filament regulation is a promising target for novel therapeutics in heart disease. Abstract Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin Filaments that allows the head or motor domains of Myosin from the thick Filaments to bind to them and induce Filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick Filaments, although the molecular mechanisms underlying thick Filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to Myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the Myosin regulatory light chain in the thick Filaments and on troponin C in the thin Filaments. The results show that BS inhibits contractility and actin-Myosin ATPase by stabilizing the OFF state of the thick Filament in which Myosin head domains are more parallel to the Filament axis. In contrast, OM stabilizes the ON state of the thick Filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-Myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and Filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick Filaments.
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sarcomere length dependence of Myosin Filament structure in skeletal muscle fibres of the frog
The Journal of Physiology, 2014Co-Authors: Massimo Reconditi, Malcolm Irving, Vincenzo Lombardi, Luca Fusi, Elisabetta Brunello, Marco Linari, Manuel Fernandez Martinez, Gabriella PiazzesiAbstract:X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the Myosin heads and Myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick Filament is maintained by an interaction between MyBP-C and the thin Filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the Myosin heads along the Filaments decreased in proportion to the overlap between thick and thin Filaments, with no change in its interference fine structure. Thus, Myosin heads in the regions of the thick Filaments that do not overlap with thin Filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed Myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick Filament involving changes in both the organisation of the Myosin heads on its surface and the axial periodicity of the Myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin Filaments.
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interference fine structure and sarcomere length dependence of the axial x ray pattern from active single muscle fibers
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Theyencheri Narayanan, Natalia A Koubassova, Ian M Dobbie, Olivier Diat, Vincenzo LombardiAbstract:Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2–3.2 μm, the M3 x-ray reflection, associated with the repeat of Myosin heads along the Filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between Myosin and actin Filaments, showing that it comes from Myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere length and was quantitatively explained by x-ray interference between Myosin heads in the two overlap regions of each sarcomere. The relative intensity of the M3 peaks was independent of sarcomere length, showing that the axial periodicities of the nonoverlap and overlap regions of the Myosin Filament have the same value, 14.57 nm, during active contraction. In resting fibers the periodicity is 14.34 nm, so muscle activation produces a change in Myosin Filament structure in the nonoverlap as well as the overlap part of the Filament. The results establish x-ray interferometry as a new tool for studying the motions of Myosin heads during muscle contraction with unprecedented spatial resolution.
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the stiffness of skeletal muscle in isometric contraction and rigor the fraction of Myosin heads bound to actin
Biophysical Journal, 1998Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Natalia A Koubassova, Ian M Dobbie, Vincenzo LombardiAbstract:Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin Filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of Myosin cross-bridges in the region of overlap between the Myosin Filament and the actin Filament remains constant, and only the length of the nonoverlapped region of the actin Filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin Filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the Myosin Filament compliance is 1.3 nm microm(-1) T0(-1). With these values for Filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of Myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.
Gabriella Piazzesi - One of the best experts on this subject based on the ideXlab platform.
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Myosin Filament activation in the heart is tuned to the mechanical task
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Massimo Reconditi, Vincenzo Lombardi, Theyencheri Narayanan, Marco Linari, Marco Caremani, Francesca Pinzauti, Joseph D Powers, Ger J M Stienen, Gabriella PiazzesiAbstract:The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the Myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of Myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick Filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.
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sarcomere length dependence of Myosin Filament structure in skeletal muscle fibres of the frog
The Journal of Physiology, 2014Co-Authors: Massimo Reconditi, Malcolm Irving, Vincenzo Lombardi, Luca Fusi, Elisabetta Brunello, Marco Linari, Manuel Fernandez Martinez, Gabriella PiazzesiAbstract:X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the Myosin heads and Myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick Filament is maintained by an interaction between MyBP-C and the thin Filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the Myosin heads along the Filaments decreased in proportion to the overlap between thick and thin Filaments, with no change in its interference fine structure. Thus, Myosin heads in the regions of the thick Filaments that do not overlap with thin Filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed Myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick Filament involving changes in both the organisation of the Myosin heads on its surface and the axial periodicity of the Myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin Filaments.
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structural changes in the Myosin Filament and cross bridges during active force development in single intact frog muscle fibres stiffness and x ray diffraction measurements
The Journal of Physiology, 2006Co-Authors: Massimo Reconditi, Gabriella Piazzesi, Elisabetta Brunello, Marco Linari, Pasquale Bianco, Pierre Panine, Theyencheri NarayananAbstract:Structural and mechanical changes occurring in the Myosin Filament and Myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4°C and 2.15 μm sarcomere length, using sarcomere level mechanics and X-ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 Myosin-based reflections were recorded with 5 ms frames using the gas-filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T0) with a half-time of 40 ms, and the spacings of the M3 and M6 reflections (SM3 and SM6) increase by 1.5% from their resting values, with time courses that lead that of force by ∼10 and ∼20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by ∼10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half-sarcomere)−1 shortening at the velocity (V0) that maintains zero force. Shortening at V0 transiently reduces SM3 following the latent period and delays the subsequent increase in SM3, but only delays the SM6 increase without a transient decrease. Shortening at V0 imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (IM3), whereas the intensity of the M6 reflection (IM6) is only slightly reduced. The changes in half-sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of Myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the Myosin heads attached to the actin Filament and the M6 reflection originates mainly from a fixed structure in the Myosin Filament signalling Myosin Filament length changes during the tetanus rise.
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interference fine structure and sarcomere length dependence of the axial x ray pattern from active single muscle fibers
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Theyencheri Narayanan, Natalia A Koubassova, Ian M Dobbie, Olivier Diat, Vincenzo LombardiAbstract:Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2–3.2 μm, the M3 x-ray reflection, associated with the repeat of Myosin heads along the Filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between Myosin and actin Filaments, showing that it comes from Myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere length and was quantitatively explained by x-ray interference between Myosin heads in the two overlap regions of each sarcomere. The relative intensity of the M3 peaks was independent of sarcomere length, showing that the axial periodicities of the nonoverlap and overlap regions of the Myosin Filament have the same value, 14.57 nm, during active contraction. In resting fibers the periodicity is 14.34 nm, so muscle activation produces a change in Myosin Filament structure in the nonoverlap as well as the overlap part of the Filament. The results establish x-ray interferometry as a new tool for studying the motions of Myosin heads during muscle contraction with unprecedented spatial resolution.
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the stiffness of skeletal muscle in isometric contraction and rigor the fraction of Myosin heads bound to actin
Biophysical Journal, 1998Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Natalia A Koubassova, Ian M Dobbie, Vincenzo LombardiAbstract:Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin Filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of Myosin cross-bridges in the region of overlap between the Myosin Filament and the actin Filament remains constant, and only the length of the nonoverlapped region of the actin Filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin Filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the Myosin Filament compliance is 1.3 nm microm(-1) T0(-1). With these values for Filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of Myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.
Vincenzo Lombardi - One of the best experts on this subject based on the ideXlab platform.
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Myosin Filament activation in the heart is tuned to the mechanical task
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Massimo Reconditi, Vincenzo Lombardi, Theyencheri Narayanan, Marco Linari, Marco Caremani, Francesca Pinzauti, Joseph D Powers, Ger J M Stienen, Gabriella PiazzesiAbstract:The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the Myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of Myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick Filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.
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sarcomere length dependence of Myosin Filament structure in skeletal muscle fibres of the frog
The Journal of Physiology, 2014Co-Authors: Massimo Reconditi, Malcolm Irving, Vincenzo Lombardi, Luca Fusi, Elisabetta Brunello, Marco Linari, Manuel Fernandez Martinez, Gabriella PiazzesiAbstract:X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the Myosin heads and Myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick Filament is maintained by an interaction between MyBP-C and the thin Filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the Myosin heads along the Filaments decreased in proportion to the overlap between thick and thin Filaments, with no change in its interference fine structure. Thus, Myosin heads in the regions of the thick Filaments that do not overlap with thin Filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed Myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick Filament involving changes in both the organisation of the Myosin heads on its surface and the axial periodicity of the Myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin Filaments.
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interference fine structure and sarcomere length dependence of the axial x ray pattern from active single muscle fibers
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Theyencheri Narayanan, Natalia A Koubassova, Ian M Dobbie, Olivier Diat, Vincenzo LombardiAbstract:Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2–3.2 μm, the M3 x-ray reflection, associated with the repeat of Myosin heads along the Filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between Myosin and actin Filaments, showing that it comes from Myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere length and was quantitatively explained by x-ray interference between Myosin heads in the two overlap regions of each sarcomere. The relative intensity of the M3 peaks was independent of sarcomere length, showing that the axial periodicities of the nonoverlap and overlap regions of the Myosin Filament have the same value, 14.57 nm, during active contraction. In resting fibers the periodicity is 14.34 nm, so muscle activation produces a change in Myosin Filament structure in the nonoverlap as well as the overlap part of the Filament. The results establish x-ray interferometry as a new tool for studying the motions of Myosin heads during muscle contraction with unprecedented spatial resolution.
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the stiffness of skeletal muscle in isometric contraction and rigor the fraction of Myosin heads bound to actin
Biophysical Journal, 1998Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Natalia A Koubassova, Ian M Dobbie, Vincenzo LombardiAbstract:Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin Filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of Myosin cross-bridges in the region of overlap between the Myosin Filament and the actin Filament remains constant, and only the length of the nonoverlapped region of the actin Filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin Filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the Myosin Filament compliance is 1.3 nm microm(-1) T0(-1). With these values for Filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of Myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.
Leanne M Redman - One of the best experts on this subject based on the ideXlab platform.
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maternal cold exposure induces distinct transcriptome changes in the placenta and fetal brown adipose tissue in mice
BMC Genomics, 2021Co-Authors: Sujoy Ghosh, Chul-hong Park, Jisu Lee, Nathan Lee, Rui Zhang, Clara Huesing, Dorien Reijnders, Jennifer Sones, Heike Münzberg, Leanne M RedmanAbstract:Brown adipose tissue (BAT) is specialized to dissipate energy in the form of heat. BAT-mediated heat production in rodents and humans is critical for effective temperature adaptation of newborns to the extrauterine environment immediately after birth. However, very little is known about whether and how fetal BAT development is modulated in-utero in response to changes in maternal thermal environment during pregnancy. Using BL6 mice, we evaluated the impact of different maternal environmental temperatures (28 °C and 18 °C) on the transcriptome of the placenta and fetal BAT to test if maternal cold exposure influences fetal BAT development via placental remodeling. Maternal weight gain during pregnancy, the average number of fetuses per pregnancy, and placental weight did not differ between the groups at 28 °C and 18 °C. However, the average fetal weight at E18.5 was 6% lower in the 18 °C-group compared to the 28 °C-group. In fetal BATs, cold exposure during pregnancy induced increased expression of genes involved in de novo lipogenesis and lipid metabolism while decreasing the expression of genes associated with muscle cell differentiation, thus suggesting that maternal cold exposure may promote fetal brown adipogenesis by suppressing the myogenic lineage in bidirectional progenitors. In placental tissues, maternal cold exposure was associated with upregulation of genes involved in complement activation and downregulation of genes related to muscle contraction and actin-Myosin Filament sliding. These changes may coordinate placental adaptation to maternal cold exposure, potentially by protecting against cold stress-induced inflammatory damage and modulating the vascular and extravascular contractile system in the placenta. These findings provide evidence that environmental cold temperature sensed by the mother can modulate the transcriptome of placental and fetal BAT tissues. The ramifications of the observed gene expression changes warrant future investigation.
Marco Linari - One of the best experts on this subject based on the ideXlab platform.
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Myosin Filament activation in the heart is tuned to the mechanical task
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Massimo Reconditi, Vincenzo Lombardi, Theyencheri Narayanan, Marco Linari, Marco Caremani, Francesca Pinzauti, Joseph D Powers, Ger J M Stienen, Gabriella PiazzesiAbstract:The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the Myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of Myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick Filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.
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sarcomere length dependence of Myosin Filament structure in skeletal muscle fibres of the frog
The Journal of Physiology, 2014Co-Authors: Massimo Reconditi, Malcolm Irving, Vincenzo Lombardi, Luca Fusi, Elisabetta Brunello, Marco Linari, Manuel Fernandez Martinez, Gabriella PiazzesiAbstract:X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the Myosin heads and Myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick Filament is maintained by an interaction between MyBP-C and the thin Filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the Myosin heads along the Filaments decreased in proportion to the overlap between thick and thin Filaments, with no change in its interference fine structure. Thus, Myosin heads in the regions of the thick Filaments that do not overlap with thin Filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed Myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick Filament involving changes in both the organisation of the Myosin heads on its surface and the axial periodicity of the Myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin Filaments.
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structural changes in the Myosin Filament and cross bridges during active force development in single intact frog muscle fibres stiffness and x ray diffraction measurements
The Journal of Physiology, 2006Co-Authors: Massimo Reconditi, Gabriella Piazzesi, Elisabetta Brunello, Marco Linari, Pasquale Bianco, Pierre Panine, Theyencheri NarayananAbstract:Structural and mechanical changes occurring in the Myosin Filament and Myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4°C and 2.15 μm sarcomere length, using sarcomere level mechanics and X-ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 Myosin-based reflections were recorded with 5 ms frames using the gas-filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T0) with a half-time of 40 ms, and the spacings of the M3 and M6 reflections (SM3 and SM6) increase by 1.5% from their resting values, with time courses that lead that of force by ∼10 and ∼20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by ∼10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half-sarcomere)−1 shortening at the velocity (V0) that maintains zero force. Shortening at V0 transiently reduces SM3 following the latent period and delays the subsequent increase in SM3, but only delays the SM6 increase without a transient decrease. Shortening at V0 imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (IM3), whereas the intensity of the M6 reflection (IM6) is only slightly reduced. The changes in half-sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of Myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the Myosin heads attached to the actin Filament and the M6 reflection originates mainly from a fixed structure in the Myosin Filament signalling Myosin Filament length changes during the tetanus rise.
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interference fine structure and sarcomere length dependence of the axial x ray pattern from active single muscle fibers
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Theyencheri Narayanan, Natalia A Koubassova, Ian M Dobbie, Olivier Diat, Vincenzo LombardiAbstract:Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2–3.2 μm, the M3 x-ray reflection, associated with the repeat of Myosin heads along the Filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between Myosin and actin Filaments, showing that it comes from Myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere length and was quantitatively explained by x-ray interference between Myosin heads in the two overlap regions of each sarcomere. The relative intensity of the M3 peaks was independent of sarcomere length, showing that the axial periodicities of the nonoverlap and overlap regions of the Myosin Filament have the same value, 14.57 nm, during active contraction. In resting fibers the periodicity is 14.34 nm, so muscle activation produces a change in Myosin Filament structure in the nonoverlap as well as the overlap part of the Filament. The results establish x-ray interferometry as a new tool for studying the motions of Myosin heads during muscle contraction with unprecedented spatial resolution.
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the stiffness of skeletal muscle in isometric contraction and rigor the fraction of Myosin heads bound to actin
Biophysical Journal, 1998Co-Authors: Marco Linari, Malcolm Irving, Massimo Reconditi, Gabriella Piazzesi, Natalia A Koubassova, Ian M Dobbie, Vincenzo LombardiAbstract:Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin Filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of Myosin cross-bridges in the region of overlap between the Myosin Filament and the actin Filament remains constant, and only the length of the nonoverlapped region of the actin Filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin Filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the Myosin Filament compliance is 1.3 nm microm(-1) T0(-1). With these values for Filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of Myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.
