Filamentous State Research Articles

Cryo-EM structures of cardiac muscle α-actin mutants M305L and A331P give insights into the structural mechanisms of hypertrophic cardiomyopathy

Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.

Myosin Isoform-Dependent Effect of Omecamtiv Mecarbil on the Regulation of Force Generation in Human Cardiac Muscle.

Omecamtiv mecarbil (OM) is a small molecule that has been shown to improve the function of the slow human ventricular myosin (MyHC) motor through a complex perturbation of the thin/thick filament regulatory state of the sarcomere mediated by binding to myosin allosteric sites coupled to inorganic phosphate (Pi) release. Here, myofibrils from samples of human left ventricle (β-slow MyHC-7) and left atrium (α-fast MyHC-6) from healthy donors were used to study the differential effects of μmolar [OM] on isometric force in relaxing conditions (pCa 9.0) and at maximal (pCa 4.5) or half-maximal (pCa 5.75) calcium activation, both under control conditions (15 °C; equimolar DMSO; contaminant inorganic phosphate [Pi] ~170 μM) and in the presence of 5 mM [Pi]. The activation state and OM concentration within the contractile lattice were rapidly altered by fast solution switching, demonstrating that the effect of OM was rapid and fully reversible with dose-dependent and myosin isoform-dependent features. In MyHC-7 ventricular myofibrils, OM increased submaximal and maximal Ca2+-activated isometric force with a complex dose-dependent effect peaking (40% increase) at 0.5 μM, whereas in MyHC-6 atrial myofibrils, it had no effect or-at concentrations above 5 µM-decreased the maximum Ca2+-activated force. In both ventricular and atrial myofibrils, OM strongly depressed the kinetics of force development and relaxation up to 90% at 10 μM [OM] and reduced the inhibition of force by inorganic phosphate. Interestingly, in the ventricle, but not in the atrium, OM induced a large dose-dependent Ca2+-independent force development and an increase in basal ATPase that were abolished by the presence of millimolar inorganic phosphate, consistent with the hypothesis that the widely reported Ca2+-sensitising effect of OM may be coupled to a change in the state of the thick filaments that resembles the on-off regulation of thin filaments by Ca2+. The complexity of this scenario may help to understand the disappointing results of clinical trials testing OM as inotropic support in systolic heart failure compared with currently available inotropic drugs that alter the calcium signalling cascade.

Complex state transitions of the bacterial cell division protein FtsZ.

The key bacterial cell division protein FtsZ can adopt multiple conformations, and prevailing models suggest that transitions of FtsZ subunits from the closed to open state are necessary for filament formation and stability. Using all-atom molecular dynamics simulations, we analyzed state transitions of Staphylococcus aureus FtsZ as a monomer, dimer, and hexamer. We found that monomers can adopt intermediate states but preferentially adopt a closed state that is robust to forced reopening. Dimer subunits transitioned between open and closed states, and dimers with both subunits in the closed state remained highly stable, suggesting that open-state conformations are not necessary for filament formation. Mg2+ strongly stabilized the conformation of GTP-bound subunits and the dimer filament interface. Our hexamer simulations indicate that the plus end subunit preferentially closes and that other subunits can transition between states without affecting inter-subunit stability. We found that rather than being correlated with subunit opening, inter-subunit stability was strongly correlated with catalytic site interactions. By leveraging deep-learning models, we identified key intrasubunit interactions governing state transitions. Our findings suggest a greater range of possible monomer and filament states than previously considered and offer new insights into the nuanced interplay between subunit states and the critical role of nucleotide hydrolysis and Mg2+ in FtsZ filament dynamics.

Impact of Bacterization with Anabaena flos-aquae and Pseudomonas putida and Salicylic Acid Treatment on Cold Resistance in Leguminous Plants

The paper presents the results of studying the eff ects of exposure of pea (Pisum sativum) and been (Phaseolus vulgaris) seeds or young sprouts to low temperature (4°C) with preliminary treatment of seeds with salicylic acid (SA), or with pure cultures of Anabaena flos-aquae or Pseudomonas putida, or with their mixture. Cold exposure lasting 24 hrs was carried out immediately or 7 days after SA treatment or bacterization of seeds. Morphometric parameters of roots and sprouts, permeability of cell membranes in leaves, activity of polyphenol oxidase and state of actin filaments in root cells were measured. Pea and bean plants differ in cold resistance, and their growth responses to low temperature exposure were multidirectional. Treatment with SА immediately before exposure to cold had a positive effect on cold resistance of pea plants. Under the delayed cold stress, seed bacterization with single bacteria suspensions or their mixture contributed to the development of cold resistance in beans. Bacterization of seeds presumably reduced the permeability of cell membranes in leaves of both plant species and improved the state of actin filaments in root cells. In general, some protective effect for peas was observed due to either bacterial or SА treatment of seeds. In bean plants, the meaningful protective effect occurred after seed bacterization only.

On the presence of an unexpected meadow of Penicillus capitatus Lamarck (Bryopsidales: Chlorophyta) in Mallorca, Balearic Islands, Western Mediterranean

An unexpected meadow of the warm-water alga Penicillus capitatus was found thriving in sandy bottoms between 16 and 21 m depth in Mallorca, Balearic Islands. Plant densities and benthic coverage were very high when compared to most Mediterranean meadows of this species. As it is common in tropical meadows of this species, all the individuals were found in a Penicillus morphological state, whereas most Mediterranean individuals are always found in the filamentous (Espera) state. Due to the high biomass of the populations, the contribution to the primary productivity of the sandy bottoms it colonizes should be far from negligible. If the expansion of these species continues, the annual calcium carbonate production of the Balearic continental shelf will increase and previous estimated budgets will have to be reconsidered. Since no genetic analysis of the specimens have been performed so far, it is still early to know if the studied populations have been originated by native Mediterranean specimens or from a cryptic lineage of Western Atlantic or Macaronesian origin. Whatever the answer is, the current bloom of this population in an environment completely devoid of this species in the recent past, provides new evidence of the current tropicalization of the Mediterranean caused by global warming.

Thermal dependence of the current in TiN/Ti/HfO2/W memristors at different intermediate conduction states

The dependence of the current in TiN/Ti/HfO2/W devices on the temperature is investigated in the range from 78 K to 340 K. Resistive switching cycles at 78 K are conducted to explore the thermal dependence in filament configurations with different intermediate resistance states. The less conductive states show an increase of the current as the temperature rises, while the fully formed filament displays a metallic-like behavior. A comprehensive model, based on the Stanford Model including a series resistance, is proposed and successfully validated by experimental data. The interplay between the ohmic and non-linear components in the model for different filament states is analyzed, emphasizing the dominance of the non-linear component (and its corresponding thermal dependence) in partially formed filaments and the prevalence of the ohmic component in the fully formed filament, which shows a decreasing current as the temperature rises. A complete compact model for simulation of circuits including the thermal dependence of these devices is developed.

Monitoring of extrusion filament state for fused filament fabrication: A super-resolution image monitoring approach based on degradation pattern learning

Extrusion filament is the fundamental unit of the fused filament fabrication process (FFF). Its state stability directly affects the product quality. Therefore, a reliable method is required to monitor the state of the extrusion filament. This work presents an innovative approach involving super-resolution algorithms for conveniently monitoring the state of the extrusion filament during the printing process using cost-effective cameras. Firstly, this work captures high-resolution images during the printing ending and low-resolution images during the printing processing. Secondly, a generative adversarial network (GAN) is employed to adaptively learn the degradation patterns in the printing environment. Subsequently, the trained generator of the GAN is used to construct pixel-level paired super-resolution training dataset. The work then utilizes this dataset to train a super-resolution model, getting a specialized super-resolution model for the degradation patterns specific to the printing scenario. Finally, this super-resolution model is employed to enhance the resolution of the images captured during the production process, resulting in improved performance in downstream image-related tasks.

The biochemically defined super relaxed state of myosin—A paradox

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76μM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Effects of specific disease mutations in non-muscle myosin 2A on its structure and function

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state, in which its regulatory light chain (RLC) is dephosphorylated, and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts and deafness. Approximately two thirds of these mutations occur in the coiled-coil tail. These mutations could destabilise the 10S state and/or disrupt filament formation, or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyse 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behaviour of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilisation are key to understanding their contributions to NM2A in disease.

DYRK-family kinases regulate Candida albicans morphogenesis and virulence through the Ras1/PKA pathway.

Candida albicans is an opportunistic human fungal pathogen that frequently causes life-threatening infections in immunocompromised individuals. To cause disease, the fungus employs several virulence traits, including its ability to transition between yeast and filamentous states. Previous work identified a role for the kinase Yak1 in regulating C. albicans filamentation. Here, we demonstrate that Yak1 regulates morphogenesis through the canonical cAMP/PKA pathway and that this regulation is environmentally contingent, as host-relevant concentrations of CO2 bypass the requirement of Yak1 for C. albicans morphogenesis. We show a related kinase, Pom1, is important for filamentation in the absence of Yak1 under these host-relevant conditions, as deletion of both genes blocked filamentous growth under all conditions tested. Finally, we demonstrate that Yak1 is required for filamentation in a mouse model of C. albicans dermatitis using genetic and pharmacological approaches. Overall, our results expand our understanding of how Yak1 regulates an important virulence trait in C. albicans.

Myosin and tropomyosin-troponin complementarily regulate thermal activation of muscles.

Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy. Rapid heating (25 to 41.5°C) within 2 s induced reversible sarcomere shortening in relaxing solution. Further, we investigated the temperature-dependence of the sliding velocity of reconstituted fast skeletal or cardiac thin filaments on fast skeletal or β-cardiac myosin in an in vitro motility assay within the body temperature range. We found that (a) with fast skeletal thin filaments on fast skeletal myosin, the temperature dependence was comparable to that obtained for sarcomere shortening in fast skeletal myofibrils (Q10 ∼8), (b) both types of thin filaments started to slide at lower temperatures on fast skeletal myosin than on β-cardiac myosin, and (c) cardiac thin filaments slid at lower temperatures compared with fast skeletal thin filaments on either type of myosin. Therefore, the mammalian striated muscle may be fine-tuned to contract efficiently via complementary regulation of myosin and tropomyosin-troponin within the body temperature range, depending on the physiological demands of various circumstances.

Hfq C-terminal region forms a β-rich amyloid-like motif without perturbing the N-terminal Sm-like structure

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution. Many aspects of the role and the structure of this region remain unclear. For instance, in its truncated form it can promote amyloid-like filament assembly. Here, we show that a minimal 11-residue motif at the C-terminal end of Hfq assembles into filaments with amyloid characteristics. Our data suggest that the full-length Hfq in its filamentous state contains a similar molecular fingerprint than that of the short β-strand peptide, and that the Sm-core structure is not affected by filament formation. Hfq proteins might thus co-exist in two forms in vivo, either as isolated, soluble hexamers or as self-assembled hexamers through amyloid-reminiscent interactions, modulating Hfq cellular functions.

The strength of a constrained lithium layer

A constrained compression test is developed to replicate the mechanical state of a lithium filament within a solid state battery. Lithium microspheres are compressed between parallel quartz plates into a pancake shape of thickness on the order of 15 µm. Full adhesion with no slip exists between the lithium and platens, and the attendant mechanical constraint implies that the average pressure on the pancake-shaped specimens increases with increasing aspect ratio of radius to height. In addition to mechanical constraint, a thickness-dependant size effect is observed whereby the apparent flow strength of the lithium increases from 0.7 MPa in the bulk to 2.0 MPa at a thickness of 15 µm. The lithium deforms in a power-law creeping manner at room temperature, and to simplify interpretation of the results, the relative velocity of the loading platens is adjusted to ensure that the true compressive strain rate is held fixed at 10−3 s−1. Additional measurements of lithium flow strength are obtained by subjecting the pancake-shaped specimens to simple shear. The size effect under shear loading is comparable to that in constrained compression. The observed size effect for lithium is consistent with that reported in the literature for lithium in indentation tests and in single pillar compression tests. Finally, the size effect of lithium in the power law creep regime is compared with that for rate-independent plasticity (for copper).

Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and β subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Negative Charges Introduced Near the IT Helix of Cardiac Troponin T Stabilize the Active State of Actin Filaments.

The disordered and basic C-terminal 14 residues of human troponin T (TnT) are essential for full inhibition of actomyosin ATPase activity at low Ca2+ levels and for limiting activation at saturating Ca2+. In previous studies, stepwise truncation of the C-terminal region of TnT increased activity in proportion to the number of positive charges eliminated. To define key basic residues more closely, we generated phosphomimetic-like mutants of TnT. Phosphomimetic mutants were chosen because of reports that phosphorylation of TnT, including sites within the C terminal region, depressed activity, contrary to our expectations. Four constructs were made where one or more Ser and Thr residues were replaced with Asp residues. The S275D and T277D mutants, near the IT helix and adjacent to basic residues, produced the greatest activation of ATPase rates in solution; the effects of the S275D mutant were recapitulated in muscle fiber preparations with enhanced myofilament Ca2+ sensitivity. Actin filaments containing S275D TnT were also shown to be incapable of populating the inactive state at low Ca2+ levels. Actin filaments containing both S275D/T284D were not statistically different from those containing only S275D in both solution and cardiac muscle preparation studies. Finally, actin filaments containing T284D TnT, closer to the C-terminus and not adjacent to a basic residue, had the smallest effect on activity. Thus, the effects of negative charge placement in the C-terminal region of TnT were greatest near the IT helix and adjacent to a basic residue.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Following the entangled state of filaments.

California blackworms serve as a template for the topological design of active matter.

Coexistence of Write Once Read Many Resistive Switching with Negative Differential Resistance in Bi‐Rich Bi12FeO20 Ideal Sillenite

The materials showing write once read many (WORM) types of resistive switching characteristics are potential candidates for permanent storage. The negative differential resistance‐based memory devices have the potential to perform like nonlinear systems together with resistive switching simultaneously. The successful synthesis of Bi‐rich Bi12FeO20 sillenite material as phase pure thin film on fluorine tin oxide (FTO) using a solution process together with thermally deposited copper (Cu) as the top contact is demonstrated. Cu/Bi12FeO20/FTO/Glass resistive random‐access memory (RRAM) devices show the WORM characteristics with Ion/Ioff ratio103 and negative differential resistance simultaneously. Further, Bi12FeO20 active material also shows nonvolatile characteristics with analog switching for 200 cycles or more with large retention time, ≈5 × 103 s, and endurance for 103 cycles, substantiating the stability of the device. The space charge limited conduction and Schottky mechanisms explain the formation of a conductive filament in a high resistance state (HRS) and rupturing of a conductive filament in a low resistance state (LRS). The observed negative differential resistance is attributed to trapping, detrapping, and Schottky barrier interface between the top electrode and active layer together with ambient humidity.

Dissecting the mechanisms by which a pathogenic variant in cardiac TnT tail domain leads to disturbed myofilament performance.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and high rate of sudden cardiac arrest at juvenile age. cTnT-I79N is located in the cTnT N-terminal (TNT1) loop region and is well-known for its pathological and prognostic relevance. Recent structural study revealed that I79 is part of hydrophobic interface between TNT1 loop and actin which stabilizes the OFF state of the cardiac thin filament. Given the importance of understanding: i) the role of TNT1 loop region on the regulation of the cardiac thin filament, and ii) the underlying mechanisms of cTnT-I79N-linked pathogenesis; we sought to investigate the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased passive force and passive sinusoidal stiffness, which could be due in part to an increase in force-generating cross-bridges during diastole. Tg-I79N also displayed increased myofilament Ca2+-sensitivity, shorter myofilament lattice spacing, and slower cross-bridge kinetics. These findings can be attributed to the destabilization of cardiac thin filament's inhibitory property, and the increase in the overall number of cross-bridges during Ca2+ activation. Finally, we show that cTnT-I79N dysregulates SRX↔DRX equilibrium, which can be explained by the excessive binding of myosin heads to the dysregulated thin filament that causes the SRX↔DRX equilibrium to shift towards the ON state. Altogether, biochemical and functional measurements, and structural data points to the following mechanism: cTnT-I79N weakens the interaction of the TNT1 loop with the actin filament, which in turn destabilizes the OFF state of the cardiac thin filament.

The effect of physiological and pharmacological inotropic interventionson the regulatory state of the thick filament in intact cardiac trabeculae.

In the cardiac muscle in diastole myosin motors are packed on the surface of the thick filament in three-stranded helical tracks, folded toward the centre of the sarcomere and unable to bind actin and hydrolyse ATP (OFF state; Woodhead et al. 2005, Nature 436:1195). In the X-ray diffraction pattern of intact trabeculae from rat heart this OFF state is marked by the intensity of the corresponding myosin-based layer lines and axial reflections (Reconditi et al. 2017, PNAS 114:3240). In trabeculae electrically paced at 0.5 Hz (temperature 27 °C), during diastole none of the X-ray signals related to the OFF state of the myosin motors are affected by inotropic interventions which increase by twofold the systolic peak force, like increase in sarcomere length (1.95-2.25 μm) or addition of 10−7 M isoprenaline, indicating that these inotropic interventions acts downstream with respect to Ca2+-activation (Caremani et al. 2019, J. Gen. Physiol. 151:53). Instead, the addition of the cardiac activator omecamtiv mecarbil (OM, 1 μM), a small molecule that binds to the catalytic domain of cardiac myosin and has been found to increase the systolic ejection fraction in animal models (Malik et al. 2011, Science 331:1439) and the Ca2+ sensitivity of force in demembranated cardiac myocytes (Nagy et al. 2015, Brit. J. Pharmacol. 172:4506), induces a partial switching ON of motors in diastole, mainly at the expense of the filament regions outside the central 1/3 containing the Myosin Binding Protein C. The specific effect that different inotropic interventions exert on the regulatory state of the thick filament in diastole reflects on the different mechanisms by which these interventions potentiate the systolic performance. Supported by ECR, University of Florence and MUR (Italy) and ESRF (France).