Cooperative folding of muscle myosins: I. Mechanical model

Mechanically induced folding of passive cross-linkers is a fundamental biological phenomenon. A typical example is a conformational change in myosin II responsible for the power-stroke in skeletal muscles. In this paper we present an athermal perspective on such folding by analyzing the simplest purely mechanical prototype: a parallel bundle of bi-stable units attached to a common backbone. We show that in this analytically transparent model, characterized by a rugged energy landscape, the ground states are always highly coherent, single-phase configurations. We argue that such cooperative behavior, ensuring collective conformational change, is due to the dominance of long- range interactions making the system non-additive. The detailed predictions of our model are in agreement with experimentally observed non-equivalence of fast force recovery in skeletal muscles loaded in soft and hard devices. Some features displayed by the model are also recognizable in the behavior of other biological systems with passive multi-stability and long-range interactions including detaching adhesive binders and pulled RNA/DNA hairpins

Conformational Dynamics of Actin: Effectors and Implications for Biological Function

Actin is a protein abundant in many cell types. Decades of investigations have provided evidence that it has many functions in living cells. The diverse morphology and dynamics of actin structures adapted to versatile cellular functions is established by a large repertoire of actin-binding proteins. The proper interactions with these proteins assume effective molecular adaptations from actin, in which its conformational transitions play essential role. This review attempts to summarise our current knowledge regarding the coupling between the conformational states of actin and its biological function

THE MECHANICAL PROPERTIES OF NON-FAILING AND FAILING HUMAN MYOCARDIUM

Heart failure is a clinical syndrome that manifests when there are structural and functional impairments to the heart that reduces the ability of the ventricles to fill or eject blood. The syndrome affects ~6 million Americans and is responsible for nearly 300,000 deaths annually. At the core of the syndrome are dysfunctional sarcomeres, the machinery that drives cardiac contraction and relaxation. By assessing the mechanical properties of human cardiac tissue, the information provided in this dissertation will provide data that demonstrates how sarcomeric dysfunction contributes to heart failure in the left and right ventricles. Additionally, these data will supply information on how probable therapeutics impact the mechanical properties of the heart and the clinical implications. Thus, the overall objective of this project is to assess the mechanical properties of failing and non-failing human myocardium while concomitantly studying the molecular mechanisms contributing to heart failure and work towards therapy. Mechanical experiments were performed with human cardiac samples obtained from patients who were receiving heart transplants and from organ donors who did not have a history of heart failure. Cardiac samples were homogenized and chemically permeabilized (pores in the membrane). Multicellular preparations from failing and non-failing hearts were attached between a force transducer and a motor to determine the mechanical properties. In the first study, we compared the mechanical properties of cardiac samples from the right and left ventricles of non-failing and failing hearts, as well as determined the relative phosphorylation levels of specific sarcomeric proteins. The results show that in non-failing hearts, calcium sensitivity was higher in the left ventricle, and in failing hearts, calcium sensitivity was higher in the right ventricle. The shift in the pattern of the calcium sensitivity data from non-failing samples to failing samples underpin a statistical interaction between heart failure status and the ventricles of the heart for calcium sensitivity. This interaction suggests that heart failure is altering the sensitivity of the myofilament to Ca2+ differently in the right ventricle. The mechanical data also demonstrated that heart failure significantly reduced isometric force and maximum power in both ventricles. Biochemical assays suggest that the cause of the interaction observed in the calcium sensitivity data is driven by the phosphorylation profile of sarcomeric proteins. We then determined the effects of two small molecules (omecamtiv mecarbil and para-Nitroblebbistatin) on the mechanical properties of human myocardium. The results of those studies demonstrate that omecamtiv mecarbil increases calcium sensitivity and slows the rate of force development in a dose-dependent manner without altering maximum isometric force. Conversely, para-Nitroblebbistatin reduced isometric force, power, and calcium sensitivity without changing shortening velocity or the rate of force development. Lastly, we measured the effects of engineered troponins on the mechanical function of failing tissue. The results show that troponin C and troponin I designed to either increase or decrease calcium sensitivity can significantly increase or decrease calcium sensitivity without altering maximum force, shortening velocity or the rate of tension development. The findings reported in this dissertation have revealed novel mechanical data from non-failing and failing human cardiac tissue. These data present three significant results. First, the right vs. left ventricular comparison data shows that heart failure in humans reduces maximum force and power in both ventricles equally while altering myofilament calcium sensitivity of the left and right ventricles in different ways. The change in calcium sensitivity may reflect ventricle specific post-translational modifications of sarcomeric proteins. Second, the use of myosin modulators revealed that molecules like omecamtiv mecarbil and para-Nitroblebbistatin that directly target myosin function can modify calcium sensitivity and the rate of force development in human cardiac tissue. Third, the engineered troponin study showed that engineered troponins C and I can alter myofilament calcium sensitivity without affecting myosin kinetics. Clinically, the results of the small molecules and engineered protein studies suggest that small molecules and engineered proteins could potentially serve as therapy for patients suffering from heart disease

The mechanics and regulation of rat aortic smooth muscle contraction: implications of cytoskeletal remodeling, protein phosphorylations, and microtubule-based kinase transport

The exact nature of the mechanisms and the regulation of vascular smooth muscle contraction is not well understood. To better understand these processes, we examined two systems involved in smooth muscle contraction, the cytoskeleton and the protein kinases. In order to study the role of the cytoskeleton in smooth muscle contraction, we examined the contractile and mechanical effects of cytoskeleton disruption. We found that the relationship between passive tension applied to aortic rings and the resulting increase in tissue length was nearly linear over the range of 1 g to 15 g. However, even with increasing tissue length, within the range of 1 g to 10 g passive tension, the total active force generated upon stimulation was not significantly changed. These observations emphasize the great flexibility of the mechanism(s) underlying the contractile response of vascular smooth muscle with regard to changes in tissue preload and length. Neither the blockade of microtubule polymerization by colchicine nor actin polymerization by cytochalasin B significantly changed the slope of the tissue length-passive tension preload curve indicating no effect on the tissues\u27 capacity to stretch at a given preload. With stimulation of the tissue at different levels of stretch, colchicine caused an increase in the initial fast component of active tension development, but partially blocked the secondary slow rise in tension. Cytochalasin B dramatically reduced the total contractile response at each preload studied, and this effect was confined almost exclusively to the secondary slow increase in tension. When tissues were cooled to cause complete dissolution of the microtubule network and then warmed in the presence of colchicine to prevent repolymerization of both the active and stable populations of microtubules, there was also a significant reduction in the slow component of contraction with no effect on the fast response. The partial blockade of synthesis of the microtubule-associated motor protein kinesin by application of an antisense oligonucleotide to aortae in situ or to aortic rings in tissue culture significantly reduced the contractile response to potassium depolarization. These results suggest that the microtubules and the actin filaments of the cytoskeleton play an active role in slow force development as opposed to a solely passive role based on the effect of the static, structural properties of these filaments on mechanical resistance. We propose that a tension-bearing element of the actin-containing cytoskeleton undergoes remodeling to adjust tension within the system. The microtubules could act through either the direct action of kinesin-mediated intracytoskeletal interactions in force development that involve a remodeling of the tension-bearing elements of the cytoskeleton or through the directed movement of the molecules involved in the transduction process. Because the cytoskeleton and the protein kinases of smooth muscle are intimately linked, we examined the potential role of protein kinases in vascular smooth muscle contraction. We began by assessing the effects of a panel of specific kinase inhibitors on smooth muscle contraction. We found reductions in contraction with inhibition of myosin light chain kinase (MLCK), calcium-dependent calmodulin kinase (CaMKII), mitogen activated protein (MAP) kinase, and protein kinase C (PKC). Protein kinase C (PKC) is translocated in an isoform-specific manner to distinct subcellular locations after stimulation of cells. It is thought that translocation is essential for PKC activation and that cellular localization underlies the PKC isoform-specific phosphorylation of substrate in the intact cell that is largely absent in in vitro assays. In the present studies, it was shown using Western blot analysis that the ratio of particulate to cytosolic PKC-α was reduced in rat aortic segments treated with colchicine to disrupt microtubular structure prior to stimulation with phorbol 12, 13 dibutyrate (PDB). Subsequent studies using laser confocal microscopy revealed that within thirty seconds after stimulation with PDB, PKC-α in cultured rat aortic smooth muscle cells changed from a diffuse cytoplasmic distribution to a highly structured filamentous pattern of staining. Dual immunostaining further indicated that the stimulation-induced filamentous pattern was due to colocalization of PKC-α with cell microtubules. At longer time intervals after PDB stimulation, PKC-α was observed to translocate to the perinuclear region of the cell. Disruption of the microtubular but not the actin-containing component of the cytoskeleton blocked the translocation of PKC-α to the perinuclear membrane. It was further shown that slow tension development, which has been reported to be selectively blocked by PKC antagonists in vascular smooth muscle, was also blocked by disruption of the cell microtubules. The results provide further evidence for the involvement of PKC in slow tension development by smooth muscle and indicate that PKC translocation may involve microtubular transport

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments

The Amino Terminal Region of Cardiac Myosin Binding Protein-C Is Necessary for Cardiac Function

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that has been suggested to regulate cardiac contraction via its amino terminal (N’) region. Following ischemic injury to the heart, cMyBP-C is cleaved into a predominant N’ fragment consisting of domains C0 through C1 and the first 17 residues of the M-domain that is referred to as C0-C1f. However, the necessity of the N’-C0-C1f region of cMyBP-C in regulating cardiac function in vivo has not been elucidated. I hypothesized that the N’-C0-C1f region of cMyBP-C is critical for normal cardiac function in vivo. To test this hypothesis, transgenic (TG) mice with 81 ± 2.2% expression of a truncated cMyBP-C missing the N’-C0-C1f region (cMyBP-C110kDa) were generated and characterized in comparison to their non-transgenic (NTG) littermates between 3- and 8-months of age. Echocardiography at 3- and 6-months of age showed a significant reduction in percent fractional shortening (FS) in cMyBP-C110kDa hearts compared to NTG hearts at both time-points indicating progressive cardiac dysfunction in cMyBP-C110kDa animals. I further observed cardiac enlargement, determined by whole-heart confocal imaging, and an elevation in cardiac pathological hypertrophy markers, determined by quantitative real-time PCR (qPCR) and RNA-Seq, in cMyBP-C110kDa compared to NTG animals. Histopathological and second harmonic generation (SHG) analyses on myocardial sections indicated increased cardiac fibrosis (p \u3c 0.0001) and increased sarcomere area (p \u3c 0.01) in cMyBP-C110kDa hearts compared to NTG hearts. Crucially, immunofluorescence analysis of isolated cardiac myocytes from cMyBP-C110kDa and NTG hearts revealed that the cMyBP-C110kDa TG protein localized properly at the C-zone within the cardiac sarcomere. Intriguingly, increased phosphorylation of the cMyBP-C110kDa TG protein within the M domain was observed in cMyBP-C110kDa myofilament fractions compared to those from NTG controls. Finally, using isolated, permeabilized papillary muscle fibers from cMyBP-C110kDa and NTG hearts, I determined that a significant elevation in maximum force (p \u3c 0.01) and rate of tension redevelopment (ktr) (p \u3c 0.05) were produced from cMyBP-C110kDa fibers compared NTG fibers. Based upon this data, I conclude that the N’-C0-C1f region of cMyBP-C regulates cardiac contractility and is necessary for maintaining normal cardiac function in vivo

Anti-S2 Peptides and Antibodies Binding Effect on Myosin S2 and Anti-S2 Peptide's Ability to Reach the Cardiomyocytes in vivo and Interfere in Muscle Contraction

The anti-S2 peptides, the stabilizer and destabilizer, were designed to target myosin sub-fragment 2 (S2) in muscle. When the peptides are coupled to a heart-targeting molecule, they can reach the cardiomyocytes and interfere with cardiac muscle contraction. Monoclonal antibodies, MF20 and MF30, are also known to interact with light meromyosin and S2 respectively. The MF30 antibody compared to anti-S2 peptides and the MF20 antibody is used as a control to test the central hypothesis that: Both the anti-S2 peptides and antibodies bind to myosin S2 with high affinity, compete with MyBPC, and possibly interact with titin, in which case the anti-S2 peptides have further impact on myosin helicity and reach the heart with the aid of tannic acid to modulate cardiomyocytes' contraction in live mice. In this research, the effects of anti-S2 peptides and antibodies on myosin S2 were studied at the molecular and tissue levels. The anti-myosin binding mechanism to whole myosin was determined based on total internal reflectance fluorescence spectroscopy (TIRFS), and a modified cuvette was utilized to accommodate this experiment. The binding graphs indicated the cooperative binding of the peptides and antibodies with high affinity to myosin. Anti-myosin peptides and antibodies competition with Myosin Binding Protein C (MyBPC) was revealed through the super-resolution expansion microscopy using wildtype skeletal and cardiac myofibrils, and MyBPC knock-out cardiac myofibril. This new emerging technique depends on using the regular confocal microscope in imaging expanded myofibril after embedding in a swellable hydrogel polymer and digestion. A decrease in the fluorescent intensity at the C-zone was observed in myofibrils labeled with fluorescently labeled anti-S2 peptides or antibodies supporting the competition with MyBPC, which further was confirmed by the absence of this reduction at the C-zone in the knockout MyBPC cardiac tissue. The anti-S2 peptide's ability to reach inside the cardiomyocytes was tested by injecting fluorescently labeled anti-S2 peptides bound to tannic acid in live mice, the destabilizer peptide reached the heart 6X more than the stabilizer peptide. Some of the peptides labeled cardiac arterioles and T-tubules as detected by super-resolution microscopic images, meanwhile some peptides reached inside the cardiomyocytes and labeled some sarcomeres. This dissertation demonstrates the ability of anti-S2 peptides and antibodies in modifying myosin as they bind cooperatively with high affinity to myosin and compete with the regulatory protein MyBPC, in addition to the possible interaction between the stabilizer peptide and titin. Lastly, the peptides succeeded in labeling some cardiac sarcomeres in live mice