Nanosurgical Manipulation of Titin and Its M-Complex

Titin is a multifunctional filamentous protein anchored in the M-band, a hexagonally organized supramolecular lattice in the middle of the muscle sarcomere. Functionally, the M-band is a framework that cross-links myosin thick filaments, organizes associated proteins, and maintains sarcomeric symmetry via its structural and putative mechanical properties. Part of the M-band appears at the C-terminal end of isolated titin molecules in the form of a globular head, named here the “M-complex”, which also serves as the point of head-to-head attachment of titin. We used high-resolution atomic force microscopy and nanosurgical manipulation to investigate the topographical and internal structure and local mechanical properties of the M-complex and its associated titin molecules. We find that the M-complex is a stable structure that corresponds to the transverse unit of the M-band organized around the myosin thick filament. M-complexes may be interlinked into an M-complex array that reflects the local structural and mechanical status of the transversal M-band lattice. Local segments of titin and the M-complex could be nanosurgically manipulated to achieve extension and domain unfolding. Long threads could be pulled out of the M-complex, suggesting that it is a compact supramolecular reservoir of extensible filaments. Nanosurgery evoked an unexpected volume increment in the M-complex, which may be related to its function as a mechanical spacer. The M-complex thus displays both elastic and plastic properties which support the idea that the M-band may be involved in mechanical functions within the muscle sarcomere.

In silico identification of potential calcium dynamics and sarcomere targets for recovering left ventricular function in rat heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a complex disease associated with multiple co-morbidities, where impaired cardiac mechanics are often the end effect. At the cellular level, cardiac mechanics can be pharmacologically manipulated by altering calcium signalling and the sarcomere. However, the link between cellular level modulations and whole organ pump function is incompletely understood. Our goal is to develop and use a multi-scale computational cardiac mechanics model of the obese ZSF1 HFpEF rat to identify important biomechanical mechanisms that underpin impaired cardiac function and to predict how whole-heart mechanical function can be recovered through altering cellular calcium dynamics and/or cellular contraction. The rat heart was modelled using a 3D biventricular biomechanics model. Biomechanics were described by 16 parameters, corresponding to intracellular calcium transient, sarcomere dynamics, cardiac tissue and hemodynamics properties. The model simulated left ventricular (LV) pressure-volume loops that were described by 14 scalar features. We trained a Gaussian process emulator to map the 16 input parameters to each of the 14 outputs. A global sensitivity analysis was performed, and identified calcium dynamics and thin and thick filament kinetics as key determinants of the organ scale pump function. We employed Bayesian history matching to build a model of the ZSF1 rat heart. Next, we recovered the LV function, described by ejection fraction, peak pressure, maximum rate of pressure rise and isovolumetric relaxation time constant. We found that by manipulating calcium, thin and thick filament properties we can recover 34%, 28% and 24% of the LV function in the ZSF1 rat heart, respectively, and 39% if we manipulate all of them together. We demonstrated how a combination of biophysically based models and their derived emulators can be used to identify potential pharmacological targets. We predicted that cardiac function can be best recovered in ZSF1 rats by desensitising the myofilament and reducing the affinity to intracellular calcium concentration and overall prolonging the sarcomere staying in the active force generating state.

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein–protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0–C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.

Myosin motors that cannot bind actin leave their folded OFF state on activation of skeletal muscle

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament. To test whether motor unfolding is triggered by the availability of nearby actin binding sites, we measured changes in the x-ray reflections that report motor conformation when muscles are activated at longer sarcomere length, so that part of the thick filaments no longer overlaps with thin filaments. We found that the intensity of the M3 reflection from the axial repeat of the motors along the thick filaments declines almost linearly with increasing sarcomere length up to 2.8 µm, as expected if motors in the nonoverlap zone had left the folded state and become relatively disordered. In a recent article in JGP, Squire and Knupp challenged this interpretation of the data. We show here that their analysis is based on an incorrect assumption about how the interference subpeaks of the M3 reflection were reported in our previous paper. We extend previous models of mass distribution along the filaments to show that the sarcomere length dependence of the M3 reflection is consistent with <10% of no-overlap motors remaining in the folded conformation during active contraction, confirming our previous conclusion that unfolding of myosin motors on muscle activation is not due to the availability of local actin binding sites.

Integration of proteomic and genetic approaches to assess developmental muscle atrophy

Muscle atrophy, or a decline in muscle protein mass, is a significant problem in the aging population and in numerous disease states. Unraveling molecular signals that trigger and promote atrophy may lead to a better understanding of treatment options; however, there is no single cause of atrophy identified to date. To gain insight into this problem, we chose to investigate changes in protein profiles during muscle atrophy in Manduca sexta and Drosophila melanogaster. The use of insect models provides an interesting parallel to probe atrophic mechanisms since these organisms undergo a normal developmental atrophy process during the pupal transition stage. Leveraging the inherent advantages of each model organism, we first defined protein signature changes during Manduca intersegmental muscle (ISM) atrophy and then used genetic approaches to confirm their functional importance in the Drosophila dorsal internal oblique muscles (DIOMs). Our data reveal an upregulation of proteasome and peptidase components and a general downregulation of proteins that regulate actin filament formation. Surprisingly, thick filament proteins that comprise the A band are increased in abundance, providing support for the ordered destruction of myofibrillar components during developmental atrophy. We also uncover the actin filament regulator Ciboulot (Cib) as a novel regulator of muscle atrophy. These insights provide a framework towards a better understanding of global changes that occur during atrophy and may lead to eventual therapeutic targets.

Abstract P315: Shortening The Thick Filament By Partial Deletion Of Titin’S C-zone Alters Cardiac Function By Reducing The Operating Sarcomere Length Range Of The Heart.

Titin’s C-zone is the inextensible part of titin that binds along the thick filament at its cMyBP-C -containing region. Previously it was shown that deletion of titin’s super-repeats C1 and C2 ( Ttn ΔC1-2 mouse model) results in shorter thick filaments and contractile dysfunction, but LV chamber stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the left ventricle (LV) and from cellular work loops of intact loaded cardiac myocytes. Ca 2+ transients were also measured as well as crossbridge cycling kinetics and Ca 2+ sensitivity of force. It was found that intact cardiomyocytes of Ttn ΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance of the LV chamber showed that the kinetics of LV activation are normal but that relaxation is slower in Ttn ΔC1-2 mice. The slowed relaxation was, in part, attributable to an increased myofilament Ca 2+ sensitivity and slower early Ca 2+ reuptake. Dynamic stiffness at the myofilament level showed that cross-bridge kinetics are normal, but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements in the mid-wall region of the LV revealed that the operating SL range is shifted in Ttn ΔC1-2 mice towards shorter lengths. This normalizes the apparent cell and LV chamber stiffness but reduces the number of force generating cross-bridges due to suboptimal thin and thick filament overlap. Thus the contractile dysfunction in Ttn ΔC1-2 mice is not only due to shorter thick filaments but also to a reduced operating sarcomere length range. Overall these results reveal that for normal cardiac function, thick filament length regulation by titin’s C-zone is critical.

Abstract 112: The 3CL Protease Of SARS-CoV2 Induces Sarcomeric Degradation Through Cleavage Of The Giant Protein Obscurin

We hypothesized that the 3CL protease (3CLPro) is responsible for the sarcomere degradation observed in cardiomyocytes infected by SARS-COV2. Overexpression of 3CLPro, but not a catalytically inactive mutant, resulted in breakdown of sarcomeres characterized by intact Z-disk/thin filament subunits that has been recently reported (Perez-Bermejo, et al, 2021) in SARS-COV2 infection. To identify potential host protein targets of 3CLPro in an unbiased fashion we screened the human proteome using a cut-site scoring algorithm that we developed. Scoring, ie likelihood of 3CL protease cleavage, was based off experimental data (Chuck et al, 2010) previously published on the highly homologous (96%) 3CL protease from SARS-COV. This scoring was followed by refinement by secondary structure prediction to identify cut-sites that lie in unstructured regions that are thus thus more likely to be accessible to the protease. Using this method, we identified >1000 potential high-likelihood cut sites across the proteome. Further filtering by proteins with cardiomyocyte expression showed 5 high-likelihood sites within the giant sarcomeric protein, Obscurin (OBSCN), as well as many other structural and signaling proteins which we experimentally validated. Expression of 3CLPro in IPSC cardiomyocytes resulted in significantly reduced OBSCN staining without alterations in Z-disk, thin filament, or thick filament proteins by both western blot and immunocytochemistry. In addition, imaging showed loss of OBSCN at sites with intact Z-disk/thin filament subunits. Thus we propose that activity of 3CLPro is a significant contributor to sarcomere breakdown in SARS-COV2 infection via degradation of Obscurin.

Abstract P494: Truncated Titin Protein In Dilated Cardiomyopathy

Truncating variations in the gene coding for titin (TTNtv) have been known to cause dilated cardiomyopathy for nearly 20 years. Efforts to detect direct evidence of either haploinsufficiency or dominant negative mechanisms have thus far failed, leaving the mechanism open to controversy. By analyzing a collection of 184 post-transplant human hearts, 22 of which bear TTNtv’s, we show evidence supporting both haploinsufficient (lack of sufficient full length titin to maintain normal cardiomyocyte contractility) and dominant-negative (toxic gain of function due to truncated titin) mechanisms. Using allele specific proteomics as well as epitope specific agarose gel immunoblotting we show that TTNtv are present in human myocardium at the expected molecular weight and bear only the epitopes expected to be present in TTNtv protein. TTNtv associate with the sarcomere bearing insoluble fraction of human myocardium but are more weakly attached to the sarcomere than full length titin, consistent with their lack of thick filament and M-line attachment sites. We further show that DCM hearts bearing TTNtv have less full length titin than non-TTNtv bearing DCM hearts, by both total protein and in ratio to sarcomeric proteins, indicating TTN haploinsufficiency is also present in TTNtv hearts. This unambiguous detection of TTNtv protein in the myocardium of DCM combined with a reduction in full length titin supports a combined dominant negative and haploinsufficient mechanism of pathogenesis of TTNtv induced DCM.

Abstract P505: Rv Sarcomeres From Lv-hfref Patients With Low Papi Have Abnormal Rv Thick Filament Structure

Patients with left heart failure and reduced ejection fraction (HFrEF) have variable RV failure that, if present, drastically worsens outcomes. In a cohort of 21 HFrEF patients from two hospital sites, we have previously shown (Aslam et al, Eur J HF; 2020: volume 23, pages 339-341) that like global function, RV myocyte maximum calcium-activated myocyte tension (T max ) is quite variable (COV 27%). To determine if a relationship between RV myocyte function and indices of RV chamber function exists, we trained a random forest classifier based on 41 clinical variables, including hemodynamic, laboratory, and echocardiographic data, and queried the importance of each. This revealed that the most predictive model for reduced T max was based on the pulmonary artery pulsatility index (PAPi), an established clinical index of RV failure. To gain insight into potential mechanisms for depressed T max in HFrEF patients with a low PAPi, we obtained small angle x-ray diffraction patterns in 5 HFrEF patients with depressed PAPi and T max and compared this to 5 non-failing (NF) controls. The equatorial intensity ratio I(1,1)/I(1,0) was reduced in low T max RV muscle fibers vs. controls (0.250.06 vs. 0.180.02, P<0.0001), suggesting myosin heads are more associated with the thick filament backbone. In meridional reflections, we find a significant decrease in M3 band spacing (14.340.03 nm in NF vs. 14.300.01 nm in HFrEF; P=0.0013) suggesting more myosin heads are in the “OFF” configuration. The latter may underly tension reduction in RV myocytes from failing RV HFrEF patients. Ongoing studies will examine these structural changes in HFrEF patients with a broader range of PAPi and T max to test if this association applies. These findings focus attention on thick filament structural and configuration abnormalities as potential culprits underlying RV disease in HFrEF. Further studies using novel sarcomere enhancers will test if these changes can be remedied, and if so, in which patients.