Cardiac Titin Research Articles

Structural basis of domain destabilization by dilated-cardiomyopathy-causing missense mutations in titin.

Truncating variants in the titin gene are the most common cause of familial dilated cardiomyopathy (DCM), accounting for 25% of the genotype-positive cases. Similar to other genetic diseases triggered by gene loss of function, it has been proposed that destabilizing titin missense mutations can also result in DCM. Indeed, the highly destabilizing C3575S mutation targeting immunoglobulin-like (Ig) 21 domain of cardiac titin has been recently identified as a cause of DCM. However, the effects of the mutation on the structure of the parent domain and whether altered disulfide bond formation contribute to the destabilizing properties of the mutation remain unexplored. Here, using high-resolution X-ray crystallography, we report that the structure of the C3575S mutant domain is largely unperturbed (RMSD: 0.538 Å). In addition, using gel-shift assays, we have ruled out that the lower thermal stability of the mutant domain results from its inability to establish intramolecular disulfide bonds. Instead, we propose that the lower hydrophobicity of serine is not well accommodated in the core of mutant domains, causing their destabilization. Finally, we have observed that similar missense mutations, which cause a decrease in hydrophobicity in buried positions of titin Ig domains, are enriched in DCM patients. Altogether, our results illustrate that subtle physicochemical changes induced by titin missense mutations can cause domain destabilization, even in the absence of remarkable structural alterations, resulting in development of DCM.

Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease.

Identification of the domains within the N2A region of titin that regulate binding to actin

Titin, the largest muscle protein, plays an important role in passive tension, sarcomeric integrity and cell signaling within the muscle. Recent work has also highlighted a role for titin in active muscle and the N2A region found in skeletal muscle titin and in some isoforms of cardiac titin has been linked to this function. The N2A region is a multi-domain region composed of four immunoglobulin domains (I80–I83) and a disordered region called the insertion sequence. Previously, our lab has shown that the N2A region binds F-actin in a calcium dependent manner, but it is not known which domains within this region are critical for this binding to occur. In this work, we have used co-sedimentation to demonstrate that only constructs containing the I80 domain are capable of binding F-actin. In addition, binding was only observed in constructs containing at least 3 immunoglobulin domains suggesting a length-dependence to binding. Finally, the calcium-dependence of N2A binding is lost when I83 is not present, consistent with the calcium stabilization that has been reported for this domain. Based on these results, we propose that I80 is critical for initiating binding to F-actin and that I83 is responsible for the calcium dependence.

E3-ligase knock down revealed differential titin degradation by autophagy and the ubiquitin proteasome system

The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.

Increased Expression of N2BA Titin Corresponds to More Compliant Myofibrils in Athlete's Heart.

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete’s heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin’s role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete’s heart. The athlete’s heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete’s heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

Single-Molecule Force Spectroscopy Studies of Missense Titin Mutations That Are Likely Causing Cardiomyopathy.

The giant muscle protein titin plays important roles in heart function. Mutations in titin have emerged as a major cause of familial cardiomyopathy. Missense mutations have been identified in cardiomyopathy patients; however, it is challenging to distinguish disease-causing mutations from benign ones. Given the importance of titin mechanics in heart function, it is critically important to elucidate the mechano-phenotypes of cardiomyopathy-causing mutations found in the elastic I-band part of cardiac titin. Using single-molecule atomic force microscopy (AFM) and equilibrium chemical denaturation, we investigated the mechanical and thermodynamic effects of two missense mutations, R57C-I94 and S22P-I84, found in the elastic I-band part of cardiac titin that were predicted to be likely causing cardiomyopathy by bioinformatics analysis. Our AFM results showed that mutation R57C had a significant destabilization effect on the I94 module. R57C reduced the mechanical unfolding force of I94 by ∼30–40 pN, accelerated the unfolding kinetics, and decelerated the folding. These effects collectively increased the unfolding propensity of I94, likely resulting in altered titin elasticity. In comparison, S22P led to only modest destabilization of I84, with a decrease in unfolding force by ∼10 pN. It is unlikely that such a modest destabilization would lead to a change in titin elasticity. These results will serve as the first step toward elucidating mechano-phenotypes of cardiomyopathy-causing mutations in the elastic I-band.

Unusual Mobility of the Elastic Protein Titin in Thirteen-Lined Ground Squirrel Hearts

Small mammalian hibernators, such as thirteen-lined ground squirrels, undergo rapid, 100-fold reductions in heart rate, leading to a substantial prolongation of the cardiac cycle when entering torpor. Initial studies using high Urea solubilization buffers and Vertical Agarose Electrophoresis suggest no seasonally dependent change in the ratio of the longer, compliant N2BA to the shorter, stiff N2B cardiac titin isoform (N2BA:N2B). However, an unusually large protein was observed. Previously, this band was observed in preparations using urea-free solubilization buffers and agarose-acrylamide gel systems.

Evaluation of cardiac titin expression after long-term exercise in a rat model

Abstract Introduction The main role of the giant elastic protein titin is to provide passive stiffness to striated (i.e. cardiac and skeletal) muscles. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B isoforms. Titin reduces passive stiffness in cardiac muscle by increased expression of the more compliant N2BA isoform (elevated N2BA:N2B ratio). Moreover, decreased passive stiffness is associated with increased exercise tolerance. Long-term exercise induces physiological adaptation of the heart, termed athlete's heart. Currently, there is limited data of titin's role in the athlete's heart. Aims Our aim is to evaluate exercise-induced morphological and functional changes of the heart. Furthermore, to determine the N2BA:N2B ratio in the rat model of athlete's heart. Methods Rats were divided into exercised (n=12) and control (n=12) groups. Athlete's heart was induced by a 12-week-long swim training (200min/day). The control group swam 5 min/day. Following the training period cardiac changes were assessed by echocardiography. In vivo cardiac function was examined by left ventricular (LV) pressure-volume (P-V) analysis. Titin isoform expressions were detected by sodium-dodecyl-sulfate (SDS)-agarose gel electrophoresis. Results Echocardiography and post-mortem measured cardiomyocyte diameters confirmed athlete's heart and LV hypertrophy in exercised rats. P-V analysis showed improved contractility, active relaxation and mechanoenergetics in the exercised group. The N2BA:N2B titin ratio was significantly increased in exercised rats compared to controls (0.28 vs. 0.21, p<0.05). Conclusions Our results confirm the morphological and functional changes of the athlete's heart. The increased ratio of N2BA:N2B titin corresponds to a more compliant heart in the exercised rats. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Research, Development and Innovation Office of Hungary (K 120277) and the ÚNKP-19-3-I New National Excellence Program of The Ministry For Innovation and Technology.

Matrix metalloproteinase-12 cleaved fragment of titin as a predictor of functional capacity in patients with heart failure and preserved ejection fraction

Objectives Serum levels of matrix metalloproteinase-12 cleaved fragment of titin (TIM), a novel circulatory biomarker specific for cardiac titin degradation, has emerged as a potential biomarker in cardiovascular diseases. In this work, we aimed to evaluate the association between TIM and maximal functional capacity assessed by the percentage of predicted peak exercise oxygen uptake (pp-peakVO2) in patients with heart failure and preserved ejection fraction (HFpEF). Design. In this post-hoc study, we included 46 stable symptomatic (New York Heart Association II-III) HFpEF patients enrolled in the TRAINING-HF study (NCT02638961). pp-peak-VO2 was calculated from baseline values. Baseline circulating levels of TIM were measured by competitive ELISA in serum from the TRAINING-HF patients. The independent association between TIM and pp-peakVO2 was evaluated by multivariate linear regression analysis. Results. The mean age of the sample was 73.8 ± 8.7 years, 56.5% were females, and 76.1% were on NYHA II. The medians of pp-peakVO2 and TIM were 60.9% (50.4–69.3), and 130.1 ng/mL (98.1–159.5), respectively. The median of NT-proBNP was 912 pg/mL (302–1826). pp-peakVO2 was significant and inversely correlated with TIM (r= −41, p = .005). In multivariate analysis, after adjusting for NYHA class, hypertension, body mass index, and glomerular filtration rate, higher TIM was significantly associated with lower pp-peak VO2 (p = .029). Conclusions. In this sample of stable and symptomatic HFpEF patients, higher serum levels of TIM identified patients with worse functional status.

Residual and passive force enhancement in skinned cardiac fibre bundles

In skeletal muscle, steady-state force is consistently greater following active stretch than during a purely isometric contraction at the same length (residual force enhancement; RFE). Similarly, when deactivated, the force remains higher following active stretch than following an isometric condition (passive force enhancement; PFE). RFE and PFE have been associated with the sarcomere protein titin, but skeletal and cardiac titin have different structures, and results regarding RFE in cardiac muscle have been inconsistent and contradictory. Therefore, the purpose of this study was to determine if cardiac muscle exhibits RFE and PFE. Skinned fibre bundles (n = 10) were activated isometrically at a sarcomere length of 2.2 μm and actively stretched by 15% of their length. The resultant active and passive forces were compared to the corresponding forces obtained for purely isometric contractions at the long length. RFE was observed in all fibre bundles, averaging 5.5 ± 2.5% (ranging from 2.3 to 9.4%). PFE was observed in nine of the ten bundles, averaging 11.1 ± 6.5% (ranging from −2.1 to 18.7%). Stiffness was not different between the active isometric and the force enhanced conditions, but was higher following deactivation from the force-enhanced compared to the isometric reference state. We conclude that there is RFE and PFE in cardiac muscle. We speculate that cardiac muscle has the same RFE capability as skeletal muscle, and that the most likely mechanism for the RFE and PFE is the engagement of a passive structural element during active stretching.

CaMKIIδ differentially alters the passive mechanics of myocardium

Abnormal diastolic function as seen in failing hearts with preserved ejection fraction (HFpEF) has been linked to alterations in the passive material property of myocardium. Specifically, the non‐crossbridge forming protein named titin contributes a stiffness that varies with fluctuating calcium concentration and has been thought to dominate the passive stiffness of myocardium throughout normal physiological range.Titin is expressed in two isoforms in the heart: the N2B, which has a single spring‐like element, and a more compliant isoform, N2BA, which contains two spring‐like domains (the N2B and N2A). The N2B element exists in all isoforms and is cardiac specific and consists of a large 575 residue unique sequence (N2B‐Us), three flanking immunoglobulin domains, and two other smaller unique sequences. Titin‐based stiffness of myocardium has been shown to be modulated by altered ratio expression of titin isoforms as well as post‐translational modification with unbalanced phosphorylation of cardiac titin characterizing HFpEF. Several proteins can alter titin stiffness including protein kinase (PK) G, extracellular signal‐regulated kinase 2 (ERK‐2), PKC, PKA and calcium calmodulin‐dependent protein kinase II (CaMKII) with varied effects depending on the target site location on titin.CaMKII is a dodecameric enzyme consisting of subunits encoded by four different genes known as CaMKII α,β,γ, and δ. CaMKIIδ is the predominant isoform in the heart and is alternatively spliced in the heart to generate δb and δc differing only by an 11‐amino acid nuclear localization signal. CaMKIIδ heteromultimerization is permissive, however, in that the enzyme may contain subunits from multiple CaMK genes and splice variants of those genes. Thus, it is hypothesized that the general ratio of CaMKIIδ subtypes determine its exact localization within the cardiomyocyte.CaMKIIδ knock‐out has recently been shown to ameliorate heart failure development in response to pressure overload, Gαq expression, and isoproterenol infusion while CaMKIIδ subtypes show a differential regulation of calcium handling in‐vitro and infarct formation following ex‐vivo myocardial ischemia/reperfusion. The idea that the specific CaMKIIδ subtypes differentially regulate cardiac mechanics is interesting yet remains unresolved.In our work, 5–6 weeks old mice models overexpressing CaMKIIδb and CaMKIIδc were bred against a CaMKIIδ knock‐out background, called KOB and KOC respectively. Results indicate that KOB mice show increased passive stiffness (12 mN/mm^2 +− 3 mN/mm^2) compared to KOC passive stiffness (6 mN/mm^2 +− 1 mN/mm^2) at 5–6 weeks prior to heart failure phenotype. Furthermore, as the mice progress into heart failure, the heart passive mechanics of KOB and KOC are consistent, thus, highlighting CaMKII as a precursor to heart failure development.Support or Funding InformationPresident's Disseration Year FellowshipFigure 1

MMP inhibitors attenuate doxorubicin cardiotoxicity by preventing intracellular and extracellular matrix remodelling.

Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix. Eight-week-old male C57BL/6J mice were treated with DXR weekly with or without MMP inhibitors doxycycline or ONO-4817 by daily oral gavage for 4 weeks. Echocardiography was used to determine cardiac function and left ventricular remodelling before and after treatment. MMP inhibitors ameliorated DXR-induced systolic and diastolic dysfunction by reducing the loss in left ventricular ejection fraction, fractional shortening, and E'/A'. MMP inhibitors attenuated adverse left ventricular remodelling, reduced cardiomyocyte dropout, and prevented myocardial fibrosis. DXR increased myocardial MMP-2 activity in part also by upregulating N-terminal truncated MMP-2. Immunogold transmission electron microscopy showed that DXR elevated MMP-2 levels within the sarcomere and mitochondria which were associated with myofilament lysis, mitochondrial degeneration, and T-tubule distention. DXR-induced myofilament lysis was associated with increased titin proteolysis in the heart which was prevented by ONO-4817. DXR also increased the level and activity of MMP-2 in human embryonic stem cell-derived cardiomyocytes, which was reduced by ONO-4817. MMP-2 activation is an early event in DXR cardiotoxicity and contributes to myofilament lysis by proteolyzing cardiac titin. Two orally available MMP inhibitors ameliorated DXR cardiotoxicity by attenuating intracellular and extracellular matrix remodelling, suggesting their use may be a potential prophylactic strategy to prevent heart injury during chemotherapy.

Compliant Titin Isoform Content Is Reduced in Left Ventricles of Sedentary Versus Active Rats.

A sedentary lifestyle is associated with increased cardiovascular risk factors and reduced cardiac compliance when compared to a lifestyle that includes exercise training. Exercise training increases cardiac compliance in humans, but the mechanisms underlying this improvement are unknown. A major determinant of cardiac compliance is the compliance of the giant elastic protein titin. Experimentally reducing titin compliance in animal models reduces exercise tolerance, but it is not known whether sedentary versus chronic exercise conditions cause differences in titin isoform content. We hypothesized that sedentary conditions would be associated with a reduction in the content of the longer, more compliant N2BA isoform relative to the stiffer N2B isoform (yielding a reduced N2BA:N2B ratio) compared to age-matched exercising controls. We obtained left ventricles from 16-week old rats housed for 12 weeks in standard (sedentary) or voluntary running wheel (exercised) housing. The N2BA:N2B ratio was decreased in the hearts of sedentary versus active rats (p = 0.041). Gene expression of a titin mRNA splicing factor, RNA Binding Motif 20 protein (RBM20), correlated negatively with N2BA:N2B ratios (p = 0.006, r = −0.449), but was not different between groups, suggesting that RBM20 may be regulated post-transcriptionally. Total phosphorylation of cardiac titin was not different between the active and sedentary groups. This study is the first to demonstrate that sedentary rats exhibit reduced cardiac titin N2BA:N2B isoform ratios, which implies reduced cardiac compliance. These data suggest that a lack of exercise (running wheel) reduces cardiac compliance and that exercise itself increases cardiac compliance.

Seipin Knockout Mice Develop Heart Failure With Preserved Ejection Fraction

The lean diabetic patients with heart failure with preserved ejection fraction (HFpEF) in Asia suffer from adverse clinical outcomes and poor life quality. The suitable animal models are urgently needed for mechanistic study and therapeutic innovations. Our study reports that lipodystrophic mice with seipin depletion are lean, diabetic, and recapitulate major manifestations of clinical HFpEF, thereby clarifying that lean diabetes per se may produce HFpEF characteristics. We further demonstrate that increased cardiac titin phosphorylation and reactive interstitial fibrosis associated with neutrophil extracellular traps lead to left ventricular stiffness and suggest that both pathways may be potential therapeutic targets in Asian HFpEF patients.

Posttranslational modifications of titin from cardiac muscle: how, where, and what for?

Titin is a giant elastic protein expressed in the contractile units of striated muscle cells, including the sarcomeres of cardiomyocytes. The last decade has seen enormous progress in our understanding of how titin molecular elasticity is modulated in a dynamic manner to help cardiac sarcomeres adjust to the varying hemodynamic demands on the heart. Crucial events mediating the rapid modulation of cardiac titin stiffness are post‐translational modifications (PTMs) of titin. In this review, we first recollect what is known from earlier and recent work on the molecular mechanisms of titin extensibility and force generation. The main goal then is to provide a comprehensive overview of current insight into the relationship between titin PTMs and cardiomyocyte stiffness, notably the effect of oxidation and phosphorylation of titin spring segments on titin stiffness. A synopsis is given of which type of oxidative titin modification can cause which effect on titin stiffness. A large part of the review then covers the mechanically relevant phosphorylation sites in titin, their location along the elastic segment, and the protein kinases and phosphatases known to target these sites. We also include a detailed coverage of the complex changes in phosphorylation at specific titin residues, which have been reported in both animal models of heart disease and in human heart failure, and their correlation with titin‐based stiffness alterations. Knowledge of the relationship between titin PTMs and titin elasticity can be exploited in the search for therapeutic approaches aimed at softening the pathologically stiffened myocardium in heart failure patients.

Influenza and sudden unexpected death: the possible role of peptide cross-reactivity

Abstract This study investigates the hypothesis that cross-reactions may occur between human cardiac proteins and influenza antigens, thus possibly representing the molecular mechanism underlying influenzaassociated sudden unexpected death (SUD). Using titin protein as a research model, data were obtained on (1) the occurrence of the titin octapeptide AELLVLLE or its mimic AELLVALE in influenza A virus hemagglutinin (HA) sequences; (2) the immunological potential of AELLVLLE and its mimic AELLVALE; (3) the possible role of the flanking amino acid aa) context of the two octapeptide determinants in eliciting cross-reactivity between the human cardiac titin protein and HA antigens.

Abstract 440: The Cardiomyocyte Diastolic Stiffness and Contractility are Inversely Related to the Size of Titin: A Cellular Work Loop Study of Single Intact Cardiomyocytes

Studying cardiac titin under physiological conditions can be at the level of the left ventricular (LV) chamber and the multicellular muscle level. However, those preparations are influenced by neurohormonal reflex, preload-afterload status and extracellular matrix. In this study, cellular work loop force measurement of the single intact cardiomyocyte was conducted under physiological conditions. Intact cell experiments were performed at 37°C under field-stimulation at 2 Hz frequency. The cell was attached to the force transducer and the piezo length controller which modulates the cell length by the feedback control system based on developed force. The force length relation (cellular work loop) of the single cell was generated. The end diastolic force length relation (EDFLR), the end systolic force length relation (ESFLR) and the preload recruitable stroke work (PRSW) were obtained. The Ttn ΔIAjxn -/- ( IA -/- ) mice, in which the I-band/A-band region of titin’s spring has been deleted were studied in comparison to α-MHC-Cre ; cRbm20 ΔRRM +/- ( cRbm20 +/- ) mice which are deficient in titin splicing and express large titin isoforms in the heart. The cellular diastolic stiffness (slope of EDFLR) was increased in IA -/- and was reduced in cRbm20 +/- (151.3, 104.0 and 91.5 μN/mm 2 in IA -/- , WT and cRbm20 +/- respectively). The contractility (slopes of ESFLR and PRSW) were 233.5 and 1215.6 uN/mm 2 in WT, and were increased in IA -/- (371.2 and 2076 μN/mm 2 ) but attenuated in cRbm20 +/- (202.9 and 907.7 μN/mm 2 ). Cellular work loop studies were also conducted in intact cells isolated from the mice that underwent transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) implantation. TAC DOCA mice developed diastolic dysfunction at the LV chamber level. The result showed that the cells from TAC DOCA hearts had increased slopes of EDFLR, ESFLR and PRSW ( 157.9, 378.9 and 1699.0 μN/mm 2 ) compared to the cells from sham mice (98.6, 230.3, and 1203.9 μN/mm 2 ). Conclusions: Cellular work loops recapitulated the cardiac physiology at the LV chamber level. The result showed that the cellular diastolic stiffness and contractility were inversely related to the size of titin and that titin stiffness plays an important roles in both diastolic and systolic function.

Matrix Metalloproteinase Inhibitors Attenuate Doxorubicin‐Induced Heart Failure by Preventing Cardiac Titin Proteolysis

Heart failure remains a major, long‐term complication with doxorubicin (DXR) cancer chemotherapy. We investigated the mechanism of DXR cardiotoxicity in order to develop new strategies to prevent heart injury when treating cancer patients. The cardiotoxicity of DXR is associated with increased oxidative stress in cardiomyocytes. Intracellular matrix metalloproteinase‐2 (MMP‐2) activity is enhanced by oxidative stress by two means: a) direct activation of 72 kDa MMP‐2 by peroxynitrite‐mediated S‐glutathiolation and, b) de novo expression of an N‐terminal truncated isoform (NTT‐MMP‐2). We have shown that MMP‐2 is enriched in the Z‐disc of the sarcomere where it cleaves titin following oxidative stress caused by ischemia‐reperfusion injury. We determined whether MMP‐2 mediated titin proteolysis contributes to DXR‐induced heart failure in vivo. 8‐week old male C57BL/6J mice were treated with DXR weekly (6 mg/kg, i.p.) or saline vehicle (control) ± MMP inhibitors (by daily gavage) doxycycline (15 mg/kg) or ONO‐4817 (60 mg/kg) for 4 weeks. Cardiac function was assessed by M‐mode echocardiography before and after treatment (n=10 mice per group) and then the hearts were collected for biochemical analysis. MMP‐2 mRNA expression, protein levels, and activity were measured by qPCR, Western blot and gelatin zymography, respectively. N2BA and N2B titin isoforms and the primary titin degradation product levels were measured by agarose gel electrophoresis. DXR caused systolic and diastolic dysfunction marked by a significant reduction in left ventricular ejection fraction (64±2% vs. 45±2%, p<0.05), fractional shortening (35±2% vs. 22±1%, p<0.05), and E′/A′ ratio (1.07±0.04 vs. 0.85±0.06, p<0.05) compared to control. Doxycycline or ONO‐4817 prevented these changes. DXR caused significant cardiac remodeling including the thinning of the posterior wall (0.99±0.03 vs 1.16±0.04 mm, p<0.05) and increased left ventricular internal diameter (2.93±0.09 vs 2.55±0.09 mm, p<0.05) during systole, an effect that was prevented by doxycycline, but not ONO‐4817. DXR upregulated NTT‐MMP‐2 mRNA in the heart to 195±16% (p<0.05) of control and increased MMP‐2 protein levels (149±19% vs. control, p<0.05), accompanied by a trend to increased MMP‐2 gelatinolytic activity (200±14% vs. control, p=0.06). These biochemical changes were prevented by doxycycline or ONO‐4817. DXR did not alter cardiac titin isoform expression (N2BA:N2B) in the heart but increased titin degradation (T2/T1 ratio) to 258±7% (p<0.05) which was reduced by ONO‐4817 (169±10%) but not doxycycline (203±16%). MMP‐2 activation contributes to DXR‐induced heart failure in mice by proteolyzing cardiac titin in an MMP‐2‐dependent manner. Moreover, two orally available MMP inhibitors prevented heart failure, suggesting that their use may be a potential prophylactic strategy to prevent heart during cancer chemotherapy.Support or Funding InformationCanadian Institutes of Health Research (to RS); Women and Children's Health Research Institute Graduate Studentship (to BC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanical responses of the mechanosensitive unstructured domains in cardiac titin.

Titin is one of the three main filaments in cardiac sarcomere. Besides a chain of Ig domains, cardiac titin also contains a proline (P), glutamate (E), valine (V), lysine (K) (PEVK) domain and a cardiac-specific N2B domain, both are largely unstructured. While they are believed to be involved in the elastic (PEVK and N2B) and the trophic (N2B) functions of the heart, their mechanical responses in physiological level of forces remains poorly understood. In order to gain understanding on their mechanical responses, we used magnetic tweezers to investigate their force responses from 1 to 30pN. We confirmed that in vitro the PEVK domain is intrinsically disordered within the force range. Surprisingly, we discovered a mechanosensitive folded element in the disordered region of N2B, ∼84 amino acids in length, which has a large folding energy of approximately -10 kB T. Based on the force responses of PEVK and N2B domains, as well as an approximated force-dependent unfolding and refolding rates of titin Ig domains, we show that the tension in cardiac titin fluctuates within 5pN during cardiac contraction and extension cycle using Gillespie simulation algorithm. Exceptionally, the simulation shows that deletion of N2B domain results in 10-fold increase in peak force. Our results highlight a critical role that N2B may potentially play in regulating tension on cardiac titin. The study provides new insights into the tension regulatory role of unstructured domains in the elastic function of the heart, which has broad implication in diastolic dysfunction and cardiac trophic mechanisms. In addition, the method can be applied to probing other unstructured mechanosensitive proteins/domains.

The Important Roles of Water in Protein Folding: an Approach by Single Molecule Force Spectroscopy

The single-chain elasticity of a completely unfolded protein ((I27)8, modules of human cardiac titin) is studied in different liquid environments by the atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS). The experimental results show that there is a clear deviation between the force curves obtained in the aqueous and nonaqueous environments. Such a deviation can be attributed to the additional energy consumed by the rearrangement of the bound water molecules around the chain of the completely unfolded (I27)8 chain upon stretching in aqueous solution, which is very similar to the partial dehydration process from a denatured/unfolded to a native/folded protein. Through the analysis of the free energy changes involved in protein folding, we conclude that it is due to the weak disturbance of water molecules and the special backbone structures of proteins that the self-assembly of proteins can be achieved in physiological conditions. We speculate that water is likely to be an important criterion for the selection of self-assembling macromolecules in the prebiotic chemical evolution.