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TNNT2 Mutations in the Tropomyosin Binding Region of TNT1 Disrupt Its Role in Contractile Inhibition and Stimulate Cardiac Dysfunction

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TNNT2 Mutations in the Tropomyosin Binding Region of TNT1 Disrupt Its Role in Contractile Inhibition and Stimulate Cardiac Dysfunction TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction Aditi Madana, Meera C. Viswanathana, Kathleen C. Woulfeb, William Schmidta, Agnes Sidora, Ting Liua, Tran H. Nguyena, Bosco Trinhc, Cortney Wilsonb, Sineej Madathild, Georg Voglerc, Brian O’Rourkea, Brandon J. Biesiadeckie,f, Larry S. Tobacmand, and Anthony Cammaratoa,g,1 aDepartment of Medicine, Division of Cardiology, Johns Hopkins University, Baltimore, MD 21205; bDepartment of Medicine, Division of Cardiology, University of Colorado Denver, Aurora, CO 80045; cDevelopment, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037; dDepartment of Medicine, University of Illinois College of Medicine, Chicago, IL 60612; eDepartment of Physiology and Cell Biology, The Ohio State University, Columbus, OH 43210; fThe Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210; and gDepartment of Physiology, Johns Hopkins University, Baltimore, MD 21205 Edited by Edwin W. Taylor, The University of Chicago, Chicago, IL, and approved June 15, 2020 (received for review January 28, 2020) Muscle contraction is regulated by the movement of end-to-end- “closed” C-state position, which partially uncovers myosin binding linked troponin−tropomyosin complexes over the thin filament sur- sites. Subsequent binding of a small population of myosin cross- face, which uncovers or blocks myosin binding sites along F-actin. bridges further displaces Tpm to an “open” M-state position, The N-terminal half of troponin T (TnT), TNT1, independently pro- completely exposing neighboring binding sites, leading to coop- motes tropomyosin-based, steric inhibition of acto-myosin associa- erative, full filament activation. tions, in vitro. Recent structural models additionally suggest TNT1 This three-state model of contractile regulation is strongly sup- may restrain the uniform, regulatory translocation of tropomyosin. ported by biochemical and structural data (3, 4). However, it was Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of developed in the absence of high-resolution structures of thin this noncanonical role remain unclear. Impaired relaxation is a hall- filament-bound Tn, and therefore the model has been incomplete in mark of hypertrophic and restrictive cardiomyopathies (HCM and important respects. Fortunately, a great deal of Tn’s structure on PHYSIOLOGY RCM). Investigating the effects of cardiomyopathy-causing muta- the thin filament has recently been revealed for the first time, via an tions could help clarify TNT1’s enigmatic inhibitory property. We incisive cryo-electron microscopy (cryo-EM) study by Yamada et al. tested the hypothesis that coupling of TNT1 with tropomyosin’s (9). Tn’s effects on the shifting position of Tpm along the regulated end-to-end overlap region helps anchor tropomyosin to an inhibi- thin filament are markedly more apparent. Three Tn regions—1) tory position on F-actin, where it deters myosin binding at rest, and an extended TnI C terminus, 2) the Tn tail (i.e., the TnT N-terminal that, correspondingly, cross-bridge cycling is defectively suppressed region), and 3) the Tn core domain (consisting of parts of all three under diastolic/low Ca2+ conditions in the presence of HCM/RCM subunits)—all interact directly with F-actin−Tpmsoastoinfluence lesions. The impact of TNT1 mutations on Drosophila cardiac perfor- mance, rat myofibrillar and cardiomyocyte properties, and human Tpm position. Furthermore, these regions seemingly have po- TNT1’s propensity to inhibit myosin-driven, F-actin−tropomyosin sitions on F-actin that, depending upon conditions, may impact motility were evaluated. Our data collectively demonstrate that re- moving conserved, charged residues in TNT1’s tropomyosin-binding Significance domain impairs TnT’s contribution to inhibitory tropomyosin posi- tioning and relaxation. Thus, TNT1 may modulate acto-myosin ac- Roughly two decades ago, troponin T (TnT) of the trimeric thin − tivity by optimizing F-actin tropomyosin interfacial contacts and by filament regulatory troponin complex, was shown to contribute ’ binding to actin, which restrict tropomyosin s movement to activating directly to the inhibition of skeletal and cardiac muscle contrac- ’ configurations. HCM/RCM mutations, therefore, highlight TNT1 ses- tion, independent of troponins I and C. The molecular basis of sential role in contractile regulation by diminishing its tropomyosin- this enigmatic role for TnT remains unknown. Hypertrophic and anchoring effects, potentially serving as the initial trigger of pathol- restrictive cardiomyopathies (HCM and RCM) are characterized ogy in our animal models and humans. by impaired relaxation and, thus, an inability of the heart muscle to properly “turn off.” Based on data gleaned from multiple troponin T | tropomyosin | Drosophila | cardiomyopathy | diastolic model systems used to investigate HCM and RCM TnT muta- dysfunction tions, and recently published thin filament structural models, we propose a mechanism that potentially underlies TnT’s heretofore triated muscle contraction is regulated by Ca2+- and myosin- poorly understood role in muscle relaxation, which, when com- Sdependent changes in the location of troponin (Tn) and promised, may cause disease. tropomyosin (Tpm) over the surface of the actin-based thin filament (1–4). Tn consists of a Ca2+ binding subunit, TnC, an inhibitory Author contributions: A.M., K.C.W., W.S., T.L., G.V., B.O., B.J.B., L.S.T., and A.C. designed subunit, TnI, and a subunit that connects the complex tightly to research; A.M., M.C.V., K.C.W., A.S., T.L., T.H.N., B.T., C.W., S.M., G.V., B.J.B., and A.C. performed research; A.M., M.C.V., K.C.W., A.S., S.M., G.V., and B.J.B. contributed new Tpm, TnT. Tpm is a semirigid, coiled-coil dimer that binds seven reagents/analytic tools; A.M., M.C.V., K.C.W., W.S., T.L., G.V., B.O., B.J.B., L.S.T., and A.C. successive actin protomers (5, 6). Tpm molecules link end to end to analyzed data; and A.M., K.C.W., W.S., T.L., S.M., B.J.B., L.S.T., and A.C. wrote the paper. form continuous strands that track along the winding, long-pitch The authors declare no competing interest. 2+ F-actin helix (5, 7, 8). In resting muscle, when intracellular Ca This article is a PNAS Direct Submission. is low, Tpm is constrained to an inhibitory, “blocking” B-state po- Published under the PNAS license. sition over myosin binding sites on F-actin (1–4). Hence, contrac- 1To whom correspondence may be addressed. Email: [email protected]. 2+ 2+ tion is suppressed. As Ca rises, Ca -bound TnC draws a TnI This article contains supporting information online at https://www.pnas.org/lookup/suppl/ regulatory region away from actin, releasing a steric constraint on doi:10.1073/pnas.2001692117/-/DCSupplemental. Tpm. This enables azimuthal movement of Tpm across F-actin to a www.pnas.org/cgi/doi/10.1073/pnas.2001692117 PNAS Latest Articles | 1of10 Downloaded by guest on September 25, 2021 myosin binding. To allow contraction, both Tpm and Tn must been investigated in vivo, however, nor have their effects been reposition properly. assessed across a full range of experimental models, and then Significantly for the current report, information regarding the considered in light of the new structural advances (9, 11). conformation of the end-to-end overlap of successive Tpms is Here, we posit that the K124N, R130C, and E136K hcTnT now available from the above work (9), and also from concur- substitutions diminish hcTNT1’s inhibitory properties by com- ring, independent, in silico studies (10, 11). The overlap domain promising its Tpm-anchoring role in contractile regulation, serving has a relatively fixed position on F-actin that approximates the as a trigger for cardiac remodeling. Specifically, we test the hy- Tpm B-state location, whether or not Ca2+ is present (9). Notably, pothesis, based on the aforementioned structural and biochemical the overlap region includes a TnT helix that closely interacts with results (9, 11–13, 18, 19, 23, 25, 34), that the TNT1−Tpm overlap both Tpm ends, and also, potentially, with actin. It thus may help region is essential for relaxation, and that, correspondingly, sup- anchor the overlap domain on actin and restrain its motion during pression of cross-bridge cycling under diastolic/low Ca2+ condi- regulatory repositioning. In light of these findings, the Tn tail, tions is defective when these missense mutations are present. First, from which this helix derives, takes on particular interest and is the we developed several Drosophila melanogaster models to scrutinize subject of the present study. the mutations’ impact on organ-, cellular-, and myofibrillar-level The primary switch regulating muscle contraction involves the function. We next ascertained the effects of the equivalent E136K TnI C terminus and the Tn core domain, which interact with each hcTnT variant on activation and, importantly, relaxation proper- other and with F-actin−Tpm in a Ca2+-sensitive manner. No ties of rat ventricular myofibrils and cardiomyocytes. Finally, we matter its primacy, this switch is considered insufficient for con- employed a reductionist in vitro approach, using hcTNT1 pep- tractile activation; actions of myosin cross-bridges are also re- tides, to identify the immediate and completely Ca2+-independent quired. The switch is also insufficient for inhibition, which requires consequences of all three mutations on hcTNT1−Tpm-mediated other features of Tn, Tpm, and actin (9, 12–17). In particular, the inhibition of contraction. Our data collectively demonstrate that N-terminal half of TnT, otherwise known as TNT1, may play a removing conserved, charged residues in the TNT1−Tpm binding role in relaxation, to a degree that is an open subject of investi- domain impairs TnT’s involvement in inhibitory Tpm positioning, gation. This domain, corresponding to residues 1 to 156 of human muscle relaxation, and diastole across phyla and across multiple cardiac TnT (hcTnT), couples with Tpm through an evolutionarily levels of organization.
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