Meera Cozhimuttam Viswanathan1, Gaurav Kaushik, Adam J Engler, William Lehman, Anthony Cammarato. 1. From the Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD (M.C.V., A.C.); Department of Bioengineering, University of California, San Diego, La Jolla, CA (G.K., A.J.E.); and Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA (W.L.).
Abstract
RATIONALE: Regulation of striated muscle contraction is achieved by Ca2+ -dependent steric modulation of myosin cross-bridge cycling on actin by the thin filament troponin-tropomyosin complex. Alterations in the complex can induce contractile dysregulation and disease. For example, mutations between or near residues 112 to 136 of cardiac troponin-T, the crucial TnT1 (N-terminal domain of troponin-T)-tropomyosin-binding region, cause cardiomyopathy. The Drosophila upheld(101) Glu/Lys amino acid substitution lies C-terminally adjacent to this phylogenetically conserved sequence. OBJECTIVE: Using a highly integrative approach, we sought to determine the molecular trigger of upheld(101) myofibrillar degeneration, to evaluate contractile performance in the mutant cardiomyocytes, and to examine the effects of the mutation on the entire Drosophila heart to elucidate regulatory roles for conserved TnT1 regions and provide possible mechanistic insight into cardiac dysfunction. METHODS AND RESULTS: Live video imaging of Drosophila cardiac tubes revealed that the troponin-T mutation prolongs systole and restricts diastolic dimensions of the heart, because of increased numbers of actively cycling myosin cross-bridges. Elevated resting myocardial stiffness, consistent with upheld(101) diastolic dysfunction, was confirmed by an atomic force microscopy-based nanoindentation approach. Direct visualization of mutant thin filaments via electron microscopy and 3-dimensional reconstruction resolved destabilized tropomyosin positioning and aberrantly exposed myosin-binding sites under low Ca2+ conditions. CONCLUSIONS: As a result of troponin-tropomyosin dysinhibition, upheld(101) hearts exhibited cardiac dysfunction and remodeling comparable to that observed during human restrictive cardiomyopathy. Thus, reversal of charged residues about the conserved tropomyosin-binding region of TnT1 may perturb critical intermolecular associations required for proper steric regulation, which likely elicits myopathy in our Drosophila model.
RATIONALE: Regulation of striated muscle contraction is achieved by Ca2+ -dependent steric modulation of myosin cross-bridge cycling on actin by the thin filament troponin-tropomyosin complex. Alterations in the complex can induce contractile dysregulation and disease. For example, mutations between or near residues 112 to 136 of cardiac troponin-T, the crucial TnT1 (N-terminal domain of troponin-T)-tropomyosin-binding region, cause cardiomyopathy. The Drosophila upheld(101) Glu/Lys amino acid substitution lies C-terminally adjacent to this phylogenetically conserved sequence. OBJECTIVE: Using a highly integrative approach, we sought to determine the molecular trigger of upheld(101) myofibrillar degeneration, to evaluate contractile performance in the mutant cardiomyocytes, and to examine the effects of the mutation on the entire Drosophila heart to elucidate regulatory roles for conserved TnT1 regions and provide possible mechanistic insight into cardiac dysfunction. METHODS AND RESULTS: Live video imaging of Drosophila cardiac tubes revealed that the troponin-T mutation prolongs systole and restricts diastolic dimensions of the heart, because of increased numbers of actively cycling myosin cross-bridges. Elevated resting myocardial stiffness, consistent with upheld(101) diastolic dysfunction, was confirmed by an atomic force microscopy-based nanoindentation approach. Direct visualization of mutant thin filaments via electron microscopy and 3-dimensional reconstruction resolved destabilized tropomyosin positioning and aberrantly exposed myosin-binding sites under low Ca2+ conditions. CONCLUSIONS: As a result of troponin-tropomyosin dysinhibition, upheld(101) hearts exhibited cardiac dysfunction and remodeling comparable to that observed during human restrictive cardiomyopathy. Thus, reversal of charged residues about the conserved tropomyosin-binding region of TnT1 may perturb critical intermolecular associations required for proper steric regulation, which likely elicits myopathy in our Drosophila model.
Authors: Nicholas M P King; Methajit Methawasin; Joshua Nedrud; Nicholas Harrell; Charles S Chung; Michiel Helmes; Henk Granzier Journal: J Gen Physiol Date: 2011-01 Impact factor: 4.086
Authors: Anthony Cammarato; Christian H Ahrens; Nakissa N Alayari; Ermir Qeli; Jasma Rucker; Mary C Reedy; Christian M Zmasek; Marjan Gucek; Robert N Cole; Jennifer E Van Eyk; Rolf Bodmer; Brian O'Rourke; Sanford I Bernstein; D Brian Foster Journal: PLoS One Date: 2011-04-25 Impact factor: 3.240
Authors: Parvin Shahrestani; Molly K Burke; Ryan Birse; James N Kezos; Karen Ocorr; Laurence D Mueller; Michael R Rose; Rolf Bodmer Journal: Physiol Biochem Zool Date: 2016-11-21 Impact factor: 2.247
Authors: Ayla O Sessions; Gaurav Kaushik; Sarah Parker; Koen Raedschelders; Rolf Bodmer; Jennifer E Van Eyk; Adam J Engler Journal: Matrix Biol Date: 2016-10-25 Impact factor: 11.583
Authors: Gaurav Kaushik; Alice Spenlehauer; Ayla O Sessions; Adriana S Trujillo; Alexander Fuhrmann; Zongming Fu; Vidya Venkatraman; Danielle Pohl; Jeremy Tuler; Mingyi Wang; Edward G Lakatta; Karen Ocorr; Rolf Bodmer; Sanford I Bernstein; Jennifer E Van Eyk; Anthony Cammarato; Adam J Engler Journal: Sci Transl Med Date: 2015-06-17 Impact factor: 17.956
Authors: Aditi Madan; Meera C Viswanathan; Kathleen C Woulfe; William Schmidt; Agnes Sidor; Ting Liu; Tran H Nguyen; Bosco Trinh; Cortney Wilson; Sineej Madathil; Georg Vogler; Brian O'Rourke; Brandon J Biesiadecki; Larry S Tobacman; Anthony Cammarato Journal: Proc Natl Acad Sci U S A Date: 2020-07-20 Impact factor: 11.205