A Drosophila melanogaster model of diastolic dysfunction and cardiomyopathy based on impaired troponin-T function.
BACKGROUND
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.


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.
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.).
Meera Cozhimuttam
MC
Viswanathan
Gaurav
G
Kaushik
Adam J
AJ
Engler
William
W
Lehman
Anthony
A
Cammarato
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D009202:Cardiomyopathies / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D003971:Diastole; D004195:Disease Models, Animal; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D056966:Excitation Contraction Coupling; D005260:Female; D005838:Genotype; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D008297:Male; D008854:Microscopy, Electron; D018715:Microscopy, Video; D008969:Molecular Sequence Data; D009154:Mutation; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010641:Phenotype; D013599:Systole; D014335:Tropomyosin / Q000378:metabolism; D020107:Troponin T / Q000235:genetics / Q000378:metabolism*; D018754:Ventricular Dysfunction / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D016276:Ventricular Function; D020257:Ventricular Remodeling
D029721:Drosophila Proteins; D006576:Heterocyclic Compounds, 4 or More Rings; D014335:Tropomyosin; D020107:Troponin T; C472645:blebbistatin; D002118:Calcium
Drosophila; myosins; tropomyosin