Modulation of titin-based stiffness by disulfide bonding in the cardiac titin N2-B unique sequence.
The giant protein titin is responsible for the elasticity of nonactivated muscle sarcomeres. Titin-based passive stiffness in myocardium is modulated by titin-isoform switching and protein-kinase (PK)A- or PKG-dependent titin phosphorylation. Additional modulatory effects on titin stiffness may arise from disulfide bonding under oxidant stress, as many immunoglobulin-like (Ig-)domains in titin's spring region have a potential for S-S formation. Using single-molecule atomic force microscopy (AFM) force-extension measurements on recombinant Ig-domain polyprotein constructs, we show that titin Ig-modules contain no stabilizing disulfide bridge, contrary to previous belief. However, we demonstrate that the human N2-B-unique sequence (N2-B(us)), a cardiac-specific, physiologically extensible titin segment comprising 572 amino-acid residues, contains up to three disulfide bridges under oxidizing conditions. AFM force spectroscopy on recombinant N2-B(us) molecules demonstrated a much shorter contour length in the absence of a reducing agent than in its presence, consistent with intramolecular S-S bonding. In stretch experiments on isolated human heart myofibrils, the reducing agent thioredoxin lowered titin-based stiffness to a degree that could be explained (using entropic elasticity theory) by altered extensibility solely of the N2-B(us). We conclude that increased oxidant stress can elevate titin-based stiffness of cardiomyocytes, which may contribute to the global myocardial stiffening frequently seen in the aging or failing heart.
Grützner
Anika
A
Physiology and Biophysics Unit, University of Münster, Münster, Germany.
Garcia-Manyes
Sergi
S
Kötter
Sebastian
S
Badilla
Carmen L
CL
Fernandez
Julio M
JM
Linke
Wolfgang A
WA
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D003198:Computer Simulation; D064211:Connectin; D004220:Disulfides / Q000737:chemistry*; D004548:Elasticity; D004926:Escherichia coli; D006801:Humans; D018625:Microscopy, Atomic Force; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics*; D050505:Mutant Proteins / Q000737:chemistry; D009210:Myofibrils / Q000737:chemistry; D010084:Oxidation-Reduction; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry
D064211:Connectin; D004220:Disulfides; D009124:Muscle Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; C577285:TTN protein, human; D013879:Thioredoxins; D011494:Protein Kinases