Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB027

Poster Communications

Stretch promotes oxidation of elastic I-band titin but not inextensible A-band titin in heart and skeletal muscles

M. Breitkreuz2, Y. Li1, K. Toischer3, L. Leichert4, N. Hamdani2, W. A. Linke1

1. Institute of Physiology II, University of Muenster, Muenster, Germany. 2. Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany. 3. Dept. of Cardiology and Pneumology, University Hospital, Goettingen, Germany. 4. Institute for Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany.


Background. The mechanical function of the giant muscle protein titin is modulated by reversible oxidation. In vitro, titin oxidation is promoted by unfolding of immunoglobulin-like (Ig) domains from titin's spring region. Ig unfolding exposes previously hidden cysteines, which can then be S-glutathiolated (1). This modification hinders Ig-domain refolding, thus altering titin-based stiffness. The unfolded Ig domains risk aggregation causing increased myocardial stiffness, e.g., in failing hearts. However, it is unknown whether titin oxidation occurs in vivo and whether there is differential oxidation of sarcomeric I-band (extensible) and A-band (inextensible) titin. Therefore, we measured in vivo titin oxidation as a function of stretch and oxidative stress. Methods and Results. Titin oxidation was studied in 3 models. Mouse hearts were perfused in a Langendorff apparatus with physiological buffer containing 0.1 mM H2O2 or 1 mM DTT. Mouse skeletal (leg) muscles were stretched ex vivo in presence of 2 mM GSH/0.5 mM diamide (oxidant) and compared to non-stretched muscle. The third model was the aorto-caval shunt mouse heart, which is under chronic volume overload (preload-increase) and develops oxidative stress (2); sham-operated animals served as controls. Titin oxidation was quantified by isotope-coded affinity tag labelling followed by mass spectrometry. In all models, oxidative stress significantly increased the ratio of oxidized (GSSG) to reduced (GSH) glutathione. Several hundred cysteines in titin became more oxidized under oxidative stress. I-band and A-band titin sites showed similar increases in oxidation level under non-stretch conditions. However, stretch or preload-increase under oxidative stress increased the proportion of oxidized cysteines more in I-band titin than in A-band titin, in both shunt:sham hearts and stretched:non-stretched leg muscles. This differential oxidation was not observed in hearts perfused in the Langendorff apparatus, probably because of insufficient stretching. Several Ig domains from elastic titin found to be preferentially oxidized were recombinantly expressed. Thermal unfolding of these Ig domains, followed by oxidation, consistently resulted in increased aggregation. Interestingly, the aggregation could be prevented by the presence of chaperone αB-crystallin, which is known to bind to titin Ig domains (3). Conclusion. Titin oxidation occurs in vivo and elastic titin becomes relatively more oxidized than A-band titin following a stretch. This effect is a consequence of the increased Ig-domain unfolding in the stretched state. Unfolded, oxidized titin is prone to aggregation, which can be prevented by αB-crystallin. Understanding the mechanisms of oxidative stress-induced titin modifications may help design strategies for the treatment of heart failure patients with overly stiff hearts.

Where applicable, experiments conform with Society ethical requirements