Introduction: In striated muscle the filament protein titin (3-3.7 MDa) spans a half sarcomere from Z-disc to M-line and acts as an important scaffold protein. With its elastic I-band region it also serves as a molecular spring whose stiffness defines myofilament extensibility. In the heart, titin thereby largely determines ventricular stiffness and diastolic properties. After myocardial infarction, increased passive stiffness of titin, mediated by increased PKCα-dependent phosphorylation in the I-band section, plays an important role in early functional adaptation of the viable myocardium. In order to maintain sarcomere function in such situations with increased mechanical strain, protein quality control and subsequent turnover of sarcomeric proteins have to be accurately managed. Recent data indicate that the proteasomal and the autophagosomal-lysosomal system participate in degradation of titin. Here, we studied the effects of ischemia and reperfusion (IR) on titin modification and the mechanisms involved in protein quality control. To this end, we established a cell culture model of IR to reproduce posttranslational modification in titin I-band, detected in in vivo and ex vivo models of myocardial infarction.

Methods: Hearts from adult Wistar rats were quickly excised and washed in perfusion buffer before digestion by retrograde perfusion with collagenase type II. Isolated cardiomyocytes were cultured for 24 h before simulation of ischemia (sI) was performed in a hypoxic chamber for 45 min or 16 h followed by simulated reperfusion (sR) for 2 h or 4 h. Hypoxia was induced by floating the chamber with 1% O2, 5% CO₂ and 94% N₂. During ischemia cardiomyocytes were cultured in ischemic buffer (125 mM NaCl, 8 mM KCl, 1.2 mM KH₂PO₄, 1.25 mM MgSO₄, 1.2 mM CaCl₂, 6.25 mM NaHCO₃, 20 mM HEPES, 5 mM sodium L-lactate, pH = 6.6) or control buffer (110 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.25 mM MgSO₄, 1.2 mM CaCl₂, 25 mM NaHCO₃, 20 mM HEPES, 15 mM glucose, pH = 7.4). Reperfusion was performed in culture medium. Western blot analyses were performed to analyze titin phosphorylation (S11878, S12022), titin ubiquitination, kinase activities and proteasomal and autophagy associated proteins. Statistical significance was tested using Student`s t-test. P<0.05 was set as threshold of significance.

Results: Already after 45 min of sI, relative titin ubiquitination significantly decreased (sI: -18.84% ± 5.00%), suggesting increased titin turnover. After 2 h of sR relative titin phosphorylation at S11878 significantly increased (sR 2 h: +16.54 ± 4.02%), but no significant activation of PKCα was determined. After 16 h of sI, the relative phosphorylation of titin at S11878 and S12022 and of PKCα remained unchanged. After 16 h of sI and after 4 h of sR significant decreases in relative titin ubiquitination (sI: -47.37 ± 7.83 %; sR 4 h: -37.85 ± 10.89 %), p62 level (sI: -36.29 ± 6.57%; sR 4 h: -39.51 ± 7.69) and LC3-II level (sI: -26.01 ± 8.87%; sR 4 h: -48.05 ± 6.86) were observed, indicating increased levels of titin degradation and autophagosomal activity during longer periods of ischemia.

Conclusion: Simulated ischemia had minor effects on titin I-band phosphorylation but had a strong impact on protein quality control mechanisms. The decrease in relative titin ubiquitination level and autophagy associated proteins indicates that ischemia induced higher titin turnover via the autolysosomal system.