Question: 

Titin-based myofilament stiffness is an important determinant of myocardial distensibility and diastolic function. Cardiac titin stiffness can be modulated by two main mechanisms: i) alterations in the relative expression levels of the cardiac titin isoforms, N2B (3.0 MDa, stiff) and N2BA (> 3.2 MDa, more compliant), and ii) phosphorylation of elastic I-band domains in both titin isoforms. Importantly, phosphorylation of the N2-B unique sequence (N2-Bus) by cGMP- or cAMP-dependent protein kinases decreases titin stiffness, whereas phosphorylation of the titin PEVK domain by protein kinase C increases stiffness. To what extent titin phosphorylation affects myofilament stiffness in failing hearts had not been investigated in detail. Here we used novel phosphosite-directed antibodies to study the phosphorylation status of titin in isoform pattern-matched samples from human donor and failing hearts. We also investigated potential phosphorylation-induced changes in passive myofilament stiffness.

Methods and Results: 

2% SDS-PAGE was used to analyze titin expression and confirm a similar isoform composition in all samples. Passive force measurements on skinned fiber preparations from human failing hearts showed a significant increase in the passive sarcomere length-tension relationship, compared to donor tissue. Western blot analysis of failing heart samples showed an average reduction in N2-Bus phosphorylation by 28% in N2B-titin and by 7% in N2BA-titin, whereas phosphorylation of the PEVK-domain was increased by 23% in N2B-titin and by 12% in N2BA-titin.

Conclusion: 

In summary, our data indicate that concerted changes in titin phosphorylation levels dynamically modulate myofilament stiffness and may play an important role in the development of diastolic dysfunction in failing hearts.