Protein kinase-A (PKA) mediated phosphorylation lowers the passive (diastolic) stiffness of cardiomyocytes. Here we show by

2% SDS-PAGE and autoradiography that PKA phosphorylates both the N2B and N2BA titin isoforms in human donor and end- stage failing dilated cardiomyopathy hearts. Phosphorylation signals were generally much stronger when titin was first de- phosphorylated by protein-phosphatase-1 (PP-1), suggesting high inherent titin phosphorylation levels. A recombinant construct comprising the human cardiac N2B-domain was strongly phosphorylated by PKA in vitro, confirming that a primary PKA- responsive element in titin is the 572-residue N2B-unique sequence, which contains up to five Ser/Thr phosphorylation sites. Titin phosphorylation by PKA substantially reduced the passive stiffness of skinned cardiac strips from human donor and end-stage failing hearts and the effect was confirmed in mechanical measurements on isolated cardiac myofibrils. The average stiffness drop was relatively higher at short than at long sarcomere lengths (~25% at 2.1 mm versus ~15% at 2.4 mm) and was larger when samples were pre-treated with PP-1 (~40% at 2.1 mm versus ~25% at 2.4 mm). We conclude that titin phosphorylation is important for the basal passive-tension level in human heart. Beta-adrenergic stimulation may further phosphorylate titin via activation of PKA, thereby decreasing myocardial passive stiffness and benefitting diastolic function.