Question:

Titin's elastic I-band region is a protein-protein interaction hotspot and important for myocyte extensibility and passive stiffness. I-band titin can be phosphorylated at a cardiac-specific domain, the N2B-unique sequence (N2Bus), by protein kinases (PK)A or PKG, which affects passive stiffness. We searched for protein phosphatase(s) acting on N2Bus.

Methods:

For detecting interactions, we performed yeast-2-hybrid (Y2H) screens and GST-pulldown assays. Neonatal rat cardiomyocytes (NRCMs) and paraffin sections of serine/threonine protein phosphatase-5 (PP5) transgenic (TG) mouse hearts were studied by immunofluorescence microscopy. Titin phosphorylation was analyzed by autoradiography and Western-blot. Passive stiffness (F_passive) was measured on single skinned human and PP5 TG cardiomyocytes.

Results:

We detected and confirmed the catalytic domain of PP5 (PP5c) as a novel binding partner of N2Bus. In NRCMs, PP5 was mainly cytosolic but also in the nucleus and at the sarcomeric I-bands, whereas in PP5 TG hearts regular striated I-band localization was most prominent. Recombinant PP5c (c, catalytic domain) was found to dephosphorylate recombinant (PKG-/PKA-)phosphorylated N2Bus, and PP5 bound more strongly to (PKG-/PKA-)phosphorylated N2Bus than to non-phosphorylated N2Bus. Phosphorylation of titin could be reduced in human heart tissue treated ex vivo with recombinant PP5(c). PP5 overexpressing TG mice revealed reduced cardiac titin phosphorylation levels compared to wildtype. F_passive of PP5 TG cardiomyocytes was decreased compared to wildtype. PP5c treatment of human cardiomyocytes significantly reduced F_passive, explainable by dephosphorylation of the PEVK-domain adjoining the N2Bus.

Conclusions:

PP5 is a novel N2Bus-titin interaction partner and acts to reduce F_passive by dephosphorylating titin. PP5 may participate in mechanical signaling pathways converging on the titin springs.