Spiked-in Pulsed in Vivo Labeling Identifies a New Member of the CCN Family in Regenerating Newt Hearts

The newt Notophthalmus viridescens , which belongs to the family of salamanders (Urodela), owns remarkable regenerative capacities allowing efficient scar-free repair of various organs including the heart. Salamanders can regrow large parts of the myocardium unlike mammals, which cannot replace lost cardiomyocytes efficiently. Unfortunately, very little is known about the molecules and the regulatory circuits facilitating efficient heart regeneration in newts or salamanders. To identify proteins that are involved in heart regeneration, we have developed a pulsed SILAC-based mass spectrometry method based on the detection of paired peptide peaks after ¹³C₆-lysine incorporation into proteins in vivo. Proteins were identified by matching mass spectrometry derived peptide sequences to a recently established normalized newt EST library. Our approach enabled us to identify more than 2200 nonredundant proteins in the regenerating newt heart. Because of the pulsed in vivo labeling approach, accurate quantification was achieved for 1353 proteins, of which 72 were up- and 31 down-regulated with a (|log 2 ratio|1) during heart regeneration. One deregulated member was identified as a new member of the CCN protein family, showing a wound specific activation. We reason that the detection of such deregulated newt-specific proteins in regenerating hearts supports the idea of a local evolution of tissue regeneration in salamanders. Our results significantly improve understanding of dynamic changes in the complex protein network that underlies heart regeneration and provides a basis for further mechanistic studies.