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.