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.

隶属于有尾目（Urodela）蝾螈科的东部红点蝾螈（Notophthalmus viridescens）具备卓越的再生能力，可实现包括心脏在内的多种器官的无瘢痕高效修复。与无法高效替换丢失心肌细胞的哺乳动物不同，蝾螈能够再生心肌的大片区域。然而，目前对于蝾螈高效心脏再生所依赖的分子与调控通路，我们仍知之甚少。为鉴定参与心脏再生的蛋白质，我们开发了一种基于脉冲式稳定同位素标记氨基酸培养（SILAC）的质谱分析方法，该方法通过检测体内将13C6-赖氨酸掺入蛋白质后产生的配对肽峰来完成分析。通过将质谱得到的肽序列与新近构建的标准化蝾螈表达序列标签（EST）文库进行比对，我们实现了蛋白质的鉴定。本方法可在再生阶段的蝾螈心脏中鉴定出超过2200种非冗余蛋白质。得益于该脉冲式体内标记策略，我们对1353种蛋白质实现了准确定量，其中72种在心脏再生过程中呈上调表达，31种呈下调表达（|log₂比值|>1）。其中1个差异表达蛋白被鉴定为CCN蛋白家族的新成员，其表达呈现伤口特异性激活模式。我们认为，在再生心脏中检测到这类蝾螈特异性差异表达蛋白，佐证了蝾螈组织再生存在局部演化的假说。本研究结果显著加深了我们对心脏再生背后复杂蛋白质网络动态变化的认知，并为后续的机制研究提供了重要基础。