A ratiometric catalog of protein isoform shifts in the cardiac fetal gene program

Pathological cardiac remodeling is associated with the reactivation of fetal genes, yet the extent of the heart's fetal gene program and its impact on proteome compositions remain incompletely understood. Here, using a proteome-wide protein ratio quantification strategy with mass spectrometry, we identified pervasive isoform usage shifts in fetal and postnatal mouse hearts, involving 145 pairs of highly homologous paralogs and alternative splicing-derived isoform proteins. Proteome-wide ratio comparisons readily rediscovered hallmark fetal gene signatures in muscle contraction and glucose metabolism pathways, while revealing what we believe to be previously undescribed isoform usage in mitochondrial and gene-expression-regulating proteins, including PPA1/PPA2, ANT1/ANT2, and PCBP1/PCBP2 switches. Paralogs with differential fetal usage tend to be evolutionarily recent, consistent with functional diversification. Alternative splicing adds another rich source of fetal isoform usage differences, involving PKM M1/M2, GLS1 KGA/GAC, PDLIM5 long/short, and other spliceoforms. When comparing absolute protein proportions, we observed a partial reversion toward fetal gene usage in pathological hearts. In summary, we present a ratiometric catalog of paralogs and spliceoform pairs in the cardiac fetal gene program. More generally, the results demonstrate the potential of applying the proteome-wide ratio test concept to discover new regulatory modalities beyond differential gene expression.