Updated project metadata. Mammalian cardiac troponin I (cTnI) contains a highly conserved N-terminal extension harboring protein kinase A targets (Ser23/24) which are phosphorylated during ß-adrenergic stimulation to increase cardiomyocyte relaxation rate. Here, we show that the Ser23/24 encoding exon 3 of TNNI3 was pseudoexonized multiple times in shrews and moles to mimic Ser23/24 phosphorylation without adrenergic stimulation, thereby facilitating the evolution of exceptionally high resting heart rates (~1000 beats min-1). We further reveal alternative splicing of TNNI3 exon 3 in distantly related bat families with both cTnI isoforms being incorporated into cardiac myofibrils.