Endogenous retroelements (REs) are pervasive in mammalian genomes, and consist mostly of silenced mobile genetic sequences long considered to be ‘junk DNA.’ In a few known instances, mammalian genomes have captured and repurposed endogenous retroviral sequences to play important roles in regulating mammalian cell functions, notably cell fusion. Our previous work has shown that some mammalian transcripts misannotated as lncRNAs can be translated into small functional peptides, called micropeptides. Here we report the discovery of a cardiac-specific micropeptide encoded by a misannotated lncRNA transcribed from a murine endogenous retroviral element. We named this transcript CARDI, and the encoded micropeptide CARDI-B, since translation of the micropeptide occurs from the second ATG start codon. We show that CARDI-B shares homology with a singular domain of a retroviral protein and localizes to the nucleus. Genetic deletion of CARDI-B in mice from an isogenic background significantly increased the frequency of binucleated cardiomyocytes, at the expense of mononucleated cardiomyocytes. In line with previous reports that the frequency of mononucleated cardiomyocytes is correlated with cardiac regeneration, we show that CARDI-B-deficient hearts perform worse following myocardial infarction injury. These data reveal that functional micropeptides can be concealed within mammalian retroelements, and suggests that RE-derived micropeptides may contribute to the natural variation in heart development and regeneration observed across species.

Voltage-gated potassium (K + ) channels (VGKCs) are essential regulators of the cardiac action potential and are required to maintain normal heart rhythm and contractility. Voltage-dependent activation, conductance and membrane trafficking of K + channels are tightly controlled by a family of single transmembrane regulatory cofactors (KCNE1-5), which co-assemble with pore forming K + channels in cell membranes. Human mutations in voltage-gated K + channels and their regulatory subunits are associated with Long QT syndrome (LQTS), a condition in which the repolarization of the heart is delayed and can lead to arrhythmias, syncope and sudden death. Here we identify a cardiac-enriched transmembrane micropeptide encoded by a small open reading frame, which we named KCNEmini, due to its sequence and structural homology with members of the KCNE family of VGKC regulators. We show that KCNEmini directly binds to and co-localizes with VGKCs in the plasma membrane and functions as a novel regulator of K + channel kinetics. Disruption of KCNEmini in mice resulted in QT interval prolongation and cardiac dysfunction. These studies shed light on a previously unrecognized regulator of K + handling in the heart, which may be an important future therapeutic target for the early diagnosis and treatment of human lethal cardiac arrhythmias.