Modulating Neuregulin/ErbB4 Signaling to Promote Cardiomyocyte Replacement

Introduction: The goal of regenerative therapy for heart failure is to replace cardiomyocytes. Whether differentiated cardiomyocytes can contribute to myocardial regeneration is a controversial question of great biomedical importance. Here we address whether and how differentiated cardiomyocytes can be induced to proliferate. Hypothesis: We hypothesized that the growth factor neuregulin1 (NRG1) and its tyrosine kinase receptor ErbB4 control post-natal proliferation of differentiated cardiomyocytes. Methods: We applied NRG1 systemically by intraperitoneal injection into three different lines of mice: a line that allowed for inducible genetic inactivation of ErbB4, a transgenic line over-expressing ErbB4, and wild type mice (C57BL6). We used uptake of BrdU, a thymidine analog, as readout of DNA synthesis and staining for phosphorylated histone H3 and aurora B kinase as readouts of karyokinesis and cytokinesis, respectively. Results: Inactivation of ErbB4 reduced postnatal cardiomyocyte proliferation, resulting in 19% less cardiomyocyte nuclei compared to littermate controls. Increasing ErbB4 expression enhanced proliferation, resulting in 16% more cardiomyocyte nuclei. Injecting NRG1 in adult wild-type animals increased cycling of differentiated cardiomyocytes. NRG1 induced mononucleated, but not binucleated cardiomyocytes to divide. Using two different thymidine analogs, we demonstrate that under NRG1 stimulation, differentiated cardiomyocytes entered the cell cycle twice and completed the cell cycle at least once. Using sparse genetic labeling of differentiated cardiomyocytes, we determined that NRG1 induced proliferation of individual cardiomyocytes, resulting in a 35-fold increase in the number of genetically labeled clusters of four or more differentiated cardiomyocytes in vivo. To divide, differentiated cardiomyocytes completely disassembled their contractile apparatus in the cleavage furrow. Using genetic fate mapping of differentiated cardiomyocytes, we demonstrate that undifferentiated progenitor cells did not contribute to NRG1-induced cardiomyocyte proliferation. Conclusions: Differentiated mononucleated cardiomyocytes have proliferative potential. ErbB4 is required and NRG1 or ErbB4 are sufficient to induce proliferation of differentiated cardiomyocytes. Thus, controlling NRG1/ErbB4 signaling may provide a promising strategy to promote myocardial regeneration.