Abstract 20451: Hedgehog Signaling and Cardiomyocyte Proliferation

Abstract

Background: In contrast to adult mammalian heart, lower vertebrates such as newt exhibit a dramatic capacity for regeneration in response to injury. Understanding the underlying mechanisms and signaling pathways in newt could form the basis for regenerative therapies in mammals. In the present study, we explored the role of hedgehog (HH) signaling during cardiac regeneration. Methods and Results: To investigate cardiac regeneration (CR) in vivo, we performed ventricular resection studies in adult newt heart. Whole mount and histochemical studies revealed CR within 30 d of resection injury. Functional assessment by echocardiographic analysis showed improved ejection fraction from 22 ± 5% at 4 d to 42 ± 3% at 30 d of regeneration relative to control (60 ± 3%). Gene clustering and qRT-PCR analysis at 7d post injury indicated enrichment of hedgehog (HH) signaling factors including Shh and ptch1 by 3- and 2-fold respectively, suggesting a critical requirement of HH signaling at early stages of CR. We determined HH signaling is essential as pharmacological inhibition of the HH pathway resulted in complete ablation of CR. Using EdU-labeling and immunohistochemical analyses, we showed that HH signaling regulates proliferation of both epicardial and myocardial cells, as inhibition of the HH pathway reduced EdU-positive nuclei from 15 ± 2% to 5 ± 3%. In contrast, qRT-PCR analysis from murine hearts postnatal day (P) 2 to P14, showed reduced levels of HH signaling factors and cell-cycle associated mRNAs by 3.5-fold with concomitant increase of p21 by 2-fold. This implies HH signals are required for the proliferative process. Consistent with the newt studies, activation of HH in murine neonatal ventricular cardiomyocytes (NVCM) promoted cardiomyocyte proliferation, increasing positive nuclei from 10 ± 2% to 25 ± 3%, while inhibition of HH signaling reduced positivity to 6 ± 3%. qRT-PCR analysis established that activation of HH signaling in NVCM resulted in up regulation of transcripts associated with cardiomyocyte proliferation. Conclusion: These data indicated that HH signaling pathways modulate cardiomyocyte proliferation providing potential therapeutic targets for achieving mammalian cardiac regeneration.