Notch signaling regulates cardiomyocyte proliferation during zebrafish heart regeneration.

The human heart's failure to replace ischemia-damaged myocardium with regenerated muscle contributes significantly to the worldwide morbidity and mortality associated with coronary artery disease. Remarkably, certain vertebrate species, including the zebrafish, achieve complete regeneration of amputated or injured myocardium through the proliferation of spared cardiomyocytes. Nonetheless, the genetic and cellular determinants of natural cardiac regeneration remain incompletely characterized. Here, we report that cardiac regeneration in zebrafish relies on Notch signaling. Following amputation of the zebrafish ventricular apex, Notch receptor expression becomes activated specifically in the endocardium and epicardium, but not the myocardium. Using a dominant negative approach, we discovered that suppression of Notch signaling profoundly impairs cardiac regeneration and induces scar formation at the amputation site. We ruled out defects in endocardial activation, epicardial activation, and dedifferentiation of compact myocardial cells as causative for the regenerative failure. Furthermore, coronary endothelial tubes, which we lineage traced from preexisting endothelium in wild-type hearts, formed in the wound despite the myocardial regenerative failure. Quantification of myocardial proliferation in Notch-suppressed hearts revealed a significant decrease in cycling cardiomyocytes, an observation consistent with a noncell autonomous requirement for Notch signaling in cardiomyocyte proliferation. Unexpectedly, hyperactivation of Notch signaling also suppressed cardiomyocyte proliferation and heart regeneration. Taken together, our data uncover the exquisite sensitivity of regenerative cardiomyocyte proliferation to perturbations in Notch signaling.

Cerebral cavernous malformation is a vascular disease associated with activated RhoA signaling.

Cerebral cavernous malformation (CCM) involves the homozygous inactivating mutations of one of three genes, ccm1, -2, or -3 resulting in hyperpermeable blood vessels in the brain. The CCM1, -2, and -3 proteins form a complex to organize the signaling networks controlling endothelial cell physiology including actin dynamics, tube formation, and adherens junctions. The common biochemical defect with the loss of CCM1, -2, or -3 is increased RhoA activity leading to the activation of Rho-associated coiled coil-forming kinase (ROCK). Inhibition of the ROCK rescues CCM endothelial cell dysfunction, suggesting that the inhibition of RhoA-ROCK signaling may be a therapeutic strategy to prevent or arrest the progression of the CCM lesions.

Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.

Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, -2, or -3, are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, -2, or -3 proteins causes a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, -2, or -3, with an especially pronounced RhoA activation in both the cytosol and the nucleus with loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin light chain 2. Functionally, loss of CCM1, -2, or -3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or short hairpin RNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, -2, and -3 in CCM pathology, whereby loss of CCM1, -2, or -3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from loss of CCM protein function.