Significance Heart failure is a term used to describe the heart’s inability to pump sufficient oxygen-rich blood throughout the body. This condition is commonly caused by the loss of heart muscle cells termed cardiomyocytes that results from a heart attack. As one of the least regenerative organs in the human body, the heart fails to regenerate damaged cardiomyocytes, forms a noncontractile scar instead, and progressively fails. Unlike mammals, adult zebrafish robustly regenerate their hearts following injury through division of uninjured cardiomyocytes, thus providing an opportunity to dissect innate cardiac regenerative mechanisms. Here, we provide evidence that Notch signaling is required for cardiomyocyte division and heart regeneration in zebrafish and, therefore, highlight a genetic determinant of natural heart renewal.

Cerebral cavernous malformations (CCM)3 are clusters of leaky, dilated capillaries in the central nervous system that occur in ϳ0.5% of the general population and up to 1.5% of the Hispanic population (1). CCM frequently lead to clinical sequelae such as hemorrhage, epilepsy, and neurological deficits (1). The disease is associated with both germline and somatic mutations in one of three genes, ccm1, -2, or -3 (2). The three CCM proteins form a common complex, indicating that they function coordinately (3, 4); each lacks defined catalytic domains, indicating that they are scaffold-or adaptor-like proteins for organization of protein complexes (3-5). The identical disease phenotype produced upon loss of any one of the three CCM proteins suggests that they coordinately regulate a common mechanism required for vascular integrity (3,4,6,7).It was recently shown that loss of endothelial cell expression of CCM2 resulted in activation of the GTPase RhoA (7,8). Crose et al. (8) demonstrated that CCM2 knockdown in brain microvascular endothelial cells resulted in defective RhoA degradation because of the dysregulation of Smurf1, a CCM2 binding partner, and an ubiquitin-protein isopeptide ligase (E3) that controls RhoA degradation. RhoA overabundance induced by loss of CCM2 was shown to increase cytoskeletal stability, inhibit vessel-like tube formation, and increase endothelial cell monolayer permeability (7,8). Herein, we show that loss of CCM1, -2, or -3 expression results in a common phenotype associated with RhoA overexpression and activation. We define ROCK as a critical RhoA effector whose increased activation dysregulates endothelial cell function. ROCK is activated by RhoA and phosphorylates several substrates, including myosin light chain, myosin light chain phosphatase, and LIM kinase for the regulation of actin cytoskeletal dynamics (9). ROCK has also been shown to regulate vascular permeability and has been a drug discovery target for regulation of vascular bed diseases (10). Our findings show that ROCK inhibition rescues extracellular matrix invasion and vessel-like tube formation, two endothelial cell functions disrupted by loss of CCM protein expression. EXPERIMENTAL PROCEDURESEstablishment of Knockdown Cell Lines-Lentiviral genespecific shRNAs in pLKO.1 were obtained from the University of North Carolina -Chapel Hill Lenti-shRNA Core Facility.RhoA Biosensor-Imaging and image processing were performed as described (11).Tube Formation Assay and Live Cell Imaging-7.0 ϫ 10 Ϫ4 cells were incubated for 15 h on Matrigel (BD Biosciences) and stained with rhodamine phalloidin as described previously (8).Imaging was performed on either a Pathway (BD Biosciences) or a Cellomics ArrayScan (Thermo Scientific). For live cell imaging, six fields of cells were imaged via transmitted light every 10 min for 15 h. Cellomics ArrayScan software was used to quantitate mean tube area. Statistical Significance-Where indicated, statistical significance was calculated using the two-tailed Student's t test. See supplemental mater...

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.

The vertebrate heart forms through successive phases of cardiomyocyte differentiation. Initially, cardiomyocytes derived from first heart field (FHF) progenitors assemble the linear heart tube. Thereafter, second heart field (SHF) progenitors differentiate into cardiomyocytes that are accreted to the poles of the heart tube over a well-defined developmental window. Although heart tube elongation deficiencies lead to life-threatening congenital heart defects, the variables controlling the initiation, rate and duration of myocardial accretion remain obscure. Here, we demonstrate that the AP-1 transcription factor, Fos-like antigen 2 (Fosl2), potentiates the rate of myocardial accretion from the zebrafish SHF. fosl2 mutants initiate accretion appropriately, but cardiomyocyte production is sluggish, resulting in a ventricular deficit coupled with an accumulation of SHF progenitors. Surprisingly, mutant embryos eventually correct the myocardial deficit by extending the accretion window. Overexpression of Fosl2 also compromises production of SHFderived ventricular cardiomyocytes, a phenotype that is consistent with precocious depletion of the progenitor pool. Our data implicate Fosl2 in promoting the progenitor to cardiomyocyte transition and uncover the existence of regulatory mechanisms to ensure appropriate SHF-mediated cardiomyocyte contribution irrespective of embryonic stage.

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