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(Circulation. 2003;108:2723-a.)
© 2003 American Heart Association, Inc.

Late-Breaking Science

H11 Kinase Promotes or Inhibits Cardiac Cell Growth and Survival, Depending on its Subcellular Distribution
Christophe Depre, Anna Zajac, Nadia Hedhli, Chull Hong, Jing Liu, Guiping Yang, Li Wang, Huacheng Dai, Univ. Medicine New Jersey, Newark, NJ; Thomas Wagner, Greenville Hosp System, Greenville, SC; Stephen F Vatner; Univ. Medicine New Jersey, Newark, NJ

H11 kinase is a protein of unknown function mainly expressed in heart and skeletal muscle. Its expression is markedly increased in the ischemic heart together with an array of cytoprotective and anti-apoptotic genes, which supposes that H11 kinase promotes cell survival. Paradoxically, overexpression of H11 kinase in other cell types induces apoptosis. Therefore, the goal of the present study was to determine how H11 kinase affects cardiac cell survival and death, using both a cardiac-specific transgenic (TG) mouse model and cardiac cell culture. After 45 min coronary occlusion in vivo, infarct size in hearts from TG mice (7-fold overexpression) was reduced by 80% compared to wild type (P<0.01). This protection was related to a significant (P<0.05) increase in the expression of proteins promoting cell survival, including heat-shock proteins (HSP70, mmDJA4), phospho-Akt, the mTOR targets p70S6K and 4EBP-1, and the glucose transporter GLUT1. Surprisingly, although overexpression of H11 kinase is protective in the TG animal, ischemia reduced H11 kinase expression by 60% in wild type mice (P<0.01 versus sham). To reconcile this paradox, we performed a subcellular fractionation, which revealed that H11 kinase is expressed in both the cytosol and the nucleus. In wild type mice, the cytosolic expression of H11 kinase during ischemia was reduced by more than 80%, whereas the nuclear expression was unaffected. In the TG mouse, overexpression of H11 kinase accumulated as a nuclear protein, which therefore mediates the survival effect. To understand the role of the cytosolic form of the protein, we transfected isolated cardiac myocytes with increasing doses of adenovirus (3 MOI to 100 MOI) harboring the H11 kinase sequence. Overexpression of H11 kinase in isolated myocytes accumulated in the cytosol and did not migrate to the nucleus, which was followed by a significant inhibition of the activity of both Akt and p70S6K (P<0.05), and by increased myocyte apoptosis up to 5-fold (P<0.01) in a dose-dependent manner. Therefore, H11 kinase represents a novel case of a dual-specificity kinase with reciprocal effects on cell growth and survival depending on its subcellular localization, acting as a promoter of cell survival in the nucleus and as a tumor suppressor in the cytosol.

Myostatin Regulates Cardiomyocyte Growth In Vitro and In Vivo
Stuart A Cook, Michael Morissette, Takashi Matsui, Tomohisa Nagoshi, Massachusetts General Hosp, Charlestown, MA; Jeffery D Molkentin, Div of Molecular Cardiovascular Biology, Cincinnati Children’s Hosp Med Cntr, OH; Anthony Rosenzweig; Massachusetts General Hosp, Charlestown, MA

Myostatin (MSTN) is a potent negative regulator of skeletal muscle hypertrophy, though its mechanisms of action are poorly understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of MSTN, not previously recognized to play a role in the heart. To determine the biological effects of MSTN in cardiomyocytes, we used adenoviral gene transfer of MSTN or a truncated mutant that functions as a dominant negative (dnMSTN) and studied the effects in an in vitro model of cardiomyocyte hypertrophy. Expression of MSTN abrogated cardiomyocyte hypertrophy in response to phenylephrine (PE) stimulation. In contrast, dnMSTN expression was sufficient to recapitulate many features of the hypertrophic response. Furthermore, PE-stimulated activation of the serine-threonine kinase, Akt, a critical determinant of cardiomyocyte cell size, was inhibited by MSTN. Restoration of Akt signaling by somatic gene transfer rescued MSTN-mediated inhibition of PE-induced cardiomyocyte hypertrophy. In contrast, expression of dnMSTN led to Akt activation in cardiomyocytes while co-expression of dominant negative Akt blocked dnMSTN-induced cardiomyocyte growth. To investigate whether myostatin also regulates cardiomyocyte growth in vivo, we analyzed mice with targeted deletion of myostatin (MSTN-/-). Interestingly, MSTN-/- hearts (p<0.02) and isolated cardiomyocytes (p<0.001) were smaller than those of wild-type (MSTN+/+) littermates at baseline. However, in response to low-dose in vivo PE infusion, heart weights in MSTN+/- or -/- mice increased 10 and 12% respectively (p<0.04 for both compared to sham) compared with 6% in MSTN+/+ littermates, suggesting an exaggerated response to PE in the absence of myostatin. Akt phosphorylation (activation) was also enhanced in MSTN-/- mice after PE treatment. Together these data suggest that myostatin is a dynamically regulated, novel modulator of cardiomyocyte growth in vitro and in vivo.

Symmetric and Asymmetric Division of Cardiac Stem Cells in the Mouse Heart
Annarosa Leri, Claudia Bearzi, Stefano Cascapera, Daniele Torella, Daniela Cesselli, Raffaella Rastaldo, Federico Quaini, Konrad Urbanek, Piero Anversa; New York Med College, Valhalla, NY

Cardiac stem cells (CSCs) and early committed cells are clustered in atrial and apical niches. The question concerns whether the CSC pool is regulated by critical proteins determining symmetric or asymmetric division, stemness or differentiation. BrdU long-term retaining experiments indicated that the slowly cycling atrial CSCs are the likely candidate of stem cell homeostasis while CSCs in the apex undergo rapid division and lineage commitment. When stem cells divide symmetrically, two self-renewing daughter cells or two committed daughter cells are generated. During asymmetric division, one stem cell and one committed cell are obtained. To identify the mechanism of stem cell growth in the atria, the localization of Numb and -adaptin was analyzed. These two endocytic proteins form vesicles that internalize and inhibit the Notch receptor. The activation or degradation of Notch conditions cell fate. EGFP-labeled primitive cells in the atria were analyzed in vivo by two-photon microscopy and after fixation and staining by confocal microscopy. CSCs divided symmetrically in 80% of the cases and asymmetrically in 20%. During asymmetric division, Numb was restricted to one side of metaphase or anaphase-telophase chromosomes, in a crescent half-moon shape. Numb was strictly associated with -adaptin and Notch was distributed across the mitotic cell. At completion of asymmetric division, the daughter cell positive for Numb and -adaptin had Notch inactivated and dispersed in the cytoplasm. Conversely, when Numb and -adaptin were absent, functional Notch was detected in the nucleus of the daughter cell. The transcription factor GATA-4, that reflects a cardiac cell commitment, was identified only in Numb and -adaptin negative cells that had Notch localized in the nucleus. The characteristic compartmentalization of Numb, -adaptin and Notch was confirmed in primary cultures of cardiac c-kit positive cells in which 40 and 60% were undergoing symmetric and asymmetric division, respectively. In conclusion, the degradation of Notch maintains CSC stemness and the activation of Notch promotes CSC differentiation mimicking the behavior of neural stem cells.

Lentivirus-Mediated Genetic Modifications of Human Embryonic Stem Cells and Their Cardiac Derivatives
Tian Xue, Camie W Chan, Charles A Henrikson, Dongpei Sang, Eduardo Marbán, Ronald A Li; Johns Hopkins Univ, Baltimore, MD

Human embryonic stem cells (hESC) can propagate indefinitely in culture while maintaining pluripotency, including the ability to differentiate into cardiomyocytes (CMs); therefore, hESC may provide an unlimited ex vivo source for transplantation and other cell-based therapies. Genetic engineering of hESC to create "custom-tailored" CMs may further provide a novel and flexible approach to repair the impaired myocardium and modify cardiac excitability. Cardiac differentiation was induced by forming embryoid bodies (hEBs) from dispersed hESC in suspension. To achieve stable genetic modification, we employed the third-generation self-inactivating HIV1-based lentiviral vectors (LV) for delivering transgene(s) to hESC. We first created LV-CAG-GFP, which directs the expression of GFP under the control of the composite constitutive promoter CAG. Undifferentiated LV-CAG-GFP-transduced hESC remained green for >100 days, propagated normally (doubling time10 days), and retained the ability to differentiate into CMs. Spontaneously-contracting hEBs containing CMs derived from control and GFP-transduced hESC could be routinely maintained in culture (>60–90d); both groups behaved similarly when their percentage in the entire differentiated hEB population (12%) and beating frequency (50bpm) were assessed. GFP expression persisted throughout and after cardiac differentiation without transgene silencing. Interestingly, LV-mediated gene transfer of hyperpolarization-activated HCN1-encoded pacemaker channels increased both the beating activity (by 20%) and the percentage of hEBs which express visibly beating primordial hearts (by 2.5-fold). In conclusion, we have achieved persistent genetic modification of hESC and their cardiac derivatives, making possible the creation of a self-renewable ex vivo library of genetically-engineered hESC-derived CMs (e.g. pacemaker cells with a range of firing rates) for different therapeutic purposes.

Discovery of a Novel Pro-Vasculogenic Small Molecule via a Chemical Genetic Screen for Suppressors of Zebrafish Gridlock Mutants
Stanley Y Shaw, Travis A Peterson, David J Milan, Cardiovascular Rsch Cntr, Massachusetts General Hosp, Charlestown, MA; Tao P Zhong, Vanderbilt Sch of Medicine, Nashville, TN; Calum A MacRae, Cardiovascular Rsch Cntr, Massachusetts General Hosp, Charlestown, MA; Stuart L Schreiber, Dept. of Chemistry and Chemical Biology, Harvard Univ, Cambridge, MA; Mark C Fishman, Novartis Institutes for BioMed Rsch, Cambridge, MA; Randall T Peterson; Cardiovascular Rsch Cntr, Massachusetts General Hosp, Charlestown, MA

Zebrafish developmental phenotypes may serve as models for a variety of human cardiovascular disease states, ranging from altered cardiac contractility to complex dysmorphic syndromes. Zebrafish gridlock mutants (grl^m145) have a malformed origin of the dorsal aorta that blocks blood flow and anatomically resembles aortic coarctation. The grl gene (also known as Hey2/HRT2/CHF1/HERP1/HESR2) encodes a basic helix-loop-helix transcriptional repressor belonging to the hairy/Enhancer-of-split-related family, and may participate in arterial vs. venous cell fate decisions. We explored the hypothesis that a small molecule suppressor of grl^m145 would identify signaling pathways that interact with grl during aortic development, and more broadly, contribute to vasculogenesis. To identify suppressors, grl^m145 embryos were arrayed in 96-well plates, and individual small molecules (total 5000) were added to each well. Suppression was scored visually as restored normal blood flow throughout the aorta, and confirmed by microangiography. A novel compound, GS4012, was identified that rescues grl^m145 at an EC50 of 4µM. GS4012 is required during the period of angioblast cell fate determination, and transient exposure limited to the first 30 hours of development corrects the aortic defect through adulthood. GS4012 treatment increases expression of VEGF, but not sonic hedgehog, flt-4, ephrin-B2, or grl in whole embryos as assayed by real-time PCR. Grl^m145 embryos are also partially rescued by expression of murine VEGF in the absence of GS4012 (32% of embryos rescued vs. 5% control, p<0.001). GS4012 and VEGF both enhance HUVEC tubule formation on Matrigel (p<0.001 vs. control for both). In conclusion, a chemical suppressor screen of grl^m145 mutants has revealed an interplay between grl and VEGF signaling, and yielded a small molecule with novel properties: upregulation of VEGF expression and enhanced vasculogenic activity.

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