Abstract 15965: Disruption of Striated Preferentially Expressed Gene Locus Impedes the Differentiation of Cardiac Progenitor Cells into Cardiomyocytes
Introduction: Myosin light chain kinases are a family of proteins known to be important for myocyte function, and a unique member of this family is the striated preferentially expressed gene (Speg). Disruption of the Speg gene locus in mice leads to death in the immediate neonatal period, with evidence of a dilated cardiomyopathy (ejection fraction of 23±3% in Speg-/- compared with 74±2% in Speg+/+ hearts). Hearts of Speg-/- mice show a reduced number of cells per mm3 of tissue and a less differentiated appearance by transmission electron microscopy, suggesting an alteration in the development of cardiac parenchymal cells.
Hypothesis: Speg is important for differentiation of cardiac progenitor cells (CPCs) into cardiomyocytes during heart development.
Methods: c-kit+ CPCs were isolated from Speg+/+ and Speg-/- mice using fluorescence-activated cell sorting (FACS) or magnetic beads. CPC differentiation in vitro and in vivo was assayed using flow cytometry and immunostaining. CPCs were also fluorescently labeled and injected into the left ventricular free wall of embryonic day (E) 14.5 hearts using micro-ultrasound guidance.
Results: Alpha-sarcomeric actin staining of cells under differentiation conditions revealed that 80% of Speg+/+ CPCs differentiated into cardiomyocytes in vitro, while 3% of Speg-/- CPCs were able to acquire a cardiomyocyte phenotype. A less severe defect in smooth muscle cell differentiation was noted in Speg-/- CPCs, and no defect in endothelial cell differentiation. Immunostaining of hearts one day after birth, injected with fluorescently labeled CPCs at E14.5, showed evidence of these exogenous cells engrafting into the developing heart. Exogenous CPCs either differentiated into alpha-sarcomeric actin+ cardiomyocytes, or remained undifferentiated. An additional in vivo assay using flow cytometry after enzymatic dissociation of heart tissue demonstrated that differentiation of exogenous CPCs into troponin T+ cardiomyocytes was 63% in Speg+/+ hearts, and 32% in Speg-/- hearts.
Conclusion: Disruption of the Speg gene locus in CPCs leads to a notable block in cardiomyocyte differentiation, both in vitro and in vivo, which may contribute to the phenotype of Speg mutant mice.
- © 2011 by American Heart Association, Inc.