signaling and the repression of vascular endothelial growth factor in myocardium of the valve-forming region.49 Micro-Rna Regulation of Cardiac Development The transcriptional regulation of cardiac development, and its modulatory and instructive signaling pathways, are well studied, and their biology is becoming well understood Less clear is the translational control of cardiac morphogenesis by small noncoding RNAs, such as microRNAs MicroRNAs are genomically encoded 20–22 nucleotide RNAs that function by targeting mRNAs either for translational inhibition or for degradation, leading to an effective reduction in quantity of the protein product Several hundred human microRNAs have been identified, and some of these have important roles in development that may be eminently relevant to congenitally malformed hearts The best characterized example is the microRNA-1 family, comprising miR-11 and miR-1-2 These microRNAs are highly conserved from worms to humans, and are specifically expressed in the progenitor cells of developing cardiac and skeletal muscle as they differentiate.50 Both are highly expressed in the cells of the outflow tract derived from the second heart field Interestingly, expression of these microRNAs is directly controlled by well-studied transcriptional regulatory networks that promote muscular differentiation Consistent with a role in differentiation, overexpression of miR-1 in the developing mouse heart results in a decrease in expansion of ventricular myocytes, with fewer proliferating cardiomyocytes remaining in the cell cycle Defects caused by mutations in microRNA genes range from benign to severe Disruption of the single fly orthologue of miR-1 had catastrophic consequences, resulting in uniform lethality at embryonic or larval stages, with a frequent defect in maintaining cardiac gene expression.51 Targeted deletion of miR-1-2 in the mouse resulted in ventricular septal defects, although with incomplete penetrance.52 In surviving adults, disruption of normal cardiac conduction and cell cycle control were also observed As miRNAs can be highly redundant, deletion of all copies of redundant miRNAs must be accomplished to uncover their underlying function Combined loss of miR-1-1 and miR-1-2 in the mouse indeed unveiled a profoundly important function of the miR-1 pair in broadly repressing a smooth muscle gene program, while promoting sarcomere formation in the developing heart.53 Many other miRNAs are enriched in specific cardiovascular cell types and play important roles in cardiogenesis, but in each case, they appear to be embedded in critical transcriptional networks, typically reinforcing the cellular actions of those networks (reviewed in Cordes and Srivastava54) Hemodynamics and Formation of the Heart As the heart forms, it soon begins to beat and pump blood This occurs at the early stages of cardiac looping, well before chambers have formed and separations between segments of the heart are established It would seem intuitive that the physical forces of a beating heart would affect its morphologic development, but until recently this has been but a concept In fact, hemodynamic forces are indeed important, and shape not only the normal development of the heart but produce the secondary defects associated with major structural congenital cardiac malformations The initial identification of a role for hemodynamics was in zebrafish, a simple model of cardiac development in which a single atrium connects to a single ventricle By altering the flow at the inflow or outflow of the zebrafish heart, and imaging the structure and function of the heart, it was determined that altering flow within the heart led to abnormal cardiac looping and defects in formation of the cardiac cushions, indicating that normal intracardiac flow is a key regulator of cardiac morphogenesis.55 This was confirmed using zebrafish and mouse models in which embryonic contractility is impaired or absent.56–58 More recent experiments, again in zebrafish, have clearly shown that intrinsic defects in contractility of cardiac myocytes, in combination with external hemodynamic forces, are essential for the normal development of the heart.59 The most intriguing results on hemodynamics and cardiac development have a direct connection to congenital malformations It had been observed in a mouse model of laterality defects that include anomalies of the outflow tracts that formation of the arteries in the branchial arches is randomized between the left and right sides.60 Normally, the arches form initially in bilateral fashion, but the left-sided brachiocephalic artery regresses, leaving only a right-sided artery It was determined that, in this mouse model, the flow to the branchial arches was randomized to both left and right sides, unlike the usual situation where flow is preferentially directed to the right side With a combination of surgical and morphologic manipulations, it was shown that the altered flow was the likely culprit for the abnormal presence of a left-sided brachiocephalic artery Thus a genetic defect, in this case involving Pitx2, leads not only to direct defects in regions of the heart where the mutated gene is expressed, but produces morphologic defects secondary to altered flow This finding has important implications for the understanding of the origin of congenital cardiac malformations