Rationale: Calcification of heart valve structures is the most common form of valvular disease and is characterized by the appearance of bone-like phenotypes within affected structures. Despite the clinical significance, the underlying etiology of disease onset and progression is largely unknown and valve replacement remains the most effective treatment. The SRY-related transcription factor Sox9 is expressed in developing and mature heart valves, and its function is required for expression of cartilage-associated proteins, similar to its role in chondrogenesis. In addition to cartilage-associated defects, mice with reduced sox9 function develop skeletal bone prematurely; however, the ability of sox9 deficiency to promote ectopic osteogenic phenotypes in heart valves has not been examined. Objective: This study aims to determine the role of Sox9 in maintaining connective tissue homeostasis in mature heart valves using in vivo and in vitro approaches. Methods and Results: Using histological and molecular analyses, we report that, from 3 months of age, Sox9 fl/؉ ;Col2a1-cre mice develop calcific lesions in heart valve leaflets associated with increased expression of bone-related genes and activation of inflammation and matrix remodeling processes. Consistently, ectopic calcification is also observed following direct knockdown of Sox9 in heart valves in vitro. Furthermore, we show that retinoic acid treatment in mature heart valves is sufficient to promote calcific processes in vitro, which can be attenuated by Sox9 overexpression. Conclusions: This study provides insight into the molecular mechanisms of heart valve calcification and identifies reduced Sox9 function as a potential genetic basis for calcific valvular disease. (Circ Res. 2010;106:712-719.)Key Words: heart valves Ⅲ calcification Ⅲ Sox9 Ⅲ extracellular matrix Ⅲ mouse model C alcification of heart valve structures affects more than 27% of the US population over 65 years of age and is the major contributor of heart valve malfunction. 1 Despite the clinical significance, little is known about the mechanisms that underlie this multifactorial disease. Treatment options for valve calcification are limited, and no known therapies prevent disease progression. 2 Normal heart valve function requires organization of differentiated cell types and specialized extracellular matrix (ECM) within the valve leaflet is arranged according to blood flow. 3 This defined tissue architecture provides the mechanical resilience and compressibility required to open and close the valve orifices effectively during the cardiac cycle. 4 In diseased heart valves, loss of ECM organization is associated with changes in mechanical properties, ultimately leading to dysfunction. 5,6 One of the most striking alterations in valve ECM homeostasis is ectopic bone-like matrix mineralization observed in calcific valve disease. 7,8 At the functional level, this histopathologic alteration results in stiffened leaflets, narrowing of the valve opening, and impaired blood flow. 9 The mechanis...

Abstract-Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx Ϫ/Ϫ mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilageassociated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx Ϫ/Ϫ mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures. (Circ Res. 2008;103:948-956.)

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Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations.