Similarly, when actin polymerization was blocked via cytochalasin,17,18leaflet force generation did not occur

Similarly, when actin polymerization was blocked via cytochalasin,17,18leaflet force generation did not occur. VIC cytoplasmic processes appearing to attach to the collagen fibers. Functional studies of isometric force development demonstrated that while control samples developed force in response to KCl, either blocking alpha2beta1integrins or blocking actin polymerization via cytochalasin abolished KCl-induced force development (p<0.001). These results strongly suggest that VIC-collagen coupling, mediated by alpha2beta1integrins, is necessary for KCl-induced force generation in the mitral leaflet. This functional coupling between collagen and VICs via alpha2beta1integrins may play a role forin vivomitral valve function. Keywords:extracellular matrix, force generation, cell contraction, integrin, cytochalasin, actin, heart valve, second harmonic generation, confocal microscopy == Introduction == While mitral valves were once regarded as passive flaps, a growing body of evidence continues to revise this notion. Mitral leaflets have their own vasculature,2,13,31,42innervation,3,10,13,28,38remodel in response to various stresses,6,11,39,40and recent studies suggest that there are multiple contractile systems within the leaflets.23Ex vivostudies have demonstrated that mitral leaflets generate force in response to vasoactive agents.16,38Valvular interstitial cells (VICs) are a Afegostat D-tartrate dynamic, active component within leaflets that enable valves to remodel and mitral leaflet VICs are known to contract in response to vasoactive agents.16Given the contractile properties of VICs and evidence that the same vasoactive agents that cause mitral VIC contraction also result in force generation in mitral leaflets,16,38it appears that mitral VIC contraction could be one component of mitral leaflet force generation. However, the means by which mitral VIC contraction is transferred into mitral force generation has not been studied. Transmission electron micrographs of mitral leaflets show VICs closely associated with collagen fibers, 16suggesting an interaction between VICs and collagen in the leaflet, perhaps even acting Afegostat D-tartrate together as a functional unit. Given that integrins are the primary proteins attaching cells to the surrounding matrix,48a potential interaction between VICs and collagen likely occurs through one of the integrins. Of the known collagen binding integrins, only alpha2beta1and alpha11beta1show higher affinity for type I collagen (the predominant collagen in heart valves9) than type IV.1Based on fibroblast-collagen gel contraction12and fibroblast collagen fiber transport30studies, we hypothesized that alpha2beta1integrins may be a mechanism by which VICs attach to collagen within the mitral valve leaflet. Given the critical role that alpha2beta1integrins play in fibroblast-collagen interactions,12,30their demonstrated presence on mitral VICs,26and the tools available to probe alpha2beta1integrin function, this study chose to investigate alpha2beta1integrins. While a variety of integrins have been shown to be present on enzymatically isolated mitral VICs, including alpha2beta1,26,27the potential functional role of integrins in valves, however, has not been studied. Based on the hypothesized roles of cellular contraction and alpha2beta1integrins in this proposed VIC-collagen mechanotransduction within the mitral valve, the aim of this study was to test the specific hypothesis that actin-mediated VIC contraction coupled to collagen via alpha2beta1integrins was necessary for force generation in mitral leaflets. == Materials and Methods == == Immunofluorescence == Porcine hearts (Yosemite Meat, Modesto, CA) were obtained within one hour of animal slaughter and transported in cold saline (0.9% sodium chloride, Hospira, Lake Forest, IL). The central bare area (central region devoid of chordal attachments) Afegostat D-tartrate of each mitral anterior leaflet was dissected within 4 hours of animal slaughter and stained to visualize VIC cytoplasm, nucleus, and F-actin. VIC cytoplasm was stained using calcein AM (Invitrogen Molecular Probes, Eugene, OR) and VIC nuclei were stained using Syto85 (Invitrogen Molecular Probes). Cytoplasmic F-actin Rabbit polyclonal to XPO7.Exportin 7 is also known as RanBP16 (ran-binding protein 16) or XPO7 and is a 1,087 aminoacid protein. Exportin 7 is primarily expressed in testis, thyroid and bone marrow, but is alsoexpressed in lung, liver and small intestine. Exportin 7 translocates proteins and large RNAsthrough the nuclear pore complex (NPC) and is localized to the cytoplasm and nucleus. Exportin 7has two types of receptors, designated importins and exportins, both of which recognize proteinsthat contain nuclear localization signals (NLSs) and are targeted for transport either in or out of thenucleus via the NPC. Additionally, the nucleocytoplasmic RanGTP gradient regulates Exportin 7distribution, and enables Exportin 7 to bind and release proteins and large RNAs before and aftertheir transportation. Exportin 7 is thought to play a role in erythroid differentiation and may alsointeract with cancer-associated proteins, suggesting a role for Exportin 7 in tumorigenesis was stained using AlexaFluor 488 phalloidin (Invitrogen Molecular Probes) in tissues fixed in 10% formalin for 30 minutes and permeabilized with 0.5% Triton-X100 (Sigma-Aldrich, St. Louis, MO). Tissue was only fixed for actin staining; all other staining and force generation experiments utilized fresh tissues without fixation or permeabilization. == Confocal Microscopy == A Zeiss Leica 510 LSM.