CapZ dynamics in response to Endothelin‐1

Cardiac myocytes hypertrophy in response to both mechanical load (e.g. pressure, stretch, osmotic stress) and agonist stimulation (e.g. β‐adrenergic, endothelin‐1). This process involves remodeling of the cytoskeleton, and sarcomere A, I and Z‐bands. We hypothesize that altered actin capping affinity facilitates the hypertrophic response by allowing sarcomeric remodeling to occur. We test this by observing the dynamics of the actin capping protein, CapZ□1 with or without ET‐1 stimulation in cultured rat neonatal cardiac myocytes. Cells adenovirally infected with either wild‐type or one of two actin binding deficient mutant (L262R or c‐terminal deletion) CapZ‐GFP fusion constructs were utilized. Proper localization of the exogenous protein to the Z‐disk was observed by confocal imaging. Cell surface area measurements increase for all 3 infection vectors after ET‐1 treatment indicating a responsiveness of cells over expressing CapZ to hypertrophic stimulation. FRAP analysis up to 20min postbleach showed that CapZ wild‐type infected cells treated with ET‐1 recovered both faster and more completely than untreated controls(71±10% vs. 48±3%, p ≤ 0.02, n=7). Initial studies of mutant protein recovery demonstrated that at 20min L262R recovery (46±4%, n=4) was similar to that of untreated cells, while C‐terminal deletion recovery (78±13%, n=2) was similar to that of ET‐1 treated cells. Our results suggest that ET‐1 treatment leads to altered CapZ actin affinity and that this alteration is effectively mimicked by utilizing an actin binding mutant of CapZ. Funded by HL 62426, HL64956.