Abstract 183: Cardiac Specific Deletion of Tbx5 Causes Left Ventricular Diastolic Dysfunction that can be Rescued by Increased Serca2a Activity
Tbx5 mutations cause congenital heart defects and limb abnormalities. To investigate the mechanisms involved in this pathology, we have generated Tbx5del/+ and ventricular specific deletion Tbx5LDN/+; Nkx2.5-Cre (Tbx5V−del/+) mice. In these mice, cardiac function was impaired and, specifically, the E/A ratio was decreased and isovolumic relaxation time was prolonged compared to wild-type littermates, suggesting a direct role for Tbx5 in regulating LV diastolic function. The molecular basic of ventricular diastolic dysfunction appears to result from down-regulation of Serca2a expression with a subsequently reduced Ca2+ uptake during diastole in both Tbx5del/+ and Tbx5V-del/+mice. Intracellular Ca2+ transients were measured in ventricular myocytes isolated from Tbx5V-del/+mice and littermates controls. The amplitude of Ca2+ transients was no different between two groups while the decay of Ca2+ transients was slower in Tbx5V-del/+ myocytes as indicated by an increased time for 50% relaxation compared to control myocytes(12.5±0.5 vs. 9.5±0.42 msec, P±0.05). To determine the role of Tbx5 in regulating SERCA gene transcription, we cloned the mouse Serca2a promoter region, which possesses multiple Tbx5 binding sites, into a luciferase reporter vector and performed transactivation assays in 10T1/2 cells. We determined that Tbx5 co-transfection increases Serca2a transcription significantly. In parallel experiments, we have been attempting to correct the defect seen in the Tbx5 deleted mice. Giving our finding of decreased Serca2a expression, one potential strategy involves relieving Serca2a inhibition in vivo. To achieve this, we mated the Tbx5V-del/+ transgenic line with mice lacking phospholamban, is a reversible inhibitor of Serca2a. In Tbx5V-del/+; PLN−/ − mice, we found heart diastolic function such as E/A ratio and isovolumic relaxation time/deceleration time (IVRT/DT) significantly recovered. Together, our results suggest that Tbx5 directly regulates the LV diastolic function via Serca2a expression and suggest that diastolic dysfunction is a primary defect in some patients with congenital heart defects.