Introduction: Timothy syndrome (TS) is caused by a de novo gain of function missense glycine to arginine mutation (G406R) in both the 8 and 8a human splice variants of the Cav1.2 channel (TS1 and TS2 respectively). Hypothesis: TS mice will exhibit prolongation of the action potential and will develop heart failure due to calcium overload. Methods: Normal expression of the TS2 as a knock in defect is lethal to mice, but the defect is tolerated when suppressed though transcriptional interference by the NEO promoter (TS2-NEO mouse). By crossing TS2-NEO with a mouse carrying a cardiac-specific tamoxifen-inducible CRE we removed the transcriptional suppression and up-regulated expression of mutant channels. We extracted mRNA from cardiac left ventricle and probed with PCR primers to exons 7 and 9. Individual PCR products were incorporated into plasmid DNA and individual clones were isolated. Only 1 in 56 TS2-NEO colonies expressed mutant channels in heart demonstrating that the NEO cassette suppressed mutant isoforms. With tamoxifen, the ratio of mutant to non-mutant exons increased from 1:34 to 29:72 demonstrating a robust upregulation of the mutant TS2 channel. We performed electrophysiology, EKG analysis, and echocardiography. Results: TS2 mice showed a significant increase in QT duration before (33.1±1.8 ms) and after (74.0±5.5 ms) tamoxifen-activation of the mutant (n=5, p<0.01). Echocardiography demonstrated a decrease in ejection fraction from 95±2 to 53±6% (n=9) in activated TS2 mice, indicative of development of heart failure, compared to with 95±1 to 89±7% (n=12) for controls (P<0.0001). At the single myocyte level, action potentials were markedly prolonged, and both fast and slow components of inactivation of the calcium current were slowed. Fast inactivation in TS2 mice was 18.8±2.9ms (n=18) vs. 12.5±1.4 in controls (n=21,p<0.05), and slow inactivation in TS was 98.2±4.4ms vs. 77.6±1.7 ms in controls (p<0.0001). Conclusion: TS2 mice have QT prolongation, decreased ejection fraction, and altered calcium channel behavior. Calcium overload through the TS2 mutation can cause a rapid development of heart failure. This emphasizes the critical role of the L-type calcium channel inactivation in the physiology of repolarization and heart disease.