Figure 3. Transient Notch induction leads to prolonged sinus bradycardia.

Quantification of N1ICD levels after 3-week doxycycline induction demonstrates approximately 8-fold activation (A), as well as up-regulation of known direct Notch targets Hrt1 and Hes1 (B). Mice were 8 weeks of age at the start of doxycycline administration, controls were αMHC-rtTA; tetO_NICD (n=2 females, n=4 males) never administered doxycycline, experimental mice were αMHC-rtTA; tetO_NICD iNICD (n=1 female, n=5 males) fed doxycycline chow. (C) N1ICD and Hrt1 levels return to baseline after 2 days of doxycycline induction followed by a 1-year washout period. Interestingly, Hes1 remains persistently up-regulated one year after the brief 2-day induction followed by a 1 year washout. (D) Heart weight/tibia length ratio of iNICD mice with varying lengths of Notch induction followed by a 1-year washout indicate no difference in heart size compared with controls. (E) The HR remains persistently depressed in iNICD mice after transient Notch induction for 0.5 days (n=3 females, n=3 males), 2 days (n=2 females, n=3 males), or 6 weeks (n=3 females, n=2 males) followed by a 40 week washout period and cessation of Notch induction. In C–E, control mice were αMHC-rtTA; tetO_NICD never administered doxycycline (n=3 females, n=3 males) and iNICD mice were αMHC-rtTA; tetO_NICD mice with periods of Notch induction beginning at 8 weeks of age for the length of time indicated within the figure. A–C statistics were performed using an unpaired t test with a Welch’s correction. D,E statistics were performed using a one-way ANOVA followed by a Dunnett’s multiple comparisons test. Values of P<0.05 were considered statistically significant. Relative fold changes were in comparison with α-actinin.