Files in this Data Supplement:

• Supplemental Methods - (Microsoft Word) (87 kb)
• Table - (Microsoft Word) (64.5 kb)
• Figure I - (Power Point) (102 kb) Genotyping, RNA, and protein expression in binary tetracycline-repressive system. A), Genotyping by PCR shows: (a) wildtype (WT) band only, (b) tTA band only, (c) sPRKAG band only, (d) or both the tTA and sPRKAG band; (e) negative control with water, (f) Marker IX. B, RNA quantification by Northern analysis shows that Tg^ON mice expressed the transgene less than the original TGγ2^N488I mice 1 (Tg^ON: 7.4±1.2 arbitrary units, N=5; original TGγ2^N488I: 10.6±0.8, N=5, P=0.03). Oral doxycycline administration resulted in a significant decrease of transgene expression, as compared to Tg^ON mice (Tg^OFF 4.3±0.5 arbitrary units, N=10, P=0.001 vs. Tg^ON), and was similar to the level of transgene expression of transgenic sPRKAG2^N488I mice not carrying the tTA transgene (sPRKAG2 without tTA: 4.3±1.5). After discontinuing dietary tetracycline, transgene expression levels significantly increased and was similar to expression levels of Tg^ON mice (Tg^OFF after transgene reexpression: 6.4±2.0 arbitrary units, N=4, P=0.02 vs. Tg^OFF). (a) through (e) show Northern blot results of 5 representative mice: (a): WT, (b) Tg^ON , (c) Tg^OFF(E-8wks) at 8 wks of age, (d) Tg^OFF(4-16wks)at 12 weeks of age, (e) Tg^OFF(E-8wks) at 16 weeks of age (transgene reexpressed for 8 weeks). First line: sPRKAG2 transgene, second line: GAPDH loading control (approx 5 μg/μl total RNA each). C) Western blot analyses using antibodies specific for γ2 AMPK subunit reacted with cardiac extracts from one Tg^OFF(E-8wks) at 8 weeks of age (a), Tg^ON at 6 weeks of age, (b) and Tg^OFF(8-20) at 20 weeks of age (c) mouse. Whereas Tg^ON mice showed robust AMPK γ2 expression, the signal was almost undetectable in Tg^OFF mice. GAPDH antibody was used as internal control.
• Figure II - (Power Point) (386 kb) Histopathology of one exemplary Tg^ON mouse is illustrated using Masson trichrome stained sections in a 4-chamber view. Collagen–rich tissue, such as the annulus fibrosis (arrows) and the valves are depicted in blue. Distinct locations within the atrioventricular region are shown. Disruption of the annulus fibrosis, which separates the interatrial (IAS) from the interventricular (IVS) septum, is demonstrated in (A). A different view of the same heart shows glycogen-containing vacuolated cells enriched within the annulus fibrosus which presumably causes loss of electrical insulation between the atria and the ventricles at different sites, leading to multiple accessory pathways (B) through (D).