Online SupplementManuscript # HR-080154
Sood S et al.Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.
Figure S1.
Fig. S1. Representative lead II surface ECG complex showing the marking of various wave forms and the corresponding ECG intervals (denoted as horizontal arrows) which were measured according to the following definitions1: The ‘PR’ interval was measured from the onset of P wave to the peak of R wave, the ‘QRS’ interval from the onset of Q wave to the peak negative deflection of the S wave (first major deflection), and the ‘QT’ interval was measured from the onset of the Q wave to the end of a second slower and lower amplitude deflection. QT intervals were corrected according to QTc=QT/(RR/100)1/2.2 P, P wave; R, R wave; Q, Q wave, T, T wave.
Supplemental Methods
Cardiac electrophysiology
ECG intervals were measured using the automated ECG Auto software v2 (Emka Technologies), which averaged data of at least 20 ECG complexes for each mouse or condition. QT intervals were corrected according to: QTc=QT/[SQRT (RR/100)] 2, 3. Sinus node recovery time (SNRT), measured after a 15 s pacing train with a basic cycle length (BCL) of 100 ms, was defined as the interval between the last stimulus in the pacing train and the onset of first sinus return beat. Atrial effective refractory period (AERP), measured at a BCL of 100 ms, was defined as the longest S1-S2 coupling interval for atria that failed to capture. Atrioventricular nodal ERP (AVERP) was defined as the maximal S1S2 coupling interval that failed to propagate to the ventricle 4. AF was defined as rapid and fragmented atrial electrograms with irregular AV-nodal conduction and ventricular rhythm for at least one second.
Western blotting
Atrial samples (100 μg) were separated on 6% (for RyR2), 8% (for Cav1.2, NCX, SERCA2a), or 15% (for PLB) acrylamide gels, and transferred onto polyvinyl difluoride membranes (PVDF). Membranes were probed with anti-Cav1.2 (1:100; Alomone Labs, Israel), anti-NCX (1:200; Swant, Switzerland), anti-RyR2 (1:10,000; Affinity BioReagents), anti-SERCA2a (1:200; Santa Cruz), anti-PLB (1:5,000; ABR), anti-PLB-pSer16 (1:5,000; Research Diagnostics), anti-PLB-pThr17 (1:5,000; Badrilla), and anti-GAPDH (1:5,000; Abcam) antibodies overnight at 4oC. The RyR2-pSer2808 phosphoepitope-specific antibody was custom generated using the peptide C-RTRRI-(pS)-QTSQV corresponding to the PKA phosphorylation site region at serine 2808 on RyR2. Secondary anti-mouse, anti-rabbit or anti-goat antibodies were conjugated to Alexa-Fluor 680 (Invitrogen Molecular Probes) or IR800Dye (Rockland Immunochemicals), respectively. Bands were visualized using the Odyssey system (Li-COR Biosciences), quantified using densitometry (NIH software, Image J), and normalized to GAPDH levels.
References
1.Appleton GO, Li Y, Taffet GE, et al.: Determinants of cardiac electrophysiological properties in mice. J. Intervent. Cardiol. Electrophysiol. 2004; 11: 5-14.
2.Mitchell GF, Jeron A, Koren G: Measurement of heart rate and Q-T interval in the conscious mouse. Am J Physiol 1998; 274: H747-51.
3.Schrickel JW, Bielik H, Yang A, et al.: Induction of atrial fibrillation in mice by rapid transesophageal atrial pacing. Basic Res Cardiol 2002; 97: 452-60.
4.Berul CI, Aronovitz MJ, Wang PJ, et al.: In vivo cardiac electrophysiology studies in the mouse. Circulation 1996; 94: 2641-8.