JCS01559 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Distribution of voltage-operated Ca²⁺ channels, RyRs, SERCA, sarcolemma and mitochondria in atrial myocytes. Panels A and B show immunolocalisation of L-type VOCs and type 2 RyRs in ventricular (A) and atrial (B) myocytes. Similar results were observed in >50 cells from 5 hearts. The distribution of RyRs in the centre of atrial cells (Bb) shows a similar striated pattern to ventricular myocytes (Ab). However, there was an additional distinct band of immunoreactivity that ran around the periphery of the atrial cell (marked by arrows in Bb), which was separated from the central RyRs by a gap of 1-2 µm. This peripheral band of staining represents the fraction of junctional RyRs that closely appose L-type VOCs and are responsible for sensing the influx of Ca²⁺ during the initiation of EC-coupling. Panel C shows a 3-dimensional reconstruction of a portion of a living atrial myocyte stained with di-8-ANNEPPS. This fluorophore preferentially accumulates in the outer leaflet of the membrane and highlights sarcolemmal distribution. A similar peripheral-only membrane staining was found in all atrial myocytes examined (n=50). Panel D depicts an atrial myocyte stained for SERCA2a distribution. The red arrows indicate the SERCA staining in the region where junctional RyRs are expressed. These distributions were typical for all cells analysed (n>30; from 5 hearts). Panel E shows a portion of an atrial myocyte that was double-labelled for mitochondria (TMRE; red) and plasma membrane (di-8-ANNEPPS; green).

  Materials and Methods. The subcellular distribution of RyR was examined using methods described previously [Lipp et al. (2000) Curr. Biol. 10, 939-942]. Confocal microscopy (PerkinElmer Life Sciences, UK) was used to image the distribution of fluorescein-conjugated secondary antibodies. The polyclonal rabbit anti-type 2 RyR antibodies and the polyclonal rabbit anti-SERCA2a antibodies were kind gifts from V. Sorrentino (Siena, Italy) and F. Wuytack (Leuven, Belgium), respectively. The polyclonal rabbit anti-L-type (a_1c) voltage-operated Ca²⁺ channel antibodies were from Calbiochem. Specific labelling of the sarcolemmal was performed by incubating the cells for 2 minutes in EM containing 5 µM di-8-ANEPPS. The 3-dimensional reconstruction of the plasma membrane was obtained by acquiring z-stacks of confocal images (200 nm spacing per image plane), and volume rendering the images using the ImageSuite software (PerkinElmer Life Sciences, UK).

• Supplemental Figure 2 -

  Fig. S2. Depolarisation of atrial myocyte mitochondria under non-paced conditions does not lead to a reduction in cellular ATP. Panel A depicts changes in mitochondrial membrane potential due to addition of oligomycin + antimycin. The downward deflection of tetramethylrhodamine ethyl ester (TMRE) fluorescence indicates mitochondrial depolarisation. The cell was not electrically paced in this experiment. The trace is representative of 11 other cells (from 4 hearts). Panel B shows changes in mag-fura-2 ratio measured in rat atrial myocytes. Since most of the cellular Mg²⁺ is bound to ATP, and Mg²⁺ ions display a more than 10-fold higher affinity for binding to ATP than to ADP, reduction of cellular ATP can be acutely monitored as an increase in Mg²⁺ concentration. The figure illustrates that when the cells were electrically paced (red trace) antimycin + oligomycin caused cellular ATP levels to decline (i.e. free magnesium increased). The brief upwards deflections indicate intracellular Ca²⁺ transients since Ca²⁺ transiently displaces Mg²⁺ from intracellular binding sites. When cells were not paced (black trace), depolarisation of the mitochondrial membrane potential did not induce a rapid decline in cellular ATP concentration. Similar time courses were found in all cells analysed under paced and non-paced conditions (n=10; from 3 hearts).

  Materials and Methods. The potentiometric indicator TMRE was used to measure the mitochondrial membrane potential (DY_mit). TMRE accumulates in energised mitochondria in a roughly Nernstian manner. Reduction of DY_mit leads to loss of TMRE fluorescence. Rat atrial myocytes were loaded with TMRE (0.1 µM in EM) for 20 minutes at room temperature. Changes in cytosolic ATP levels were monitored by measuring the free cytosolic magnesium concentration. Rat atrial myocytes were loaded with mag-fura-2 (1 µM mag-fura-2 AM in EM for 45 minutes, followed by 30 minutes in EM for de-esterification). Mag-fura-2 fluorescence ratios were calculated from the fluorescence emission monitored at >510 nm following alternation excitation at 340 nm and 380 nm. Imaging was performed on a MIRACAL system (PerkinElmer Life Sciences, UK).