High resolution structural evidence suggests the Sarcoplasmic Reticulum forms microdomains with Acidic Stores (lysosomes) in the heart

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

Of 19 cells imaged in this data set 3 cells exhibited the partial staining shown in B, whilst 16 exhibited no evidence of lysosomal Ned-19 as shown in C, demonstrating that BZ194 pre-incubation prevents Ned-19 localising to lysosomes. D) As a control for possible fluorescent interference of BZ194, cells were incubated with BZ194 and LysoTracker Red alone. These results show that BZ194 is not in itself fluorescent under the imaging conditions used for this study.
Ned-19 does not stain acidic stores after pre-incubation with another known inhibitor of NAADP signalling (BZ194), suggesting that Ned-19 is binding at the lysosomal membrane and not accumulating in acidic compartments as part of a degradative pathway. Scale bar: 10 µm. There is not always a very good Pearson correlation because the images we have used are detailed enough to show that the signals are adjacent but not 'on top of each other'. In this Figure we show how the cross-correlation varies from positive (signal lag = 0) to more strongly positive when a phase shift (lag) in one signal is introduced, to negative when the signals are out of phase. This correlation varies from positive to negative, as expected, over the sarcomere length. So the Pearson correlation is maximised when a lag is introduced equivalent to the mean distance between RyR/PMB and LAMP, and there is no incongruity between the Pearson and Fourier analyses. A) Generated 'ideal' example signals with appropriate analysis of those signals in B. C) Phase-lag cross-correlation analysis of the data presented in this paper.

Supplementary Video 1
Dual axis electron tomography used to image, reconstruct and model lysosomes, T-tubules, mitochondria and sarcoplasmic reticulum (snap shots seen in Figure 5A&B). Plane-by-plane 3D electron tomography (00:00 to 00:30) is followed by zoomed video of nanojunction region.

Immuno-Gold Staining for Electron Microscopy
Samples were prepared from chemically fixed intact mouse ventricular tissue. Approximately 1 mm 3 pieces of tissue from ventricles were chemically fixed (paraformaldehyde 4%, glutaraldehyde 0.1%, phosphate buffer, pH 7.4), embedded in LR White resin and cut into ultrathin sections. For staining sections were washed in distilled water followed by PBS/chicken egg albumin (EA) twice for 5 minutes and blocked in EA for 30 minutes. Sections were incubated in LAMP-2 (rabbit-anti-LAMP-2, Thermofisher PA1-655, Lot: PA189864) primary antibody made up in PBS/EA (2 hours) and washed several times in PBS/EA followed by incubation with protein A gold (1 hour, British Biocell). Finally, sections were washed in PBS/EA and distilled water several times. Prior to viewing the section under an electron microscope (TEM, Joel 1200EX II), sections were post-stained with uranyl acetate and lead citrate.

Controls for Ned-19 Staining in Live Cells
The derivatized nicotinic acid BZ194 has been reported to antagonize NAADP-mediated Ca 2+ signalling 1 . Cells were incubated with BZ194 (100 µM, 1 h) and Ned-19 (100 µM, 40 minutes) at room temperature with addition of 500 nM LysoTracker red for the final 2 min. Cells were allowed to adhere to a glass coverslip and superfused with Tyrode's solution containing (in mM): 135 NaCl, 4.5 KCl, 11 glucose, 20 HEPES, 1 MgCl2, 1.8 CaCl2, pH 7.4 with NaOH. Ned-19 fluorescence was excited at 355 nm and detected at 415±30 nm, whilst LysoTracker red was excited at 514 nm and collected at 590-747 nm. Imaging was performed with a Leica SP5 inverted confocal microscope with a 63x oil-immersion objective.