Abstract
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.
Highlights
The traditional view that lysosomes function solely as a terminal end of cellular catabolic pathways has been challenged recently by various lines of evidence showing that this tiny acidic organelle plays a central role in cell function via lysosomal calcium signalling[1]
Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) no longer produces a response in cardiac myocytes after abolition of the H+ gradient required for lysosomal calcium loading[20], and cells derived from Two-Pore Channel-2 (TPC2) knock-out mice do not respond to NAADP application[3]
In order to investigate the proximity of lysosomal membranes to other subcellular organelles, we carried out transmission electron microscopy (TEM) on rabbit isolated ventricular myocytes and fixed ventricular tissue
Summary
The traditional view that lysosomes function solely as a terminal end of cellular catabolic pathways has been challenged recently by various lines of evidence showing that this tiny acidic organelle plays a central role in cell function via lysosomal calcium signalling[1]. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) is a second messenger which induces the release of calcium from acidic stores such as the lysosome and lysosome-related reserve granules in the sea urchin egg[4] The importance that this signalling molecule plays in the mobilisation of calcium in many species of animal and plant has been established in recent decades[5]. Calcium entering the cytosol via voltage-gated L-type calcium channels is sensed at the cytosolic face of ryanodine receptors (RyR), which open, leading to massive calcium release from the sarcoplasmic reticulum (SR) This amplification from the cell’s intracellular calcium store is known as calcium induced calcium release (CICR) and is terminated by closure of ryanodine receptors and clearance of calcium from the cytosol back into the SR. That effects of NAADP in the heart involve both calcium re-uptake and calcium release mechanisms simultaneously
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