ABSTRACT
AIMS: In the heart, a period of ischaemia followed by reperfusion evokes powerful cytosolic Ca(2+) oscillations that can cause lethal cell injury. These signals represent attractive cardioprotective targets, but the underlying mechanisms of genesis are ill-defined. Here, we investigated the role of the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which is known in several cell types to induce Ca(2+) oscillations that initiate from acidic stores such as lysosomes, likely via two-pore channels (TPCs, TPC1 and 2). METHODS AND RESULTS: An NAADP antagonist called Ned-K was developed by rational design based on a previously existing scaffold. Ned-K suppressed Ca(2+) oscillations and dramatically protected cardiomyocytes from cell death in vitro after ischaemia and reoxygenation, preventing opening of the mitochondrial permeability transition pore. Ned-K profoundly decreased infarct size in mice in vivo. Transgenic mice lacking the endo-lysosomal TPC1 were also protected from injury. CONCLUSION: NAADP signalling plays a major role in reperfusion-induced cell death and represents a potent pathway for protection against reperfusion injury.
Subject(s)
Calcium Channels/metabolism , Calcium Signaling/drug effects , Carbolines/pharmacology , Mitochondria, Heart/drug effects , Mitochondrial Membrane Transport Proteins/antagonists & inhibitors , Myocardial Infarction/prevention & control , Myocardial Reperfusion Injury/prevention & control , Myocytes, Cardiac/drug effects , NADP/analogs & derivatives , Piperazines/pharmacology , Animals , Calcium Channels/deficiency , Calcium Channels/genetics , Cytoprotection , Disease Models, Animal , Dose-Response Relationship, Drug , Male , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Mitochondrial Swelling/drug effects , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , NADP/antagonists & inhibitors , NADP/metabolism , Rats, Sprague-Dawley , Time FactorsABSTRACT
Surface patterning (micro-moulding) of dense, biomimetic collagen is a simple tool to produce complex tissues using layer-by-layer assembly. The aim here was to channelise three-dimensional constructs for improved perfusion. Firstly, collagen fibril accumulation was measured by comparative image analysis to understand the mechanisms of structure formation in plastically compressed collagen during µ-moulding. This showed that shape (circular or rectangular) and dimensions of the template affected collagen distribution around moulded grooves and consequently their stability. In the second part, this was used for effective fabrication of multi-layered plastically compressed collagen constructs with internal channels by roofing the grooves with a second layer. Using rectangular templates of 25/50/100 µm widths and 75 µm depth, grooves were µ-moulded into the fluid-leaving surface of collagen layers with predictable width/depth fidelities. These grooves were then roofed by addition of a second plastically compressed collagen layer on top to produce µ-channels. Resulting µ-channels retained their dimensions and were stable over time in culture with fibroblasts and could be cell seeded with a lining layer by simple transfer of epithelial cells. The results of this study provide a valuable platform for rapid fabrication of complex collagen-based tissues in particular for provision of perfusing microchannels through the bulk material for improved core nutrient supply.
