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Active propagation of waves of mitochondrial membrane potential collapse across the intact heart
  1. AR Lyon1,
  2. FG Akar2
  1. 1Imperial College, London, UK
  2. 2Mount Sinai School of Medicine, New York, USA

Abstract

Polarisation of the mitochondrial membrane potential (Δψm) is critical for normal mitochondrial function and cellular energetics. A variety of disease processes initiate Δψm collapse in isolated cells, but spatiotemporal changes at the tissue level have not previously been reported.

Methods Δψm changes in intact ex-vivo rat hearts were studied using high-resolution optical mapping and the Δψm-sensitive dye tetramethylrhodamine methylester (TMRM). Hearts were excited with filtered light (525 ± 20 nm), and emitted fluorescence (585 ± 20 nm) was focused onto a high resolution CCD camera. Background fluorescence intensity following 250 nmol TMRM staining and washout was measured across a 4 × 4 mm region of the anterior rat ventricular epicardium with 50 μm spatial resolution. Hearts were subjected to global (n  =  17) or regional (n  =  4) ischaemia followed by reperfusion at 7.5 or 15 minutes. A subset of hearts (n  =  5) was studied following focal overexpression of PKBS, the peripheral benzodiazepine receptor, using adenoviral transfection in vivo. Ad.GFP injected hearts (n  =  4) served as controls.

Results Adjacent zones of Δψm hyperpolarisation and depolarisation emerged during the first minute of global myocardial ischaemia. Progressive ischaemia initiated waves of Δψm depolarisation, which propagated across the epicardial surface with a mean velocity of 19.6 ± 3.9 μm/s. Ripples of fluctuating Δψm precede final collapse, supporting organised functional coupling of mitochondrial function across hundreds of cells within the intact ischaemic heart. At reperfusion transient Δψm hyperpolarisation was followed by Δψm collapse in the ischaemic myocardium. Reperfusion-induced depolarisation was reversible after brief ischaemic periods (7.5 minutes), but became permanent in hearts ischaemic for 15 minutes. PKBS overexpression resulted in prolonged Δψm hyperpolarisation during both ischaemia and reperfusion.

Conclusions These findings indicate novel spatial patterns of coordinated mitochondrial dysfunction within ischaemic mammalian tissue. Complex spatiotemporal gradients of Δψm developed during metabolic stress produced by ischaemic or reperfusion injury in the normal rat heart. Δψm depolarisation waves propagate slowly across the epicardial surface of the ischaemic myocardium, with alternating depolarisation and polarisation before collapse. Ischaemia duration influences the reversibility of Δψm collapse after reperfusion, and PKBS overexpression modifies the spatial patterning during oxidative stress with Δψm hyperpolarisation the dominant response. The generation, amplification and propagation of mitochondrial dysfunction through an intact tissue present novel opportunities to target and limit injury within the diseased mammalian heart.

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