Involvement of the Mitochondrial Calcium Uniporter in Cardioprotection by Ischemic Preconditioni

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Background

Ischemic preconditioning (IPC, which was originally identified by Murry et al.) provides powerful protection to the ischemic myocardium. Although a variety of intracellular signaling pathways have been implicated in this protection, the precise mechanisms remain elusive.

An important phenomenon:

The close relationship among IPC , the mitochondrial calcium concentration and the improved contractile function of heart.

A guess

There might be certain relationship between ischemic preconditioning and calcium transport system of mitochondrial.

Mitochondrial permeability transition pore (MPTP) and ischemic preconditioning

Mitochondrial calcium uniporter plays important role in mediating the enter of calcium into the mitochondrial.

Questions:

1.Whether activity of mitochondrial calcium uniporter is involved in ischemic preconditioning?

2.And how the relationship between the mitochondrial calcium uniporter and the mitochondrial permeability transition pore?

Part one: Isolated heart experiment

Perfusion protocols used for hemodynamic measurements in isolated rat hearts.

Contractile function investigation:

• Left ventricular developed pressure (LVDP)

• Left ventricular end diastolic pressure (LVEDP)

• +dP/dtmax (the maximal left ventricular pressure increase rate)

• - dP/dtmax (the maximal left ventricular pressure decrease rate)

Infarct size measurement:Infarct size was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) staining method. Infarct size was expressed as percentage of risk zone.

Lactate dehydrogenase measurement

• Coronary effluent at 5, 10, 15, 20 and 30 min during reperfusion was collected

• The activity of lactate dehydrogenase (LDH) was spectrophotometrically assayed and expressed as units per liter.

ResultsContractile functional change of isolated rat hearts:

Effects of ischemia/reperfusion (I/R), ruthenium red (RR), Ru360, spermine (Sper), cyclosporin A (CsA) and ischemic preconditioning (IPC) on LVDP (A), dP/dtmax (B and C) and LVEDP (D) at 30 min of reperfusion following 30 min of ischemia in isolated rat hearts.

Effects of ischemia/reperfusion (I/R), ruthenium red (RR, 5 M), Ru360 (10 M), spermine (Sper, 20 M), cyclosporin A (CsA, 0.2 M) and ischemic preconditioning (IPC) on infarct size in isolated rat hearts after 120 min of reperfusion following 30 min of ischemia

Effects of ischemia/reperfusion (I/R), ruthenium red (RR, 5 M), Ru360 (10 M), spermine (Sper, 20 M), cyclosporin A (CsA, 0.2 M), ischemic preconditioning (IPC) and IPC+Sper ( 20 M) on lactate dehydrogenase (LDH) release during reperfusion in isolated rat hearts.

Part two: experiment in isolated heart mitochondrial

Experiment procedure of Isolated mitochondria

• Isolation of mitochondrial:the extraventricular tissue was removed, was centrifuged at 8000 g for 10 min at 2oC to obtain the original mitochondrial pellet, which was resuspended by use of the homogenizer in suspension buffer containing sucrose (320 mM) and Tris-HCl (10 mM), and centrifuged again at 8000 g for 10 min at 2oC to get the final mitochondrial pellet.

• Measurement of mitochondrial permeability transition pore opening : Pore opening was determined by following the absorbance decrease in light scattering at 520 nm that accompanies mitochondrial swelling. Swelling was initiated by addition of CaCl2 (200 ), and the extent of pore opening w

as expressed in terms of the maximal rate of mitochondrial swelling

Results

The effects of ruthenium red (RR), Ru360, spermine (Sper), cyclosporin A plus spermine (CsA + Sper), ischemic preconditioning (IPC) and IPC plus spermine treatment, on pore opening.

Conclusion

• These results indicate that the mitochondrial calcium uniporter is involved in the cardioprotection conferred by ischemic preconditioning, and may act via inhibiting opening of the mitochondrial permeability transition pore during reperfusion.