Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart

Abstract

Opening of the mitochondrial permeability transition pore (MPTP) is thought to be a critical event in mediating the damage to hearts that accompanies their reperfusion following prolonged ischaemia. Protection from reperfusion injury occurs if the prolonged ischaemic period is preceded by short ischaemic periods followed by recovery. Here we investigate whether such ischaemic preconditioning (IPC) is accompanied by inhibition of MPTP opening. MPTP opening in Langendorff-perfused rat hearts was determined by perfusion with 2-deoxy[3H]glucose ([3H]DOG) and measurement of mitochondrial [3H]DOG entrapment. We demonstrate that IPC inhibits initial MPTP opening in hearts reperfused after 30 min global ischaemia, and subsequently enhances pore closure as hearts recover. However, MPTP opening in mitochondria isolated from IPC hearts occurred more readily than control mitochondria, implying that MPTP inhibition by IPC in situ was secondary to other factors such as decreased calcium overload and oxidative stress. Hearts perfused with cyclosporin A or sanglifehrin A, powerful inhibitors of the MPTP, also recovered better from ischaemia than controls (improved haemodynamic function and less lactate dehydrogenase release). However, the mitochondrial DOG entrapment technique showed these agents to be less effective than IPC at preventing MPTP opening. Our data suggest that protection from reperfusion injury is better achieved by reducing factors that induce MPTP opening than by inhibiting the MPTP directly.

Figure 1. Perfusion protocols

Perfusion protocols used for measuring the effects of IPC, SfA and CsA on opening of the MPTP using mitochondrial DOG-entrapment with either pre-loading (A) or post-loading (B).

Figure 2. The effects of IPC and SfA treatment of hearts on the [Ca²⁺] sensitivity of MPTP opening in isolated de-energised mitochondria

Mitochondria were isolated from hearts that had been subjected to IPC (A) or perfusion with and without 0.2 μm CsA or SfA (B) and pore opening determined under de-energised conditions at the free [Ca²⁺] shown. C, mean data (+s.e.m., error bars) are shown for control and IPC hearts isolated immediately after the pre-conditioning protocol (n = 8), at the end of ischaemia (n = 5) and after reperfusion (n = 6). In the latter experiments the recovery of LVDP as a percentage of the pre-ischaemic value was 66.7 ± 2.6 % for control hearts and 94.4 ± 4.5 % for IPC hearts (P < 0.01)

Figure 3. The effects of IPC treatment of hearts on the [Ca²⁺] sensitivity of MPTP opening in isolated energised mitochondria

Mitochondria were isolated from control hearts and those subjected to IPC and pore opening determined under energised conditions as described under Methods. Additions of [Ca²⁺] were made as indicated.

Figure 5. Correlation between the percentage recovery of LVDP during reperfusion of hearts subject to 30 min global ischaemia and the mitochondrial DOG entrapment

Data are presented for control hearts and those subjected to IPC or treated with 0.2 μm CsA or SfA prior to ischaemia. Separate control experiments were performed for the IPC experiments (filled symbols) and the SfA/CsA experiments (open symbols). Mean data for all relevant parameters are shown in Tables 1 and 2.

Figure 4. Correlation between the release of LDH and the EDP (A) or percentage recovery of LVDP (B) during reperfusion of hearts subject to 30 min global ischaemia

Data are shown for control hearts and those treated with 0.2 μm CsA or SfA prior to ischaemia using both the pre-loading (open symbols) and post-loading (filled symbols) protocols shown in Fig. 1. Mean data for all relevant parameters are shown in Table 2.

Figure 6. The amount of DOG taken up by hearts in the post-loading protocol correlates with the percentage recovery of LVDP during reperfusion

Total [³H]DOG uptake was measured for hearts that had been loaded using either the pre-loading (A) or post-loading (B) protocol. Data are presented for control hearts and those subjected to IPC or treated with 0.2 μm CsA or SfA prior to ischaemia. Note that the total loading of hearts was different by a factor of 5 between the IPC experiments and those with SfA and CsA as explained in the text.