Energy status determines the distinct biochemical and physiological behavior of interfibrillar and sub-sarcolemmal mitochondria

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Abstract

Reports about the effect of ischemia and reperfusion on specific activities of the respiratory chain are often discrepant. One of the factors that govern this discrepancy is that typical mechanical procedures for mitochondrial isolation yield largely sub-sarcolemmal mitochondria (SSM), while the interfibrillar mitochondria (IFM), which provide most of the energy for the contractile apparatus, are under-represented. Here we investigated the impact of myocardial ischemia and reperfusion on SSM and IFM separately. Thirty-two Wister rats were randomly divided into four groups: control groups, ischemia groups, reperfusion groups and precondition groups. SSM and IFM were isolated from the rats’ hearts from all the groups. The mitochondrial membrane potential (Δψ) and swelling were assessed at energized (using either 5 mM succinate or 5 mM glutamate and 5 mM malate (GM) as a substrate) and non-energized conditions, where IFM showed better resistance to change in both conditions. Results showed that IFM have a higher coupling efficiency than SSM when energized by GM, but lower than SSM when energized with succinate. Preconditioning the rats’ hearts prior to ischemia or reperfusion preserved the physiological and biochemical functions of both IFM and SSM and are energy dependent. The distinct physiological–biochemical functions of the mitochondrial sub-populations during ischemia and reperfusion depend on the overall energy status of the mitochondrial sub-population.

Highlights

► Myocardial ischemia and reperfusion (I/R) affect SSM and IFM differently. ► Energized IFM showed better resistance to I/R injury than SSM. ► Higher coupling efficiency was shown in IFM with glutamate malate energization. ► Higher coupling efficiency was shown in SSM with succinate mediated energization. ► Myocardial protection by ischemic preconditioning requires energized mitochondria.

Introduction

Reperfusion injury is caused by the increased cellular Ca2+ and the reactive oxygen species (ROS) generation, both initiated in ischemia and amplified upon reperfusion. This injury originates mainly from the mitochondria [1]. Overall, it is thought that the combined effects of ROS generation and elevated Ca2+ play a critical role in the transition from reversible to irreversible reperfusion injury. In particular, they lead to the opening of the mitochondrial permeability transition pore (MPTP), now widely accepted to play a critical role in reperfusion injury [2]

Depending on their location in cardiomyocytes, heart mitochondria are classified as either sub-sarcolemmal (SSM) or interfibrillar (IFM) [3]. The two sub-populations are different with respect to both their inherited properties and their response to disease. An increased susceptibility of cardiac SSM to ischemic damage, as compared to IFM, has been observed in rat [4].

Lucas and Szweda [5] reported decreased respiration using NAD as a substrate after ischemia or reperfusion in the mitochondria isolated from the perfused Langendorff rats’ hearts. On the other hand, other studies showed that there is no decrease in the respiratory activity in mitochondria after exposure to a 30 min ischemia [6], and NMR studies indicate that any putative post ischemic damage is not sufficient to slow down coupled electron flow [7]. Thus, it has been shown that after ischemia/reperfusion in heart, respiration may fall, rise or remain the same. We show that this uncertainty can be resolved by studying the effect of ischemia and reperfusion on distinct mitochondrial sub-populations, which can elucidate the contribution of mitochondrial dysfunction to cardiac failure.

Section snippets

Animals and experimental design

Sprague–Dawley male rats (250–300 g) housed under standard conditions and fed regular ad libitum diet and water were used. All the experimental protocols were approved by the ‘Institutional Animal Care and Use Committee’ of the Hebrew University of Jerusalem, conforming to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Perfusion protocols

Hearts were removed and mounted on the Langendorff apparatus as previously

Cardiac function and infarct size

The functional capacity of the heart during the experiment was assessed by the rate pressure product (RPP, product of the heart rate and left ventricular developed pressure). RPP of ischemia and I/R hearts were significantly lower and indicate poor recovery during the experimental procedure. The insult of ischemia reperfusion to IPC hearts significantly recovered the hearts as, compared to ischemia reperfusion controls (Table 1).

In order to show that the IPC procedure was effective as

Discussion

The main information obtained from our experiments is that: (a) distinct oxidative phosphorylation capacities are exhibited in mitochondrial subpopulations during ischemia and reperfusion and are dependent on substrate specific respiration. For example, IFM showed better coupling capacity (both ADP/O ratio and RCR) when respiration was driven by the complex I substrate GM, while SSM exhibit better oxidative phosphorylation coupling (ADP/O ratio) than IFM with succinate as a respiration

Acknowledgments

Lady Davis foundation is acknowledged for their financial support to GAK. Mottie Chevion is the incumbent of the Dr. William Ganz Chair of Heart Studies at the Hebrew University of Jerusalem.

This study was supported by grants from the Pepka and Dr. Moshe Bergman Memorial Fund, Joels Landovsky Fund (Hebrew University) and German Israeli Fund (GIF).

References (26)

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