Omega-3 polyunsaturated fatty acid-enriched diet differentially protects two subpopulations of myocardial mitochondria against Ca²⁺-induced injury

Abstract

Omega-3 polyunsaturated fatty acids (PUFA) confer protection against myocardial injury after ischemia-reperfusion. There are two subfractions of mitochondria located in different regions of the cell: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The present study explored possible differences between Ca²⁺-induced mitochondrial swelling in rat SSM and IFM fractions under control conditions (control group [CG]) and after dietary supplementation with omega-3 PUFA (experimental group [EG]). Changes in mitochondrial matrix volumes were measured using the light-scattering technique. In the CG, the time courses of swelling were comparable in both mitochondrial fractions, with no difference in Ca²⁺-induced swelling between the two mitochondrial fractions. In the SSM fraction, no difference in the time course of swelling in Ca²⁺-free solution between CG and EG was detected. In the EG, both SSM and IFM fractions demonstrated a decreased sensitivity to Ca²⁺; IFM fractions, however, exhibited significantly less pronounced swelling following Ca²⁺ addition. The authors conclude that IFM and SSM fractions do not differ in their sensitivity to Ca²⁺-induced swelling. While dietary omega-3 PUFA protected both mitochondrial fractions against Ca²⁺-evoked swelling, the protective effect appeared to be more pronounced for the IFM fraction than for the SSM fraction.

Consumption of omega-3 polyunsaturated fatty acids (PUFA) has long been known to reduce the incidence of cardiovascular disease and decrease the risk for heart failure (1). A large body of evidence has demonstrated that omega-3 PUFA protect the myocardium against ischemia-reperfusion injury (2). Although the underlying mechanisms are not yet fully elucidated, experimental data indicate that such protection is at least partially explained by a positive effect of omega-3 PUFA on functional parameters of cardiac mitochondria (2,3) and involve changes in Ca²⁺ transport mechanisms. Numerous studies have shown that dietary omega-3 PUFA are protective against Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP) (4), a main requirement for elevated levels of Ca²⁺ in mitochondria (5).

In heart muscle, two morphologically distinct subpopulations of mitochondria located in different regions of the cell exist: the subsarcolemmal mitochondria (SSM), which are located immediately underneath the sarcolemmal membrane; and intermyofibrillar mitochondria (IFM), located within the myofibrils (6–8). These two mitochondrial subpopulations differ in protein and lipid composition, biochemical properties and function (6). However, little is known about the role of mitochondrial functional heterogeneity in health and disease and, specifically, about myocardial protection against ischemia-reperfusion injury. The IFM fraction accounts for up to 80% of total mitochochondrial content (6,9); however, the majority of studies investigating isolated mitochondria do not discriminate between the two subpopulations.

There is uncertainty regarding the sensitivity of the two mitochondrial fractions to Ca²⁺-induced injury. It has been reported that IFM exhibit higher Ca²⁺ uptake capacity than SSM. Ca²⁺ loading in SSM leads to the release of cytochrome c, whereas no cytochrome c release was observed when IFM were challenged with a similar amount of Ca²⁺ loading (8), indicating that SSM are more susceptible to Ca²⁺-induced MPTP opening. Progression of ischemic damage is more rapid in SSM than in IFM (10,11). These data are in contrast to the findings obtained from mitochondrial subpopulations isolated from skeletal muscles, in which IFM were reported to be more sensitive to Ca²⁺(6). The present study aimed to determine possible differences in the susceptibility of SSM and IFM fractions to Ca²⁺ under control conditions and following in vivo supplementation with omega-3 PUFA for one month. We explored Ca²⁺-induced swelling in SSM and IFM fractions under control conditions and the impact of dietary omega-3 PUFA on the magnitude of Ca²⁺-induced mitochondrial swelling in SSM and IFM fractions. We show that supplementation with dietary omega-3 PUFA for relatively short periods of time confers differential protection against Ca²⁺-induced injury in the two mitochondrial fractions. Preliminary data from the current study have been presented in abstract form elsewhere (12).

METHODS

Animals and diet

Animal use and handling were in accordance with the guidelines of the Biomedical Ethics Committee of Bogomoletz Institute of Physiology (Kiev, Ukraine). Three- to four-month-old male Wistar rats weighing 240 g to 300 g were randomly divided into two groups: the control group (n=5) and the experimental group (n=6), both of which were provided ad libitum access to chow and tap water. In the experimental group, rats were administered (orally using a syringe) epadol (0.1 mL/100 g) containing 45% omega-3 PUFA (Kyiv Vitamin Factory, Kiev Ukraine) daily for four weeks.

Different mitochondrial fractions were isolated as described previously (8), with slight modifications. Briefly, following cervical dislocation, hearts were rapidly removed and rinsed with ice-cold 0.9% NaCl and minced in buffer A containing 250 mM sucrose, 20 mM Tris HCl (pH 7.2), 1 mM EGTA and 0.5% bovine serum albumin. The minced tissue was homogenized using a Teflon-glass homogenizer and centrifuged at 700 g at 2°C for 10 min. The supernatants from these preparations were pooled and centrifuged at 5000 g at 2°C for 10 min to obtain the sub-SSM fraction. The resulting pellet was resuspended to its original volume in buffer A without EGTA and bovine serum albumin, and centrifuged again at 5000 g at 2°C for 10 min. The isolated SSM fraction was kept on ice until used.

The remaining tissue pellet obtained during SSM isolation was resuspended in buffer A. To these samples, Nagarse (Sigma Chemical Co, USA) was added to a final concentration of 2.5 mg/g wet weight of tissue and homogenized immediately. This homogenate was diluted twofold with buffer A and centrifuged at 5000 g for 10 min. The remaining pellet after Nagarse treatment was resuspended in buffer A and sedimented at 700 g at 2°C. The resulting supernatants were pooled and centrifuged at 5000 g at 2°C for 10 min. IFM were washed twice and resuspended in buffer A. Protein content was estimated using the Lowry method (13).

Mitochondrial swelling

MPTP opening, which is facilitated under conditions of elevated Ca²⁺ concentration, causes massive mitochondrial swelling, outer membrane rupture and the release of proapoptotic factors (7). MPTP opening was measured by monitoring the decrease in light scattering associated with mitochondrial swelling at 540 nm. Isolated mitochondria (0.1 mg protein/mL) were resuspended in buffer B (10 mM KH₂PO₄, 120 mM KCl, 10 mM Tris-HCl, 10 mM succinate, pH 7.2), and supplemented with 5 μM rotenone. Data were collected every 60 s over a 15 min interval. SSM and IFM were treated with 100 μM CaCl₂. Experimental data points were normalized to the data points obtained during the first minute of recording. Data are expressed as mean ± SEM, with the statistical significance of each effector being analyzed using the unpaired Student’s t test; P<0.05 was considered to be statistically significant and is indicated in each figure by an asterisk.

RESULTS

Control group: IFM and SSM exhibit similar sensitivities to Ca²⁺

Initially, the sensitivity of SSM and IFM fractions to Ca²⁺-induced swelling was studied (Figure 1). In the absence of external Ca²⁺, the time course of decrease in light scattering, which corresponded to mitochondrial swelling, did not differ significantly between the SSM and IFM fractions (up to 0.81±0.01 and 0.80±0.02, respectively), estimated at 15 min. Administration of external Ca²⁺ (100 μM) caused a similar increase in mitochondrial swelling in SSM (decrease in light scattering up to 0.63±0.05) and IFM fractions (up to 0.65±0.06). Mitochondrial swelling in response to Ca²⁺ was prevented by the addition of 10 μM cyclosporine A (data not shown), indicating that Ca²⁺-induced mitochondrial swelling reflects MPTP opening.

Figure 1).

Time course of swelling of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) in the control group. Swelling was measured spectrophotometrically. Ca²⁺ (100 μM) was added at 5 min where indicated for inducing mitochondrial permeability transition pore opening. IFM and SSM of the control group were compared. Every data point represents mean values for three to five individual experiments. 1: Time course of swelling of IFM from control group in Ca²⁺-free medium; 2: Time course of swelling of SSM from control group in Ca²⁺-free medium; 3: Time course of swelling of IFM from control group after Ca²⁺ addition at 5 min; 4: Time course of swelling of SSM from control group after Ca²⁺addition at 5 min. A Absorbance; t Time

Dietary supplementation with omega-3 PUFA reduces the sensitivity of SSM and IFM fractions to Ca²⁺

Next explored was whether dietary omega-3 PUFA influence mitochondrial swelling in the absence and presence of Ca²⁺. Figure 2 illustrates the impact of omega-3 PUFA supplementation on the time course of SSM swelling in a Ca²⁺-free and Ca²⁺-containing solutions. In Ca²⁺-free medium, the SSM fraction isolated from the control (Figure 2, trace 1) and experimental (Figure 2, trace 2) groups showed no difference in the time course of swelling (decrease in light scattering up to 0.81±0.01 and 0.81±0.01, respectively). However, SSM from the experimental group (Figure 2, trace 3) demonstrated significantly (P<0.05) decreased swelling after the addition of Ca²⁺ (decrease in light scattering to 0.71±0.06) compared with the corresponding Ca2⁺-induced swelling of SSM from the control group (0.63±0.05) (Figure 2, trace 4). Remarkably, unlike the SSM fraction, omega-3 PUFA supplementation resulted in significantly decreased swelling of the IFM fraction even in Ca²⁺-free solution (Figure 3, traces 1 and 2). Furthermore, in the IFM fraction isolated from the treatment group, mitochondrial swelling in response to Ca²⁺ addition was significantly suppressed compared with the same fraction isolated from the control group (Figure 3, traces 3 and 4 for the control and experimental group, respectively). Omega-3 PUFA supplementation suppressed mitochondria swelling of the IFM fraction from 0.65±0.06 to 0.83±1.60 (P<0.05). In both mitochondrial fractions isolated from the experimental group, cyclosporine A suppressed Ca²⁺-induced mitochondrial swelling (data not shown).

Figure 2).

Time course of swelling of subsarcolemmal mitochondria (SSM) from the control and experimental groups. Swelling was measured spectrophotometrically. Ca²⁺ (100 μM) was added at 5 min where indicated for inducing mitochondrial permeability transition pore opening opening. The control and experimental groups were compared. Each point represents the mean vales of three to five individual experiments. *P<0.05 versus respective control values. 1: SSM from control group in Ca²⁺-free solution; 2: SSM from the experimental group in Ca²⁺-free solution; 3: SSM from the experimental group after the addition of 100 μM Ca²⁺ at 5 min; 4: SSM from the control group after addition of 100 μM Ca²⁺ at 5 min. A Absorbance; t Time

Figure 3).

The time course of swelling of interfibrillar mitochondria (IFM) isolated from control and experimental groups. Swelling was measured spectrophotometrically. Ca²⁺ (100 μM) was added at 5 min where indicted for inducing mitochondrial permeability transition pore opening. The control and experimental groups were compared. Every data point represents mean values for three to five individual experiments. *P<0.05 versus respective control values. 1: IFM from control group in Ca²⁺-free solution; 2: IFM from the experimental group in Ca²⁺-free solution; 3: IFM from the control group after the addition of 100 μM Ca²⁺ at 5 min; 4: IFM from the experimental group after the addition of 100 μM Ca²⁺ at 5 min. A Absorbance; t Time

Protective effect of dietary omega-3 PUFA was more pronounced in the IFM fraction

In the control group, mitochondrial swelling in Ca²⁺-free solution did not differ between the two fractions. However, in the IFM fraction isolated from the experimental group, a decrease in light scattering in Ca²⁺-free solution (0.81±0.01) was significantly less pronounced (Figure 4, trace 1) (0.91±0.02) than that observed in the SSM fraction (Figure 4, trace 2) (P<0.05). Furthermore, although omega-3 PUFA supplementation resulted in protection against Ca²⁺-induced mitochondrial swelling in both SSM and IFM fractions, the degree of protection appeared to be different. For the IFM fraction isolated from omega-3 PUFA-treated rats, mitochondrial swelling in response to the addition of Ca²⁺ was significantly (P<0.05) blunted (decrease in light scattering up to 0.83±0.01) compared with the SSM fraction (0.71±0.06). Collectively, the data suggest that dietary supplementation with omega-3 PUFA significantly decreases IFM swelling in both Ca²⁺-free and Ca²⁺-containing solutions, while having no impact on SSM swelling in Ca²⁺-free solution and protecting, albeit to a lesser extent, SSM swelling in Ca²⁺-containing solution.

Figure 4).

The time course of swelling for interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) isolated from control and experimental groups. Swelling was measured spectrophotometrically. Ca²⁺ (100 μM) was added at 5 min where indicated for inducing mitochondrial permeability transition pore opening. IFM and SSM from the experimental group were compared. Every data point represents mean values for three to five individual experiments. *P<0.05 versus respective IFM values. 1: IFM from the experimental group in Ca²⁺-free solution; 2: SSM from the experimental group in Ca²⁺-free solution; 3: IFM from the experimental group after the addition of 100 μM Ca²⁺ at 5 min; 4: SSM from the experimental group after the addition of 100 μM Ca²⁺ at 5 min. A Absorbance; t Time

DISCUSSION

To determine the sensitivity of mitochondria to Ca²⁺ loading, changes in light scattering of mitochondrial suspensions, which detect mitochondrial matrix volume, has become the technique of choice. Ca²⁺-induced mitochondrial swelling is frequently used as a marker for MPTP opening. In the current study, we explored the impact of one month of epadol treatment on SSM and IFM swelling both in Ca²⁺-free and Ca²⁺-containing solutions. Dietary supplementation with omega-3 PUFA ranging from 2 g to 8 g per day has reportedly been beneficial in the treatment of hypertriglyceridemia and conditions accompanied by chronic inflammatory processes (14). According to manufacturer’s instructions, the recommended dose of epadol is 4 g per day. We used a rather high dose (approximately five times the recommened dose for humans) of dietary omega-3 PUFA supplementation over a short period of time of to detect possible differences in Ca²⁺ sensitivity of two mitochondrial fractions. Previously, supplementation with lower doses of omega-3 PUFA for two months was shown to modulate nitric oxide synthase activity and connexion-43 expression in spontaneously hypertensive rats (15).

In the control group, no difference in Ca²⁺-induced mitochondrial swelling was observed between the two subpopulations, indicating that SSM and IFM do not differ in terms of Ca²⁺ sensitivity. In this group, SSM and IFM exhibit gradual swelling with a similar time course in Ca²⁺-free solution. Because prolonged incubation in K⁺-free medium during mitochondrial preparation results in a loss of matrix K⁺, it is conceivable that gradual mitochondrial swelling in Ca²⁺-free solution upon reconstitution of K⁺ may be explained by an uptake of K⁺ ions coupled to the inward movement of water (16,17) via mechanisms described below. Hence, our data may indicate that in control rats, the rates of K⁺ uptake in the two subpopulations of myocardial mitochondria do not differ significantly. However, we observed that in the IFM fraction isolated from the treatment group, the decrease in light scattering in Ca²⁺-free solution was significantly less pronounced compared with the SSM fraction. An obvious reason for mitochondrial swelling in Ca²⁺-free solution is K⁺ transport.

Because extramitochondrial Ca²⁺ ions are required for the activity of large conductance mitochondrial Ca²⁺-dependent K⁺ channels (mitoBCa), these channels are highly unlikely to mediate a gradual supply of K⁺ responsible for mitochondrial swelling in Ca²⁺-free solution. Opening of mitochondrial ATP-sensitive K⁺ (mitoKATP) channels has been implicated in the mechanism of ischemic preconditioning (17,18), although the existence of this channel remains debated (19). Remarkably, mitoKATP channel openers or blockers elicit either no changes in light scattering (20) or rather small (up to 5%) changes (19), suggesting that the decrease in light scattering in Ca²⁺-free solution in the IFM fraction isolated from the treatment group is unlikely to be related to inhibition of mitoKATP channels. Our own unpublished observations indicate that KATP channel openers with different chemical structures (diazoxide and flocalin, a fluorine-containing derivative of pinacidil) differentially affect mitochondrial swelling, confirming earlier reports that the majority of modulators of mitoKATP channels have a number of nonspecific effects on mitochondrial ion transport mechanisms (19,20), thus making the results difficult to interpret. Another mechanism responsible for gradual mitochondrial swelling is passive permeability to protons (ie, H⁺ leak) and cations driven by negative mitochondrial membrane potential. Although we did not directly measure mitochondrial membrane potential in the current study, it is likely that decreased light scattering of the IFM fraction isolated from the treatment group, which we observed in Ca²⁺-free solution, could result from a decreased K⁺ influx, presumably due to depolarized membrane potential. Our observation that omega-3 PUFA treatment significantly suppresses light scattering in the IFM, but not SSM fraction in Ca²⁺-free solution, may indicate differential effects of such a treatment on mitochondrial membrane potential in two mitochondrial fractions, highlighting possible intrinsic differences in the mechanisms of electro-genesis between the two mitochondrial fractions. Partial mitochondrial depolarization is well known to be beneficial for cardioprotection, a phenomenon explained by the strict voltage dependency of mitochondria for Ca²⁺ uptake. In fact, our observation that the IFM fraction exhibited higher protection against Ca²⁺-induced injury following omega-3 PUFA treatment supports our assumption that in this group, mitochondria of the IFM fraction are likely depolarized.

Differences in the sensitivity to protection elicited by dietary omega-3 PUFA may indicate that the mechanisms involved in omega-3-induced protection against Ca²⁺-induced MPTP opening in IFM and SSM differ. Several mechanisms may underpin the protective effect of dietary omega-3 PUFA against Ca²⁺-induced MPTP opening. It has been suggested that the beneficial effects of a diet rich in omega-3 PUFA are mediated by incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into cell membranes (21). It was previously reported that the intake of fish oil containing DHA and EPA for eight weeks delays Ca²⁺-induced MPTP opening both in SSM and IFM (7). This supplementation resulted in an increase in EPA and DHA content and a decrease in arachidonic acid (AA) content in cardiac mitochondrial phospholipids. Although AA was identified as an MPTP inducer (22), in vivo supplementation with AA appeared to delay MPTP opening (23), making it unlikely that modulation of free AA fully explains the beneficial effect of omega-3 PUFA on MPTP opening. Furthermore, despite increasing DHA and decreasing AA content in cardiac phospholipids, changes in MPTP opening were not observed in rats with heart failure, suggesting that changes in phospholipid composition are unlikely to be responsible for the delay in MPTP opening observed in sham-treated rats.

The same group addressed possible differences between the individual effects of EPA and DHA in normal and hypertrophied myocardium (24). It was found that supplementation with DHA alone effectively increased both DHA and EPA in cardiac mitochondrial phospholipids. These changes were accompanied by a delay in MPTP opening in both normal and hypertrophied myocardium (24). Hence, changes in the phospholipid composition of cardiac mitochondria in different pathological settings may (24) or may not (7) positively correlate with MPTP opening. The answer to the apparent paradox may lie in mitochondrial morphological and functional heterogeneity, their differential sensitivity to Ca²⁺-induced MPTP opening and, perhaps, phospholipid composition following different modalities of dietary PUFA treatment.

Recently, it was reported that cardiac IFM, but not SSM, become increasingly vulnerable to Ca²⁺-induced MPTP opening during aging (25). Considering the relatively greater abundance of IFM (80%) compared with SSM (20%), our finding that the protective effect of dietary omega-3 PUFA against Ca²⁺-induced injury was more pronounced in the IFM fraction may indicate that omega-3 PUFA supplementation may be especially beneficial in older individuals. In support of this, it was shown that a diet rich in omega-3 PUFA reverses the age-associated membrane omega-3:omega-6 PUFA imbalance, thereby improving tolerance to ischemia and reperfusion (26).

Under controlled conditions, we showed that the IFM and SSM fractions have similar sensitivities to Ca²⁺-induced MPTP opening, although the protective effect of omega-3 PUFA was noticeably more pronounced for the IFM than for the SSM fraction. Different sensitivities to MPTP opening after omega-3 PUFA treatment observed in the present study may have functional implications. While SSM are likely to play a role in signal transduction from the extracellular space, the IFM are more suitable for control of cell viability and protection against excessive Ca²⁺ overload. Different functional loads of the two mitochondrial fractions are likely to be ensured by specific signalling mechanisms. Further study is required to understand the more fundamental mechanisms of cardioprotective signalling mediated by mitochondria under controlled and pathological settings.