Although mitochondrial oxidative catabolism of fatty acid (FA) is a major energy source for the adult mammalian heart, cardiac lipotoxity resulting from elevated serum FA and enhanced FA use has been implicated in the pathogenesis of heart failure. To investigate the effects of intermediates of FA metabolism [palmitoyl-l-carnitine (Pal-car) and palmitoyl-CoA (Pal-CoA)] on mitochondrial function, we measured membrane potential (ΔΨm), opening of the mitochondrial permeability transition pore (mPTP), and the production of ROS in saponin-treated rat ventricular myocytes with a laser scanning confocal microscope. Our results revealed that 1) lower concentrations of Pal-car (1 and 5 μM) caused a slight hyperpolarization of ΔΨm [tetramethylrhodamine ethyl ester (TMRE) intensity increased to 115.5 ± 5.4% and 110.7 ± 1.6% of baseline, respectively, P < 0.05] but did not open the mPTP, 2) a higher concentration of Pal-car (10 μM) depolarized ΔΨm (TMRE intensity decreased to 61.9 ± 12.2% of baseline, P < 0.01) and opened the mPTP (calcein intensity decreased to 70.7 ± 2.8% of baseline, P < 0.01), 3) Pal-CoA depolarized ΔΨm without opening the mPTP, and 4) only the higher concentration of Pal-car (10 μM) increased ROS generation (2′,7′-dichlorofluorescein diacetate intensity increased to 3.4 ± 0.3-fold of baseline). We concluded that excessive exogenous intermediates of long-chain saturated FA may disturb mitochondrial function in different ways between Pal-car and Pal-CoA. The distinct mechanisms of the deteriorating effects of long-chain FA on mitochondrial function are important for our understanding of the development of cardiac diseases in systemic metabolic disorders.
- mitochondrial membrane potential
- mitochondrial permeability transition pore
- reactive oxygen species production
metabolic syndrome and insulin resistance are risk factors for cardiovascular diseases and are frequently accompanied by high serum fatty-acid (FA) levels. Recent studies (10, 35) have suggested that these metabolic disorders are also related to the development of clinical heart failure without any coronary heart disease (1). High serum FA levels are accompanied by an increased prevalence of serious arrhythmia and could be linked to higher mortality in patients with heart failure (22, 35). Opie proposed the concept of a “metabolic vicious cycle” (24) where increasing insulin resistance and serum FA levels, as a consequence of the hyperadrenergic state, could reduce myocardial energy efficiency in a failing heart (1).
In studies (5, 38) using transgenic mice or rat models of obesity and diabetes, it has been shown that the accumulation of lipids and related intermediates in cardiomyocytes can be a cause of cardiac lipotoxicity or cardiac cell death resulting in an impaired contractile function and cardiac hypertrophy. Although the role of metabolic alteration in the development of heart failure is not yet fully understood, it is suggested that cardiac lipotoxity as a result of enhanced FA metabolism plays an important role in the onset and development of clinical heart failure (15).
An overload of FA, specifically saturated long-chain FA such as palmitic acid (PA), affects mitochondrial function and induces cardiomyocytes apoptosis (32). PA is converted to its CoA ester form [palmitoyl-CoA (Pal-CoA)] in the cytosol (12), and Pal-CoA is taken up by mitochondria as a carnitine ester form [palmitoyl-l-carnitine (Pal-car)] immediately (33) and reconverted to Pal-CoA by carnitine palmitoyl-transferase (CPT)-II in the mitochondrial matrix (12). In previous studies using isolated mitochondria (13, 25) or cultured cells (32, 37), PA and its intermediates (Pal-car and Pal-CoA) have been shown to affect mitochondrial membrane potential (ΔΨm), the mitochondrial permeability transition pore (mPTP), activity of the respiratory chain, and the generation of ROS. Among these, the accumulation of Pal-car and Pal-CoA in myocytes may have an impact on mitochondrial function under physiological conditions. However, little is known about whether Pal-car and Pal-CoA cause distinct responses in ΔΨm, mPTP, and ROS generation in vivo. The aim of this study was to investigate the differences in the effects of FA intermediates on mitochondrial function at the myocyte level. For this purpose, we perfused permeabilized ventricular myocytes with Pal-car or Pal-CoA and monitored ΔΨm, opening of the mPTP, and the generation of ROS.
MATERIALS AND METHODS
Cell isolation and permeabilization.
This investigation conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). This investigation was approved by the Hamamatsu University School of Medicine Animal Care and Use Committee. Isolated myocytes were obtained by enzymatic dissociation from male Sprague-Dawley rats (250–350 g), and cells were kept in modified Kraft-Brühe solution (9), which contained (in mM) 40 KCl, 20 KH2PO4, 3 MgCl2, 50 glutamic acid, 10 glucose, 10 HEPES, and 0.5 EGTA (pH was adjusted to 7.4 with KOH). Just before the experiments, cells were placed in a chamber and incubated with normal Tyrode solution, which was composed of (in mM) 143 NaCl, 5.4 KCl, 0.5 MgCl2, 0.25 NaH2PO4, 1 CaCl2, 5.6 glucose, and 5 HEPES (pH 7.4 with NaOH). All experiments were conducted at room temperature (25°C) within 6 h of cell isolation, and all samples were perfused with the solutions (∼3.0 ml/min) according to the protocol. Sarcolemmal membranes of cardiac myocytes were permeabilized by saponin (0.05 mg/ml) for 30 s in a Ca2+-free internal solution, which contained (in mM) 50 KCl, 80 K-asparate, 2 Na-pyruvate, 20 HEPES, 3 MgCl2·6H2O, 2 Na2ATP, and 3 EGTA (pH 7.3 with KOH), and saponin was then removed by the continuous perfusion of a Ca2+-free internal solution for 2 min. Experiments were performed with a laser scanning confocal microscope (LSM 510, Karl Zeiss) coupled to an inverted microscope (Axiovert S100, Karl Zeiss) with a ×63 water-immersion objective lens (numerical aperture = 1.3, Karl Zeiss).
Measurement of ΔΨm.
For the measurement of ΔΨm, permeabilized myocytes were loaded with a continuous perfusion of the fluorescent indicator tetramethylrhodamine ethyl ester (TMRE; 10 nM). TMRE was excited at 543 nm with a helium-neon laser, and emission signals were collected through a 560-nm long-pass filter. Data are presented as percentages of the TMRE signal before the application of drugs. Fluorescence intensity was integrated over regions of interest (25 × 25 pixels) excluding the nuclei and the edge of the cell.
Imaging of mPTP opening.
To evaluate mPTP opening, myocytes were loaded with calcein AM (1 μM) for 25 min, and the sarcolemmal membrane was then permeabilized to remove cytoplasmic dyes. This method allowed the selective loading of calcein in mitochondria. When the mPTP opens, entrapped calcein is released from the mitochondrial matrix. Calcein was excited at 488 nm, and emission was collected through a 505- to 550-nm bandpass filter. After the experiments with Pal-car and Pal-CoA had been concluded, pore-forming antibiotic alamethicin (10 μg/ml) was applied to induce maximal calcein release from the mitochondrial matrix, and the minimum calcein fluorescence after alamethicin is regarded as 0% for the normalization of calcein fluorescence.
Measurement of ROS in skinned myocytes.
For the measurement of ROS generation, permeabilized myocytes were loaded with a continuous perfusion of the fluorescent indicator 2′,7′-dichlorofluorescin diacetate (DCF; 10 μM). Cells were excited at 514 nm with an argon laser, and emission signals were collected through a 530-nm long-pass filter. The fluorescent intensity at identical regions of interest (25 × 25 pixels) was monitored every 1 min to analyze changes in DCF signals.
Measurement of FAD oxidation.
The mitochondrial redox state of single myocytes was assessed by measuring their FAD-linked protein fluorescence. Endogenous FAD autofluorescence was excited at 488 nm with an argon laser, and fluorescence was collected through a 505-nm long-pass filter. Two-dimensional images were acquired at 1-min intervals. The FAD signal decreased to a minimum in the presence of cyanide (CN−; 4 mM), an inhibitor of cytochrome oxidase, and increased upon exposure to the uncoupler of mitochondria 2,4-dinitrophenol (DNP; 100 μM). CN− and DNP were expected to cause maximum reduction and oxidation (9), respectively. Each signal was calibrated as 0% for CN−-induced complete reduction and as 100% for DNP. Fluorescence intensity was integrated over regions of interest (25 × 25 pixels) excluding the nuclei and the edge of the cell.
Chemicals and data analyses.
All chemicals were obtained from Sigma (St. Louis, MO), and fluorescent dyes were purchased from Molecular Probes (Eugene, OR). Data are presented as means ± SE, and the number of cells or experiments is shown as n. Statistical analyses were performed using two-way repeated-measures ANOVA, followed by a Bonferroni test. A level of P < 0.05 was accepted as statistically significant.
Fatty acyl-carnitine and fatty acyl-CoA and inhibitors of CPT.
Pal-car was dissolved in the internal solution, and Pal-CoA was dissolved in ethanol. Ethanol was present in the perfused solution at a final concentration of 0.2% and had no individual effect on TMRE, calcein, DCF, or FAD intensity. In this study, we did not apply BSA to the solution to avoid attenuating the activities of these fatty acyl intermediates in the solution (13). Perhexiline, a CPT-I and CPT-II inhibitor, and oxfenicine, a CPT-I inhibitor (11), were dissolved in the internal solution.
Effects of Pal-car on ΔΨm and mPTP.
We investigated the effects of Pal-car on ΔΨm and mPTP. Figure 1A shows the time course of the changes in TMRE intensity, which represent the changes in mitochondrial ΔΨm, during and after the application of Pal-car (1–10 μM) for 10 min. Pal-car (1 and 5 μM) slightly increased TMRE intensity (115.5 ± 5.4% and 110.7 ± 1.6% of baseline, respectively, P < 0.05). However, a higher concentration of Pal-car (10 μM) depolarized ΔΨm significantly (61.9 ± 12.2% of baseline, P < 0.01). These results indicated that when appropriate concentrations of Pal-car were added in the internal solution and taken up by mitochondria, mitochondrial basal metabolism was accelerated and ΔΨm was hyperpolarized slightly. On the other hand, the application of higher concentrations of Pal-car (and possibly higher concentrations of Pal-CoA inside the mitochondrial matrix and/or excessive Pal-car in the cytosol) resulted in ΔΨm depolarization. To differentiate this effect, the same experiments were conducted with perhexiline, a CPT-I and CPT-II inhibitor. When cells were pretreated with perhexiline (0.1 μM), the subsequent application of Pal-car (1 μM in Fig. 2A and 10 μM in Fig. 2B) did not alter ΔΨm, indicating that the observed changes in ΔΨm were related to the uptake of Pal-car into the mitochondrial matrix and to its metabolism.
The opening of the mPTP has been suggested to be a mechanism of FA-induced mitochondrial damage (2, 13). We hypothesized that the opening of the mPTP could be involved in Pal-car-induced ΔΨm depolarization. We (9) have previously reported that the reduction of mitochondria-trapped calcein intensity indicates the opening of the mPTP in permeabilized myocytes. Figure 1B shows changes in calcein intensity during and after a 10-min perfusion of Pal-car. The pore-forming antibiotic alamethicin was applied after the perfusion of Pal-car to complete the maximal release of calcein from the mitochondrial matrix, providing a positive control. The application of 1 μM Pal-car did not cause any changes in calcein intensity (95.4 ± 2.9% of baseline) compared with the control. However, 10 μM Pal-car decreased calcein intensity significantly (70.7 ± 2.8% of baseline, P < 0.01 vs. control). The mPTP inhibitor cyclosporine A (CsA; 0.1 μM) attenuated the Pal-car-induced reduction of the calcein signal (84.5 ± 0.2% of baseline). From these results, it was shown that there were biphasic effects of Pal-car on ΔΨm and mPTP, i.e., only higher concentrations of Pal-car depolarized ΔΨm and opened the mPTP.
Effects of Pal-CoA on ΔΨm and mPTP.
Under conditions of increased plasma free FA levels, the concentration of Pal-CoA in the cytosolic space is also increased (30). We next investigated the effects of Pal-CoA on ΔΨm and the mPTP. In this series of experiments, cells were permeabilized, and l-carnitine did not exist in the internal solution. Therefore, this suggested that Pal-CoA applied to the internal solution could not enter into the mitochondrial matrix since the conversion of Pal-CoA to Pal-car might not occur. Figure 3A shows the time course of the changes in TMRE intensity during a 10-min perfusion of Pal-CoA (0.1–10 μM) and after the wash out. In contrast to Pal-car, Pal-CoA (0.1–5 μM) decreased the TMRE signal at 10 min (90.8 ± 3.8% with 0.1 μM, 84.7 ± 2.8% with 0.5 μM, 65.8 ± 4.2% with 1 μM, and 56.9% ± 13.0% with 5 μM, P < 0.05 and P < 0.01 vs. baseline) dose dependently. However, when cells were exposed to a higher concentration of Pal-CoA (10 μM), ΔΨm dissipated rapidly and completely within a few minutes. These results suggest that the accumulation of Pal-CoA in the cytosolic space altered ΔΨm, and the higher concentration of Pal-CoA might be harmful to the maintenance of mitochondrial function.
We next examined whether Pal-CoA could open the mPTP by measuring the reduction of calcein intensity. The time course of the changes in calcein intensity in the presence of Pal-CoA is shown in Fig. 3B. In contrast to the effects of Pal-car on the mPTP (Fig. 1B), the reduction of calcein intensity by Pal-CoA (1–10 μM), which were the concentrations used to depolarize ΔΨm, was the same as that of the control perfusion. These results suggest that the depolarization or dissipation of ΔΨm by Pal-CoA is not related to the opening of the mPTP.
Effects of Pal-car and Pal-CoA on the generation of ROS.
It has been suggested that ROS generation is related to FA-induced mitochondrial damage and apoptosis (29) and that ROS are a potent inducer of mPTP opening (7). Figure 4 shows the time course of the changes in DCF intensity during and after a 10- min perfusion of Pal-car (1 and 10 μM) and Pal-CoA (10 μM). The data show that 1 μM Pal-car promoted a gradual and modest increase (1.6 ± 0.2-fold of baseline, P < 0.05 vs. baseline) in DCF fluorescence, and the application of 10 μM Pal-car caused a rapid and larger increase (3.4 ± 0.3-fold of baseline, P < 0.01 vs. baseline) in DCF intensity, and DCF fluorescence decreased rapidly after the withdrawal of Pal-car. Since the decrease in the DCF signal cannot be due to the reduction of DCF by intracellular reductases and the efflux of oxidized DCF from mitochondria is dependent on ΔpH (membrane potential and proton gradient), a modest recovery of ΔΨm after Pal-car washout may have affected the efflux of this dye. In addition, the constant perfusion of internal solution during protocols could wash out the leaked DCF immediately.
When cells were exposed to 10 μM Pal-car for 5 min and the ROS scavenger N-acetylcysteine (NAC; 400 μM) (21) was added to the internal solution in the continuous presence of 10 μM Pal-car, the increase in DCF intensity was completely eliminated, confirming that these changes in DCF fluorescence were attributed to the generation of ROS. In contrast to Pal-car, when cells were exposed to 10 μM Pal-CoA for 10 min, there was only a transient and slight increase in DCF signal (1.8 ± 0.2-fold of baseline, P < 0.05 vs. baseline) followed by the reduction of the signal. Thus, the mechanisms of ROS generation by Pal-car and Pal-CoA were shown to be different and only the perfusion of Pal-car generated ROS significantly in the mitochondrial matrix.
mPTP inhibitor and ROS scavenger prevented Pal-car-induced ΔΨm depolarization.
The changes in TMRE intensity with Pal-car (10 μM) in the presence of CsA, a mPTP inhibitor, are shown in Fig. 5A. When cells were pretreated with CsA (0.1 μM), the reduction of TMRE intensity by Pal-car (10 μM) was notably inhibited (87.7 ± 6.2% of baseline, P < 0.01 vs. Pal-car alone). To further confirm that the mPTP-mediated depolarization of ΔΨm can be related to the generation of ROS, the effects of NAC on Pal-car-induced ΔΨm depolarization were investigated. As shown in Fig. 5B, pretreatment with NAC (400 μM) slightly increased (107 ± 3.0% of baseline, P < 0.01 vs. Pal-car alone) rather than decreased TMRE signals after the application of Pal-car (10 μM). These results indicate that both a CsA-sensitive opening of the mPTP and ROS generation are related to the depolarization of ΔΨm induced by Pal-car under our experimental conditions.
Extramitochondrial Pal-CoA did not alter the mitochondrial redox state.
As shown in Fig. 3, A and B, Pal-CoA depolarized ΔΨm without opening the mPTP. We then applied Pal-CoA and l-carnitine simultaneously to encourage the uptake of Pal-CoA into the mitochondrial matrix. When cells were pretreated with l-carnitine (2.5 mM) (6) and then exposed to 10 μM Pal-CoA simultaneously, the Pal-CoA-induced depolarization of ΔΨm was prevented significantly. In addition, this ΔΨm-preventing effect of l-carnitine was inhibited by the CPT-I inhibitor oxfenicine (10 μM; Fig. 6A). These results indicated that the stimulation of mitochondrial Pal-CoA uptake with l-carnitine prevented the deleterious effects of extramitochondrial Pal-CoA.
Finally, we investigated how Pal-CoA depolarized ΔΨm. A previous study (2) has proposed several mechanisms for the depolarization of ΔΨm induced by Pal-CoA, such as the direct inhibition of the respiratory chain, the generation of ROS, and increased proton leak. We have already shown that the application of Pal-CoA caused only small and transient ROS generation (Fig. 4). In this study, we assessed the activity of the respiratory chain by measuring FAD autofluorescence. Acceleration of the respiratory chain increases the oxidation of FADH to FAD, resulting in the elevation of FAD autofluorescence. When myocytes were perfused with 10 μM Pal-CoA for 10 min, there were no changes in FAD autofluorescence (Fig. 6B). These results suggest that, under our experimental conditions, a direct effect of Pal-CoA on the respiratory chain was unlikely.
It is well established that an accumulation of excess FA in cardiac myocytes leads to cardiac lipotoxity, which is associated with the development of heart failure (1, 38). Since lipotoxity from the accumulation of long-chain FA is specific for saturated FA (15), we investigated the effects of Pal-car and Pal-CoA, intermediates of the most common saturated FA, on ΔΨm, opening of the mPTP, and the generation of ROS in saponin-treated permeabilized cardiac myocytes. In this study, we demonstrated that 1) lower concentrations of Pal-car caused a slight hyperpolarization of ΔΨm but did not open the mPTP, 2) the higher concentration of Pal-car depolarized ΔΨm and opened the mPTP, 3) Pal-CoA depolarized ΔΨm without opening the mPTP, and 4) only the higher concentration of Pal-car increased ROS generation. These results indicate that there are different actions between Pal-car and Pal-CoA on mitochondrial function.
Previous studies examining the effects of long-chain FA or their intermediates on mitochondrial function have been conducted mainly using isolated mitochondria (13, 25) or cultured cells (32, 37). Compared with those experiments with isolated mitochondria, the use in this study of permeabilized myocytes provides an opportunity to examine mitochondrial function under more physiological circumstances (14) and allows the cytosolic concentration of Pal-car or Pal-CoA to be controlled without changing the conditions of the extramitochondrial medium (i.e., cytosolic Ca2+, ATP, or other substrates) and intracellular architectures (14, 27). The concentrations of Pal-car (18, 20) or Pal-CoA (31) used in this study were within the ranges reported in previous studies.
Effects of Pal-car on ΔΨm and the mPTP.
We demonstrated the biphasic effect of Pal-car on ΔΨm, where only the higher concentration of Pal-car (10 μM) depolarized ΔΨm, whereas lower concentrations of Pal-car (1 and 5 μM) hyperpolarized ΔΨm (Fig. 1A). These effects of Pal-car were prevented with the CPT inhibitor perhexiline (Fig. 2, A and B), which has been reported to inhibit both CPT-I and CPT-II (11). Since the inhibition of CPT-II blocked the conversion of Pal-car to Pal-CoA in the inner membrane, it is suggested that the observed changes in ΔΨm were indeed related to the uptake of Pal-car into the mitochondrial matrix and to its metabolism. It has been reported that, in the cytosolic space of rat ventricular myocytes, the concentration of Pal-car was 2–2.5 μM (8). After Pal-car enters the mitochondrial matrix and is reconverted to Pal-CoA, it can be oxidized and stimulates electron transport. The hyperpolarization of ΔΨm could be due to the acceleration of the respiratory chain achieved from the optimal supply of Pal-CoA as an energy substrate (17). In the experiments on isolated mitochondria, 10 μM Pal-car has been used as the substrate for mitochondrial respiration. Differences in experimental conditions may explain these discrepancies.
We also demonstrated that 10 μM Pal-car decreased calcein intensity significantly, which was blocked by CsA (Fig. 1B), suggesting that Pal-car (10 μM) opened the mPTP. Since CsA also inhibited Pal-car-induced depolarization of ΔΨm (Fig. 5A), the opening of the mPTP could explain the depolarization of ΔΨm by a higher concentration of Pal-car. A direct action of Pal-car on the mPTP has not been reported, although a previous study (16) reported that much higher concentrations of Pal-car (at least 25 μM) were required to enhance its amphiphilic effect. However, when 10 μM Pal-car was introduced in the cytosolic space and the uptake of Pal-car into mitochondria matrix was inhibited by perhexiline, there were no changes in ΔΨm under our experimental conditions (Fig. 2B). When a large amount of Pal-car enters the matrix, concentrated Pal-car itself could serve as an amphiphile to damage mitochondria. Since most Pal-car is converted rapidly to Pal-CoA, existing amounts of Pal-car are estimated to be <15% of total carnitine in the mitochondrial matrix (8). It is therefore unlikely that excessive Pal-car, either inside or outside mitochondria, altered ΔΨm or opened the mPTP directly.
Effects of Pal-CoA on ΔΨm and the mPTP.
Pal-CoA plus l-carnitine was applied to the internal solution to facilitate Pal-CoA transportation across the inner mitochondrial membrane, as cytosolic l-carnitine may not exist by itself after permeabilization of the plasma membrane. In this study, we showed that Pal-CoA depolarized ΔΨm (Fig. 3A) and that the application of l-carnitine (6) with Pal-CoA inhibited Pal-CoA-induced depolarization (Fig. 6A), indicating that an excessive amount of extramitochondrial Pal-CoA impairs mitochondrial function. These results are in good agreement with a previous study (26) that demonstrated that l-carnitine has protective effects against the mPTP and ischemia-reperfusion injury. It has been reported that when chronic substrate supply exceeds the capacity of oxidation or when the oxidation process is compromised, intracellular lipid metabolites accumulate, which leads to arrhythmia and contractile dysfunction (22, 23).
Several mechanisms could be involved in the depolarization of ΔΨm induced by fatty acyl-CoA: 1) membrane peroxidation, 2) direct inhibition of the respiratory chain, 3) opening of the mPTP, and 4) proton-leaking effects (2). Fatty acyl-CoA inserts its hydrophobic moiety and binds to biological membranes because of its amphiphilic effect (2). Pal-CoA induces a peroxidation of the membrane at concentrations as low as 10 μM (16) and depolarizes ΔΨm due to amphiphilic properties outside mitochondria (4, 16). Pal-CoA promotes mPTP opening by either 1) its interaction on adenine nucleotide translocase (ANT) increasing the probability of mPTP opening (direct effect) (28, 36) or 2) its protonophoric effects depolarizes ΔΨm and it opens the mPTP (indirect effect) (2, 13).
In this study, Pal-CoA did not accelerate calcein leakage from mitochondria despite the complete dissipation of ΔΨm (Fig. 3, A and B). In addition, we did not observe any changes in FAD autofluorescence from Pal-CoA (Fig. 6B), indicating that Pal-CoA did not alter the mitochondrial redox state significantly and that the inhibitory effect of Pal-CoA on the respiratory chain was not significant. Previous studies (13, 16, 36) have reported on the protonophoric properties of long-chain FA, and the strongest activity was found for C12-C16 saturated FA (2). However, fatty acyl-CoA does not exert protonophoric activity or uncouple oxidative phosphorylation, because it is unable to cross the inner mitochondrial membrane and it does not flip-flop because of the large hydrophilic CoA head (2). Taken together, it is likely that Pal-CoA-induced depolarization or dissipation of ΔΨm could be due to the proton-leaking effects of Pal-CoA acting from outside the mitochondria.
Effects of Pal-car and Pal-CoA on ROS generation.
In this study, we demonstrated that Pal-car increased ROS generation (Fig. 4) and that NAC prevented the depolarization of ΔΨm (Fig. 5B), suggesting the contribution of Pal-car-induced ROS generation to the opening of the mPTP. On the other hand, Pal-CoA caused ROS generation only for a short period, and total amounts of ROS were significantly smaller compared with those of Pal-car. The distinct responses of ROS generation caused by FA intermediates have not previously been reported, and this is the first study to demonstrate that there are significant differences between Pal-car- and Pal-CoA- induced ROS generation in skinned myocytes.
The mechanism of mitochondrial ROS generation induced by FA remains to be clarified. Several studies (13, 32) have reported that direct inhibition of the respiratory chain could be attributed to ROS generation by FA. Schönfeld and Wojtczak (29) reported that FA abolishes ROS generation in rat heart mitochondria when electron transport was reversed. As Pal-car supplies electrons and stimulates (rather than inhibits) the mitochondrial respiratory chain (29), it is postulated that the acceleration of electron transport might contribute, at least in part, to Pal-car-induced ROS generation under our experimental conditions. It has been reported that a transient increase in ROS triggers a burst generation of ROS in neighboring mitochondria (ROS-induced ROS release) (39). This ROS-induced ROS release may explain the rapid and larger increase in DCF fluorescence caused by Pal-car observed in this study. Pal-CoA may alter the mitochondrial membrane structure by its amphiphilic properties (3), and generated ROS may cause peroxidation of the mitochondrial membrane (16). These structural and functional changes could be responsible for the observed Pal-CoA-induced ROS generation. It has also been reported that Pal-CoA could stimulate ROS generation by inhibiting ANT (13). Further studies are required to identify the distinct mechanism of mitochondrial ROS generation between Pal-car and Pal-CoA.
In patients with metabolic disorders, such as metabolic syndrome, obesity, and diabetes mellitus (19), cardiac lipotoxity has been implicated in the pathogenesis of clinical heart failure as a result of enhanced FA metabolism secondary to elevated serum FA (15). Recent clinical studies using positron emitted tomography techniques have provided evidence of higher myocardial FA uptake ratios in patients with heart failure (34) and an increased FA utilization and accumulation of FA that could lead to myocardial dysfunction (1, 2). Our results suggest that the excessive exogenous intermediates of long-chain saturated FA may disturb mitochondrial function in different ways between Pal-car and Pal-CoA. These findings may be useful to establish strategies for the treatment of heart failure caused by the impairment of FA metabolism.
This study demonstrates that excessive amounts of Pal-car, which could result in an inappropriate increase of Pal-CoA in the mitochondrial matrix, are harmful to mitochondrial function by accelerating ROS production and by increasing the probability of opening of the mPTP. On the other hand, excessive cytosolic Pal-CoA itself may affect mitochondrial function by causing a loss of ΔΨm from outside the mitochondria. These distinct mechanisms of the deteriorating effects of long-chain FA on mitochondrial function are important for our understanding of the development of cardiac diseases in systemic metabolic disorders.
This work was supported by Japan Grants-In-Aid 13670703 (to H. Katoh) and 50135258 (to H. Hayashi) and by Grant Aid for the Center of Excellence from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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