Statistical differences were assessed by one way-ANOVA. P < 0.05 was considered statistically significant. Data were expressed as the mean
± SEM.
30
Results
Palmitic acid (PA)-induced apoptosis, and cells death.
To clarify palmitic acid induced cytotoxicity in cardiacmyocyte, H9c2 were treated with different concentrations of PA for 24 and 48 h. The result of MTT showed that after treatment with various concentrations of PA for indicated time peroid significantly decreased the cell viability in a dose-dependent manner (Fig.1A). The cell viability is lower then 50% in concentration of PA on 0.5mM treated with H9c2 cells ,therefore 0.5 mM was used for the following experiments.We also used TUNEL analysis for observing cells undergoing apoptosis. After incubation with PA for 24 h, we observed a significant increase apoptosis bodies (Fig.1B).
Palmitic acid increased generation of mitochondrial reactive oxygen species (ROS).
Previous investigation demonstrated that free fatty acid (FFA) induced-oxidative stress plays an important key role in development of cardiovascular disease in metabolic syndrome (Madamanchi and Runge 2007). We therefore, examined the cellular ROS levels after treatment with 0.5 mM PA for 24 h by fluorometric assay using DCF-AM and DHE.
As shown in Fig 2A and 2B, an approximately three-fold and two-fold increase of ROS and superoxide was observed in cells incubated with PA compared with untreated cells. NADPH oxidase and mitochondrion are known major sources of superoxide (Land 2012), so we measured the expression levels of NADPH oxidase subunits by using Western blot (Fig.2D) and generation of superoxide in mitochondira by using
31
MitoSOX™ Red (Fig.2C). In Fig 2C, an approximately three-flod increase of mitochondrial superoxide was observed in cells incubated with 0.5 mM PA for 24 h compared with normal condition. However, the protein levels of gp91phox, p47phox, Rac-1 protein in H9c2 cells in a time dependent manner (0-24h) (Fig.2D).
Intracellular ROS levels are regulated by the balance between ROS generation and antioxidant enzymes such as catalase or SOD. Besides, the involved ROS are able to inactivate antioxidative enzymes that additionally increase the imbalance in favor of oxidative stress. Therefore, we investigated the expression of its isoforms in H9c2 cells in response to PA. Our results showed that the antioxidant enzymes SOD1 and SOD2 decreased in H9c2 cells treatment with PA for 0.5mM (Fig.2E).
Palmitic acid led to collapse of mitochondria member potential
To examine whether influence of mitochondrial disruption accounts for the apoptosis effect of PA, we tested the effect of PA on mitochondrial permeability. When H9c2 cells were exposed to PA (0.5 mM), the △Ψm was depolarized, quantitative analysis from flow cytometry supported these findings (Fig.3A).
Palmitic acid induced-apoptosis involved in a mitochondrial- dependent pathway.
BCL2 family proteins are upstream regulators of mitochondrial membrane potential. Since PA depolarized △Ψm, whether PA also influenced Bcl2 family protein was investigated. After treated PA for different times (0-24 h), the immunobolotting studies demonstrated that
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PA downregulated the antiapoptoic (BCL2 and p-AKTser473) and upregulated the proapoptoic (Bax) proteins, also increased caspase 3 activity in H9c2 cells (Fig.3B).
It is known that disruption of mitochondrial membrane function results in the discharge of the mitochondrial enzyme cytochrome c into the cytosol. Consequently, mitochondria were separated from the cytosolic fraction and detected by Western blotting. As show in Fig.3C, the amount of cytochrome c released into the cytosolic fraction was much greater in H9c2 cells that had been incubated with PA for 24 h than in control cells.
The results show that PA significantly induced release of cytochrome c.
Role of MAPK family proteins, NFκB signaling pathway in PA induced apoptosis.
To investigate whether MAPK family protiens were involved in the apoptosis-related signaling pathways activated in H9c2 treated with PA, we examined the expression levels of MAPK family proteins by Western blot. Our results showed that the phosphorylation of JNK, but not ERK or P38 was increased after treatment with PA for 24 h (Fig.4A).
Accumulated evidne indcaed that IκB kinase/NF-κB (IKK/NFκB) signaling pathways play critical roles in a variety of physiological and pathological processes many stimuli activate NF-κB, mostly through IκB kinase–dependent (IKK-dependent) phosphorylation and subsequent degradation of IκB proteins. The liberated NFκB dimers enter the nucleus, where they regulate transcription of diverse genes encoding cytokines, growth factors, cell adhesion molecules, and pro- and antiapoptotic proteins. In accordance with previous findings, our results showed protein
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level of NFκB was increasing in the nuclear fraction (Fig.4B). In addition, cells were transfected with construct containing the NFκB-responsive luciferase reporter gene (NFκB Luc) to further confirm the effects of PA on NF-κB activation. The result of lucisferase assay indicated that NFκB promoter activity was increasing in a time-dependent manner (Fig.4C). In order to identify whether JNK is an upstream regulator of NF-κB, we knockdown JNK and NF-κB by using si-RNA (10nM) to advance study PA-induced apoptosis pathway. Cells were transfected with si-RNAs for 24 h and then treated with PA plus anti-oxidant (NAC) (500μM) for 24 h, apoptosis could be markedly suppressed by JNK, NFκB, and NAC. The data showed NFκB si-RNA had no effect on PA-induced JNK activity (Fig.4D). These observations indicate that JNK/NFκB pathway could mediate cardiomyocyte cell apoptosis induced by palmatic acid, but NFκB has no influence on JNK activity.
HDL downregulated palmitic acid-induce apoptosis
HDL is a complex, bioactive particle, containing multiple acute phase response proteins, protease inhibitors, and complement regulatory proteins. So, I would like to know whether HDL can downregulated palmitic acid-induce apoptosis in H9c2 cardiomyocytes cells.
The viability of cells incubated with PA in the absence or presence of indicated concentrations of HDL was assessed using the MTT assay (Fig.5A). The result showed that PA significantly reduced viability in H9c2 cells after 24h of incubation; however, pretreatment with HDL inhibited PA-induced cytotoxicity of H9c2 cells dose dependently. Next, we examined whether HDL possesses antiapoptoic effects in PA-treated
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H9c2 cells. To further determine whether HDL could protect against PA-induced apoptosis, PA-treated cells were analyzed biochemically via Annexin V binding assay (Fig.5B) and TUNEL and DAPI staining assay (Fig.5C) and evaluated by flow cytometry (Fig.5D) and microscopic observation (Fig.5C). Our results showed that the cells showed typical features of apoptosis, including the formation of compressed nuclei after treated with PA for 24h, which were, however, not observed in the HDL-pretreated H9c2 cells and also reduced the phenomenon of apoptosis in dose dependently. Phase-contrast microscopy was performed to examine the protective effects of HDL on morphological features of H9c2 cells after exposure to PA, the number of shrunken cells of cells with blebbing membranes was significantly reduced by the presence of HDL (Fig.5C) and anti-oxidant (NAC,500μM) (Fig.5D).
HDL inhibits palmitic acid-induce ROS and superoxide generation.
Studies demonstrated that PA induce elevate the concentration of cellular ROS, which subsequently led to the change the cell signaling pathway to mediate cell dysfunction. Therefore, we investigated the effects of HDL on generation of ROS, a potential factor related to PA-induced H9c2 cells injury, by using hydroxyl radical sensitive probe 2’,7’-dichlorofluorescein acetoxymethyl ester (DCF-AM)(Fig.6A) and MitoSOX™ Red mitochondrial superoxide indicator (Fig.6B). The levels of ROS and mitochondrial superoxide generation have significantly decreased in H9c2 cells after pretreatment with HDL (25-100μg/ml) for 2h before exposure to PA (0.5 mM) in a dose-dependent manner (Fig.6C), the result were measured by flow cytometry. Intracellular ROS levels are
35
regulated by the balance between ROS generation and antioxidant enzyme. Besides, the involved ROS are able to inactivate antioxidative enzymes that additionally increase the imbalance in favor of oxidative stress. Therefore, we clarify the expression of SOD isoforms in H9c2 cells in response to PA. The results showed that HDL significantly reduced the suppression of SOD activity caused by PA in dose dependent manner, expression was diminished after treatment with PA for 24h and could be significantly rescued by pretreatment with HDL (25-100μg/ml) (Fig.6D).
Sustain exposures HDL can reduce mitochondrial ROS in neonatal cardiomyocytes treated with PA.
To verify lipotoxicity induced cardic induced cellular ROS generate, and wether HDL could attenuate the phenomenon. We examined the cellular of mitochondrial superoxide generation in cultured primary rat neonatal cardiomyocytes. Pretreatment of neonatal cardiomyocytes with HDL (25-100μg/ml) for 2h before exposure to PA for 24h. We used MitoSOX™ Red mitochondrial superoxide indicator to confirm by microscopic observation (Fig.7A) and flow cytometry (Fig.7A). As show in Fig.7, PA enhance mitochondrial superoxide generation returned to levels close to those seen in control cells when neonatal cardiomyocytes were treated with HDL (25-100μg/ml) to sitmulation with PA, there were also reversed by anti-oxidant (NAC,500μM) and mitochondrial superoxide inhibitor (Rotenone,5μM).
36
HDL stabilized on mitochondrial transmembrane permeability transition.
To examine whether inhibition of mitochondrial disruption accounts for the anti-apoptotic effect of HDL, we examined the effects of HDL on mitochondrial permeability. When H9c2 cells were exposed to PA (0.5mM), the △Ψm was depolarized, as shown by the increase in green fluorescence. Pretreatment with HDL reduced the change in △Ψm, as indicated by repression of green fluorescence and restoration of red fluorescence. As shown in Fig.8, PA caused a marked increase in JC-1 green fluorescence (middle) compared with the control (left).
Preatreatment with HDL (100μg/ml) caused marked inhibition of this PA-induced apoptotic index (right).
HDL restores surivial protein expression and suppresses caspase 3 activity.
Since PA depolarized △Ψm whereas HDL maintained it, whether HDL also influenced the equilibrium of Bcl-2 family proteins was investigated.
Immunoblotting studies demonstrated that PA downregulated the antiapoptotic and survival protein (Bcl2, p-AKTser473), also upregulated the proapoptoic protein (Bax), whereas HDL pretreatment effectively repressed these PA-induced proapoptoic events (Fig.9). Therefore, activated caspase 3 is a key factor in the execution of mithchondrial apoptosis (Narula, Pandey et al. 1999). Wether PA and HDL ultimately influence this factor to modulate apoptosis, we subsequently determined the pro-form and active-form of caspase 3 by immunoblotting (Fig.9).
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The data showed that active caspase 3 was significantly increased in cells that had been treated with PA was suppressed in cells that had been pretreated with HDL.
HDL decreased p-JNK and p-NFκB protein expression, and down-regulation of promoter activity in H9c2 cardiomyoblast cells.
It has been shown that the transcriptional factor NFκB can be induced by a multitude of atimuli, including cytokines and ROS. However, in cardiomyocytes, NFκB activation has been found to produce cell apoptosis instead of preventing the cells from apoptosis. Therefore, we sought to determine whether HDL inhibits NFκB-triggered downstream inflammatory proteins in H9c2 cells. We investigated the immunostaining of p-NFκB, p-JNK and NFκB-triggered downstream inflammatory proteins, to determine whether HDL inhibits the phenomenon. As show in Fig.10A, pretreated with HDL (25-100μg/ml) significantly inhibited p- NFκB, p-JNK, COX2, and MMP-3 protein expression in a dose-dependent manner. Further, the activation of NFκB was measured in terms of its ability to promote of target gene expression. The result of the luciferase assay were used to represent the activity of NFκB regulate the expression of its target genes. Cells transiently transfected with NFκB lucisferase plasmid after 24h were exposed to PA with different concentrations of HDL (25-100μg/ml) for 24h. As shown in (Fig.10B), there was an approximately threefold increase in lucisferase activity in H9c2 cells stimulated with PA as compared with control. Pretreatment with HDL (25-100μg/ml) inhibited PA-induced luciferase activity in a dose-dependent manner. These finding indicate that PA causes activation
38
of NFκB, and HDL could significantly inhibit NFκB activity.
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Discussion
In the present study we found that palmitic acid induced significant apoptosis and ROS generation in H9c2 at the concentrations above 0.5mM. Although some papers were obtained palmitic acid with this ratio, it is different from the most often used 2/1 ratio (de Vries, Vork et al.
1997; Hickson-Bick, Buja et al. 2000; Ostrander, Sparagna et al. 2001).
Dietary fats modify the composition of cellular and mitochondrial membranes (Innis and Clandinin 1981; Lemieux, Blier et al. 2008), affecting their susceptibility to peroxidation by ROS (Pamplona, Portero-Otin et al. 2000). Oxidative stress is recognized as an important trigger in the development of cardiovascular disease (Fearon and Faux 2009). Mitochondrial β-oxidation of fatty acids is the major source of energy for the heart. Mitochondria are also central to stress-induced programmed cell death. In addition, in nonphagocytic cells, these organelles are the principal site of ROS production, via the electron transport chain (Hickson-Bick, Sparagna et al. 2002). Our results showed that plamitic acid-induced ROS generation through mitochondria not from NADPH complex (Fig.2C.D). Antioxidant defense enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase.
There are three types of SOD expressed in blood vessel: cytosolic Cu/Zn-SOD, Mn-SOD localized in mitochondria, and an extracellular form of Cu/Zn-SOD (Faraci and Didion 2004). SOD protects against superoxide-mediated cytotoxicity by rapidly dismutating O2– to H2O2.
Cu/Zn-SOD, after treated with palmitic acid, decreased Cu/Zn- SOD and Mn-SOD protein expression level (Fig.2E). Palmate-induced apoptosis in
40
the neonatal cardiomyocyte is associated with a decrease in the mitochondrial membrane potential, also decreasing the ability of the mitochondria to produce ROS (Hickson-Bick, Sparagna et al. 2002). ROS have been implicated in signal transduction pathways leading to a modulation of the DNA-binding activities of the transcription factor NFκB (Maulik, Sasaki et al. 2000), implying a role for alterations in gene transcription as a response to oxidative stress, p38 MAPK, NH2-terminal Jun kinases/stress-activated protein kinases (JNK/SAPK), advanced glycosylation end-products (AGE)/receptor for AGE (RAGE), and protein kinase C (PKC) (Evans, Goldfine et al. 2002).Our results showed that palmitic acid induced ROS generation and decreased mitochondria membrane potential, also downregulated the antiapoptoic (BCL2 and p-AKTser473) and upregulated the proapoptoic (Bax) proteins, furthermore, increased caspase 3 activity in H9c2 cells (Fig.3B). Palmitic acid disruption of mitochondrial membrane function results in the discharge of the mitochondrial enzyme cytochrome c into the cytosol. In our study, we found that palmitic acid increased JNK/SAPK protein expression, but not p38 MAPK and ERK (Fig.4A).We assumed that palmitic acid induced-damage through JNK/SAPK dependent pathway. The stress-activated protein kinases JNK1 and IKK are central signal transducers in innate immunity and stress responses that control the expression of several proinflammatory genes (Solinas and Karin 2010).
Recently it has become evident that interference with either JNK1 or IKK activity improves insulin signaling in mouse models of obesity and lipid-induced glucose intolerance (Yuan, Konstantopoulos et al. 2001;
Hirosumi, Tuncman et al. 2002). Moreover, JNK and IKK are also
41
downstream of pathways activated by toxic lipids and excessive glucose levels (Solinas and Karin 2010). In addition to JNK activation (Kamata, Honda et al. 2005), oxidative stress was proposed to activate NFκB (Schreck, Rieber et al. 1991; Sen and Packer 1996; Manna, Zhang et al.
1998). However, the link between NFκB and ROS has become complex because NFκB activation has anti-oxidant functions (Li and Karin 1999;
Pham, Bubici et al. 2004; Kamata, Honda et al. 2005). JNK activation may, however, promote ROS accumulation (Ventura, Cogswell et al. 2004) and link ROS production to insulin resistance and loss of β-cell function (Kaneto, Matsuoka et al. 2007; Temkin and Karin 2007; Matsuzawa and Ichijo 2008). We therefore used siRNA to figure out whether JNK/ NFκB pathway could mediate cardiomyocyte cell apoptosis induced by palmitic acid. As shown in Fig.4D NFκB has no influence on JNK activation.
Epidemiological and clinical studies have demonstrated the inverse association between HDL cholesterol levels (HDL-C) and the risk of coronary heart disease (CHD) (Gordon and Rifkind 1989; Assmann, Schulte et al. 1996). Low HDL-C is the most frequent dyslipoproteinemia in patients with premature infarction (Genest, Martin-Munley et al. 1992) and is an independent predictor of recurrent coronary events(Bolibar, von Eckardstein et al. 2000; Ridker 2001). Furthermore raising HDL, decreases the incidence of coronary artery disease (Robins 2001). Several different actions are attributed to HDLs, which taken all together, have an anti-atherogenic effect. The primary action is the reverse cholesterol transport, mentioned above. Other actions have been described in vitro and in animals, such as: antioxidant, anti-inflammatory, platelet antiaggregant, anticoagulant, profibrinolytic, and endothelial protection
42
effects (von Eckardstein and Assmann 2000; Nofer, Kehrel et al. 2002;
Alsheikh-Ali, Kuvin et al. 2005). In summary, the present results indicated that HDL attenuates palmitic acid-induced cardiomyocyte lipotoxicity and oxidative dysfunction via modulating mitochondria dependent pathway and p-JNK/NFκB signaling (Fig.3, 4). Therefore, reduce the downstream of superoxide-induced ROS generation and impairment of antioxidant enzymes, and inflammatory protein expression (Fig.10). In addition, HDL inhibited palmitic acid-induced cell death and apoptosis in cardiomyocytes (Fig.9). Further studies are required to confirm the effect of HDL on the inhibition of palmitic acid mediated pro-atherogenic effects and the effectiveness in vivo. Our findings may be a relevant therapeutic molecular mechanism in the improvement of cardiovascular disease. In H9c2 cells, several lines of evidence demonstrated that palmitic acid are taken up by the heart either via CD36/FATP transporters (Lopaschuk, Ussher et al. 2010). Whether HDL protects the cells against palmitic acid-induced apoptosis via Apo A-1, SR-B1 or other receptors will be another issue we can identify in the future study.
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