Introduction
Hypertension is the most common risk factor for congestive heart failure (1), which can induce apoptotic changes in heart function and peripheral vascular resistance (2). Evidence indicates higher levels of activated cardiomyocyte apoptosis and cardiac apoptosis in hypertensive models (3). Since the side-effects of western drugs in the treatment of cardiac diseases cannot be avoided, the investigation of natural products or dietary supplements which can protect cardiac abnormalities and injuries is essential (4).
Apoptosis is a recognized mechanism for the elimination of redundant cells, although it may also inhibit cell proliferation. In fact, it has been suggested that apoptosis plays a critical role in the pathogenesis of cardiac disorders (3,5,6). An increase in the levels of the pro-apoptotic protein, angiotensin II (Ang II), has been observed in hypertension, coronary artery disease, left ventricular hypertrophy and heart failure and may function as a regulator of apoptosis in cardiac tissues (7,8). Both Fas-dependent and mitochondrial-dependent apoptotic pathways are considered as major pathways which directly cause cardiac apoptosis (9,10).
The ‘extrinsic’ Fas-dependent apoptotic pathway is initiated by binding the Fas ligand to the Fas death receptor or by binding tumor necrosis factor (TNF)-α to TNF receptor 1, which results in the clustering of receptors, initiating an extrinsic pathway (11). A previous study reported that cardiac Fas receptor-dependent apoptotic pathways were activated in obese rat hearts, which is the one of the possible mechanisms behind cardiac abnormalities in obesity (12). The ‘intrinsic’ mitochondrial-dependent apoptotic pathway is mediated by internal factors, particularly in the mitochondria, the main site of action for apoptosis-regulating proteins, exemplified by the Bcl-2 family, such as Bax, Bad, t-Bid and Bak (11). Pro-apoptotic and anti-apoptotic Bcl-2 family members can homodimerize or heterodimerize to each other and appear to interact with and neutralize each other, so that the relative balance of these effectors strongly influences the release of cytochrome c. When cytochrome c is released from the mitochondria into the cytosol, it is responsible for activating caspase-9, which further activates caspase-3 and executes the apoptotic program (13). In a recent study, we observed cardiac mitochondrial-dependent but not Fas-dependent apoptosis in hamsters fed a hyper-cholesterol diet, and demonstrated the cardioprotective effects induced by the activation of the insulin-like growth factor-I receptor (IGF-IR) survival pathway by treatment with Li-Fu formula (4).
Previous studies have indicated that insulin-like growth factor (IGF)-1 signaling contributes to the modulation of survival responses in cardiomyocytes and that low IGF-I levels are associated with a high risk of myocardial infarction and heart failure (11,14). IGF-1 is the survival factor through which IGF-1R activates the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (PKB) pathway, thus preventing myocyte apoptosis (15). In particular, activated PI3K enhances the levels of phosphorylated Akt (p-Akt) (16), which in turn regulates the activity of phosphorylated-Bad (p-Bad) and Bcl-2 to prevent the apoptosis of cardiomyocytes (11).
The consumption of γ-aminobutyric acid (GABA)-enriched fermented milk has been reported to depress elevated blood pressure in spontaneously hypertensive rats (SHRs) and mildly hypertensive humans (17–19). In a previous study, we demonstrated that purple sweet potato (PSP) [Ipomoea batatas (L.) Lam.] contains high levels of anthocyanin and can easily grow in subtropical areas through fermentation by 3 lactic acid bacteria (LAB), with high levels of GABA (20). However, the protective effects of probiotic-fermented milk and its derivatives, such as GABA on cardiac apoptosis have not been reported. This study was therefore undertaken to examine the potential benefits of GABA-enriched PSP yogurt (PSPY) on the cardiac apoptotic pathways in SHR hearts. We demonstrate that PSPY attenuates cardiac apoptosis by activating the PI3K/AKT survival signaling pathway.
Materials and methods
Bacterial strains and growth conditions
In this study, the 3 experimental LAB strains used were purchased from the Food Industry Research and Development Institute of the Biological Resources Conservation and Research Center: Lactobacillus acidophilus BCRC 14065; L. delbrueckii subsp. lactis BCRC 12256; and L. gasseri BCRC 14619. The stock culture was maintained at −80°C in 20% glycerol prior to use. The bacteria were propagated twice in Lactobacilli MRS broth (Difco, Sparks, MD, USA) containing 0.05% L-cysteine overnight at 37°C before the experimental procedures.
Preparation of PSPY
PSP was acquired from the Taiwan Agricultural Research Institute and stored at 4°C after being cleaned with tap water. To prepare the PSPY, the potatoes were first peeled, cut into 1-cm-thick slices and steamed at 100°C for 20 min. The cooked spuds were then homogenized and then 0.05% α-amylase, 10% skimmed milk powder, 0.05% protease and 3% whey protein were added to the mixture before pasteurization (121°C, 15 min). The 3 different LAB strains were inoculated at a concentration of 109 CFU/ml to the PSP milk and incubated at 37°C for 24 h until the fermented PSPY was obtained. The final product was stored at 4°C in a refrigerator for use in subsequent experiments.
Animals and experimental groups
Twenty-two male SHRs and 12 male Wistar Kyoto (WKY) rats were purchased from BioLASCO Taiwan Co., Ltd, Taipei, Taiwan. The animals (aged 6 weeks) were housed individually in a temperature (20±2°C)- and humidity (55±5%)-controlled environment. The rats were maintained on a 12 h dark-light cycle with lights on from 8 a.m. to 8 p.m. The rats were allowed access to food [chow pellets (MF-18; Oriental Yeast Co. Ltd., Tokyo, Japan)] and water ad libitum. An acclimatization period of 1 week after delivery by the supplier was allowed before the SHRs were randomly divided into the following 4 groups: i) the SHR control group (rats administered 2.5 ml distilled water); ii) the anti-hypertensive captopril group [rats administered captopril, 15.6 mg/kg, body weight (BW)/day]; iii) the 10% PSPY group (rats administered a 10-fold dilution of PSPY, 1.068 μg GABA/2.5 ml PSPY); and iv) the 100% PSPY group (rats administered undiluted PSPY, 10.68 μg GABA/2.5 ml PSPY). Experimental feeding concentrations of PSPY were calculated on the basis of GABA concentrations by the high-performance liquid chromatography (HPLC) method. Captopril and PSPY were orally administered daily to the rats until the rats were sacrificed on the 8th week of the experimental period. WKY rats were used as the negative control. The entire experimental procedure was performed according to the NIH Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Institutional Animal Care and Use Committee of HungKuang University, Taichung, Taiwan.
4′,6-diamidine-2-phenylindole dihydrochloride (DAPI) staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)
After the hearts were excised, the hearts were soaked in formalin, dehydrated through graded alcohols and embedded in paraffin wax. From the heart tissues, the 3-μm-thick paraffin sections were cut from the paraffin-embedded tissue blocks. The sections were deparaffinized by immersing them in xylene (5 min, thrice), rehydrated and incubated in phosphate-buffered saline (PBS), pH 7.4, with 3% H2O2 to inactivate endogenous peroxidase. The sections were then incubated with proteinase K (20 μg/ml) for 30 min, washed in 0.1 M PBS, and incubated with TUNEL reaction mixture for 60 min at 37°C using an apoptosis detection kit (Roche Applied Science, Indianapolis, IN, USA). After washing with PBS twice, the sections were stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) for 5 min to detect cell nuclei by UV light microscopic observations (blue). TUNEL-positive nuclei (fragmented DNA) fluoresced bright green at 450–500 nm, whereas DAPI-positive nuclei (intact DNA) fluoresced blue at 360 nm. The mean numbers of TUNEL-positive and DAPI-labeled cells were counted for at least 3 separate fields from 2 slides excised from the rat hearts in each group. All counts were performed by at least 2 independent individuals in a blinded manner.
Tissue extraction
The left ventricle was cut into 8 sections. One section from the left ventricle was minced with scissors; then lysis buffer (20 mM Tris, 2.0 mM EDTA, 50 mM 2-mercaptoethanol, 10% glycerol, pH 7.4), a proteinase inhibitor cocktail tablet and phosphatase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) were added at a concentration of 100 mg tissue/ml buffer and the mixture was homogenized on ice using a Model PT l0/35 Polytron homogenizer for 2 cycles of l0 sec each. The homogenate was then placed on ice for 10 min and then centrifuged at 12,000 × g for 40 min. The supernatant was collected and stored at −70°C for further western blot analysis.
Protein content
The protein content of the left ventricle extracts was determined using the Bradford protein assay using the protein-dye kit (Bio-Rad, Richmond, CA, USA). Commercially available bovine serum albumin (Sigma-Aldrich) was used as a standard. Changes in absorption were monitored at 595 nm.
Electrophoresis and western blot analysis
The left ventricle extracts were prepared as described above. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed using 10% polyacrylamide gels. Equal amounts (20 mg) of the samples were electrophoresed at 100 V for 3 h and equilibrated for 15 min in transfer buffer [25 mM Tris-HCl, pH 8.3, containing 192 mM glycine and 20% (v/v) methanol]. Subseqently, the electrophoresed proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 μm pore size) (Millipore, Bedford, MA, USA) using a Bio-Rad Scientific Instruments Transphor Unit at 100 V with transfer buffer for 3 h. The PVDF membranes were incubated at room temperature for 1 h in blocking buffer containing 100 mM Tris-Base, 0.9% (w/v) NaCl, 0.1% (v/v) Tween-20 (pH 7.4) and 5% non-fat milk. Monoclonal antibodies against Akt (BD Pharmingen, San Jose, CA, USA), p-Akt (Cell Signaling Technology, Inc., Danvers, MA, USA) and polyclonal antibodies against Fas, Bid, t-Bid, Bcl-xL, PI3K, phoshorylated PI3K (p-PI3K), Bak, Bax, caspase-3, α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), cleaved caspase-9, p-Bad (Cell Signaling Technology, Inc.) and IGF-IR and phosphorylated-IGF-IR (p-IGF-IR) (Abcam, Taipei, Taiwan) were diluted in an antibody-binding buffer containing 100 mM Tris-Base, pH 7.5, 0.9% (w/v) NaCl and 0.1% (v/v) Tween-20. The immunoblots were washed 3 times in binding buffer for 10 min and then immersed in secondary antibody solution containing goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, or donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Inc.) for 1 h and diluted 500-fold in binding buffer. The filters were then washed 3 times (10 min each) in blotting buffer. The immunoblotted proteins were visualized by using enhanced chemiluminescence (ECL) Western Blotting Luminal Reagent (Santa Cruz Biotechnology, Inc.) and quantified using a Fujifilm LAS-3000 chemiluminescence detection system (Fujifilm, Tokyo, Japan). Color was developed in a 20 ml mixture consisting of 7 mg nitro blue tetrazolium, 5 mg 5-bromo-4-chloro-3-indolyl-phosphate, 100 mM NaCl, and 5 mM MgCl2 in 100 mM Tris-HCl, pH 9.5. The immunoblot with antibody against α-tubulin, which was prepared with the same procedure, was used as an internal control.
Statistical analysis
All statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data were compared between the animal groups using one-way analysis of variance (ANOVA). Dunnett’s test was used to determine significant differences. P-values <0.05 were considered to indicate statistically significant differences. Significant differences are indicated with symbols, as shown in the tables and figures.
Results
TUNEL-positive apoptotic cells in cardiac tissues
We wished to determine the effects of PSPY on hypertension-induced apoptosis in cardiac cells. We examined the TUNEL-positive cardiac cells in the excised hearts from the WKY rats, and from the SHRs in the control, captopril and PSPY (both concentrations, 10 and 100%) groups by TUNEL assay. Following TUNEL staining, an increased number of TUNEL-positive cardiac cells was observed in the left ventricle in the SHR-control group compared with the WKY group (Fig. 1A). Notably, a significantly reduced number of TUNEL-positive cardiac cells was found in the left ventricle of the SHR hearts in the captopril, and 10 and 100% PSPY groups compared with the SHR-control group (Fig. 1A). The percentage of TUNEL-positive cardiac cells was calculated and the quantified results are shown in Fig. 1B.
Changes in the levels of Fas death receptor-related components in the hearts of SHRs fed different concentrations of PSPY
We examined the variations in the levels of Fas death receptor-associated proteins in the hearts of SHRs administered captopril and various concentrations of PSPY by western blot analysis (Fig. 2). The protein products of Fas ligands and Fas extracted from the left ventricles of the excised hearts in the SHR-control group were significantly increased compared with the WKY group (Fig. 2). By contrast, significantly decreased levels of protein products of Fas ligands were detected in the SHR hearts from the captopril, and 10 and 100% PSPY groups compared with the SHR-control group (P<0.05) (Fig. 2B). The levels of the protein products of Fas were significantly decreased in the SHR-100% PSPY group, compared with the SHR-control group (P<0.05) (Fig. 2C). The levels of protein products of Fas were slightly decreased in the SHR hearts in the captopril and 10% PSPY groups, and these levels did not differ significantly from those in the SHR-control group (Fig. 2C). The ratios of the protein products of Fas ligands and Fas relative to α-tubulin were calculated and are shown in Figs. 2B and C.
Changes in the levels of mitochondrial-dependent apoptotic components in the hearts of SHRs fed different concentrations of PSPY
We examined the variations in the levels of mitochondrial-dependent apoptotic components in the cardiac tissues of SHRs fed PSPY. The protein levels of Bcl-2 family members (t-Bid, Bcl-xL, Bak, Bax and p-Bad) were examined by western blot analysis (Figs. 3 and 4). Significantly decreased levels of the protein products of t-Bid were detected in the left ventricles of excised SHR hearts from the captopril, and 10 and 100% PSPY groups, compared with the SHR-control group (P<0.05); however, the SHR-control group showed no significant difference to the WKY group (Fig. 3B). In addition, the levels of anti-apoptotic proteins (Bcl-xL) were significantly increased in the SHR hearts in the 100% group compared with the WKY and SHR-control groups (Fig. 3C). However, the levels of mitochondrial related pro-apoptotic proteins (Bak and Bax) were significantly increased in the SHR-control group, compared with the WKY group (P<0.05) (Fig. 4), whereas significantly decreased levels of Bak and Bax protein were detected in the SHR hearts from the 100% PSPY group, compared with the SHR-control group (Fig. 4B and C). Moreover, the protein levels of activated caspase-9 did not differ significantly among the groups (Fig. 5B). By contrast, the levels of activated caspase-3 were significantly increased in the SHR-control group compared with the WKY group (P<0.05) (Fig. 5C), and were significantly decreased in the SHR hearts from the 10 and 100% PSPY groups compared with the SHR-control group (Fig. 5C).
Change in the levels of cardiac survival signaling components in the hearts of SHRs fed different concentrations of PSPY
In order to determine the effects of PSPY on cardiac IGF-IR-dependent survival pathways, we examined the protein levels of IGF-IR, p-IGF-IR and IGF-IR signaling components, including PI3K, p-PI3K, AKT and p-AKT in the rat hearts. The protein levels of IGF-IR were significantly decreased in the SHR hearts from the SHR-control, captopril, and 10 and 100% PSPY groups compared with the WKY group (P<0.05) (Fig. 6). However, the protein levels of p-IGF-1R were significantly increased in the SHR hearts from the SHR-control group compared with the WKY group. The SHR-10 and 100% PSPY groups showed even higher levels of p-IGF-IR than the SHR-control group (P<0.05) (Fig. 6C). Notably, we found significantly increased levels of PI3K, p-PI3K, AKT and p-AKT in the SHR hearts from the 10 and 100% PSPY groups compared with the SHR-control group (P<0.05) (Figs. 7 and 8). By contrast, no significant increase in the levels of cardiac PI3K, p-PI3K and p-AKT was observed in the SHR hearts from the control and captopril groups compared with the WKY group (Figs. 7 and 8).
Discussion
The major findings of the present study can be summarized as follows: i) a higher number of TUNEL-positive apoptotic cells was observed in the SHR-control group than in the WKY group, whereas a significantly reduced number of TUNEL-positive cells was observed in the 10 and 100% PSPY groups. ii) The levels of Fas receptor- and mitochondrial-dependent apoptotic signaling pathway components, such as Fas ligand, Fas, t-BID, Bak, Bax and activated caspase-3 were significantly reduced and those of anti-apoptotic proteins (Bcl-xL and p-Bad) were increased in the left ventricle tissues in the SHR hearts from the 10 and 100% PSPY groups compared with the SHR-control group. iii) A significant decrease in the levels of IGF-IR downstream-associated components was detected in the excised ventricle tissues from the SHR-control group even with the increase in the compensatory activated form (p-IGF-IR). However, the increased levels of p-IGF-IR significantly increased IGF-IR signaling components, such as p-IGF-IR, p-PI3K and p-AKT, as observed in the SHR hearts from rats fed 10 and 100% PSPY. After integrating our current findings into previously proposed apoptotic theories, a hypothesized path diagram was created (Fig. 9), which suggested that cardiac Fas receptor-dependent and mitochondrial-dependent pathways may be activated in hypertensive rats and may be suppressed by the supplementation of PSPY. By contrast, the compensatory IGF-IR pathway was activated, but the levels of the downstream survival components were still decreased in the hypertensive rats; these levels increased by the supplementation of PSPY; PSPY completely restored the survival mechanisms. Our findings demonstrate the novel therapeutic effects of PSPY in hypertensive rat hearts; PSPY can be used to prevent apoptosis and enhance survival. It seemed to be more effective than captopril.
Chronic hypertension is a major risk factor for the development of cardiovascular diseases (1). Hypertension induces pathological cardiac hypertrophy secondary to the pressure overload, thereby contributing to decreased cardiac function and increased cardiac apoptosis (21,22). In a previous study, we demonstrated that PSPY exerted anti-hypertrophic effects in the hearts of SHRs by interfering with the IGF-II signaling pathway and the IL-6-related-ERK5 pathway (23). In the present study, the results of cardiomyocyte loss, as shown by TUNEL assay are consistent with those of previous studies describing the gradual cardiac decompensation in SHR hearts (24). Similar results have also been reported in a recent study, where the authors observed that the left ventricles of SHR hearts had more TUNEL-positive cardiac cells than those in the WKY group (25).
The balance between cell death and survival is a tightly controlled process, particularly in terminally differentiated cells, such as cardiomyocytes (21). The Fas receptor-dependent apoptotic pathway is mediated by the Fas ligand, the Fas receptor, TNF-α, the TNF receptor, Fas-associating death domain-containing protein (FADD) and the activation of caspase-8 (5,11). In our findings, PSPY significantly prevented the activation of the Fas receptor-dependent apoptotic pathways in SHR hearts, as evidenced by the decrease in the levels of hypertension-upregulated Fas ligand and Fas receptor after the oral administration of 10 and 100% PSPY. To our knowledge, the present study is the first to illustrate that PSPY prevents the activation of cardiac Fas receptor-dependent apoptotic pathways in hypertensive rats.
The mitochondrial-dependent apoptotic pathway is tightly controlled by the Bcl-2 family. Pro-apoptotic and anti-apoptotic members of the Bcl-2 family seem to interact with and neutralize each other, so that the relative balance of these effectors strongly influences cell fate (26). Shifting the balance of Bcl-2 family members toward pro-apoptotic members activates caspase-9, which further activates caspase-3 and executes the apoptotic program (13). In the present study, PSPY prevented the activation of pro-apoptotic members of the Bcl-2 family in SHR hearts, as evidenced by the decrease in the levels of hypertension-upregulated components, t-Bid, Bak and Bax, after the supplementation of PSPY in the 10 and 100% groups. The supplementation of PSPY significantly increased the levels of anti-apoptotic components due to the elevation of Bcl-xL and p-Bad levels, and thus decreased activated caspase-3 levels in the hypertensive rat hearts. Therefore, our results strongly suggest that the oral administration of PSPY in SHRs may prevent the activation of cardiac apoptotic pathways.
The cardiac survival pathway can be mediated by IGF-I-related survival pathways, such as IGF-I, IGF-IR, p-PI3K and p-Akt. Previous studies have indicated that increased Bcl-xL levels in the mitochondria were observed in IGF-I pre-treated rats and that cardiac-specific IGF-I overexpression has an anti-apoptotic function; however, increased apoptosis followed by myocardial infarction was observed in IGF-I-deficient mice (27,28). Consistent with previous findings, our results demonstrated that a significant decrease in the levels of IGF-IR pathway-associated components was detected in the ventricles from the excised SHR hearts. By contrast, the oral administration of 10 and 100% PSPY activated the compensatory cardiac survival pathway in SHR hearts, evidenced by the increased levels of p-IGF-IR, and restored the levels of PI3K, p-PI3K, Akt and p-Akt. Similar results were also reported in a previous study, demonstrating that the IGF-I/PI3K/Akt survival pathway was activated in the SHR-exercise training groups compared with SHR and WKY groups (25). These findings suggest that PSPY attenuates cardiac apoptosis and facilitates the activation of the compensatory IGFI/PI3K/Akt survival pathway.
The 2 concentrations (10 and 100%) of PSPY exerted a significant effect on cardiac apoptosis and the IGF-I survival pathway in our study, although there was no significant concentration-response effect observed between the 10-fold differences in PSPY concentrations. We speculate that the component of PSPY may contain other elements apart from GABA, whose biological function has yet been elucidated, regardless of the original presumption that the cardiac anti-apoptotic effects came from the presence of GABA. Various compounds of plants with antioxidant properties have been shown to exert a number of therapeutic effects in animal models of hypertension, which supports the hypothesis that reactive oxygen species (ROS) are involved in the progression of hypertension (29). In a previous study, we demonstrated that the fermentation procedure could further elevate anthocyanin levels and antioxidative activity significantly in PSPY with multi-strain probiotics (20). Additionally, the supplementation of probiotics may influence host intestinal microflora and subsequently improve cardiovascular functions. Lam et al (30) demonstrated that the oral administration of the commercially available probiotic juice (L. plantarum 299v) reduced myocardial infarct size in Dahl S rats. Sobol et al (31) considered that LAB and their metabolic products in particular may positively affect the calcium signal in cells in the cardiovascular system, which may result in increased contractile activity of blood vessels and cardiac cells. Based on previous findings, we hypothesized that anthocyanidin-enriched purple potato and the probiotics in PSPY may also be the components that potentially contribute to the activation of the PI3K/AKT survival pathway and the attenuation of cardiac apoptosis, and account for the insignificant difference between the 2 PSPY dosages.
In conclusion, hypertension is considered a major risk factor for the development of heart failure. Our current findings indicate that impaired cardiac IGF-I/PI3K/Akt survival and Bcl-2 family anti-apoptotic pathways in hypertensive rats may provide an important mechanism to explain the development of hypertensive heart diseases. Additionally, the supplementation of PSPY in the diet, found to be beneficial by enhancing cardiac survival and activating anti-apoptotic pathways in hypertensive hearts, may be considered as a potential novel therapeutic strategy to prevent the development of apoptosis-related cardiac diseases in hypertension. Further clinical experiments are required to clarify the survival and apoptotic mechanisms responsible for the beneficial effects of PSPY in human hypertensive hearts.