The Toxicity Mechanisms of Action of Aβ25–35 in Isolated Rat Cardiac Myocytes

β-Amyloid (Aβ) is deposited in neurons and vascular cells of the brain and is characterized as a pathologic feature of Alzheimer’s disease (AD). Recently studies have reported that there is an association between cardiovascular risk factors and AD, however the mechanism of this association is still uncertain. In this study we observed Aβ had an effect on cardiovascular cells. We represent as a major discovery that Aβ25–35 had toxicity on isolated rat cardiac myocytes by impacting the cytoskeleton assembly and causing ER stress, ultimately contributing to the apoptosis of the myocytes. Importantly, the activation of ER stress and subsequent cellular dysfunction and apoptosis by Aβ25–35 was regulated by the MAPK pathway, which could be prevented by inhibition of p38 via pharmacological inhibitors. It was noteworthy that Aβ25–35 played a critical role in cardiac myocytes, suggesting that Alzheimer’s disease (AD) had a relation with the heart and understanding of these associations in future will help search for effective treatment strategies.


Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disease all over the world, representing more than 60% of all cases. It is estimated that approximately 35 million people are being affected by AD worldwide, and the number of affected individuals is expected to grow dramatically [1]. The two major pathologic features of AD are extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles (NFT). They are abnormally folded and accumulated in the brains of AD patients [2]. The mutation in the genes of amyloid precursor protein (APP) and presenilin (PS) contribute to the excessive amount of misfolded amyloid peptides and the mutation in the gene of apolipoprotein E (ApoE) leads to altered clearance and transport of Aβ, resulting in the amyloid plaque deposit [3].
Among the amyloid plaques, Aβ as a full length peptide with 40-43 amino acids, is the critical component. Aβ comes from the amyloidogenic processing of the β-amyloid precursor protein (APP). APP is a kind of ubiquitously single-pass transmembrane protein, that is sequentially cleaved by the sequential action of β-secretase/BACE1 and γ-secretase [4]. However, the interesting property of Aβ peptide is that there are various fragments including residues 1-28 [5], 25-35 [6], and 34-42 [7], and they also show similar biophysical and biochemical properties as full-length Aβ peptide [8].
Recently many evidences suggest that there is close relationship between Alzheimer's disease and cardiovascular disease, especially cardiac insufficiency. Compared to the wild rat, the APPswe/PS1dE9 mouse with Alzheimer's disease has obvious cardiomyocyte contractile dysfunction, suggesting that Aβ maybe affects the mouse cardiomyocytes and leads to the cardiomyocyte contractile dysfunction [9]. Intracellular accumulation of β-amyloid were seen by using ultrastructural tests in a cardiac biopsy taken from a heart with amyloidosis, suggesting that heart issue may be another organ where amyloidogenic peptide leads to cardiomycocyte destruction and heart dysfunction [10,11]. Liao reported in 2009 that amyloidogenic light chain (AL-LC) proteins provoked oxidative stress, cellular dysfunction, and apoptosis in isolated adult cardiomyocytes [12]. At present the mechanism whereby β-amyloid (Aβ) could damage the organism is still unclear. However, it has been reported that human amyloidogenic precursor proteins directly impair cell function without forming amyloid fibrils.
Based on the above study we demonstrated for the first time that Aβ 25-35 as one of the active fragments of Aβ could have an impact on rat cardiac myocytes in vitro. Using isolated rat cardiac myocytes, we proved Aβ 25-35 directly caused cardiac myocyte ER stress and cytoskeletal changes, ultimately leading to apoptosis. These observations agreed with the report that Aβ peptide induced neuronal ER stress leading to the activation of the mitochondrial apoptotic pathway. It has been reported that mitogen-activated protein kinase (MAPK) could be activated in AD disease [13]. We, therefore, set out to determine whether MAPK could be a key mediator involved in the regulation of Aβ induced apoptosis and the ER stress on rat cardiac myocytes in vitro.

Effect of Aβ 25-35 on the Viability of Rat Cardiac Myocyte in Vitro
The Aβ 25-35 fragment of the Alzheimer amyloid β-peptide, like its full-length peptide Aβ , has shown toxic activities in cultured cells [8,14]. The effect of Aβ (1-42) on cultured neurons is concentration-dependent, and significant cell loss was detected after treatment at concentrations of 20 μM. Although these Aβ concentrations are higher those seen in pathological conditions, low plasma Aβ levels could still cause a big negative impact due to extended stimulation [14][15][16][17]. We studied whether the fragment Aβ [25][26][27][28][29][30][31][32][33][34][35] has the ability to inhibit the growth of rat cardiac myocytes. The cell growth-inhibitory activity was determined by colorimetric measurement of cell viability. Firstly the rat cardiac mycocytes were cultured overnight and then treated with different concentrations of Aβ 25

Aβ 25-35 Induced Cardiac Myocytes Apoptosisfigure
Aβ 25-35 affected the viability of cardiac mycocytes. In order to detect Aβ 25-35 -mediated apoptosis in myocytes, the Hoechst and Annexin V/PI staining methods were applied. In Hoechst staining, control cells emitted a blue fluorescence with consistent nucleus intensity and presented a typical homogeneous distribution of chromatin in the nucleus. In contrast, cells treated with Aβ 25-35 for 24 h presented the morphological features of early apoptotic cells, especially apoptotic bodies and nuclei pyknosis. These features appeared more frequently with increasing concentrations. To further study the effect of Aβ 25-35 on cardiac myocytes, a quantitative analysis of apoptotic cells was performed by flow cytometry. As Figure 2E-H show, Aβ 25-35 treatment resulted in a significant, dose-dependent induction of apoptosis compared with control. Treatments with 40 μM Aβ 25-35 for 24 h induced more than 46.1% of cells to total apoptosis and more than 15.68% of cells to early apoptosis ( Figure 2I). In order to corroborate these results, we determined some well-established biochemical markers of apoptosis by immunoblotting. Indeed, Aβ [25][26][27][28][29][30][31][32][33][34][35] increased the levels of cleaved caspase 3, 7/PARP in a concentration dependent fashion ( Figure 3A,C). The expression of Bax (an apoptosis promoter) was dramatically higher and Bcl-2 (an apoptosis inhibitor) was significantly decreased compared with control in the cardiac mycocytes. It was confirmed that Aβ 25-35 could induced cardiac myocyte apoptosis by an enhanced Bax/Bcl-2 signal pathway and showed toxicity on these normal cells.  [25][26][27][28][29][30][31][32][33][34][35] . Values are expressed as mean ± SEM of three independent experiments, each in triplicate. ** p < 0.01, *** p < 0.001 vs. the control group.

Aβ 25-35 Induced ER Stress in Cardiac Myocytes Cell
Previously it was reported that Aβ could lead to endoplasmic reticulum stress in cultured cortical neurons [18]. Now we further sought to detect by western blot (WB) whether Aβ 25-35 would induce the ER stress in cardiac myocytes by measuring the protein levels of ER stress markers. XBP-1 is a key mediator of the ER unfolded protein response (UPR), and GRP78 is the chaperone [19]. Results showed that after treatment with different concentrations of Aβ 25-35 the levels of XBP and Grp78 were significantly increased and the effect started at 10 μM after 24 h incubation. Prolonged ER stress would promote the up-regulation of a transcription factor C/EBP homologous protein (CHOP), which down-regulates the level of anti-apoptotic protein Bcl-2, further leading to apoptosis [20]. Indeed, in our results the ER stress induced by Aβ 25-35 could contribute to the high level of CHOP, decreasing the Bcl-2 protein as seen in Figure 3B. These results suggested that Aβ 25-35 led to ER stress, which in turn reduced Bcl-2 activation in the cardiac myocytes.

Aβ 25-35 Affects the Cytoskeleton Assembly in Cardiac Myocyte Cells
Microtubules and actin filaments play important roles in mitosis, cell signaling and cell-motility. The amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease, and the amyloid precursor protein intracellular domain (AICD) could disrupt actin dynamics and mitochondrial bioenergetics [21]. However, Aβ 25-35 is the cleaved product from APP. In order to confirm the role Aβ 25-35 plays in cardiac myocytes, we applied immunofluorescence assays for the determination of cytoskeletal assembly. When the cardiac myocytes were treated with Aβ 25-35 for 24 h, actin filaments became dramatically destabilized compared with the control (Figure 4). In addition ROCKs control actin-cytoskeleton assembly and cell contractility, and thereby contribute to several physiological processes [22]. We also detected the level of ROCKs protein on the cardiac myocyte cells. As Figure 3F shows Aβ 25-35 increased the expression of phosphorylated ROCK protein (phospho T249) in a concentration dependent way without any effect on the total protein. Evidence suggested that the damaged cell's cytoskeleton could contribute cell with the poor viability and even apoptosis fates. It is possible that there is a relationship between Aβ 25-35 -induced cytoskeleton assembly activity and ROCK protein, however further study is needed to conform this.

Discussion
A novel and critical conclusion of this paper is that Aβ [25][26][27][28][29][30][31][32][33][34][35] has an important toxicity toward cardiac myocytes. We have demonstrated that Aβ 25-35 causes ER stress and affects cytoskeletal assembly, leading to the apoptosis of cardiac myocytes through activation of p38 and inhibition of Erk1/2. Through the current study reports an IC 50 ~20 μM for cardiac effects whereas the plasma levels are low, Aβ 25-35 could have a tiny and long term effect of a concentration-dependent removal from the brain and ultimately contribute to heart problems.
Alzheimer's disease (AD) is the most common type of dementia. Accumulation of amyloid-beta (Aβ) peptides is considered as the most important cause associated with AD pathogenesis, which is cleaved from the amyloid precursor protein (APP) [27]. Cardiovascular risk is also prevalent and increases in the elderly AD patient [28]. By applying the genome-wide association studies (GWAS) method to AD pathogenesis it is found that cardiovascular disease contributes to AD [29]. Recently there have been more literature reports of associations between cardiovascular risk factors and AD [30][31][32][33]. Although the mechanisms for these associations are uncertain we hypothesize Aβ may affect the cardiovascular system and heart. Interestingly, our results provide clues as to the link between these diseases. Although Aβ or other amyloid precursor proteins have the ability to activate a programmed nervous cell death pathway and contribute to the pathology of the Alzheimer's disease, our study demonstrates that Aβ [25][26][27][28][29][30][31][32][33][34][35] can also trigger such cell death in cardiac myocytes isolated from rat. We believe that these finding will be very useful for future studies of AD and heart disease.
It is known that the MAPK signaling pathway plays an important role in cell ER stress response, apoptosis, cytoskeletal reorganization, and transcriptional regulation of genes in differentiation, proliferation, and inflammation [40]. Abundant literature has reported that MAPK was activated to reply to growth stimuli, promoting cell growth [41]. ER-mediated MAPK activation exists in the cardiovascular system [42]. In search of the mechanism underlying ER-stress and the apoptosis induced by Aβ 25-35 , we found there is an increase in p38 and a decrease in ERK phosphorylation in cardiac myocytes during exposure of Aβ 25-35 for 24 h. The literature has reported that Aβ 25-35 induced significant ERK activation after 5 min that gradually weakened after 6 h in a time-dependent manner in neonatal cardiomyocytes and in neonatal cardiomyocytes and in α 1A -AR harboring CHO cells [43]. However, in our results Aβ [25][26][27][28][29][30][31][32][33][34][35] inhibited ERK phosphorylation during the exposure of Aβ 25-35 for 24 h, suggesting the length of exposure time of Aβ 25-35 had great influence on the ERK response. Aβ [25][26][27][28][29][30][31][32][33][34][35] could regulate the levels of ERK and p38 phosphorylation, suggesting that the MAPK pathway might be involve in its cardiac myocyte-protective effects. However, P38 phosphorylation may trigger cell apoptosis by differentially regulating the expression and activity of pro-and anti-apoptotic Bcl-2 family proteins. SB203580 as a selective p38 MAPK inhibitor that can inhibit the activity of p38. SB203580 exerts its inhibitory effect by binding the ATP binding pocket of p38 and has been used to identify the p38 phosphorylation in cultured cells in several previous reports [44]. P38 are a class of mitogen-activated protein kinases that are activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), and ultraviolet light, and are involved in adaptation to stress, apoptosis or cell differentiation [45]. The ERK1/2 could phosphorylate a number of substrates important for cell proliferation, cell cycle progression, cell division and differentiation [46]. Our results have shown that Aβ [25][26][27][28][29][30][31][32][33][34][35] has different effects on p38 and ERK1/2, suggesting Aβ 25-35 -induced ER stress will result in the high p38 expression and inhibit myocyte proliferation by decreasing the ERK1/2 expression.

Cells and Culture Conditions
Rat cardiac myocytes were isolated from the heart of young rats as described previously [9]. Briefly, the rats were anesthetized of sodium pentobarbital (150 mg/kg) and heparin (300 U/kg). After waiting until the rat is not responsive, the abdomen is sprayed with 70% EtOH, the thorax cut open and the heart removed above the aortic arch. The rat heart was excised and retrogradely perfused on a Langendorff apparatus with Ca 2+ -Tyrode's solution(in mM NaCl 135 mM, KCl 5.4 mM, MgCl 2 1.0 mM, NaH 2 PO 4 0.33 mM, glucose 5 mM, and HEPES 10 mM, pH 7.4) via the aorta at a perfusion rate of 6 mL/min for 5 min. Then, the heart was perfused with Tyrode's solution containing CaCl 2 (34 mM) and collagenase II (300 mg/L) for 20 min. The temperature was at 37 °C. Finally, the heart was removed and cut into smaller pieces of 1-3 mm 3 in PBS solution containing 0.1% tyrisin for 25 min at 37 °C. After the digestion, the cells were centrifuged for 7 min at 1500 r/min, and the pellet was resuspended. Then single myocytes were harvested after filtration through a nylon mesh (pore size 200 mm) and stored at room temperature for at least 20 min, then the supernatant were changed twice. Finally cells were placed in DMEM culture with 10% FBS without moving for 24 h, which was changed once every two days. The animal care and the experimental protocol were approved by the Animal Ethics Committee of China Medical University.

TUNEL-Based Assay
TUNEL staining was performed using an In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science), according to the manufacturer's directions. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, then washed three times in PBS for 5 min each. Then, slides were were rinsed twice in PBS buffer and immersed in 0.1% (v/v) Triton X-100 supplemented with 0.1% (w/v) sodium citrate in ice-cold PBS, for 2 min, to permeabilize the cells. Slides were rinsed again three times with PBS and incubated with TUNEL mixture for 1 h at 37 °C, in the dark. Finally, slides were rinsed three times with PBS and mounted with Dako Cytomation Fluorescent solution (Dako Cytomation, Carpinteria, CA, USA) onto a microscope slide for visualization in fluorescence microscope (Olympus).

Immunofluorescence Assays
After the treatment, rat cardiac myocytes in coverslips were washed three times with ice-cold PBS and fixed with freshly prepared 4% paraformaldehyde in PBS for 15 min. After the fixation, cells were soaked with PBS three times for 5 min and then permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature. Subsequently, the cells were again washed with PBS twice for 5 min and blocked with 1% phalloidin for 60 min at room temperature in the dark. Then the phalloidin were washed and cells were incubated with DAPI. Finally the DAPI were washed and a drop of anti-fluorescence quencher was added. To visualize actin filaments, the treated cells were directly labeled with phalloidin conjugated with Alexa Fluor 488 and analyzed under a fluorescence microscope after counterstaining with DAPI.

Statistical Analysis
Nonlinear mixed models were used to obtain IC 50 . All data represent at least three independent experiments and are displayed as the mean ± SEM. Two-tailed student's t-test was used to evaluate statistical significance of difference between treated and control groups.

Conclusions
In summary, our data suggest that Aβ 25-35 plays a critical role by causing the ER stress and apoptosis mediated by p38 and ERK1/2 phosphorylation in cardiac myocyte cells isolated from rat. Because the relationship between cardiac cardiovascular disease and AD is unclear, our finding represents a major step forward toward understanding the molecular mechanisms underlying Aβ and could contribute to the study of cardiovascular and AD disease as well as treatment strategies for these two groups of patients.