Hyperphosphate-induced myocardial hypertrophy through calcineurin/NFAT3 signal pathway is attenuated by ERK inhibitor treatment
Authors and Affiliations:
Yao-Lung Liu1,2, Chiu-Ching Huang1,2, Jiung-Hsiun Liu1,2, Che-Yi Chou1,2, Shih-Yi Lin1, I-Kuan Wang 1,2, Dennis Jine-Yuan Hsieh 3,4, Gwo-Ping Jong 4, Chih-Yang Huang 5,6*, Chao-Min Wang 7*
1. Division of Nephrology and Kidney Institute, China Medical University Hospital, Taichung, Taiwan
2. School of Medicine, China Medical University, Taichung, Taiwan 3. School of Medical Laboratory and Biotechnology, Chung Shan Medical
University, Taichung , Taiwan
4. Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung , Taiwan
5. Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
6. Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan
7. Research Center for Biodiversity, China Medical University, Taichung, Taiwan 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Running title: Cardiac hypertrophy is attenuated by ERK inhibitor
Conflict of interest statement: No competing interests exist.
Word count: 3099
*Author for correspondence: Chih-Yang Huang, Ph.D
Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
Address: No. 91, Hsueh-Shih Road, Taichung, 404, Taiwan E-mail: [email protected]
Phone number: +886-4-2205-3366, ext. 3313. FAX number: +886-4-2207-0465
Chao-Min Wang, Ph.D
Research Center for Biodiversity, China Medical University, Taichung, Taiwan Address: Room 721, 7F, Lifu Hall, No. 91, Hsueh-Shih Road, Taichung, 404, Taiwan. E-mail: [email protected]
Phone number: +886-4-2205-3366, ext. 1631. FAX number: +886-4-2207-1500
Key Words: Hyperphosphate, cardiomyocyte hypertrophy, calcineurin, ERK 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
inhibitor 43
Abstract
Background/Aims: Numerous epidemiological studies have associated elevated
serum phosphorus levels with cardiovascular disease and the risk of death in the general population as well as in chronic kidney disease (CKD) and dialysis patients. In this study, we explored whether elevated phosphate conditions induce cardiac hypertrophy, and attempted to identify the molecular and cellular mechanisms in the hypertrophic response.
Methods: H9c2 myocardial cells were incubated in high-phosphate conditions to
induce hypertrophy. Pathological hypertrophic responses were measured in terms of cell size, arrangement of actin filaments, and hypertrophy markers such as atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in myocardial cells. Several transcriptional factors involved in cardiac hypertrophy development were measured to investigate the molecular pathways involved in elevated phosphate-induced cardiac hypertrophy.
Results: High-phosphate conditions induced cellular hypertrophy, marked by
increased cell size, reorganization of actin filaments, and upregulation of both, ANP and BNP in H9c2 cells. Both upstream calcineurin and downstream transcription factors, including GATA-4 and NFAT-3, were significantly increased under
hyperphosphate conditions. Moreover, MEK1/2-ERK1/2 expression both increased 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
significantly and cellular hypertrophy was markedly attenuated by U0126, an ERK1/2 inhibitor.
Conclusion: These results suggest that hyperphosphate induces myocardiac
hypertrophy through ERK signaling pathway in H9c2 cells. Our findings provide a link between the hyperphosphate-induced response and the ERK/NFAT-3 signaling pathway that mediates the development of cardiac hypertrophy. In view of the potent and selective activity of ERK inhibitor U0126, this agent warrants further
investigation as a candidate for preventing hyperphosphate-induced cardiac hypertrophy in CKD and dialysis patients.
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Introduction
Phosphate levels are strongly associated with poor outcomes in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) . Several epidemiological studies have documented a link between serum phosphorus and adverse outcomes in CKD . In addition, a high serum phosphate level is also highly correlated with the extent of vascular calcification and vascular disease . Many of persons worldwide have combined cardiovascular disease (CVD) and CKD .
Cardiovascular complications are the major cause of death in patients with end-stage renal disease . More recent observational data have associated hyperphosphatemia with increased cardiovascular mortality among dialysis patients . Phosphorus levels are also associated with subclinical atherosclerosis in the general population and young adults . Recent reports have demonstrated that conventional hemodialysis is associated with significant left ventricular hypertrophy (LVH) . Abnormal mineral metabolism, especially hyperphosphatemia, is now a novel cardiovascular risk factor among dialysis patients. However, the reasons and mechanisms responsible for phosphorus dampening are only partially understood, because the putative receptor mediating phosphorus toxicity in target organs has not yet been identified.
Intracellular signaling pathways of the cardiac hypertrophy response are typically induced by active membrane-bound receptors including multiple GTPase 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
proteins, kinases, and phosphatases . In the heart, mitogen-activated protein kinases (MAPK) signaling pathways and Ca2+-calmodulin-activated phosphatase calcineurin have been reported to participate in the development of cardiac hypertrophy in response to stimuli . However, no experiments have been conducted to establish a causal relationship between hyperphosphate and hypertrophy of myocardial cells. In the present study, we first examined whether hyperphosphate induces cardiac hypertrophy and have subsequently identified the precise molecular and cellular mechanisms involved in the hypertrophic response induced by hyperphosphate in myocardiac cells. 91 92 93 94 95 96 97 98 99
Materials and Methods
Elevated phosphate-induced hypertrophy in myocardiac cells
Cardiomyoblast cells (H9c2) were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (Clontech, USA), 2 mM glutamine, 1 mM HEPES buffer, and antibiotics (100 μg/ml penicillin, 100 μg/ml streptomycin) in 5% CO2 at 37°C. For treatment with elevated phosphate, the H9c2 cells were incubated in different concentrations of NaH2PO4. Cell sizes were calculated at various time intervals. Finally, the best condition was used to induce cellular hypertrophy in myocardiac cells.
Cell-size measurement
Cell surface area was determined after imaging by fluorescence microscopy. H9c2 cells were fixed with 4% paraformaldehyde, washed with ice-cold phosphate buffered saline (PBS), permeabilized with 0.5% Triton X-100, and blocked with PBS containing 2% bovine serum albumin as described previously. Actin filaments were stained using rhodamine-labeled phalloidin (Molecular Probe, USA). Surface areaswere quantified by visualizing the boundary of individual cells by using Zeiss Axio Vision software. For each treatment condition, 30 cells were counted, in triplicate. 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
Immunoblotting
Crude proteins of cultured myocardiac cells were isolated using lysis buffer (Roche Molecular Biochemicals, USA). Nuclear protein was extractedas per a protocol reported previously . Protein concentration in the supernatant was
determined by the colorimetric assay (Bio-Rad, USA). Samples containing 50 µg of protein were analyzed by western blot. Antibody against ANP, BNP, GATA-4, phosphorylated GATA-4, and NFAT-3, as well as goat anti-mouse IgG antibody conjugated to horseradish peroxidase, goat anti-rabbit IgG antibody conjugated to horseradish peroxidase, and goat anti-rabbit IgG horseradish peroxidase conjugate were obtained from Santa Cruz Biotechnology, Inc. (California, USA). We used α-tubulin (Lab Vision Corporation, Fremont, California, USA) as the loading control.
Inhibitor treatments
P38 MAPK inhibitor (SB203680), JNK inhibitor (SP600125), ERK1/2 inhibitor (U0126), and calcineurin inhibitor (cyclosporine A [CsA]) were obtained from TOCRIS (Ellisville, Missouri, USA). H9c2 cells were preincubated with U0126, SB203580, SP600125, and CsA for 1 h, followed by NaH2PO4 for 6 h. Actin-immunofluorescence was performed to determine the effect of these inhibitors on cardiomyocyte hypertrophy induced by elevated phosphate conditions.
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Statistical analysis
All statistical analyses were conducted using SPSS 13.0 software. Each
experiment was replicated at least three times. Results were presented as the average mean ± standard error (SE), and statistical comparisons were made using the
Student’s t-test. A P value <0.05 was considered statistically significant. 140 141 142 143 144 145
Results
Hyperphosphate conditions induce cellular hypertrophy and upregulate pathologic hypertrophy markers ANP and BNP in myocardial cells
Myocardial cells showing hypertrophy, induced by elevated levels of phosphate, were analyzed by performing immunofluorescence to examine actin fibers and cell size. Results showed that the surface area of H9c2 cells increased significantly, approximately 1.3-, 1.8-, 1.5-, and 1.2-fold in response to 1.2, 1.4, 1.6, and 1.8 mM NaH2PO4 treatment, respectively, as compared to the control. In addition, H9c2 cell surface area increased significantly at different time intervals under 1.4 mM NaH2PO4 treatment conditions (Fig. 1).
Cell lysates were analyzed for pathologic hypertrophy markers, ANP and BNP. As shown in Fig. 2, H9c2 cell cultures were treated with 1.4 mM NaH2PO4 for 24 h and analyzed by western blot using antibodies against ANP and BNP. ANP
significantly increased within 1 h following treatment and levels were maintained for up to 24 h. BNP levels increased 6 h after treatment and reached maximum at 24 h.
Elevated phosphate conditions induce calcineurin level, GATA-4 activation and NFAT-3 nuclear localization in myocardiac cells
As shown in Fig. 3, elevated phosphate conditions induced the calcineurin level 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
within 6 h, phosphorylation/activation of GATA-4 within 3 h and the active state of GATA-4 was maintained for up to 24 h. NFAT-3 levels increased in a time-dependent manner during the first 12 h. (Fig. 3). These results suggest that elevated phosphate conditions induce the development of myocardiac hypertrophy through calcineurin and the activation of GATA-4 and nuclear localization of NFAT-3.
MAPKs mediate elevated phosphate-induced myocardial hypertrophy
To further elucidate the signal transduction pathway involved in the mechanism of elevated phosphate-induced myocardial hypertrophy, MAPKs such as ERK, MEK, JNK, and p38 were analyzed by western blot. Elevated phosphate-induced myocardial hypertrophy increased the expression of ERK only at 6 h post-treatment. In addition, the level of MEK expression increased within 1 h and decreased after 6 h as compared to the control (Fig. 4). These results show that the MAPK pathway is involved in elevated phosphate-induced myocardial hypertrophy.
Potential inhibitors of elevated phosphate-induced myocardial hypertrophy
To confirm the involvement of MAPK signal transduction pathway and identify potential inhibitors of elevated phosphate-induced myocardial hypertrophy, signal transduction inhibitors, CsA (calcineurin inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK1/2 inhibitor) were used to 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185
block MAPK and calcineurin-mediated pathways in H9c2 cells. Results showed that the ERK1/2 inhibitor, U0126, suppressed elevated phosphate-induced cell size hypertrophy significantly (Fig. 5). CsA (calcineurin inhibitor) and the other MAPK inhibitors, namely SB203580 (p38 MAPK inhibitor) and SP600125 (JNK1/2 inhibitor), all showed moderate levels of suppressive influence (Fig. 5). Results indicating that Ca2+-calmodulin-activated protein phosphatase, calcineurinis not majorly involved in regulation of elevated phosphate-induced hypertrophy. These results confirm that ERK1/2 play an important role in elevated phosphate-induced myocardial hypertrophy response.
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Discussion
High serum phosphate is a major risk factor for cardiovascular disease and vascular calcification. Numerous epidemiological studies have associated serum phosphorus levels with different markers of cardiovascular disease and the risk of death in the general population as well as in CKD and dialysis patients . Therefore, establishing a causal relationship between elevated phosphate serum levels and hypertrophy of myocardial cells is essential. In this study, we first explored the molecular and cellular mechanisms behind the hypertrophic response in myocardial cells induced by elevated phosphate conditions. We demonstrated that elevated phosphate conditions induce hypertrophic response in H9c2 cells, marked by increased cell surface area, reorganization of actin filaments, and upregulation of hypertrophy markers (ANP and BNP). Both transcription factors, GATA-4 and NFAT-3 are important for the development of cardiac hypertrophy. Finally, our results demonstrated that the characteristic features of cardiac hypertrophy in H9c2 cells are majorly mediated by the ERK1/2 signaling pathway.
The MAPK pathway is important in transferring external stimuli to the nucleus via phosphorylation and regulation of several transcription factors. Serine-threonine kinases have been shown to phosphorylate important downstream mediators that participate in regulation of cellular functions such as proliferation, differentiation, 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213
apoptosis, and growth processes including cardiac hypertrophy . MAPKs can be divided into three subfamilies on the basis of sequence homology, namely, ERKs, JNKs, and p38. Growing evidence shows that environmental stress (osmotic stress, DNA damage, and ultraviolet radiation) stimulates cardiomyocyte hypertrophy and activates protein kinase cascades including ERK, p38 MAPK, and JNK .
Interestingly, the significant role for these pathways in hypertrophic signaling was confirmed by overexpression of MAPK phosphatase 1, which inhibits all three major factors of MAPK signaling, blocks cardiac hypertrophy, both in vitro and in vivo. In this study, pretreatment with ERK, but not JNK or p38 MAPK inhibitor, showed suppressive effects on hypertrophic changes such as reorganization of actin filaments. These results indicate that hypertrophy is specifically suppressed by an ERK inhibitor.
Previous studies have demonstrated that calcineurin/NFAT signaling pathway plays an important role in the development of cardiac hypertrophy . It was also demonstrated that calcineurin activity is increased in compensated, hypertrophic human myocardium and end-stage heart failure . However, pretreatment with calcineurin inhibitor, CsA, showed slightly effects on hypertrophic features such as reorganization of actin filaments in this study. These results suggest that Ca2+ -calmodulin-activated protein phosphatase, calcineurin is not majorly involved in elevated phosphate-induced hypertrophic responses in H9c2 myocardiac cells. 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232
Previous studies indicated that poor outcomes in patients with CKD and ESRD were associated with phosphate levels in serum . Additionally, observational studies have correlated the use of phosphate binders as a tool for controlling
hyperphosphatemia, with better survival rates in CKD and ESRD . However, the use of phosphate binders in CKD is based on observational rather than clinical trial data and these suggest only a limited effect of phosphate binders . This study revealed that elevated phosphate levels induce myocardial hypertrophy in H9c2 cells via the ERK signaling pathway. To prevent cardiac hypertrophy in CKD and dialysis patients, we propose that blocking the ERK/NFAT-3 signaling pathway, using a pharmacological ERK inhibitor, such as U0126, may be a good therapeutic approach, thus preventing pathological hypertrophy and heart dysfunction. Considering the growing incidence of dialysis and CKD, we believe that this area should be prioritized for future research. Further studies are required to explore candidate ERK inhibitors for clinical
applications. 233 234 235 236 237 238 239 240 241 242 243 244 245 246
Acknowledgements:
This study was financially supported by grants from the China Medical
University Hospital to Y. L. Liu and supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004).
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Figure legends
Fig. 1 Cell size measurements in elevated phosphate-induced myocardial cells, performed using actin immunofluorescence staining. H9c2 myocardial cells were cultured with different concentrations of NaH2PO4 and treated for various time-periods (1, 2, 6, 12, and 24 h). Cell surface area was observed using fluorescence microscopy. Results are expressed as the mean ± SE values of 3 experiments.
Asterisks indicate significant difference (*p < 0.05, **p < 0.01) in comparison to the control.
Fig. 2 Expression of pathologic markers (ANP and BNP) in elevated phosphate-induced myocardial cells. The expression levels of ANP and BNP were measured by immunoblotting with antibodies against proteins as indicated. Results are expressed as the mean ± SE values of 3 experiments. Asterisks indicate significant difference (*p < 0.05, **p < 0.01) in comparison to control.
Fig. 3 Expression of calcineurin, GATA-4 and nuclear localization of NFAT-3 in elevated phosphate-induced myocardial cells. The protein levels of calcineurin, GATA-4, phosphorylated GATA-4, and the nuclear fraction of NFAT-3 were
determined by western blot analysis. Results are expressed as the mean ± SE values of 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348
3 experiments. Asterisks indicate significant difference (**p < 0.01) in comparison to control.
Fig. 4 Expression of MAPK markers in elevated phosphate-induced myocardial cells. The protein levels of MEK1/2, phosphorylated-ERK1/2, phosphorylated-MEK1/2, phosphorylated-JNK, and phosphorylated-p38 were determined by western blot analysis. Results are expressed as the mean ± SE values of 3 experiments. Asterisks indicate significant difference (*p < 0.05, **p < 0.01) in comparison to control.
Fig. 5 Potential inhibitors of elevated phosphate-induced myocardial hypertrophy. H9c2 myocardial cells were pre-treated with potential inhibitors, namely CsA
(calcineurin inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK1/2 inhibitor) for 1 h and subsequently elevated phosphate (1.4 mM) treatment for 6 h. Cells cultured without pre-treatment were used as control. Inhibitory effect of various inhibitors on elevated phosphate-induced myocardial hypertrophy was determined by actin-immunofluorescence assay. Relative cell size in response to different inhibitors was analyzed, where 30 cells were counted in each experiment, performed in triplicate. Results are expressed as the mean ± SE values of 3 experiments. Asterisks indicate significant difference (*p < 0.05, **p < 0.01) in comparison to control, and (#p < 0.05, ##p < 0.01) in comparison to phosphate 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368
treatment. 369
