PKD deletion promotes autophagy and inhibits hypertrophy in cardiomyocyte

Journal Pre-proof PKD deletion promotes autophagy and inhibits hypertrophy in cardiomyocyte Di Zhao, Yan Gao, Wei Wang, Hui Pei, Chunxiao Xu, Zhuo Zhao PII:

S0014-4827(19)30625-1

DOI:

https://doi.org/10.1016/j.yexcr.2019.111742

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YEXCR 111742

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Experimental Cell Research

Received Date: 9 April 2019 Revised Date:

17 November 2019

Accepted Date: 19 November 2019

Please cite this article as: D. Zhao, Y. Gao, W. Wang, H. Pei, C. Xu, Z. Zhao, PKD deletion promotes autophagy and inhibits hypertrophy in cardiomyocyte, Experimental Cell Research (2019), doi: https:// doi.org/10.1016/j.yexcr.2019.111742. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

CRediT author statement Di Zhao: Data curation, Investigation, Writing- Original draft preparation. Yan Gao: Visualization, Investigation, Software. Wei Wang: Formal analysis, Supervision. Hui Pei: Formal analysis, Validation. Chunxiao Xu: Data Curation, Software Zhuo Zhao: Conceptualization, Methodology, Resources, Writing- Reviewing and Editing.

PKD deletion promotes autophagy and inhibits hypertrophy in cardiomyocyte Di Zhao1,2,3*, Yan Gao3*, Wei Wang4, Hui Pei5, Chunxiao Xu3, Zhuo Zhao3

1. Shandong University of Traditional Chinese Medicine, Postdoctoral Station, Shandong, China 2. Department of Cardiology, Affiliated Hospital of Shandong Academy of Medical Sciences, Shandong, China. 3. Department of Cardiology, Jinan Central Hospital Affiliated to Shandong University, Shandong, China. 4. Department of Cardiology, Shandong Provincial Chest Hospital, Shandong, China. 5. Taian Central Hospital, Taian City, Shandong, China

This work was supported by grants from National Science Foundation of China (NO. 81670245 to Z.Z) and Shandong Provincial Natural Science Foundation (NO. ZR2019BH033 to D.Z.).

* These authors contributed equally to this work. Corresponding author: Zhuo Zhao, MD, PhD Department of Cardiology Jinan Central Hospital, Affiliated to Shandong University 105 Jiefang Rd, Ji’nan, Shandong Province, China 250014 Tel: (+86) 531-82965022 Fax: (+86) 531-82506006 E-mail: [email protected]

Abstract Protein kinase D (PKD) plays an important role in the development of cardiac hypertrophy induced by pressure overload. However, the mechanism involved is unclear. This study, using primary cardiomyocyte culture, PKD knockdown and overexpression, and other molecular techniques, tested our hypothesis that PKD pathway mediates cardiac hypertrophy by negatively regulating autophagy in cardiomyocyte. Neonatal cardiomyocytes were isolated from Wistar rats and cell hypertrophy was induced by norepinephrine treatment (PE, 10-4mol/L), and divided into the following groups: (1) Vehicle; (2) PE; (3) PE + control siRNA; (4) PE + Rapamycin (100nM); (5) PE + PKD-siRNA (2 × 108 U/0.1ml); (6) PE + PKD siRNA + 3MA (10mM). The results showed that PE treatment induced cardiomyocyte hypertrophy, which were confirmed by cell size and biomarkers of cardiomyocyte hypertrophy including increased ANP and BNP mRNA. PKD knockdown or Rapamycin significantly inhibited PE-induced cardiomyocyte hypertrophy. In addition, PKD siRNA increased autophagy activity determined by electron microscopy, increased biomarkers of autophagy by Western blot, accompanied by down-regulated AKT/mTOR/S6K pathway. All the effects of PKD knockout were inhibited by co-treatment with 3-MA, an autophagy inhibitor. Oppositely, the autophagy in cardiomyocytes was inhibited by PKD overexpression. These results suggest that PKD participates in the development of cardiac hypertrophy by regulating autophagy via AKT/mTOR/S6K pathway. Keywords: PKD, AKT/mTOR signaling pathway, autophagy, cardiomyocyte

Introduction Cardiac hypertrophy is an early compensatory change of the heart in response to various stresses. However, progressive cardiac hypertrophy may lead to severe heart failure and sudden death [1]. At the cellular and molecular levels, cardiac hypertrophy is characterized by increased cell size, increased protein synthesis, and re-expression of fetal genes including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). However, the exact molecular mechanisms of cardiac hypertrophy still remain unclear. A number of recent studies have shown a relationship between cardiomyocyte autophagy and cardiomyocyte hypertrophy [2]. Autophagy is a lysosomal-dependent intracellular degradation system that degrades damaged proteins, organelles, and pathogens in a physiological state, and helps to stabilize the internal environment of the cells [3]. Cardiac hypertrophic stimulating factor activates multiple autophagic signaling pathways in cardiomyocytes, including AMP-dependent protein kinase (AMPK), MAPK, PI3K/AKT, and growth factor signaling pathways. The classical regulation of autophagy is governed by the mammalian target of rapamycin (mTOR) pathway, which negatively regulates this process in cardiomyocytes [4-6]. Treatment with the mTOR inhibitor, Rapamycin, can significantly attenuate cardiac hypertrophy by activating AKT pathway in animal models, while increased mTOR induces cardiac hypertrophy [7]. To date, how to maintain the balance of autophagy in cardiomyocyte and the specific molecular mechanism of autophagy in hypertrophy is still unclear [8]. Our previous study demonstrated that the protein kinase D (PKD) gene knockdown activated the AKT/mTOR signaling pathway, promoted autophagy, and ultimately inhibited cardiac hypertrophy caused by pressure overload in animal models [6]. Considering that approximately 70% of cells in rat heart are noncardiomyocytes, the functional roles of autophagy in cardiomyocyte during cardiac hypertrophy remains unclear. Therefore, this study was designed to further investigate that, in cardiomyocytes, whether PKD knockdown attenuates hypertrophy of cardiomyocytes through AKT/mTOR pathway and autophagy regulation in a phenylphrine (PE)-induced cardiomyocyte hypertrophy model. Compared to PKD knockdown, the opposite effects of PKD overexpression on autophagy in cardiomyocytes was also demonstated in this study.

Methods Neonatal cardiomyocyte culture The study using animals conforms with the Guide for the Care and Use of Laboratory Animals published by the National Academy Press (NIH Publication No 85–23, revised 1996) and was approved by the Institutional Animal Research Committee of Shandong University. Neonatal cardiomyocytes were isolated from the hearts of 2-to 4-day-old Wistar rats and cultured as described previously.[4] Briefly, neonatal hearts were separated and washed three times in D-Hank’s balanced salt solution (0.4 g/L KCl, 0.06 g/L KH2PO4, 8.0 g/L NaCl, 0.35 g/L NaHCO3, pH 7.2) at 4°C. They were then minced and digested with 200U/mL of collagenase II at 37°C. After digestion and determination with Dulbecco’s modified Eagle’s medium (DMEM), cells were isolated by centrifugation at 700 rpm for 5min. Cardiomyocytes were then seeded at 5×105 cells/cm2 in culture dishes, and cultured in DMEM with heat-inactivated 10% FBS at 37°C in a humidified atmosphere containing 5% (v/v) CO2. Cell treatment and siRNA transfection After twenty-four hours of culture, almost all the cardiomyocytes attached and spread on the surface of the dishes. The cells were then treated for 48 hours with: (1) Vehicle; (2) PE (10-4mol/L, Sigma, St Louis); (3) PE + Control siRNA; (4) PE + Rapamycin (100 nM); (5) PE + PKD-siRNA; (6) PE + PKD siRNA + 3MA (10mM). All experiments were performed in triplicate. For siRNA transfection, the cells were seeded in 6-well plates (5×104cells/well). PKD siRNA at 2×108 units/0.1 ml was transduced with 20 ml lentivirus (109TU/ ml) as suggested by the provider (Genechem Co., China). The PKD siRNA transduction with lentivirus was repeated in two weeks. PKD overexpression in neonatal cardiomyocytes The recombinant adenvirus vector of overexpressing PKD was designed and synthesized by Weizhen Biotechology Ltd. (Jinan, China). Transfections with PKD or control vectors were performed following the manufacturer’s instructions when the cell density reached about 80%. At 24 hours after transfections, the cells were treated for 48 hours with: (1) Control vector; (2) PKD overexpression; (3) Control vector + Rapamycin (100 nM); (4) PKD overexpression + Rapamycin (100 nM). Cells were then collected for experiments. Real-time quantitative reverse transcription PCR RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. After purification, RNA was subjected to real-time RT-PCR analysis for the expression of ANP, BNP and β-MHC. The primer sequences used for PCR are: 5’GGGGGTAGGATTGACAGGAT-3’ (forward) and 5’-GGATCTTTTGCGATCTGCTC-3’ (reverse) for ANP; 5’-GCTGCTTTGGGCAGAAGATA-3’ (forward) and 5’-GGAGTCTGCAGCCAGGAGGT-3’ (reverse) for BNP; 5’-ACTGTCAACACTAAGAGGGTCA-3’ (forward) and 5’TTGGATGATTTGATCTTCCAGGG-3’ (reverse) for β-MHC; 5’-TGGTCTACATGTTCCAGTATGACT3’ (forward) and 5’-CCATTT-GATGTTAGCGGGATCTC-3’ (reverse) for GAPDH. Western blot analysis Western blot analysis was performed as previously described. Proteins were separated by electrophoresis through an 8%–12% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membranes. The membranes were probed with antibodies against LC3B (1:1000, Cell Signaling, USA ), Beclin1 (1:1000, Cell Signaling, USA), SQSTM1/p62 (1:1000, Cell Signaling, USA ), total AMPK and phosphorylated AMPK (1:2000 dilution, Cell Signaling USA), total Akt and phosphorylated Akt (Ser473) (1:1000, Cell Signaling, USA), total and phosphorylated-mTOR (Ser2448) (1:1000 dilution, Cell Signaling, USA), total and phosphorylated S6K (Ser235/236) (1:1000 dilution; Cell Signaling, USA). Glyceraldehyde-

3-phosphate dehydrogenase (GAPDH; 1:5000; Cell Signaling, Danvers, MA, USA) was used as a loading control. The bands were digitized using MCID image analysis software (Imaging Research, Inc., Ontario, Canada). Each band was expressed in arbitrary units and normalized to its own GAPDH. Measurement of the cell surface area Twenty-four hours after treatment, the cardiomyocytes were fixed with 4% paraformaldehyde. Images were captured using Olympus BX51 microscope and software (Olympus, Tokyo, Japan). The relative cell surface area was analyzed using the Image-Pro Plus 6.0 (Media Cybemetics, US). 50–100 cardiomyocytes in each group were randomly selected and examined in each experiment. MDC staining Monodansylcadaverine (MDC, Sigma) was applied to stain autophagic vacuoles as previously described. Briefly, cells were fixed in 2% para-formaldehyde-PBS at room temperature for 15 minutes and incubated with the auto-fluorescent dye MDC with a final concentration of 0.05mM for 10 minutes at 37°C. Similarly, treated cells are used for fluorescent microscopy for qualitative analysis of autophagy. Autophagy was measured by determining the increased intensity of fluorescence in MDC-stained autophagosomes. GFP-mRFP- LC3 expression analysis and autophagosome quantitative analysis Autophagy is a dynamic process that includes autophagosome formation and degradation defined as autophagic flux [4]. The fluorescent-tagged LC3 reporters (GFP-mRFP-LC3) have been widely used to detect autophagic flux in cells vasulized using confocal microscopy [5]. The green LC3 dots mainly represent autophagosomes, whereas red LC3 dots indicate both autophagosomes and autolysosomes. The colocalization of GFP and mRFP, which merges yellow, indicates the autophagosomes. In this experiment, 5×103 cardiomyocytes were seeded into 24-well plates. Cultured cardiomyocytes were transduced with the adenovirus GFP-mRFP -LC3 (Hanbio Biotechnology Co,Ltd China) at a multiplicity of infection of 100 and then cultured in serum-free medium for 72 h. Then cultured in Opti-MEM with different treatments for 6 h. After removing the culture media, cardiomyocytes were fixed with 4% para-formaldehyde prepared in PBS for 10 min 37°C. The slides were covered with coverslips and then observed with a laser scanning confocal microscope (Carl Zeiss LSM780. Shandong University University). The numbers of fluorescent puncta were counted from at least three independent experiments, while at least 20 randomly selected cells were scored in each experiment. Fields were chosen from various sections to ensure objectivity of sampling. Statistics The results are expressed as the mean±SEM, and the statistical analysis was performed using the SPSS (Statistical Package for the Social Sciences) software package, version 16.0 (SPSS Inc, Chicago, IL, USA). All statistical significance was determined by one-way ANOVA, followed by Tukey post-hoc tests. A value of P<0.05 was considered statistically significant.

Results PKD knockdown inhibits PE-induced cardiomyocyte hypertrophy PE-induced cardiomyocyte hypertrophy was used as an in vitro cell model in this study. As shown in Fig. 1A 100 mM of PE treatment for 24 hours significantly increased cell size in Ad-GFP-infected cardiomyocytes. The mRNA levels of the biomarkers of cardiomyocyte hypertrophy, ANP, BNP, and βMHC were also increased by 2.5-3.5 folds with PE treatment (Fig. 1B). Control siRNA did not affect cell size or the biomarkers of cardiomyocyte hypertrophy. However, PKD siRNA and Rapamycin, an autophagy inducer, markedly inhibited PE-induced cardiomyocyte hypertrophy, as reflected by decreased cardiomyocyte size and the mRNA levels of ANP, BNP, and β-MHC. These effects of PKD siRNA were attenuated by the treatment of 3-MA, an autophagy inhibitor.

Monodansylcadaverine (MDC) staining MDC staining has been widely used as a marker of autophagic vacuoles due to its accumulation in mature autophagic vacuoles. As shown in Fig. 2, the MDC-positive dots were obviously increased in rapamycin and PKD siRNA treated groups compared with control siRNA or Vehicle treated groups. The PKD nockdown-induced MDC accumulation was attenuated by 3-MA treatment (Fig. 2). mRFP-GFP-LC3 Tandem Fluorescent Protein Quenching Assay As shown in Figure 3A, in PE-treated cells, there were several GFP and RFP dots which were overlapped in the merged images, indicating the basic level of autophagy in normal cardiomyocytes. The numbers of GFP and RFP dots were significantly higher after PKD-siRNA and Rapamycin treatment accompanied by the increased yellow puncta in the merged images, suggesting that that the autophagic flux was enhanced by PKD-siRNA and Rapamycin treatments. However, the treatment of 3MA markedly reduced the PKD siRNA-induced increase of yellow dots but not the red dots, suggesting that the formation of autophagosomes was inhibited by 3MA. The quantification of GFP and mRFP dots in cardiomyocytes under these conditions was shown (Fig. 3B). The results suggest that PKD related pathway induce the autophagic flux in cardiomyocytes. Autophagy markers in cardiomyocyte by Western blot analysis To determine whether PKD siRNA in cardiomyocytes interfere with autophagic processes in vitro, we investigated the expression of three autophagic marker genes: LC3-II/LC3-I, Beclin-1, and P62. Compared to control cells, PE treatment significantly increased LC3-II/LC3-I and Beclin-1 expression levels (Fig. 4A). Similarly, both Rapamycin treatment and PKD siRNA transfection increased the expression level of LC3II/LC3-I, and Beclin-1, compared to their individual controls, suggesting that PKD siRNA and Rapamycin was sufficient to induce autophagy in cardiomyocytes. The effects of PKD-siRNA on the protein expression of LC3-II/LC3-I, Beclin-1 were attenuated by the treatment of 3MA (Fig. 4B). Activation of AKT/mTOR/ p70S6k signaling pathway in PKD-siRNA induced autophagy. Western blot showed that the phosphorylated AKT (Ser 473), phosphorylation of mTOR (Ser2448) and phosphorylation of p-70S6K (Thr389) significantly increased in the cardiomyocytes treated with PE vs Vehicle cells. These increases were inhibited by either Rapamycin or PKD siRNA treatment. However, the inhibitory effects of PKD siRNA on the phosphorylation of AKT, mTOR, and S6K were blocked by cotreatment with 3MA (Fig. 5). There is no difference among groups for AMPK phosphorylation. Rapamycin or PKD siRNA did not affect AMPK phosphorylation in the cells. PE treatment increased PKD phosphorylation in comparison to vehicle treated cells. As expected, PKD siRNA decreased PKD expression ~80% in the cardiomyocytes (Fig. 5). Effect of PKD overexpression on cardiac hypertrophy and autophagy PKD overexpression after transient transfection of PKD vectors was significantly increased at both protein and mRNA levels, determined by Western blot and PCR, respectively (Fig. 6A). Results from realtime PCR showed that the mRNA levels of the biomarkers of cardiomyocyte hypertrophy, ANP, BNP, and β-MHC were also increased in PKD vs. control vector-transfected cells (Fig. 6B). In addition, PKD overexpression decreased biomarkers of autophagy determined by Western blot. As shown in Figure 6C, compared with control cells, PKD overexpression decreased the expression levels of LC3-II/LC3-I and Beclin-1, and increased p62. The effects of PKD overexpression was inhibited by the treatment of rapamycin. Discussion

The main finding of this study is that PKD knockdown activates the AKT/mTOR pathway, enhances autophagy in cardiomyocytes, and inhibits PE-induced cardiomyocyte hypertrophy, while the autophagy was inhibited by PKD overexpression. These results from primary cardiomyocytes help us to better understand the involvement of PKD/AKT/mTOR pathway and autophagy in the development of cardiac hypertrophy. More and more evidence suggests that PKD regulates a variety of physiological and pathological processes, which might be involved in cardiac hypertrophy and heart failure through different signaling pathways. It has been reported that PKD knockout significantly attenuates pressure overload or Ang II-induced cardiac hypertrophy, remodeling and dysfunction in animal models. The transgenic mice with persistently high expression of PKD in the heart had significant cardiac hypertrophy, and eventually caused the ventricular wall thinning, enlarged heart chamber, and heart failure [1]. This study was designed to investigate the roles of PKD in cardiomyocytes, using PE-induced cardiomyocyte hypertrophy as a research model. PE successfully induced cardiomyocyte hypertrophy which was demonstrated by cell size and the biomarkers of cardiomyocyte hypertrophy. PKD knockdown by siRNA significantly inhibited PE-treated cardiomyocytes associated with increased cardiomyocyte autophagy, demonstrating that PKD plays a critical role in the development of cardiomyocyte hypertrophy by regulating autophagy. Autophagy in cardiomyocytes has been shown to play an important role in maintaining cardiac function in response to multiple types of stresses [9.10]. Studies have shown that induction of autophagy protects heart by eliminating damaged organelles and proteins. Activated autophagy has been reported to attenuate cardiac hypertrophy caused by pressure overload [11] , while inhibition of autophagy can cause cardiac hypertrophy, diastolic dysfunction, and promote cardiac aging [12] . However, excessive autophagy is also harmful to the heart, leading to cell death and ultimately heart failure [13]. Recent studies have shown a significant increase in autophagy in cardiomyocytes in a variety of heart diseases, including dilated cardiomyopathy, valvular heart disease, hypertensive heart disease, and chronic ischemia[1]. However, the exact role of autophagy in the development of cardiac hypertrophy and its transition to heart failure is unclear. The roles of myocardial autophagy in cardiac hypertrophy and heart failure may be different. The results of this study suggest that the autophagy in cardiomyocytes has protective function against PE stimulation. However, the roles of autophagy in cardiac hypertrophy induced by other stresses or stimuli need further investigations. The rapamycin target protein (mTOR), a serine/threonine kinase, is a typical autophagy inducer, which can prevent cardiac hypertrophy and improve heart function[20]. Rapamycin attenuates both compensatory and decompensated cardiac hypertrophy induced by pressure-overload, while it is effective in improving compensatory cardiac hypertrophy [21]. Under certain stimuli, intracellular PI3K is activated, which increases PI3K, activates AKT, and ultimately inhibits autophagy by increasing mTOR [22]. The involvement of PI3K/AKT pathway in cardiac hypertrophy is supported by previous findings from other investigators showing that PI3K/Akt pathway was activated in a mouse model of pressure overload-induced cardiac hypertrophy, while the treatments with PI3K inhibitor LY294002 or AKT inhibitor 1701-1 attenuated cardiac hypertrophy [23,24]. In the present study, we found that knockout of PKD significantly down-regulated p-Akt, p-S6K and pmTOR in hypertrophic cardiomyocytes, suggesting that PKD is involved in the regulation of cardiac hypertrophy by directly affecting the mTOR autophagy pathway. In order to further confirm whether PKD deletion induces autophagy through the mTOR pathway, 3-MA was used in this study, which is an inhibitor of autophagy. It inhibits the conversion of cytosolic LC3I to the autophagosome membrane protein LC3II, and therefore inhibits autophagy activation. In this study, 3-MA increased the phosphorylation of Akt, S6k and mTOR, and inhibited PKD siRNA-induced autophagy, suggesting that the Akt/mTOR pathway is a key mechanism by which PKD regulates cardiomyocyte autophagy and participates in cardiac hypertrophy. We also realized the limitations of this study. The process of cardiomyocyte autophagy is complicated which could be protective or harmful to the heart depending on the conditions, various stages of heart

diseases, systemic and local environments, and other factors. Future studies based on the present findings including in vivo studies using PKD knockout or overexpressing animal models are needed to further clarify the relationships among PKD pathway, cardiomyocyte autophagy, and heart failure. In summary, the results of this study have shown that PKD knockout protects cardiomyocytes from PE-induced hypertrophy, which was mediated by the activated AKT/mTOR pathway and autophagy in cardiomyocytes.

Acknowledgments This work was supported by grants from National Science Foundation of China (NO. 81670245 to Z.Z) and Shandong Provincial Natural Science Foundation (NO. ZR2019BH033 to D.Z.). References [1] Mizushima, Noboru, and M. Komatsu. Autophagy: Renovation of Cells and Tissues. Cell. 147(2011)728-741. [2] Lemon DD, Harrison BC, Horn TR, et al. Promiscuous actions of small molecule inhibitors of the protein kinase D-class IIa HDAC axis in striated muscle. FEBS Letters. 589(2015)1080-1088. [3] Zhao D, Wang W, Wang H, et al. PKD knockdown inhibits pressure overload-induced cardiac hypertrophy by promoting autophagy via AKT/mTOR pathway. International Journal of Biological Sciences. 13(2017)276-285. [4] Wang W, Wang H, Geng QX, et al. Augmentation of autophagy byatorvastatin via Akt/mTOR pathway in spontaneously hypertensive rats.Hypertens Res. 813(2015) 1102-9. [5] Liang N, Zhang C, Dill P, et al. Regulation of YAP by mTOR and autophagy reveals a therapeutic target of tuberous sclerosis complex. Journal of Experimental Medicine. (2015) 2249-2263. [6] Dai S N, Hou A J, Zhao S M, et al. Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway. Chinese Journal of Integrative Medicine. (2018)1-8. [7] Zhao D, Wang W, Wang H, et al. PKD knockdown inhibits pressureoverload-induced cardiac hypertrophy by promoting autophagy via AKT/mTORpathway. International journal of biological sciences. 13(2017) 276-85. [8] Kheloufi M, Rautou PE, Boulanger CM. Autophagy in the cardiovascular system. Medecine sciences. 33(2017)283-9 [9] Gatica D, Chiong M, Lavandero S, et al. Molecular Mechanisms of Autophagy in the Cardiovascular System. Circulation Research.116(2015)456-467. [10] Ola R K, Meena C B, Ramakrishnan S, et al. Detection of Left Ventricular Remodeling in Acute ST Elevation Myocardial Infarction after Primary Percutaneous Coronary Intervention by Two Dimensional and Three Dimensional Echocardiography. Journal of Cardiovascular Echography. 28(2018)39-44 [11] Xue R, Zeng J, Chen Y, et al. Sestrin 1 ameliorates cardiac hypertrophy via autophagy activation. Journal of Cellular & Molecular Medicine. 21(2017) 1193-1205. [12] Le L, Chao W, Yan L, et al. Suppression of calcium-sensing receptor ameliorates cardiac hypertrophy though inhibition of autophagy. Molecular Medicine Reports. (2016)111-120. [13] Le L, Chao W, Yan L, et al. Suppression of calcium-sensing receptor ameliorates cardiac hypertrophy though inhibition of autophagy. Molecular Medicine Reports. (2016)111-120. [14] Zhu H, Tannous P, Johnstone J L. Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest. 7(2007)1782-1793 [15] Rotter D, Rothermel BA, Targets,trafficking,and timing of cardiac autophagy.Pharmacol Res. 66(2012) 494-504 [16] Zhu H, Tannous P, Johnstone J L, et al. Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest. 117(2007)1782-1793.

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Figure legends: Figure 1. The cardiomyocyte size in each group. (A) Cardiomyocyte morphology and size subjected to Ad-GFP-infected cells (green) and stained with DAPI (blue for nuclei). Scale bar: 10µm. (B) mRNA levels of ANP, BNP, and β-MHC in cardiomyocytes, evaluated by real-time RT-PCR and corrected by internal control, β-actin. Values are mean + SEM; * P<0.05 vs. vehicle; # P<0.05 vs. PE; & P<0.05 vs. PE+PKD siRNA. Figure 2. MDC staining. Autophagy was also characterized by the formation and promotion of autophagosomes. The treated cardiomyocytes were stained with 50 mM MDC for 15 min. After incubation, the cells were immediately analyzed using a confocal microscope (Upper: 400X magnification; Lower: 40X magnification). Green color shows the acidic vesicular organelle. Fluorescence intensity of MDC was measured at an excitation wavelength of 380 nm, emission wavelength of 530 nm. Figure 3. GFP-mRFP- LC3 expression in cardiomyocytes. (A) Representative imagines showing GFPmRFP- LC3 expression in cardiomyocytes. The colocalization of GFP and mRFP, which appears yellow in merged image, indicates autophagosome, whereas the free mRFP signal that does not overlay with the GFP in the merged image is indicative of autolysosomes. (B) Quantification of GFP/mRFP double-positive and mRFP single-positive puncta cell in cells with various treatments. Data were the mean value of three independent experiments with each count. Values are expressed as the mean ± SEM. * P<0.05 vs. vehicle; # P<0.05 vs. PE; & P<0.05 vs. PE+PKD siRNA.

Figure 4. Western blot for the biomarkers of autophagy in cardiomyocytes. (A) Representative images of Western blot for LC3, Beclin-1, and p62 expressions in cardiomyocytes. (B) Quantification of the signal densities from Western blot. Values are mean + SEM. * P<0.05 vs. vehicle; # P<0.05 vs. PE; & P<0.05 vs. PE+PKD siRNA. Figure 5. Western blot analysis for the signaling pathways related to cardiac autophagy. (A) Representative images of Western blot for phosphorylated and total Akt, mTOR, S6K, AMPK, and PKD in the the cardiomyocytes with various treatments. (B) Quantification of the signal densities from Western blot. Values are mean + SEM. * P<0.05 vs. vehicle; # P<0.05 vs. PE; & P<0.05 vs. PE+PKD siRNA. Figure 6. PKD overexpression induced cardiomyocyte hypertrophy. (A) Western blot and RT-PCR confirmed that PKD protein and mRNA levels were significantly increased in the cells overexpressing PKD vs. control cells. (B) mRNA levels of ANP, BNP, and β-MHC in cardiomyocytes, evaluated by real-time RT-PCR, were increased in cells overexpressing PKD compared with control cells. (C) Effects of PKD overexpression and rapamycin on cardiomyocyte autophagy. Values are mean ± SEM; # P<0.05 vs. Control; & P<0.05 vs. PKD.