OBG-like ATPase 1 inhibition attenuates angiotensin II-induced hypertrophic response in human ventricular myocytes via GSK-3beta/beta-catenin signaling

Abstract

Obg-like ATPase 1 (OLA1) that possesses both GTP and ATP hydrolyzing activities has been shown to involve in translational regulation of cancer cell growth and survival. Also, GSK3β signaling has been implicated in cardiac development and disease. However, the role of OLA1 in pathological cardiac hypertrophy is unknown. We sought to understand the mechanism by which OLA1 regulates GSK3β-β-Catenin signaling and its functional significance in angiotensin-II (ANG II)-induced cardiac hypertrophic response. OLA1 function and its endogenous interaction with GSK3β/β-catenin signaling in cultured human ventricular cardiomyocytes (AC16 cells) and mouse hearts (in vivo) was evaluated with/without ANG II-stimulated hypertrophic response. ANG II administration in mice increases myocardial OLA1 protein expression with a corresponding increase in GSK3β phosphorylation and decrease in β-Catenin phosphorylation. Cultured cardiomyocytes treated with ANG II show endogenous interaction between OLA1 and GSK3β, nuclear accumulation of β-Catenin and significant increase in cell size and expression of hypertrophic marker genes such as ANF (atrial natriuretic factor; NPPA) and MYH7 (β-myosin heavy chain). Intriguingly, OLA1 inhibition attenuates the above hypertrophic response in cardiomyocytes. Taken together, our data suggest that OLA1 plays a detrimental role in hypertrophic response via GSK3β/β-catenin signaling. Translation: Strategies to target OLA1 might potentially limit the underlying molecular derangements leading to left ventricular dysfunction in patients with maladaptive cardiac hypertrophy.

Introduction

Compensatory myocardial hypertrophy is a physiological response to hemodynamic overload. However, sustained pathological stimuli and hypertrophy, if untreated could transition to maladaptive response that significantly increases the risk of cardiovascular disease and mortality [1–3]. Cardiac muscle growth and adaptation is a complex, integrative biological process characterized by enhanced rate of protein synthesis, cardiomyocyte cell growth and thickening of ventricular walls. Angiotensin II (ANG II), a powerful vasoconstrictor or α/β-adrenergic signaling agonists that stimulate growth signals may also activate hypertrophic gene expression [4]. These pathways have been shown to lead to the transcription of immediate-early genes and re-expression of cardiac fetal genes [5]. However, understanding the intracellular downstream signaling pathways involved in the hypertrophic response might aid in strategies to limit pathological transition of cardiac hypertrophy.

OBG-like ATPase 1 (OLA1), which belongs to translation-factor-related (TRAFAC) class, YchF subfamily of P-loop GTPases and obg family, possesses both GTPase and ATPase activities that has been shown to be involved in the translational regulation of cell proliferation, growth and protein synthesis in both mammals and yeast, saccharomyces cerevisiae [6–8]. OLA1 is essential for normal progression of mammalian development. In a previous report [6], OLA1 null mice shows growth retardation and developmental delay due to reduced rate of cell proliferation through translational regulation of p21 [6]. In addition, OLA1 binds to eukaryotic initiation factor 2 (eIF2), hydrolyzes GTP and interferes with ternary complex formation, thus enhances the integrated stress response (ISR) by supressing protein synthesis. In this study, OLA1 was reported to control protein synthesis and cell fate decisions in stressed cells as well as impacts the growth and progression of tumor [7]. Another study demonstrates the role of OLA1 in translation regulation thus identifying OLA1 as one of the three candidate genes important for cytoplasmic protein biosynthesis in yeast [8].

TRAFAC GTPases not only include translation factors, but also proteins involved in intracellular transport, signal transduction and stress responses. However, the exact signaling pathway involved in OLA1-mediated regulation of translation and more specifically during hypertrophic stimuli is not known. Experimental evidence show that GSK3β is an essential negative regulator of cardiac hypertrophy and that the inhibition of GSK3β by hypertrophic stimuli is an important mechanism contributing to the development of cardiac hypertrophy [9]. Previous reports show that OLA1 inhibits GSK3β activity by mediating its Ser9 phosphorylation in lung cancer [10]. However, role of OLA1 in cardiomyocyte hypertrophic response and whether the response is mediated through GSK3β signaling is not understood yet.

In this study, our data demonstrates that ANG II-mediated stimulation of hypertrophic response in cultured human ventricular cardiomyocytes or mouse heart significantly increases OLA1 expression with a corresponding increase in GSK3β phosphorylation, nuclear accumulation of β-Catenin leading to transcription of fetal cardiac genes and hypertrophic response. OLA1 inhibition ameliorates the above effects suggesting its role in modulating hypertrophic response via GSK3β/β-Catenin pathway.

Results

Hypertrophic stimuli upregulates OLA1 expression in cultured human cardiomyocytes or mouse hearts after ANG II administration

OLA1 upregulation has been reported in clinical progression and poor prognosis of lung cancer patients and also its knockdown inhibits breast cancer cell migration and invasion [10,11]. To determine the effect of hypertrophic response on OLA1 signaling in cardiomyocytes, human ventricular myocytes (AC16 cells) were treated with ANG II (500nM) for 48h and OLA1 mRNA and protein expression was evaluated using RT-PCR and Western blotting, respectively. After ANG II treatment, we observed a significant increase in OLA1 protein expression as compared to untreated controls (Figure 1A, *P<0.05).

Fig. 1. ANG II-induced cardiac hypertrophic response stimulates OLA1 expression.

A. Western blot and densitometry data showing significant increase in OLA1 protein expression in ANG II (48h)-treated cultured human ventricular myocytes (AC16 cells). n=3, *P<0.05 vs control. B. Representative Western blot showing OLA1 protein expression in mouse heart at 3d, 14d and 28d after ANG II administration. C. Scatter plot showing the relationship between ANG II administration time and the relative protein expression of OLA1. n=3, *P<0.05 vs control. All values are group means ± SEs.

To validate the biological significance of the above findings in an in vivo model of cardiac hypertrophy, we evaluated OLA1 expression in the hearts of mouse at 3d, 14d and 28d after ANG II administration. Western blotting analyses shows that myocardial OLA1 protein expression significantly and gradually increases at 3d and 14d after ANG II administration (Figure 1B, *P<0.05). Interestingly, the protein expression restores to baseline (control) levels on 28d after ANG II administration (Figure 1C). These data suggests that OLA1 is essential during the initial stages of ANG II- induced hypertrophic response where it plays a compensatory role (translation of fetal genes such as NPPA and MYH7 and increase in cardiomyocyte size and heart weight to tibia length ratio). While, a subsequent decrease in OLA1 could be a cause/effect of molecular switch for the transition to decompensated hypertrophy leading to maladaptive response. Under chronic ANG II exposure, cardiomyocytes are under stress, and hence several other factors influence the hypertrophied heart (such as oxidative stress) that might downregulate the multifunctional molecular switch like OLA1. Molecular studies during chronic ANG II stimulation could delineate the possible mechanism by which OLA1 is downregulated on 28d post treatment. However, we believe that the initial up-regulation of this ATPase is sufficient to activate the downstream GSK3β/β-Catenin signalling. In contrast, OLA1 mRNA expression remained relatively unchanged upon ANG II exposure (Data not shown).

Endogenous OLA1 and GSK3β protein-protein interactions and altered GSK3β/β-Catenin signaling in ANG II-treated cardiomyocytes

Previous report has shown that OLA1 inhibits GSK3β activity by mediating its Ser9 phosphorylation in lung cancer patients [10]. Also, GSK3β/β-Catenin interaction and β-Catenin-TCF/LEF transcription complex controls numerous cellular processes, including cellular growth and proliferation [12,13]. GSK3β has been shown to regulate cardiac hypertrophic response [9], however, whether OLA1 interacts with GSK3β and its effect on GSK3β/β-Catenin signaling during hypertrophic stimulation is not yet known. To first demonstrate the endogenous interaction between OLA1 and GSK3β during hypertrophic stimuli, protein extracts of cultured cardiomyocytes with/without ANG II exposure were subject to OLA1 immunoprecipitation and GSK3β immunoblotting. Figure 2 shows protein-protein interaction between OLA1 and GSK3β, both in untreated and ANG II-treated cells (Figure 2A). To demonstrate its implications on downstream mechanisms, we evaluated effects of ANG II on GSK3β/β-Catenin signaling in both cultured cardiomyocytes and mouse heart tissue. We observed a significant increase in GSK3β protein expression and its phosphorylation at Ser 9 in ANG II-treated cardiomyocytes as compared to untreated controls (Figure 2B, *P<0.05). GSK3β is inactivated when phosphorylated at Ser9. The above observations were coupled with a significant reduction in β-Catenin phosphorylation in ANG II treated cells (Figure 2C and 2F, *P<0.05). Similar trend in p-GSK3β and p-β-Catenin was observed in mouse hearts extracted on 3d and 14d after ANG II administration (Figure 2D and 2E, *P<0.05).

Fig. 2. ANGII-treated cardiomyocytes show endogenous interaction between OLA1 and GSK3β, and alteration of GSK3β/β-catenin phosphorylation.

A. Immunoprecipitation (IP) with OLA1 antibodies and Western blotting (WB) against GSK3β show OLA1 and GSK3β endogenous interaction in control and ANG II-treated ventricular myocytes. B. Western blot and densitometry data showing significant increase in GSK3β phosphorylation C. Western blot and F. densitometry showing a corresponding decrease in β-Catenin phosphorylation in ANG II-treated human cardiomyocytes. D. Representative Western blot showing significant increase in GSK3β phosphorylation and reduction in β-Catenin phosphorylation in mouse hearts at 3d and 14d after ANG II administration. Scatter plot showing the relationship between ANG II administration time and relative phosphorylation of E. GSK3β (Upper thick solid black line) andβ-Catenin (lower thin grey line). n=3, *P<0.05. All values are group means ± SEs.

OLA1 knockdown in cardiomyocytes abrogates ANG II-induced alterations in GSK3β and β-catenin phosphorylation

To investigate the role of OLA1 in ANG II-induced hypertrophic signaling response, we studied the effect of siRNA-mediated OLA1 knockdown on GSK3β and β-catenin phosphorylation. After 72h of transfection, we achieved efficient knockdown of OLA1 in cultured cardiomyocytes at both, mRNA and protein levels (Figures 3A and 3B, *P<0.05).

Fig. 3. OLA1 knockdown in ventricular myocytes reduces GSK3β phosphorylation.

A. qRT-PCR and B. Western blot/densitometry data showing efficient knockdown of OLA1 after 72hrs of siRNA transfection in human ventricular myocytes. C. Western blot and densitometry data showing a significant decrease in GSK3β phosphorylation and D. no change in β-catenin phosphorylation in OLA1 knockdown cardiomyocytes. n=3, *P<0.05. All values are group means ± SEs.

Interestingly, OLA1 knockdown significantly reduces p-GSK3β (Figure 3C, *P<0.05) without changing the phosphorylation of β-Catenin (Figure 3D, *P<0.05). Interestingly, we observed a significant decrease in OLA1 expression at both mRNA and protein levels (Figures 4A and 4B, *P<0.05) even after stimulation with ANG II in OLA1 knockdown cardiomyocytes compared to ANG II. Also, reduction in p-GSK3β (Figure 4C, *P<0.05) and an increase in p-β-Catenin was observed in these cells with ANG II stimulation.

Fig. 4. OLA1 knockdown reverses ANG II-induced modulation of GSK3β/β-catenin signaling in ventricular myocytes.

A. qRT-PCR and B. Western blot/densitometry data showing a significant decrease in OLA1 expression in human ventricular myocytes treated with OLA1 SiRNA + ANG II vs ANG II alone. C. Western blot and densitometry data showing a decrease in GSK3β phosphorylation and D. increase in β-catenin phosphorylation in OLA1 knockdown cardiomyocytes with ANG II stimulation vs ANG II alone. n=3, *P<0.05. All values are group means ± SEs.

OLA1 inhibition in cardiomyocyte reduces ANG II-induced nuclear accumulation of β-Catenin and hypertrophic response

We further elucidated the effect of OLA1 knockdown on β-Catenin nuclear localization upon ANG II treatment in cultured cardiomyocytes by evaluating cytoplasmic and nuclear compartmentalization by western blotting (Figure 5A). Nuclear extracts of ANG II-treated cells show a significant increase in β-Catenin expression, while there was no change in cytoplasmic extracts. Interestingly, upon OLA1 knockdown, we observed a significant reduction in nuclear β-catenin protein expression (Figure 5A). Furthermore, intriguingly, OLA1 knockdown cells show a significant decrease in ANG II-induced cell growth (Figure 5B and 5C; *^#P<0.05). Also, we observed a decrease in expression of fetal genes such as NPPA and MYH7 in OLA1 knockdown cells (Figure 5D and 5E).

Fig. 5. OLA1-deficiency decreases nuclear translocation of β-Catenin and hypertrophic response in ANG II-treated ventricular cardiomyocytes.

A. Western blot and densitometry showing an increase in β-Catenin protein accumulation in nuclear fractions of ANG II-treated (48h) human ventricular cardiomyocytes as compared to control cells. B. Representative immunofluorescence images of human cardiomyocytes treated with/without ANG II after OLA1 knockdown (OLA1 siRNA). Cells stained with Flash Phalloidin Green 488 (for cytoskeleton) and DAPI (for nuclei). Magnification 20X, bar 100 μm. C. Bar graph showing attenuation of ANG II-induced increase in cell area (μm²) in OLA1 knockdown cardiomyocytes compared to control (n= 27–65, *P<0.05). D. and E. qRT-PCR data showing decrease in mRNA expression of NPPA and MYH7 respectively in control, ANG II and OLA1 siRNA+ANG II-treated cardiomyocytes. n=3, *P<0.01. All values are group means ± SEs.

Discussion

Cardiac hypertrophy is an adaptive mechanism, seen in response to hemodynamic overload to reduce ventricular wall stress and increase contractility. At the cellular level, the process involves increase in cardiomyocyte cell size and volume. However, its molecular mechanism is complex and intersects with numerous signaling pathways that might cause or play a role in transition to maladaptive response [14]. Previous reports on OLA1 is majorly focused on its role in translation, cancer cell migration and proliferation [7,10]. Surprisingly, its role in cell growth as seen in cardiac hypertrophy is not explored so far. In the present study, we report that ANG II-treated cultured human cardiomyocytes (AC16 cells) or mouse hearts show upregulation of OLA1 expression with a corresponding increase in GSK3β phosphorylation, decrease in β-Catenin phosphorylation and subsequent increase in β-Catenin translocation to the nucleus. Also, our data shows an endogenous interaction between OLA1 and GSK3β in the cardiomyocytes. Interestingly, blockage of OLA1 decreases GSK3β phosphorylation, upregulates β-Catenin phosphorylation, diminishes β-Catenin nuclear accumulation leading to reduction in hypertrophic response. Based on the above findings, we demonstrate that OLA1 mediates ANG II-induced hypertrophic response via GSK3β/β-Catenin signaling (proposed mechanism illustrated in Figure 6). To the best of our knowledge, this is the first report to demonstrate the role of OLA1 in cardiomyocyte hypertrophic response.

Fig. 6. Proposed pathway for the role of OLA1 in ANG II-induced cardiac hypertrophy.

Illustration of mechanism depicts that ANG II treatment increases OLA1 expression, which endogenously interacts with and phosphorylates GSK3β, rendering it inactive. Thus, β-catenin phosphorylation decreases (degradation reduces) leading to cellular accumulation and translocation to nucleus from cytosol resulting in transcription of cardiac hypertrophy response genes such as NPPA and MYH7. Sustained activation of these genes, which is a feature of cardiac hypertrophy, ultimately leads to heart failure. HW/TL- Heart Weight/Tibia Length.

To investigate the functional role of OLA1 in cellular hypertrophic response, we studied the effect of loss-of-function on ANG II-induced increase in cardiomyocyte cell size and expression of hypertrophic genes like NPPA and MYH7. While our data suggests that OLA1 knockdown decreases ANG II-induced hypertrophic response, it is not clear how ANG II induces and regulates OLA1 expression. OLA1, also known as DOC45 has been shown as a downstream target of PI3K signalling in human cancer cell lines [15]. Hence, we would expect OLA1 to be regulated via PI3K/Akt signalling, in cardiomyocytes as well. Although, the exact mechanism by which OLA1 is regulated has to be elucidated during chronic ANG II exposure (beyond 14 days), OLA1 could be a target of ANG II via PI3K signalling in an indirect mechanism. Also, PI3K/Akt signalling (essential for physiological cardiomyocyte growth) is reported to be increased in physiological hypertrophy while this signalling is decreased during decompensatory hypertrophy which could possibly influence OLA1 expression [16,17]. However, our findings establish the downstream signaling mechanisms that are involved in OLA1-mediated decrease in hypertrophic response. OLA1 possesses GTPase and ATPase activities and has been shown to play a role in protein translation and synthesis [6–8]. Previous reports have shown that OLA1 mediates GSK3β phosphorylation (Ser9) in lung cancer [10]. GSK3β along with its complex including Axin and APC phosphorylate β-Catenin at Ser33, Ser37 and Thr41 [12,18–20]. β-Catenin, when phosphorylated, is specifically recognized by β-TrCP (a subunit of E3 ubiquitin ligase complex) and undergoes degradation by proteasome pathway [18,21]. In the present study, we found that ANG II induces GSK3β phosphorylation (Ser9) in both AC16 cell and mouse heart tissue, thus rendering it inactive. Concurrently, we observed a reduction in β-Catenin phosphorylation at Ser33/37/Thr41, thus leading to cellular accumulation of β-Catenin and its translocation to the nucleus, where it is involved in Tcf-lef (T-cell factor/lymphocyte enhancer factor)-mediated transcription of hypertrophic response genes [22]. OLA1 knockdown reverses the above molecular changes suggesting that the effect of OLA1 on hypertrophic response is mediated through GSK3β/β-Catenin signaling.

In the present study, immunoprecipitation and western blotting data shows OLA1 and GSK3β protein-protein interaction, however, we did not observe such interaction with β-Catenin (data not shown). While we demonstrate the effect of OLA1 knockdown on p-GSK3β, the possible interaction with other kinases or with Axin/APC complex is not clear. Furthermore, β-Catenin in the nucleus could promote Tcf-lef (T-cell factor/lymphocyte enhancer factor)-mediated transcription of hypertrophic response genes. In the present study, we show that OLA1 knockdown decreases NPPA and MYH7 hypertrophic gene expression. However, it is not clear how OLA1/GSK3β/β-catenin signaling upregulates hypertrophic gene expression. Previous reports have shown that GSK3β phosphorylates NFAT proteins and antagonizes the action of calcineurin, a potent transducer of hypertrophic stimuli [23]. GSK3β is also been reported to interact with GATA4, which plays an important role in NPPA transcription [24]. It might be interesting to determine other binding partners and signaling mechanisms that might serve as potential targets for OLA1-mediated hypertrophic response, which is our on-going and future research interests. Study limitation: While the expression of OLA1 increases in the mouse hearts after ANG II administration, limitation of the present study is that it is not clear which cell type in the heart contributes to OLA1 increase and how it could affect other biological processes that might contribute to pathogenesis of hemodynamic overload-induced cardiac pathology. Conclusions: In summary, our data suggest that OLA1 mediates ANG II-induced cardiac hypertrophic response via GSK3β/β-Catenin pathway. To our knowledge this is the first report to demonstrate the role of OLA1 in cardiac hypertrophy. Strategies to inhibit OLA1 might attenuate underlying molecular derangements in cardiac hypertrophic response, thus might limit transition from adaptive to maladaptive cardiac hypertrophy.

Methods

Vertebrate animals and osmotic minipump implantation for Angiotensin II administration in mice

All experiments conform to the protocols approved by UAB Institutional Animal Care and Use Committee. Eight-weeks-old C57BL/6J mice (Jackson lab, Bar Harbor, ME) were subcutaneously implanted with osmotic minipump (models 1007D, 2002 and 2004, ALZET DURECT, CA, USA) to continuously deliver ANG II (1000 ng.kg−1.min−1; cat# 05-23-0101, EMD Millipore, MA, USA) or saline (vehicle) for 3d, 14d and 28d as previously described [4].

Cell Culture and OLA1 knockdown (siRNA transfection)

Human cardiomyocyte cell line (AC16; SCC109, EMD Millipore, MA, USA) was cultured in DMEM/F12 (D6434, Sigma Aldrich, MO, USA) with 12.5% FBS, 2 mM L-Glutamine (cat# TMS-013-B, K0283-BC,EMD Millipore, MA, USA) and 1% Penicillin-Streptomycin (cat# 516106, EMD Millipore, MA, USA) and were incubated in a humidified chamber at 37°C with 5% CO₂. Cells in 60 mm culture dishes (~80% confluence) were transfected with either siRNA (SASI_Hs01_00244684, Sigma-Aldrich, MO, USA) against human OLA1 (siOLA1) or siRNA negative control (SIC001) at the final concentration of 50 nM using Lipofectamine RNAiMax Reagent (cat# 13778150, Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions and incubated for 72 hours.

Total protein isolation from cardiomyocytes and heart tissue

Protein isolation from cultured cells or homogenized mouse heart tissue was performed in lysis buffer (cat# 9803, Cell Signaling, MA, USA) containing protease and phosphatase inhibitors (cat# 78440, Thermo Fisher Scientific, MA, USA) as per manufacture’s instruction. Protein content was measured by Bradford assay using Quick Start™ Bradford 1x Dye Reagent (cat# 500–0205, Bio-Rad, CA, USA).

Cytoplasmic and nuclear protein extraction and western blot analysis

AC16 cells were fractionated into cytosolic and nuclear fractions using a NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (cat# 78833, Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. Protein quantification was performed by Bradford assay. Equal amounts of protein were separated by 10% SDS-PAGE and blotted onto PVDF membranes (cat# 1704156, Bio-Rad, CA, USA). The blots were incubated with antibodies against OLA1 (cat# 16371–1-AP, Proteintech, IL, USA), GSK-3β (cat# 12456, Cell Signaling Technology, MA, USA), P-GSK3β (S9) (cat# 9323, Cell Signaling Technology, MA, USA), β-Catenin (cat# 9562S, Cell Signaling Technology, MA, USA), Phospho-β-Catenin (Ser33/37/Thr41) (cat# 9561, Cell Signaling Technology, MA, USA), GAPDH (cat# MA5–15738, Thermo Fisher Scientific, MA, USA) and blots developed with an enhanced chemiluminescence detection system (Gel Doc EZ Imager, Bio-Rad, CA, USA). Densitometry of the bands from the western blot was analyzed using NIH-ImageJ software.

Immunoprecipitation and western blotting

AC16 cells were washed with ice-cold PBS (pH 7.4) and then lysed in the lysis buffer (cat# 895347, R&D systems, MN, USA). OLA1 protein was immunoprecipitated from the total protein extract following the manufacturer’s protocol as indicated in “Dynabeads Protein A” user manual (cat# 10001D, 10002D and 10008D, Life Technologies, CA, USA). Briefly, Dynabeads were bound to antibody for the protein of interest and immunoprecipitated the target antigen in total protein extracts. The target antigen was eluted from the immunocomplex and run on 10% SDS-PAGE gel and western blotting was performed.

RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted using RNA extraction kit (Qiagen), reverse transcribed to cDNA using qScript cDNA SuperMix (Quanta Biosciences) and qRT-PCR was performed using Fast SYBR Green Master Mix and Quanta bio-studio system (Applied Biosystems, NY). Relative mRNA expression of target gene was normalized to GAPDH and data was represented as fold change versus respective control.

Fluorescence staining and cell area measurement

Cells were fixed in 4% PFA for 10 min at room temperature at the end of treatment, permeabilized for 10 min with 0.5% Triton X-100 and washed twice with PBS. The cells were blocked for 30 min with 5% FBS at room temperature. Cells were then stained with Flash Phalloidin Green 488 (cat# 42420, Biolegend, CA, USA). At the end, coverslips mounted on glass slides with VECTASHIELD antifade mounting medium with 4’, 6-diamidino-2-phenylindole-DAPI (cat# H-1200, Vector Laboratories, CA, USA) and observed under inverted microscope (IX83, Olympus, TYO, JP). Images were analyzed for cell area measurements using NIH’s image J software.

Statistical analyses

Analyses were performed using SigmaPlot 12.0 (Systat Software Inc, San Jose, CA) using t-test and non-parametric statistics. Mann–Whitney’s test was used to compare between two groups. When more than two groups were involved, analysis of variance with a Bonferroni multiple comparison test was used to analyze the data. P-values less than 0.05 were considered to be statistically significant.

Supplementary Material

Supp figS1–2

Fig. S1. Heart weight to tibia length ratio in ANG II administered mice Bar graph showing an increase in heart weight to tibia length ratio after 14d and 28d of ANG II administration in mice in comparison to control mice. n=3, *P<0.05.

Fig. S2. OLA1 mRNA expression in ANG II administered mice heart qRT-PCR data showing no change in OLA1 mRNA expression in mice hearts on 3d, 14d and 28d after ANG II administration.