Cardioprotective Effect of Ulmus wallichiana Planchon in β-Adrenergic Agonist Induced Cardiac Hypertrophy

Ulmus wallichiana Planchon (Family: Ulmaceae), a traditional medicinal plant, was used in fracture healing in the folk tradition of Uttarakhand, Himalaya, India. The present study investigated the cardioprotective effect of ethanolic extract (EE) and butanolic fraction (BF) of U. wallichiana in isoprenaline (ISO) induced cardiac hypertrophy in Wistar rats. Cardiac hypertrophy was induced by ISO (5 mg/kg/day, subcutaneously) in rats. Treatment was performed by oral administration of EE and BF of U. wallichiana (500 and 50 mg/kg/day). The blood pressure (BP) and heart rate (HR) were measured by non-invasive blood pressure measurement technique. Plasma renin, Ang II, NO, and cGMP level were estimated using an ELISA kit. Angiotensin converting enzyme activity was estimated. BP and HR were significantly increased in ISO group (130.33 ± 1.67 mmHg vs. 111.78 ± 1.62 mmHg, p < 0.001 and 450.51 ± 4.90 beats/min vs. 347.82 ± 6.91 beats/min, respectively, p < 0.001). The BP and HR were significantly reduced (EE: 117.53 ± 2.27 mmHg vs. 130.33 ± 1.67 mmHg, p < 0.001, BF: 119.74 ± 3.32 mmHg vs. 130.33 ± 1.67 mmHg, p < 0.001); HR: (EE: 390.22 ± 8.24 beats/min vs. 450.51 ± 4.90 beats/min, p < 0.001, BF: 345.38 ± 6.79 beats/min vs. 450.51 ± 4.90 beats/min, p < 0.001) after the treatment of EE and BF of U. wallichiana, respectively. Plasma renin, Ang II, ACE activity was decreased and NO, cGMP level were increased. The EE and BF of U. wallichiana down regulated the expression of ANP, BNP, TNF-α, IL-6, MMP9, β1-AR, TGFβ1 and up regulated NOS3, ACE2 and Mas expression level, respectively. Thus, this study demonstrated that U. wallichiana has cardioprotective effect against ISO induced cardiac hypertrophy.


INTRODUCTION
Cardiac hypertrophy is the abnormal growth, or thickening, of the heart muscle, often due to chronic hypertension that leads to HF. While in left ventricular hypertrophy, the myocardial oxygen consumption is increased which results in decreased coronary blood flow reserve which results in angina pectoris, myocardial infarction, and sudden death (Levy et al., 1990). It is estimated that nearly 23 million people are affected by HF worldwide (McMurray et al., 1998). In India, the prevalence of HF is around 4.6 million with an annual occurrence of nearly 1.8 million (Huffman and Prabhakaran, 2010). Cardiac hypertrophy is a physiological adaptation which causes increased workload on the myocardium. Hypertrophic growth is due to many types of heart disease, including ischaemic heart disease, hypertension, heart failure, cardiac hypertrophy, and valvular disease (Frey et al., 2004).
Ulmus wallichiana is used traditionally as diuretic (Khare, 2008). A related species U. rhynchophylla, native to China, known as Gou Teng in Chinese medicine, is used for eclampsia, headache, dizziness, convulsions, high fever, and hypertension (WHO). U. macrocarpa has antihypertensive and vasorelaxant activity (Oh et al., 2008). Therefore due to diuretic property of U. wallichiana it is used in hypertension as it contains quercetin (a polyphenolic flavonoid) and its major metabolites modulate NO/cGMP dependent vasorelaxation and regulates BP (Suri et al., 2010). Due to prolong increase in BP (hypertension) there is direct haemodynamic stress, leading to hypertrophy (Mehta et al., 2014). Therefore, we have explored the effect of EE and BF of U. wallichiana in cardiac hypertrophy.
The effective dose of EE ranges from 500 to 750 mg/kg and that of BF ranges from 25 to100 mg/kg as reported in patent by Maurya et al. (2014) (co-author of this article). We have selected the above dose of EE (500 mg/kg) as it showed optimal decrease in BP. As this dose is high enough to be translated from rat to human, BF was isolated and used with effective dose of 50 mg/kg. BF enriched with high content of quercetin analog flavonoids compared to EE (Sharan et al., 2010). Therefore the effective dose of EE is 500 mg/kg and that of BF is 50 mg/kg (Maurya et al., 2014).
The anti-hypertrophic activity of U. wallichiana was remained unexplored in cardiac hypertrophy model. In our lab, we have explored the antihypertensive activity of EE and BF of U. wallichiana (Syed et al., 2016). Therefore, in the present work, we have investigated the anti-hypertrophic activity of EE and BF of U. wallichiana in ISO induced cardiac hypertrophy.

Plant Material
Stem bark of U. wallichiana was collected and authenticated by Arya et al. (2013) of CSIR-Central Drug Research Institute (CDRI), Lucknow, India and extraction procedure was reported (Sharan et al., 2010;Arya et al., 2013;Maurya et al., 2014). EE and BF from stem bark of U. wallichiana were obtained from Medicinal and Process Chemistry division of CSIR-CDRI, Lucknow, India. The fingerprinting of EE and BF of U. wallichiana was done by us using LC-MS/MS -Mass spectrometer QTrap 5500-AB (Applied Biosystems/MDS Sciex, Canada), using an electrospray ionization source in negative ion mode (ESI) and published (Syed et al., 2016).

Animals
The experiments were carried out with male normotensive wistar rats (180-220 g) procured from the Laboratory

Study Protocols
Male wistar rats were injected subcutaneously (s.c.) with ISO 5 mg/kg/day for 10 days (Yeh et al., 2010) with or without 500 mg/kg/day of EE and 50 mg/kg/day of BF of U. wallichiana. The rats were randomly divided into four groups and each group contains six animals each.
(i) Control group: olive oil (vehicle) was given s.c. for 10 days.
(ii) ISO group: Isoprenaline, 5 mg/kg/day was administered subcutaneously for 10 days, to induce cardiac hypertrophy. (iii) ISO + EE group: EE was administered orally for 14 days at a dose of 500 mg/kg/day of U. wallichiana after development of cardiac hypertrophy. (iv) ISO + BF group: Oral administration of BF for 14 days at a dose of 50 mg/kg/day of U. wallichiana after development of cardiac hypertrophy.

NIBP Measurement
To measure changes in arterial blood pressure in conscious rat, NIBP technique was used (Columbus instrument, Model NIBP-8). Conscious rats were acclimatized in the restrainer for 15 min three times a day for a period of 1-week before the starting of the testing schedule. The trained animals were placed in a restrainer and tails of rats were exposed to hot air blower. The rats were allowed to acclimatize inside the cage for 15 min before starting the actual blood pressure measurement. Occlusion and sensor cuffs were placed around the base of the tail and the cuffs were inflated and deflated several times to measure tail arterial blood pressure. A minimum of 8-10 cycles of inflation and deflation were made and the measurement of tail arterial blood pressure was recorded and the data was averaged. The data were recorded and analyzed with software.

IBP Measurement
Arterial blood pressure was measured by invasive method in the urethane (1.2 g/kg) anesthetized rats. The tracheotomy was performed. The catheter was inserted into the carotid artery to monitor the alteration in the arterial blood pressure. The other end of the catheter was connected to the computerized Data Acquisition System (DAS), AD-Instruments with compatible software.
Determination of Heart Weight to Body Weight Ratio, Heart Weight to Tail Length Ratio, and Heart Weight to Tibia Length Ratio After the measurement of IBP the rats were sacrificed and heart weight, tail length, and tibia length were measured. The heart weight index was calculated by dividing the heart weight by the body weight (Yeh et al., 2010), the heart tail index was calculated by dividing the heart weight by tail length (Chowdhury et al., 2013), and the heart tibia index was calculated by dividing the heart weight by tibia length (Oh et al., 2015).

Renin Inhibition Assay
The renin inhibition assay was performed by Renin Inhibitor Screening Assay Kit (catalog number: 10006270; Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions.
The renin inhibition was expressed as percentage inhibition or percent initial activity as a function of the inhibitor concentration to determine the IC 50 value.

Estimation of Plasma Renin Level in Plasma
The plasma renin assay was measured by rat Renin (Renin) ELISA kit (catalog number: E02R0022; BlueGene Biotech, Shanghai, China) according to the manufacturer's instructions.
To determine renin level, the blood was collected from the retroorbital plexus of rat in EDTA containing tubes. Plasma was harvested by using centrifugation at 1000 × g for 15 min at 4 • C. Plasma (100 µL) was used for estimation of renin concentration.

Angiotensin Converting Enzyme (ACE) Assay
Angiotensin converting enzyme activity was determined by a spectrophotometric mono-reagent assay. The reaction mixture containing serum (10 µL) and the substrate solution (200 µL) were kept at 37 • C in Elisa plate reader (BIOTEK, USA) and reaction was monitored at 340 nm for 30 min. Serum ACE activity was expressed as unit/L (Hanif et al., 2009).

Estimation of cGMP Level in Plasma
The cGMP concentrations were estimated using an enzyme immunoassay (EIA) kit (catalog number: 581021; Cayman Chemical, Ann Arbor, MI, USA), as per the manufacturer's instructions. To determine the cGMP levels in plasma, 2 mL of ice-cold ethanol was added to 500 µL of plasma, and the solution was centrifuged at 1500 × g for 10 min. The supernatant was evaporated by vacuum centrifugation (Turbo Vap LV, Caliper, USA) and used for the cGMP estimation.

Determination of Reactive Oxygen Species (ROS)
Reactive oxygen species was estimated in heart tissue. Heart tissue was homogenized in ice-cold 40 mMtris buffer (pH 7.4) and samples were centrifuged at 100,000 × g at 4 • C in an Eppendorf centrifuge (5819 R Germany) for 15 min. The resulting heart homogenate was incubated with DCFDA (5 µM) for 30 min at 37 • C. Formation of fluorescent product DCF was monitored by fluorometer at an excitation wavelength of 488 nm and emission wavelength of 530 nm.

Histopathological Examination
After measurement of hemodynamic parameters of rats, their hearts were quickly and filled with 4% paraformaldehyde solution, and kept in it for 24 h. The heart samples were embedded in paraffin blocks. Samples were sliced of 5 µm thickness and stained with hematoxylin and eosin (H&E) for assessing myocardial fibrosis. Myocardial fibrosis and relative cell surface area were evaluated in each of the heart tissue sections using a morphometric procedure using ImageJ software.

Total RNA Extraction
Total RNA was extracted from 0.1 to 0.2 g of rat heart tissues by using TRIzol reagent as per manufacturer's protocol. The quality and quantity of the isolated RNA were assessed using the 260/280 nm absorbance ratio (1.8-2.0 indicates a highly pure sample) using NanoDrop 2000c spectrophotometer (USA). Total RNA was stored at −80 • C until used.

Synthesis of cDNA and RT qPCR Quantification
RNA was transcribed into cDNA in a 20 µL reaction using a High Capacity RNA to cDNA Reverse Transcriptase kit, analyzed, and amplified. 2x master mixes were prepared using 2 µl 10x Reverse Transcription buffers, 0.8 µL 25x dinucleotide triphosphate (dNTP) mix (100 mM), 2 µL 10x Reverse Transcription random primers, and 1 µL MultiScribe Reverse Transcriptase. For a 20 µL reaction mixture under the following conditions: 5.8 µL master mix; 2 µL of 2 µg RNA sample; 12.2 µL nuclease free water. Following was the program for reverse transcriptase PCR using SureCycler 8800 (Agilent Technologies, USA): 25 • C for 10 min; 37 • C for 120 min; 85 • C for 5 min; hold at 4 • C. The product obtained was diluted 4× for real-time PCR. Samples were seeded in triplicate in 96-well reaction plates (LightCycler 480). RT qPCR was performed on Light Cycler 480 II (Roche Diagnostics) with SYBR Green fluorescent label. Samples (10 µL final volume) contained the following: 2x SYBR Premix Ex Taq 1 µL of each primer; 3 µL of cDNA sample, and 5 µL of nuclease free water. Cycle threshold (Ct) values were used to calculate the amount of amplified PCR product relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control (reference gene).

Statistical Analysis
Statistical analysis was performed with Prism software version 5.01 (Graph Pad Software, San Diego, CA, USA) and Curve Expert Professional version 2.0.3. Results are expressed as mean ± SEM. Statistical significance was evaluated by oneway analysis of variance (ANOVA) followed by Newman-Keuls Multiple comparison test and t-test. A value of p < 0.05 was considered to be statistically significant.

U. wallichiana Attenuated Heart Weight to Body Weight Ratio, Heart Weight to Tail Length Ratio, and Heart Weight to Tibia Length Ratio
Heart to body weight, heart weight to tail length, and heart weight to tibia length ratio were significantly increased in the ISO groupas compared to control group. The treatment of extract and fraction of U. wallichiana significantly decreased heart to body weight, heart weight to tail length, and heart weight to tibia length ratio in ISO+EE and ISO+BF group compared to disease control (Figures 1A-C), respectively. The images of whole heart have been provided in the Supplementary Figure 3.

Hemodynamics
Blood pressure and HR were significantly increased in ISO induced cardiac hypertrophy as compared to normotensive Wistar group (Figures 2A-C). The treatment of extract and fraction of U. wallichiana significantly decreased the SBP in ISO+EE and ISO+BF compared to diseased control (Figure 2A) and it also decreased the HR in ISO+EE and ISO+BF compared Frontiers in Pharmacology | www.frontiersin.org FIGURE 1 | Effect of Ulmus wallichiana on (A) Heart weight to body weight ratio, (B) Heart weight to tail length ratio, and (C) Heart to tibia length ratio in ISO induced cardiac hypertrophy in wistar rats. Data were analyzed by using one-way ANOVA followed by Newman-Keuls Multiple comparison test. Data are shown as Mean ± SEM; α, control vs. ISO; β, ISO vs. EE; γ, ISO vs. BF; * P < 0.05, * * P < 0.01, * * * P < 0.001.
to diseased control (Figure 2D), respectively. (In support IBP data is given in Supplementary Figures 2A-D.)

Renin Inhibition
Renin inhibition assay in vitro was conducted to assess the renin inhibitory properties of EE and BF of U. wallichiana. The IC 50 value for EE was found to be 12.5 µg/mL and that of BF was 5.5 µg/mL (Figures 3E,F)

U. wallichiana Reduced Plasma Renin Concentration
The plasma renin concentration was increased significantly in ISO group. Administration of EE and BF of U. wallichiana significantly decreased plasma renin concentration in ISO+EE and ISO+BF group compared to diseased control ( Figure 3A) group, respectively.

U. wallichiana Decreased ACE Activity
The plasma ACE activity was increased significantly in ISO group. Further administration of EE and BF of U. wallichiana significantly decreased ACE activity in ISO+EE and ISO+BF group compared to diseased control ( Figure 3B) group, respectively.

U. wallichiana Attenuated AG II Concentration in Plasma
The plasma Ang II level was increased significantly in ISO group. Further administration of EE and BF of U. wallichiana significantly decreased ACE activity in ISO+EE and ISO+BF compared to diseased control ( Figure 3D) group, respectively.

U. wallichiana Increased Plasma Nitrite/Nitrate (NO) Concentration
The concentration of NO was decreased significantly in ISO group. Further administration of EE and BF of U. wallichiana significantly increased NO in ISO+EE and ISO+BF group compared to diseased control ( Figure 3C) group, respectively.

U. wallichiana Increased cGMP Concentration
The concentration of cGMP was decreased significantly in ISO group. Further administration of EE and BF of U. wallichiana significantly increased cGMP concentration in ISO+EE and ISO+BF group compared to diseased control ( Figure 4A) group, respectively.

U. wallichiana Attenuated ROS Level
The ROS activity was increased significantly in ISO group. Further administration of EE and BF of U. wallichiana significantly increased ROS activity in ISO+EE and ISO+BF group compared to diseased control ( Figure 4B) group, respectively.

U. wallichiana Reduced Expression of Cardiac Hypertrophic Markers
To investigate whether EE and BF of U. wallichiana confers cardio-protection in ISO treated group. We have determined the cardiac gene expression of ANP, BNP, TNF-α, IL-6, TGFβ1, MMP9, β1-AR, ACE2, Mas relative to ISO treated group. ISO caused significant up regulation of ANP, BNP, TNF-α, IL-6, TGFβ1, MMP9, β1-AR and down regulation of NOS3, ACE2, Mas as compared to control group. On treatment with the EE and BF of U. wallichiana the ANP, BNP, TNF-α, IL-6, TGFβ1, MMP9, β1-AR was significantly reduced in ISO+EE and ISO+BF group and significantly increased in NOS3, ACE2, Mas expression as compared to diseased control group, respectively, ( Figure 4E).

U. wallichiana Decreased Cardiac Fibrosis and Relative Cell Surface Area
The regularly arranged myocardial fibers with clear striations were observed in the control group ( Figure 5A). The photomicrograph of heart from normal group shows no pathological alteration. The histological sections of the heart obtained from ISO group showed enlargement of cardiomyocyte showing increased cell radius with eccentric positioning of nucleus indicating hypertrophy. Fibrocytic proliferation was also invariably observed along with cellular degenerative changes characterized by scanty cytoplasm and vacuolation. Focal infiltration of inflammatory cells is observed in few animals. There was significant increase in myocardial fibrosis in ISO group ( Figure 5B). There was also significant increase in relative cell surface area in ISO group. On administration of EE and BF of U. wallichiana significantly decreased myocardial fibrosis in ISO+EE and ISO+BF group compared to diseased control (Figures 4C and 5C,D); decreased relative cell surface area in ISO+EE and ISO+BF group as compared to diseased control ( Figures 4D and 5C,D) group, respectively. A representative photo has also been given in the Supplementary Figure 4.

DISCUSSION
In the present study, the U. wallichiana attenuated cardiac hypertrophic response induced by ISO, a non-selective β-adrenoceptor agonist. The EE and BF of U. wallichiana attenuated cardiac hypertrophy by decreasing SBP and HR and inhibiting plasma renin, ACE, Ang II and augmented plasma NO and cGMP concentration (Supplementary Table 2). The EE and BF of U. wallichiana have antihypertensive activity (Syed et al., 2016). ISO in chronic dose not only produced cardiac hypertrophy but also altered the gross structure of the heart. There was an increase in weight of the heart, but no significant effect on weight of the body. The EE and BF of U. wallichiana decreased the heart rate, renin release, and Ang II level there by decreasing the cardiac output. ISO, a directly acting catecholamine, binds with the β-adrenergic receptor in heart and causes significant increase in heart rate (Arnold and McDevitt, 1983), increased plasma renin and Ang II level (Leenen et al., 2001). ISO, a sympathetic β-adrenergic receptor agonist, stimulates renin release via adenylate cyclase-cyclic AMP system (Suzuki and Hashiba, 1986). The stimulatory effect of ISO on renin release was inhibited by the EE and BF of U. wallichiana due to the presence of quercetin analog flavonoids. Renin overproduction increases the level of Angiotensin I in the blood, leading to high BP. The IC 50 value for EE was found to be 12.5 µg/mL and that of BF was 5.5 µg/mL, showing renin inhibition in the present study.
Cardiac mast cell secretes renin initiating the formation of angiotensinogen. Cardiac mast cells releases renin on degranulation by ROS. Renin initiates the activation of a local renin-angiotensin system which culminates in the formation of Ang II (Reid et al., 2011). The quercetin analog flavonoids acts as antioxidant and inhibits release of pro-inflammatory cytokines, IL-6 from mast cell. Therefore the EE and BF of U. wallichiana attenuates the ROS production, due to its antioxidant activity, inhibiting the TNF-α and IL-6 and it has renin inhibition property (Figures 3E,F). Thus EE and BF may be responsible to inhibit the release of renin from the cardiac mast cells.
Angiotensin converting enzyme, an enzyme that regulates RAAS pathway, contains zinc and a zinc ion (Ojeda et al., 2010). The flavonoids quercetin analog forms chelate complex with zinc and inhibit the ACE enzyme (Actis- Goretta et al., 2006;Guerrero et al., 2012). The ACE2 and Mas receptors are involved in the cardiac hypertrophy as their reduced expression is found in hypertrophied heart (Zhang et al., 2014). ACE2, by degradation of Ang II, forms Ang (1-7) which opposes actions of Ang II by binding to the Mas receptor leading to vasodilation, antihypertrophic and anti-fibrotic effect (Dias-Peixoto et al., 2012). The EE and BF of U. wallichiana increased the ACE2 and Mas receptor gene expression as compared to the ISO group.
In RAS pathway, increased renin and ACE enzyme causes increased level of Ang II. Angiotensin II activates NAD(P)H oxidase leading to oxidative stress (Veresh et al., 2008) causing decrease in bioavailability of NO resulting in endothelial dysfunction and vasoconstriction of arterioles leading to hypertension. Emerging evidence suggests that the nitricoxide (NO)/guanosine 3 5 -cyclic monophosphate (cGMP) pathway plays an important role in the modulation of cardiac hypertrophy (Ozaki et al., 2002). Therefore the EE and BF of U. wallichiana increased the bioavailability of NO/cGMP leading to vasodilation and act as anti-hypertrophic (Tirziu and Simons, 2008). As NO has anti-hypertrophic effect which is mediated by cGMP which involves cGMP-dependent protein kinase I activation. Plasma NO level was decreased by ISO, an indirect indicator of endogenous NO production.
NO/sGC/cGMP/cGMP dependent protein kinase G acts as cardioprotective. U. wallichiana causes increase in the NO level, probably via signal transduction pathway which results in cGMP generation. Cardiovascular diseases are associated with the impairment of Nitric oxide/soluble guanylate cyclase/cGMP signaling (Korkmaz et al., 2009). NO/cGMP/ANP act as antihypertrophic. ANP and BNP are present in cardiac tissues (London, 2006), and act as cardiac hypertrophic markers (Althurwi et al., 2013).
The cardiac mast cell induces MMP activation due to oxidative stress as well as TNF-α which causes degradation of collagen causing cardiac fibrosis in cardiac hypertrophy (Lee et al., 2010;Parthasarathy et al., 2014). ROS also have potent effects on the extracellular matrix, stimulating cardiac fibroblast proliferation  and activating MMPs. EE and BF of U. wallichiana attenuates the increased expression of TGFβ1, MMP9, and ROS activity. TNF-α, IL-6 are cytokines which mediates various physiological and pathological functions like inflammation, cellular survival, growth, differentiation, and apoptosis. TNF-α in the myocardium produce inflammation and apoptosis via TNFR1 receptor (Sack et al., 2000) where as IL-6 also produce cardiac hypertrophy, fibrosis, and inflammation (Diaz et al., 2009;Meléndez et al., 2010). The flavonoids and quercetin analogs present in EE and BF of U. wallichiana act as antioxidant (LaManna et al., 2011;Brunetti et al., 2013) thus decreasing ROS activity.

CONCLUSION
We suggest that the quercetin analog flavonoids present in the EE and BF of U. wallichiana attenuated cardiac hypertrophy (Figure 6) showing cardioprotective effect. The BF showed pharmacological response and potent at a lower dose (50 mg/kg/day) as compared to EE (500 mg/kg/day) as BF contains higher concentration of flavonoids than EE (Sharan et al., 2010).
Thus, the EE and BF of U. wallichiana showed cardioprotective effect against ISO induced cardiac hypertrophy. Based on these findings the marker compounds present in U. wallichiana can be isolated to study the mechanism of action and it may be a novel therapeutic agent for treatment of cardiac hypertrophy.

AUTHOR CONTRIBUTIONS
AS, KH, and JG designed the research work; AS, SL, DM, GV, AG, and SK performed the research experiments; RM, HB, KH, and JG contributed new reagents/analytic tools; AS, SL, DM, KH, HB, RM, and JG analyzed data; AS, KH, and JG wrote the paper.

FUNDING
JG is thankful to Department of Science and Technology, SERB (Govt. of India), Department of Biotechnology (Govt. of India), and CSIR (Govt. of India) for financial support [GAP0114, BSC0103, BSC0201].