Lycium barbarum polysaccharides restore adverse structural remodelling and cardiac contractile dysfunction induced by overexpression of microRNA‐1

Abstract MicroRNA‐1 (miR‐1) stands out as the most prominent microRNA (miRNA) in regulating cardiac function and has been perceived as a new potential therapeutic target. Lycium barbarum polysaccharides (LBPs) are major active constituents of the traditional Chinese medicine based on L. barbarum. The purpose of this study was to exploit the cardioprotective effect and molecular mechanism of LBPs underlying heart failure. We found that LBPs significantly reduced the expression of myocardial miR‐1. LBPs improved the abnormal ECG and indexes of cardiac functions in P‐V loop detection in transgenic (Tg) mice with miR‐1 overexpression. LBPs recovered morphological changes in sarcomeric assembly, intercalated disc and gap junction. LBPs reversed the reductions of CaM and cMLCK, the proteins targeted by miR‐1. Similar trends were also obtained in their downstream effectors including the phosphorylation of MLC2v and both total level and phosphorylation of CaMKII and cMyBP‐C. Collectively, LBPs restored adverse structural remodelling and improved cardiac contractile dysfunction induced by overexpression of miR‐1. One of the plausible mechanisms was that LBPs down‐regulated miR‐1 expression and consequently reversed miR‐1‐induced repression of target proteins relevant to myocardial contractibility. LBPs could serve as a new, at least a very useful adjunctive, candidate for prevention and therapy of heart failure.

function and progression of HF. 4,5 And notably microRNA-1 (miR-1), a cardiac-enriched miRNA, is in most close relation to heart conditions, and the changes in its expression have been discovered in a variety of heart diseases. [6][7][8][9][10] Our previous report revealed that outcomes of miR-1 overexpression induced adverse structural remodelling, which impaired cardiac contractile and diastolic function and even caused HF. 11 MiR-1 could result in an epigenetic defect as cardiac hypertrophy by microinjecting fragments of miR-1. 12 The functional significance of the findings uncovered that miR-1 might be a trigger and sustainer for the structural remodelling and dysfunction of HF. Down-regulation of miR-1 may produce cardioprotective effects 7,8,13 and miR-1 has been perceived as a new therapeutic target for heart diseases. Therefore, it is valuable to develop new candidates regulating miR-1 by which to treat relevant heart diseases.
Lycium barbarum polysaccharides (LBPs) are important active constituents extracted from the traditional Chinese herb L. barbarum. There are scientific proofs of its pharmacological and biological functions including anti-oxidative properties, 14 immunomodulation, 15 antitumor activity, 16 anti-ageing effect, 17 neuroprotection, 18 hypoglycaemic and hypolipidemic effects 19 and male fertility-facilitating, 20 indicating extensive application prospects on relevant diseases. To the best of our knowledge, there are only a few studies concerning the effect of LBPs on cardiovascular system. Electrocardiographic and biochemical evidence were found that LBPs elicit a typical cardioprotective effect against Doxorubicin-related oxidative stress in rats and dogs. 21,22 LBPs reduced myocardial apoptosis and injury in ischemia/reperfusion process of rat heart and could prevent the development of cardiovascular diseases. 23,24 However, until now, no available information has addressed the effects of LBPs on cardiac structure and function in HF. Given its biological property, it is conceivable that LBPs may produce beneficial actions in preventing the development of HF and act as a potential therapy option. This study was aimed to investigate the effects of LBPs on impaired cardiac function and structural remodelling induced by overexpression of miR-1 and to unravel the underlying molecular mechanism for exploiting therapeutic potential on HF.

| Preparation of Lycium barbarum
polysaccharides Lycium barbarum polysaccharides were prepared using the extraction procedure optimized by Jingcheng Tang. 25

| Generation of miR-1 transgenic (Tg) mice
All experimental procedures and protocols used in this investigation received approval by the Institutional Animal Care and Use Committee of Harbin Medical University, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). We generated Tg mouse line for cardiac-specific overexpression of miR-1 driven by the a-myosin heavy chain (a-MHC) promoter as previously reported. 11 Briefly, sexually immature female C57BL/6 mice (4-5 weeks of age) were applied to obtain sufficient quantity of eggs (>250) for microinjection. The mice used in this study were the fifth generation or later.

| Administration of LBPs to mice
Animals were divided into three groups: C57BL/6 male wild-type (WT) littermate mice, miR-1 Tg mice, miR-1 Tg mice received LBPs. LBPs were delivered into miR-1 Tg mice at 3 months of age through intragastric administration at a dosage of 200 mg/kg/d 22,26 for 1 month.
The age-matched WT mice and miR-1 Tg mice were received 0.9% NaCl (0.2 mL/d) as the vehicle. In the next 2 months, LBPs was mixed in water and administered to the mice by drink at the same dose. Monitoring of drinking quantity manifested no change in all mice. Measurements were made when the mice were at an age of 6 months.

| Culture of neonatal rat ventricular cardiomyocytes (NRVCs)
Hearts from 1-to 3-day-old Wistar rats were excised, and the ventricular myocardium was minced in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) and cells were dissociated with 0.25% trypsin-EDTA solution (Beyotime, China). After centrifugation, the collected isolated cells were plated onto 25 cm 2 cell culture flask (Corning Incorporated, USA) for 100 minutes to separate ventricular myocytes from the faster attaching non-myocytes. The re-collected cells were then seeded in a six-well plate (2 9 10 5 /well) in DMEM containing 10% foetal bovine serum (FBS) and 0.1 mmol/L bromodeoxyuridine (sigma). Cells were used for experiments 48-72 hours after isolation when demonstrating rhythmic contractions.

| Detection of heart function
Mice were anaesthetized with sodium pentobarbital (60 mg/kg, intraperitoneal). After recording ECG for 10 minutes, pressurevolume (PV) loops (Scisense, Ontario, Canada) measurements were performed with a 1.2F mouse pressure-volume catheter (Pressure-Volume Control Unit FV896B) which was retrogradely inserted into the left ventricle cavity through the right carotid artery to measure baseline arterial pressure haemodynamics in the closed chest. All data were analysed with iWork Labscribe2 Data Recording and Analysis software. Baseline haemodynamic values were obtained by averaging 300 beats recorded during steady-state periods. The main measured parameters included ejection fraction (EF), cardiac output (CO), end-systolic pressure (ESP), end-diastolic pressure (EDP), endsystolic volume (ESV), end-diastolic volume (EDV), maximum derivative of change in systolic pressure over time (dP/dt max ) and maximum derivative of change in diastolic pressure over time (dP/dt min ). Haematoxylin and eosin (H&E) staining was performed by routine method. Hearts were taken and fixed in zinc formalin for 24-48 hours then processed using a Sakura Tissue Tek VIP5 processor.

| Evaluation of morphological remodelling
Samples were embedded in paraffin and sectioned longitudinally from the identical plane of the initial portion of the ascending aorta at 4 lm using a microtome. Sections were stained with haematoxylin and eosin for identification. Heart-to-bodyweight ratio (HW/BW) was calculated for each group. The length of sarcomeres was evaluated by Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MA).

NRVCs
The total RNA samples were isolated using Trizol and phenol/chloroform extraction procedures. MiR-1 level was quantified by the

| Western blot analysis
The

| Data analysis
Data were calculated as mean AE SEM (standard error of the means) except the length of sarcomeres as mean AE SD (standard deviation).
The ANOVA test was performed for statistical comparisons among multiple groups. Differences were considered statistically significant at P < .05. SPSS13.0 was used for all statistical analyses.

| LBPs reduced the expression of myocardial miR-1 in vitro and in vivo
We quantified the miR-1 levels in neonatal rat ventricular cardiomyocytes (NRVCs) with incremental doses of LBPs. Excitingly, our data from qRT-PCR showed that LBPs significantly reduced the expression of endogenic miR-1 ( Figure 1A). Similar results were observed in the overexpression model of miR-1 by transfecting miRNA mimics in NRVCs ( Figure 1B) (Figure 2A,B). TEM examination found that intercalated discs were dissolved markedly with vacuolar degeneration of gap junctions and decreased density of the macula adherents in the hearts of Tg mice but not in those treated with LBPs ( Figure 2C,D). Figure 2D presented the quantitative analysis of percentage of damaged length in intercalated disc organization.  Figure 3A). In addition, the impaired cardiac contractile and diastolic functions of Tg mice were indicated by decreased CO, ESP, dP/dt max and dP/dt min , as well as increased EDP, ESV and EDV ( Figure 3B-H). Interestingly, in the treatment of LBPs, the indexes of cardiac contractile function including CO, ESP, ESV and dp/dt max were seen significant restorations.
However, except EDV, the indexes of cardiac diastolic function including EDP and dP/dt min were not improved significantly in mice treated with LBPs ( Figure 3B-H). These implied the beneficial effect of LBPs on contractile function and at least partial recovery on diastolic function.

| LBPs reversed the reductions of target proteins of miR-1 and key contractile proteins
In the present study, the repressive effects of miR-1 on the target proteins calmodulin (CaM) and cardiac myosin light chain kinase (cMLCK) are shown in Figure 5A

| DISCUSSION
MiRNA-based therapy has been recognized to be a promising novel therapeutic strategy for the treatment of cardiovascular diseases. 29,30 The principal finding of this study is the first to identify that LBPs protect the hearts from adverse structural remodelling and Lycium barbarum (also known as Goji berry or wolfberry), a famous Chinese medicinal herb and also a functional food, has a long history of use in a broad spectrum of diseases to nourish liver, kidneys and eyes and becomes increasingly popular in Europe and North America. 19,41,42 Polysaccharides are the most important functional ingredient that is approximately 40% of dry mass in L. barbarum fruits. 18 Till recent several years LBPs are given increasing attention in the field and most researches focus on its anti-oxidative property as a free radical scavenger for immunomodulation and anticancer. [14][15][16] The studies of LBPs on cardiovascular aspect are only limited to the protective phenomena (no discussion of mechanism) on doxorubicin-induced cardiotoxicity and ischaemia/reperfusion injury of rat heart. [21][22][23][24] In the present study, we used the model of HF with complicated pathogenesis is associated with loss of cardiac contractility, abnormalities in Ca 2+ handling and altered phosphorylation states of cardiac contractile regulatory protein. 43,44 Our previous study has elucidated that overexpression of miR-1 repressed potential target proteins CaM and cMLCK, which attenuated the phosphorylation of CaMKII, cMyBP-C and MLC2v, leading to impaired sarcomeric assembly and consequent heart dysfunction. 11 CaM, a known transducer of Ca 2+ signal, activates CaMKII 45 to directly phosphorylate cMyBP-C, which is a thick filament protein with physiological significance for normal myocardial contractility and stability and serves as a convergent node for signalling processes in the cardiomyocyte. 46,47 Activation of cMLCK, also regulated by CaM, appears to be pivotal to maintain the phosphorylation of MLC2v, which functions as an essential component of thick myofilament assembly and plays a critical role in maintaining normal myocardial contractility and function. 48 there is still a lack of in-depth study on the pharmacological effects of its active ingredients, especially those that exert cardioprotection.
Our research addressed the protection of LBPs on cardiac contraction and conduction dysfunction and adverse structural remodelling induced by overexpression of miR-1 via direct and indirect improvement of key proteins essential for cardiac contractile function, and provided new insights into the significant role of LBPs as a new, at least a very useful adjunctive, candidate for prevention and therapy of HF. Herein, we have accumulated scientific evidence for LBPs' cardioprotective role and hope that these novel findings will develop its potential as an evidence-based medicine and expand the application of LBPs on heart disease.