Regular paper Vol. 59, No 2/2012

Insulin resistance (IR) plays a critical role in metabolic syndrome (MS). Previous studies have demonstrated that activated ROCK is increased in MS patients. However, the effect of Rho-kinase (ROCK) on IR has not been definitely determined. Thus, the aims of the present study were to determine whether ROCK activation induces IR or affects myocardial structure and function, as well as the possible mechanisms underlying this process. Wistar rats fed high fat, high glucose and high salt diet sewed as model of MS and we used transmission electron microscopy, echocardiogram technology, and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling staining to identify any myocardial damage. The protein levels of MYPT-1 (characteristic of ROCK activation), IRS-1 and AKT were analyzed by immunohistochemistry and Western blotting. In hearts from MS rats, we found increased protein levels of phospho-MYPT-1 and phospho-IRS-1 (Ser307) and decreased phospho-AKT compared to levels in normal rats. In conclusion, the results suggest that ROCK-mediated IR is involved in the development of myocardial impairments in MS rats and that this effect is mediated probably via the IRS-1/PI3-kinase/AKT pathway.


INTRODUCTION
Insulin resistance (IR) plays an important role in the pathophysiology of metabolic syndrome (MS) (Bonora, 2006;Fan & Peng, 2007).The insulin signaling pathway is activated once insulin binds to its membrane-bound tyrosine kinase receptor, leading to subsequent phosphorylation of intracellular insulin receptor substrates (IRSs).The phosphorylated IRSs are then used as docking sites for downstream proteins to further transduce the insulin signal through the PI3-kinase/AKT pathway (Ferre, 2007).PI3-kinase/AKT activation is essential and collectively controls programmed cell death, cellular metabolism and organ protection (Amaravadi & Thompson, 2005;Asano et al., 2007).The IR mechanism involves activation of several serine/threonine kinases, reduction in tyrosine phosphorylation of IRS-1/2, and impairment of the IRS/PI3-kinase pathway of insulin signaling (Boden, 2006;Sesti, 2006).
Rho-associated kinase (ROCK) is a serine/threonine protein kinase that is activated by RhoA, a member of the Rho family of small G-proteins (Noma et al., 2006).Abnormal RhoA/ROCK pathway activation has been observed in major cardiovascular disorders, such as atherosclerosis, restenosis, hypertension and cardiac hypertrophy (Chitaley et al., 2001;Fonseca, 2003;Loirand et al., 2006).ROCK was activated in rats with spontaneous and angiotensin II-induced hypertension, and treatment with ROCK inhibitors effectively reduced blood pressure (Mukai et al., 2001;Uehata et al., 1997).Furthermore, increased RhoA/ROCK pathway activation has been demonstrated in both type 2 diabetic db/db mice and streptozotocin-induced diabetic rats ((Bivalacqua et al., 2004;Xie et al., 2006).Previous studies have already shown that ROCK activity is increased in the serum from MS patients (Liu et al., 2007) and in arteries from obese Zucker rats (a model of MS) (Naik et al., 2006).Additionally, ROCK-mediated IR either positively or negatively regulates insulin signaling, depending on cell type and stimulus (Shi & Wei, 2007).It is currently unknown, however, whether increased ROCK activity is also present in the hearts of MS patients.
Therefore, we induced MS in rats with special diets that cause insulin resistance.The present study show that ROCK was activated in hearts from MS rats, and that activation of ROCK-mediated IR induced changes of cardiac structure and function during chronic MS possibly through the IRS/PI3-kinase/AKT pathway in vivo.

Animals.
All experiments were carried out in accordance with the recommendations of the Universities Federation for Animal Welfare Handbook on the Care and Management of Laboratory Animals.Twenty-two male Wistar rats from the animal center of Shandong University (6 weeks of age; mean body weight 135 ± 10 g) were used.The rats were caged under standard light con-ditions (alternating 12 h light/dark cycles), temperature (22 ± 0.5°C) and humidity (60 ± 10%) during all experiments.The rats were then randomly divided into two groups.Twelve were fed with free access to a high-fat, high-glucose and high-salt diet with sucrose solution to induce MS (MS group).Concomitantly, ten age-matched Wistar rats were fed normal diet as controls (NC group).After 28 weeks, rats with MS (n=10) were identified using criteria analogous to ATP III as ≥3 of the following: hypertriglyceridemia, low HDL-C, high fasting glucose, excessive waist circumference, and hypertension.
Basic characteristics and plasma data analysis.Body weight (BW) was measured every two weeks in the morning, together with systolic blood pressure (SBP) by the tail-cuff method using a Rat Tail Manometer provided by the Japanese and Chinese Friendly Hospital (RBT-1, Beijing, China).Samples of venous blood were collected every two weeks to detect triglycerides (TG), cholesterol (CHO), HDL-C, LDL-C, fasting blood glucose (FBG) and insulin (FINS) levels after 12 hours of fasting.The insulin resistance index (IRI) was calculated as follows: IRI= (FBG*FINS)/22.5.FBG, CHO, HDL-C, LDL-C, TG and FINS were measured in a clinical laboratory of Qilu Hospital, which is affiliated with Shandong University (Jinan, China).
Echocardiographic evaluation.At the begining of the study and before sacrifice, each rat was anaesthetized with chloral hydrate (0.3 ml/100 g) through intraperitoneal injection, and placed in the left lateral decubitus position.Echocardiography was performed with Hewlett-Packard equipment, Sonos 7500 model, and an electronic 10-MHz transducer.M-mode recordings were obtained of the left ventricle (LV) at the level of mitral valve in the parasternal view using two-dimensional echocardiographic guidance in both the short and long axis views.Pulsed wave Doppler was used to examine mitral diastolic inflow from the apical four chamber view.Integrated backscatter images in the parasternal long-axis view also were recorded that quantifies the intensity of the backscattered echoes returning from myocardial cells within a user-defined region of interest.For each measurement, data from three consecutive cardiac cycles were averaged.All measurements were made from digital images captured at the time of the study by use of inherent analysis software.
Evaluation of collagen deposition.Paraffin-embedded tissue sections (5 μm) of the median part of the left ventricle were hydrated and stained with Masson Accustain Trichrome stain (Sigma, St. Louis, MO).The proportion of blue staining, indicative of interstitial collagen, was measured in 10 fields randomly selected on each of the three non-consecutive serial sections from each heart, and the values were averaged.Additionally, five-μm-thick paraffin sections were stained with the collagen-specific Sirius Red for measurement of the interstitial fibrosis.
TUNEL stainning.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique was used to evaluate apoptotic activity.Each section was deparaffinized and rehydrated.Proteinase K (20 mg/L) was applied to the section for 1 h with the intention of producing optimal proteolysis.The endogenous peroxidase was inhibited with 3% hydrogen peroxide for 10 min.A commercial apoptosis detection kit (Roche, Germany) was used.The TdT reaction was carried out for 1 h at 37°C, and then DAB chromogen was applied.The sections were analyzed with a computerassisted color image analysis system (Image-ProPlus 5.1, Media Cybernetics, Silver Spring, MD, USA).
Immunohistochemical (IHC) staining.Myocardial tissues were collected from both groups and immediately fixed in 4% paraformaldehyde solution for 24 hours.The tissues were then dehydrated and embedded in paraffin using standard histological procedures.Paraffin sections were dewaxed and rehydrated before microwave antigen retrieval.To retrieve the antigen, slides were soaked in 1% citric acid buffer, pH 6.0 and kept at 92-98°C for 10-15 min.The slides were cooled at room temperature for 30 min, rinsed with PBS, and blocked in 5% bovine serum albumin for 20 min.Slides were then incubated overnight at 4°C with 1:100 rabbit polyclonal anti-rat phospho-AKT antibody (Cell Signaling Technology), and with 1:200 rabbit polyclonal anti-rat total-AKT antibody (Cell Signaling Technology), followed with 1:200 biotinylated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology).DAB substrate kits were used to develop the immunohistochemical reaction.PBS was substituted for the primary antibody as a negative control.
Transmission electron microscopy (TEM).A 0.5×1×5 mm 3 cross section from cardiac muscle tissues was fixed with 2% glutaraldehyde for electron microscopic examination.The tissues were examined for any cardiocyte changes using an H-7000FA transmission electron microscope (Hitachi Co. Ltd.) at the Academy of Medical Science (Jinan, Shandong, China).
Statistical analysis.Data was expressed as mean ± S.D. Experimental means were subjected to either Student's t test for two groups, or one-way ANOVA with Newman-Keuls multiple comparison test for more than two groups.A probability value of P<0.05 was considered statistically significant.The above statistical analyses were performed using SPSS 13.0.

Characteristics of MS rats
MS rats were found to develop insulin resistance, hypercholesterolemia, low HDL-C, hyperglycemia, hypertension and obesity compared to the NC group (Fig. 1).

Echocardiographic features of MS rat heart
As shown in Table 1, the intraventricular septal wall (IVSW) and left ventricular posterior wall thickness (LVPW) increased in the left ventricle (LV) geometry of MS rats (vs control rats, P<0.01), demonstrating eccentric LV hypertrophy.In comparison, the left ventricular enddiastolic dimension (LVEDd), left ventricular end-systolic dimension (LVESd) and fractional shortening (FS, %) were not significantly changed in the MS rats (P＞0.05).Moreover, the E/A ratio and the E-wave deceleration time (DT) was significantly higher in MS group compared with the NC group, suggesting reduced diastolic function in the MS rat heart.There was also a significnat difference of the integrated backscatter percent (IBS%) and cyclic variation of IBS (CVIB) in LVPW and IVS between the NC and MS groups (P <0.01), demonstrating changes of collagen contents in MS rat heart.

Changes of myocardial ultrastructure in MS rats
TEM showed disruption of myofibers and swollen mitochondria in the MS rat heart (Fig. 2).

Collagen contents measured by Sirius and Masson staining
As shown in Fig. 3, Sirius Red and Masson staining revealed an increase of interstitial fibrosis in the myocardium of MS rats compared with the NC group.

IHC analysis of myocardial phospho-AKT and total AKT
Phospho-AKT was present only sporadically in the myocardium of the MS group, but phospho-AKT staining was predominatly located in the endochylema of cardiocytes in the NC group (Fig. 4B).Total AKT was present similarly in the myocardium of two groups (Fig. 4A).

Localization of apoptosis by TUNEL assay
Figure 4C shows TUNEL positive cells.Compared with the NC group, the percentage of positive cells relative to normal cardiocytes was significantly increased in the MS group (P<0.01)(Fig. 4D).

DISCUSSION
MS includes many metabolic disorders, such as obesity, low HDL-C, high triglycerides and impaired glucose tolerance ( (Norenberg & Rao, 2007;Tsujimoto & Shimizu, 2007).In the current study, we created a rat model of MS with the following features: 1) significantly increased body weight, 2) increased systolic blood pressure, 3) increased cholesterol and decreased HDL-C, and 4) increased glucose and insulin levels.Because those trends resembled those reported in human MS patients, those rats thus represent a good model of human MS.
Myocardial impairment in MS patients ultimately results from abnormal cellular metabolism in response to insulin resistance.As myocytes rarely proliferate in adult cardiac muscles, the loss of cardiocytes and the irreversible changes of their morphology would eventually compromise overall heart function.In our study, TUNEL staining demonstrated considerable apoptosis of cardiocytes, and TEM analysis showed pronounced interruption of myofibers and swollen mitochondria, all of which have been implicated in the development of cardiac dysfunction.Mitochondria are vital for cell life, as they represent the principal source of cellular energy (Norenberg & Rao, 2007;Vial G et al., 2010).Recent studies have demonstrated that improving mitochondrial permeability and swelling could inhibit apoptosis and mediate cardioprotection (Hadzimichalis et al., 2007;Tsujimoto & Shimizu, 2007).Thus, mitochondrial swelling can be regarded as a characteristic of irreversibly injured myocytes (Jennings & Reimer, 1991).Furthermore, disrupted myofibers directly affect cardiac diastolic and systolic dysfunction, manifested by decreased compliance, prolonged  myocardial relaxation, and left ventricular ejection inability (Lin et al., 2007).At the same time, we found the eccentric hypertrophy and reduced diastolic function of LV by echocardiogram study.These changes are probably due to the inhibition of the PI3-kinase/AKT pathway, an intracellular signaling pathway previously demonstrated to play significant roles not only in insulin sensitivity, but also in any potential protection against myocardial injury, such as ischaemia/reperfusion injury and diabetes ( (Hausenloy & Yellon, 2004;Mocanu & Yellon, 2007;Tsang et al., 2005).However, few studies have linked the effects of PI3-kinase/AKT pathway activation to cardioprotection in MS.Here, we have demonstrated apoptosis of cardiocytes, changes in sarcomeric and mitochondrial morphology, cardiac hypertrophy, and reduced diastolic function by inhibition of the PI3-kinase/AKT pathway in MS rats.
MS is an early indicator of diabetes and cardiovascular disease that is associated with major mortality and morbidity (Rasouli et al., 2007).However, the exact pathophysiological events leading to the development of MS remain unknown.ROCK is the immediate downstream target of RhoA, and is involved in diverse cellular functions, including smooth muscle contraction, actin cytoskeletal organization, and gene expression.Recent studies suggest that the RhoA/ROCK pathway plays a significant pathophysiological role in various aspects of cardiovascular disease.Indeed, ROCK was activated in patients with cardiovascular disease or its associated risk factors, and inhibition of ROCK served as a useful therapeutic approach (Noma et al., 2006;Mustafa S et al., 2010).Although many studies have shown that ROCK activity is increased in the serum of MS patients as well as in smooth muscle cells from MS rats (Liu et al., 2007;Wingard et al., 2007;Lee DH et al., 2009), we demonstrate that ROCK activation was increased in cardiocytes of MS rats and associated with insulin resistance.
Phosphorylated IRS-1 (Ser307) is a key protein involved in insulin resistance, but it has a controversial relationship with ROCK activation.Some studies have shown that ROCK phosphorylates IRS-1, thereby enhancing PI3-kinase activation in adipocytes and muscle cell lines ex vivo (Furukawa et al., 2005).In contrast, phosphorylated IRS-1 led instead to the inhibition of both insulin-induced IRS-1 tyrosine phosphorylation and PI3-kinase/AKT activation in fibroblasts derived from p190B RhoGAP-null mice and in vascular smooth muscle cells ( (Begum et al., 2002;Loirand et al.,2006;Sordella et al., 2002).Similarly, our results also indicate that ROCK activity was increased in the hearts of MS rats and that it negatively modulated insulin signaling.Increased ROCK activation was accompanied by IRS-1 serine phosphorylation, which inhibited AKT kinase activation and ultimately resulted in cardiocyte apoptosis and changes to the myocardial structure and function.Thus, insulin signaling in the hearts of MS rats could be attenuated by ROCK activation, implying a negative role for ROCK in insulin resistance.
In summary, this is the first demonstration that ROCK is activated in hearts from rats with MS.Furthermore, ROCK-associated serine phosphorylation of IRS-1 plays a critical role in insulin resistance, suggesting that ROCK may be a promising future therapeutic target in the treatment of MS.At the same time, there were some deficiencies in our study.As only ten MS rats were investigated in this study, we should confirm our findings  with a larger sample size.Additionally, we did not inhibit ROCK activation with either siRNA technology or specific inhibitors, such as Y27632 or fasudil.We plan to assess the effects of ROCK inhibition in vivo and in vitro in future studies.

Figure 2 .
Figure 2. Electron microscopy of myocardium in rats.(A) NC group.The sarcomere arranged regularly, and the mitochondria of normal size and abundance.(B) MS group.Note the interrupted myofibers and swollen mitochondria shown by arrows.Original magnification: 10 000×.

Table 1 . Echocardiographic findings in NC and MS rats at week 1 and 28
Results are shown as mean ± S.D. LVEDd, left ventricular end-diastolic dimension; LVESd, left ventricular end-systolic dimension; LVPW, left ventricular posterior wall thickness; IVSW, intraventricular septal wall; FS, fractional shortening; E/A, ratio of peak early diastolic filling velocity to peak velocity at atrial contrations; DT, E-wave deceleration time; IBS%, the integrated backscatter percent in LVPW and IVS; CVIB, cyclic variation of IBS.*P<0.01 compared to 1st week in the MS group.# P<0.05, # # P<0.01 compared to NC group.Each group comprised ten rats.