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Cardiovascular Research Advance Access first published online on April 14, 2008
This version [Corrected Proof] published online on May 12, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn095
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Role of inducible nitric oxide synthase in induction of RhoA expression in hearts from diabetic rats

Hesham Soliman, Graham P. Craig, Prabhakar Nagareddy, Violet G. Yuen, Guorong Lin, Ujendra Kumar, John H. McNeill and Kathleen M. MacLeod*

Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3

* Corresponding author. Tel: +1 604 822 3830; fax: +1 604 822 3035. E-mail address: kmm{at}interchange.ubc.ca

Received 4 October 2007; revised 20 March 2008; accepted 8 April 2008

Time for primary review: 19 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Recent studies from our laboratory demonstrated that increased expression of the small GTP-binding protein RhoA and activation of the RhoA/rho kinase (ROCK) pathway play an important role in the contractile dysfunction associated with diabetic cardiomyopathy in hearts from streptozotocin (STZ)-induced diabetic rats. Nitric oxide (NO) has been reported to be a positive regulator of RhoA expression in vascular smooth muscle, and we have previously found that the expression of inducible NO synthase (iNOS) is increased in hearts from STZ-diabetic rats. Therefore, in this study, we investigated the hypothesis that induction of iNOS positively regulates RhoA expression in diabetic rat hearts.

Methods and results: To determine whether NO and iNOS could increase RhoA expression in the heart, cardiomyocytes from non-diabetic rats were cultured in the presence of the NO donor sodium nitroprusside (SNP) or lipopolysaccharide (LPS) in the absence and presence of the selective iNOS inhibitor, N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL). In a second study, 1 week after induction of diabetes with STZ, rats were treated with L-NIL (3 mg/kg/day) for 8 more weeks to determine the effect of iNOS inhibition in vivo on RhoA expression and cardiac contractile function. Expression of iNOS was elevated in cardiomyocytes isolated from diabetic rat hearts. Both SNP and LPS increased RhoA expression in non-diabetic cardiomyocytes. The LPS-induced elevation in RhoA expression was accompanied by an increase in iNOS expression and prevented by L-NIL. Treatment of diabetic rats with L-NIL led to a significant improvement in left ventricular developed pressure and rates of contraction and relaxation concomitant with normalization of total cardiac nitrite levels, RhoA expression, and phosphorylation of the ROCK targets LIM (Lin-11, Isl-1, Mec-3) kinase and ezrin/radixin/moesin.

Conclusion: These data suggest that iNOS is involved in the increased expression of RhoA in diabetic hearts and that one of the mechanisms by which iNOS inhibition improves cardiac function is by preventing the upregulation of RhoA and its availability for activation.

KEYWORDS RhoA; iNOS; Diabetic cardiomyopathy; Contractility; Rho kinase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Cardiomyopathy, characterized by impaired cardiac function that can be detected in the absence of hypertension or ischaemic heart disease, is a common complication of both type 1 and type 2 diabetes. This condition is associated with a series of morphological, biochemical, and functional abnormalities seen in both human patients and animal models of both type 1 and type 2 diabetes, including changes in metabolism, contractile protein composition, and intracellular Ca2+ transients.1,2 Despite intensive investigation, the precise mechanisms underlying the contractile dysfunction are still not completely understood.

The small GTPase RhoA and its effector rho kinase (ROCK) play important roles in physiological and pathophysiological functions in the cardiovascular system (reviewed in references 3–5). Recently, we found evidence suggesting that the RhoA/ROCK pathway is activated in hearts from diabetic rats.6 Increases in the expression and activity of RhoA, and increased phosphorylation of the downstream ROCK target, LIM (Lin-11, Isl-1, Mec-3) kinase (LIMK), were detected in hearts from rats with chronic streptozotocin (STZ)-induced diabetes. This was associated with an increase in actin polymerization that was blocked by inhibition of ROCK. Most importantly, acute administration of ROCK inhibitors improved the function of hearts from diabetic rats, both in vivo, as assessed by echocardiography, and in vitro, in the isolated working heart.6 These data suggest that the RhoA/ROCK signalling pathway plays an important role in the development of diabetic cardiomyopathy.

The regulation of RhoA activity through its interaction with guanine-nucleotide dissociation inhibitors (RhoGDIs), guanine-nucleotide exchange factors (RhoGEFs), and GTPase-activating proteins (GAPs) and its role as a ‘molecular switch’ controlling cell signalling pathways has been well characterized (reviewed in reference 7). On the other hand, relatively few studies have addressed the factors that regulate the expression of RhoA. However, recent evidence points towards an interaction between nitric oxide (NO) and RhoA. In particular, it has been shown that the expression of RhoA in rat aortic smooth muscle cells is highly inducible by NO.8 We have previously shown that inducible NO synthase (iNOS) expression was significantly elevated in STZ diabetic rat hearts9 and once induced, iNOS is known to generate large amounts of NO until the enzyme is degraded.10 We hypothesized that NO produced by iNOS is responsible for the upregulation of RhoA expression observed in the heart during diabetes and therefore is a contributing factor in the RhoA/ROCK-mediated contractile dysfunction by increasing the total pool of RhoA available for activation. To test this hypothesis, we first investigated whether there was an interaction between NO and RhoA in the heart by determining whether an NO donor or induction of iNOS by lipopolysaccharide (LPS) could induce increased expression of RhoA in isolated cardiomyocytes from non-diabetic rats. In addition, we investigated the role of iNOS-mediated regulation of RhoA expression in diabetic cardiomyopathy by determining the effects of chronic treatment with a selective iNOS inhibitor on the expression and phosphorylation of RhoA, phosphorylation of the ROCK downstream targets ezrin/radixin/moesin (ERM) and LIMK, and left ventricular contractile function in hearts from diabetic rats. The results of this investigation support a role for iNOS in the upregulation of RhoA and consequently, RhoA/ROCK pathway-mediated contractile dysfunction in rat diabetic hearts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Induction of diabetes mellitus in rats and drug treatment
Male Wistar rats weighing 250–300 g were obtained from the UBC Animal Care Facility. To induce diabetes, rats were treated with a single tail vein injection of STZ dissolved in saline (60 mg/kg). Control rats were administered equivalent volumes of saline via the same route. Blood glucose was measured 72 h after STZ administration, and induction of diabetes was confirmed by the presence of hyperglycaemia (blood glucose ≥ 18 mmol/L). To determine the effects of chronic inhibition of iNOS, 1 week after injection of STZ, control and diabetic rats were subdivided into two groups, one of which received the selective iNOS inhibitor, N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) dissolved in water at a dose of 3 mg/kg/day by oral gavage.11,12 The other group received equivalent volumes of vehicle by the same route. Eight weeks later, rats were weighed and blood was taken for measurement of glucose levels. Then rats were deeply anaesthetized with sodium pentobarbital (65 mg/kg, IP) and hearts were excised rapidly. The hearts were used either for ex vivo isolated working heart studies, as described previously,6 or the left ventricles were frozen rapidly in liquid nitrogen and stored at –70°C until processed for biochemical analysis.

This investigation conforms with the Canadian Council on Animal Care Guidelines on the Care and Use of Experimental Animals, and the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85–23, revised 1996). All protocols were approved by the UBC Animal Care Committee.

2.2 Isolated working heart studies
The contractile function of isolated working hearts from vehicle and L-NIL-treated control and diabetic rats was measured as described in Lin et al.6 Hearts were initially perfused through the aorta at a rate of 17 mL/min with Chenoweth–Koelle (CK) solution (composition in mM: NaCl 120, KCl 5.6, CaCl2 2.18, MgCl2 2.1, NaHCO3 19.2, and glucose 10) maintained at 37°C and bubbled with 95% O2/5% CO2. Following cannulation of the pulmonary vein, cardiac work was initiated by switching the perfusion system from the retrograde mode to the working heart mode. Left ventricular developed pressure (LVDP), and the rates of contraction and relaxation (+dP/dT and –dP/dT) in response to increases in left atrial filling pressure, produced by pre-determined stepped increases in the rate of perfusion of the CK buffer, were measured with a pressure transducer attached to a 20 gauge needle inserted through the apex of the heart into the left ventricle. The afterload was kept constant throughout the perfusion. The heart was paced at 300 b.p.m. using a Grass model SD 90 stimulator connected to a stainless steel electrode placed on the left atrium.

2.3 Preparation of isolated rat ventricular cardiomyocytes
Ca2+-tolerant cardiomyocytes were isolated as described in Lin et al.6 Cardiomyocytes were either snap-frozen in liquid nitrogen or were maintained in primary culture in order to assess the effects of drug treatment. For the latter, cardiomyocytes were resuspended in medium 199 supplemented with 1% BSA, 100 units/mL penicillin, 100 µg/mL streptomycin, 1.2 mM L-carnitine, and 25 mM HEPES (pH 7.4). Cells prepared from a single rat heart were plated on laminin-coated culture dishes and allowed to recover for 3 h. Cultured cells were then incubated for 18 h at 37°C in supplemented medium 199 containing the various treatments and then snap-frozen in liquid nitrogen.

2.4 Western blot analysis
Frozen ventricles and isolated cardiomyocytes were homogenized as described in Lin et al.6 Both ventricular and myocyte homogenates were spun at 700 g for 5 min, and the protein content of the supernatants was determined by the Bradford protein assay. Equal amounts of protein (40 µg) from each sample were separated by 8 or 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h in a solution of 5% skim milk and then incubated overnight at 4°C with primary antibodies against RhoA (1:2000), phospho-RhoA (1:400), β-actin (1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000) (all from Santa Cruz Biotechnology Inc., CA, USA), iNOS (1:1000, Abcam Inc., MA, USA), phospho-LIMK (1:1000), and phospho-ERM (1:1000) (the latter two antibodies were from Cell Signaling Technology Inc., MA, USA). Membranes were washed, incubated with goat anti-mouse or anti-rabbit horse-radish peroxidase-conjugated secondary antibodies (1:10 000, Santa Cruz Biotechnology Inc.) for 1 h then exposed to chemiluminescence reagents (Amersham Inc., Québec, Canada), and developed on photographic film. Densitometric analysis was performed to quantify band optical densities.

2.5 Assay of nitric oxide production
A commercially available assay kit (Cayman Chemical, MI, USA) was used to measure the nitrite/nitrate (NOx–) levels in whole-heart homogenates as an index of NO production.13 Samples were incubated with nitrate reductase enzyme for 3 h at room temperature to convert all nitrates to nitrites, which were quantified using the Griess reaction. Proteins were extracted from ventricular tissue according to the manufacturer's protocol and protein concentration was determined using the Bradford method.14 Samples were measured in triplicate.

2.6 Statistical analysis
All values are expressed as means ± SEM; n denotes the number of animals in each group. Statistical analysis of all data except LVDP, +dP/dt, and –dP/dt was performed using one-way ANOVA followed by the Newman–Keuls test when more than two groups were compared using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). LVDP, +dP/dt, and –dP/dt were analysed by repeated measures ANOVA (general linear models approach) followed by the Newman–Keuls test, using NCSS 2000 (NCSS, Kaysville, UT, USA). For all results, the level of significance was set at P < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Expression of inducible nitric oxide synthase and RhoA in cardiomyocytes isolated from diabetic rats
We previously reported that RhoA expression was upregulated in cardiomyocytes from diabetic rats.6 In the present study, we examined whether this was associated with an increase in expression of iNOS in the same cells. As shown in Figure 1, both RhoA and iNOS expression levels were significantly increased in cardiomyocytes isolated from 12–14 week diabetic rat hearts compared with their age-matched controls.


Figure 1
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  		Figure 1 RhoA and inducible nitric oxide synthase expression in cardiomyocytes isolated from diabetic rat hearts. (A) Representative western blot showing RhoA expression, with actin shown as a loading control, in freshly isolated control and diabetic cardiomyocytes. (B) Representative western blot showing inducible nitric oxide synthase expression, with actin shown as a loading control, in freshly isolated control and diabetic cardiomyocytes. (C) inducible nitric oxide synthase band optical densities were corrected by the optical densities of their corresponding actin band and expressed relative to the mean control value (n = 8 in each group). Data are expressed as mean ± SEM. *P < 0.05 compared with control.

 
3.2 Effect of sodium nitroprusside and lipopolysaccharide on RhoA expression in isolated cardiomyocytes
We next determined whether the NO donor, sodium nitroprusside (SNP), could increase the expression of RhoA in cardiomyocytes from non-diabetic rats, as was reported in vascular smooth muscle cells.8 Treatment of isolated cardiomyocytes in primary culture with 10 µM SNP for 18 h resulted in a significant increase in RhoA expression compared with control myocytes that were incubated for the same time in the absence of SNP (Figure 2A and B).


Figure 2
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  		Figure 2 Effect of sodium nitroprusside (SNP) and lipopolysaccharide treatment on inducible nitric oxide synthase and RhoA expression. (A) Representative western blot showing RhoA expression, with actin shown as a loading control, in cultured control and sodium nitroprusside-treated cardiomyocytes. (B) RhoA band optical densities were corrected by the optical densities of their corresponding actin band and expressed relative to the mean control value (n = 8 in each group). (C) Representative blot showing inducible nitric oxide synthase and RhoA, with glyceraldehyde-3-phosphate dehydrogenase as a loading control, at different lipopolysaccharide concentrations in cardiomyocytes. (D) Inducible nitric oxide synthase band optical densities were normalized by the optical densities of their corresponding glyceraldehyde-3-phosphate dehydrogenase band and expressed relative to the mean control value (n = 8 in each group). (E) RhoA band optical densities were corrected by the optical densities of their corresponding glyceraldehyde-3-phosphate dehydrogenase band and expressed relative to the mean control value (n = 8 in each group). Data are expressed as mean ± SEM. *P < 0.05 compared with control.

 
In order to determine whether iNOS induction was also associated with an increase in RhoA expression, the effect of treatment of isolated myocytes with LPS (Salmonella enterica serotype typhimurium) on expression of both RhoA and iNOS was determined. Treatment of myocytes with 20 µg/mL LPS for 18 h had no effect on the expression of either iNOS or RhoA (Figure 2C–E). However, a higher concentration of LPS (40 µg/mL) produced a significant increase in the expression of both iNOS and RhoA (Figures 2C–E and 3). The phosphorylation of RhoA at serine 188 was also increased in LPS-treated cardiomyocytes (Figure 3A), such that the ratio of phosphorylated to total RhoA remained unchanged compared with untreated myocytes (Figure 3B). The LPS-induced increases in both the expression and phosphorylation of RhoA were prevented by the selective, irreversible iNOS inhibitor, L-NIL (Figure 3), suggesting that they were secondary to the induction of iNOS by LPS.


Figure 3
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  		Figure 3 Effect of N6-(1-iminoethyl)-L-lysine dihydrochloride treatment on lipopolysaccharide-induced increase in RhoA and inducible nitric oxide synthase expression in isolated cardiomyocytes. (A) A representative blot showing inducible nitric oxide synthase and RhoA expression and phosphorylation (p-RhoA), with glyceraldehyde-3-phosphate dehydrogenase as a loading control. (B) Inducible nitric oxide synthase, RhoA, and p-RhoA/RhoA ratio band optical densities were normalized by the optical densities of their corresponding glyceraldehyde-3-phosphate dehydrogenase band and expressed relative to the mean control value (n = 5–7 in each group). Data are expressed as mean ± SEM. *P < 0.05 compared with control.

 
3.3 Effect of 8 week N6-(1-iminoethyl)-L-lysine dihydrochloride treatment on RhoA and inducible nitric oxide synthase expression and RhoA, Lin-11–Isl-1–Mec-3 kinase, and ezrin/radixin/moesin phosphorylation
To investigate whether the increased expression of iNOS detected in hearts from diabetic rats was responsible for the associated increase in RhoA expression, diabetic rats were treated with L-NIL (3 mg/kg/day) by oral gavage for 8 weeks. At the end of this period, STZ-treated rats had significantly elevated blood glucose levels and reduced body weights compared with their age- and gender-matched controls, which were not altered by chronic L-NIL treatment (Table 1). The expression of iNOS was significantly elevated (Figure 4A and B), whereas the expression of eNOS was reduced and no change in levels of nNOS was detected (data not shown) in hearts from vehicle-treated diabetic rats. Immunohistochemical detection of iNOS in ventricular slices from diabetic rat hearts confirmed that it was expressed in cardiomyocytes (see Supplementary material online, Figure S1). To determine the effectiveness of L-NIL, total NOx– levels were also measured in hearts from L-NIL and vehicle-treated animals as an index of NO production. In vehicle-treated diabetic rats, NOx– levels in ventricular homogenates were significantly elevated (Figure 4C), consistent with the increased expression of iNOS. Although L-NIL had no significant effect on the expression of iNOS, it prevented the increase in NOx– levels in diabetic hearts, suggesting that it was effectively inhibiting the enzyme (Figure 4C). Furthermore, in control rats, L-NIL treatment did not affect either mean arterial blood pressure (data not shown) or total cardiac NOx– levels, suggesting that at the dose used, L-NIL was selective for iNOS. RhoA expression was also significantly elevated in the STZ-treated rat hearts, and this increase was also prevented by treatment of diabetic rats with L-NIL (Figure 4F). However, the increased RhoA expression in hearts from untreated diabetic rats was not accompanied by a change in the phosphorylation of RhoA at serine 188 (Figure 4D), resulting in a significant decrease in the ratio of phospho-RhoA to total RhoA (Figure 4F). By reducing RhoA expression without affecting its phosphorylation, L-NIL treatment normalized the phospho-RhoA to total RhoA ratio (Figure 4D–F). L-NIL had no effect on the expression or phosphorylation of RhoA in hearts from control rats.


Figure 4
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  		Figure 4 Effect of chronic N6-(1-iminoethyl)-L-lysine dihydrochloride treatment on RhoA and inducible nitric oxide synthase expression, and NOx– level in control and diabetic cardiac ventricular tissue. (A) Representative blot showing inducible nitric oxide synthase, with glyceraldehyde-3-phosphate dehydrogenase as a loading control, in the ventricular tissue of the different groups. (B) Inducible nitric oxide synthase band optical densities were normalized by the optical densities of their corresponding glyceraldehyde-3-phosphate dehydrogenase band and expressed relative to the mean control value (n = 8 in each group). (C) Cardiac ventricular NOx– level in the different groups (n = 6–7 in each group). (D) Representative blot showing RhoA and phosphorylated RhoA (p-RhoA), with glyceraldehyde-3-phosphate dehydrogenase as a loading control, in the ventricular tissue of the different groups. (E) RhoA band optical densities were corrected by the optical densities of their corresponding glyceraldehyde-3-phosphate dehydrogenase band and expressed relative to the mean control value (n = 6–7 in each group). (F) Ratio of p-RhoA to total RhoA optical densities, expressed relative to the mean control value (n = 7–8 in each group). Data are expressed as mean ± SEM. *P < 0.05 compared with control. C, control; D, diabetic; CT, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated control; DT, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated diabetic.

 


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  		Table 1 Effect of N6-(1-iminoethyl)-L-lysine dihydrochloride on body weight and blood glucose level in control and diabetic rats

 
LIM kinase is an immediate downstream target of ROCK, and in our previous investigation, we found that the phosphorylation of LIMK was increased in hearts from diabetic rats, and that this could be abolished by inhibition of ROCK.6 In the present study, we confirmed the increase in LIMK phosphorylation in hearts from untreated diabetic rats and found that it was attenuated by treatment of the rats with L-NIL (Figure 5A and B). Similarly, the elevated phosphorylation of ERM, another downstream target of ROCK, in the diabetic rat hearts was significantly reduced by L-NIL treatment (Figure 5C and D).


Figure 5
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  		Figure 5 Effect of chronic N6-(1-iminoethyl)-L-lysine dihydrochloride treatment on Lin-11–Isl-1–Mec-3 kinase and ezrin/radixin/moesin phosphorylation in control and diabetic cardiac ventricular tissue. (A) Representative blot showing phosphorylated LIMK 1/2 (p-LIMK 1/2; two bands represent p-LIMK 1 at 72 kDa and p-LIMK 2 at 65 kDa), with glyceraldehyde-3-phosphate dehydrogenase as a loading control, in the ventricular tissue of the different groups. (B) p-LIMK 1/2 band optical densities were corrected by the optical densities of their corresponding glyceraldehyde-3-phosphate dehydrogenase band and expressed relative to the mean control value (n = 8 in each group). (C) Representative blot showing phosphorylated ezrin/radixin/moesin (p-ERM; two bands represent moesin at 75 kDa and ezrin and radixin at 80 kDa, with glyceraldehyde-3-phosphate dehydrogenase as a loading control, in the ventricular tissue of the different groups. (D) p-ERM bands optical densities were corrected by the optical densities of their corresponding glyceraldehyde-3-phosphate dehydrogenase band and expressed relative to the mean control value (n = 7–8 in each group). Data are expressed as mean ± SEM. *P < 0.05 compared with control. C, control; D, diabetic; CT, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated control; DT, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated diabetic.

 
3.4 Effect of 8 week N6-(1-iminoethyl)-L-lysine dihydrochloride treatment on contractile function of the diabetic hearts
As shown in Figure 6, compared with controls, hearts from vehicle-treated diabetic rats showed a significant reduction in LVDP, +dP/dT, and –dP/dT at LAFP above 5 mmHg, which is characteristic of the left ventricular dysfunction of diabetic cardiomyopathy. Administration of L-NIL to diabetic rats prevented the deterioration of cardiac contractile function and significantly improved LVDP, +dP/dT, and –dP/dT compared with the diabetic untreated rats. L-NIL did not significantly alter the contractile function of hearts from control rats except for a small decrease in LVDP at the highest filling pressure (10 mmHg).


Figure 6
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  		Figure 6 Effect of chronic N6-(1-iminoethyl)-L-lysine dihydrochloride treatment on the contractile function of isolated working hearts from control and diabetic rats. Increases in left ventricular developed pressure (LVDP, upper panel), +dP/dt (middle panel), and –dP/dt (lower panel) in response to increases in left atrial filling pressure (LAFP). Black squares represent untreated control hearts; open squares, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated control hearts; black triangles, untreated diabetic hearts; open triangles, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated diabetic hearts (n = 6–7 in each group). Data are expressed as mean ± SEM. *P < 0.05 compared with all other groups; **P < 0.05 compared with control group; ***P < 0.05 compared with control and diabetic groups; ****P < 0.05 compared with control and N6-(1-iminoethyl)-L-lysine dihydrochloride-treated control groups. C, control; D, diabetic; CT, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated control; DT, N6-(1-iminoethyl)-L-lysine dihydrochloride-treated diabetic.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
The importance of the RhoA/ROCK pathway in cardiovascular physiology and pathophysiology has recently become clear. Extensive research is ongoing to elucidate role of this pathway in cardiovascular pathologies including hypertension,15 arteriosclerosis,16 and left ventricular hypertrophy and failure.17 We have previously reported that the RhoA/ROCK pathway is activated in diabetic cardiomyopathy and contributes to the pathogenesis of this complication.6 The present work provides evidence to support a role for iNOS in enhancing RhoA expression both in isolated cardiomyocytes and in diabetic rat hearts and highlights an important role for iNOS in mediating cardiac contractile dysfunction in the diabetic heart through upregulation of RhoA expression, thus contributing to increased activation of the RhoA/ROCK pathway.

A role for NO in positively regulating the expression of RhoA was first suggested by the observation that treatment of cultured vascular smooth muscle cells with the NO donor SNP produced significant increases in the expression of RhoA.8 Similarly, we have shown that a significant increase in RhoA expression can be induced in cultured cardiomyocytes by the same concentration of SNP, suggesting that NO can also positively regulate the expression of RhoA in the heart. Furthermore, treatment of cardiomyocytes with LPS induced concurrent expression of iNOS and RhoA, and the latter was prevented by L-NIL, an L-arginine analogue which acts as a selective, irreversible iNOS inhibitor,18 confirming that it is iNOS that is responsible for increasing RhoA expression.

The results of the present study confirm our previous reports of increased expression of RhoA6 and iNOS9 in hearts from chronically diabetic rats. The detection of elevated levels of iNOS in cardiomyocytes by immunoblotting of protein from isolated cardiomyocytes and by immunohistochemical detection in whole hearts suggests that cardiomyocytes rather than other cells such as inflammatory or endothelial cells are the major site of iNOS expression in the diabetic heart. Since expression of eNOS was reduced and no change in nNOS expression could be detected, it is likely that iNOS is responsible for the increase in NOx– levels found in diabetic hearts. This is supported by the observation that L-NIL, at a dose previously reported to be selective for iNOS in vivo,12 completely prevented the increase in cardiac NOx– levels and further suggests that iNOS activity was effectively inhibited in these hearts. The normalization of RhoA expression by L-NIL treatment suggests that in vivo, as well as in vitro, the increased levels of NO produced by iNOS are responsible for RhoA upregulation. Interestingly, we have also found that the cardiac NOx– level and RhoA expression were elevated in the Goto-Kakizaki rat, a non-obese model of spontaneous type 2 diabetes (unpublished observations) at an age at which these animals have been reported to exhibit cardiomyopathy,19 suggesting that regulation of RhoA expression by iNOS may be common to both type 1 and 2 diabetes.

The mechanism by which iNOS/NO might regulate RhoA expression in the heart is not known. However, the elevated levels of RhoA induced by NO production in vascular smooth muscle appear to be due to a combination of transcriptional and translational upregulation of the RhoA gene and decreased degradation of the RhoA protein. A number of reports have demonstrated that NO, acting through cGMP-dependent protein kinase (PKG), leads to phosphorylation of serine 188 in RhoA, thus protecting the protein from ubiquitin/proteasome-mediated degradation by increasing its interaction with RhoGDI.8,20,21 In addition, stimulation of the NO/PKG pathway by SNP or the cGMP analogue 8-pCPT-cGMP has also been shown to lead to increased RhoA gene transcription in cultured vascular smooth muscle cells.8 Chronic treatment of rats with a non-selective NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA), was associated with a decrease in RhoA mRNA and protein expression in the aorta and pulmonary artery, suggesting that in vivo as well as in vitro, NO regulates RhoA expression at the level of gene transcription.8 The decrease in RhoA mRNA and protein expression induced by chronic hypoxia in the rat pulmonary artery was reversed by treatment with the phosphodiesterase 5 inhibitor, sildenafil, implicating the cGMP/PKG pathway in this process.

In addition to increasing RhoA expression in vascular smooth muscle cells by decreasing its degradation, phosphorylation at the Ser188 residue was reported to inactivate RhoA by enhancing the ability of RhoGDI to interact with and extract RhoA from the cell membrane.21,22 In the present study, consistent with studies in cultured vascular smooth muscle cells, iNOS upregulation was associated with an increase in RhoA expression and Ser188 phosphorylation in cultured cardiomyocytes treated with LPS. In contrast, in hearts from diabetic rats, the increase in RhoA expression was not associated with a corresponding increase in Ser188 phosphorylation. As a result, the ratio of phosphorylated to non-phosphorylated RhoA was significantly lower in hearts from untreated diabetic than control rats and was normalized by treatment of diabetic rats with L-NIL. This implies that iNOS induction, by positively regulating RhoA expression, may contribute to the increased RhoA activity that we previously observed in diabetic hearts,6 leading to an increase in the RhoA/ROCK pathway activity and contractile dysfunction. In agreement with this, we found that the diabetes-induced increase in phosphorylation of the ROCK targets, LIMK and ERM, which were increased in hearts from untreated diabetic rats, were normalized by L-NIL treatment. Similarly, Sauzeau et al.8 showed that treatment of rats with L-NNA, in addition to reducing RhoA expression in the aorta and pulmonary artery, also inhibited RhoA-mediated calcium sensitization of the contractile apparatus. Expression and calcium sensitization were restored using the phosphodiesterase 5 inhibitor, sildenafil. On the basis of this, these authors concluded that RhoA expression is a limiting factor for RhoA-dependent functions, which include ROCK activation.

The discrepancy between our findings of a positive association between iNOS expression and RhoA/ROCK activity in diabetic rat hearts and the results of studies demonstrating that NO negatively regulates the activity of this pathway23–27 could arise from a number of different factors. However, the results of the present study and those of Sauzeau et al.8 suggest it may arise at least in part from differences between relatively acute studies in cultured cells vs. chronic studies in whole animals. In isolated cultured cells, the NO-mediated increase in RhoA phosphorylation that accompanies its increased expression has been shown to lead to accumulation of a pool of inactive GTP-bound RhoA in the cytosol that is available for translocation to its active site on the membrane.21,22 It has been pointed that this pool of RhoA may be activated independently of RhoGEF, although the mechanisms remain unknown.28 It is possible that under physiological and/or pathophysiological conditions in vivo, the mechanisms responsible for activation of this pool of cytosolic GTP-RhoA are functional.

If iNOS mediates increased RhoA expression in the diabetic hearts, then, on the basis of our previous findings that hyperactivity of the RhoA/ROCK pathway is implicated in diabetic cardiomyopathy and that normalizing this pathway significantly improves contractile function, L-NIL treatment would also be expected to improve contractile function in diabetic rat hearts. This possibility was investigated in isolated working hearts. Coinciding with the elevation of iNOS and RhoA expression as well as LIMK and ERM phosphorylation, a significant drop in LVDP, +dP/dt, and –dP/dt occurred in hearts from untreated diabetic rats. On the other hand, inhibition of iNOS activity with L-NIL, in addition to normalizing the expression of RhoA and phosphorylation of LIMK and ERM, significantly improved the abovementioned variables. We have reported previously that the contractile dysfunction seen in hearts of diabetic rats was accompanied by elevation of RhoA expression and activity, and that acute inhibition of ROCK normalized the contractile function.6 Taken together, these data further support our hypothesis that iNOS mediates an increase in RhoA expression which contributes to an increased activity of the RhoA/ROCK pathway and contractile dysfunction. Inhibiting iNOS activity limits its ability to upregulate RhoA expression, thus diminishing the availability of the latter for subsequent activation of ROCK.

It is generally accepted that in diabetes, there is a marked decrease in the bioavailability of NO as a result of decreased eNOS function.29,30 Although the elevated levels of nitrites detected in the diabetic heart are consistent with increased production of NO from iNOS, this NO is generally thought to exert pathophysiological effects.31,32 In fact, studies have shown that increased expression of iNOS, through increased NO production, contributes to impaired function of eNOS.31,33

It is possible that, besides its effect on RhoA expression, inhibition of iNOS might have improved contractile function in the diabetic rat hearts through other mechanisms involving NO or its reactive metabolites such as peroxynitrite. Nevertheless, our previous finding that normalizing the RhoA/ROCK pathway activity leads to complete recovery of left ventricular contractile function6 strongly suggests that preventing the upregulation of RhoA is one of the major mechanisms by which iNOS inhibition produced significant improvement in contractile function of the hearts of the diabetic rats in this study. Surprisingly, L-NIL treatment in control non-diabetic animals seems to have produced a slight depression in LVDP that reached statistical significance at 10 mmHg LAFP, compared with the control untreated rats. We have no explanation for this effect, but it is probably an NO-independent effect since we did not observe any differences in the cardiac NOx– level between the L-NIL-treated and the untreated groups.

In conclusion, the results of the present study demonstrate that iNOS induces increased expression of RhoA in isolated cardiomyocytes and that the increased expression and activity of the RhoA/ROCK pathway in the diabetic heart are secondary to induction of iNOS in these hearts. Reduction of the activity of the RhoA/ROCK pathway appears to be one of the important mechanisms by which iNOS inhibition improves contractile function in diabetic cardiomyopathy.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
Canadian Institutes of Health Research (MOP 68942 to K.M.M. and J.H.M.) and the Heart and Stroke Foundation of BC & Yukon (to K.M.M.). P.N. was the recipient of Doctoral Research Awards (Heart and Stroke Foundation of BC & Yukon, Michael Smith Foundation for Health Research).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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