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Endocrinology Vol. 149, No. 7 3286-3293
Copyright © 2008 by The Endocrine Society

Relaxin Ameliorates Fibrosis in Experimental Diabetic Cardiomyopathy

Chrishan S. Samuel, Tim D. Hewitson, Yuan Zhang and Darren J. Kelly

Howard Florey Institute (C.S.S.) and Department of Biochemistry and Molecular Biology (C.S.S.), University of Melbourne, Victoria 3010, Australia; Department of Nephrology (T.D.H.), Royal Melbourne Hospital and Department of Medicine (T.D.H), Royal Melbourne Hospital, University of Melbourne, Victoria 3050, Australia; and Department of Medicine (Y.Z., D.J.K.), University of Melbourne, St. Vincent’s Hospital, Victoria 3065, Australia

Address all correspondence and requests for reprints to: Dr. Chrishan Samuel, Howard Florey Institute and Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia. E-mail: chrishan.samuel{at}florey.edu.au; or Associate Professor Darren Kelly, Department of Medicine, University of Melbourne, St. Vincent’s Hospital, Victoria 3065, Australia. E-mail: dkelly{at}medstv.unimelb.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fibrosis (extracellular matrix accumulation) is the final end point in diabetic cardiomyopathy. The current study evaluated the therapeutic effects of the antifibrotic hormone relaxin (RLX) in streptozotocin-treated transgenic mRen-2 rats, which undergo pathological and functional features similar to human diabetes. Twelve-week-old hyperglycemic mRen-2 rats, normoglycemic control rats, and animals treated with recombinant human gene-2 (H2) RLX from wk 10–12 were assessed for various measures of left ventricular (LV) fibrosis, hemodynamics, and function, while the mechanism of RLX’s actions was also determined. Hyperglycemic mRen-2 rats had increased LV collagen concentration (fibrosis) and gelatinase activity (all P < 0.05 vs. controls) but equivalent levels of interstitial collagenase and tissue inhibitor of metalloproteinase-1 to that measured in control rats. The increased LV fibrosis associated with diabetic animals led to significant alterations in the E/A wave ratio and E-wave deceleration time (both P < 0.05 vs. controls) in the absence of blood pressure changes, reflective of myocardial stiffness and LV diastolic dysfunction. H2-RLX treatment of diabetic rats led to significant decreases in interstitial and total LV collagen deposition (both P < 0.05 vs. diabetic group), resulting in decreased myocardial stiffness and improved LV diastolic function, without affecting nondiabetic animals. The protective effects of H2-RLX in diabetic rats were associated with a reduction in mesenchymal cell differentiation and tissue inhibitor of metalloproteinase-1 expression in addition to a promotion of extracellular matrix-degrading matrix metalloproteinase-13 (all P < 0.05 vs. diabetic group) but were independent of blood pressure regulation. These findings demonstrate that RLX is an antifibrotic with rapid-occurring efficacy and may represent a novel therapy for the treatment of diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DIABETES IS A chronic, debilitating, and costly disease caused by glucose-induced damage to cells, tissues, and eventually whole organ systems (1). One of the most frequent and serious complications of diabetes is the accumulation of excessive connective tissue (pathological fibrosis). This is a major feature in diabetic cardiomyopathy (2), in which the increased connective tissue leads to myocardial stiffness and eventually cardiac dysfunction, ultimately leading to cardiac failure. Diabetic patients have a 2- to 5-fold increased risk of developing heart failure. An important component of this risk is the development of a fibrotic cardiomyopathy independent of coronary artery disease, age, weight, and blood pressure (2). The pathological alterations associated with diabetic cardiomyopathy involve myocardial hypertrophy and impaired contraction, increased deposition of extracellular matrix (ECM) proteins, abnormal glycosylation, and cross-linking of these proteins, resulting in altered diastolic compliance (2, 3), the ultimate consequence of these events being myocardial fibrosis with reduced myocardial elasticity and overt cardiac dysfunction.

However, despite the availability of modern therapies to control hyperglycemia and blood pressure, many patients with diabetes mellitus continue to show progressive organ damage (4). Thus, further examination of the mechanisms underlying the development of elevated blood glucose and more direct treatment strategies that target connective tissue (collagen) turnover are warranted.

Relaxin (RLX) is a naturally occurring hormone (5, 6, 7) that inhibits organ fibrosis (8, 9, 10). Normally associated with reproduction (5, 6, 7, 11), RLX has been implicated in a number of pregnancy related functions, including softening the cervix and vagina at delivery, inhibiting cell apoptosis, and decreasing the total peripheral resistance through vasodilation. The physiological actions of RLX may also have important implications elsewhere, having been shown repeatedly to inhibit excessive collagen accumulation in various cell culture and animal models of fibrosis (5, 6, 8, 9, 12, 13). Collagen accumulation is often a balance between synthesis and degradation by collagenases/gelatinases, RLX having a role in both processes. Secretion and activity of a variety of matrix metalloproteinases (MMPs), including MMP-1 and its rat analog MMP-13, MMP-2, and MMP-9, are increased by RLX in several situations, whereas expression of their tissue inhibitors [tissue inhibitor of metalloproteinases (TIMPs)] is decreased (14, 15, 16, 17, 18, 19, 20, 21). In addition, RLX limits the de novo synthesis of collagen by inhibiting both fibroblast proliferation and expression of smooth muscle actin ({alpha}-SMA), a surrogate marker of mesenchymal cell (myofibroblast) differentiation in vitro (19, 20), and in an experimental model of hypertension in vivo (22).

Despite its potential to limit fibrogenesis and remodel the ECM, it is unknown if RLX can limit the progression of diabetic cardiomyopathy. Therefore, this study specifically examined the therapeutic potential of exogenous recombinant human gene-2 (H2)-RLX in the streptozotocin (STZ)-treated mRen-2 rat model of diabetes (23), which mimics several pathological features of human diabetic cardiomyopathy (24, 25).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design
The therapeutic potential of H2-RLX in diabetic cardiomyopathy was explored by examining its effect in treating the STZ transgenic [(mRen-2)27] rat, an accelerated rodent model of STZ-induced diabetes with elevated angiotensin II activity (23, 24, 25). The major advantage of the mRen-2 rat model is that it meets most of the key criteria set by the Animal Models of Diabetes Complications Consortium, and develops structural and functional changes that closely mimic the pathological features of human diabetic cardiomyopathy (24, 25). The hypothesis tested was that exogenous H2-RLX ameliorates the pathophysiology of these diabetic complications by reducing the aberrant deposition of the myocardial collagen, the basis of cardiac fibrosis.

Materials
Recombinant H2-RLX was generously provided by BAS Medical (San Mateo, CA). H2-RLX is the major-stored and circulating form of RLX in humans and is bioactive in rats (18, 21, 22, 26, 27).

Animals
Because male diabetic Ren-2 rats develop malignant hypertension (28), female animals were used in the current study to avoid any confounding variables. Six-week-old female heterozygous Ren-2 rats were randomized to receive either 55 mg/kg STZ (Sigma-Aldrich, St. Louis, MO) diluted in 0.1 M citrate buffer (pH 4.5) (n = 12) or citrate buffer alone (n = 12; for nondiabetic controls) by tail-vein injection after an overnight fast. In each subsequent week, rats were weighed, and blood glucose levels were determined (AMES Glucometer; Bayer Diagnostics, Melbourne, Australia). Diabetic rats were defined as those with a blood glucose more than 16 mmol/liter, 2 wk after STZ injection. At 10 wk of age (4 wk after STZ administration), diabetic and nondiabetic rats (n = 6 per treatment group) were further randomly allocated to (vehicle and H2-RLX) treatment groups. In each case, osmotic minipumps (model 2ML2; Alzet, Cupertino, CA) were filled with either 0.5 mg/kg·d H2-RLX in vehicle [20 mM sodium acetate buffer (pH 5.0)] or vehicle alone and implanted sc on the dorsal surface of rats under inhalational anesthesia (Isoflurane; Rhodia Australia Pty Ltd., Notting Hill, Victoria, Australia; by nosecone) for a 2-wk period (from wk 10–12; when diabetic mRen-2 rats demonstrate organ fibrosis) (25). These pumps provided continuous infusion of H2-RLX or vehicle for the 2-wk treatment period. The concentration of H2-RLX used was previously shown to successfully ameliorate other models of organ fibrosis in rats (21, 22, 26) and produce circulating RLX levels of approximately 20 ng/ml (21), which is within the circulating RLX levels of pregnant rats (5, 6). All rats were housed in a controlled environment at constant room temperature (21 ± 1 C) with a 12-h light, 12-h dark cycle, and had free access to standard rat chow (Barastock Stockfeeds, Packenham, Victoria, Australia) and water throughout the experiment. Diabetic animals sc received 2–4 U isophane insulin (Humulin NPH; Eli Lilly, West Ryde, New South Wales, Australia) three times per week to maintain body weight and avoid ketonuria without achieving euglycemia. These experiments were approved by the University of Melbourne, St. Vincent’s Hospital Animal Ethics Committee, which adheres to the Australian Code of Practice for the care and use of laboratory animals for scientific purposes.

Cardiovascular functional measurements
Just before animals were killed, transthoracic echocardiography, including Doppler examination, was performed on anesthetized rats, using a Vivid 7 Dimension echocardiograph (GE Vingmed, Horten, Norway) with a 10-MHz phased array probe, as described previously (29). M-mode echocardiography was performed using a parasternal short axis view at the level of the papillary muscles. Left ventricular (LV) posterior and anterior wall thickness was obtained during diastole and systole, as were the LV internal diameter at end diastole and end systole. From the parasternal short axis view, the end-diastolic and end-systolic cross-sectional blood pool areas were measured. Fractional area change (FAC) was then calculated as: [(end-diastolic area – end-systolic area)/end-diastolic area] x 100. The apical four-chamber view was used to assess early and late transmitral peak diastolic flow velocity (E and A waves), using pulsed wave Doppler with a sample volume of 2 mm placed at the tips of the mitral valve leaflets. All Doppler spectra were recorded for 10 cardiac cycles at a sweep speed of 200 mm/sec. Tissue Doppler imaging was performed in the apical four-chamber view, and used to assess peak systolic as well as early and late (E' and A') diastolic tissue velocity at the septal side of the mitral annulus. The sample volume was 1–3 mm, with gain limited to avoid superimposition of multiple amplitudes. Frame rates greater than 300 fps were obtained for all rats using a dedicated rodent software package (GE Vingmed). The depth was set at 2.5 cm and the Nyquist limit 12 cm/sec. The sweep speed was set at 200 mm/sec. All parameters were assessed using an average of three beats, and calculations were made in accordance with the American Society of Echocardiography guidelines. All data were acquired and analyzed by a single blinded observer using EchoPAC (GE Vingmed) offline processing. Systolic blood pressure (SBP) was also recorded in trained, preheated conscious rats by tail cuff plethysmography at wk 12. An average SBP was calculated from three consecutive readings, as detailed previously (25).

Tissue collection
At 12 wk of age (6 wk after STZ administration and 2 wk after vehicle or H2-RLX treatment), all diabetic (n = 12) and nondiabetic (n = 12) animals were then killed by an overdose of anesthetic [by ip injection of sodium pentobarbitone (Virbac; Australia Pty. Ltd., Peakhurst, New South Wales, Australia); 10 mg/100 g body weight] before the heart was removed, the left ventricle separated and sliced transversely. One portion of the left ventricle was stored at –80 C for subsequent biochemical analyses, and the remainder was fixed in neutral-buffered formalin for histological evaluation. Fixed tissue was routinely processed, embedded in paraffin, and sectioned.

Histology and morphometric analysis
Transverse (5 µm) sections of paraffin-embedded LV tissues were cut and stained with 0.1% picrosirius red to identify fibrillar collagen. Images of five nonoverlapping fields from at least six to eight randomly selected LV sections (from six rats per group) were captured and digitized using a BX50 microscope (Olympus, Hamburg, Germany) attached to a Fujix HC5000 digital camera (Fujifilm Corp., Tokyo, Japan). All images were collected with an Apochromat 20 x 0.6 Ph2 objective (Nikon Corp., Tokyo, Japan). Interstitial collagen content (a measure of cardiac fibrosis) in the left ventricle was determined by image analysis of the digitized images (version 6.0; AIS, Analytical Imaging Station, Ontario, Canada) as described before (24) and expressed as "interstitial cardiac collagen (% of area)."

Hydroxyproline analysis
Portions of the left ventricle from each rat were treated as described previously (22, 30) to determine the hydroxyproline content of each tissue, respectively. Hydroxyproline values were converted to collagen content by multiplying by 6.94 (based on hydroxyproline representing ~14.4% of the amino acid composition of collagen, in most mammalian tissues) (31). To correct for discrepancies in the size of the portions analyzed, collagen content was then expressed as a proportion (percentage) of the tissue dry weight (collagen concentration).

Western blot analysis
Total protein from similar portions of LV tissue was extracted using the TRIZOL reagent (according to the manufacturer’s instructions; Life Technologies, Gaithersburg, MD) and analyzed by the Bio-Rad dye-binding protein assay (Bio-Rad Laboratories Inc., Richmond, CA). Protein extracts (in 1% sodium dodecyl sulfate; 10–15 µg total protein per lane) were analyzed by SDS-PAGE under nonreducing conditions on 12.5% acrylamide gels, as previously described (20, 22). Western blot analyses were performed with monoclonal antibodies to {alpha}-SMA (a marker for mesenchymal cell differentiation; 1:1000 dilution; Dako Corp., Carpinteria, CA) and to MMP-13 (1:1000 dilution; Calbiochem, San Diego, CA), whereas the housekeeping protein β-tubulin (monoclonal antibody kindly provided by Dr. Zhonglin Chai, Baker Heart Research Institute, Victoria, Australia) was also performed to demonstrate equivalent loading of the protein samples. Blots were detected using the ECL detection kit (Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK), according to the manufacturer’s instructions, before being quantitated by densitometry, using a Bio-Rad GS710 Calibrated Imaging Densitometer and Quantity-One software (Bio-Rad Laboratories).

Gelatin zymography and reverse zymography
Gelatin zymography was performed to determine the effects of diabetes and H2-RLX treatment on the latent and active forms of MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) in the heart, as described previously (20, 32). MMPs were extracted from LV tissue using the method described by Woessner (32), and total protein was quantified from these extracts using the Bio-Rad dye-binding protein assay (Bio-Rad Laboratories) to ensure equal loading of samples. Reverse zymography of the same cardiac tissue extracts from diabetic and nondiabetic animals, with or without H2-RLX treatment, was also performed as described before (33) for measurement of TIMP-1 and TIMP-2 expression. Equal loading of samples was verified by Coomassie blue staining of proteins, separately run on 10% acrylamide gels, whereas densitometry of the MMP and TIMP bands was performed as described previously.

Statistical analysis
The results were analyzed by a one-way ANOVA using the Bonferroni post hoc test for multiple comparisons between groups. All data in this paper are presented as the mean ± SEM, with P < 0.05 considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of the experimental model
Transgenic mRen-2 rats treated with STZ developed sustained hyperglycemia and pathological changes characteristic of diabetic complications. At the time the rats were killed, both plasma glucose and glycosylated hemoglobin levels were approximately 5-fold higher in diabetic animals than their nondiabetic counterparts (both P < 0.01) (Table 1Go). Although the body weight of diabetic animals at the time the rats were killed was not significantly different from untreated control animals, after correction for body weight, heart weight was significantly greater in diabetic animals vs. nondiabetic rats (both P < 0.05. However, H2-RLX treatment of control and diabetic animals had no effects on any of these parameters (Table 1Go).


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  		TABLE 1. Characteristics of the experimental model

 
Functional responses of the experimental model and H2-RLX treatment
Using M-mode traces (echocardiography) crossing the left ventricle and measurements of Doppler flow velocities across the mitral valves during the diastolic phase to assess LV diastolic function, it was observed that the E/A wave ratio (a measure of LV diastolic function) was significantly decreased in both the diabetic and diabetic plus H2-RLX treated groups vs. the control group (both P < 0.05; Table 2Go), whereas several other measures of LV dimensions, wall thickness, and FAC were similar between control and diabetic groups (data not shown). Of note, E-wave deceleration time (a measure of myocardial stiffness and associated with LV hypertrophy and diastolic dysfunction) was significantly increased in the diabetic group (P < 0.05 vs. control group) but was significantly decreased by 2-wk H2-RLX treatment (P < 0.05 vs. diabetic group) to levels measured in the control group (Table 2Go), consistent with RLX’s antifibrotic/matrix remodeling actions. Of further note, H2-RLX treatment of diabetic animals had no significant effect on SBP (Table 2Go).


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  		TABLE 2. Echocardiographic parameters

 
Effects of the experimental model and H2-RLX treatment on cardiac fibrosis
Analysis of 12-wk-old diabetic rats 6 wk after STZ treatment indicated an 18.1% increase in LV collagen concentration (P < 0.05 vs. age-matched control group; Fig. 1AGo). Two weeks of H2-RLX treatment had no effects on basal LV collagen levels (Fig. 1Go, A, B, and D), but in diabetic rats, significantly decreased LV collagen concentration (P < 0.05 vs. diabetic group; Fig. 1AGo) and interstitial LV collagen staining (P < 0.05 vs. diabetic group; Fig. 1Go, B and F) in the heart.


Figure 1
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  		FIG. 1. The mean ± SE cardiac collagen concentration (A) and interstitial cardiac collagen content (B) were used as measures of the effects of hyperglycemia and H2-RLX therapy on cardiac fibrosis from control, control plus H2-RLX treated, diabetic, and diabetic plus H2-RLX treated rats (n = 6 animals per group). *, P < 0.05 vs. control group. #, P < 0.05 vs. diabetic group. Also shown are representative images of picrosirius red-stained collagen within the left ventricle of control (C), control plus H2-RLX treated (D), diabetic (E), and diabetic plus H2-RLX treated (F) rats. Quantitative analysis of the interstitial collagen content within the left ventricle is shown in B. Magnification of C–F, x200.

 
Effects of the experimental model and H2-RLX on {alpha}-SMA, MMP, and TIMP expression and activity
STZ-treated diabetic mRen-2 rats had increased LV expression of {alpha}-SMA (P < 0.01 vs. control group; Fig. 2Go), consistent with the observed increase in cardiac collagen concentration (Fig. 1AGo) that was measured in these animals. H2-RLX administration to control rats had no effect on basal {alpha}-SMA expression but significantly decreased LV expression of {alpha}-SMA in diabetic animals (P < 0.05 vs. diabetic group; Fig. 2Go).


Figure 2
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  		FIG. 2. Western blot analysis of {alpha}-SMA (43 kDa) was used to detect changes in cardiac mesenchymal cell differentiation in control (lanes 1 and 2), control plus H2-RLX treated (lanes 3 and 4), diabetic (lanes 5 and 6), and diabetic plus H2-RLX treated (lanes 7 and 8) rats. Shown are representative blots of cardiac {alpha}-SMA expression from two animals per group, whereas separate blots from a total of six animals per group gave similar results (data not shown). In addition, a blot of β-tubulin expression (in the left ventricle) was also included to demonstrate quality and equivalent loading of protein samples. The mean ± SE densitometry levels of cardiac {alpha}-SMA expression (from six animals per group) were expressed as a relative ratio to the mean value of the control group, which was expressed as one. **, P < 0.02 vs. control group. #, P < 0.05 vs. diabetic group.

 
Diabetic animals had equivalent levels of latent and active MMP-13 (collagenase-3; Fig. 3AGo) but increased levels of latent and active MMP-2 (gelatinase-A; P < 0.05) and MMP-9 (gelatinase-B; P < 0.05; Fig. 3BGo) to that measured in the left ventricle of control rats by 12 wk of age. H2-RLX administration over a 2-wk period increased the expression of the interstitial collagenase MMP-13 in the left ventricle of both normoglycemic (P < 0.05 vs. control group) and hyperglycemic animals (P < 0.02 vs. diabetic group), although the magnitude of this increase was greater in the diabetic group (Fig. 3AGo). Continuous infusion of exogenous H2-RLX for 2 wk also increased LV MMP-9 and MMP-2 expression and activity in normoglycemic rats (all P < 0.05 vs. control group) (Fig. 3BGo). However, H2-RLX administration to hyperglycemic animals did not have any statistically significant effects on LV MMP-9 or MMP-2 expression and activity, compared with that measured in diabetic rats.


Figure 3
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  		FIG. 3. Western blot analysis of latent (L) and active (A) MMP-13 (A) was used to detect changes in interstitial collagenase activity in the heart of control (lanes 1 and 2), control plus H2-RLX treated (lanes 3 and 4), diabetic (lanes 5 and 6), and diabetic plus H2-RLX treated (lanes 7 and 8) rats. In addition, a blot of β-tubulin expression (in the left ventricle) was also included to demonstrate quality and equivalent loading of protein samples (A). Gelatin zymography was used to detect changes in latent and active MMP-2 and -9 in the heart of control (lane 1), control plus H2-RLX treated (lane 2), diabetic (lane 3), and diabetic plus H2-RLX treated (lane 4) rats (B), whereas a molecular mass marker (lane 5) is also included. A Coomassie blue-stained protein sample from the corresponding MMP extracts (bottom row) was used to verify equal loading of the samples. Shown is a representative blot of MMP-13 (A) or zymograph of MMP-2 and -9 (B) from one to two animals per group, whereas separate blots from a total of six animals per group gave similar results, respectively (data not shown). The mean ± SE (combined latent and active) levels of LV MMP-13 (A) in addition to that of MMP-9 and MMP-2 (B) were determined by densitometry (from six animals per group) and expressed as a relative ratio to the mean value for each respective control group, which was expressed as one. *, P < 0.05; **, P < 0.02 vs. control group. #, P < 0.05; ##, P < 0.02 vs. diabetic group.

 
In addition, diabetic animals had equivalent levels of TIMP-1 expression to that measured in the left ventricle of control rats by 12 wk of age (Fig. 4Go). In contrast to its effects on MMP expression and activity, H2 RLX administration to diabetic animals was able to significantly decrease LV TIMP-1 expression (P < 0.05 vs. diabetic group) but had no marked effects on basal TIMP-1 levels over a 2-wk treatment period. In comparison, changes in TIMP-2 expression could not be accurately measured because it was lowly expressed in all four groups studied (data not shown).


Figure 4
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  		FIG. 4. A representative reverse zymograph of TIMP-1 expression in control (lane1) control plus H2-RLX treated (lane 2), diabetic (lane 3), and diabetic plus H2-RLX treated (lane 4) rats, with a TIMP-1 standard (lane 5) also shown. A Coomassie blue-stained protein sample from the corresponding MMP/TIMP extracts (bottom row) was used to verify equal loading of the samples. Separate zymographs from a total of six animals per group gave similar results, respectively (data not shown). The mean ± SE levels of LV TIMP-1 were determined by densitometry (from six animals per group) and expressed as a relative ratio to the mean value of TIMP-1 in the control group, which was expressed as one. #, P < 0.05 vs. diabetic group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated for the first time that the administration of RLX (H2-RLX) to an experimental model of diabetes significantly reversed established cardiac fibrosis, which resulted in decreased myocardial stiffness and improved diastolic function of the diabetic heart. The therapeutic effects of RLX were associated with its ability to inhibit mesenchymal cell differentiation and, presumably, the ability of these cells to synthesize collagen, whereas also increasing collagenase (MMP-13) activity and reducing the levels of TIMP-1, thus promoting a collagen degradation phenotype in the diabetic myocardium. These findings suggest that RLX may represent a novel therapy for limiting the progression of cardiac fibrosis associated with diabetes. However, importantly, the effects of RLX were shown to be independent of blood pressure regulation, the level of hyperglycemia and basal matrix turnover, confirming its status as a specific antifibrotic with rapid-occurring efficacy.

Fibrosis is increasingly recognized as a ubiquitous cause of organ dysfunction and failure in a diverse range of circumstances (34). Therefore, the study of diabetic fibrosis is relevant to our understanding of both diabetic complications and progressive organ failure in general. However, despite the significance of progressive fibrosis, therapeutic strategies for its treatment remain elusive. Although renin-angiotensin system blockers are now viewed as first-line treatment for diabetic nephropathy and cardiomyopathy (35) and have clearly been shown to confer organ protection, these treatments fail to halt the progression of diabetes-induced fibrosis in most patients (36). Even more elusive has been the search for therapies that may modify or reduce existing scarring, the final outcome in many diabetic complications. The clinical reality is that most patients present with established pathology. Currently, there are no treatments that consistently and effectively reverse established collagen deposition. Therefore, it is imperative that novel interventions that can halt the progression of diabetic cardiomyopathy are identified.

This study specifically examined the antifibrotic properties of RLX in experimental diabetes, in which experimental groups were matched with similar levels of glycemia. Clinically relevant techniques were used to measure the effect of exogenous RLX on cardiac function. Echocardiographic (M-mode and Doppler flow) measures of mitral inflow demonstrated a decrease in arterial E/A wave ratio and an increase in E-wave deceleration time (a measure of LV diastolic dysfunction) in diabetic animals, consistent with the loss of cardiac function that occurs in diabetes. However, continuous short-term exposure of RLX to diabetic mRen-2 rats resulted in reduced myocardial stiffening and improved LV diastolic function, highlighting the functional significance of RLX in this model.

The functional changes observed were paralleled by proportionally similar changes in cardiac structure. The findings of this study are consistent with H2-RLX’s previously documented collagen-inhibitory effects in vitro (14, 15, 19, 20, 21) and in other experimental models of cardiac fibrosis in vivo (8, 9, 12, 13), including models of β2-adrenergic receptor-induced fibrotic cardiomyopathy (20) and hypertensive heart disease (22).

The means by which RLX is cardioprotective are less clear and likely to involve a number of mechanisms. Mesenchymal cells in the heart (vascular smooth muscle cells, fibroblasts) are central to the pathogenesis of diabetic fibrosis through their prodigious production of ECM components. Aberrant and de novo expression of proteins associated with smooth muscle differentiation is widely used as a marker of mesenchymal cell activation. Our analysis of {alpha}-SMA expression suggests that RLX reduces collagen accumulation by inhibiting the differentiation of mesenchymal cells such as fibroblasts, consistent with our previous findings in other disease models (22). Several studies have also shown that RLX inhibits TGF-β (14, 15, 19, 20) or angiotensin II (20)-induced myofibroblast differentiation, which may be the case for the actions of RLX in the mRen-2 rat model because both these profibrotic factors are up-regulated during the pathogenesis of diabetes (2, 24).

In addition, RLX was shown to promote interstitial collagenase (MMP-13) expression and activity, while reducing the levels of TIMP-1 in the diabetic heart, which would enhance collagen degradation in the diabetic myocardium (37). Although these findings are consistent with RLX’s previously reported effects in increasing the expression and activity of MMPs (8, 9, 14, 15, 16, 19, 20, 22) and decreasing the levels of TIMPs (14) in several other models, somewhat surprisingly, they differ from the increased expression of LV MMP-2 that was observed upon RLX administration to spontaneously hypertensive rats (22). This discrepancy in LV MMP-2 expression/activity between experimental models may be explained in part by the different pathogenesis of cardiomyopathy in spontaneously hypertensive rats and diabetic rats. In each case only a single time point was examined, making temporal comparisons between models problematic. Furthermore, controversy surrounds the role of metalloproteinases in the pathogenesis of fibrosis associated with diabetes. Although dogma suggests that the balance between collagen synthesis and degradation determines the extent of fibrosis, it is becoming apparent that several MMPs have multifactorial roles (38), as in some cases, the activation of MMPs may be profibrotic through generation of active peptides and facilitation of migration through breakdown of basement membranes. Furthermore, confusion has often occurred because investigators have failed to consider the different specificities of collagenases/gelatinases. Although any temporal changes are unclear, our experimental model per se (in the absence of RLX) was associated with higher levels of MMP-2 and MMP-9, even though collagen deposition was increased. These findings are consistent with the increased circulatory MMP-2 and -9 levels and activities that were measured in human type 1 diabetics (39), and to a certain extent, with the increased MMP-2 mRNA and activity, but decreased MMP-9 expression and activity that was reported in the kidneys of younger (8 wk old) male Ren2 diabetic rats (40). However, they differed from the decreased MMP-2 activity that was measured in the diabetic (and fibrosed) mouse heart after STZ administration (41), suggesting that gelatinase activity may vary in diabetics depending on the organ, species, and gender studied. Conversely, the induction of diabetes in mRen-2 rats has diminished MMP-7 (matrilysin) expression (42). Further work will be required to determine if this is a temporal factor in the pathogenesis of diabetes in general or, more simply, a reflection of the heart’s efforts to maintain tissue homeostasis when faced with chronic injury.

In addition to its antifibrotic effects, RLX also possesses potent vasodilatory properties (7, 11) that have been demonstrated in humans (43) and are potentially relevant in this model. Previous studies have shown that the renoprotection afforded by RLX in hypertensive models is sometimes (27), but not usually (22, 26, 27), accompanied by a reduction in blood pressure. In the present study, the RLX-induced cardiac functional and structural improvements occurred independently of changes in blood pressure, suggesting that the benefits of RLX were not hemodynamically related.

In conclusion, whereas a whole series of conditions may act in synergy to contribute to diabetic cardiomyopathy, the delineation of RLX specific effects may offer an important adjunctive therapy to existing clinical interventions. Importantly, the current study indicated that as little as 2-wk infusion of H2-RLX was sufficient to affect structural and functional changes in the diabetic heart. Together, these studies highlight that RLX may be a valuable therapeutic strategy for limiting the progression of established fibrosis in diabetic cardiomyopathy. Being a naturally occurring physiological hormone, RLX has an excellent safety profile (43, 44) with potentially fewer side effects than conventional treatments.


   Acknowledgments
 
We thank Ms. Chongxin Zhao for technical assistance.


   Footnotes
 
This study was supported by a National Heart Foundation of Australia/National Health & Medical Research Council of Australia R. D. Wright Career Development Award (to C.S.S.), a Juvenile Diabetes Research Foundation Career Development Award (to D.J.K.), and a Diabetes Australia Research Trust Research Grant (to T.D.H., C.S.S., and D.J.K.).

Disclosure Summary: The authors have nothing to declare.

First Published Online April 3, 2008

Abbreviations: ECM, Extracellular matrix; FAC, fractional area change; H2, human gene-2; MMP, matrix metalloproteinase; RLX, relaxin; LV, left ventricular; SBP, systolic blood pressure; SMA, smooth muscle actin; STZ, streptozotocin; TIMP, tissue inhibitor of metalloproteinase.

Received February 22, 2008.

Accepted for publication March 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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