Cardiovascular Research

Cardiovascular Research 2003 57(2):395-404; doi:10.1016/S0008-6363(02)00663-6
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype

Xiao-Jun Du^a,*, Chrishan S Samuel^b, Xiao-Ming Gao^a, Ling Zhao^b,1, Laura J Parry^b and Geoffrey W Tregear^b

^aExperimental Cardiology Laboratory, Baker Medical Research Institute, PO Box 6492, St Kilda Road Central, Melbourne, Vic. 8008, Australia
^bHoward Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Melbourne, Vic. 3010, Australia

^* Corresponding author. Tel.: +61-3-8530-1294; fax: +61-3-8530-1100. xiaojun.du{at}baker.edu.au

Received 19 March 2002; accepted 10 September 2002

	Abstract

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

 
Objective: To investigate cardiac phenotypes in mice deficient in the peptide hormone relaxin by gene targeting. Methods: Echocardiography and cardiac catheterization were performed on male and female relaxin deficient (Rlx^–/–) mice as well as heterozygous (Rlx^+/–) and wildtype (Rlx^+/+) littermates aged between 8 and 24 months. Collagen expression and content in the heart were analysed by real-time PCR, hydroxyproline assay and histology. Results: Heart rate, blood pressures, left ventricular (LV) dimensions, fractional shortening and maximal and minimal dP/dt did not differ significantly between the three genotypes of either gender at any age. However, 8–10-month-old Rlx^–/– males exhibited a greater transmitral flow velocity (A-wave) at the late LV diastolic phase. Male Rlx^–/– mice aged between 12 and 24 months had significantly higher LV end-diastolic pressures, a 30% increase in atrial weight and 10–30% increases in lung and liver weights. Male mice also showed an age-dependent increase (P<0.01) in LV collagen content that was more pronounced in Rlx^–/– than control littermates (P<0.01). Procollagen type-1 expression was also significantly higher in the LV of Rlx^–/– males compared with either Rlx^+/– or Rlx^+/+ males at 6, 9 and 12 months of age. Age-matched female Rlx^–/– mice did not display any of these cardiac phenotypes seen in Rlx^–/– males. Conclusions: Male Rlx^–/– mice had impeded LV diastolic filling and increased atrial weights, most likely due to an increase in ventricular collagen content and chamber stiffness. These phenotypes in the Rlx^–/– males were not observed in Rlx^–/– females, indicating the importance of other gender-related factors in cardiovascular function.

KEYWORDS Fibrosis; Gender; Gene expression; Hormones; Ventricular function

	1. Introduction

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

 
Relaxin is a peptide hormone structurally similar to insulin and insulin-like growth factors, with two chains linked by disulfide bridges [1]. Relaxin concentrations are highest in the ovary, placenta or serum of females during pregnancy [1]. The main functions of relaxin are associated with female reproductive physiology and in particular connective tissue remodeling in the interpubic symphysis and uterine cervix to facilitate birth [1–3]. Relaxin also stimulates growth and differentiation of the mammary gland and nipple, both important in lactating females [1]. These effects of relaxin are achieved through its inhibition of collagen synthesis and activation of matrix metalloproteinases (MMPs) that degrade collagen [4,5]. The mouse has two relaxin genes, M1 and M3, equivalent to the human relaxin genes H2 and H3, respectively [6,7]. Late-pregnant mice rendered deficient in M1 relaxin (Rlx^–/–) by gene targeting appear to deliver normally, although the pups are unable to suckle and do not survive [8]. This is due to insufficient development of the mammary gland and nipple caused by an increased collagen content in these tissues [8,9]. Male Rlx^–/– mice also show phenotypes of higher collagen content in the prostate gland, poor tubular development and a reduction in prostate growth [10].

In recent years, evidence has accumulated to show that relaxin acts on the cardiovascular system in both females and males. High affinity relaxin binding sites are localized to the atria of male and female rats [11,12] and studies in vitro and in vivo have demonstrated positive chronotropic and probably inotropic responses to exogenous relaxin in both genders [13–15]. Relaxin stimulates atrial natriuretic peptide secretion from isolated perfused rat hearts [16] and also causes a dose-dependent increase in coronary blood flow via a nitric oxide-mediated mechanism [17]. Cultured atrial cardiocyctes derived from adult male rats produce and secrete immunoreactive relaxin [18], and a recent study has demonstrated relaxin mRNA in the human heart [19]. Data also show that chronic heart failure patients have increased myocardial relaxin gene expression and elevated plasma relaxin concentrations [19], suggesting that cardiac relaxin may regulate heart function by an autocrine/paracrine mechanism.

To investigate the physiological significance of endogenous relaxin in the heart, we determined cardiac function in both male and female Rlx^–/– mice. The ventricular myocardium was examined for chemical and morphological abnormalities, with emphasis on interstitial collagen content and myocyte hypertrophy. To address the age-dependent development of cardiac phenotypes in these mice, animals were studied between 8 and 24 months of age for male mice and 12 and 24 months of age for female mice. The results from this study point to a cardiac phenotype in male mice deficient in relaxin. Specifically, left ventricular diastolic filling is impeded which is very likely related to an increase in collagen content in the left ventricle (LV).

	2. Methods

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

 
2.1 Animals
All animal experiments were approved by the Baker Institute and Howard Florey Institute Animal Experimental Ethics Committees. The relaxin gene knockout (Rlx^–/–) strain was generated at the Howard Florey Institute by targeted deletion of the relaxin gene as described previously [8]. Heterozygous (Rlx^+/–) mice were mated to obtain wildtype (Rlx^+/+), Rlx^–/– and Rlx^+/– littermates. All mice used in these studies had the same mixed background (129SV/C57Blk6J) and were aged between 9 and 24 months for males and 12 and 24 months for females. Tail biopsies and PCR analysis of genomic DNA [8] confirmed the genotype of each animal at weaning.

2.2 Echocardiography
Transthoracic echocardiography was performed using a Sonos 5500 ultrasound machine with a 15-MHz linear-array transducer (Hewlett-Packard), as described previously [20]. The first experiment was conducted on a total of 60 male and female Rlx^+/+, Rlx^+/–, Rlx^–/– mice aged between 12 and 24 months. Mice were anesthetized (mixture of ketamine–xylazine–atropine at 60–12–0.6 mg/kg, respectively) and 2-dimensional guided M-mode traces crossing the LV were acquired at a sweep speed of 100 mm/s. The following parameters were measured, using the leading-edge technique: LV internal dimensions of diastole and systole (LVIDd, LVIDs), LV external dimension of diastole (LVEDd) and wall thickness at diastole and systole. Measurements were taken, in blind fashion, from two cardiac cycles and the data averaged. Fractional shortening (FS%) was calculated as (LVIDd–LVIDs)/LVIDd. Wall thickening index (WTI) was the net increase in LV wall thickness in systole versus diastole. LV mass was calculated as (LVEDd³–LVIDd³)x1.05.

A second echocardiographic experiment used 27 male Rlx^+/+, Rlx^+/– and Rlx^–/– mice aged at 9 months (between 8 and 10 months). After completion of M-mode image recording, the ventricular diastolic function was analyzed by long-axis 2-D image guided color Doppler recordings of blood flow velocities across the mitral valves from the apical position while a standard lead-II ECG was continuously recorded. Peak flow velocities, isovolumic-relaxation time (IVRT) and E-wave deceleration time (DT) were measured digitally in standard fashion [21,22]. All measures were taken from seven to ten cardiac cycles and averaged.

2.3 Cardiac catheterization
Arterial blood pressures and LV pressures were measured in groups of seven to 12 male and female mice, as described previously [23]. Mice were anesthetized (pentobarbitone 80 mg/kg and atropine 0.6 mg/kg) and a 1.4 F catheter (Millar Instruments, Houston) was inserted into the LV via the right carotid artery. Aortic blood pressures, heart rate, LV pressures and the maximal rate of increase or decay of LV pressures, dP/dt_max or dP/dt_min, were acquired digitally. After recording baseline parameters, a β-adrenergic agonist isoproterenol was infused intravenously as a bolus at increasing doses of 50–2000 pg per mouse and functional responses were monitored.

2.4 Morphological analysis
Mice were killed at the end of the experiments by pentobarbitone overdose. The hearts were isolated and the LV, right ventricle and atria were separated and weighed. The lungs, liver and kidney were also weighed and tibia length was measured. Ventricles were either frozen for biochemical assay or fixed in 10% formalin in PBS. Tissues were embedded in paraffin and transverse sections (5 µm) were cut and stained with hematoxylin and eosin or 0.1% picrosirius red to stain collagen. Images of LV sections were gathered with a CCD video camera (Optimas, BioScan, Edmonds, WA, USA), digitized and quantified using Optimas 6.5 program. Interstitial collagen content in the LV was determined following the method described previously [23]. The LV sections were sampled in a systematic fashion and 15–20 fields in each LV were analyzed. The picrosirius red stained area was calculated as a percentage of the total area within a field and the averaged percentage was used. One section at the LV equator was used for measuring myocyte transverse cross-sectional area. The average of 60–70 cells from randomly selected fields was used.

2.5 Collagen expression
Total collagen content and concentrations in the LV myocardium were determined by hydroxyproline assay as described previously [24]. Male mice (n=44) aged between 9 and 24 months and female mice aged between 12 and 24 months (n=18) were studied. The results were expressed as µg hydroxyproline/mg dry weight. In a separate experiment, heart tissues were collected from 12-month-old male Rlx^+/+ and Rlx^–/– mice. The atria were separated from the ventricles and all tissues frozen in liquid nitrogen. Total RNA was extracted using RNA Wiz (Ambion; Geneworks, Adelaide, South Australia). Real time PCR was used to quantify gene expression of procollagen type 1 (COL-1) and proMMPs (proMMP-2, proMMP-9 and proMMP-13). First strand cDNA synthesis used 1 or 1.5 µg total RNA and 1.25 U/µl MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA) in a 30-µl reaction as described previously [25]. A second reverse transcription reaction used 30 ng RNA and a series of ventricle RNA dilutions (100–0.001 ng) for the endogenous reference (18S ribosomal RNA) PCR reactions. Mouse-specific COL-1 and proMMP primers and probes were designed using Primer Express (Applied Biosystems). The probes were labelled at the 5' end with FAM (6-carboxy fluorescein; Keystone Division, Biosource International, Foster City, CA). The PCR reactions were carried out in triplicate 25-µl volumes in an ABI PRISM 7700 Sequence Detector using the relative standard curve method, 96-well optical reaction plates and TaqMan^® PCR components (all Applied Biosystems) as outlined previously [25]. In a further experiment, COL-1 expression was analysed in 6- and 9-month-old male Rlx^+/+ and Rlx^–/– mice.

The presence of relaxin gene transcripts in the mouse atria and ventricles was assessed by reverse transcription (RT)-PCR with mouse-specific relaxin oligonucleotide primers (100 ng/µl: forward 5'-GTGAATATGCCCGTGAATTGATC and reverse 5'-AGCGTCGTATCGAAAGG CTCT) and 5-µl cDNA template in a 50-µl PCR reaction. Primers were selected to span the intron/exon junction, thus favoring PCR products (150-bp) arising only from cDNA rather than genomic contamination. The quality of first strand cDNA synthesis was demonstrated in parallel RT-PCR reactions that assessed expression of glyceraldehyde phosphate dehydrogenase (GAPDH).

2.6 Statistics
Results are expressed as mean±S.E.M. Between-group comparisons were made using one- or two-way analysis of variance (ANOVA) and unpaired Student's t-test as post-hoc analysis. Data from the real time PCR did not show homogeneity of variance and were log transformed before analysis by one-way ANOVA. Linear correlation was performed for echo-derived LV mass and actual weights. P<0.05 was considered statistically significant.

	3. Results

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

 
3.1 Echocardiographic changes
There were no gross abnormalities in the cardiovascular system and no mortality in male or female Rlx^–/– mice of any age. Using echocardiography and catheterization techniques, we found no significant difference in heart rate, dP/dt_max, dP/dt_min, LV dimensions, wall thickness, FS or WTI between the three genotypes of males at 8–24 months of age or females at 12–24 months of age (Table 1). Aortic blood pressures and LV pressures in the Rlx^–/– mice were also similar to those of the Rlx^+/– and Rlx^+/+ mice. LV end-diastolic pressure (LVEDP) was unchanged in male Rlx^–/– mice at 8–10 months of age but elevated at 12–24 months of age (P<0.05, Table 1). Although there was a slight trend for an increase in LVEDP in the female Rlx^–/– mice, it was not significant (Table 1). While the catheter was positioned in the LV, the heart was challenged by intravenous injection of isoproterenol. The responses of heart rate, dP/dt_max and dP/dt_min were similar in all three genotype groups of both genders (Fig. 1). These findings indicate normal chronotropic, inotropic and lusitropic function in Rlx^–/– mice.

View this table: [in this window] [in a new window]   Table 1 Functional parameters determined by echocardiography and catheterization in male and female mice

 

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Heart rate, left ventricular (LV) dP/dtmin and dP/dtmax (mmHg/s) measured at baseline and during isoproterenol administration (50–2000 ng/mouse, intravenous in bolus) in 12–24-month-old male and female mice of each genotype (n=8–12 per group). dP/dtmax and dP/dtmin are measures of the maximal rates of LV pressure rise or decline.

 
Diastolic function of the LV was further examined in male mice aged 8–10 months by measuring Doppler flow velocities across the mitral valves during the diastolic phase. All three genotype groups of mice had similar early filling flow velocity (E-wave). However, the late filling flow velocity (A-wave) was higher in the Rlx^–/– males, leading to a significant reduction in the ratio of E/A waves (E/A ratio; Fig. 2). IVRT was similar between the three groups in keeping with an unchanged dP/dt_min in Rlx^–/– mice measured by catheter. However, Rlx^–/– mice had a significant lengthening of DT compared to other two groups (P<0.01; Fig. 2), indicating a lower passive compliance of the LV [21].

View larger version (62K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Echocardiographic measurement of transmitral flow velocities in 9-month-old male mice of three genotypes. Upper panel: Representative traces of Doppler detected transmitral flow velocities and electrocardiogram in an Rlx+/+ and two Rlx–/– mice with early (E) and late or atrial (A) flow waves indicated. Lower panel: mean±S.E.M. of E and A flow velocities, E/A ratio, isovolumic relaxation time (IVRT), and E-wave deceleration time (DT) in each of the genotype groups (seven to 11 mice per group). *P<0.01 versus Rlx+/+. Other echocardiographic parameters are given in Table 1.

 
3.1.1 Morphometry
In 8–10-month-old Rlx^–/– males, heart weight was similar between the three genotype groups except for an increase in atrial weight (P<0.05, Table 2). In Rlx^–/– males aged 12–24 months, whilst LV weight did not increase, the overall heart and atrial weights were significantly greater (P<0.05) compared to those of Rlx^+/+ and Rlx^+/– littermates (Table 2). However, histological analysis of atrial tissues showed no evidence of interstitial fibrosis. The lack of LV hypertrophy in male Rlx^–/– mice between 12 and 24 months was further indicated by unchanged cross-sectional areas of LV myocytes (Rlx^+/+: 233±6; Rlx^+/–: 232±10; and Rlx^–/–: 230±6 µm²; P0.05).

View this table: [in this window] [in a new window]   Table 2 Body weight (BW), tibial length (TL) and organ wet weights normalized by TL mice of three genotypes

 
Organ weights in the 8–10-month-old male Rlx^–/– mice were similar among the three genotype groups. A trend for a higher lung weight was observed in Rlx^–/– but this was statistically insignificant (Table 2). In the 12–24-month-old Rlx^–/– males, the wet weights of the lungs and liver, but not kidney, were significantly (P<0.05) higher than both Rlx^+/+ and Rlx^+/– mice (Table 2).

There were no significant differences in atrial weights or other organ weights between 12- and 24-month-old female Rlx^+/+, Rlx^+/– and Rlx^–/– mice (Table 2).

In this study, the LV mass from echocardiography correlated well with the LV weight (r=0.851, n=78, P<0.001).

3.1.2 Myocardial collagen expression
The LV collagen content was determined using two methods, hydroxyproline assay and quantitative histology. For hydroxyproline assay, data from Rlx^+/+ (n=13) and Rlx^+/– (n=9) were very similar and therefore combined (n=22) for comparison with the data from Rlx^–/– group (n=22) between 9 and 24 months of age. The collagen content correlated significantly with age (P<0.001 by ANOVA, r=0.744 for the combined Rlx^+/+/Rlx^+/– group, r=0.885 for Rlx^–/– group, P<0.01 for both correlation coefficients). Rlx^–/– mice had significantly higher hydroxyproline content than controls (P<0.01 by ANOVA). When data were assigned into three different age groups of 9–11, 12–16 and 18–24 months and analysed separately, hydroxyproline content in the LV was higher in the Rlx^–/– compared with control mice in each age group (Fig. 3A). In contrast, hydroxyproline concentrations in the LV were not significantly different in 12–24-month-old female Rlx^–/– and Rlx^+/+ mice (Fig. 3B). The area of the LV stained for collagen was also significantly (P<0.05) greater in male Rlx^–/– mice compared with the other two genotypes at 12–24 months of age (Fig. 3C).

View larger version (67K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Hydroxyproline concentrations (µg/mg dry weight) in the left ventricular (LV) myocardium of male (panel A) and female (panel B) mice, 9–24 months of age. Collagen content (% total area) as determined by histological analysis in the LV of 12–24-month-old male mice (panel C). In panel A, data from control mice (Rlx+/+ and Rlx+/–, n=22) and Rlx–/– mice (n=22) were compared within each age group. Numbers in brackets indicate sample size. Sirus-red stained LV sections showing increased interstitial collagen (pink color) in a male Rlx–/– mouse compared to a male Rlx+/+ mouse (panels D and E). *P<0.05 versus both Rlx+/+ and Rlx+/– mice. Bar represents 30 µm.

 
Quantitative analysis of procollagen type-1 (COL-1) gene expression demonstrated a significant increase (P<0.05) in COL-1 mRNA levels in the ventricles of male Rlx^–/– compared with Rlx^+/+ mice at 6, 9 and 12 months of age (Fig. 4). There was no increase in COL-1 gene expression in the atria of the Rlx^–/– mice in the 12-month age group (Fig. 4B). Concentrations of proMMP-2, proMMP-9 and proMMP-13 mRNA did not differ significantly in either the ventricles or atria of the Rlx^–/– compared with Rlx^+/+ mice at 12 months of age (data not shown).

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Expression of procollagen type 1 mRNA in the ventricles of 6- and 9-month-old male Rlx+/+ and Rlx–/– mice (panel A). Expression of procollagen type 1 mRNA in the atria and the ventricles of 12-month-old male Rlx+/+ and Rlx–/– mice (panel B). Results are mean±S.E.M. Numbers in brackets indicate sample size. Different amounts of RNA were used in the cDNA synthesis (panel A, 1.5 µg; panel B, 1.0 µg), hence the different y-axis scales. (a) P<0.05 and (b) P<0.01 versus age-matched Rlx+/+ values.

 
3.1.3 Relaxin gene expression
Analysis of relaxin gene expression in the mouse heart by RT-PCR demonstrated the presence of specific M1 relaxin gene transcripts in the atria and ventricles of 12-month-old Rlx^+/+ mice, but not in Rlx^–/– mice (Fig. 5).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 RT-PCR analysis of mouse M1 relaxin gene transcripts in the atria and ventricles of 12-month-old male Rlx+/+ (n=3) and Rlx–/– mice (n=1). Brain cDNA from an Rlx+/+ mouse was used as a positive control (+ve CTL), whereas water replaced cDNA in negative control reactions.

 

	4. Discussion

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

 
In this study, we demonstrated cardiac phenotypes in male relaxin deficient mice. Biochemically, myocardial collagen concentrations and procollagen type 1 mRNA were higher in the ventricles of Rlx^–/– male aged between 9 and 24 months. Expression of precollagen type-1 gene was already increased in Rlx^–/– males as early as 6 months of age. Functionally, while arterial blood pressures, heart rate, contractility and isovolumic relaxation of the LV were normal in male Rlx^–/– mice, these mice had elevated LVEDP, transmitral flow velocity at the late diastolic phase and lengthening of E-wave deceleration time. These functional changes in the male Rlx^–/– mice were associated with a significant hypertrophy in the atria but not in ventricles. Thus, the overall physiological effect of relaxin deficiency on cardiac function was a moderate impediment of LV diastolic filling. These changes, however, did not lead to more pronounced cardiac dysfunction or mortality. The cardiac phenotypes were not observed in aged-matched female Rlx^–/– littermates.

In this study, we show that both atria and ventricles are putative sources of relaxin in the mouse heart, similar to the human [19]. High affinity relaxin binding sites are also localised to the rodent atria [11,12], implying that relaxin has a direct action on the heart to influence cardiovascular activity. These include chronotropic and inotropic effects stimulated by administration of relaxin both in vivo and in vitro [13–15]. In this study, we did not observe any significant difference between Rlx^+/+ and Rlx^–/– mice in blood pressures, heart rate and ventricular inotropy and lusitropy (dP/dt_max and dP/dt_min) under basal and isoproterenol-stimulated conditions. Other cardiovascular effects of exogenous relaxin are coronary, pulmonary and renal vasodilatation [17,26,27] and stimulation of release of vasoactive peptides such as atrial natriuretic peptide [16] and vasopressin [28]. The vascular action of relaxin is mediated by nitric oxide and endothelin (ET-1) through endothelin ET_B receptors [19,27,29]. We found that endothelium-dependent vasodilatation and norepinephrine-induced vasoconstriction in perfused aortic rings were comparable in Rlx^+/+ and Rlx^–/– mice of both sexes (our unpublished data), suggesting that relaxin deficiency does not have a profound effect on vascular tone. Thus, although the cardiovascular system responds to exogenous relaxin, the long-term absence of endogenous relaxin in male and non-pregnant female mice does not result in significant alterations in hemodynamics.

The functional measurement revealed a greater late-phase flow velocity of LV diastolic filling due to atrial contraction and lengthening of DT in 8–10-month-old male Rlx^–/– mice, and a modestly elevated LVEDP in 12–24-month-old male Rlx^–/– mice. The atrial weight was increased in both age groups. Histological analysis of atrial tissues showed no evidence of interstitial fibrosis. Therefore, the weight increase in the atria is likely due to hypertrophy of atrial myocytes. These changes are best explained by an increased ventricular impedance at the late diastolic phase, resulting in atrial hypertrophy. An age-dependent increase in collagen content in the LV was observed in Rlx^–/– males from 9 to 24 months of age. The LV diastolic function was studied in the 8–10-month-old but not in the 12–24-month-old male Rlx^–/– mice. However, considering an age-dependent build-up of collagen in the LV, it is likely that the aged Rlx^–/– mice would exhibit a more severe diastolic dysfunction than that seen in their younger counterparts. Further experiments showed increased procollagen type-1 gene expression in the ventricles of 6-, 9- and 12-month-old male Rlx^–/– mice. However, there was no indication of LV hypertrophy in these mice as neither LV weight nor myocyte cross-sectional areas were significantly different from wildtype mice. Thus, long-term disruption of endogenous relaxin in male mice resulted in elevated LV interstitial collagen that may have led to a moderate increase in LV chamber stiffness and ventricular diastolic dysfunction.

The reason for the increased lung and liver weights in male Rlx^–/– mice is not clear. This might be secondary to organ congestion due to impeded ventricular filling. However, this possibility is unlikely considering a well preserved dP/dt_max and dP/dt_min and only a modest increase in LVEDP in the aged male Rlx^–/– mice. A recent study reported that the Rlx^–/– mice had an age-related increase in collagen content and histological abnormalities in the lung [30], providing an explanation for the increased lung weights in the Rlx^–/– males in the current study. It remains to be established whether a collagen build-up also occurs in the liver of these mice. Thus, the increased weights of lungs and liver are very likely the consequence of elevated organ collagen content due directly to relaxin deficiency.

There is now good evidence to show that relaxin acts directly on human fibroblasts of different organs [5,31,32] to decrease the synthesis and secretion of interstitial collagens. Relaxin also increases the expression of proMMP-1, proMMP-2 and proMMP-3 and decreases production of tissue inhibitors of metalloproteinases (TIMPs) in the human and porcine uterus [5,33]. These data suggest a mechanism by which relaxin regulates collagen concentrations in tissues. In our study, an increase in procollagen gene expression and collagen content in the ventricles of Rlx^–/– mice was not accompanied by a significant alteration in MMP expression. However, MMPs are secreted in a proMMP form and are activated by cleavage of the propeptide sequence. It has been demonstrated that relaxin treatment increases MMP activity without affecting MMP protein expression in the porcine uterus [33]. We did not examine MMP activity in the ventricles of Rlx^–/– mice, so it is possible that changes in MMP activity did occur without changes in MMP gene expression. A second explanation is that the level of MMP gene expression was very low in the hearts of Rlx^–/– mice so we were unable to detect reductions in mRNA levels. Additionally, the low levels of MMPs present may not have been sufficient to inhibit collagen deposition observed in the Rlx^–/– mice. This possibility is supported by our data showing progressive increases in LV collagen with aging despite the presence of proMMP mRNA.

Recent studies using experimental animal models of fibrosis in various organs have shown that administration of relaxin reduces the extent of fibrosis [30–35]. The cardiac phenotypes observed in male Rlx^–/– mice are in line with these studies and further support the view that relaxin also regulates collagen synthesis and breakdown in non-reproductive tissues [30–35]. Dschietzig et al. reported that myocardial relaxin expression and plasma relaxin concentrations increase profoundly in patients with moderate to severe congestive heart failure [19]. Although the implications of this finding remain unclear, relaxin may function as a compensatory mediator and a marker for heart failure. Relaxin dilates coronary, renal and pulmonary arteries by stimulating NO generation [29,36] and antagonising vasoconstrictive peptides [26], and protects the myocardium from ischemia/reperfusion injury [36]. With all documented actions of exogenous relaxin, the cardiac phenotypes reported in the present study, and reported changes in relaxin levels in the failing heart [19], it is feasible to speculate that relaxin plays an important compensatory role in cardiovascular diseases. Furthermore, relaxin or relaxin-like compounds might form a class of anti-fibrotic agents in pathological conditions, including heart disease.

Although the mouse has two relaxin genes (mRlx-1 and mRlx-3) [6,7], it is unlikely that there is an upregulation in mRlx-3 expression in the hearts of mRlx-1 knockout mice. mRlx-3 transcripts are detected in the hearts of Rlx^+/+ mice but only by RT-PCR [6], suggesting that mRlx-3 expression is extremely low in this organ. We have found that mRlx-3 expression is not upregulated in the lungs of 12-month-old Rlx^–/– mice [30]. Furthermore, the reproductive and cardiac phenotypes associated with increased collagen and tissue fibrosis in mice lacking mRlx-1 are still clearly evident [8–10], despite the probable presence of the mRlx-3. This implies that mRlx-3 does not compensate for the lack of mRlx-1 and does not influence the phenotypes associated with mRlx-1 deficiency in mice.

Unexpectedly, the cardiac phenotype was only observed in male but not female Rlx^–/– mice. It has been reported that matrix-remodeling activity of relaxin depends on or is potentiated by female hormones, particularly estrogen [1,2]. The lack of cardiac phenotypes in female Rlx^–/– mice suggests that other female hormones or factors compensate for the lack of relaxin in female Rlx^–/– mice. Interestingly, recent studies in other gene-targeted mouse strains have shown that gender influences the development of cardiac phenotypes [37–40]. In these mouse strains, males usually display more pronounced phenotypes than females. Thus, gender is an important factor in the development of cardiac specific phenotypes in mice.

In summary, our study provides evidence that deficiency of endogenous relaxin results in an age-dependent increase in myocardial collagen content, atrial hypertrophy and impeded ventricular diastolic filling but preserved myocardial function. These data indicate that under physiological conditions, relaxin is required for regulation of myocardial collagen content and hence maintenance of a normal ventricular diastolic function. These cardiac phenotypes in Rlx^–/– males were not observed in Rlx^–/– females, indicating the importance of other gender-related factors in cardiovascular function.

Time for primary review 29 days.

	Acknowledgements

 
The authors thank Chongxin Zhao for mouse breeding and genotyping, Helen Gehring, Dr Martyn Lewis and Elodie Percy for technical assistance, Dr Ross Bathgate for discussion and Dr Elaine Unemori for providing some of the real-time PCR primers and probes. This study was supported by Australian National Health and Medical Research Council Block Grants to the Baker Medical Research Institute and Howard Florey Institute. Dr Laura Parry holds an Australian Research Council QEII Fellowship.

	Notes

 
¹ Present address: Laboratory of Genetics and Physiology, NIDDK, NIH, Bethesda, MD 20892, USA.

	References

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

 

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