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Endocrinology Vol. 144, No. 6 2695-2703
Copyright © 2003 by The Endocrine Society

Shortened Life Span, Bradycardia, and Hypotension in Mice with Targeted Expression of an Igf2 Transgene in Smooth Muscle Cells

Silvio Zaina1, Linda Pettersson, Anne Bloch Thomsen, Chun-Ming Chai, Zhongquan Qi, Johan Thyberg and Jan Nilsson

Departments of Medicine (Experimental Cardiovascular Research) (S.Z., L.P., A.B.T., J.N.) and Experimental Research (C.-M.C., Z.Q.), University of Lund, Malmö General Hospital, 205 02 Malmö, Sweden; and Department of Cell and Molecular Biology (J.T.), Karolinska Institutet, 171 77 Stockholm, Sweden

Address all correspondence and requests for reprints to: Silvio Zaina, Department of Clinical Biochemistry, Sector 3014, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark. E-mail: zailund{at}yahoo.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF2 is known to affect the normal development and pathology of the cardiovascular system. We previously created mutant mice with targeted expression of an Igf2 transgene in the smooth muscle cells and showed that these mice spontaneously develop aortic intimal cushions. In the present work, we provide a general description of the phenotype of two independent lines of heterozygous transgenics. These mice showed organomegaly and a shortened life span. The latter trait was stronger in the line with a relatively more marked organomegaly and more pronounced in males than females in both lines. Postmortem histology revealed gross abnormalities of the cardiac architecture, suggesting that transgenic mice may accumulate lethal cardiovascular defects. Accordingly, apparently normal transgenic mice had mild cardiomegaly, an enlarged left ventricle, bradycardia, and hypotension. These observations are discussed in the light of the proposed therapeutic use of IGF2 in human cardiac diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGF2 PEPTIDE is a fetal promoter of cell growth, survival, and differentiation (1, 2). The Igf2 gene is under a tight epigenetic control dictating exclusive expression of the paternal allele during fetal and early postnatal life (imprinting) (1). In adulthood, IGF2 generally disappears or decreases to very low levels in nearly all tissues (1). IGF2 is an important factor in the determination of body size; mice with biallelic or paternal disruption of the Igf2 gene are 30–40% smaller than wild-type mice (3). In addition, IGF2 has been implicated in certain forms of tumors and overgrowth syndromes in humans (4, 5, 6, 7). These diseases are associated with a relaxation of the normal epigenetic control of the Igf2 gene (loss of imprinting) or with changes in the levels of receptors mediating signaling or turnover of IGF2 (4). IGF2 binds to at least four receptors at the cell surface. Three of them are signaling receptors mediating the biological effects of IGF2: the type 1 IGF receptor, the insulin receptor, and the product of the GPC3 gene (1, 8, 9). The type 1 IGF receptor mediates the effects of both IGF2 and the structurally and functionally related IGF1 peptide (1, 10). A fourth receptor, the type 2 IGF receptor, also known as IGF2/mannose 6-phosphate receptor and cation-independent mannose 6-phosphate receptor, mediates IGF2 turnover by binding and internalization of the peptide (11). In addition, IGFs (IGF1 and IGF2) bind to at least six types of soluble binding proteins (IGFBPs) with different biological functions (12).

Numerous observations suggest that IGF2 may play an important role in the physiology and pathophysiology of the circulatory system (reviewed in Ref. 13). IGFs promote proliferation, migration, and a delayed spontaneous dedifferentiation of cultured smooth muscle cells (SMC) (14). Furthermore, targeted expression of either IGF1 or an IGFBP type known to inhibit the bioactivity of IGFs (IGFBP4) in the SMC of transgenic mice results either in an increase or decrease in size and contractility of blood vessels, respectively (15, 16, 17).

Opposite effects of IGFs have been described in cardiovascular diseases. IGFs can delay infarction and generally improve postinfarction healing in animal models (18, 19, 20, 21). These effects are explained by the proliferative and antiapoptotic activity of IGFs on cardiomyocytes. Furthermore, a recent study established an association between increased risk of ischemic heart disease and low-circulating IGF1 (22). On the other hand, proatherogenic effects of IGFs have been documented, and high levels of these peptides may therefore be regarded as possible risk factors for myocardial infarction. IGF1 has been shown to accelerate the development of vascular lesions in animal models of atherosclerosis induced by transplantation or hyperlipidemia (23, 24). Previously, we have provided direct genetic evidence that IGF2 plays a pivotal role in the development of atherosclerotic lesions in vivo in a mouse model (25). Furthermore, we showed that expression of an Igf2 transgene controlled by the {alpha}-smooth muscle actin promoter (Smaigf2 transgene) in mice results in the spontaneous formation of aortic focal intimal thickenings (25).

The present work provides a general description of the phenotype of two independent lines (Isac and Igor) of Smaigf2 heterozygous mice and shows that sustained expression of IGF2 in SMC affects the life span and has distinct effects on the cardiovascular system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
All animal work conformed to the requirements of the local Ethical Animal Research Committee (Malmö/Lunds Djurförsöksetiska Nämnd, license no. M89-01).

Animal material and analysis of organ size
The generation of two independent lines (Igor and Isac) of Smaigf2 heterozygous mice (indicated as Smaigf2 mice) and part of their phenotype were previously described (25). Smaigf2 and wild-type littermate mice were at 5th–7th-generation breeding on to a C57BL/6 background. Bleeding and measurement of organ size, dry weight, and DNA content were performed exactly as described (26). For the morphometric analysis of the aorta, the thoracic segment of the perfused vessel was fixed and sectioned as described (25). The medial and luminal area and the medial thickness were measured in sections corresponding to the middle point of the thoracic aorta, by computer-assisted morphometry, as described (25). The thickness of the ventricular walls and the sectional area of the ventricles were measured in sagittal sections of the heart by using the same method. Values for individual mice represent the average of two to three sections. The density of cell nuclei and cell size was measured in 150 x 125-mm fields of specimens stained with hematoxylin and eosin, viewed with a x40 objective. Fields containing cells with similar morphology were chosen. Values for individual mice represent the average of two to three fields. All histology was performed by standard techniques. Monocytes and macrophages were detected by immunohistochemistry, with a specific antibody (MOMA-2; BMA Biomedicals, Augst, Switzerland), according to the manufacturer’s instructions. Electron microscopy was performed as described (25). Whole mount mammary glands were obtained as described (27). Females were typed for the phase of the oestral cycle by vaginal smear as described (28).

Analysis of transgene, Igf1, Igfbp4 RNA
The expression of the Smaigf2 transgene was studied by an ribonuclease protection assay as described (25). Relative levels of Smaigf2 RNA were determined by comparison with the intensity of the Gapd control band in digital images of autoradiography films as described (25). Tissue levels of Igf1 and Igfbp4 RNA were measured by a semiquantitative RT-PCR as described (29, 30). The following primers, amplifying a 123-bp fragment of the mouse 18S rRNA (nucleotides 1536–1658, accession no. X56974), were used as the internal control: 5'-tcgaacgtctgccctatcaac-3' (forward); 5'-ccttggatgtggtagccgttt-3' (reverse). All oligonucleotides used in this study have been synthesized by DNA Technology (Aarhus, Denmark).

Analysis of serum levels of IGFs and IGFBPs
The levels of the IGF2 peptide were measured by RIA as described (26). IGF1 was assayed with a commercial RIA system (Diagnostic System Laboratories, Webster, TX) (31). The latter assay produced values of serum IGF1 concentration in the 270 ng/ml range in wild-type mice. These are lower than the corresponding plasma values obtained by I. Ueki and co-workers (31) (1000 ng/ml range) but comparable to other published plasma IGF1 levels (for example, see Ref. 32). The levels of IGFBPs were measured by Western ligand blotting using IGF2 as labeled ligand as described (33). The specificity of ligand labeling was determined by coincubation with excess unlabeled IGF2. This method allows for the measurement of IGFBP2 and IGFBP4 polypeptide levels.

Measurement of cardiac activity and arterial blood pressure (BP)
Needle electrodes were placed sc, to obtain a standard lead II electrocardiogram (ECG) to monitor the heart rate and the presence of arrhythmia in anesthetized mice. The ECG signals were continuously collected by using the AcqKnowledge III acquisition software (Bipac Systems, Santa Barbara, CA) for Macintosh. Traces were recorded for 10 min, starting 5 min after the application of the electrodes. P–P (two consecutive P waves) intervals were measured manually in printed fields covering 30–35 heartbeats. Individual heart rates were calculated as the average of two to three field averages. Arterial pressure was measured in anesthetized mice by adapting a method designed for large-size laboratory animals. A catheter filled with 20 IE/ml heparin, saline was introduced in the carotid artery and connected to a BP transducer (model PMKIT 1DT-XX; Ohmeda, Murray Hill, NJ). The signal was continuously collected by using the AcqKnowledge III software. The arterial pressure stabilized within 5 min from the start of the acquisition, and mean values were calculated by averaging values collected over fields covering 40 sec of recording at 10 min.

Statistics
Pairs of mice (matched by sex, age, and litter) were compared by using the nonparametric Wilcoxon paired test with the Statview program (Abacus Concepts, Berkeley, CA) for Macintosh (25). The Mann-Witney U test was used for the comparison of heart rates, P–R (two adjacent P and R waves) intervals, and arterial pressure. The survival analysis was conducted with the Gehan’s generalized Wilcoxon test using the STATISTICA program (StatSoft, Tulsa, OK) for Macintosh.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transgene expression
The distribution and levels of transgene RNA were studied in the Isac line (Table 1Go). Smaigf2 transgene RNA was detectable in the embryo from at least embryonic day 12.5 on, and in the placenta and amniotic sac at embryonic day 16.5. In the adult, transgene RNA was present at variable levels in all organs examined but was distinctively abundant in organs rich in SMC (aorta, alimentary canal, uterus, vesicular and salivary gland; Table 1Go). No difference in transgene expression levels in selected organs (heart, intestine) was noticed between males and females (not shown). The levels of serum total (free and bound to circulating proteins) IGF2 peptide were measured in 8-wk-old transgenic mice of both transgenic lines (Isac and Igor) and matched wild-type mice. IGF2 was greatly elevated in the Isac line (107.1 ± 20.5 ng/ml) and less markedly so in the Igor line (36.7 ± 38.4 ng/ml) relative to wild-type (16.2 ± 1.4 ng/ml; P = 0.0006 and P = 0.041 in the comparison with Isac and Igor transgenes, respectively).


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  		Table 1. Levels of Smaigf2 RNA in the embryo and adult organs

 
Size of body and organs
Transgenic mice of both Igor and Isac lines displayed a characteristic abdominal enlargement that became readily visible during the first week after birth. This trait was more pronounced in the Isac line, and adult males of this line could be mistaken for pregnant females (Fig. 1AGo). The body weight at the age of 8 wk was significantly increased in the order: Isac>Igor>wild-type, as previously reported (25).



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  		Figure 1. Organomegaly in Smaigf2 mice of the Isac line. A, Abdominal view of a transgenic male and a matched wild-type mouse. Mice were anesthetized and photographed. T, Transgenic; NT, nontransgenic. B, View of the spleen (left) and vesicular gland (right); symbols as in A. C and D, Length of the small intestine in a transgenic (C) and a matched wild-type (D) mouse. Horizontal bar, 1 cm. E and F, Whole mount mammary gland of a transgenic (E) and matched wild-type (F) mouse stained with hematoxylin. Arrows indicate examples of the additional ramifications present in transgenic glands. Original magnification, x20. All pictures refer to 8- to 10-wk-old mice.

 
A quantitative analysis of organ size at 8 wk revealed a general organomegaly in transgenic mice of both Igor and Isac lines. Organs rich in SMC (alimentary canal, vesicular gland, aorta) tended to be particularly enlarged (Table 2Go; Fig. 1Go, B–D). In addition, the examination of selected organs (heart, small intestine) showed that small peripheral vessels were enlarged. The organomegaly was generally more severe in the Isac than in the Igor line (Table 2Go). The dry weight measured in selected organs of Isac transgenics was elevated to an extent similar to wet weight (P > 0.05 in all cases), suggesting that edema did not substantially contribute to organomegaly in Smaigf2 mice (Table 2Go). Total organ DNA content was also elevated proportionately to wet weight in the stomach and the vesicular gland in the Isac line (P > 0.05 for both organs). By contrast, the increase in DNA content was more than twice the increase in wet weight in the spleen (Table 2Go; P = 0.037). The Smaigf2 transgene therefore increased organ size by causing an increase in the number of cells. The study of the morphology of intestinal SMC by electron microscopy revealed that no phenotypic changes occurred in transgenics. SMC of both Igor and Isac lines were contractile and indistinguishable from cells of matched wild-type tissue (not shown).


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  		Table 2. Effect of Smaigf2 transgene on organ size, dry weight, and DNA content

 
The uterus was significantly enlarged in both lines at diestrus, but not in other phases of the oestral cycle (Table 2Go). The Smaigf2 transgene had marked effects on female fertility. Isac females became pregnant, but were generally unable to deliver their pups, and died 1–2 d after term (seven out of eight mated females; the female that completed delivery died 12 h later). Postmortem inspection revealed dead (but apparently well-developed) fetuses. By contrast, transgenic males were fertile. Both lines were therefore propagated by paternal transmission.

In addition, the inspection of whole mount mammary glands of the Isac line revealed a more arborized pattern of ducts in transgenic, compared with wild-type, females matched for the oestral cycle phase. In particular, additional short buds were visible on the ductal tree and occasionally on the terminal end buds (Fig. 1Go, E and F).

IGF1 and IGFBP4 are potent regulators of growth, with marked effects on the cardiovascular system; and deranged levels of these factors may therefore be associated with the organomegaly of Smaigf2 mice (15, 16, 17). The analysis of aortic RNA by a semiquantitative RT-PCR revealed that the levels of Igf1 and Igfbp4 transcripts were not grossly changed in Smaigf2 mice, compared with wild-type, suggesting that the contribution of these factors to the observed phenotype, if any, was not substantial (not shown). Likewise, the serum levels of the IGF1 peptide were not statistically different in wild-type and Smaigf2 mice (wild-type, 270.4 ± 48.6 ng/ml, n = 5; Igor line, 278.2 ± 29.0 ng/ml, n = 5; Isac line, 297.4 ± 28.3 ng/ml, n = 5). Furthermore, a preliminary analysis of the abundance of serum IGFBP2 and IGFBP4 polypeptides, by Western ligand blotting, revealed no gross difference between wild-type and Smaigf2 mice (not shown).

Reduced viability and cardiac abnormalities
Smaigf2 mice were born at the expected 50% Mendelian frequency (n = 118, representing 22 litters; not shown). Postnatally, male mice of both the Isac and Igor lines showed a significantly shortened life span, compared with sex-matched wild-type mice (Isac, P < 0.0001; Igor, P = 0.038; Fig. 2Go). This trait was more severe in the Isac line, whose males did not survive beyond the age of 5 months, whereas the mortality rate among Igor males was approximately 20% within the first year of life (Fig. 2Go). Among females, only Isac transgenics had a mortality rate significantly higher than sex-matched wild-type mice (P = 0.0376; Fig. 2Go). Deaths were not consistently preceded by any sign of illness. In some cases, trembling and lethargy were noticed for variable lengths of time, but deaths were apparently sudden in other cases.



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  		Figure 2. Reduced life span of Smaigf2 mice. Survival times vs. cumulative proportion surviving plot for Isac males ({square}, n = 19) and females ({circ}, n = 18), Igor males ({bullet}, n = 20) and females (, n = 22), wild-type males ({triangleup}, n = 20) and females ({diamond}, n = 19).

 
The postmortem inspection of organs of a total of nine informative mice consistently revealed cardiac abnormalities. Generally, the heart showed an adhering large blood clot and an increase in wet weight (Fig. 3Go, A–D). Sagittal sections showed that the general architecture of the heart was grossly altered in both Isac and Igor lines (Fig. 3Go, A–D). The ventricles were extremely enlarged, particularly at the apical region. In one extreme case, the septum was apparently absent and the heart was univentricular, with a very thin ventricular wall. The cardiac blood vessel did not show any sign of occlusion in any of the mice examined. No signs of gross cellular abnormalities, fibrosis, or inflammatory cell infiltration were detected in histological sections stained with hematoxylin and eosin, Masson trichrome, or the monocyte/macrophage-specific MOMA-2 antibody (Fig. 3Go, E–J). Likewise, no significant difference in cardiomyocyte size (Smaigf2 mice, 488.4 ± 39.0 mm2, n = 8; wild-type mice, 501.0 ± 29.4 mm2, n = 7; P > 0.05) or density of cell nuclei (Smaigf2 mice, 39.4 ± 2.6 nuclei/150 x 125-mm field, n = 8; wild-type mice, 38.9 ± 3.1, n = 7; P > 0.05) was observed.



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  		Figure 3. Postmortem histology of hearts from Smaigf2 mice. A, Sagittal section from a 23-d-old Smaigf2 male mouse of the Isac line; B, 23-d-old matched wild-type; C, as in A, but aged 5 months; D, 5-month-old matched wild-type. Bar, 1 mm. The wet weight is indicated in each case. E–J, Representative hematoxylin, eosin (E and F), Masson trichrome (G and H), and monocyte/macrophage (I and J) staining. The specimens shown are from sections adjacent to C (E, G, and I) and to D (F, H, and J). Vessels are shown in the lower part of G and H as an internal control for staining of connective/fibrous tissue. Bar, 50 µm. Original magnification, x200.

 
The cardiac abnormalities were not associated with any gross perturbation of the local levels of IGF1 or IGFBP4 in Smaigf2 mice, compared with wild-type controls, as shown by the analysis of heart RNA with a semiquantitative RT-PCR (not shown).

Heart morphometry, ECG, and arterial BP
The above observations suggested that Smaigf2 mice may develop cardiac abnormalities that eventually result in premature death. To understand the morphological and physiological changes underlying these abnormalities, we performed a quantitative morphometry of the ventricular wall and lumen and analyzed the cardiac activity by measuring ECGs in apparently normal transgenic and wild-type mice.

Morphometric studies, conducted at the age of 8 wk, showed an approximately 20% enlargement of the sectional area of the left ventricle in the Isac line, compared with wild-type (P = 0.028, n = 7), associated with a increased thickness of the left free ventricular wall (+32%, compared with wild-type; P = 0.043; n = 6). Likewise, the septum and the right free ventricular wall were thicker in the Isac line, but the difference with wild-type did not reach statistical significance (not shown).

ECG traces showed that transgenic mice had a significantly reduced heart rate (Table 3Go; Fig. 4Go, A–C). The heart rate, measured as the number of P–P intervals per minute, was 18–37% slower in mice of the Isac line than in wild-type (Table 3Go). A decrease in heart rate was detected at all ages analyzed (8 and 16 wk) in this line and tended to be more pronounced in older mice (Table 3Go). In the Igor line, a relative bradycardia was detected in 16-wk-old (but not in younger) mice (Table 3Go). By contrast, no significant difference was observed in the length of the P–R intervals in either line at the age of 16 wk (Table 3Go). In addition, 5 males out of 9 (mix of males and females) analyzed transgenic mice of the Isac line showed an irregular spacing between consecutive P waves. Typically, P–P intervals suddenly increased, and returned to regularity after 8–10 gradually accelerating heart beats (Fig. 4Go, B and C). In all cases, such patterns were observed once during 5- to 10-min recordings. These irregularities closely resembled an intermittent sinoatrial block (34). The heart rate was regular in all the wild-type mice examined (Fig. 4AGo). The observed abnormalities of the cardiac activity are not likely to be experimental artifacts caused by the use of anesthetics during the recording of ECGs, because the heart rate was not detectably affected by the use of either Avertin or a standard mixture of Hypnorm and Dormicum (not shown).


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  		Table 3. Effects of the Smaigf2 transgene on the cardiac activity

 


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  		Figure 4. Bradycardia and hypotension in Smaigf2 mice. A–C, ECG traces of 16-wk-old wild-type male (A) and matched transgenics of the Isac line (B and C). The positions of P–P and P–R, respectively, are shown in A. The length of the P–P intervals (sec) is shown below each trace. Horizontal bars, 0.5 sec. D, Mean arterial pressure in wild-type females ({blacksquare}) and matched Isac transgenics ({square}). The median and 95% confidence interval are shown for each distribution. The P value refers to the comparison between the two groups. E and F, Examples of representative recordings of the arterial pressure in a wild-type female (E) and a matched Isac transgenic (F). Horizontal bars, 5 sec.

 
The concomitant presence of vasculomegaly, bradycardia, and intermittent sinoatrial block suggests that Smaigf2 mice may fail to maintain normal levels of BP. Arterial BP was markedly reduced in apparently normal Isac females, relative to matched wild-type mice, indicating that Smaigf2 mice have a constitutive hypotension (Fig. 4Go, D–F). In addition, the analysis of ECG and BP in a moribund 6-month-old Isac male revealed a severe hypotension (mean arterial BP, 79 mm Hg; compare with values in Fig. 4DGo) associated with bradycardia (base line heart rate, 315.8 beats/min; compare with values in Table 3Go) and intermittent sinoatrial block (P–P intervals, up to 0.26 sec; compare with values in Fig. 4Go, A–C). Episodes of heart block were apparent every 5–10 sec during a 10-min recording.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We present evidence that two independent lines (Isac and Igor lines) of mice expressing an Igf2 transgene targeted to the SMC by the {alpha}-smooth muscle actin promoter have organomegaly, a reduced life span, abnormalities in cardiac structure and activity, and hypotension.

We observed an increase in size in nearly every organ examined and a widespread presence of transgene RNA, in accordance with the idea that blood vessels are ubiquitous sources of IGF2 in Smaigf2 mice. Noticeably, all the measured parameters related to growth (serum levels of the IGF2 peptide, body weight, organ size) were consistently higher in the Isac than the Igor line. Furthermore, the cell number was increased in three out of three organs examined (stomach, vesicular gland, spleen). Generally, the pattern of organ growth in Smaigf2 mice closely mirrored the phenotype of mice with an Igf1 transgene driven by the same {alpha}-SMC actin promoter, with differences in relative transgene expression levels likely to explain the more widespread organomegaly observed in the present study (15). The effects of transgene IGF2 were not associated with any major changes in the levels of Igf1 or Igfbp4 RNA in the aorta and the heart or of the corresponding circulating polypeptides, suggesting that neither of these factors contributed substantially to the observed phenotype. Furthermore, transgene IGF2 acted mainly by autocrine or paracrine mechanisms, as shown by the relatively more marked increase in growth of organs rich in SMC. Accordingly, we previously showed that the body and tibial length were normal in Smaigf2 mice (25). These observations are consistent with several reports showing mainly local effects of tissue-specific Igf2 transgenes despite greatly elevated levels of the circulating peptide (32, 35, 36, 37, 38). Vasculomegaly was extensive in Smaigf2 mice and included major, as well as small, peripheral vessels. Organs containing nonvascular SMC beds were particularly enlarged. One exception was the uterus, showing an increase in wet weight only in the diestrus phase. This observation suggests that, in other phases of the oestral cycle, the potent growth stimulation by ovarian hormones may overshadow the contribution of transgene IGF2 to organ size. In addition, transgene expression affected the functionality of the uterus, given that transgenic females could not deliver their otherwise apparently normally developed fetuses. The targeted expression of IGF1 in SMC in mice was shown to increase vascular contractility, and it is possible that elevated levels of IGF2 cause an aberrant response of uterine SMC to hormones regulating their contractile activity, thus interfering with delivery (17). As for the increased growth observed in transgenic mammary glands, functional studies assessing lactating and nursing abilities were hampered by the inability of Smaigf2 females to complete delivery.

A shortened life span was an unexpected trait of Smaigf2 transgenic mice, particularly the much higher mortality rate among males, compared with females. Persistent expression of IGF2 in the SMC during adulthood must trigger specific catastrophic events, because localized expression of Igf2 transgenes driven by tissue or cell type specific promoters does not generally cause early postnatal death, although, in some cases, produces tumors developing at a relatively old age (32, 36). On the other hand, mice with an adult ubiquitous transactivation of the endogenous Igf2 gene, after transgene integration or disruption of the H19 gene, show a high mortality within early postnatal life (6, 39). These animal models show cardiac abnormalities in some cases reminiscent of the defects accumulated by Smaigf2 mice (cardiomegaly, thickening of ventricular walls, dilated chambers), and heart failure has been suspected to be among the causes of death. Likewise, mice with elevated IGF2 levels, as a consequence of the disruption of the Igf2r gene, die prematurely, probably because of heart failure (40, 41, 42). The severely compromised cardiac developmental program observed in these animal models complicates their use in the interpretation of cardiovascular phenotypes in human overgrowth syndromes associated with elevated levels of IGF2. Cardiomegaly and several cardiac defects, including arrhythmia, have been described in patients with the Beckwith-Wiedemann syndrome, although the cardiovascular involvement seems to be limited in the disease (43, 44).

The above observations remand to the question whether the described cardiovascular abnormalities play a major role in determining the reduced life span of Smaigf2 mice. It is conceivable that the combination of vasculomegaly and bradycardia exacerbates the already constitutive hypotension during intermittent episodes of sinoatrial block by causing syncope and ischemia. The presence of blood clots adhering to the heart in postmortem specimens suggests that embolism could be an alternative or concurrent cause of death. Noticeably, syncope, embolism, and bradyarrhythmia can be associated in sudden heart failure in humans (45). Although the mechanisms by which an excess IGF2 may cause bradycardia and hypotension are not clear, it is interesting that mice with reduced levels of IGF1 show a tendency toward the opposite cardiocirculatory abnormalities, e.g. tachycardia and hypertension (46). Furthermore, hypotension was among the effects of the administration of recombinant IGF1 in at least two clinical works (47, 48). Tachycardia was also reported in the latter studies, confirming the intuitive idea that the effects of IGFs strongly depend on the concurrent pathophysiological conditions. The aberrant architecture and activity of the myocardium may be either a consequence of vasculomegaly and other abnormalities of the circulatory system or a direct effect of locally delivered IGF2 on the cardiac developmental program, or a combination of the two mechanisms. The levels of transgene RNA were relatively low in the postnatal heart in Smaigf2 mice, suggesting that the effects of locally produced peptide, if any, were limited. Nonetheless, the endogenous {alpha}-SMC actin promoter is active in the myocardium prenatally (discussed in Ref. 15); and if a wave of transgene expression occurs in the embryonic heart, it may produce long-term developmental effects. Several observations suggest that the levels of IGF2 are tightly regulated in the myocardium and that the cardiac developmental program may be more sensitive to the alteration of the levels of IGF2 than most other organs (discussed in Ref. 49). This implies that the local transgene levels may concur in causing the observed cardiac phenotype of Smaigf2 mice.

Our observations may be important, in light of the proposed attempts to improve cell survival and functionality after myocardial infarction, by raising local levels of IGFs (18, 50). The rationale for the therapeutic use of IGFs in cardiac diseases is based on the promising results obtained in animal models of myocardial infarction. These include the spectacular inhibition of myocardial cell death in mice with targeted expression of an Igf1 transgene in the heart and the cardioprotective activity of infused or locally delivered IGF2 (19, 20, 21). Our results complement the above observations by showing that sustained expression of IGF2 in selected cell types may have distinct effects on the cardiac development and activity and that the target for delivery of IGFs to the cardiovascular system should be chosen carefully.

The pathophysiological basis for the increased mortality rate among males is not clear. The present expression data do not support the idea that males constitutively express higher transgene levels than females. It is possible that sustained expression of IGF2 in the gonadal tissue results in secondary endocrine effects, possibly including sustained deranged levels of male hormones. The latter condition may contribute to the observed cardiac abnormalities, given that abnormal levels of androgens are associated with cardiovascular diseases in humans (51). Alternatively, estrogens may play a protective role, possibly acting directly on the expression level of signaling receptors for IGF2 (52). At any rate, no other endocrine alterations can be presently ruled out as alternative or complementary explanations. Work is in progress to further understand the physiopathological effects of the Smaigf2 transgene.


   Acknowledgments
 
We thank A. Bassim Hassan and Isabel Gonçalves for reading the manuscript.


   Footnotes
 
This work was supported by the Crafoordska stiftelsen, the Swedish Heart and Lung Foundation, Kungliga Fysiografiska Sällskapet i Lund, Lundströms Stiftelse, Albert Påhlsson Foundation, the King Gustaf V 80th Birthday Fund, the Swedish Medical Research Council (Grant No. 8311 to J.N., 6537 to J.T.), and Malmö University Hospital Research Fund.

1 Present address: Department of Clinical Biochemistry, Rigshospitalet, 2100 Copenhagen, Denmark. Back

Abbreviations: BP, Blood pressure; ECG, electrocardiogram; IGFBP, IGF-binding protein; P–P, two consecutive P waves; P–R, two adjacent P and R waves; SMC, smooth muscle cells.

Received September 6, 2002.

Accepted for publication February 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S. Rodriguez, T. R. Gaunt, S. D. O'Dell, X.-h. Chen, D. Gu, E. Hawe, G. J. Miller, S. E. Humphries, and I. N.M. Day
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