Nature Medicine
9, 68 - 75 (2002)
Published online: 23 December 2002; | doi:10.1038/nm805
Melusin, a muscle-specific integrin
 1−interacting protein, is required to prevent cardiac failure in response to chronic pressure overloadMara Brancaccio1, 5, Luigi Fratta2, 5, Antonella Notte2, Emilio Hirsch1, 3, Roberta Poulet2, Simona Guazzone1, Marika De Acetis1, Carmine Vecchione2, Gennaro Marino2, Fiorella Altruda1, 3, Lorenzo Silengo1, 3, Guido Tarone1, 3
& Giuseppe Lembo2, 41 Department of Genetics, Biology, and Biochemistry, Turin University, 10126 Turin, Italy 2 Department of Angiocardioneurology, IRCCS 'Neuromed', 86077 Pozzilli (IS), Italy 3 Experimental Medicine Research Center, San Giovanni Battista Hospital, 10126 Turin, Italy 4 Department of Experimental Medicine and Pathology, University of Rome 'La Sapienza' Rome, Italy 5 M.B. and L.F. contributed equally to this work.
Correspondence should be addressed to Guido Tarone guido.tarone@unito.it or Giuseppe Lembo lembo@neuromed.itCardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin  1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload—a condition that induces a hypertrophic response in wild-type controls—they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure—that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3 (GSK-3) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.Arterial hypertension imposes a biomechanical stress on the left ventricle, requiring the heart to work harder than under normal circumstances. Under these conditions, the heart undergoes hypertrophy resulting from increased synthesis and assembly of the contractile proteins of the actomyosin fibrils. Cardiac hypertrophy is considered beneficial because it allows the generation of greater contractile force, but it may also result in activation of pathways that reduce the efficiency of structural adaptation, promoting the transition toward cardiac dilation and failure1. Identifying the molecular mechanisms involved in cardiac hypertrophy and the transition toward dilation and dysfunction is thus an important challenge of cardiovascular biology and medicine2.
Cardiac hypertrophic remodeling is triggered by a combined action of mechanical stretching of cardiac walls and activation of neurohumoral growth factors1,
3,
4. Mechanical stress is considered the primary trigger to induce the growth response in the overloaded myocardium. Indeed, cardiomyocytes possess an intrinsic mechanosensing mechanism, as indicated by the ability of cultured cardiomyocytes to undergo hypertrophy when subjected to mechanical stretching in vitro5. Stretching is thought to activate the release of a cascade of autocrine and paracrine humoral factors, such as angiotensin II, endothelin 1, insulin like growth factor (IGF-1), transforming growth factor- (TGF-), fibroblast growth factor (FGF) and cardiotrophin-1 (CT-1) (refs. 3,6,
7,
8), that in turn promote cardiomyocyte growth and heart tissue remodeling. The intracellular signaling pathways triggered by mechanical stress and by humoral trophic factors are probably temporally and mechanistically distinct; however, whether these pathways can act independently is still under debate. In vitro and in vivo studies have identified several signaling molecules and pathways involved in cardiac hypertrophy, including the  Gq subunit of the heterotrimeric G protein coupled to the -adrenergic receptors9,
10,
11; the phospholipase C and protein kinase C, acting downstream of the G proteins12; the phosphoinositide 3-kinase; the calcineurin/NF-AT3 pathway; the Ras cascade, including Raf-1 and ERK1/2 MAP kinases; the stress kinases Jnk and p38; and the Jak-STAT pathway13,
14. Although most of these molecules are activated by both biomechanical and humoral factors, some are specifically involved in cardiac hypertrophy induced by humoral factors. In fact, inactivation of the genes encoding angiotensin II type 1 (AT-1) and the 1b adrenergic receptor (1b-AR) abrogates cardiac hypertrophy induced by sub-pressor doses of angiotensin II or phenylephrine in the absence of mechanical stress, but does not affect the hypertrophic response to mechanical overload imposed by aortic coarctation15,
16. This indicates the existence of a pathway triggered by angiotensin II or norepinephrine via AT-1 and an 1b-AR receptors, independently of mechanical stress.
In contrast, it is not known which molecules are required to couple mechanical stress to the induction of intracellular signals responsible for the hypertrophic response. Integrins and the associated signaling machinery are likely candidates, because of their role in linking the force-generating actin cytoskeleton inside the cell to the extracellular matrix17. Integrins are crucial to heart development and function, as indicated both by in vitro and in vivo studies. Both chimeric mice and embryoid bodies constructed from integrin 1−null cells show delayed development and differentiation of 1-deficient cells along the cardiac lineage, as well as abnormal sarcomerogenesis18. In addition, dominant-negative disruption of integrin 1 function19 as well as selective cardiac excision of the integrin 1 gene20 result in myocardial fibrosis and cardiac failure.
We recently identified melusin, a protein binding to the integrin 1 cytoplasmic domain and specifically expressed in striated muscle tissue, where it localizes at costameres in proximity of the Z line along with integrins and vinculin21. By inactivating the gene encoding melusin, we show here that the absence of melusin does not affect cardiac development or basal function, but leads to a reduced left ventricular hypertrophy and favors the transition toward dilated cardiomyopathy and contractile dysfunction in response to chronic pressure overload. Yet the absence of melusin does not influence the development of cardiac hypertrophy to humoral factors such as angiotensin II or phenylephrine in the absence of biomechanical stress. Thus, melusin is most likely a specific mechanosensor transducing signal that acts in preventing the transition to heart failure in conditions of biomechanical stress.
Generation of melusin-null mice
We generated a targeting construct to replace exons 1−4 of the melusin gene, with a cassette containing LacZ and neomycin-resistance genes (see Methods and Fig. 1a). In mice derived from two different recombinant clones, the mutant allele was inherited according to normal segregation ratios for an X-linked gene (Fig. 1b). Hemizygote males and homozygote mutant females developed normally, were fertile and appeared healthy up to 18 month of age. The body weights of wild-type and null mice were comparable (Table 1). Western blot analysis with polyclonal antibodies against melusin (Fig. 1c) showed that the protein was absent from skeletal and cardiac muscles of mutant mice, whereas integrin 7B1D was normally expressed (Fig. 1c) and localized to the plasma membrane (Fig. 1d).
 | | Figure 1. Targeted disruption of the mouse melusin gene. | | |  | a, Restriction map of the wild-type melusin (Itgb1bp2) locus, the targeting construct and the targeted locus. A PstI fragment containing exons 1−4 was replaced with a cassette containing IRES sequences linked to the lacZ gene followed by the neomycin-resistance gene driven by a PGK promoter. b, Southern blot analysis: when analyzed using the probe shown in a, the targeted locus contains a 9.5 kb EcoRV fragment, whereas the intact allele generates a 15 kb band. Because the melusin gene maps to the X chromosome21, one homologous recombination event was sufficient to target the single allele in male ES R1 cells. c, Western blot analysis: protein extracts from heart and skeletal muscle of wild-type (WT) and melusin-null (null) male mice were separated by SDS-PAGE and probed with an affinity-purified polyclonal antibody against melusin (top row), 1D and integrin 7B subunits (middle and bottom rows). d, Immunohistochemical analysis of 1D integrin expression. Frozen sections of left ventricle from wild-type (left) and melusin-null (right) hearts were stained with affinity-purified polyclonal antibodies to 1D integrin followed by peroxidase-labeled secondary antibody. Scale bars, 40  m.
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Absence of melusin does not affect basal cardiac function
Both male and female melusin-null mice had normal lifespans and no evidence of morphogenic defects in cardiac or skeletal muscle. Histological analysis of mutant cardiac and skeletal muscles derived from mice up to 1 year old showed no evidence of necrosis, fibrosis or myofibrillar disarray (data not shown).
We examined basal cardiac dimensions and function in male mice 10−16 weeks old and found them to be unaffected by the absence of melusin. In particular, left ventricular diameters, wall thickness and contractile function, as evaluated by echocardiography, were similar in melusin-null mice and their wild-type littermates (Table 1). Left ventricular contractility and diastolic function, assessed by left ventricle dP/dt, were similar in melusin-null and wild-type mice (left ventricular dP/dt max, 9,610  750 versus 9,700 600 mmHg/sec, n.s.; left ventricular dP/dt min, -9,930 700 versus -10,530 850 mmHg/sec, n.s.) and increased to a similar extent in response to an acute pressure overload evoked by transversal aortic coarctation (left ventricular dP/dt max, 15,860 890 versus 15,535 920 mmHg/sec, n.s.), indicating that the absence of melusin does not affect basal inotropic function and its reserve. During left ventricular hemodynamic evaluations, the basal heart rate (HR) was similar in melusin-null mice and wild-type controls (HR, 475 8 versus 491 10 b.p.m., n.s.) and during acute transversal aortic coarctation (TAC) (HR, 513 10 versus 521 9 b.p.m., n.s.). Finally, melusin-null mice did not show hypertrophy or heart failure up to 18 months of age. Thus, under physiological conditions melusin is not required for the development and organization of heart tissue or for basal cardiac function.
Absence of melusin leads to heart dilation after TAC
Genetic mutations have been reported that do not affect basal cardiac function but cause heart failure under conditions of high blood pressure22,
23. Therefore, we assessed the effects of exposing melusin-null mice to chronic pressure overload induced by transversal aortic coarctation (TAC). These and subsequent studies described here were carried out using 10−16-week-old male SV129 mice. Seven days after TAC, wild-type mice developed left ventricular hypertrophy as identified by left ventricular weight/body weight ratio (LVW/BW) (Fig. 2a and b). In contrast, the left ventricular hypertrophic response to TAC was markedly impaired in melusin-null mice (Fig. 2a and b), though they were exposed to a similar degree of biomechanical stress, as estimated from the systolic pressure gradient across the surgical aortic stenosis (mean values, 50 4 mmHg in melusin-null versus 46 4 mmHg in wild-type, n.s.; Fig. 2a).
| | Figure 2. Left ventricle growth response 1 wk after TAC. | | |  | Both wild-type (WT, empty circles and bars) and mutant mice (null, filled circles and bars) were subjected to surgical TAC. a, At 7 d after TAC, the LVW/BW ratio was determined and plotted against the systolic pressure gradient as a difference between right and left carotid systolic blood pressure. b, Mean value of LVW/BW. c, Left ventricles were processed for histology to measure the cardiomyocyte cross-sectional areas. Scale bars, 40 m. d, Mean values from 200 determinations per mouse. e and f, Left ventricle expression of atrial natriuretic factor (e) and -myosin heavy chain (f) was determined by northern blot analysis of left ventricles from wild-type and melusin-null mice 7 d after TAC. Quantitative values were obtained by densitometric scanning of the bands of 8 different mice (4 WT and 4 null) after normalization with 28S RNA. Means s.e.m. Statistical significance was measured by the Student's t test; §, P < 0.001 versus sham; *, P < 0.001 versus wild type.
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Histological analysis showed that increase of the cardiomyocyte cross-sectional area was substantially lower in melusin-null mice subjected to TAC (Fig. 2c and d), whereas no differences were detectable between both genotypes in fibrosis (data not shown) and apoptosis (0.8 0.2 and 0.75% 0.3 apoptotic cardiomyocytes in wild-type and melusin-null mice, respectively). In addition, induction of ANF and -MHC expression, markers of left ventricular hypertrophy, was significantly lower in melusin-null mice (Fig. 2e and f).
To evaluate the geometric remodeling that occurred in response to chronic pressure overload, we subjected mice to TAC for 4 weeks, during which we examined them by serial echocardiographic analysis (Table 1). As expected, wild-type mice showed increased interventricular septum and posterior wall thickness and reduced end-systolic and end-diastolic left ventricular diameters, with a consequent increase in left ventricular relative wall thickness (Table 1 and Fig. 3). Such remodeling represents a typical pattern of concentric hypertrophy, an adaptive compensatory mechanism that occurs in response to sustained hypertensive conditions. In contrast, 7 days after TAC, melusin-null mice developed only modest thickening of ventricular walls and a substantial chamber enlargement without change in relative wall thickness. This represents a typical pattern of eccentric hypertrophic remodeling (Table 1), an unfavorable geometric adaptation to sustained hypertensive conditions. At 2 weeks after TAC, melusin-null mice showed a further enlargement of left ventricular chamber as compared to that in wild-type mice (Table 1 and Fig. 3). After 4 weeks, left ventricular dilation was even more evident and was associated with a marked deterioration of contractile function, as detected by fractional shortening (Table 1 and Fig. 3). Finally, lethality rates at 4 weeks after TAC were greater in mutant than in wild-type mice (53.3% versus 30.7%). Thus, whereas wild-type mice developed a typical concentric compensatory hypertrophy in response to prolonged pressure overload, in the absence of melusin the pressure overload resulted in eccentric cardiac remodeling, which accelerates the transition toward heart failure.
| | Figure 3. Left ventricle remodeling and function 2 and 4 wk after TAC. | | |  | Both wild-type (WT, empty bars) and mutant mice (Null, filled bars) were subjected to TAC. Cardiac structure and function were evaluated non-invasively by transthoracic echocardiography in basal condition and at 2 and 4 wk after TAC. All measurements were determined in a short-axis view at the level of papillary muscles. a, Representative M-mode left ventricular echocardiographic recording of wild-type (top) and melusin-null mice (bottom) in the basal state (left), or 2 wk (middle) or 4 wk (right) after TAC. b, Left ventricular end-diastolic diameter (LVEDD). c, Interventricular septum thickness in end-diastole (IVSTD). d, Percent fractional shortening (%FS) as parameter of left ventricle contractile function. e, Representative gross morphology of whole hearts (upper rows) and transversal sections at base level of the left ventricles of wild-type (left) and melusin-null (right) mice after 4 wk after TAC. §, P < 0.01 versus basal; *, P < 0.01 versus wild type; #, P < 0.05 versus wild type.
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Normal hypertrophy in the absence of mechanical stress
Angiotensin II and norepinephrine induce heart hypertrophy by exerting a direct trophic action on cardiomyocytes via G protein−coupled seven-transmembrane-domain receptors24. To explore the role of melusin in cardiac hypertrophy caused by these trophic factors in the absence of biomechanical stress, mice were exposed chronically (for 21 days) to doses of angiotensin II or phenylephrine which did not affect arterial blood pressure. Systolic and diastolic blood pressure (Fig. 4a and c) and heart rate (data not shown), evaluated by radiotelemetry during the infusion period, were not affected by either treatment. After 21 days of infusion, left ventricular mass, as measured by LVW/BW, increased at similar extent in wild-type and melusin-null mice in response to both angiotensin II and phenylephrine (Fig. 4b and d). These data indicate that melusin is not required for cardiac hypertrophy induced by humoral stimulation that does not involve a biomechanical stress.
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Absence of melusin impairs GSK3- signaling after TAC
To investigate the effect of melusin on cardiac intracellular signaling, we analyzed the phosphorylation of p38, ERK 1/2 and GSK3-, proteins reported to be involved in cardiac hypertrophy14,
25,
26,
27. In wild-type mice, all three molecules were strongly phosphorylated 10 minutes after TAC (Fig. 5a−c) and were appreciably phosphorylated even at 5 minutes after TAC (data not shown). This is consistent with the hypothesis that these molecules respond to a mechanical event. In melusin-null mice, however, the degree of GSK3- serine 9 phosphorylation was much lower (Fig. 5c). Notably, no significant differences were observed between wild-type and melusin-null mice in p38 and ERK1/2 phosphorylation in response to TAC (Fig. 5a and b). As a comparison, we analyzed the phosphorylation state of the kinase AKT, which regulates phosphorylation of serine 9 of GSK3-. AKT is rapidly phosphorylated in response to TAC in wild-type mice, but this response is much weaker in melusin-null mice (Fig. 5d).
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Because GSK3- is well known to be a target of insulin receptor signaling28, we also tested whether the absence of melusin affects GSK3- phosphorylation in response to insulin. Western blot analysis of heart extracts from mice treated with insulin for 10 and 20 min showed that GSK3- was phosphorylated to a comparable extent in both mouse genotypes (Fig. 5e).
Attenuation of cardiac hypertrophy in melusin-null mice was seen 7 days after TAC; we therefore tested GSK3- signaling at this time point. Phosphorylation of GSK3- serine 9 occurred to a lesser extent, however, in melusin-null mice than in wild-type mice 7 days after TAC (Fig. 5f). In addition, kinase activity was greater in melusin-null mice, as would be predicted given the inhibitory effect of serine 9 phosphorylation (Fig. 5g). Thus altered GSK3- signaling persists in melusin-null mice exposed to the effects of TAC for 7 days. Together, these data indicate that the absence of melusin selectively impairs left ventricular GSK3- phosphorylation in response to biomechanical stress.
Discussion
We previously identified melusin as a protein that binds to the integrin 1 cytoplasmic domain and specifically expressed in striated muscle21. The phenotype of melusin-null mice reported here demonstrates that melusin, though dispensable for cardiac muscle development and for cardiac function under physiological conditions, is required to sustain compensatory left ventricle hypertrophy in response to pressure overload. Notably, the absence of melusin does not influence the cardiac hypertrophy evoked by trophic stimuli such as angiotensin II or phenyl-ephrine at sub-pressor doses that do not impose a mechanical overload. As melusin interacts with integrin 1 cytoplasmic domain and localizes at costameres21, where contractile filaments are anchored laterally to the sarcolemma, we suggest that melusin senses high threshold levels of mechanical stress and consequently activates signaling pathways required to support cardiomyocyte hypertrophy.
Our analysis of the signaling events occurring in response to mechanical stress indicated that phosphorylation of p38 and ERK1/2 MAP kinases, as well as GSK3-, is strongly stimulated within 5 minutes of induction of the stress in wild-type mice. These molecules are phosphorylated in response to integrin stimulation by matrix ligands in vitro17,
29,
30, supporting the hypothesis that integrin stimulation could be responsible for their phosphorylation in heart after mechanical stress. The absence of melusin expression specifically reduced phosphorylation of GSK3- serine 9, but did not substantially affect phosphorylation of p38 and ERK 1/2. In addition, phosphorylation of AKT was greatly impaired in aortic-banded heart of melusin-null mice. As AKT is the principal kinase that phosphorylates the inhibitory site of GSK3-, this result points to an important role of melusin in regulating this pathway. GSK3- is also phosphorylated in response to hormonal stimuli not involving mechanical stress, such as insulin28; this pathway, however, was unaffected in the melusin-null background. This observation, together with the very rapid kinetics of GSK3- phosphorylation in response to aortic coarctaction, supports the hypothesis that melusin selectively senses the biomechanical stimuli. In contrast to other kinases, GSK3- is highly active in unstimulated cells and becomes inactivated by phosphorylation of serine 9 in response to several stimuli28. Recent studies have shown that this inactivation is required for cardiac hypertrophy. In cardiomyocytes, active GSK3- phosphorylates the transcription factors NF-AT and GATA4, inducing their translocation from the nucleus to the cytoplasm and thus inhibiting their transcriptional activity25,
26,
27. In addition, active GSK3- also induces inhibition of elF2B, a principal initiation factor regulating protein synthesis. Thus, the phosphorylation of serine 9 seen during aortic coarctation may allow the activation of both protein synthesis and transcriptional events required for the hypertrophic response. Indeed, expression of a constitutively active form of GSK3- in transgenic mice causes an impaired cardiac hypertrophy in response to pressure overload27. It is thus likely that the reduced GSK3- phosphorylation in response to pressure overload that we observed in melusin-null hearts can account, at least partially, for the defective hypertrophic response in these mice.
The impaired left ventricle remodeling of melusin-null mice in response to mechanical overload shows the typical pattern of eccentric left ventricular hypertrophy, characterized by an enlargement of the left ventricular chamber and an impaired wall thickening. This is an unfavorable geometric adaptation to hypertensive conditions that markedly accelerates the evolution toward heart failure31. Indeed, in contrast to the compensatory hypertrophy developed by control animals, melusin-null mice undergo a progressive left ventricle dilation followed by the onset of cardiac failure over a period of 4 weeks after TAC, and this is associated with an increased mortality rate. We conclude that the absence of melusin causes a deleterious left ventricular remodeling in conditions of sustained biomechanical stress, such as eccentric hypertrophy, thus accelerating the transition toward heart failure. In contrast, other studies32 have reported that a reduced left ventricular hypertrophy is protective from the onset of cardiac dilation and dysfunction in two strains of genetically modified mice defective either for Gq signaling (Gq-transgenic) or for norepinephrine synthesis (Dbh null). A possible explanation for this conflicting evidence could be the difference in genetic background between the mice used in the two laboratories: we used inbred SV129, whereas the other researchers probably used different strains. Another possible explanation is that in both Gq-transgenic and Dbh-null mice, the genetic mutations affect G protein−coupled receptor (GPCR) signaling that is unaltered in melusin-null mice, as judged from the normal hypertrophy response to angiotensin II or phenyl-ephrine. It is therefore likely that the differences in cardiac remodeling in response to long-standing pressure overload between melusin-null and Gq-transgenic or Dbh-null mice are attributable to the perturbation of two independent pathways that have opposing effects on heart remodeling. Whereas the GPCR pathway, when active, favors left ventricle dilation, the integrin-linked melusin pathway supports concentric compensatory hypertrophy.
Several genetically modified mouse models of dilated cardiomyopathy have been described, and have enabled different structural and signaling proteins involved in this process to be identified1,
33,
34,
35. In most cases, however, altered expression of these molecules causes basal alteration in cardiac structure and function, with spontaneous development of dilated cardiomyopathy. Fewer examples have been reported in which abrogation of protein function by gene inactivation does not affect basal cardiac physiology, but causes dilated cardiomyopathy and failure in response to mechanical overload. The proteins involved in these cases include the gp130 cytokine receptor22 and CD95/Fas receptor23. Mice in which gp130 is specifically inactivated in the heart have normal cardiac structure and function, but develop rapid-onset dilated cardiomyopathy, resulting from massive myocyte apoptosis, within 7 days after aortic pressure overload. The mechanism underlying cardiac dilation in these mice is distinct from that seen in melusin-null mice, as the latter do not develop cardiomyocyte apoptosis after pressure overload. The CD95/Fas receptor is a well-studied molecule that controls apoptosis in several cell types. In cardiomyocytes, however, Fas induces the pro-hypertrophic transcription factor AP-1 rather than inducing apoptosis36. Mice without a functional Fas receptor develop rapid-onset left ventricular dilation and failure in response to aortic coarctation, but no apoptotic mechanism is involved23. The possibility that the absence of melusin expression prevents normal of gp130 and/or CD95/Fas expression was excluded by western blot analysis of mutant and wild-type hearts (M.B., unpublished observations). Notably, the absence of compensatory hypertrophy in CD95/Fas mutant mice is accompanied by impaired GSK3- phosphorylation, indicating that the pathway controlled by melusin and Fas receptor converges on GSK3-. Because integrins can cooperate with several growth-factor and cytokine receptors37, it is tempting to speculate that melusin may be involved in integrin−Fas receptor cross-talk.
Finally, whereas mice with mutant gp130 or Fas receptors show cardiac dilation and dysfunction after 1 week of biomechanical stress22,
23, melusin-null mice showed cardiac dilation and then dysfunction only 4 weeks after aortic banding. The time-course in melusin-null mice is thus closer to that of the slowly developing process of dilated cardiomyopathy. Therefore, melusin-null mice may be a powerful model to clarify the molecular mechanisms linking the cytoskeleton to intracellular signaling pathways and their role in cardiac remodeling as a response to biomechanical stress.
Methods
Generation of melusin-null mice.
Using the murine cDNA probe21, we isolated a genomic fragment of 14.8 kb encompassing the first exon, which contains the ATG start codon, and three other exons. A PstI fragment containing exons 1−4 was replaced with a cassette containing IRES sequences linked to the LacZ gene followed by the neomycin-resistance gene driven by a PGK promoter. We identified by Southern blot analysis three different ES R1 cell clones in which homologous recombination had occurred. Two generated chimeras that produced germline transmission. All studies presented in this work were performed using 10−16-wk-old male SV129 mice and were confirmed in female mice. The use of animals was in compliance with the guidelines of the European Community and was approved by the Animal Care and Use Committee of the University of Torino.
Left ventricle hemodynamics.
Mice were anesthetized, as previously described16, with an intraperitoneal injection of tribromoethanol (Avertin, 350 mg/kg), and inotropic and lusitropic function was evaluated by measuring the maximum rate of left ventricular pressure developed (dP/dt max) and left ventricular pressure decay (dP/dt min) with a micromanometer catheter (Millar 1.4 F, SPR 671, Millar Instruments, Houston, Texas) positioned in the left ventricle via right common carotid artery cannulation. To evaluate inotropic cardiac reserve, left ventricular dP/dt max was measured after an acute increase (60 mmHg) of cardiac afterload induced by temporary aortic coarctation, as described below.
In vivo biomechanical stress.
Mechanical stress was imposed on the left ventricle through transverse aortic coarctation (TAC) between the truncus anonimus and left carotid artery, as previously described38. A separate group of mice underwent the same surgical procedures but without aortic stenosis (sham procedure). To evaluate the degree of biomechanical stress imposed on the left ventricle, the systolic pressure gradient (SPG) was measured by selective cannulation of left and right carotid arteries38. After hemodynamic evaluation, the mice were weighed, then the hearts were excised and the ratio of left ventricular weight to body weight (LVW/BW) was calculated.
Transthoracic echocardiography.
Echocardiographic analysis was performed using a commercially available echocardiograph (System Five Performance, General Electric Vingmed, Waukesha, Wisconsin) equipped with a 10 MHz imaging transducer. After good quality two-dimensional short-axis images of left ventricle were obtained, M-mode freeze frames were printed on common echocardiographic paper. End-diastolic and end-systolic interventricular septum (IVSTd, IVSTs), posterior wall thickness (PWTd, PWTs) and left ventricular internal diameters (LVEDD, LVESD) were measured using a computed NIH Image analysis system (National Institutes of Health, Bethesda, Maryland). Percent fractional shortening (%FS) and relative wall thickness (RWT) were calculated using standard formulas:
Reproducibility, calculated as the difference between two determinations divided by the mean of the two determinations and expressed as percentage of error, was 3.7 0.6 for left ventricular diameters and 7.2 0.9 for wall thickness.
Chronic infusions of angiotensin II or phenylephrine at sub-pressor doses.
Conscious blood pressure and heart rate were measured under unrestrained conditions by a radiotelemetric device (TA11PA-C20, Data Sciences International, St. Paul, Minnesota) whose sensing catheter was inserted into the femoral artery, as previously described16. After implantation, mice were housed in a single cage and allowed 10 d of recovery from surgical procedures. Blood pressure and heart rate were continuously recorded for 4 h daily (from 8:00 AM to 12:00 AM), in basal conditions (4 d) and during 21 d of active treatment with sub-pressor doses of angiotensin II (0.15 mg/kg/d) or phenylephrine (100 g/kg/d), infused subcutaneously with osmotic minipumps. Telemetered pressure signals were stored and analyzed by a dedicated computed data acquisition system (Dataquest Acquisition and Analysis System, DQ ART 1.1 Gold, Data Sciences International) that calculated the mean value during the acquisition time.
Antibodies.
Polyclonal antibodies against melusin 7B and against 1D were obtained as described21,
39,
40. Rabbit polyclonal antibodies against phospho-Ser9-GSK3-, phospho-Ser473-AKT, AKT and phospho-ERK1/2 were from New England BioLabs (Beverly, Massachusetts); those against GSK3-, ERK1 (C16) and FAS (M-20) were from Santa Cruz Biotechnology (Santa Cruz, California); those against p38 and phospho-p38 were from Calbiochem (Damstadt, Germany); and those against gp130 were from Upstate Biotechnology (Lake Placid, New York). Mouse monoclonal antibodies against GSK-3 were from BD Biosciences (Heidelberg, Germany).
Histological analysis and immunohistochemistry.
Paraffin-embedded hearts were cut into 5 m slices, which were stained with H&E for morphological analysis or with Picrosirius red (Fluka, Buchs, Switzerland) for detection of fibrosis. To measure myocyte area, suitable cross-sections were defined as those having nearly circular capillary profiles and nuclei. Detection of apoptotic cells and immunohistochemistry were performed as previously described40,
41.
Analysis of signaling pathways and hypertrophic markers.
Mice were anesthetized with sodium pentobarbital (30 mg/kg injected intraperitoneally) and subjected to TAC as described above. After anesthesia was induced, the animals were maintained under controlled temperature and ventilation. A customized adjustable clamp was placed around the transverse aorta, after which the thoracic cavity was closed. After stabilization (20 min), pressure overload was initiated with adjustments of the aortic clamping while blood pressure signals from above and below the constriction were monitored. The experimental protocol included sustained increase (60 mmHg for 10 min) of systolic pressure gradient across the aortic coarctation. The ventricles were then rapidly removed, frozen and triturated in liquid nitrogen, and then extracted by sonication and boiling in a solution containing 60 mM Tris-Cl, pH 6.8, 1% SDS, 1 mM Na3VO4, 10 mM NaF, 10 g/ml leupeptin, 0.4 g/ml pepstatin and 0.1 TIU/ml aprotinin. Proteins were than analyzed by SDS-PAGE and western blotting using standard procedures.
To evaluate kinase activity, total GSK-3 was immunoprecipitated from ventricular protein extracts (in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-Cl, pH 8, 1 mM Na3VO4, 10 mM NaF, 10 g/ml leupeptin, 0.4 mg/ml pepstatin, 0.1 TIU/ml aprotinin) using the monoclonal antibody mentioned above. The immunocomplexes were then incubated in kinase assay buffer42 with 1 g protein phosphatase inhibitor-2 (Sigma), as substrate, 100 nM nonradioactive ATP (Sigma) and 5 Ci [ -32P]ATP (6,000 Ci/mM) for 10 min at 30 °C. Reactions were analyzed by 15% SDS-PAGE and autoradiography.
Left ventricular ANF and -MHC expressions were evaluated in sham-operated and 7-d post-TAC mice, as previously described38. The optical density (OD) of ANF and -MHC bands were normalized with 28S RNA.
Statistical analysis.
Data, expressed as mean s.e.m., were analyzed with two-way ANOVA and Bonferroni's correction for multiple comparisons (SYSTAT).
Received 19 September 2002; Accepted 22 November 2002; Published online: 23 December 2002.
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