(Received for publication, February 27, 1995; and in revised form, July 25, 1995)
From the
p21 has been implicated in the
hypertrophic response of cultured cardiac myocytes to defined growth
stimuli. To determine if activation of ras-dependent
intracellular signaling pathways is sufficient to induce in vivo hypertrophy, transgenic mice were created that express oncogenic ras in the cardiac ventricular chamber. Mice homozygous for
the transgene displayed morphological, physiological, and genetic
markers of marked cardiac muscle hypertrophy. Miniaturized
catheterization technology documented a selective prolongation of
cardiac relaxation, similar to that seen in early human hypertrophic
heart disease. An increase in left atrial mass, in the absence of
transgene expression in that chamber, further supported physiologically
abnormal left ventricular diastolic function. Histological analysis
revealed myofibrillar disarray, indistinguishable from that in
hypertrophic cardiomyopathy in man. These studies establish a ras-dependent pathway for hypertrophic heart disease and
document the feasibility of mapping in vivo signaling pathways
for cardiac hypertrophy and dysfunction by applying in vivo microphysiological assays to genetically manipulated mice. ras-dependent pathways may also be a rational target for
developing new approaches to inhibit the genesis of hypertrophy in
certain pathological settings.
Cardiac muscle hypertrophy is one of the most important compensatory responses of the heart(1, 2) . Long standing hypertension, myocardial injury, or increased cardiac work elicit a hypertrophic response, which permits the myocardium to adapt appropriately by increasing cardiac muscle mass. This increase in mass is due to an enlargement of individual muscle cells and is not accompanied by a proliferative response. Although this process is initially compensatory, there can be a pathological transition in which the myocardium becomes dysfunctional. In this regard, the onset of hypertrophy is a well accepted prognostic indicator for subsequent cardiac dysfunction and morbid events(3) . Although the signaling pathways that mediate the development of hypertrophy and the transition to overt heart failure have been the subject of numerous descriptive studies, proof of their involvement has been lacking due to the absence of in vivo animal model systems that, by altering intracellular signal transduction in the myocyte itself, faithfully reproduce key features of human cardiac hypertrophy.
To approach this problem, we have recently characterized mouse model systems that allow use of genetically based approaches to study this physiological response in the in vivo context. Recent studies have established that the mouse is a valid model system to study cardiac muscle hypertrophy and dysfunction in vivo(4, 5, 6, 7) , which conserves many features of larger mammalian model systems. By fusing constitutively active mutants of candidate signaling proteins to cardiac muscle promoters that can target expression to specific cardiac chambers in transgenic mice, the generation of genetically based mouse models of cardiac muscle hypertrophy and failure appears to be entirely feasible(8, 9) , as has been shown for cardiac myocyte proliferation(8, 10) . The availability of miniaturized technology, microsurgical approaches, and techniques to quantitatively assess in vivo murine cardiac physiology, offers the further possibility of discriminating among various forms of hypertrophy and categorizing their effects on function(9) . In addition to the in vivo physiological techniques used here, other investigators have demonstrated the feasibility of applying other physiological assays to the mouse heart, including the isolated heart preparation (11) and the analysis of isolated myocytes(12) .
Utilizing a well characterized cultured
cardiac myocyte model system (13, 14, 15, 16, 17) ,
three independent signaling pathways (ras(18) ,
G(13) , and gp130(19) ), (
)have
recently been implicated in the activation of key features of the
hypertrophic response in vitro(19) . The question
remains as to whether the activation of any of these signaling
molecules is sufficient to generate a hypertrophic response in
vivo, and whether it would induce concomitant cardiac dysfunction
and/or specific subsets of cardiac hypertrophy and cardiomyopathy that
have been well defined in clinical human disease. To examine whether ras-dependent pathways are sufficient to activate a
hypertrophic response in the in vivo context, in the current
study the transgenic approach was used to target the expression of
oncogenic ras into the mouse cardiac ventricular chamber under
the control of the MLC-2v promoter.
Transient transfection studies in neonatal rat ventricular myocytes
were performed by calcium phosphate precipitation as
described(22) . 8 µg of plasmid were transfected with 2
µg of ANF luciferase reporter plasmid and 1 µg of
cytomegalovirus promoter--galactosidase plasmid (to correct for
differences in transfection efficiency). Cells were harvested 72 h
after transfection and analyzed for luciferase activity. Fold induction
is expressed as the ratio of the luciferase value (corrected for
-galactosidase) for the active plasmid construct to that for the
backbone vector pMLCSVOA. Each experiment was performed in triplicate,
and the results of three experiments were pooled.
A 250-bp fragment of the MLC-2v promoter has been shown to
confer cardiac ventricular chamber-specific expression to a luciferase
reporter gene in transgenic mice(26, 27) . Oncogenic
(Val mutant) human Ha-ras cDNA was subcloned into
a eukaryotic expression vector that contains the 250-bp MLC-2v
promoter, a polylinker, and a polyadenylation signal derived from a
firefly luciferase cDNA (pMLCSVOA, Fig. 1A). To
determine whether the MLC-2v-ras construct would be capable of
directing the expression of a functional, constitutively active Ras protein, co-transfections were performed in cultured
ventricular muscle cells with an ANF luciferase reporter gene which is
up-regulated by the parental Ha-ras vector, as it is by other
defined hypertrophic stimuli. The studies employing the MLC-ras construct induced a 4.2 ± 0.6-fold (mean ± S.E.)
increase in luciferase activity compared to the vector alone, similar
to the increase seen with activated Ha-ras under its native
promoter (HRV; (18) ) in parallel experiments (4.1 ±
2.4-fold); neither ras construct had any effect on the
promoterless pSVOAL
5` luciferase vector (data not shown).
Co-transfection of a wild type Ha-ras cDNA in the cardiac
muscle cell context did not lead to an induction of the ANF luciferase
reporter gene (1.0 ± 0.2-fold), as seen in Fig. 1B. These data document that the MLC-ras transgene construct can direct expression of a functional,
constitutively active molecule that can drive the expression of a
genetic marker of the hypertrophic response in cardiac muscle cells.
Figure 1:
MLC-ras transgene and evidence
for in vitro functional activity. A, structure of the
transgene cassette and location of selected restriction sites and PCR
primers. H, HindIII; E, EcoRI; N, NotI; X, XbaI; K, KpnI. The transgene (1.1 kilobase pair) was excised from the
vector as a HindIII-KpnI fragment prior to
microinjection. B, cotransfection experiments in cultured
neonatal rat ventricular myocytes. HRV, plasmid
containing the human [Val]Ha-ras cDNA
under the control of its own promoter(13) ; MLCHRV,
transgene construct in A (above); MLCHRG, plasmid
analogous to MLCHRV, but containing the wild type Ha-ras cDNA.
Data shown are mean values ± S.E.; HRV and MLCHRV values are
both significantly different from control (*, p < 0.05,
Student's t test).
Subsequently, the MLC-ras construct was microinjected into
fertilized C57BL/6JxSJL mouse oocytes using standard
procedures(28) . Sixty founder generation pups were obtained,
of which 5 had integration of the transgene detectable by screening
tail DNA by PCR using a 5` primer specific for the MLC-2v promoter and
a 3` primer in the Ha-ras cDNA. PCR screening of the founders
was confirmed by genomic Southern blotting, which also was utilized to
identify mice homozygous for the MLC-ras transgene in
subsequent progeny; homozygosity was confirmed in several animals by
outcrosses with wild type breeders. Three founders transmitted the
transgene. One line (F21) was chosen for analysis, and a second line
(F48) showed an identical phenotype to F21, while that of a third line
(F26) displayed a qualitatively similar, but less prominent phenotype.
As assessed by RT-PCR analysis (Fig. 2, A and B), the expression of the MLC-ras transgene in line
F21 was predominantly restricted to the cardiac ventricles; the RV
level from a +/+ animal was approximately 85% of that seen in
the LV, while the atria displayed <5% of the ventricular level. No
detectable expression was present in uterus, lung, or spleen; the level
in skeletal muscle was slightly greater than that in the atrium (all
corrected for -tubulin cDNA); liver, kidney, and brain had lower
but detectable levels (all corrected for
-tubulin cDNA).
Expression in the LV of a +/- animal was 25% of that in the
+/+ LV.
Figure 2:
Tissue expression of the transgene. A, chamber-restricted expression of the transgene. RT-PCR
products amplified from transgene-specific primers were transferred to
a nylon membrane and probed with a transgene-specific DNA probe. LV
samples are shown from a homozygote (+/+) animal, a
heterozygote (+/-), and a wild type (-/-); RV
and atrial (A) samples are from the homozygote. B,
tissue-restricted expression. Using the same RT-PCR as in panelA, comparison is made between LV and other tissues from a
homozygote. Sk, skeletal muscle; Ut, uterus; Lu, lung; Ki, kidney; Li, liver; Sp, spleen; Br, brain. The exquisite detectability of
cDNA by Southern blotting of RT-PCR products explains the apparent
difference in tissue selectivity between these transgenic mice and
those assayed for the presence of the reporter protein luciferase (26) or -galactosidase (R. Ross and K. Chien, unpublished
observations); these lines have demonstrated that at the protein level,
gene expression directed by the MLC2v promoter is higher in the
ventricular compartment by at least 2 orders of magnitude (26) .
To monitor for the presence of a hypertrophic phenotype, we utilized three independent criteria: an increase in chamber mass (normalized to body weight), an enlargement in myocardial cell size, and the activation of a marker of the embryonic gene program (the reactivation of ANF gene expression in the ventricular chamber). These represent conserved features of the hypertrophic response found in large mammalian species (including humans), as well as in the hypertrophied mouse ventricular chamber following pressure overload in vivo(4, 5, 6, 7) . Mice (line F21) heterozygous (+/-) for the MLC-ras transgene showed a modest yet statistically significant 7.8% increase in LV mass (3.75 ± 0.07 mg/g body weight in +/- (n = 49), versus 3.48 ± 0.13 mg/g in -/- (n = 27)), while mice homozygous for the MLC-ras transgene (+/+) exhibited a marked 57.5% increase (5.48 ± 0.14 mg/g (n = 4)) (Fig. 3). The magnitude and pattern of increase in chamber mass was similar in line F48, although the increase in LV mass in line F26 +/+ was approximately 30% (data not shown). There were no differences in body weight among any of the experimental groups, indicating that the increase in LV/body weight ratio primarily reflected an increase in the mass of the left sided cardiac chambers. This increase in LV mass was substantially greater than that seen in wild type mice exposed to in vivo pressure overload (average 41% increase in whole heart weight; (4) and (5) ) and is consistent with the activation of a hypertrophic response in the left ventricular chamber. In contrast to the LV, only a trend toward an increase in RV mass, which did not reach statistical significance, was observed in the homozygous MLC-ras mice, indicating that the effect of ras was selective for LV chamber. Interestingly, left atrial mass was also significantly increased (175% versus wild type, 0.37 ± 0.05 mg/g versus 0.13 ± 0.01), while right atrial mass was not different between groups. Since the MLC-ras transgene was not significantly expressed in the atrial compartment, the increase in LA mass is most likely not secondary to a direct effect of ras on the atrial hypertrophic response. However, this finding raised the possibility that the increase in LA mass was secondary to chronic exposure of the left atrial chamber to elevated LV filling pressure, due to the effects of ras on ventricular chamber function.
Figure 3: Chamber wet weights. Data are shown from 3 litters; n = 4 for each group. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. Data shown are mean ± S.E. and were analyzed with single factor analysis of variance with posthoc tests based on t test with Bonferroni correction for three comparisons (*, p < 0.05 versus both other groups; , p < 0.05 versus wild type; , p < 0.001 versus both other groups).
In order to exclude secondary hypertrophy, heart rate and arterial blood pressure were measured in the closed chest mouse prior to further hemodynamic studies. Anesthetized heart rate was similar between -/- (n = 13) and +/+ (n = 15) animals (226 ± 16 versus 205 ± 15 beats/min), as were systolic (90.1 ± 8.2 versus 89.2 ± 4.7 mm Hg), diastolic (57.6 ± 4.8 versus 62.3 ± 3.4 mm Hg), and mean (68.4 ± 5.8 versus 71.3 ± 3.7 mm Hg) arterial pressures (p = not significant for all groups, by Student's t test).
Average myocyte cross-sectional area from hearts fixed at constant pressure was increased by 22% in +/+ compared with -/- and +/- (+/+: 196.6 ± 5.3 (n = 4); -/-: 161.2 ± 8.9 (n = 8); +/-: 158.8 ± 6.1 (n = 5); p < 0.05 by single factor ANOVA, post hoc analysis by Scheffé's test); this indicates that the increase in LV mass is due to cardiac myocyte hypertrophy. There was no significant difference in cell area between +/- and -/- animals. Histological analysis of transverse sections of hearts from the various experimental groups were stained with hematoxylin-eosin and Masson trichrome stains. Fig. 4A presents comparable sections from -/- and +/+ littermates, demonstrating concentric chamber hypertrophy with generally normal myocyte architecture. Fig. 4B demonstrates the significant increase in ventricular chamber cross-sectional area seen in the +/+ animals (n = 8) compared to -/- (n = 4). No evidence for tumorigenesis was seen, and there was no significant perivascular, interstitial, or subendocardial fibrosis. However, as seen in Fig. 4C, focal areas of myocyte disarray were evident in 2 of 8 +/+ hearts studied (one sectioned completely from base to apex, the others only sectioned at 100-µm intervals from midwall to base); this histologic picture is virtually indistinguishable from that found in human hypertrophic cardiomyopathy.
Figure 4:
Histology. A, transverse sections
of hearts fixed at constant pressure (11) at the papillary
muscle level, showing gross left and right ventricular hypertrophy in a
+/+ animal (right), compared to a -/-
littermate (left). Hematoxylin/eosin stain was used. B, transverse sections of hearts from +/+
and -/- animals, fixed and stained as in A, were
projected at constant magnification and traced by two observers blinded
to genotype, and the area occupied by ventricular myocardium was
measured by planimetry. Data shown as mean ± S.E. and analyzed
by t test (*, p = 0.05). C, section
demonstrating focus of myocyte disarray (right) and a
comparable area of a nearby section (10 µm away) from the same
heart (as a control). Both sections are from the interventricular
septum of the +/+ mouse in Fig. 4A, above.
Hematoxylin/eosin stain, 100 magnification, is
shown.
To determine if the left ventricular hypertrophy is associated with activation of embryonic genes (a marker of the hypertrophic response; (29) and (30) ), RNA from -/-, +/-, and +/+ animals was analyzed for ANF expression by Northern blotting (Fig. 5). As in other models of hypertrophy, there was a profound increase in ANF mRNA in the +/+ group, with no significant increase in +/- animals compared to -/-.
Figure 5: Northern blot of LV total RNA, using rat ANF probe. -/-, wild type; +/-, heterozygous for transgene; +/+, homozygous for transgene. Each lane represents RNA from one animal; similarity of RNA loading is shown by methylene blue staining of the 28 S ribosomal RNA band.
To assess cardiac function, in vivo LV pressures and the maximal first derivative of LV
pressure (dP/dt) were obtained in the open-chest mouse.
Peak LV systolic pressure was not different between +/+ and
-/- littermates (Fig. 6A), and no
significant differences were observed in LV end-diastolic pressure (Fig. 6B) or LV dP/dt
(an index of
contractile state, Fig. 6C); therefore, the hypertrophy
observed in the +/+ MLC-ras mice was not simply due
to impaired cardiac contractile function. To assess the effect of
hypertrophy on relaxation, the time constant of isovolumic LV pressure
decay (
) was determined (Fig. 6D)(24) .
is the most frequently used index of human ventricular
relaxation(31) , since it is less dependent on peak aortic
pressure and left atrial pressure than other indices; although it has
been utilized in rat studies(32) , it has not been previously
described in murine models. In +/+ animals,
was
significantly prolonged, indicating prolongation of LV relaxation, a
finding typical of early human diastolic ventricular dysfunction. We
also assessed cardiac responsiveness to
-adrenergic stimulation in
+/+ and -/- animals, using graded doses of
isoproterenol. No significant differences in peak heart rate (not
shown), LV systolic pressure, LV end-diastolic pressure, or LV
dP/dt
were identified between the two groups, although a
trend toward increased LV end-diastolic pressure in +/+
animals is apparent, which does not reach statistical significance (Fig. 6). In some +/+ animals, however,
failed
to shorten or was increased by isoproterenol, in contrast to the
decrease observed in all -/- animals, further supporting
the presence of abnormalities in LV relaxation. These results indicate
that MLC-ras +/+ transgenic mice have diastolic
dysfunction evidenced not only by impaired relaxation in the basal
state, but also by increased left atrial mass, which in the absence of
significant transgene expression in that chamber is consistent with
physiologically abnormal LV diastolic function. However, systolic LV
contractility was preserved in these young adult +/+ mice.
Figure 6:
Hemodynamic measurements performed in the
open-chest animal. Responses are shown for the basal and peak dose of
isoproterenol. A, peak LV systolic pressure; B, LV
end-diastolic pressure; C, maximum dP/dt; D,
(13) . Data shown are mean ± S.E.; basal values for
are significantly different between homozygotes and wild type
mice (*, p < 0.05, t test).
In summary, the present study provides direct evidence that ras is sufficient to activate a hypertrophic response in
cardiac muscle in the in vivo context. Activation of the
hypertrophic response has been monitored by several independent
criteria, including an increase in LV mass/body weight ratio, increased
myocardial cell size, and increased expression of an embryonic genetic
marker of the hypertrophic response, ANF. The increases in these
structural, morphological, and genetic markers of hypertrophy achieved
by targeting oncogenic ras expression to the ventricular
muscle cells are qualitatively similar to those seen in hypertrophy due
to pressure overload in murine myocardium, but they occurred in the
absence of concomitant valvular disease, hypertension, or other
systemic effects. The hypertrophic response was more marked, however,
in the +/+ mice than in mice with pressure overload, except
when the overload was very severe (100 mm Hg trans-stenotic gradient
during thoracic aortic constriction; (7) ). Analysis of in
vivo cardiac physiology in these mice has demonstrated an effect
of ras expression to produce diastolic dysfunction, as
assessed by altered LV relaxation and a selective increase in left
atrial (versus right atrial) mass (consistent with a chronic
increase in LV diastolic pressure). In contrast, basal and
-adrenergic-stimulated systolic contractile function remained
normal. These effects of ras may be direct, or some of them
may be mediated, at least in part, by autocrine/paracrine factors or by
cross-talk with other signaling pathways. There also appears to be a
threshold effect, such that a certain amount of activated ras is necessary for the hypertrophic response, since animals
heterozygous for the transgene had very little hypertrophy.
These mice exhibited a functional phenotype that in most animals is qualitatively similar to that seen in compensated human hypertensive heart disease. The selective diastolic dysfunction, in association with hypertrophy resulting from a primary cardiac muscle disorder (without accompanying hypertension or valvular disease), bears functional similarity to that seen in human hypertrophic cardiomyopathy (HCM); likewise, the myocyte disarray seen in at least some +/+ mice is characteristic of HCM, although it has been described less frequently in other forms of human hypertrophy. The findings in this murine model suggest a role for ras-dependent pathways in the genesis of cardiac hypertrophy, as well as in the transition between compensatory hypertrophy and the onset of cardiac muscle dysfunction and the development of myocyte disarray. These mice should be valuable in genetically based approaches to identify further downstream signaling pathways which mediate this form of cardiac muscle dysfunction, using molecular physiological analysis to characterize the functional phenotype. While this transgenic mouse does not model the human familial HCM genotype per se, nevertheless, it may provide useful insights into the molecular mechanisms that lead to cardiac ventricular hypertrophy and the onset of diastolic dysfunction, which are important entities in the clinical disease process. While familial HCM in humans can be caused by mutations in sarcomeric protein genes, the mechanisms by which these mutations lead to the activation of the hypertrophic phenotype is unclear. Our current studies suggest the possibility that ras-dependent pathways might play a role, either directly or indirectly, in this pathological process. As a genetically based model of cardiac muscle disease, these mice should now permit dissection of the interaction of ras with retinoids and other signaling pathways, through genetic crosses with other transgenic strains, as well as physiological and pharmacological manipulations which induce or impair the development of hypertrophy. Furthermore, they identify ras and subsequent downstream signaling pathways as potential targets for interrupting the pathological process of hypertrophy in the in vivo context. In fact, based on these observations, we have recently demonstrated that agents that can block the oncogenic effects of ras, such as retinoids, can serve as suppressors of myocardial cell hypertrophy in an in vitro model system(33) , supporting the potential therapeutic importance of these observations.