Published online before print November 11, 2004, doi: 10.1161/01.RES.0000150366.08972.7f
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© 2004 American Heart Association, Inc.
Integrative Physiology |
Physiological Growth Synergizes With Pathological Genes in Experimental Cardiomyopathy
From the Heart and Vascular Center of the University of Cincinnati (F.S., A.O., H.S.H., E.W. B., R.A.L., Y.M., G.W.D) and the Division of Molecular Cardiovascular Biology (A.S., J.R.), Cincinnati Children’s Hospital Research Foundation, Ohio.
Correspondence to G.W. Dorn II, University of Cincinnati Heart and Vascular Center, University of Cincinnati Medical Center, 231 Albert B. Sabin Way, Cincinnati, OH 45267-0542. E-mail dorngw{at}ucmail.uc.edu
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Abstract |
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Hundreds of signaling molecules have been assigned critical roles in the pathogenesis of myocardial hypertrophy and heart failure based on cardiac phenotypes from
-myosin heavy chain–directed overexpression mice. Because permanent ventricular transgene expression in this system begins during a period of rapid physiological neonatal growth, resulting phenotypes are the combined consequences of transgene effects and normal trophic influences. We used temporally-defined forced gene expression to investigate synergy between postnatal physiological cardiac growth and two functionally divergent cardiomyopathic genes. Phenotype development was compared various times after neonatal (age 2 to 3 days) and adult (age 8 weeks) expression. Proapoptotic Nix caused ventricular dilation and severe contractile depression in neonates, but not adults. Myocardial apoptosis was minimal in adults, but was widespread in neonates, until it spontaneously resolved in adulthood. Unlike normal postnatal cardiac growth, concurrent left ventricular pressure overload hypertrophy did not synergize with Nix expression to cause cardiomyopathy or myocardial apoptosis. Prohypertrophic Gq likewise caused eccentric hypertrophy, systolic dysfunction, and pathological gene expression in neonates, but not adults. Thus, normal postnatal cardiac growth can be an essential cofactor in development of genetic cardiomyopathies, and may confound the interpretation of conventional -MHC transgenic phenotypes.
Key Words: apoptosis • signal transduction • transgenic mice • hypertrophy
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Introduction |
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Cardiac hypertrophy is both a compensatory response to sustained hemodynamic stress,1 and an independent risk factor for heart failure and death.2 Contractile dysfunction and "drop-out" (apoptotic or necrotic death) of hypertrophied cardiac myocytes are thought to contribute to the progression of cardiac hypertrophy to dilated cardiomyopathy.3 Accordingly, human heart failure is characterized both by massive enlargement of individual ventricular myocytes4–6 and by striking increases in the rate of their programmed death.6–8 This clinical link between cardiac hypertrophy and heart failure, and the associated pathophysiological connection between cardiac myocyte growth and programmed death, suggests a functionally significant interaction between myocardial trophic and apoptotic pathways.
Individual in vivo roles for numerous hypertrophy or apoptosis receptors, transducers, and effectors have been defined, in part, through manipulation of the mouse genome.9,10 Especially useful for directing cardiac overexpression has been the murine -myosin heavy chain (MHC) promoter, which reproducibly provides high-level cardiomyocyte-specific transgene expression in a copy number–dependent and position-independent manner.9–11 This transgenic system has been used in more than 150 published cardiac-specific overexpression models, many of which have helped to establish critical roles for the expressed transgene based on the development of striking heart phenotypes.9,10 Because it is largely silent in the ventricles until birth and then expresses robustly,11 -MHC-directed expression of toxic genes is not lethal during embryogenesis, but causes early lethality12–14 or phenotypic progression during early life.13,15
The characteristic temporal pattern of conventional -MHC–directed transgene expression, with atrial expression before and after birth and ventricular induction at approximately the time of birth, results in robust transgene expression and accumulation of transgenic protein during a period of rapid heart growth. Although there is evidence for an interaction between cardiomyocyte hypertrophic and apoptotic pathways,3,4,16,17 the interplay between normal cardiac growth and transgene-directed cardiac pathology has not previously been examined. Using a robust inducible system, we compared the consequences of expression of the hypertrophy-regulated mitochondrial death protein Nix12,18 and the hypertrophy signal transducer Gq19–21 in either rapidly growing neonatal (1 to 4 weeks) or terminally differentiated (8 to 12 weeks) adult hearts. Nix expression in the early postnatal period resulted in apoptotic cardiomyopathy, but Nix expressed at identical or higher levels in the adult heart had minimal effects on cardiomyocyte apoptosis, chamber size, or ventricular contractile function. Likewise, neonatal expression of Gq resulted in eccentric left ventricular hypertrophy with contractile depression and fetal gene expression, but adult expression was without effect. These results demonstrate a critical modulatory function of normal physiological growth on the pathological responses to these cardiac genes and provide an explanation for age-dependent phenotypic variation in cardiac transgenic models.
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Materials and Methods |
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Generation of Conditional
-MHC-Nix Transgenic MiceA tetracycline-responsive binary
-MHC transgene system allowing suppression of transgene expression with doxycycline was used for temporally defined cardiomyocyte-specific expression of the mouse Nix cDNA. tTA mice on the FVB/N background and the tet-responsive ("tet-off") attenuated -MHC promoter have been previously described.22 The mouse Nix cDNA12 was cloned into the SalI-HindIII site of the tet-off promoter to generate MHCminTetOmNix, the linearized construct was injected into male FVB/N pronuclei, and founders were identified by genomic Southern analysis of tail clip DNA.
MHCminTetOmNix mice were bred with -MHCtTA mice and the double heterozygotes (tTA/ MHCminTetOmNix) were studied under one of two conditions: (1) Mice were never administered doxycycline, so that Nix was expressed in the neonatal period consistent with unsuppressed -MHC-mediated expression (neonatal Nix); or (2) Pregnant dams, suckling dams, and weaned pups received doxycycline (0.2 mg/mL in drinking water with 2% sucrose) until 8 weeks of age, after which it was withdrawn (adult Nix). Control mice consisted either of syngenic littermates with continued doxycycline suppression or identically treated TetOmNix mice. Animals were treated in accordance with approved University of Cincinnati IACUC protocols. All animals used in the study were generated at the animal facility of the University of Cincinnati, and all cDNA injections were performed within the transgenic core facility of the University of Cincinnati.
Generation of Inducible Gq Transgenic Mice
A binary Cre-lox transgene system allowing induction of myocardial Gq expression by administration of tamoxifen was used for adult expression of Gq. The murine Gq cDNA was cloned 3' to a floxed chloramphenicol acetyl transferase (CAT) gene in a chicken ß-actin promoter construct.23 ß-Actin-CAT-Gq mice were bred with mice carrying an MHC-driven modified estrogen receptor-Cre (MER-Cre-MER) transgene,24 and cardiac-specific Gq expression was induced in double heterozygotes by intraperitoneal injection of tamoxifen citrate, 10 mg/mL in 50% ethanol (15 µg/g per day for 5 consecutive days).
Immunoblot Analysis
Analysis of Nix expression used a custom polyclonal rabbit anti-mouse Nix antibody prepared against amino acids 98 to 116 (QEDGQIMFDVEMHTSRDHS) and affinity purified with the same peptide. Gq protein was assayed with anti-Gq/11 from Santa Cruz. Proteins were visualized using enhanced chemifluorescence and quantitated using a Storm PhosphorImager.
RNA Dot Blot Analysis
Two micrograms of individual left ventricular total RNA was applied to nylon membranes using vacuum filtration and hybridized to 32P-labeled oligonucleotides specific for a panel of cardiac-expressed genes, as previously described.19 Quantitation was by phosphorimager. For each heart, data were indexed to glyceraldehyde phosphodehydrogenase (GAPDH) expression.
Functional Assessments
2-D guided M-mode echocardiography of unsedated mice measured left ventricular (LV) diastolic and systolic dimensions (LVEDD and LVESD), from which fractional shortening (FS) was derived. Pulsed wave Doppler was used to measure aortic ejection time (ET) and calculate velocity of circumferential shortening, Vcf (FS/ET). Invasive hemodynamic studies were performed on anesthetized, spontaneously breathing transgenic mice and littermate controls to measure left ventricular pressures and systolic (+dP/dt) and diastolic (–dP/dt) function at baseline and in response to graded infusions of dobutamine (4 to 128 ng/g/min).
Transverse Aortic Banding
Twelve-week-old tTA/MHCminTetOmNix mice and controls were subjected to acute pressure overloading by surgical coarctation of the transverse aorta as described.25–27 Doxycycline was withdrawn the day of surgery to result in Nix expression during the peak hypertrophy response.25 Mice underwent terminal invasive hemodynamic studies with assessment of transaortic gradient and left ventricular hemodynamics 8 weeks after aortic banding.
Histopathology and TUNEL Studies
Histopathological examination was performed on Masson trichrome-stained sections. TUNEL staining used the TACSTM 2 TdT-DAB apoptosis detection kit (Trevigen Inc). Proliferative Cell Nuclear Antigen (PCNA) staining was performed with the PCNA Staining Kit from Zymed Laboratories (cat. no 93–1143).
Statistical Analysis
Results are mean±SE. Experimental groups were compared using Student t test or one-way ANOVA. A Bonferroni test was used for post hoc comparisons, with P<0.05 significant.
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Results |
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Temporally Defined Cardiac Nix Expression
To determine whether normal postnatal cardiac growth was a phenotypic modifier of Nix-induced myocardial apoptosis, a bitransgenic
-MHC–driven tetracycline-suppressible system was used22 (Figure 1a). The -MHC promoter drives cardiac expression of the tetracycline transcriptional activator (tTA), which is necessary for Nix transgene expression by the doxycycline-suppressible -MHCminTetOmNix. Becausee both arms of this system are -MHC driven, if doxycycline is not administered, the time course for Nix expression should be similar to that for conventional -MHC-Nix mice (Figure 1b, top). Indeed, high-level Nix expression in neonates was detected on day 3 (Figure 1b, bottom), as previously reported for conventional -MHC-Nix mice.12 Nix expression was maintained for at least 10 weeks (Figure 1b, bottom). For adult Nix expression, doxycycline was administered to the dam and, after weaning, continuously to the pups until 8 weeks of age (Figure 1c, top). Withdrawal at that time resulted in detectable Nix protein after 3 days, with maximal expression at 2 weeks (Figure 1c, bottom), and was maintained for at least 8 weeks (not shown). The level of Nix expression using the bitransgenic system was slightly less in neonates than adults (Figure 1d), and was approximately 25% that obtained with the previously described high-expressing conventional -MHC-Nix transgenic line (not shown).
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Effects of Nix on Neonatal and Adult Hearts
Conventional Nix transgenic mice were reported to develop early postnatal cardiac dilation and growth retardation.12 Because these mice succumbed at approximately 2 weeks of age, the cardiac functional consequences of Nix expression have never been assessed. In this study, neonatal Nix expressors also developed a heart phenotype, although with less myocardial Nix expression lethality was only 11% at 10 weeks (n=18). Serial echocardiography demonstrated striking left ventricular enlargement (Figure 2a) and depressed systolic performance (Figure 2b, %FS [fractional shortening]) at 2 weeks of age and thereafter. Both echocardiography and gravimetric measures showed increased heart mass (Figure 2b) and an increase in the ratio of left ventricular dimension to wall thickness (r/h, Figure 2b), consistent with a dilated cardiomyopathy. In contrast, induction of Nix expression in adult myocardium resulted in only minimal changes in heart size, left ventricular dimension, and left ventricular function (Figure 2a and 2b). Cardiac gene expression was entirely normal in adult Nix expressors, whereas -skeletal actin was significantly increased 2 weeks after neonatal Nix expression (Figure 2c).
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As anticipated from the prior studies of Nix,12,18 the mechanism for cardiac chamber dilation (Figure 3a) after neonatal Nix expression appeared to be stimulation of cardiomyocyte apoptosis. TUNEL staining of transgenic and control ventricular myocardia revealed apoptotic indices (no. TUNEL-positive nuclei/ no. total cardiomyocyte nuclei) between 10% and 20% in 1-week and 4-week-old neonatal Nix expressors, compared with less than 1% TUNEL positivity in controls (Figure 3b and 3c). TUNEL positivity was rare, but higher than controls, in adult Nix expressors assessed at the same intervals after Nix induction (Figure 3b and 3c). Thus, structural, functional, and cellular phenotypes demonstrate that Nix expression in the adult heart does not have the severe consequences seen with its expression in the neonatal heart.
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We noted that the rate of cardiomyocyte apoptosis (TUNEL positivity) in neonatal Nix expressors diminished between the ages of 4 and 10 weeks, ie, during the transition into adulthood (Figure 3c). Indeed, the rate of cardiomyocyte apoptosis in full-grown neonatal Nix expressors was similar to that in adult Nix expressors. Given that Nix expression had such different phenotypes in growing neonatal versus nongrowing adult hearts, we hypothesized that the phenotype of 10-week-old neonatal Nix expressors had evolved into an adult-like Nix phenotype because growth of the heart had stopped. If this were the case, then the progressive cardiac dilation seen between 1 and 4 weeks in neonatal Nix expressors (see Figure 2b, r/h ratio) might reverse in full-grown mice. This was the case, as ventricular geometry (r/h) and mass measurements revealed reverse remodeling in 10-week-old neonatal Nix expressors (Figure 4a and 4b). These changes did not, however, normalize ventricular function as echocardiographic fractional shortening (Figure 4c) and catheterization-derived +dP/dt (Figure 4d) remained depressed, likely reflecting the deleterious functional consequences of "compensatory" hypertrophy25,26,28 and residual myocardial damage.
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Taken together, these results support a contributory effect of normal postnatal cardiac growth on cardiomyocyte apoptosis stimulated by Nix. To determine whether abnormal growth in the form of pressure overload hypertrophy would similarly synergize with Nix, the Nix transgene was activated by doxycycline withdrawal concommitantly with creation of acute pressure overload by surgical transverse aortic constriction (TAC) in adult mice.27 Importantly, nonoperated adult Nix expressors followed for 8 weeks (Figure 5, "NonNix") showed no delayed effects of adult Nix expression, and these findings were similar even after four months (not shown). Transaortic gradients for Nix expressors were 76±5 mm Hg and for controls were 75±6 mm Hg (n=4 each, P=NS). Coinduction of Nix in adult mice did not alter the normal hypertrophic response to pressure overload (Figure 5a), and actually normalized ventricular geometry (Figure 5b). Baseline ventricular function was maintained (Figure 5c), and 8 weeks after combined TAC and Nix induction, the response to dobutamine stimulation was similarly depressed in control and Nix-expressing TAC mice (Figure 5d). Thus, unlike normal postnatal cardiac growth, cardiac hypertrophy in response to acute pressure overload did not contribute to Nix-mediated apoptotic heart failure.
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We considered that the disparate results seen with neonatal versus adult Nix expression could represent either a phenomenon generally applicable to powerful cardiac signaling proteins, or a unique characteristic of this mitochondrial death gene. To distinguish between these possibilities, parallel studies were performed comparing neonatal and adult expression of the hypertrophy signaling protein, Gq.19 Based on gene knockout and in vivo myocardial inhibition studies,20,21 Gq is a critical transducer for signaling of pressure overload hypertrophy. Conventional -MHC-driven cardiac Gq overexpression results in hypertrophy with many of the characteristics of pressure overload.19,25 We compared neonatal Gq expression (-MHC-Gq) to inducible adult Gq expression using a Cre-lox–based, tamoxifen-inducible bitransgenic system23,24 (Figure 6a). Approximately 2 weeks after completion of tamoxifen treatment, myocardial Gq protein levels were similar to those in the higher-expressing (
5-fold) overexpressing Gq-40 line of conventional -MHC-Gq mouse hearts (Figure 6b). However, adult Gq overexpression failed to reproduce the hallmark phenotypic features of cardiac hypertrophy (Figure 6c), contractile depression (Figure 6c), or increased fetal cardiac gene expression (Figure 6d) that is seen with neonatal, -MHC–driven Gq expression.
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Discussion |
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These studies show that normal cardiac growth in the postnatal period is an essential cofactor for development of cardiac pathology mediated by two functionally distinct transgenes in the Gq signaling pathway of maladaptive hypertrophy, proapoptotic Nix, and prohypertrophic G
q. These findings reveal that functional synergy can occur between physiological cardiac growth and pathological hypertrophy/apoptosis, thus reinforcing the need to consider the physiological context of gene expression when interpreting the effects of in vivo cardiomyocyte signaling manipulations. In the Gq signaling pathway at least, inducible adult expression may have more relevance to conventional human cardiac disease, whereas standard -MHC–directed neonatal expression can provide additional insights into the consequences of signaling perturbations in the pediatric population.
Nix is a proapoptotic member of the Bcl-2 family of mitochondrial-targeted proteins. In vitro, Nix transfection causes apoptosis through the intrinsic pathway by stimulating mitochondrial cytochrome c release, apoptosome activation, and cleavage of caspases.12,18 First detected in the heart as a regulated gene in Gq-mediated hypertrophy,29 and subsequently in experimental and human pressure overload hypertrophy,12 Nix is necessary for the apoptotic cardiomyopathy that occurs in Gq transgenic mice.12 Conventional MHC-Nix transgenic mice exhibited growth retardation, cardiac enlargement, cardiomyocyte apoptosis, and premature death from unknown causes.12 The current study, in which Nix was expressed at somewhat lower levels that resulted in viable mice, establishes that Nix-mediated myocardial apoptosis results in a dilated cardiomyopathy, and that heart failure is the probable cause of death. Interestingly, Nix protein expression in neonatal hearts was less than in adult hearts using the same bitransgenic system. The profound neonatal phenotype is therefore not caused by less Nix expression. Indeed, the likely reason for diminished Nix in neonatal hearts is that it is a suicide transgene, which kills the cardiomyocytes that express it most robustly, and ultimately diminishes overall myocardial expression.
Because Nix upregulation and the Nix-mediated apoptotic peripartum cardiomyopathy were originally described in pressure overload and Gq-mediated cardiac hypertrophy,12,29 it is not surprising that pathological cardiotrophic stimuli can increase myocardial sensitivity to apoptosis. Indeed, a relationship between cellular proliferation and programmed death has previously been established at a number of different levels,30,31 and a critical difference between postnatal cardiac growth (which synergized with Nix) and pressure overload hypertrophy (which did not) is ongoing cardiomyocyte proliferation in the former. In rodents, cardiomyocyte proliferation is intense in the embryo and continues up to 4 days after birth, followed by a phase of cardiomyocyte hypertrophy before quiescence.32 Likewise, in the present study, PCNA staining for nuclear proliferation was high in 1-week-old mouse hearts, but progressively diminished over the following 3 weeks. Thus, cell cycling may provide a permissive environment that exaggerates the consequences of cardiomyocyte signaling, relative to quiescent cells. An attenuated effect of signaling perturbations after cardiomyocyte cell cycle exit can also explain the current findings that neonatal Nix-induced apoptosis and cardiac dilation diminished, and then reversed, after 4 weeks of age.
The enhanced effect of Gq overexpression in neonatal versus adult hearts suggests that synergism between cardiac growth and signaling transgenes may be a general phenomenon, at least in this important signaling pathway. Compared with the inducible Nix studies, Gq is a hypertrophic rather than apoptotic gene, and was expressed using a tamoxifen-stimulated system rather than by withdrawal of doxycycline. Thus, attenuated phenotypes with adult versus neonatal expression cannot be attributed to the functional class of the transgene or to the particular characteristics of the inducible expression system. A previously described phenotype of sudden death with neonatal expression of activated protein kinase Cß2, compared with mild progressive hypertrophy with adult expression,33 provides a third example of signaling transgene synergy with neonatal cardiac growth.
An exaggerated cardiac phenotype with neonatal signaling factor overexpression does not imply functional irrelevance for that factor in the adult heart. Notwithstanding absence of effect of induced Gq overexpression in adult mouse hearts, a central role for Gq signaling in mediating pressure overload hypertrophy is incontrovertible, based on in vivo loss-of-function (gene knockout and peptide inhibitor expression) studies.20,21 Lack of a major phenotypic impact of Gq and Nix expression in the adult heart likely reflects absence of concurrent activity in the growth hormone/PI3-K/Akt pathway that stimulates normal developmental cardiac growth.
Whether the differences in adult versus neonatal phenotypes for Gq, Nix, and PKC are widely generalizable or not, the current data constitute a strong argument for temporally precise and controllable cardiac transgene expression of signaling effectors. Accordingly, in vivo evaluation of cardiac signaling factors should probably combine neonatal and adult expression studies with loss of cardiac function models to properly gauge their impact on adult cardiac physiology, and by analogy, the adult human condition.
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Acknowledgments |
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This work was supported by NHLBI HL58010 and HL59888.
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Footnotes |
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Original received July 27, 2004; revision received October 21, 2004; accepted November 1, 2004.
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