Pressure-Overload Deinduction of Human α2 Na,K-ATPase Gene Expression in Transgenic Rats
The early and sustained deinduction of α2 Na,K-ATPase gene expression in both cardiac left ventricle and aorta in various pressure-overload rat models and in hypertrophied human heart suggests a common transcriptional pressure response mechanism to pressure overload in both rats and humans. To test this hypothesis, we developed transgenic rat lines expressing the chloramphenicol acetyltransferase reporter gene regulated by the human α2 Na,K-ATPase (−798 to +67) regulatory region, Hα2-CAT. Analysis of two homozygous transgenic rat lines revealed (1) parallel tissue-specific regulation of the Hα2-CAT transgene and rat α2 Na,K-ATPase gene and (2) parallel load-induced deinduction of both cardiac and vascular (aortic) Hα2-CAT transgene and rat α2 Na,K-ATPase gene expression in a 3-day model of induced pressure overload. Cardiac Hα2-CAT deinduction was detected at a systolic pressure greater than or equal to 150 mm Hg and correlated with the degree of systolic pressure elevation (r=.82, P<.0001). The data suggest a systolic pressure gradient–dependent coordinate pressure-overload transcriptional response mechanism in the heart and aorta, with one of its target genes being the α2 Na,K-ATPase gene in both humans and rats.
Analyses of cardiovascular molecular responses to in vivo pressure overload and in vitro hypertrophy/pressure-overload stimuli (mechanical stretch, shear stress, hormonal and peptide growth factor stimulation) have identified stimulus-specific and overlapping programs of gene-specific alterations of gene expression.1 2 3 4 5 6 7 However, unresolved issues highlight the challenges in the mechanistic analysis of pressure-overload transcriptional responses and point toward the necessity for in vivo corroboration in integrated biological experimental models.1 7 These issues are exemplified in the following experimental systems. (1) In a static stretch cardiomyocyte culture system, an in vitro transcriptional stretch-response region involving the c-fos oncogene (prototype pressure-overload immediate early response gene) serum response element was delineated within −356 bp of the 5′ flanking region of the c-fos gene8 ; however, in vivo corroboration remains to be done. More significantly, evidence that immediate early genes play key roles in the myocardial pressure-overload response remains to be obtained.7 (2) Elucidation of a transcriptional pressure-overload/stretch response mechanism using the ANF gene as molecular probe has been elusive. In transgenic mice, although a −500-bp ANF 5′ flanking region directed atrial-specific expression, the transgene did not exhibit the expected pressure response upregulation in vivo despite the marked upregulation of the endogenous ANF mRNA.9 Additionally, a longer 3.4-kb 5′ flanking regulatory region of the ANF gene did not exhibit transcriptional activation by stretch in a validated in vitro stretch neonatal cardiomyocyte-rich experimental system, again despite the upregulation of the endogenous ANF mRNA.8 Further study is necessary to unravel the underlying mechanism.1 7 (3) The recent identification of a shear stress response element present in vascular endothelial shear stress response genes, eg, platelet-derived growth factor-B chain gene,5 delineates a critical molecular mechanism for mechanical stress responses. In vivo corroboration remains to be done. (4) It is also recognized that the signaling mechanism for gene expression in pressure-overload cell culture models would most likely be discrepant with signaling mechanisms in vivo,7 thus indicating that the analysis of bona fide integrated biological mechanisms would require corroboration in in vivo models.
Issues regarding the relevance to human pathophysiology and identification of key players in pressure-overload responses also remain unresolved. The activation of the “fetal gene program” (β-MHC, ANF, skeletal α-actin) in response to pressure overload is a late response1 2 7 8 and would most likely not be part of early regulatory events initiating key hierarchical responses to pressure overload. Additionally, although β-MHC is the rodent hypertrophy-specific isoform, in humans, β-MHC is the constitutive MHC isoform in normal human heart9 ; thus, the consequences of the myocardial switch to β-MHC, albeit corroborated in rat in vitro and in vivo models, is irrelevant in human pressure-overload pathogenesis.
The α2 Na,K-ATPase gene provides an alternative molecular system for dissection of this problem. Studies of different rat pressure-overload models (deoxycorticosterone acetate–salt uninephrectomized,6 10 11 Ang II–infused,6 aortic constriction,12 and renovascular13 hypertension models) consistently showed the decrease of α2 Na,K-ATPase mRNA and protein levels in cardiac left ventricle. This deinduction was specific for α2 Na,K-ATPase, as α1 and β1 Na,K-ATPase, β-actin, and α-tropomyosin gene expression remained unchanged.6 Deinduction of α2 Na, K-ATPase gene expression has also been observed in human pressure-overloaded right ventricles and failing left ventricular samples.14 Concordantly, deinduction of α2 Na,K-ATPase gene expression is also detected in vascular tissue (aorta) in deoxycorticosterone acetate–salt and Ang II–induced hypertension models6 and in acute aortic constriction (V.L.M.H. et al, unpublished data, 1996). Because the deinduction of α2 Na,K-ATPase is common to different experimental rat models of pressure overload, is observed in both cardiac and vascular (aortic) myocytes, and is paralleled by similar observations in humans,14 we hypothesized that the α2 Na,K-ATPase gene deinduction is a primary pressure-overload transcriptional response mechanism distinguished from the ensuing secondary cascade of changes in late gene expression coinciding with the onset of myocyte hypertrophy.
To demonstrate a pressure-overload transcriptional mechanism, we developed a pressure-overload transgenic rat model containing the human α2 Na,K-ATPase 5′ flanking regulatory region linked to the CAT reporter function gene, Hα2-CAT. The use of the human α2 Na,K-ATPase 5′ flanking regulatory region would provide insight into pressure response mechanisms in humans through deductions made from direct observations of the regulation of human genes in validated transgenic animal models. Transcriptional activity of the human α2 Na,K-ATPase regulatory region would be directly assessed from the modulation of CAT gene products and validated by (1) parallel observations in at least two transgenic lines, (2) parallel modulation of the endogenous rat α2 Na,K-ATPase gene, and (3) consistency of results in different rats assessed individually. The accuracy of molecular biology technology would be matched by the reliability of BP measurements obtained in a continuous, nonstressed manner by radiotelemetry.
Recombinant DNA Constructs
We amplified the human α2 Na,K-ATPase gene by polymerase chain reaction to isolate the 5′ flanking regulatory region based on published sequences.15 The 865-bp polymerase chain reaction–amplified fragment, −798 bp 5′ flanking and +67 bp of the 5′ untranslated sequences, was then subcloned into the enhancerless/promoterless pCAT-basic plasmid (Promega) containing the CAT reporter gene to reduce Hα2-CAT. Nucleotide sequencing confirmed the orientation and sequence identity of the Hα2-CAT construct. The positive control consisted of the pCAT-control plasmid (Promega) that has the SV40 enhancer and promoter. The negative control consisted of the pCAT-basic plasmid (Promega) with a 1.5-kb “stuffer DNA” cloned into the multiple cloning site, keeping the plasmid enhancerless and promoterless. This plasmid had less “leaky” expression than the Promega pCAT-basic plasmid and is referred to as the modified pCAT-basic* plasmid.
Tissue Culture CAT Assays
A subclone of C2C12 mouse skeletal muscle cells (American Type Culture Collection) derived in our laboratory was used for all experiments.16 This subclone was selected from a single-cell clone exhibiting optimal myotube formation in fusing medium (Dulbecco's modified Eagle's medium with 2.5% horse serum and 2.5% fetal bovine serum; GIBCO BRL). The cells were grown in Dulbecco's modified Eagle's medium and 20% fetal calf serum and were split into p100 dishes for transfection expression experiments. For C2C12 myotube experiments, 106 cells per p100 dish were seeded; transfected the next day with 20 μg of test DNA [(+) pCAT-control, (−) pCAT-basic; Hα2-CAT], and 8 μg β-galactosidase DNA for internal control; and changed to fusing medium upon reaching confluence. Cells were harvested for CAT assays when 90% myotube formation was evident, approximately 3 days after transfection and 2 days in fusing medium. For C2C12 myoblasts, 2×105 cells were seeded onto p100 dishes, transfected the next day, and harvested 2 days after transfection, ie, before reaching confluence and before the development of significant myotube formation. Transfection was done with Lipofectin (GIBCO BRL) following the manufacturer's specifications. CAT assays were done as described16 on equal protein amounts of total cellular extracts, validating visual quantification. As an additional control, we checked β-galactosidase activities (Promega) to ascertain equivalent transfection efficiencies. Variations were less than 10%. Protein concentrations were determined by the BCA assay (Pierce Chemical Co). We used autoradiographic densitometry measurements and direct measurement of 14C-labeled CAT products to corroborate the visual comparative analysis presented.
Transgenic Rat Development
Outbred Sprague-Dawley rats were used for all transgenic experiments. For DNA microinjection, we digested the Hα2-CAT plasmid with Bgl I restriction enzyme to remove most of the vector sequences. The desired 4.06-kb Bgl I/Bgl I fragment containing the −798 to +67 Hα2-CAT–SV40 polyadenylation signal was purified and resuspended in Brinster's medium at 2 ng/μL.17 DNA microinjection was done essentially as described.17 18 Natural matings were used for both donor and recipient females for optimization of rat embryo viability. Procedures followed were in accordance with institutional guidelines. Pentobarbital (50 mg/kg IP) was used as anesthetic.
DNA Analysis of Transgenic Rats
For genotyping of founders and subsequent generations of offspring, tail DNA was purified as described17 and analyzed by slot blot analysis with the use of 5 μg DNA per sample. The radiolabeled probe used for analysis was the 4.06-kb Bgl I transgene DNA fragment. Negative controls were composed of DNA from nontransgenic rats. Homozygous F2 offspring were detected by a twofold increase in autoradiographic signals and were genetically tested by outbreeding to nontransgenic rats, with homozygosity ascertained by 100% transgene-positive heterozygous offspring.
Southern blot analysis was done as described,17 with the following specifications: 5 μg DNA was digested with HindIII restriction enzyme; nitrocellulose membranes were used; 2-day hybridization was done; and the 4.06-kb Bgl I/Bgl I transgene DNA fragment was used as the random-primed 32P-labeled probe. HindIII restriction digestion was done because this would not cut the transgene internally. Detection by Southern blot analysis of HindIII DNA restriction fragments greater than a 4.06-kb fragment would indicate an intact full-length transgene. Detection of a single HindIII DNA restriction fragment would indicate a single integration site into the rat genome.
Telemetric Measurement of BP
Surgical implantation of the BP telemetric device (Data Sciences International) was done following the manufacturer's specifications and in accordance with institutional procedures. Pentobarbital was used as anesthetic as described above. The rats were allowed to recover for 2 weeks. Baseline BPs were then measured over 3 to 5 days with the Datascience Acquisition program (average BP over 10 seconds every 5 minutes, 24 h/d [288 data points per day]). Parameters simultaneously obtained were systolic BP, diastolic BP, mean arterial pressure, heart rate and activity.
Ang II–Induced Hypertension
Control sham-infused and test Ang II–infused (Sigma Chemical Co) rats were implanted with 3-day Alzet minipumps (Alza Corp) (1 μL/h) as previously described.6 The Ang II pressor dose in saline medium was 1 ng/min per gram body weight; the sham-infused dose was normal saline at 1 μL/h. BP parameters were obtained for 24 hours for 3 to 5 days for baseline BP levels and for 72 hours during Ang II and sham infusion. After 72 hours, Ang II–infused test rats and control sham-infused rats were analyzed for Hα2-CAT activity, with identical experimental conditions maintained to validate comparative analysis. Twenty-four-hour average systolic BP, 288 data points, was used for correlation with CAT product levels.
CAT Assays on Transgenic Rat Tissues
Tissues were isolated from transgenic rats after euthanasia with intraperitoneal sodium pentobarbital (Nembutal). All tissues were homogenized with a polytron (Brinkmann); extracts were prepared as described.19 Cardiac left ventricles were analyzed individually; aortas were pooled in matched pairs except for 28-week-old rat aortas. Cardiac right ventricle, whole brain, and skeletal muscle (gluteus maximus) were analyzed as control tissues not exposed to pressure overload. Protein concentrations were measured by the BCA assay (Pierce). CAT assays were done with specifications for visual and quantitative comparative analysis as described above.
Correlation of the percent deinduction of Hα2-CAT activity and systolic BP levels on Ang II–induced pressure overload was analyzed by the Pearson product moment test (SigmaStat, Jandel Scientific).
Regulation of Human α2 Na,K-ATPase Gene
The minimal basal functional 5′ flanking regulatory region of the rat α2 Na,K-ATPase was delineated to within −175 bp as assessed in rat L6 skeletal muscle cells.20 To ensure the presence of basal as well as putative complex regulatory response sequences, we designed the transgene to be 4.5-fold longer, spanning −798 to +67 bp of the human α2 Na,K-ATPase gene sequences linked to the CAT reporter function gene, Hα2-CAT (Fig 1A⇓). Preliminary testing in mouse C2C12 skeletal muscle cells revealed that the transgene exhibited the expected skeletal myotube upregulation,21 validating its functionality (Fig 1B⇓). Hα2-CAT exhibited at least twofold upregulation of CAT gene product in C2C12 myotubes compared with C2C12 myoblasts. Fig 1C⇓ shows that induction of Hα2-CAT expression was paralleled by an increase in mouse α2 Na,K-ATPase mRNA, as previously reported.21 Myoblast-to-myotube differentiation was confirmed by light microscopy documenting fused myocytes and by RNA analysis showing α-tropomyosin isoform switching from the nonmuscle to the striated muscle isoform (Fig 1C⇓), as previously described.22 Induction was specific for the Hα2-CAT construct because both positive (pCAT-control plasmid) and negative (modified pCAT-basic) control CAT constructs did not exhibit modulation of gene expression (Fig 1B⇓). The levels of Hα2-CAT expression detected in myoblast reflect myoblast-to-myotube transition in a subpopulation of C2C12 transient transfectants or alternatively “leaky” expression caused by increased copy number of transfected Hα2-CAT over endogenous α2 Na,K-ATPase gene.
We then cut this construct with Bgl I restriction enzyme to remove most of the vector sequences and purified and microinjected the 4.06-kb fragment into single-cell rat embryos as described by Mullins et al18 for the rat on the basis of modifications of mouse microinjection techniques.17 Four transgenic founders were produced; all were germ-line integrated. DNA analysis revealed different integration sites (Fig 2⇓). As seen in Fig 2⇓, Southern blot analysis of all four founders (as well as F1 offspring; data not shown) detected different-sized HindIII restriction digestion fragments hybridizing to the full-length 4.06-kb Hα2-CAT transgene random-primed 32P-labeled probe. In all founders, the HindIII restriction DNA fragment detected by the full-length transgene probe was greater than the length of the transgene (4.06 kb), indicating the integration of the transgene as an intact DNA fragment. Furthermore, the detection of only a single HindIII restriction fragment indicates a single integration site of the transgene into the genome in all four founders. The different-sized HindIII restriction fragments detected indicate that the intact transgene was integrated into the rat genome at different sites in the different founders.
Analysis of the four hemizygous founders at 30 weeks of age revealed different levels but parallel tissue-specific patterns of CAT expression: brain>heart>aorta; lines 90≥88>68>104 (Fig 3⇓). This tissue-specific gene expression pattern is identical to that of rat α2 Na,K-ATPase,6 23 validating the use of these transgenic models for analysis of pressure-overload response mechanisms. Three homozygous transgenic lines were established from male hemizygous founders: Tg68, Tg88, and Tg104. The Tg68 and Tg88 lines were chosen for further analysis because the optimal expression levels of the Hα2-CAT transgene in these lines (Fig 3⇓) would facilitate the analysis of transcriptional deinduction of the reporter function gene.
To assess pressure-overload transcriptional modulation of the Hα2-CAT transgene, we made homozygous transgenic Tg88 and Tg68 rats hypertensive at 20 and 28 weeks of age by subcutaneous Ang II infusion as described.6 We chose this model because BP levels could be induced acutely and measured continuously in a nonstressed manner by radiotelemetry. Most importantly, this model has been validated, showing deinduction of α2 Na,K-ATPase RNA levels in both left ventricle and aorta by 48 hours (earliest time point checked)6 in contrast to brain and skeletal muscle control tissues. BP was monitored by radiotelemetry for both Ang II–infused rats (n=18) and age-matched sham-infused controls (n=11) in a nonstressed, continuous manner. As shown in Fig 4A⇓, systolic BP increased immediately with Ang II infusion and stayed elevated during 3 days. It is interesting to note that Ang II–induced BP elevation does not abrogate the diurnal variation of BP. Diastolic and mean arterial BPs were likewise elevated (data not shown). After 72 hours, CAT assays were then done comparing cardiac left ventricle and aortas of Ang II–infused hypertensive rats with those of control age-matched, sham-infused normotensive rats. As shown in Fig 4B⇓, decreased 14C-labeled acetylated chloramphenicol (CAT activity) levels were evident in both 20- and 28-week-old hypertensive rat cardiac left ventricles and aortas compared with those of age-matched normotensive controls. This decrease in CAT activity was not observed in tissues expressing α2 Na,K-ATPase but not exposed to pressure overload (brain, skeletal muscle, and right ventricle) (Fig 4B⇓). This absence of deinduction of α2 Na,K-ATPase gene expression in brain and skeletal muscle in pressure-overload rat models is consistent with previous observations. Data on right ventricles have not been reported previously. It should be noted that the 3-day hypertensive rat hearts and aortas were not morphologically hypertrophied (no increase in wall thickness and normal ratio of left ventricular weight to body weight), as observed previously.6 The decrease in Hα2-CAT expression paralleled the decrease in cardiac and aortic endogenous rat α2 Na,K-ATPase mRNA levels as detected by RNA blot analysis in hypertensive Tg88 rats compared with controls (Fig 4C⇓). The greater decrease in CAT activity noted in transgenic rats at 28 weeks compared with transgenic rats at 20 weeks (Fig 4B⇓) despite equivalent BP elevations (Fig 4D⇓) might be due to age-specific differences or other factors currently unrecognized.
Analysis of the Variation of Response
Rather than presenting means of the study groups, we present the variation in individual responses to pressure overload as measured by quantitative densitometry of autoradiographic signals of Hα2-CAT transgene expression (Fig 4D⇑). Because of the accuracy in BP monitoring by radiotelemetry based on nonstressed 24-hour monitoring and the ability to quantitatively analyze individual transgenic rat hearts for Hα2-CAT activity, the analysis of the correlation of Hα2-CAT deinduction and degree of pressure overload per rat are valid. As Fig 4D⇑ shows, a gradient of response can be noted from systolic BP (24-hour average) levels of 150 to 190 mm Hg, indicating a greater deinduction of Hα2-CAT activity with increasing BP elevation. As expected from in vivo studies, the apparent gradient is not a perfect 1:1 correlation, but the trend is unequivocal. The correlation of systolic BP level and the degree of α2 Na,K-ATPase deinduction as measured by Hα2-CAT decrease is statistically significant (r=.82, P<.0001).
Our results provide evidence identifying the α2 Na,K-ATPase as a target gene of a pressure-overload transcriptional control mechanism in the cardiovascular system activated before morphological hypertrophy. Specificity of a cardiovascular pressure-overload response rather than an isolated direct hormonal Ang II–induced response is substantiated by the following observations: (1) In noncardiovascular tissues with Ang II receptors and α2 Na,K-ATPases (brain and skeletal muscle), no decreases in Hα2-CAT expression were observed in all experimental groups (Fig 4B⇑). This is consistent with previous data on endogenous rat α2 Na,K-ATPase mRNA levels.6 (2) In hearts exposed to minimal systolic BP elevations (<150 mm Hg) despite infusion of identical pressor doses of Ang II, Hα2-CAT expression was not decreased in left ventricles (Fig 4D⇑). (3) In the cardiac right ventricle exposed to Ang II but not exposed to systemic pressure overload, Hα2-CAT expression was not decreased (Fig 4B⇑). (4) Previous observations showed that subpressor experimental protocols do not downregulate α2 Na,K-ATPase gene expression in both heart6 11 and aorta.6 (5) A threshold of response at a systolic BP greater than or equal to 150 mm Hg was delineated in our study as well as previously,11 along with a pressure-related gradient (Fig 4D⇑) simulating the specificity of a dose-response curve. (6) An in vitro study of vascular smooth muscle cells demonstrated that Ang II does not directly modulate α2 Na,K-ATPase gene expression.24 The concordance of both in vivo and in vitro data strengthens the observation of pressure-overload deinduction of Hα2-CAT transgene expression.
The consistency of the tissue-specific and developmental regulation of the Hα2-CAT transgene in four transgenic rat lines as well as the parallel basal regulation and pressure-overload modulation of Hα2-CAT transgene and rat α2 Na,K-ATPase gene expression suggest that these observations are bona fide reflections of α2 Na,K-ATPase gene regulation in humans. This is concordant with previous observations of a decrease in α2 Na,K-ATPase mRNA in human hypertrophied hearts, albeit various technical problems associated with human heart studies were not overcome.14 The absence of a serum response element7 25 and the α-adrenergic/endothelin 1–inducible hypertrophy factor-1 element26 in the Hα2-CAT transgene suggest that the pressure-overload deinduction of α2 Na,K-ATPase most likely represents a distinct response pathway from the immediate early gene and adrenergic hypertrophy response mechanisms. Nevertheless, the distinctions and correlation of findings among these different pressure-overload/mechanical stress or hypertrophy pathways will be critical in the holistic and hierarchical dissection of pressure-overload mechanisms.
The correlation of the degree of deinduction of Hα2-CAT activity to increasing levels of pressure overload (r=.82, P<.0001) is consistent with a gradient of response previously observed in vitro in isolated perfused rat hearts.27 Additionally, the variation in the extent of deinduction of the Hα2-CAT transgene in significantly hypertensive rats (24-hour average systolic BP 180 to 185 mm Hg) (Fig 4D⇑) could also be attributed to genetic heterogeneity in the outbred Sprague-Dawley rat strain in pressure-overload responses rather than to technical experimental variation because identical experimental conditions were maintained and the accuracy of molecular and physiological measurements was ascertained. Genetic modifiers of cardiovascular pressure-overload responses are logical from pathophysiological and genetic perspectives and are consistent with the multifactorial complexity of hypertension.28 Furthermore, the parallel deinduction of the Hα2-CAT transgene and endogenous rat α2 Na,K-ATPase in both heart and aorta demonstrates the involvement of coordinated pressure-overload response transcriptional regulatory mechanisms in cardiomyocytes and vascular smooth muscle cells. This suggests a unifying hypothesis for cardiac and vascular hypertrophy, as occurs in systemic hypertension. This hypothesis remains to be tested.
Identification of the cis- and trans-acting components of this α2 Na,K-ATPase pressure-overload transcriptional response mechanism could be pivotal in understanding the development of adaptive and subsequent progression to maladaptive structural and functional changes in the heart. Identification of consensus cis-acting core sequences could help elucidate the putative panel of target genes modulated by this pressure-overload response pathway. Based on the apparent parallel regulation of human and rat α2 Na,K-ATPase gene expression, a priori regions of homology between human and rat 5′ flanking sequences would facilitate the delineation of the said pressure-overload response element. The subsequent identification of trans-acting factors and their modes of activation would facilitate the investigation of the signal transduction mechanism by which pressure overload modulates nuclear-specific events.
The physiological consequences of the deinduction of α2 Na,K-ATPase gene expression in the heart and aorta become a key question to be investigated. Although species-specific differences are expected to exist, identification of parallel pathophysiological effects will contribute key concepts in cardiovascular pressure-overload responses and a priori is the strategic initial focus. Intuitively, the parallel modes of gene regulation observed would indicate that pathophysiological consequences in rat and human cardiovascular systems would likewise be parallel. Since the isoform-specific roles of Na,K-ATPase α-subunits have not been unequivocally elucidated in both rats and humans, we can validly hypothesize only that the net qualitative change in Na,K-ATPase α-subunit isoform profile and the net quantitative change in total Na,K-ATPase amount in cardiac and vascular myocytes due to the selective decrease in α2 Na,K-ATPase could alter not only the set point but also the integrated properties of the Na+-K+ gradient in cardiac and vascular myocytes. Since the deinduction of α2 Na,K-ATPase expression persists throughout different durations of pressure overload regardless of the experimental model,6 10 11 12 13 this alteration of Na,K-ATPase isoform profile and net amount could contribute to an adaptive ouabainlike cardiac inotropy normalizing cardiac output in the face of increased pressure overload. The observed gradient of deinduction of human α2 Na,K-ATPase gene expression proportional to the degree of pressure overload in the transgenic rat model studied would support this notion. It is pertinent to note that the cardiac sodium channel gene is not modulated in pressure-overloaded rat ventricles7 and that α1 and α3 Na,K-ATPase gene expression patterns are also altered, albeit variably in different pressure-overload models.6 12 14 These observations emphasize the putative significance of the deinduction of α2 Na,K-ATPase gene expression since it is observed in all pressure-overload rat models studied as well as in human pressure-overloaded ventricles. Additionally, since rat α2 Na,K-ATPase is detected as a major isoform in the rat cardiac conduction system and in Purkinje cells at levels greater than in the rest of the myocardium,29 determination of the differential effects of pressure overload on rat and human α2 Na,K-ATPase gene expression specific to the conduction system is another key question. Deinduction of α2 Na,K-ATPase in the conduction system, whether parallel to or distinct from the rest of the myocardium, might contribute to the known dysrhythmic vulnerability of the pressure-overload hypertrophied heart. Testing these hypotheses would necessitate conditional bigenic strategies in integrated biological models as well as correlative analysis with other pressure-overload response target gene systems.
Selected Abbreviations and Acronyms
|ANF||=||atrial natriuretic factor|
|Ang II||=||angiotensin II|
|MHC||=||myosin heavy chain|
This work was supported by National Institutes of Health grant HL-48903. N.R.-O. is an Established Investigator of the American Heart Association. X.H.X. performed all rat embryo microinjections. We thank John J. Mullins for advice on transgenic rat technology, and most especially Aram V. Chobanian. We thank Joseph Loscalzo for critical reading of the manuscript. We acknowledge the excellent technical support of Fang Qing, Lyle V. Lopez, and Cathy Reardon.
- Received May 8, 1996.
- Revision received June 24, 1996.
- Revision received September 3, 1996.
Komuro I, Yazaki Y. Intracellular signaling pathways in cardiac myocytes induced by mechanical stress. Trends Cardiovasc Med. 1994;4:177-121.
Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF Jr, Gimbrone MA Jr. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element. Proc Natl Acad Sci U S A. 1993;90:4591-4595.
Herrera VLM, Chobanian AV, Ruiz-Opazo N. Isoform-specific modulation of Na,K-ATPase α subunit gene expression in hypertension. Science. 1988;241:221-223.
Chien KR, Knowlton KU, Zhu H, Chien S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J. 1991;5:3037-3046.
Sadoshima J, Jahn L, Takahashi T, Kulik T, Izumo S. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. J Biol Chem. 1992;267:10551-10560.
Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Chien KR. Segregation of atrial-specific and inducible expression of an ANF transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci U S A. 1991;88:8277-8281.
Sweadner KJ, Herrera VLM, Amato S, Moellmann A, Gibbons DK, Repke KRH. Immunologic identification of Na,K-ATPase isoforms in myocardium. Circ Res. 1993;74:669-678.
Charlemagne D, Orlowski J, Oliviero P, Rannou F, Beuve CS, Swynghedauw B, Lane LK. Alteration of Na, K-ATPase subunit mRNA and protein levels in hypertrophied rat heart. J Biol Chem. 1994;269:1541-1547.
Shull MM, Pugh DG, Lingrel JB. Characterization of the human Na,K-ATPase α2 gene and identification of intragenic restriction fragment length polymorphisms. J Biol Chem. 1989;264:17532-17543.
Herrera VLM, Ruiz-Opazo N. Regulation of α-tropomyosin and N5 genes by a shared enhancer: modular structure and hierarchical organization. J Biol Chem. 1990;265:9555-9562.
Hogan B, Costantini F, Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1990.
Orlowski J, Lingrel JB. Differential expression of the Na,K-ATPase α1 and α2 subunit genes in a murine myogenic cell line. J Biol Chem. 1988;263:17817-17821.
Ikeda U, Takahashi M, Okada K, Saito T, Shimada K. Regulation of Na,K-ATPase gene expression by angiotensin II in vascular smooth muscle cells. Am J Physiol. 1994;267:H1295-H1302.
Zhu H, Garcia A, Ross RS, Evans SM, Chien KR. A conserved 28 bp element (HF-1) in the rat cardiac myosin light chain-2 gene confers cardiac specific and α-adrenergic inducible expression in cultured neonatal rat myocardial cells. Mol Cell Biol. 1991;11:2273-2281.
Shunkert H, Jahn L, Izumo S, Apstein CS, Lorell BH. Localization and regulation of c-fos and c-jun protooncogene induction by systolic wall stress in normal and hypertrophied rat hearts. Proc Natl Acad Sci U S A. 1991;88:11480-11484.
Zahler RM, Brines M, Kashgarian M, Benz EJ Jr, Gilmore-Hebert M. The cardiac conduction system in the rat expresses the α2 and α3 isoforms of the Na,K-ATPase. Proc Natl Acad Sci U S A. 1991;89:99-103.