Transgenic animal models: new avenues in cardiovascular physiology.

Application of molecular genetic tools to inherited cardiovascular disorders has provided important insights into the molecular mechanisms underlying cardiomyopathies, arrhythmias, blood pressure regulation, and atherosclerosis. In addition, alteration of gene expression has been observed under common cardiovascular conditions such as cardiac hypertrophy and heart failure. Recent advances in transgenic and gene-targeting approaches allow a sophisticated manipulation of the mouse genome by gene addition, gene deletion, or gene modifications. These transgenic models enable the dissection of in vivo pathways responsible for these complex disease phenotypes. This review describes tissue-specific promoters suitable for targeting candidate genes to the cardiovascular system as well as a number of valuable transgenic animal models of blood pressure regulation, atherogenesis, defects in the coagulation system, cardiac hypertrophy, myocarditis, cardiomyopathies, and heart failure. Limitations and difficulties associated with these transgenic approaches are discussed. Animal models which may provide a basis for future gene therapy of cardiovascular diseases are introduced. Finally, methods are described to regulate the spatial and temporal expression level of a transgene, to inactivate a target gene in a tissue-specific manner, and to introduce specific mutations into the genome. These recent advances in transgenic technology are expected to have a considerable impact on cardiovascular research in the near future.

Transcription of the rat sarcoplasmic reticulum Ca2+ adenosine triphosphatase gene is increased by 3,5,3'-triiodothyronine receptor isoform-specific interactions with the myocyte-specific enhancer factor-2a.

Thyroid hormone (T3) increases the transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) gene (SERCA 2) through three thyroid hormone response elements. The existence of repetitive cis elements with different configurations is likely to serve specific functions such as interactions with nuclear transcription factors. In addition, the presence of different T3 receptor isoforms (T3Rs) may contribute to another level of complexity in providing specificity for T3 action. In this study, we investigated T3R alpha 1-vs. T3R beta 1-specific interactions with the myocyte enhancer-specific factor-2 (MEF-2) on the expression of the SERCA 2 gene in transient transfection assays in embryonal heart-derived H9c2 cells. MEF-2a in combination with either T3R alpha 1 or T3R beta 1 isoforms resulted in a 2.5-fold increase in SERCA 2 transgene expression in the absence of T3. Addition of T3 did not induce any further increase in SERCA 2 expression when T3R alpha 1 and MEF-2a expression vectors were cotransfected. In contrast, in the presence of T3R beta 1 and MEF-2, the addition of T3 increased chlorampenicol acetyltransferase activity by an additional 2.2-fold to a total 5.5-fold increase. The interaction between MEF-2a and T3R is transcription factor specific because another factor that binds to MEF-2 consensus sites (heart factor 1b) was not able to interact with T3R. In addition, MEF-2a failed to interact with other nuclear factors (cAMP response element-binding protein and Egr-1) that stimulate SERCA 2 gene transcription. In addition, we found that a single homologous thyroid hormone response element is not able to mediate the interactions between MEF-2a and T3Rs to increase SERCA 2 gene transcription. Our findings point to T3R isoform-specific interactions with a cell type-specific transcription factor (MEF-2) in the regulation of SERCA 2 gene expression.

Phorbol myristate acetate-induced hypertrophy of neonatal rat cardiac myocytes is associated with decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2) gene expression and calcium reuptake.

The decreased expression of the sarcoplasmic reticulum Ca(2+)-ATPase associated with cardiac hypertrophy was investigated in cultured neonatal rat cardiac myocytes. Northern blot analysis indicated a significant 55-60% decrease in Ca(2+)-ATPase mRNA levels and after 12 and 24 h of treatment with the phorbol ester phorbol myristate acetate (PMA). Myocytes treated with the phorbol ester for 80 h showed a significant 34% decrease (relative to vehicle-treated control cells) in the levels of Ca(2+)-ATPase protein, and a significant 38% increase in the levels of alpha-sarcomeric actin, as assessed by Western blot analysis using specific antibodies. Immunocytochemistry of myocytes treated for 72 h with the phorbol ester revealed a hypertrophied cell morphology, and showed a marked decrease in Ca(2+)-ATPase staining intensity. Contractile calcium transients were evaluated through the use of indo-1. It was found that the t1/2 for the decline of calcium transient was significantly prolonged by PMA treatment (0.51 +/- 0.15) when compared to controls (0.38 +/- 0.17, P < 0.001). Treatment of myocytes with endothelin-1 also led to a 35% decrease in sarcoplasmic reticulum Ca(2+)-ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca(2+)-ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca(2+)-ATPase gene expression as observed in vivo in the hypertrophied and failing heart. The observed prolongation in t1/2 for [Ca2+]i decline might be due to the observed depressed levels for sarcoplasmic reticulum Ca(2+)-ATPase in PMA treated cells.

Negative regulation of the rat Na-K-ATPase alpha 3-subunit gene promoter by thyroid hormone.

Na-K-ATPase alpha 3-subunit mRNA levels are both positively and negatively controlled by thyroid hormone [3,5,3'triiodothyronine (T3)] in primary cultures of neonatal rat cardiac myocytes. In this study, transient transfection analysis indicated that two regions of the rat alpha 3 gene between nucleotides -116 and -6 and -6 and +80 conferred T3-mediated inhibition of reporter gene expression. Electrophoretic mobility shift assays showed specific binding of T3 receptor monomers and T3 receptor-retinoid X receptor heterodimers at each alpha 3 gene negative T3-response region. The alpha 3 gene region from -116 to -6 base pairs also mediates repression in response to retinoic acid (RA) and binds RA receptor. In the absence of ligand, reporter gene expression driven by the -116 to -6-base pair region is repressed with cotransfection of T3 receptor, whereas it is unaffected by overexpression of RA receptor. These data demonstrate that the proximal promoter of the rat Na-K-ATPase alpha 3 gene contains sequence motifs that mediate repression of alpha 3 gene transcription in response to either T3 or RA in neonatal rat cardiac myocytes.

Heart muscle-specific gene expression using replication defective recombinant adenovirus.

Adenoviruses are a promising vector system for future gene therapy of heart muscle diseases. The promiscuous tissue tropism of adenoviruses, however, may lead to the undesirable expression of putative therapeutic genes in nontarget cells and hence to considerable safety limitations for this vector system. To restrict gene expression to cardiomyocytes we constructed an adenoviral vector (Ad-mlcLuc) in which the luciferase gene is under the control of the ventricle-specific myosin light chain-2 (mlc-2v) promoter. For controls, we constructed a recombinant adenovirus without promoter (Ad-Luc) and one with the Rous sarcoma virus (RSV) promoter (Ad-rsvLuc). Our data demonstrate that the newly established viral vector Ad-mlcLuc was specifically active in rat neonatal cardiomyocytes in vitro but not in three established cell lines. Injections of the recombinant adenoviruses into the cardiac cavity of neonatal rats resulted in myocardial specific gene expression of Ad-mlcLuc in vivo, despite the fact that viral DNA was detected by PCR at different levels in all tissues investigated. In vitro and in vivo, Ad-mlcLuc was exclusively active in cardiac muscle cells, reaching 8-9% of the RSV-induced luciferase activity. Direct injection of Ad-mlcLuc into thigh muscle gave only background luciferase activity (0.05% of Ad-rsvLuc). Therefore, in the adenoviral system, the mlc-2v promoter allows heart-specific expression of a foreign gene thus providing a promising tool for gene transfer targeted to the myocardium.

Delineation of three different thyroid hormone-response elements in promoter of rat sarcoplasmic reticulum Ca2+ATPase gene. Demonstration that retinoid X receptor binds 5' to thyroid hormone receptor in response element 1.

Thyroid hormone (3,5,3'-triiodothyronine) positively regulates transcription of the sarcoplasmic reticulum Ca2+ATPase gene in rat heart, and sequences within 559 nucleotides upstream from the transcription start site confer thyroid hormone responsiveness upon a reporter gene. In the present study, three thyroid hormone-response elements (TREs) are identified between nucleotides -485 and -190. Each TRE is active in transient transfection assays and specifically binds 3,5,3'-triiodothyronine receptors (TRs) alpha 1 and beta 1 alone and in combination with retinoid X receptors (RXRs) alpha and beta. TRE 1 is a direct repeat of two half-sites separated by four nucleotides; TREs 2 and 3 are inverted palindromes of two half-sites separated by four and six nucleotides, respectively. Methylation interference analysis of TRE 1 showed binding of a TR alpha 1 monomer to the 3' half-site, whereas the heterodimer contacts both half-sites. Subsequent studies employed TR beta and RXR alpha mutants in which their P-boxes were replaced with the P-box of the glucocorticoid receptor. Bandshifts of wild type and mutant proteins with either wild type TRE 1 or a mutant version, in which the 5' half-site was converted to a glucocorticoid response element half-site, demonstrated preferential binding of RXR to the 5' half-site and of TR to the 3' half-site of TRE 1.

Influence of thyroid hormone and retinoic acid on slow sarcoplasmic reticulum Ca2+ ATPase and myosin heavy chain alpha gene expression in cardiac myocytes. Delineation of cis-active DNA elements that confer responsiveness to thyroid hormone but not to retinoic acid.

The mRNA encoding the sarcoplasmic reticulum (SR) Ca2+ ATPase is highly influenced by thyroid hormone (T3) in the hearts of intact animals. We show here that this effect of T3 can be mimicked in primary neonatal rat cardiocytes, both in serum-containing and in serum-free media; the expression of SR Ca2+ ATPase mRNA is myocyte-specific and is also modulated by retinoic acid (RA). RA also induces myosin heavy chain (MHC) alpha-mRNA in this system. The induction of Ca2+ ATPase mRNA is sensitive to T3 (EC50 approximately 30 pM) and less sensitive to RA (EC50 approximately 2 nM). Transient transfection experiments utilizing various segments of the Ca2+ATPase promoter fused to the reporter gene chloramphenicol acetyltransferase (CAT) indicate a minimal thyroid hormone response element (TRE) between nucleotides -262 and -322, while sequences between -322 and -559 are required for maximal trans-activation. RA is not able to regulate these constructs. Likewise, a clear effect of T3 but no effect of RA was observed when the CAT gene was driven by a TRE derived from the rat alpha-MHC gene. In contrast, CAT expression was induced by either hormone when placed under the control of a synthetic palindromic TRE. Taken together, these results indicate that T3 and RA induce gene expression in primary cardiac myocytes, but through distinct response elements and/or mechanisms.

Amiodarone reduces the effect of T3 on beta adrenergic receptor density in rat heart.

The effect of injection of 1 mg/kg triiodothyronine on cardiac beta-adrenoceptor state was investigated in hypothyroid rats and compared to the effect in hypothyroid rats pretreated with amiodarone (200 mg/kg/day for 8 days). The Kd values of iodocyanopindolol binding to the beta-receptors were not influenced by either T3 injection or by amiodarone treatment. In the absence of amiodarone, injection of triiodothyronine resulted in a small decrease in receptor density at 6 hr, followed by an increase at 24 hr. Rats treated with amiodarone showed a similar response pattern to hormone injection (i. e. a small decrease in receptor density at 6 hr, followed by an increase at 24 hr), but the amplitude of the response was significantly reduced. Moreover, in vehicle injected rats amiodarone treatment resulted in a decrease in receptor density when rats were mildly hypothyroid, but not when rats were severely hypothyroid. It is concluded that amiodarone interferes (directly or indirectly) with thyroid hormone action in the heart.

Nuclear 3,5,3'-triiodothyronine receptor occupancy, phosphoenolpyruvate carboxykinase (PEPck) messenger ribonucleic acid levels and PEPck enzyme activity in rat liver.

The expression of the gene coding for phosphoenolpyruvate carboxykinase (PEPck) is under complex multi-hormonal control. A stimulatory effect of T3 on the expression of this gene has been described, but it has not been related to nuclear T3 receptor occupancy. We therefore studied simultaneously nuclear T3 receptor occupancy, PEPck enzyme activities, and PEPck messenger RNA (mRNA) levels in the liver of thyroidectomized, T3-treated rats. A dose-dependent increase in receptor occupancy and in enzyme activities was observed 24 h after a single ip injection of T3. However, no increase in the levels of PEPck mRNA was observed, using a specific complementary DNA (pPCK10). To evaluate this further, time-course experiments were performed over a 24 h period after a single ip injection of a receptor saturating dose of T3. These experiments showed simultaneous increases of PEPck enzyme activities and PEPck mRNA levels. Receptor occupancy had increased to 95% 3 h after injection of T3, whereas maximal levels of enzyme activity and mRNA were obtained 6-12 h after T3 administration. Translational efficiency did not change during induction. Twenty-four hours after T3 administration, mRNA levels and enzyme activity had declined, but receptor occupancy was still maximal. We conclude that T3 influences PEPck gene expression primarily at a pretranslational (transcriptional) level. The transient increase in PEPck mRNA despite persisting full receptor occupancy suggests a modification in time of the interaction between the T3-receptor complex and the regulatory region of the PEPck gene.

Effects of amiodarone on thyroid hormone-responsive gene expression in rat liver.

The hypothesis that amiodarone (AM) acts by inducing a local 'hypothyroid-like' state in thyroid hormone-responsive tissues was investigated in rat liver. Hypothyroid rats, pretreated orally for 8 consecutive days with AM (200 mg/kg) or water, were given a single i.p. injection of equimolar doses of T4, T3 or rT3 (1.00 to 1.20 mg/kg). Six hours later, the rats were killed and liver nuclear T3 receptor occupancy was determined. Simultaneously, the activity of two thyroid hormone-responsive enzymes was measured, together with the levels of their respective mRNAs by hybridization to specific cDNAs. The enzymes were phosphoenolpyruvate carboxykinase and glutamine synthetase. AM showed no effect on nuclear T3 receptor occupancy in rats injected with either vehicle, rT3, or T3, but it completely blocked the increase in receptor occupancy in rats injected with T4. With regard to postreceptor effects, T4 and T3 elicited an approximately two-fold increase in the levels of the mRNAs coding for the two enzymes, whereas rT3 had no effect. The increase of the two mRNAs was potentiated by AM, but this is probably secondary to an AM-induced state of anorexia. Remarkably, this potentiating effect of AM was not observed at the protein-level: enzyme activities were lower in rats pretreated with AM. AM-pretreatment thus results in lower enzyme activity to mRNA ratios for both enzymes, irrespective of hormonal treatment. Therefore, although no conclusions can be drawn about possible effects of AM at the transcriptional level, it is concluded that AM interferes with thyroid hormone responsive gene expression in rat liver at a post-transcriptional level. As a consequence, in the present experimental design the livers of AM-treated rats resemble the liver of hypothyroid rats with regard to specific enzyme activities, but not with regard to either nuclear T3 receptor occupancy or the levels of specific mRNAs.

Nuclear reverse T3 binding sites: an artefact of isolation?

Binding of [125I]rT3 to rat liver nuclear extracts prepared in 0.25 M sucrose could be abolished by a prior wash of the nuclei with 2.4 M sucrose. Analysis of mixtures containing [125I]rT3 and nuclear extracts (0.25 M sucrose) showed that after incubation for 2 h at 22 degrees C, degradation of rT3 into 3,3'-T2 and I- had taken place. It appears that the presence of 125I- in these mixtures can account for the previously observed 'binding' of [125I]rT3 to these nuclear extracts. Further characterization of the deiodination process in nuclear extracts showed: 1) inactivation by heating, 2) production of equimolar amounts of I- and 3,3'-T2, 3) stimulation by sulfhydryl compounds and inhibition by propylthiouracil in a fashion similar to the microsomal iodothyronine 5'-deiodinase (ping-pong mechanism). We conclude that the observed deiodination of rT3 in hepatic nuclear extracts is of enzymatic nature, due to contamination of the nuclear preparation by microsomal iodothyronine 5'-deiodinase. However, since the nuclei are prepared in the presence of the non-ionic detergent Triton X-100, a nucleus associated deiodinase activity cannot be totally excluded.