The transcription factor GATA4 is activated by extracellular signal-regulated kinase 1- and 2-mediated phosphorylation of serine 105 in cardiomyocytes.

The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonist-induced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

p300 Functions as a coactivator of transcription factor GATA-4.

Transcription factor GATA-4 plays critical roles in controlling heart development and cardiac hypertrophy. To understand how GATA-4 functions under diverse conditions, we sought to identify its coactivators. We tested p300 as a coactivator in GATA-4-dependent transient transcription assays in NIH3T3 cells and found that p300 synergistically activated GATA-4-dependent transcription on both synthetic and natural promoters. Direct physical interactions between the N- and C-zinc finger domains of GATA-4 and the cysteine/histidine-rich region 3 (C/H3) of p300 were identified in immunoprecipitation and glutathione S-transferase pull-down experiments. Deletion of the C/H3 region of p300 abolished its coactivator activity indicating that the physical interaction was required for functional synergy. Through the use of a series of GATA-4 zinc finger mutants, the amino acids WRR in the C finger were identified as critical to the interaction. The adenoviral E1A protein or a peptide encoding the C/H3 region of p300 could inhibit GATA-4-dependent transcription, presumably by competing for p300 binding. Furthermore, deletion of the region of p300 encoding the histone acetyltransferase activity abolished its effect on GATA-4-dependent transcriptional activity. These results establish that p300 acts as a GATA-4 coactivator and that the p300 histone acetyltransferase activity is necessary for the functional interaction.

The transcription factors GATA4 and GATA6 regulate cardiomyocyte hypertrophy in vitro and in vivo.

The zinc finger-containing transcription factors GATA4 and GATA6 are important regulators of basal and inducible gene expression in cardiac and smooth muscle cell types. Here we demonstrate a direct functional role for GATA4 and GATA6 as regulators of cardiomyocyte hypertrophic growth and gene expression. To model the increase in endogenous GATA4 and GATA6 transcriptional activity that occurs in response to hypertrophic stimulation, each factor was overexpressed in cardiomyocytes using recombinant adenovirus. Overexpression of either GATA4 or GATA6 was sufficient to induce cardiomyocyte hypertrophy characterized by enhanced sarcomeric organization, a greater than 2-fold increase in cell surface area, and a significant increase in total protein accumulation. In vivo, transgenic mice with 2.5-fold overexpression of GATA4 within the adult heart demonstrated a slowly progressing increase in heart to body weight ratio, histological features of cardiomyopathy, and activation of hypertrophy-associated genes, suggesting that GATA factors are sufficient regulators of cardiomyocyte hypertrophy in vitro and in vivo. To evaluate the requirement of GATA factors as downstream transcriptional mediators of hypertrophy, a dominant negative GATA4-engrailed repressor fusion-encoding adenovirus was generated. Expression of GATA4-engrailed blocked GATA4- and GATA6-directed transcriptional responses and agonist-induced cardiomyocyte hypertrophy, demonstrating that cardiac-expressed GATA factors are necessary mediators of this process.

Selective opportunistic screening for hypercholesterolemia in primary care practice.

OBJECTIVES: To assess the performance of selective opportunistic screening in a primary care group practice. DESIGN: Cross-sectional survey of coronary heart disease risk factors and retrospective chart audit of cholesterol testing. SETTING: Capitation-funded primary care group practice in Ontario, Canada. SUBJECTS: 7785 enrolled patients between the ages of 20 and 69 years. INTERVENTION: Protocol-based selective opportunistic screening program for hypercholesterolemia of 45 months duration. MAIN OUTCOME MEASURES: Targeting (proportion of screening tests that were appropriate), coverage (proportion of those meeting screening criteria who had a screening test performed), over-screening (proportion of those not meeting screening criteria who had a screening test performed), and screening ratio (likelihood that a screening test was performed on an individual who met screening criteria rather than one who failed to meet screening criteria). RESULTS: 64.7% of patients tested met the practice criteria for screening. 37.7% of patients who met the practice screening criteria were tested and 24.9% of those not meeting practice screening criteria had a cholesterol test performed. The screening ratio was 1.52. CONCLUSION: Our findings bring into question the effectiveness of opportunistic approaches to preventive care.

The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression.

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.

Screening for hypercholesterolaemia in primary care: randomised controlled trial of postal questionnaire appraising risk of coronary heart disease.

OBJECTIVES: To validate a self administered postal questionnaire appraising risk of coronary heart disease. To determine whether use of this questionnaire increased the percentage of people at high risk of coronary heart disease and decreased the percentage of people at low risk who had their cholesterol concentration measured. DESIGN: Validation was by review of medical records and clinical assessment. The questionnaire appraising risk of coronary heart disease encouraged those meeting criteria for cholesterol measurement to have a cholesterol test and was tested in a randomised controlled trial. The intervention group was sent the risk appraisal questionnaire with a health questionnaire that determined risk of coronary heart disease without identifying the risk factors as related to coronary heart disease; the control group was sent the health questionnaire alone. SETTING: One capitation funded primary care practice in Canada with an enrolled patient population of about 12 000. SUBJECTS: Random sample of 100 participants in the intervention and control groups were included in the validation exercise. 5686 contactable patients aged 20 to 69 years who on the basis of practice records had not had a cholesterol test performed during the preceding 5 years were included in the randomised controlled trial. 2837 were in the intervention group and 2849 were in the control group. MAIN OUTCOME MEASURES: Sensitivity and specificity of assessment of risk of coronary heart disease with risk appraisal questionnaire. Rate of cholesterol testing during three months of follow up. RESULTS: Sensitivity of questionnaire appraising coronary risk was 87.5% (95% confidence interval 73.2% to 95.8%) and specificity 91.7% (81.6% to 97.2%). Of the patients without pre-existing coronary heart disease who met predefined screening criteria based on risk, 45 out of 421 in the intervention group (10.7%) and 9 out of 504 in the control group (1.8%) had a cholesterol test performed during follow up (P<0.0001). Of the patients without a history of coronary heart disease who did not meet criteria for cholesterol testing, 30 out of 1128 in the intervention group (2.7%) and 18 out of 1099 in the control group (1.6%) had a cholesterol test (P=0.175). Of the patients with pre-existing coronary heart disease, 1 out of 15 in the intervention group (6.7%) and 1 out of 23 in the control group (4.3%) were tested during follow up (P=0.851, one tailed Fisher's exact test). CONCLUSIONS: Although the questionnaire appraising coronary risk increased the percentage of people at high risk who obtained cholesterol testing, the effect was small. Most patients at risk who received the questionnaire did not respond by having a test.

A calcineurin-dependent transcriptional pathway for cardiac hypertrophy.

In response to numerous pathologic stimuli, the myocardium undergoes a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes. We show that cardiac hypertrophy is induced by the calcium-dependent phosphatase calcineurin, which dephosphorylates the transcription factor NF-AT3, enabling it to translocate to the nucleus. NF-AT3 interacts with the cardiac zinc finger transcription factor GATA4, resulting in synergistic activation of cardiac transcription. Transgenic mice that express activated forms of calcineurin or NF-AT3 in the heart develop cardiac hypertrophy and heart failure that mimic human heart disease. Pharmacologic inhibition of calcineurin activity blocks hypertrophy in vivo and in vitro. These results define a novel hypertrophic signaling pathway and suggest pharmacologic approaches to prevent cardiac hypertrophy and heart failure.

Health years equivalents as a measurement of preferences for dental interventions.

OBJECTIVE: To test the feasibility and importance of measuring preferences among treatment choices using Healthy Years Equivalents (HYE). DESIGN: Development of scenarios for alternative approaches to caries treatment. Completion of category rating and standard gamble questions elicited in personal interviews. SETTING: The provision of dental care to children in a public-funded dental health clinic. PARTICIPANTS: Random sample of the adult population of Hamilton, Ontario. MAIN OUTCOME MEASURES: The percentage of the sample unable to complete the interviews, time taken to perform interviews, ease of understanding of interviews, correlation between rank ordering and HYE scores. RESULTS: Ninety-six per cent of the sample were able to complete the HYE exercise. Inconsistencies between HYE scores and rank orders implying preference reversal occurred in 6% of those completing HYE scores for the two scenarios. The additional time taken by the HYE was of the order of 17 minutes but increased with the age of the subject. Where problems occurred, they were related to the method of measurement or the sensitivity of the chosen scale as opposed to additional requirements of the HYE. There was some evidence that HYEs and QALYs produced different scores even in the context of chronic constant health states. CONCLUSIONS: HYEs are a feasible and important practical method of measuring preferences among interventions. Alternative utility-based approaches, such as willingness to pay, may be required to detect differences in modest improvements in temporary health states.

cis-Acting sequences that mediate induction of beta-myosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction.

BACKGROUND: Marked alterations in the expression of specific genes occur during the development of cardiac hypertrophy in vivo. Little is known, however, about the cis-acting elements that mediate these changes in response to clinically relevant hypertrophic stimuli, such as hemodynamic overload, in intact adult animals. METHODS AND RESULTS: The left ventricular expression of a directly injected reporter gene driven by 3542 bp of rat beta-myosin heavy chain (beta-MHC) promoter was increased 3.0-fold by aortic constriction (P<.005), an increment similar to the 3.2-fold increase in the level of the endogenous beta-MHC mRNA in the same left ventricles. Subsequent analysis identified a 107-bp beta-MHC promoter sequence (-303/-197) sufficient to convert a heterologous neutral promoter to one that is activated by aortic constriction. These sequences contain two M-CAT elements, which have previously been demonstrated to mediate inducible expression during alpha1-adrenergic-stimulated hypertrophy in cultured neonatal cardiac myocytes, and a GATA element. Although simultaneous mutation of both M-CAT elements markedly decreased the basal transcriptional activity of an injected 333-bp beta-MHC promoter, it had no effect on aortic constriction-stimulated transcription (3.5-fold increase, P<.005 for both wild type and mutant). In contrast, mutation of the GATA motif markedly attenuated aortic constriction-stimulated transcription (1.6-fold, P=NS) without affecting the basal transcriptional activity. This GATA site can interact with in vitro translated GATA-4 and compete with an established GATA site for GATA-4 binding activity in nuclear extracts from aortic constricted hearts. CONCLUSIONS: Basal and aortic constriction-stimulated transcription of the beta-MHC gene is mediated, at least in part, through different mechanisms. A GATA element within beta-MHC sequences -303/-197 plays a role in the transcriptional activation of this gene by aortic constriction.

Casting the screening net: separating big fish from little fish.

Screening tests are a rapidly growing part of medical practice. If we are going to make the best use of resources, screening tests need to be considered in terms of effectiveness, efficiency and equity. We present a framework as a way to think about screening programmes. The framework expands on existing literature that recognizes two categories of screening: universal and opportunistic. By adding the dimension of 'selectivity', we identify four categories of screening: active non-selective (universal or mass screening), active selective, opportunistic non-selective and opportunistic selective. We illustrate the framework by categorizing screening recommendations for high serum cholesterol levels. We conclude there is no one ideal strategy for screening that simultaneously satisfies criteria of effectiveness, efficiency and equity. However, our framework allows a systematic consideration and balancing of these objectives in the development and assessment of screening programs. In this way, it may assist decision-makers by making this trade-off more explicit.

Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload.

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the alpha-skeletal actin promoter 1.7-fold whereas the alpha-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1. 6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

Alpha-myosin heavy chain gene regulation: delineation and characterization of the cardiac muscle-specific enhancer and muscle-specific promoter.

The alpha-myosin heavy chain (alpha-MHC) gene encodes a cardiac muscle-specific protein involved in active force generation. The mechanism responsible for restricting expression of this gene to the heart should provide clues for the identification of transcriptional regulatory events involved in the induction and maintenance of the cardiac cell lineage. In this report we dissect the alpha-MHC regulatory region to identify the components necessary for directing high levels of cardiac muscle-restricted expression. Deletion, site-specific mutant and heterologous promoter constructs were assayed for expression after injection into adult rat heart and skeletal muscle or transfection into non-muscle cells. These studies indicated that sequences from -344 to -156 directed high levels of cardiac-muscle specific expression from a heterologous promoter that was independent of position and orientation. This region includes a previously uncharacterized CArG box, alpha-MHC sequences from -86 to +16 promoted activity from two heterologous enhancers in a muscle-specific fashion. Mutational analysis of an E-box and a CArG box within the promoter revealed that they act as negative and positive regulatory elements, respectively. Based on competitive binding and supershift electrophoretic mobility shift assays, serum response factor was shown interact with the CArG boxes found in the promoter and enhancer. Similar experiments demonstrated that the E-box bound to a factor immunologically related to upstream stimulatory factor. Together, these results identify two distinct regions with different regulatory function that are critical for the tissue restricted expression of the alpha-MHC gene.

Is small really beautiful? Some thoughts on the 1995 federal budget.

The 1995 federal budget meets the financial markets' expectations by proposing deep funding cuts and defining a smaller federal presence in several sectors of Canadian society. The most significant aspect of the budget for health care is the creation of the Canada Health and Social Transfer, which merges federal funding for health, post-secondary education and social assistance into one block grant. Provinces will be free to shift funds among these programs.

Review of the multi-hospital arrangements literature: benefits, disadvantages and lessons for implementation.

The multi-hospital arrangements literature is reviewed for Canada and the United States. There is a notable lack of evaluations on the outcomes of these arrangements, especially in the Canadian context. For evaluations that do make it to the literature, generalizability of conclusions is difficult because most is based on case studies and relates to "for-profit" U.S. hospitals. We are forced to conclude, however, that there is little definitive evidence that quality of care is improved by multi-hospital arrangements or to support or refute the claims of better human resources deployment. The most striking organizational benefit appears to be that institutions considering merger or other arrangements are forced into explicit considerations of their mission and goals. Many of the potential disadvantages of multi-hospital arrangements may be ameliorated with appropriate strategic planning and attention to detail during negotiation of the arrangement. As new multi-institutional arrangements may cause harm as well as reap benefits, careful evaluation is needed.

An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo.

Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy.

Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene.

The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.

Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo.

A myocyte-specific enhancer-binding factor (MEF-2) DNA binding site was identified in the rat alpha-myosin heavy chain (MHC) gene adjacent to the E-box binding site for alpha-MHC binding factor-2 (BF-2). Mutation of the MEF-2 site, within the context of the full-length promoter, reduced activity by 85 and 80% in neonatal cardiomyocytes and the adult heart, respectively. Mutation of the BF-2 site reduced activity approximately 70% in both models. A MEF-2/BF-2 double mutant gave significantly less activity than the BF-2 mutant but not the MEF-2 mutant, suggesting the possibility that BF-2 and MEF-2 interact. Mutations in MEF-2, which decreased functional activity, also abolished MEF-2 DNA binding activity. MEF-2 DNA binding activity was present in the developing heart, reached a peak in the late fetal and early neonatal stages, and then declined to low levels in the adult heart. The adult levels were sufficient to support alpha-MHC gene expression. MEF-2 activity was increased 2-3-fold in the adult heart subjected to a pressure or volume overload. Two working models are proposed as possible explanations of the antithetic relationship between MEF-2 levels and alpha-MHC gene expression.

Clofibrate prevents the development of hypertension in Dahl salt-sensitive rats.

We have reported that cytochrome P-450-dependent omega-hydroxylation of arachidonic acid is reduced in microsomes prepared from the renal outer medulla of Dahl salt-sensitive (SS/Jr) rats, but the functional significance of this observation is unknown. The present study examined whether long-term induction of renal fatty acid omega-hydroxylase with clofibrate would alter the development of hypertension in Dahl SS/Jr rats. Dahl SS/Jr rats were placed on a high salt diet (8.0% NaCl) and given either vehicle or clofibrate (80 mg/day) in their drinking water. After 4 weeks of a high salt diet, mean arterial pressure averaged 170 +/- 3 mm Hg in vehicle-treated (n = 17) and 127 +/- 2 mm Hg in clofibrate-treated (n = 19) SS/Jr rats. Clofibrate had no effect on arterial pressure in Dahl salt-resistant rats. The antihypertensive effect of clofibrate was reversible. Mean arterial pressure rose from 131 +/- 4 to 182 +/- 8 mm Hg in the first week after clofibrate treatment (n = 6) was discontinued. Clofibrate had no effect on arterial pressure in SS/Jr rats (n = 9) in which hypertension was already established by feeding the rats a high salt diet for 4 weeks before the study. In clofibrate-treated SS/Jr rats (n = 12), the omega-hydroxylation of arachidonic and lauric acids by renal cortical and outer medullary microsomes was greater than that seen in vehicle-treated rats (n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)