Cardiovascular Diabetology BioMed Central Original investigation

Background Patients with diabetic cardiomyopathy have an impaired myocardial glucose handling and distal distribution of coronary atherosclerosis. Trimetazidine, an anti-ischemic metabolic agent, improves myocardial glucose utilization though inhibition of fatty acid oxidation. Aim of the present study was to evaluate whether the metabolic effect of trimetazidine on left ventricular function in patients with diabetic cardiomyopathy. Methods 32 patients (24 males and 8 females, mean (SE) age = 67 ± 6 years) with type 2 diabetes and ischemic cardiomyopathy were randomized to receive either trimetazidine (20 mg, t.d.s.) or placebo (t.d.s.) for six months in a randomized parallel study. Patients performed an echocardiogram at baseline and after 6 months. Results Demographic data were comparable between the two groups. After six month baseline left ventricular end-diastolic diameters increased from 62.4 ± 1.7 to 63 ± 2.1 mm in the placebo group, while decreased from 63.2 ± 2.1 to 58 ± 1.6 mm (p < 0.01 compared to baseline) in the trimetazidine group. Compared to baseline, left ventricular ejection fraction increased by 5.4 ± 0.5% (p < 0.05) in the trimetazidine group while remained unchanged in the placebo group -2.4 ± 1.1% (NS), p < 0.01 between groups. A significant improvement in wall motion score index and in the E/A wave ratio was detected in patients treated with trimetazidine, but not in those receiving placebo. Conclusion in diabetic patients with ischemic heart disease trimetazidine added to standard medical therapy has beneficial effect on left ventricular volumes and on left ventricular ejection fraction compared to placebo. This effect may be related to the effect of trimetazidine upon cardiac glucose utilization.


Conclusions:
The deletion/deletion genotype of the α 2B -AR does not emerge in this study as a risk factor for AMI among members of families with type 2 diabetes; however, it might be involved in the development of hypertension.

Background
Vascular disease is the major cause of morbidity and mortality in patients with diabetes mellitus [1]. In vivo studies in humans have shown that α 2 -adrenoceptors (ARs) mediate constriction of large and small coronary arteries [2], an effect augmented by atherosclerosis [3], and mediate peripheral vasoconstriction [4]. However, lack of α 2 -AR subtype-selective drugs has precluded the clarification of the precise roles of each subtype in these responses to α 2 -AR activation. Based on studies on genetically engineered mice lacking the α 2 -AR A or B or C subtype, it has been demonstrated that the α 2B -AR subtype mediates peripheral vasoconstriction [5].
A variant form of the human α 2B -AR gene encodes a receptor protein with deletion of three glutamate residues [6]. Studies on transfected cells have revealed that this deletion variant manifests significantly impaired agonist-promoted receptor desensitization [7]. In vivo studies in humans have associated the deletion/deletion (DD) genotype with reduced flow-mediated dilatation of the brachial artery [8], and reduced coronary blood flow and increased peripheral resistance upon adrenaline infusion [9]. In a population-based, prospective study on 912 middle-aged men, the DD genotype was associated with an increased incidence of acute myocardial infarction (AMI) in comparison to the other two genotypes [10]. In another population of men who died suddenly outside of a hospital, the DD genotype was associated with increased relative risks for AMI and sudden cardiac death [11]. The relative risks for AMI and sudden cardiac death were especially high in men who died before the age of 55 [11].
To explore the possible effect of the insertion/deletion variation in the α 2B -AR gene on the risk for AMI in a population at high risk for type 2 diabetes and cardiovascular disease, we conducted a study on 154 sibling-pairs from Finland, discordant for AMI. We hypothesized that the DD genotype will be more prevalent among patients with a history of AMI than among their phenotype-discordant sib-pairs. We also explored the possibility that the DD genotype is associated with hypertension in this study population.

Study population
This study had a phenotype-discordant sib-pair design with AMI as the selection phenotype. 154 subjects (88 men, 66 women) from eastern [12] and western [13] Fin-land who reported having had an AMI were selected from families with type 2 diabetes participating in the Botnia study [13]. Each case-subject was matched for sex and age with one of his or her siblings who had not had an AMI. Characteristics of the study population are presented in Table 1. Before participating in the study, voluntary informed written consent was obtained from each subject. The study protocol was approved by the ethics committee of Lund University.
Using a multiple-risk-factor assessment equation [14], with complete data available for 79% of the study population, global risk-assessment scoring was calculated, and the age-stratified relative risk for coronary heart disease was estimated. The majority of the subjects (60%) had above average relative risk for coronary heart disease (26% moderately above average relative risk, 34% high relative risk), 27% had below average relative risk, and 13% had an average relative risk. Taking into account the relatively old age of the subjects in this population (44% over 70 years, 63% over 65 years), the absolute risk for coronary heart disease in this population may thus be considered high.

Phenotypic characterization of the study subjects
A standardized health questionnaire was filled by a trained nurse together with the subject, covering medical history, including current medication (use of any drug prescribed by a physician at the time of examination), and smoking and alcohol consumption habits. A subject was defined as a smoker if he or she had smoked for a period of at least one year.
Myocardial infarction was defined as an acute coronary event requiring hospitalization; the information was verified against the subject's hospital records.
A subject was classified as having hypertension if he or she had systolic blood pressure (BP) ≥ 160 mmHg, or diastolic BP ≥ 90 mmHg, or was treated with antihypertensive medication [15]. Diagnosis of type 2 diabetes mellitus was based upon the WHO criteria of 1998 [15].

Measurements and assays
BP was measured three times from the right arm at 5 min intervals with the subject in seated position after a 30 min rest, and the mean of the three values was used. This procedure followed well established routines of how to measure BP [16].
Serum total cholesterol, HDL cholesterol, and triglyceride concentrations were measured on a Cobas Mira analyzer (Hoffman LaRoche, Basel, Switzerland). The LDL cholesterol concentrations were calculated using the Friedewald formula.

Genotyping
Genomic DNA was extracted from whole blood using standard methods. The method used to genotype the α 2B -AR insertion/deletion polymorphism was based on PCR amplification and DNA electrophoresis, and has been described elsewhere [10].

Statistical analyses
Paired t tests (for continuous variables) and McNemar's test (for discrete variables) were used in the characterization of the population. Since the study population was sampled as sibling pairs, analysis of the entire population according to the three genotypes must take into account the possibility that error terms are not independent but are correlated between siblings. We therefore used linear mixed models with the genotype group information as a fixed factor, and the sib-pair information as a random factor to characterize the genotype groups. The effect of the α 2B -AR insertion/deletion polymorphism on the risk for AMI was estimated by the difference in the frequency of the DD genotype between the phenotype-discordant sibling pairs, and was tested using McNemar's test. Odds ratios for hypertension and their 95% confidence intervals (95% CI) were calculated using logistic regression models. Using a univariate logistic regression model, the effect of the different genotypes of the α 2B -AR insertion/ deletion variation on the odds for hypertension was explored. In multivariate logistic regression models, age, sex, smoking, diabetes, body mass index, serum LDL cholesterol, and the α 2B -AR genotype were added as covariates in a conditional stepwise fashion with p = 0.05 for entry and 0.1 for removal. Statistical computations were performed with SPSS/Win version 11.0.1 software (SPSS Inc., Chicago, IL, USA).
The prevalence of the DD genotype did not statistically significantly differ between subjects with a history of AMI and their sib-pair controls (p = 0.5): the DD genotype was carried by 34 subjects with AMI (22%), and by 39 subjects without AMI (25%). Neither did we observe any significant difference in D allele frequencies of the α 2B -AR insertion/deletion polymorphism between patients with a history of AMI (0.44) and their sib-pair controls (0.46, p = 0.65).
To explore the possible association of the α 2B -AR insertion/deletion variation and hypertension, we compared the prevalence of hypertension in the genotype groups. In subjects with no history of AMI, the DD genotype was associated with increased odds for hypertension when compared with the II genotype group (odds ratio (OR) 3.7, 95% CI 1.2-11.1, p = 0.021), or when compared with the II + ID genotype groups combined (OR 3.2, 95% CI 1.2-8.9, p = 0.026) ( Table 2). Among subjects with AMI, there were only 16 individuals who were not classified as hypertensive according to the employed criteria, and no statistically significant associations were found between the α 2B -AR genotypes and hypertension.
In subjects that were not treated with an antihypertensive drug, a trend for increased systolic and diastolic BP was observed among those carriers of the α 2B -AR DD genotype who had not had AMI (Table 3).

Discussion
The main finding of this study is a lack of association between the α 2B -AR DD genotype and AMI among members of families with type 2 diabetes. Another finding of this study is a possible association between this genotype and hypertension.

Association with AMI
Earlier, association of the α 2B -AR DD genotype with AMI was reported in a population-based prospective study on 912 middle-aged men from eastern Finland [10], and in a series of 700 unselected sudden out-of-hospital deaths of Finnish men subjected to medico-legal autopsy [11]. The populations in these studies were younger than the population of the current study, and their relative risk for coronary heart disease was expected to be similar to the general population. The lack of association between the α 2B -AR DD genotype and AMI in high-risk subjects observed in the present study may suggest that the mechanism by which the α 2B -AR deletion variant confers its observed increased risk for AMI is not directly dependent on established coronary heart disease risk factors that lead to atherosclerosis, and that in subjects at high risk for coronary heart disease the effect of this genetic variation is Results are from univariate and conditional multivariate logistic regression models. *Data are presented for the last step of the stepwise conditional insertion of variables into the models. Adjustment was done for age, sex, smoking, diabetes, BMI, and LDL cholesterol, which were inserted into a multivariate logistic regression model in a stepwise manner (p = 0.05 for entry and 0.1 for removal). N/A denotes not applicable (all the variables were rejected by the models). I, insertion; D, deletion; BMI, body mass index   diluted. This interpretation was supported by the morphometric autopsy findings of the sudden death study [11]. In that study [11], the DD genotype was associated with increased odds for AMI and sudden cardiac death, but not with the severity of coronary atherosclerosis.

Association with hypertension and BP
In subjects that had not had an AMI, the DD genotype was associated in univariate logistic regression analysis with increased odds for hypertension. However, since this analysis was conducted as further exploration of the data rather than based on an a priori hypothesis, adjustment for multiple testing should be employed. A corrected p value corresponding to 0.05 when testing 2 independent hypotheses would be 0.0253 (based on the equation α = 1 -0.95 1/N where N is the number of hypotheses tested). The observed p value for the association between the DD genotype and hypertension (about 0.02) has therefore only borderline statistical significance.
Two previous studies explored the possible involvement of the α 2B -AR insertion/deletion variation in the development of hypertension, and reported lack of association [10,18]. However, in the study on 912 middle-aged men [10], the prevalence of cardiovascular risk factors was different from that of the current study, making it difficult to compare the results of the studies. Because of the very low frequency of the DD genotype (n = 3) among the subjects in the study on 155 sib-pairs concordant for hypertension [18], that study can be considered to have insufficient power to detect linkage between the α 2B -AR DD genotype and the studied phenotype.
Several earlier studies have suggested a role for the α 2B -AR in the development of hypertension. α 2 -adrenergic vasoconstriction in humans [4] is probably mainly mediated by the α 2B -AR subtype [5], and the observed decreased agonist-promoted desensitization property of the deletion variant [7] may suggest that the DD genotype confers increased vasoconstriction that leads to increased peripheral resistance -a common finding in hypertension [19]. Furthermore, rodent studies suggest a significant role for the α 2B -AR in acquired and hereditary hypertension [20][21][22]. Additionally, loci on human chromosome 2 have been linked with increased BP and hypertension in several recent genome-wide studies [23,24]. So far, the location of the human α 2B -AR gene (ADRA2B, GeneBank accession number M34041) on chromosome 2 has been tentatively placed at 2p13-q13, but more precise mapping will be needed to confirm or exclude the α 2B -AR gene as the hypertension risk gene involved in these genome-wide linkage study results.

Methodological considerations
Using a health questionnaire, a subject was categorized as a smoker if he or she had smoked for a period of at least one year. This information may be considered insufficient to provide a complete estimation of the impact of this risk factor on the total risk for AMI.
Interpretation of results from studies with a case-control design, where the studied risk factor may also affect survival, such as in the present study, is not straightforward. Inherently by the study design, only survivors of an acute coronary event were included as index cases in the present study -possibly creating selection bias. Such an effect was demonstrated by Hamajima et al. [17], who showed that the effects of genotypes increasing disease risk and fatality rate are underestimated, while those increasing the risk and improving prognosis are overestimated.
It should also be acknowledged that the apparently unaffected sibs were themselves at higher risk for AMI on the basis of family history, both by belonging to high-risk type 2 diabetes families and by having a sib with AMI. Thus, the unaffected sib may also become affected in a short time.

Conclusions
To conclude, the results of this study suggest that the α 2B -AR insertion/deletion variation either does not play a significant role in AMI morbidity in members of families with type 2 diabetes that are at high risk for cardiovascular diseases, or that by affecting mortality, the DD genotype could be underrepresented among the surviving AMI cases. We also propose a role for this genetic variation in the development of hypertension; however, further studies are required to replicate this finding.