Population

Thirty patients with mild heart failure (HF) were included in the study. Inclusion criteria were heart failure with a Left Ventricular Ejection Fraction (LVEF) of 35% or below, documented by echocardiography at the time of entering the study and stable NYHA class I-II. Decompensated heart failure, beta blocker intolerance, uncontrolled hypertension, hypotension and bradycardia were all exclusion criteria for entering the study. Patients were secondarily excluded from the study if they became clinically unstable or had to change medical treatment during the study period. Ten volunteers with no documented cardiovascular disease, no diabetes and no medication use were enrolled as control group for comparison on baseline measurements.

Patient recruitment was done by advertisement in newspapers and from an out-patients clinic. Informed consent was given before entering the study. The study was approved by the ethics committee of the city of Copenhagen (ref KF 02-071/03), as well as the Danish Medicines Agency (ref 2612-2423) and registered at clinical trials.gov (ref NCT00497003).

Design

Before randomization all patients were treated with carvedilol for at period of at least two months to ensure equal use of beta blockers at baseline. Patients receiving beta blocker treatment at the time they were included in the study, had their beta blocker treatment changed to carvedilol. If the patients were beta blocker naïve at the time of inclusion, they started treatment with carvedilol and titrated to the highest tolerable dose. The patients were then randomized to receive treatment with carvedilol, metoprolol tartrate or metoprolol succinate for a period of two months. The group of patients who continued on carvedilol treatment served as time-control. Patients were otherwise kept on their usual medication throughout the whole study period.

Ten patients were randomized to receive carvedilol with a target dose of 50 mg a day; ten patients were randomized to receive metoprolol succinate with a target dose of 200 mg a day and ten patients were randomized to receive metoprolol tartrate with a target dose of 200 mg a day.

The study was designed as an open parallel group study. Before and after the two months randomization period an examination of endothelial function as well as insulin stimulated endothelial function was performed by venous occlusion plethysmography as described in the following section. Forearm glucose uptake was measured during intra-arterial insulin infusion both before and after the treatment period. 24 hour blood pressure and heart rate measurements were also done before and after the two months randomization period.

Venous occlusion plethysmography

Endothelial function was measured by using the method venous occlusion plethysmography as described before [11]. Venous occlusion plethysmography is an invasive technique in which vasoactive agents are infused directly into the artery. The technique allows us to test a vasoactive response to different agents without systemic changes. Additionally we tested the vasoactive properties of insulin as well as direct insulin stimulated glucose-uptake in the forearm. Venous occlusion plethysmography has been found to be a valid method of measuring endothelial function [12].

Examinations were done after an overnight fast, in a quiet room with the temperature kept constant during the day. The patients were all abstinent from smoking for at least 8 hours and did not take their usual medication in the morning before examination. Examinations were done with the patients lying supine with the forearm at a horizontal level with the right atrium.

Endothelium-dependent vasodilatation was assessed stepwise after an intra-arterial infusion of increasing doses of serotonin (7, 21, 70 ng/min) [Serotonin (Clinalfa, Läufelfingen, Switzerland)] for 4 minutes at each dose level to achieve a dose-response profile. Serotonin is an agonist of endothelial nitric oxide (NO) production. Endothelium-independent vasodilatation was assessed by infusion of increasing doses of nitroprusside [Nitropress (Abbott Laboratories, North Chicago, IL)] (0.5, 1 and 1.5 μg/min). Nitroprusside is an external NO donor in vascular smooth muscle cells and provide vascular smooth muscle relaxation and dilatation. To assess insulin-stimulated endothelial vasodilatation, insulin [Actrapid (Novo Nordisk Scandinavia, Malmö Sweden) in a 1% human albumin solution (vehicle)] was co-infused with serotonin. Insulin was infused intra-arterially for 60 minutes prior to the vaso-reactivity studies with serotonin, at a rate of 0.05 mU/kg body weight/min. Studies with co-infusions of N^G-monomethyl-L-arginine [L-NMMA (Clinalfa, Läufelfingen, Switzerland)], serotonin and insulin were done to determine the NO-dependent fraction of the insulin-stimulated endothelium-dependent vasodilatation. L-NMMA is a non-specific NO synthase inhibitor. Flow measurements are presented as the relative blood flow, given as a proportion between the actual blood flow (ml/min) in the infused arm and the actual blood flow in the non-infused arm. All evaluation of blood flow was performed blinded to treatment allocation.

Forearm Glucose uptake

Blood samples were drawn simultaneously from a catheter placed intravenously in the infused arm, a catheter placed in the brachial artery of the infused arm, and a catheter placed intravenously in the non-infused arm. The latter served as a control for systemic changes in concentrations of insulin and glucose during intra-arterial infusion of insulin. Forearm glucose uptake was calculated as the arterio-venous difference in glucose concentration in relation to forearm blood flow in samples from the artery and vein of the infused arm, respectively, and done on both examination days-before and after the two-month treatment period with either of the three beta blockers. Plasma glucose concentrations were determined by the glucose oxidase method [Vitros Chemistry; Johnson & Johnson, Rochester New York] and serum insulin concentrations by a chemiluminescent immunometric assay [Immulite 2500; DPC, USA].

Measurements of systemic metabolic changes of insulin resistance were calculated with the use of a computerized Homeostatic model assessment calculator [(HOMA2) Diabetes Trial Units, The Oxford Centre for Diabetes, Endocrinology and Metabolism]. The used formula for the calculations: HOMA Insulin Resistance = (Fasting Plasma Insulin (mU/L) × Fasting Plasma Glucose (mmol/L))/22.5.

24 hour ambulatory blood pressure and measurements of heart rate

Changes in blood pressure and heart rate were examined in a sub-group of patients in all three treatment groups. Patients had a 24-hour ambulatory blood pressure and heart rate examination done on a separate day before and after the randomized treatment period. An ambulatory blood pressure monitor was used [Model TM-2430; A&D Instruments ltd., Oxford, UK] and computer software was used for retrieving data [EZ Doctor Software for TM-2430; Kivex A/S, Hørsholm, Denmark].

Statistics

Results are expressed as means ± standard error of mean (SEM), unless otherwise specified.

Comparisons of baseline differences between the groups were performed using unpaired Students t-test. Paired comparisons after the treatment or observation period were analyzed with students paired t-test. Differences between groups for single parameters after the treatment with either of the three beta blockers were compared with 2-way analysis of variance (ANOVA). Changes in forearm blood flow as well as changes in forearm glucose uptake were subject to analysis of variance for repeated measurements using the proc mixed procedure in the Statistical Analyses Software, version 8.0 (SAS Institute, Cary, NC, USA). Forearm glucose uptake measurements were log transformed to satisfy assumptions of normal distribution and homogeneity of variance of residuals. Subjects entered the model as random effect. The dose of vasodilator and the interaction between insulin and serotonin entered the model as fixed values.

Multivariable association between flow, treatment and other cofactors was examined in mixed variance covariance models using proc mixed from SAS. Individual and interaction between study and individual were random variables whereas cofactors and interaction between study day and treatment were entered as fixed variables.

From previous studies in our group [11] it was found that a sample size of 10 patients in each group, a difference of 20% in forearm blood flow and forearm glucose uptake can be found with a statistical significance of 5%.

Vaso-reactivity studies

The group of patients with HF had a slightly but not significantly lower response to serotonin compared to the group of healthy controls (Figure 1a), while insulin-stimulated endothelial function was markedly reduced in this group (Figure 1b).

Endothelium-dependent vasodilatation assessed with infusion of increasing doses of serotonin was equal in the three groups before randomization and did not change after switching beta blocker treatment from carvedilol to metoprolol succinate or metoprolol tartrate: In the group randomized to metoprolol succinate the relative forearm blood flow was 1.24 ± 0.11; 1.21 ± 0.12; 1.54 ± 0.19; and 1.82 ± 0.29 at each dose level of serotonin before randomization treatment and 1.38 ± 0.16; 1.43 ± 0.15; 1.68 ± 0.26; and 2.10 ± 0.37 after two months treatment with metoprolol succinate (p = 0.27) (Figure 2a). In the group randomized to metoprolol tartrate the relative blood flow was 1.17 ± 0.08; 1.36 ± 0.19; 1.79 ± 0.18 and 2.57 ± 0.33 before randomization and 1.38 ± 0.23; 1.30 ± 0.21; 1.79 ± 0.34 and 2.42 ± 0.55 after the treatment period (p = 0.74) (Figure 2b). The third group was kept on carvedilol treatment after randomization and blood flow did not change during the treatment period: The blood flow was 1.18 ± 0.10; 1.25 ± 0.11; 1.87 ± 0.16 and 2.42 ± 0.33 before randomization and 1.24 ± 0.07; 1.22 ± 0.08; 1.80 ± 0.12 and 2.14 ± 0.24 after randomization treatment period (p = 0.34) (Figure 2c).

Switching beta blocker treatment did not affect insulin-stimulated endothelial function significantly: The percentage change in forearm blood flow in patients randomized to stay on carvedilol treatment for each serotonin dose was 33.58% ± 10.97; 24.74% ± 15.21 and 24.92% ± 21.86 before randomization and 33.56% ± 12.10; 28.72% ± 17.77 and 16.45% ± 31.46 after the treatment period (p = 0.84) (Figure 3a). In the metoprolol succinate group the percentage change in flow was 28.71% ± 8.36; 10.27% ± 12.00 and 40.78% ± 20.19 before treatment with metoprolol succinate and 7.03% ± 8.69; 12.82% ± 16.13 and 17.09% ± 15.01 after two months treatment (p = 0.29) (Figure 3b). In the metoprolol tartrate group the percentage change in flow was 11.19% ± 6.45; 2.08% ± 10.38 and -9.55% ± 16.51 before randomization and 15.03% ± 9.26; 42.48% ± 9.69 and 20.22% ± 29.79 after treatment (p = 0.48) (Figure 3c).

Studies of endothelium-independent vasodilatation with sodium nitroprusside were also not affected by the change in beta blocker treatment (data not shown). L-NMMA co-infusion with both insulin and serotonin abolished the stimulated vasodilatation and was unchanged in the three treatment groups (data not shown).

Other analysis

Multivariable analysis of the relation between treatment (carvedilol, metoprolol tartrate and metoprolol succinate) and serotonin induced flow also revealed no relation when diabetes, ACE inhibitor or AT antagonist treatment, treatment with statins, smoking, NYHA class, left ventricular ejection fraction (LVEF) and body mass index (BMI) were also entered in the analysis (p = 0.36). Also insulin induced flow changes were not changed by treatment when tested with multivariable analysis (p = 0.32).

Forearm glucose uptake

The group of patients with HF had impaired forearm glucose uptake compared to healthy individuals (Figure 4). During insulin infusion, forearm glucose uptake in the carvedilol group was similar throughout the study: Glucose uptake after 30 minutes of insulin infusion, increased from 1.12 mmol/min ± 0.19 to 1.97 mmol/min ± 0.51 and to 2.22 mmol/min ± 0.42 after 60 minutes of infusion on the first examination day with a similar increase seen on the second examination day with an forearm glucose uptake of 1.19 mmol/min ± 0.16 to 1.86 mmol/min ± 0.52 after 30 minutes and 2.19 mmol/min ± 0.69 after 60 minutes (p = 0.94 between the two days) (Figure 5a). Switching carvedilol to metoprolol succinate or to metoprolol tartrate did not affect glucose uptake either, as it is shown in Figures 5b and 5c, (p = 0.58 between treatment with carvedilol and metoprolol succinate and p = 0.92 between treatment with carvedilol and metoprolol tartrate).

As expected, serum-insulin concentration increased in the infused arm during insulin infusion and this increase did not change between treatment groups or from day 1 to day 2 (data not shown). Systemic serum-insulin concentrations were kept constant during the intra-arterial insulin infusion and did not change from day 1 and 2 in either of the three treatment groups.

Ambulatory 24 hour blood pressure and pulse measurement

Data on systolic and diastolic blood pressure and heart rate on the two examination days during ambulatory 24-hour blood pressure measurement and heart rate measurement are presented in Figure 6. Both systolic, diastolic blood pressure as well as heart rate measurements were comparable in the three treatment groups at the beginning of the study and did not change two months after switching beta blocker treatment from carvedilol to metoprolol succinate or metoprolol tartrate.

Study Limitation

Our patients were already treated with carvedilol at the first examination of endothelial function. Methodologically, it would have been an advantage to the study if patients were beta blocker naïve at entering the study. We found it unethical to withdraw beta blocker treatment in patients with a proven benefit of treatment and risk of worsening of heart failure symptoms in the situation of withdrawal of beta blocker treatment.

A limitation to the study could be the relative small number of patients included. With 9 patients in each group we were not able to show any difference in endothelial function between different beta blocker treatments. The fact that we did not find any difference in endothelial function could be explained by the short treatment period and a risk of a carry over effect.