M is associated with early HIF-2α-IN-1 cardiac structural changes that occur in non-dialysis CKD. In light of this, 
we investigated if this gene variant is associated with changes in systolic and diastolic function, based on NSC305787 (hydrochloride) web detailed cardiac magnetic PubMed ID:http://jpet.aspetjournals.org/content/12/4/221 resonance imaging in non-dialysis CKD patients with no known history of heart failure. Methods Patients The study cohort consisted of patients who were initially enrolled into two completed, single centre, randomised controlled trials based at the Queen Elizabeth Hospital Birmingham. To limit the confounding effect of population stratification only white patients were included in this genetic substudy. Inclusion criteria were: baseline CMR and echocardiographic investigations, age 1880 with non-dialysis CKD, total cholesterol <5.5mmol/L, resting blood pressure controlled to <140/90 for at least 6months. Exclusion criteria were: diabetes mellitus, peripheral vascular disease, previous myocardial infarction, known heart failure, valvular heart disease and atrial fibrillation. The study was approved by East Midlands Nottingham 1 Research Ethics Committee and adhered to the Declaration of Helsinki. Study participants provided written informed consent. 2 / 10 eNOS Association with LVEF in Early CKD Genotyping Whole blood was collected in PAXgene Blood DNA Tubes which were then frozen and stored at -80C. Samples were defrosted at room temperature for 2 hours before starting the DNA extraction process. DNA extraction was undertaken using the PAXgene Blood DNA Kit as previously described. All DNA samples were then all diluted to a working stock of 4ng/ml with 2.25ml of DNA added into each reaction. eNOS SNP rs1799983 genotyping was performed using Taqman technology as previously described. 384-well plates were read using a 7900HT Fast Real-Time PCR System. Cardiovascular Magnetic Resonance Imaging CMR was performed on a 1.5-T scanner. Serial contiguous short axis cines were piloted from the vertical long axis and horizontal long axis of the right and left ventricle in accordance with previously validated methodologies. Analysis was performed off-line by two blinded observers for the assessment of ventricular volumes and ejection fraction. Heart rate and baseline brachial blood pressure was measured at the time of CMR. Echocardiography Transthoracic echocardiography was performed by an experienced echocardiographer. All parameters were measured in triplicate according to American Society of Echocardiography recommendations and analysed offline by two blinded observers. Resting left ventricular diastolic function was assessed using standard techniques. Arterial Stiffness and Distensibility Pulse wave velocity, augmentation index and ascending aortic distensibility were common measures of arterial stiffness and distensibility in the two randomised control trials, they have been included here. Pulse wave analysis was performed on the radial artery using a high-fidelity micromanometer. The peripheral arterial waveform was used to generate a central arterial waveform using a validated transfer function. The same system was used to determine aortic pulse wave velocity by sequentially recording ECG-gated carotid and femoral waveforms as previously described. CMR data was used for the ascending aortic distensibility measurement data. Outcome Measures The primary aim was to evaluate the association between LV ejection fraction with eNOS genotype. Secondary outcomes included: LV end-diastolic volume indexed to body surfa.M is associated with early cardiac structural changes that occur in non-dialysis CKD. In light of this, we investigated if this gene variant is associated with changes in systolic and diastolic function, based on detailed cardiac magnetic PubMed ID:http://jpet.aspetjournals.org/content/12/4/221 resonance imaging in non-dialysis CKD patients with no known history of heart failure. Methods Patients The study cohort consisted of patients who were initially enrolled into two completed, single centre, randomised controlled trials based at the Queen Elizabeth 
Hospital Birmingham. To limit the confounding effect of population stratification only white patients were included in this genetic substudy. Inclusion criteria were: baseline CMR and echocardiographic investigations, age 1880 with non-dialysis CKD, total cholesterol <5.5mmol/L, resting blood pressure controlled to <140/90 for at least 6months. Exclusion criteria were: diabetes mellitus, peripheral vascular disease, previous myocardial infarction, known heart failure, valvular heart disease and atrial fibrillation. The study was approved by East Midlands Nottingham 1 Research Ethics Committee and adhered to the Declaration of Helsinki. Study participants provided written informed consent. 2 / 10 eNOS Association with LVEF in Early CKD Genotyping Whole blood was collected in PAXgene Blood DNA Tubes which were then frozen and stored at -80C. Samples were defrosted at room temperature for 2 hours before starting the DNA extraction process. DNA extraction was undertaken using the PAXgene Blood DNA Kit as previously described. All DNA samples were then all diluted to a working stock of 4ng/ml with 2.25ml of DNA added into each reaction. eNOS SNP rs1799983 genotyping was performed using Taqman technology as previously described. 384-well plates were read using a 7900HT Fast Real-Time PCR System. Cardiovascular Magnetic Resonance Imaging CMR was performed on a 1.5-T scanner. Serial contiguous short axis cines were piloted from the vertical long axis and horizontal long axis of the right and left ventricle in accordance with previously validated methodologies. Analysis was performed off-line by two blinded observers for the assessment of ventricular volumes and ejection fraction. Heart rate and baseline brachial blood pressure was measured at the time of CMR. Echocardiography Transthoracic echocardiography was performed by an experienced echocardiographer. All parameters were measured in triplicate according to American Society of Echocardiography recommendations and analysed offline by two blinded observers. Resting left ventricular diastolic function was assessed using standard techniques. Arterial Stiffness and Distensibility Pulse wave velocity, augmentation index and ascending aortic distensibility were common measures of arterial stiffness and distensibility in the two randomised control trials, they have been included here. Pulse wave analysis was performed on the radial artery using a high-fidelity micromanometer. The peripheral arterial waveform was used to generate a central arterial waveform using a validated transfer function. The same system was used to determine aortic pulse wave velocity by sequentially recording ECG-gated carotid and femoral waveforms as previously described. CMR data was used for the ascending aortic distensibility measurement data. Outcome Measures The primary aim was to evaluate the association between LV ejection fraction with eNOS genotype. Secondary outcomes included: LV end-diastolic volume indexed to body surfa.
