Case: All patients presented with the diagnosis of heart failure with preserved ejection fraction (HFpEF) to Department of Cardiology, BSMMU were initially approached and then selected as case on the basis of inclusion and exclusion criteria. Heart failure diagnosis was made according to the 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. All inclusion criteria were established within 24 hours of presentation. The eligible patients were explained about the study, written informed consent was taken and demographic data were recorded.

Control group: Apparently normal healthy volunteers were initially approached. Medical records were reviewed for prevalent cardiovascular disease (stroke, coronary artery disease, heart failure, arrhythmia), cardiovascular risk factors (hypertension, diabetes mellitus, hyperlipidemia, smoking, renal dysfunction), systemic disease (such as cancer, infections, autoimmune disorders) or any pharmacotherapy Subjects were excluded if any of these were identified. The eligible patients were explained about the study. Written informed consent was taken and demographic data, Pulse, blood pressure and respiratory rate were recorded.

Clinical data, including detailed medical history, cardiovascular risk factors and associated comorbidities of patients with HFpEF were noted. Relevant physical examination was done and recorded in semi-structured designed data collection sheet. Complementary data were collected including chest radiography, ECG, Echocardiography, and relevant laboratory tests. The symptoms of patients were noted. Shortness of breath was graded according to NYHA functional classification. Venous blood samples were collected in plastic vacuum tubes at room temperature for analysis of BNP using chemilumunescent micro particle immunoassay (CMIA) on the ARCHITECT iSystem, on day 1 of presentation in HFpEF cases. Other laboratory investigations including hemoglobin, serum creatinine, HbA1c in diabetics were also done.

Echocardiography was performed by using Vivid E9 (GE Healthcare, Norway) using a 3.5 Mhz transducer. ECG leads were connected before analysis. LV diameters were calculated by M-mode and LVEF by Simpson"s modified biplane method. The LV mass was estimated by using the area length method and adjusted for body surface area. Echocardiographic LV hypertrophy was defined as an LV mass index\> 115 g/m2 for men and \> 95 g/m2 for women. LV geometry was classified based on relative wall thickness (RWT), defined as (2×diastolic posterior wall thickness)/LV end-diastolic dimension and Left Ventricular Mass Index (LVMi) as recommended by the American Society of Echocardiography (ASE): normal = RWT ≤ 0.42 and no LVH; eccentric hypertrophy = RWT ≤ 0.42 and LVH; concentric remodeling = RWT \> 0.42 and no LVH; concentric hypertrophy = RWT \> 0.42 and LVH. Right ventricular (RV) function was assessed by tricuspid annular plane systolic excursion (TAPSE) and tricuspid lateral annular systolic velocity (S') by pulsed tissue Doppler. Peak pulmonary arterial systolic pressure (PASP) was estimated as the sum of peak RV-right atrial gradient from the tricuspid valve regurgitant jet and right atrial pressure on the basis of size and collapsibility of inferior vena cava. Presence and severity of valvular heart diseases were assessed by color Doppler imaging and image guided pulsed and continuous Doppler studies according to 2014 AHA/ACC Guidelines for the Management of Patients with Valvular Heart Disease. Patients with more than mild valvular heart diseases were excluded.Diastolic function parameters were measured as follows: peak early diastolic filling (E) and late diastolic filling (A) velocities, E/A ratio, E deceleration time, early diastolic septal and lateral mitral annular velocity (e'), average E/E', peak TR jet velocity, left atrial volume index. Left atrial volume index was calculated using biplane area-length method from apical four and two chamber views at end-systole from the frame preceding mitral valve opening and was indexed to body surface area. Diastolic dysfunction was classified into three grades according to 2016 ASE/EACVI guidelines.

LV longitudinal strains were analyzed by 2D speckle tracking echocardiography for both controls and patients with HFpEF. Cardiac cycles were obtained during a breath hold in end-expiration. Special care was taken to obtain correct view and checking for foreshortening. Endocardial border was traced at end systole, with a frame rate of 50-80/second, from apical long axis, four chambers and two-chambers view. In case of poor tracking, region of interest (ROI) was readjusted. The results of all three planes were combined in a single bull"s eye summary, along with a global longitudinal strain value (GLS) for the LV which was automatically calculated by automated function imaging (AFI). All strain analysis on HFpEF, and normal control subjects was be performed by a single investigator. Two independent investigators analyzed the echocardiography recordings blinded to clinical data. The intra-observer and inter-observer variability of GLS was assessed from 10 randomly selected patients by intra-class correlation coefficient(R). The R value for intra-observer variability was 0.983 and for inter-observer variability was 0.980. This showed good reproducibility of GLS for both same and different operators.