Biomarkers for Heart Failure with Preserved Ejection Fraction and relationship to physical activity

Aims
• To discover a multi-marker panel of proteins that would distinguish heart failure from normal control patients, and stratify the heart failure patients into those with reduced (HFrEF) or preserved (HFpEF) ejection fraction.
• To develop and validate a LC-MS/MS-SRM (liquid chromatography tandem mass spectrometry selected reaction monitoring) method for the accurate quantification of multiple proteins in the plasma of HFpEF patients.
• To assess the relationship of the protein biomarkers of HFpEF to indices of fluid overload, ventricular fibrosis, and to physical activity
• To assess the effect of physical activity on these HFpEF biomarkers in separate cohorts of patients at risk of HFpEF (eg, diabetic, obese populations)

Background
Heart failure with preserved ejection fraction (HFpEF) is as common as heart failure with reduced ejection fraction (HFrEF), but disease-modifying therapies for the latter do not alter prognosis in HFpEF. HFpEF is more common in the elderly, especially female, hypertensive, obese or diabetic. There is a need to understand the pathophysiology of HFpEF in order to develop new therapies. This project aims to discover new biomarkers in plasma of HFpEF patients, and to determine how they relate to measures of fluid overload, ventricular fibrosis, and exercise capacity, and links research activities across the cardiovascular, lifestyle and precision medicine themes. These markers may indicate novel pathways involved in HF, especially HFpEF.

The cardiovascular theme has already recruited and phenotyped about 250 subjects in a heart failure study, and at least 150 of these patients are diagnosed as HFpEF, with 50 HFrEF and 50 normal control subjects. Our current proteomic discovery methods involve different fractionation and affinity methods to examine subproteomes in plasma, using a bottom-up approach of quantification of tryptic peptides in a high-definition mass spectrometer and bio-informatic processing of data with reference to in-house tissue proteome libraries. These approaches currently allow identification of about 40-45% of the human proteome. Verification of biomarkers that have been discovered will be through internal quality controls that have been labelled to enable relative quantification of targeted biomarkers.

Hypothesis
A multimarker panel of plasma proteins enables stratification of patients with heart failure from those who are normal, and also stratifies heart failure patients into those with preserved or reduced ejection fractions. Such a protein panel may be linked to measures of fluid overload, ventricular fibrosis or physical activity, and prognosis.

Experimental Methods and Research Plan
• Develop high-throughput analytical methods for quantification of proteins in sub-proteomes of plasma from normal, HFpEF and HFrEF patients.
• Utilise a suite of bioinformatics analytical platforms to define the multi-marker protein panel that distinguishes and stratifies patients into the above groups.
• Develop targeted methods for several proteins identified in these agnostic discovery experiments using LC-MS/MS-SRM (liquid chromatography tandem mass spectrometry with selected reaction monitoring) with stable isotope labelled standards.
• Validate the above method using FDA bioanalytical method validation criteria (including (not exclusively) precision, accuracy, linearity, robustness etc.)
• Examine the relationship of the biomarker panel to echocardiographic measures of fluid status, MRI measures of ventricular fibrosis, and exercise capacity in HFpEF patients.
• If time permits, the strongest candidate proteins could be developed into high-throughput immunoassays, for studies in larger cohorts.

These proteins will be investigated further in other cohorts, as follows:-
1) We will study the use of these proteins in determining outcomes in HFpEF patients, in the cardiovascular theme HFpEF cohort and another acute heart failure of about 500 patients from our group in collaboration with the GREAT (Global research on acute conditions team) consortium. Multimarker panels linked to fluid overload or exercise capacity will be tested for prediction of mortality and heart failure rehospitalisation.
2) We will also link these panels to outcomes of HFpEF patients within the Biostat-CHF cohort, for which we provided the proteomics work package, to confirm results found in (1).
3) Further validation of these findings will be performed using samples from HFpEF cohorts recruited in USA (Dr. Julio Chirinos, University of Pennsylvania, Philadelphia) to ensure findings are generalizable to other populations (include Black ethnic groups which are prevalent in USA HFpEF cohorts).
4) We will examine whether the multimarker panel linked to exercise capacity, is modifiable by exercise programmes, using existing cohorts within the Lifestyle theme.

Expected outcome and Impact
• Validated LC-MS assay for the accurate measurement of multiple proteins in a single, high-throughput assay.
• Utility of a multimarker panel (alone or in combination with existing phenotypic measurements) in distinguishing heart failure from normal subjects, and stratifying patients into reduced and preserved ejection fraction.
• Insight into the pathophysiological mechanisms involved in HFpEF and HFrEF.
• Utility of markers in relation to fluid overload, fibrosis and exercise capacity of HFpEF patients
• Utility of markers in prediction of poor outcomes in HFpEF patients.
• Behaviour of markers in response to changes in physical activity and exercise regimes, independent of ethnicity.
• Potential commercialisation of assays in partnership with biomarker SMEs.
• Potential for publication in high-impact journals that would attract widespread citations