Biological phenotypes of heart failure with preserved ejection fraction

Lewis. Biological phenotypes of HFpEF

Gavin A. Lewis, MBChB,1,2 Erik B. Schelbert, MD, MS,3-5 Simon G. Williams, MD,2 Colin Cunnington, MBChB, DPhil,1,6 Fozia Ahmed, MBChB, MD,1,6 Theresa McDonagh, MBChB, MD,7 Christopher A. Miller MBChB, PhD1,2,8

Total Word Count: (9,968)

Affiliations1. Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PL2. University Hospital of South Manchester NHS Foundation Trust, Southmoor Road, Wythenshawe, Manchester, M23 9LT3. Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA4. UPMC Cardiovascular Magnetic Resonance Center, Heart and Vascular Institute, Pittsburgh, PA, USA5. Clinical and Translational Science Institute, University of Pittsburgh, Pittsburgh, PA, USA6. Central Manchester University Hospitals NHS Foundation Trust, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL7. King’s College Hospital, Denmark Hill, London, SE5 9RS.8. Wellcome Centre for Cell-Matrix Research, Division of Cell-Matrix Biology & Regenerative Medicine, School of Biology, Faculty of Biology, Medicine & Health, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT

From University of Manchester, Manchester, UK (GAL, CC, FA, CAM); University Hospital of South Manchester, Manchester, UK (GAL, SGW, CAM); University of Pittsburgh, PA, USA (EBS); Central Manchester University Hospitals Manchester, UK (CC, FA); King’s College Hospital (TM).

Sources of fundingDr Lewis is funded by a fellowship grant from the National Institute for Health Research.  Dr Miller is funded by a Clinician Scientist Award (CS-2015-15-003) from the National Institute for Health Research. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.

DisclosuresNone declared.

Address for CorrespondenceDr. Christopher A. Miller, Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PLTelephone: 0044 161 291 2034. Fax: 0044 161 291 2389

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Email: Christopher.Miller@manchester.ac.uk

Acknowledgments None

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AbstractHeart failure with preserved ejection fraction (HFpEF) involves multiple pathophysiological mechanisms, which result in the heterogeneous phenotypes that are evident clinically, and which have potentially confounded previous HFpEF trials. A greater understanding of the in vivo human processes involved, and in particular, which are the causes and which are the downstream effects, may allow the syndrome of HFpEF to be distilled into distinct diagnoses based on the underlying biology. From this, specific interventions can follow, targeting individuals identified on the basis of their biological phenotype. This review describes the biological phenotypes of HFpEF and therapeutic interventions aimed at targeting these phenotypes.

Condensed AbstractHeart failure with preserved ejection fraction (HFpEF) involves multiple pathophysiological mechanisms, resulting in clinically evident heterogeneous phenotypes. A greater understanding of the in vivo human process involved may allow the syndrome of HFpEF to be distilled into distinct diagnoses based on underlying biology, allowing targeted intervention based on their ‘biological phenotype’. This review describes the biological phenotypes of HFpEF and therapeutic interventions aimed at targeting these phenotypes.

Key WordsHeart failure with preserved ejection fraction, heart failure, diastolic dysfunction, myocardial fibrosis, titin, ejection fraction.

Abbreviations List

CMR = cardiovascular magnetic resonanceCVF = collagen volume fractionECM = extracellular matrixEF = ejection fractionHF = heart failureHFpEF = heart failure with preserved ejection fractionHFrEF = heart failure with reduced ejection fractionLV = left ventricularLVH = left ventricular hypertrophyLA = left atrial

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Introduction

Described as the “single largest unmet need in cardiovascular medicine”, heart failure with

preserved ejection fraction (HFpEF) potentially accounts for up to half of heart failure (HF),

indeed, as the population ages and its risk factors become more prevalent, the impact of

HFpEF is set to rise considerably.

However, reflecting the ongoing lack of understanding of HFpEF, its definition, even its

name, continues to change in clinical guidelines and randomised controlled trials (1,2). While

the use of natriuretic peptides has provided greater confidence in the diagnosis of HF, which

represents progress from some early HFpEF trials that appear to have included patients who

did not have HF, the heterogeneity of HFpEF continues to frustrate clinicians and be cited as

a reason for the failure of clinical effectiveness trials (2). Even after leaving aside specific

causes of HF in the context of a normal or near normal ejection fraction (EF) (e.g.

hypertrophic cardiomyopathy, cardiac amyloidosis, Fabry disease), it is true that HFpEF

represents a broad cohort of patients with a range of comorbid conditions (3). However, it is

precisely this heterogeneity that needs to be embraced and explored if management is to

advance. Defining the diverse pathophysiological mechanisms of HFpEF will provide the

basis for the development of therapies that target each of these mechanisms, which can then

be trialled in patients identified as displaying particular mechanisms. The ‘one-size fits all’

approach to HFpEF has proved unsuccessful.

An outline of therapy based on ‘clinical phenotypes’ of HFpEF has been recently proposed

(4). Whilst this acknowledges the heterogeneity and need for individualised management, it is

the ‘biological phenotypes’, i.e. the underlying disease mechanisms of HFpEF, that ultimately

need to be addressed, and which will be the focus of the current discussion (Figure 1). When

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interpreting the presented data, it is important to note that study findings reflect the population

studied, which often differs according to data source (clinical trials vs. registries vs.

mechanistic studies), and may itself contribute to the heterogeneity.

Morphological heterogeneity

Left ventricular hypertrophy (LVH) and left atrial (LA) dilatation are considered hallmarks of

HFpEF and are included in its definition in recent guidelines (1) and in the inclusion criteria

of current randomised controlled trials, however there is considerable morphologic

heterogeneity.

In clinical trials and contemporary registries, approximately one-third to two-thirds of patients

with HFpEF do not have LVH (Figure 2A) (5,6). A further proportion of patients (for

example, 12% and 9% in the studies by Katz et al (6) and Shah et al (7) respectively) have

eccentric LVH rather than the conventional concentric pattern. Hypertrophic remodelling is

more common in HFpEF patients with hypertension, although approximately half of patients

with HFpEF and normal left ventricular (LV) mass have hypertension (6). Furthermore, LVH

is not exclusive to HFpEF, indeed the prevalence of LVH is similar in patients with

hypertension and no history of HF as compared to patients with hypertension and HFpEF (8).

LV mass, and the binary presence of LVH, are associated with adverse outcome in HFpEF on

multivariable analyses, primarily driven by an association with increased rates of

hospitalisation (5,6,9).

Similarly, a third to a half of patients in HFpEF studies have normal LA size (Figure 2B)

(5,7,10). In comparison, a third of patients with hypertension without HF have LA

enlargement (11). The relationship between LV mass and LA size is also not straightforward.

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For example, in the echocardiographic sub-study of the Treatment of Preserved Cardiac

Function Heart Failure With an Aldosterone Antagonist Trial (TOPCAT) (7,12), patients who

were enrolled on the basis of elevated natriuretic peptides had significantly larger LA volumes

than patients who were enrolled on the basis of previous hospitalisation, but had thinner LV

walls, lower mass and a lower prevalence of LVH. LA size has an inconsistent association

with adverse outcome in HFpEF; dichotomous LA enlargement was associated with adverse

outcome in the Irbesartan in Heart Failure With Preserved Ejection Fraction (I-PRESERVE)

trial on multivariable analysis (5,13), although LA area, as a continuous variable, was not. In

the echocardiographic sub-study of TOPCAT (9), LA width but not LA volume was

independently associated with outcome, and LA size was not associated with adverse outcome

in the Candesartan in Heart failure: Assessment of Reduction in Mortality and morbidity-

Preserved Echocardiographic substudy (CHARMES) (10,14).

Thus, while measurements of LV mass and LA size may be prone to error and variability, it is

clear that these parameters are inadequate for identifying patients with HFpEF and guiding

management. Other features, ideally reflecting underlying pathophysiological mechanisms,

are required.

Functional heterogeneity

Diastolic dysfunction also forms part of the guideline definition of HFpEF (1). Using invasive

conductance pressure-volume assessments, Westermann et al (15) found mean LV relaxation

time constant, end-diastolic pressure, diastolic stiffness and stiffness constant to be higher in

HFpEF patients (EF > 50%) at rest, and mean end-diastolic volume, stroke volume and

cardiac output to be reduced during tachycardic atrial pacing, compared to mean values in

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age, gender and co-morbidity-matched controls, although there was a strong trend towards a

higher prevalence of hypertension in the HFpEF group (37% vs. 20%; p=0.05).

However, consistently, a third of patients in the echocardiographic substudies of HFpEF

RCTs have normal diastolic function, even in the context of elevated natriuretic peptides, and

a further 20-30% have only mild or grade 1 diastolic dysfunction (Figure 2C) (5,7,10). In

comparison, a recent study of older people (aged 67–90 years) without HF found 96% had

abnormal diastolic function according to guideline-based definitions (16).

Advanced diastolic dysfunction was associated with adverse outcome in CHARMES

(moderate and severe diastolic dysfunction) and TOPCAT (severe diastolic dysfunction), but

diastolic function was not associated with outcome in I-PRESERVE. Katz et al (6) found

diastolic function to be worse in patients with HFpEF and concentric LVH compared to

eccentric LVH, but outcome was equivalent.

The load-dependency and variability of echocardiographic diastolic assessment are well

documented (17). In a condition in which diastolic dysfunction has been considered a defining

pathophysiology (‘diastolic heart failure’), it is unclear whether the variation in presence, and

prognostic significance, of diastolic dysfunction in HFpEF reflects the inadequacy of its

assessment with echocardiography, or a pathophysiological mechanism independent of

diastolic function in a substantial proportion of patients.

Atrial function may be abnormal in approximately 25% to 50% of patients with HFpEF,

although it has not been assessed in large cohorts, reference ranges are not well defined for all

analysis techniques and atrial dysfunction is prevalent in patients with hypertension without

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HF (18). Nevertheless, Freed et al (18) found LA reservoir strain to be independently

associated with adverse outcome in HFpEF, even after adjusting for LA volume and after

excluding patients with atrial fibrillation.

Defining what represents a ‘preserved ejection fraction’ has also been inconsistent across

guidelines, clinical trials and mechanistic studies (1,2,15) Post-hoc analysis from the

TOPCAT study, where ejection fraction (EF) ranged from 44 to 85%, showed significantly

higher event rates occurring at the lower end of the EF spectrum, particularly when EF was

less than 50% (19).

Myocyte structure and function

Titin

Elevated cardiomyocyte resting tension or passive stiffness (FPassive) has been demonstrated in

both isolated cardiomyocytes and strips of myocardium from patients with HFpEF (20-22).

Resting tension in cardiomyocytes is highly dependent on titin, a large sarcomeric protein that

functions as a molecular spring, storing energy during contraction and releasing it during

relaxation. The compliance of titin itself is dependent on post-transcriptional and post-

translational modifications, including isoform expression and phosphorylation (23).

Differential splicing results in two adult myocardial titin isoforms; N2B and N2BA. Van

Heerebeek et al (22) demonstrated a significant shift towards expression of the shorter, stiffer

N2B-isoform in myocardium from patients with HFpEF, which was hypothesised as being

responsible for the observed higher cardiomyocyte passive stiffness. RNA binding motif-20 is

a major splicing factor of titin and inhibition of RNA binding motif-20 results in expression

of highly compliant titin isoforms. Recently, in a murine HFpEF-like model (transverse aortic

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constriction and deoxycorticosterone acetate pellet implantation), Methawasin et al (24) found

that inactivating RNA binding motif-20 resulted in upregulation of compliant titin isoforms

and a large reduction in cellular passive stiffness, which translated into attenuation of

concentric LVH and normalisation of diastolic function and exercise tolerance. The

improvements occurred despite persistent, and unchanged, myocardial fibrosis, suggesting

they were cardiomyocyte-specific.

Titin stiffness is acutely modulated by phosphorylation. Titin is phosphorylated by a number

of pathways including cyclic adenosine monophosphate (cAMP)-dependent protein kinase-A

(PKA), activated in response to β-adrenergic stimulation by catecholamines, cyclic guanosine

monophosphate (cGMP)-dependent protein kinase-G (PKG), activated by nitric oxide (NO)

or natriuretic peptides, and protein kinase Cα (PKCα), calcium/calmodulin-dependent protein

kinase II (CaMKII), and extracellular signal-regulated kinase-2 (ERK2), activated by

endothelin-1 and angiotensin-II (23,25). PKA, PKG, and ERK2 signalling appear to decrease

cardiomyocyte resting tension, whereas PKCα phosphorylation increases it (Figure 3).

Hypophosphorylation of the N2B-isoform of titin has been demonstrated in myocardial tissue

from patients with HFpEF and in preclinical models, and is associated with elevated passive

stiffness of cardiomyocytes (26-28). In contrast to work by Van Heerebeek, Zile et al (21)

found no difference in N2BA:N2B isoform ratio between patients with hypertension and

HFpEF, patients with hypertension but no evidence of HF and normotensive controls, but

they did find that the ratio of phosphorylated N2BA:N2B was significantly higher in the

HFpEF group compared to the other groups, which is in keeping with findings from

preclinical models (28,29). Specifically, increased phosphorylation was seen at a PKC site on

the N2BA isoform and reduced phosphorylation at the PKA/PKG site on the N2B-isoform.

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Low PKA and PKG activity and cGMP concentration have also been demonstrated in HFpEF,

indeed administration of PKA and PKG in vitro is associated with normalisation of

cardiomyocyte passive stiffness (22,26,30).

Bishu et al (28) found augmentation of cGMP levels via natriuretic peptide stimulation and

phosphodiesterase-5 inhibition resulted in a significant relative increase in the

phosphorylation of N2B, and hence a reduction in the ratio of phosphorylated N2BA:N2B,

which was associated with a lower cardiomyocyte resting tension and improved diastolic

function. The beneficial effects were seen without changes to the phosphorylation status of

other sarcolemmic proteins and after excluding potential effects of transmembrane calcium

currents and myocardial fibrosis. Fukada et al (29) showed that the extent of the reduction in

resting tension during protein kinase A-mediated phosphorylation of titin was greater in

myocardial tissue with higher N2B-isoform expression. Thus, manipulation of myocardial

titin isoform expression and the phosphorylation state of each isoform represent attractive

therapeutic targets.

The combined angiotensin-II receptor and neprilysin inhibitor, valsartan/sacubitril, the

neprilysin inhibitor component of which augments active natriuretic peptides resulting in an

increase in cGMP, was associated with a greater reduction in serum NT-proBNP levels than

valsartan alone in patients with HFpEF after 12 weeks of treatment in the PARAMOUNT trial

(31). The ongoing PARAGON-HF is investigating the clinical effectiveness of

valsartan/sacubitril. The SOCRATES-preserved trial (NCT01951638) is investigating the

effect of augmenting cGMP levels using an orally active soluble guanylyl-cyclase stimulator.

Calcium handling

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HFpEF is associated with abnormal cardiomyocyte calcium homeostasis and ion channel

remodelling (32). Selby et al (33) found ventricular myocardium from patients with LVH and

LA dilatation has increased resting (diastolic) tension due to a persistent increase in actin-

myosin cross-bridge activation as a result of elevated diastolic cytosolic calcium

concentration, itself due to reduced sarcolemmal calcium extrusion due to abnormalities in the

function of the sodium-calcium exchanger. Calcium leak from the sarcoplasmic reticulum and

reduced sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) expression have also been

shown to contribute to the elevated diastolic cytosolic calcium concentration in preclinical

HFpEF models, although the data suggests both may be consequences of diastolic dysfunction

rather than causes (32,34).

In the study by Selby et al, diastolic tension increased further as heart rate increased, as a

result of increasing amounts of calcium entering the cell, in ventricular myocardium from

patients with LVH and LA dilatation, whereas no significant change was seen in myocardium

from patients with normal LV mass and LA size, findings which are in keeping with those

from a single cell model of HFpEF (32,33). These findings are consistent with the

aforementioned tachycardia-induced reduction in LV end-diastolic volumes observed by

Westermann et al (15), and potentially provide a mechanism for the observed exercise

limitation and intolerance to tachycardia seen in HFpEF. Heart rate restriction may therefore

be a potential therapeutic strategy. Nevertheless, in the study by Selby et al, increased

diastolic tension was seen to prevent complete cardiomyocyte relaxation even at low normal

heart rates (e.g. 60 beats/min), hence while such interventions may be beneficial for

preventing excessive tachycardias, reducing heart rate to less than physiological rates is

unlikely to be of therapeutic value (35). Indeed, two small studies investigating the short-term

impact of ivabradine (If-inhibitor) on exercise capacity and diastolic function found opposing

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results, and more recently Komajda et al showed no improvement in diastolic function,

exercise capacity or NT-proBNP after 8 months of ivabradine therapy despite a mean heart

rate reduction of 13beats/min (36-38).

Increased intracellular sodium concentration is also observed in HFpEF and may contribute to

the increased diastolic cytosolic calcium concentration via sodium-calcium exchange (2).

Ranolazine inhibits the late sodium current, minimising intracellular sodium accumulation

and hence reducing calcium concentration, and is associated with reduced diastolic tension

and improved diastolic function in human myocardium in vitro (39). In an in vivo proof of

concept study in patients with HFpEF, ranolazine was associated with an acute improvement

in LV end-diastolic pressure and pulmonary capillary wedge pressure (PCWP) but it had no

effect on invasive relaxation parameters, and it also had no effect on diastolic function,

exercise parameters or NT-pro-BNP levels after 14 days of treatment (40).

In a preclinical HFpEF model, Primessnig et al (41) demonstrated that an inhibitor of the

calcium leak from the sarcoplasmic reticulum reversed maladaptive LV remodelling (reversed

the increase in LV mass and LA size seen with placebo), improved diastolic function, and

reduced NT-proBNP levels, independent of blood pressure effects. The same inhibitor has

previously been shown to improve mortality in models of hypertensive heart disease (42).

Such work requires translation into human tissue, but nevertheless, abnormal calcium

handling is a potential therapeutic target in HFpEF.

Myocardial energetics

Ventricular diastole is an active process that utilises ATP, and excessive energy is consumed

maintaining the abnormally increased diastolic tension in HFpEF (33). In addition,

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microvascular dysfunction (see below) and myocardial extracellular matrix expansion (see

below), which increases the oxygen diffusion distance between the capillary and collagen-

encircled cardiomyocyte, potentially render the cardiomyocyte prone to hypoxia (43). In

keeping with the resultant lower resting energy reserve, myocardial phosphocreatine

(PCr)/ATP ratio, measured using phosphorus-31 magnetic resonance spectroscopy (31P-

MRS), is significantly lower in patients with hypertensive heart disease compared to healthy

controls, and in patients with HFpEF compared to controls, and is associated with diastolic

dysfunction (44,45). Furthermore, Lamb et al (45) demonstrated that PCr/ATP ratio declined

more severely in patients with hypertensive heart disease than in healthy controls during

exercise, suggesting the energy reserve equilibrium declines further during exercise, which is

consistent with the findings of Selby et al (33) and Westermann et al (15) described earlier.

Recently, distinct alterations in fatty acid beta-oxidation (FAO) have also been demonstrated

in patients with HFpEF using quantitative metabolomic profiling (46). Interventions that shift

energy substrate utilisation have led to significant improvements in PCr/ATP ratio and

diastolic function in other conditions, but have not been investigated in HFpEF (47-49).

Myocardial extracellular matrix

Changes in extracellular matrix (ECM) composition and structure appear to be important

pathophysiological mechanisms in HFpEF.

ECM expansion secondary to collagen accumulation is consistently demonstrated on a group

level in myocardial tissue from patients with HFpEF (20-22,30), the magnitude of which is

similar to that seen in HF with reduced EF (22). Zile et al (21) found myocardial collagen

volume fraction (CVF) was significantly higher in patients with hypertension and HFpEF

compared to patients with hypertension and no history of HF, suggesting a potentially crucial

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pathophysiological discriminator, although CVF in the group of patients with hypertension

and no history of HF was no different to that in non-hypertensive controls, which is contrary

to other studies (50). Nevertheless, asymptomatic patients with LVH and patients with HFpEF

have been found to display distinct fibrotic cytokine profiles, with a shift in collagen

homeostasis to a more profibrotic state seen in HFpEF (21,51).

Borbely et al found collagen volume fraction (CVF) was normal in a third of patients with

HFpEF despite evidence of diastolic dysfunction. This is in keeping with the proportion (14%

to 56% depending on the cut off used) of HFpEF patients with a normal myocardial ECM

volume measured using cardiovascular magnetic resonance (CMR) imaging in a recent large

cohort, and demonstrates the pathophysiological variation.

Collagen content in myocardial tissue from patients with HFpEF, and CMR-derived

myocardial ECM volume in patients with HFpEF, are associated with echocardiographic

parameters of diastolic function and LA size (21,52,53). In an important study, Rommel et al

(54) found CMR-derived myocardial ECM volume was the only independent predictor of

load-independent intrinsic LV stiffness, as measured using invasive pressure-volume

assessment, in patients with HFpEF. Furthermore, when patients were dichotomised

according to median ECM volume, both groups showed a pathological upward shift of the

end-diastolic pressure volume relationship during exercise, but in patients with elevated ECM

volume, the dominant pathophysiology was an increase in myocardial passive stiffness,

whereas in patients with a below median ECM volume the dominant mechanisms were

arterial stiffness and impaired active relaxation. Interestingly, both groups demonstrated

similar echocardiographic diastolic parameters. As such, this study serves to demonstrate the

variations in biological phenotypes that exist between individuals that are broadly classified

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as having HFpEF, the inadequacy of echocardiographic diastolic parameters for detecting

these phenotypic variations, and, more broadly, the inadequacy of echocardiography for

determining intrinsic LV stiffness.

In a substudy of the I-PRESERVE trial (55), circulating markers of collagen deposition

(procollagen type I amino-terminal peptide and osteopontin) were associated with adverse

outcome (death and hospitalisation for pre-specified cardiovascular causes) on univariable

analysis, but not on multivariable analysis, although circulating collagen markers are not

specific to the heart and confounded by numerous factors such as renal function (56). In a

study of HFpEF patients by Duca et al (53), CMR-derived ECM volume was associated with

adverse outcome (hospitalisation for HF or cardiovascular death) during median 24 month

follow-up on univariable analysis, but not on multivariable analysis, although the study was

relatively small given the heterogeneity of the condition (n=117). Recently Schelbert et al

(57), in a considerably large cohort of patients (n=410) with HFpEF or at risk for HFpEF

(BNP>100pg/mL but no clinical HF), found CMR-derived myocardial ECM volume was

strongly associated with adverse outcome on univariable and multivariable analyses (Figure

4). Indeed, myocardial ECM volume was more strongly associated with outcome than factors

such as age, LV mass, atrial fibrillation or previous myocardial infarction, and a clear ‘dose-

response’ relationship was observed between ECM volume and outcome. Considerable data

demonstrate both the potential for myocardial ECM to have a primary aetiological role in

HFpEF, and the adverse impact that ECM expansion has on myocardial mechanical, electrical

and microvascular function, which is in keeping the adverse impact in other organs

(21,54,58,59).

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Importantly, myocardial ECM expansion is reversible. Brilla et al (60) and Diez et al (50)

demonstrated regression of myocardial fibrosis in patients with hypertensive heart disease,

which was associated with improvement in diastolic function, after 6 months of lisinopril and

12 months of losartan respectively. Izawa et al (61) demonstrated regression of myocardial

fibrosis after 12 months of spironolactone, albeit in a different population (dilated

cardiomyopathy). In patients with HFpEF, Deswal et al (62) found 6 months of treatment with

eplerenone was associated with a significant reduction in circulating markers of collagen

deposition, compared to placebo. Interestingly, in the studies by Diez et al (50) and Izawa et

al (61), myocardial fibrosis regression was most prominent in patients with a greater burden

of myocardial fibrosis at baseline. This demonstration that antifibrotic agents may be more

effective in patients exhibiting a fibrotic phenotype may explain why angiotensin converting

enzyme inhibitors, angiotensin receptor blockers and aldosterone antagonists have not proven

beneficial in phase III trials in which recruitment has not targeted phenotypic variations

(12,63,64). The on-going PIROUETTE trial (NCT02932566) is investigating the effect of a

pure antifibrotic agent in patients with HFpEF with evidence of ECM expansion at baseline.

The angiotensin-II receptor blocker component of valsartan/sacubitril is associated with

myocardial fibrosis regression (65), and thus, given the effect of the neprilysin inhibitor

component described earlier, two HFpEF biological phenotypes are being targeted in the

PARAGON-HF trial (NCT01920711).

Lopez et al (66) found it was the degree of collagen cross-linking, rather than total collagen

volume, which was associated with LV filling pressures in patients with HFpEF and

hypertension. The same group also found torasemide reduced overexpression of lysyl oxidase

(which catalyses collagen cross-linking), collagen cross-linking and CVF, although in

hypertensive patients with HF and a preserved mean EF (54%), torasemide was not associated

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with a reduction in circulating collagen markers (67). Recently, lysyl oxidase-like 2 inhibition

was found to essentially eliminate the interstitial fibrosis, LV remodelling and HF associated

with transaortic constriction, although lysyl oxidase-like 2 inhibition has not proven beneficial

in phase II trials of pulmonary and hepatic fibrosis, possibly because once formed, collagen

crosslinks, particularly hydroxyallysine crosslinks, are much less susceptible to degradation,

thus lysyl oxidase-like 2 inhibition may need to be given earlier in the disease process

(68,69).

Zile et al (21) found myocardial fibrosis and abnormal titin function may co-exist in HFpEF,

synergistically leading to increased myocardial stiffness, although there is little evidence for

common pathophysiological pathways. Manipulation of titin isoform compliance by

Methawasin et al (24) did not alter collagen volume fraction. It is not known whether anti-

fibrotic agents influence titin compliance.

Vascular function

Paulus et al have proposed a central role for endothelial dysfunction, driven by co-morbidity

induced-systemic inflammation, in the pathophysiology of HFpEF (70). Circulating levels of

interleukin-6 and tumor necrosis factor-α are strongly and independently associated with

incident HFpEF (71), and cross sectional studies have consistently demonstrated elevated

circulating inflammatory markers in patients with established HFpEF, although this latter

finding may represent a manifestation of HFpEF rather than a cause (21). Paulus et al (70)

postulate that systemic inflammation leads to coronary microvascular endothelial

inflammation, as evidenced by abundant expression of vascular cell adhesion molecules,

which stimulates endothelial production of reactive oxygen species and impairment of

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endothelial-myocyte nitric oxide signalling, which in turn results in reduced myocyte PKG,

pro-hypertrophic signalling and increased myocyte stiffness (30,72,73).

Myocardial vascular function

Microvascular endothelial inflammation is known to be associated with endothelial

dysfunction and microvascular rarefaction (74), and, in an autopsy study that included 124

HFpEF patients (defined as HF hospitalisation and or outpatient HF diagnosis between

approximately 1980 and 2010, and a LV EF >40% within a median of 1 day of HF event),

HFpEF was associated with reduced microvascular density, which itself was associated with

myocardial fibrosis, in comparison to a control group who had died of non-cardiovascular

causes (59). However, whilst statistical adjustments were made for the between group

differences, the HFpEF group had substantially higher rates of confounders such as

hypertension (79 vs. 31%), diabetes (42 vs. 11%), epicardial coronary disease (65 vs. 0%) and

renal dysfunction. Srivaratharajah et al (75) found HFpEF was associated with a significant

reduction in myocardial flow reserve, as assessed using 82Rubidium positron emission

tomography, compared to hypertensive and normotensive controls, although natriuretic

peptides were not included in HF diagnosis, patients with infiltrative cardiomyopathies and

significant valvular heart disease were not excluded, and there were important baseline

differences between groups. Interestingly, HFpEF patients had higher resting myocardial

blood flow than normotensive controls and a trend towards higher resting myocardial blood

flow than hypertensive controls, which appears discordant with the reduced microvascular

density observed by Mohammed et al (59). Kato et al (76) found coronary flow reserve,

measured using phase-contrast CMR of the coronary sinus at rest and during adenosine stress,

showed a significant inverse correlation with serum natriuretic peptide levels in patients with

HFpEF and without significant epicardial coronary disease. Using paired arterial and coronary

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sinus blood gas sampling at rest and during supine cycle ergometry, van Empel et al (77)

found myocardial oxygen delivery, and potentially oxygen extraction, were reduced in

HFpEF and correlated with PCWP. Nevertheless, therapies that reduce inflammation and/or

improve endothelial function, (e.g. statins, angiotensin receptor blockers, phosphodiesterase-5

inhibitors), have proved ineffective in improving patient outcomes in HFpEF (63,78-80). It

may be that such therapies need to be given earlier in the disease process, before

microvascular rarefaction has occurred. A phase 2 trial of the antioxidant coenzyme-Q in

HFpEF is planned (NCT02779634).

Peripheral vascular function

HFpEF is associated with increased central arterial stiffness and increased magnitude of

arterial wave reflections in comparison to age-matched controls, although these parameters

have not been assessed in comparison to age and blood pressure-matched controls (80-82).

Increased afterload, particularly late systolic load, is associated with LVH and impaired

systolic and diastolic function in people without cardiovascular disease and in patients

undergoing invasive assessment for coronary disease with a LV EF >50%, and is strongly and

independently associated with incident HF (84,85). Arterial stiffness is associated with

decreased exercise capacity in HFpEF (83,86), and in preclinical models increased afterload is

associated with LV hypertrophy, fibrosis and HF (87). Organic nitrates have been shown to

reduce arterial wave reflections acutely (88), but in a recent randomised controlled trial in

patients with HFpEF, 6 months of isosorbide dinitrate, with or without hydralazine, did not

reduce wave reflections or improve remodelling or exercise tolerance, and were poorly

tolerated (89). Similarly in the NEAT-HFpEF trial (90), 6 weeks of isosorbide mononitrate

therapy did not improve quality of life or exercise capacity compared to placebo, indeed dose-

dependent decreases in physical activity levels were seen in patients receiving isosorbide

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mononitrate, possibly due to orthostatic hypotension. Inorganic nitrate, converted to nitric

oxide via the nitrate-nitrite-nitric oxide pathway in a process enhanced by tissue hypoxia and

acidosis, appears to be better tolerated with less risk of resting hypotension (91). Borlaug et al

(92) found sodium nitrite infusion acutely improved exercise PCWP and ventricular

performance in patients with HFpEF. Zamani et al (93) found a single dose of inorganic

nitrate-rich beetroot juice and subsequently 2 weeks of an oral preparation of inorganic nitrate

(potassium nitrate) improved exercise duration and quality of life in patients with HFpEF, and

was well tolerated. In addition to a beneficial effect on arterial wave reflections, the authors

suggested the positive findings were mediated by replacement of myocardial nitric oxide

deficiency and peripheral vasodilation. Further trials of inorganic nitrates are underway

(NCT02256345). Angiotensin-converting enzyme and phosphodiesterase-5 inhibition are not

associated with improvements in aortic distensibility in HFpEF (94) (78).

HFpEF is also associated with impaired skeletal muscle vasodilatory reserve during exercise

that results in a blunted exercise-induced reduction in systemic vascular resistance and

presumed abnormal skeletal muscle oxygen delivery, which, together with changes in skeletal

muscle fibre type and reduced capillary density, may contribute to the observed exercise

intolerance (95-97).

Pulmonary vascular function

In a population-based study of 244 patients with HFpEF, Lam et al (98) found 83% had

pulmonary hypertension, defined as a pulmonary artery systolic pressure (PASP) of over

35mmHg, derived from tricuspid regurgitation velocities on echocardiography, with a median

PASP of 48 (IQR 37-56mmHg), which is consistent with a prior invasive catheter based study

(99). PASP was significantly higher in patients with HFpEF than with hypertension without

20

HF, and was strongly associated with symptoms and mortality (hazard ratio 1.2 per 10 mmHg

increase on multivariable analysis). As expected, PASP was associated with pulmonary

venous pressure, estimated using E/e’ ratio, but interestingly, after accounting for pulmonary

venous pressure, PASP in HFpEF still exceeded that of hypertensive controls without HF,

suggesting abnormal pulmonary arterial function. Whether this pulmonary arterial

hypertension is due to pulmonary vascular remodeling secondary to sustained pulmonary

venous pressure elevation, primary abnormalities in pulmonary arterial function, abnormal

RV-PA coupling, or a combination of these factors remains unclear, but nevertheless PA

pressure is a potential therapeutic target.

However, in the Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise

Capacity in HFpEF (RELAX) trial (79), phosphodiesterase-5 inhibition with sildenafil for 24

weeks had no effect on exercise capacity, clinical status, quality of life, LV mass, diastolic

function or PASP compared to placebo. Similarly, despite an earlier trial suggesting sildenafil

may be beneficial in patients with HFpEF and pulmonary hypertension (100), Hoendermis et

al (101) recently found sildenafil for 12 weeks did not reduce PA pressures or improve other

invasive haemodynamic or clinical parameters in patients with HFpEF and pulmonary

hypertension. In the RELAX trial, plasma cGMP levels did not differ significantly between

groups, leading the authors to suggest that the negative results may have been because

sildenafil was unable to enhance cGMP sufficiently. Bonderman et al (102) found no

significant changes in mean PA pressure or other haemodynamic parameters 6 hours after

treatment with a soluble guanylate cyclase stimulator (riociguat) in patients with HFpEF and

pulmonary hypertension.

Comorbidities

21

Obesity

Multiple studies have demonstrated an association between obesity and HF, with a ‘dose-

response’ relationship observed between BMI and HF incidence in people with a BMI in the

overweight range or higher (103,104). (105). Whilst obesity is associated with a number of

HF risk factors, obesity is independently associated with HF, with a relative risk of 1.41 (95%

confidence interval 1.34–1.47) per 5-unit increment in BMI in a recent meta-analysis (106).

More than 80% of patients with HFpEF are overweight or obese and in the TOPCAT and

RELAX trials, median/mean BMI was 31kg/m2 and over 35kg/m2 respectively (107). Obokata

et al (108) recently described a distinct obese HFpEF phenotype, characterised by greater

concentric LV remodelling, higher LV filling pressures at rest and with exercise, greater

plasma volume overload (yet lower NT-pro BNP levels), a larger increase in pulmonary

arterial pressures with exercise, larger right ventricular size and more significant exercise

intolerance compared to non-obese HFpEF. Weight loss following bariatric surgery is

associated with reduced LV mass and mass-volume ratio, and improved diastolic function

(109).

Lung disease

Approximately 30-40% of patients with HFpEF have COPD (110). Whilst COPD and HF

share a number of risk factors, multiple studies have demonstrated a strong and independent

relationship between the severity of airflow limitation and incident HF (adjusted odds ratio of

up to 3.9) (111). Indeed in a large, community-based sample, Lam et al (112) found airflow

obstruction was the most prominent noncardiac predictor of incident HFpEF, and Barr et al

(113) showed emphysema and airflow limitation are linear related to impaired LV filling.

Obstructive sleep apnoea (OSA) is also common, with a prevalence of approximately 25-50%

22

in HFpEF, although in a study by Stahrenberg et al (114) OSA was not independently

associated with exercise tolerance, and its impact on outcome in HFpEF is not clear (115).

Kidney disease

Approximately 25-50% of patients with HFpEF have chronic kidney disease (CKD; defined

as an estimated glomerular filtration rate of less than 60 mL/min/1.73 m2), the prevalence of

which increases with age (116). CKD is consistently associated with adverse outcome in HF,

and appears to be of greater prognostic importance in HFpEF than HFrEF (odds ratio for all-

cause mortality 2.40 (95% CI 2.18, 2.63) with LVEF > 40% vs. 2.0 (1.81, 2.21) with LVEF

<30%) (117).

Comorbidity pathophysiology and potential interventions

The pathophysiological mechanisms underlying the associations between these comorbidities

and HFpEF remain unclear. Obesity, lung disease and kidney disease are associated with

systemic inflammation and, as described earlier, Paulus et al (70) hypothesise that chronic

inflammation leads to endothelial dysfunction, myocardial hypertrophy, fibrosis and diastolic

dysfunction.

Circulating inflammatory markers are associated with incident HFpEF, natriuetric peptide

levels and diastolic dysfunction (118). However the relationship between comorbidities,

inflammation, and HFpEF is not consistent, and therapies that reduce inflammation or

improve endothelial function have not proved effective to date (63,78,79). Other proposed

pathophysiological mechanisms include abnormal haemodynamics, metabolic dysregulation

and neurohumoral activation (119).

23

In a large cohort of obese patients without previous HF, Sundström et al (105) recently

demonstrated a graded association between increasing weight loss and decreasing risk of

incident HF, suggesting that comorbid intervention before HFPEF ensues may be beneficial.

Empagliflozin, an inhibitor of sodium-glucose transporter-2 (SGLT-2), which is located

almost exclusively in the kidney, was associated with a 35% reduction in the risk of HF

hospitalization and a 39% reduction in the risk of HF death or hospitalization compared to

placebo in patients with type II diabetes mellitus and cardiovascular disease in the EMPA-

REG trial, with benefits seen in patients with and without HF at baseline (120,121). HF

outcomes relative to EF were not assessed, although HF outcome rates in those with baseline

HF were similar to those in HFpEF trials, leading to suggestions that empagliflozin may have

been beneficial in patients with HFpEF (122). The mechanisms by which empagliflozin may

positively impact HF are unclear, but the improvement in HF outcomes was independent of

glycaemic control. Hypotheses include a blood pressure lowering effect, sodium and fluid

loss, beneficial effects on the renin-angiotensin system, weight loss, maintenance of renal

function, decreased atrial stiffness and decreased inflammation. A phase-III trial of

empagliflozin in HFpEF is currently in progress, including patients without type II diabetes

(NCT03057951).

Discussion

It is clear that HFpEF involves multiple pathophysiological mechanisms, which, to a variable

extent, likely co-exist and combine to result in the heterogeneous phenotypes that are evident

clinically (123).

24

In an eloquent study, Shah et al (124) applied machine learning techniques to a well

characterised HFpEF cohort in order to identify groups of patients based on their clinical

phenotypes (“phenomapping”). Patients were recruited following hospitalisation for HF

although elevated natriuretic peptides were not required for diagnosis. Three markedly

differing phenogroups were identified: (1) younger patients with moderate diastolic

dysfunction who had relatively normal BNP (mean 72pg/mL); (2) obese, diabetic patients

with a high prevalence of obstructive sleep apnea who had the worst LV relaxation; and (3)

older patients with significant chronic kidney disease, electric and myocardial remodeling,

pulmonary hypertension, and RV dysfunction. Outcomes (including hospitalisation and death)

varied significantly by phenogroup, with a stepwise increase in risk profile from group 1,

which had the lowest risk to group 3, which had the highest. The phenogrouping provided

more discriminatory risk profiling than BNP and results were confirmed in a validatory

cohort. The work by Shah et al demonstrates that the heterogeneity of HFpEF can, at least to

some extent, be resolved, and provides the opportunity for future trials to recruit, and

determine effect, according to phenotypic characterisation.

Nevertheless, it is the biology underlying these clinical phenotypes that ultimately needs to be

determined; in particular, which biological mechanisms are root causes and which are

downstream effects. Studies are required that simultaneously investigate multiple biological

mechanisms, in order to understand the relative contribution of each mechanism, how

different mechanisms interact and whether apparently distinct mechanisms share common

drivers. For example, in order to more fully understand myocardial stiffness, concurrent

assessments of titin, calcium handling, energetics and extracellular matrix, their interaction

and molecular drivers, are required.

25

Machine learning techniques, such as those used by Shah et al, applied to large, deeply

phenotyped (biological and clinical) datasets in conjunction with well-defined outcomes may

help to further resolve the heterogeneity of HFpEF, improve our understanding of how

biological mechanisms integrate (‘biological phenogroups’) and how biological phenotypes

integrate with clinical phenotypes. From this, specific interventions can follow, targeting

individuals identified on the basis of their biological phenotypes. Biological heterogeneity has

potentially compromised HFpEF trials previously (12-14); HFpEF now needs ‘to get

personal’.

This involves multiple challenges, not least the translation of cardiovascular molecular

biology into clinical diagnostics that are acceptable and scalable. CMR ECM volume

quantification has its limitations, but is an example of a technique that non-invasively

interrogates a pathophysiological mechanism, which can be used to identify patients and

measure the effect of interventions. A specific preclinical HFpEF model is lacking, although

this reflects the heterogeneity of the condition and our lack of understanding of the integrated

pathophysiology. Nevertheless, the ‘epidemic’ status of HF means the potential gains are

large.

Understanding the relationship between HFpEF and aging may help with understanding the

biology of HFpEF more generally. In the study by Shah et al, older patients had more severe

electrical and myocardial remodelling, more abnormal ventricular-arterial coupling, worse RV

function and higher PA pressures in comparison to younger patients, despite a similar

duration of HF. The mechanisms responsible for these age-related phenotypic differences are

not clear, but it does not appear to simply reflect accumulation of comorbidities; whilst CKD

was more common in older patients, obesity, diabetes and obstructive sleep apnea were not.

26

Diastolic dysfunction is common in older patients without HF and is generally considered a

benign manifestation of aging. However, it may reflect changes in myocardial and vascular

structure that confer vulnerability (16). Indeed, preclinical studies have demonstrated that the

myocardial response to injury is directly influenced by age (125).

Finally, natriuretic peptides increase the diagnostic confidence of HFpEF and are associated

with adverse outcomes, and therefore contemporary clinical trials often require elevated

natriuretic peptide levels for entry. However, elevation of natriuretic peptide levels potentially

reflects an advanced stage in the pathophysiological process, when decompensation has

occurred. Indeed, post hoc analyses of data from the I-PRESERVE and TOPCAT trials found

patients with low levels of natriuretic peptides derived benefit from irbesartan and

spironolactone respectively, but patients with higher levels were unresponsive to intervention

(126,127). Characterisation of the biological phenotypes, understanding the biological

differences between patients with typical co-morbidities but without HF and those with

clinically similar profiles but with HF, and identification of interventions that disrupt or

reverse this pathobiology, may pave the way for patients to targeted before the HFpEF

syndrome ensues.

27

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Legends

CENTRAL ILLUSTRATION (Figure 1). Biological phenotypes in heart failure with

preserved ejection fraction (HFpEF). HFpEF is a systemic disease with multiple biological

phenotypes contributing to a heterogeneous clinical syndrome, including cardiomyocyte,

extracellular matrix, vascular and co-morbidity-related pathophysiological mechanisms.

Figure 2. Morphological and functional heterogeneity in HFpEF clinical trials. The

prevalence of left ventricular hypertrophy (A), left atrial dilatation (B) and diastolic

dysfunction (C) is highly variable, demonstrated in major HFpEF randomised trials (I-

PRESERVE, CHARM-Preserve, and TOPCAT).

Figure 3. Cardiomyocyte-specific biological phenotypes in HFpEF. Titin phosphorylation

and isoform expression (N2B/N2BA) alter cardiomyocyte stiffness. Mitochondrial

dysfunction leads to abnormal phosphocreatinine (PCr) to adenosine triphosphate (ATP) ratio.

Abnormal calcium handling may result from calcium leak and reduced expression of the

sarcoendoplasmic reticulum calcium-transport ATPase (SERCA) pump. ADP, adenosine

triphosphate; CaMKII, Ca2+/calmodulin-dependent protein kinase-II; cAMP, cyclic adenosine

monophosphate; Cr, creatinine; ERK2, extracellular signal-regulated kinase-2; GMP,

guanosine monophosphate; GTP, guanosine triphosphase; pGC, particulate guanylyl cyclase;

PKA, protein kinase A; PKCα, protein kinase Cα; PKG, protein kinase G; sGC, soluble-

guanylyl cyclase.

Figure 4. Outcomes in patients with HFpEF or at risk of HFpEF according to

extracellular volume (ECV). ECV measurement of myocardial fibrosis provides robust risk

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stratification for the combined end point of death or hospitalisation for heart failure (HHF).

CMR, cardiac magnetic resonance imaging. Reproduced with permission from Schelbert et al

(57).

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