Myocardial Stiffness in Patients with Heart Failure and a ...circ.ahajournals.org/content/circulationaha/early/2015/01/30/... · Myocardial Stiffness in Patients with Heart Failure

DOI: 10.1161/CIRCULATIONAHA.114.013215

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Myocardial Stiffness in Patients with Heart Failure and a Preserved Ejection

Fraction: Contributions of Collagen and Titin

Running title: Zile et al.; Stiffness in HFpEF

Michael R. Zile, MD1; Catalin F. Baicu, PhD1; John Ikonomidis MD, PhD2; Robert E. Stroud,

MS2; Paul J. Nietert, PhD3; Amy D. Bradshaw, PhD1; Rebecca Slater, BS4; Bradley M. Palmer,

PhD5,6; Peter Van Buren, MD5,6; Markus Meyer, MD, PhD5; Margaret Redfield, MD7;

David Bull, MD8; Henk Granzier, PhD4; Martin M. LeWinter, MD5,6

1Division of Cardiology, Dept of Medicine, Medical University of South Carolina, and RHJ Department of Veterans Affairs Medical Center, Charleston, SC; 2Division of Cardiothoracic

Surgery, Dept of Surgery, Medical University of South Carolina, and RHJ Department of Veterans Affairs Medical Center, Charleston, SC; 3Dept of Public Health Sciences, Medical University of

South Carolina, Charleston, SC; 4Dept of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ; 5Cardiology Unit, Dept of Medicine, University of Vermont, Burlington, VT; 6Dept of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT; 7Division of

Cardiology, Mayo Clinic, Rochester, MN; 8Division of Cardiothoracic Surgery, Dept of Surgery, University of Utah Health Sciences Center, Salt Lake City, UT

Address for Correspondence:

Michael R. Zile, MD

Division of Cardiology, Department of Medicine

Medical University of South Carolina

Ashley River Towers, 25 Courtenay Drive, Room 7067

Charleston, SC 29425

Tel: 843-792-6866

Fax: 843-789-6850

E-mail: [email protected]

Journal Subject Codes: Heart failure:[110] Congestive, Hypertension:[15] Hypertrophy, Hypertension:[115] Remodeling, Myocardial biology:[104] Structure

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Abstract:

Background—The purpose of this study was to determine whether patients with heart failure

and a preserved ejection fraction (HFpEF) have an increase in passive myocardial stiffness and

the extent to which discovered changes are dependent on changes in extracellular matrix fibrillar

collagen and/or cardiomyocyte titin.

Methods and Results—Seventy patients undergoing coronary artery bypass grafting underwent

an echocardiogram, plasma biomarker determination, and intra-operative left ventricular (LV)

epicardial anterior wall biopsy. Patients were divided into 3 groups: referent control (n=17, no

hypertension or diabetes), hypertension (HTN) without(-) HFpEF (n=31), and HTN with(+)

HFpEF (n=22). One or more of the following studies were performed on the biopsies: passive

stiffness measurements to determine total, collagen-dependent and titin-dependent stiffness

(differential extraction assay), collagen assays (biochemistry or histology), or titin isoform and

phosphorylation assays. Compared with controls, patients with HTN(-)HFpEF had no change in

LV end diastolic pressure (LVEDP), myocardial passive stiffness, collagen, or titin

phosphorylation but had an increase in biomarkers of inflammation (CRP, sST2, TIMP-1).

Compared with both control and HTN(-)HFpEF, patients with HTN(+)HFpEF had increased

LVEDP, left atrial volume, NT-proBNP, total, collagen-dependent and titin-dependent stiffness,

insoluble collagen, increased titin phosphorylation on PEVK S11878(S26), reduced

phosphorylation on N2B S4185(S469), and increased biomarkers of inflammation.

Conclusions—Hypertension in the absence of HFpEF, did not alter passive myocardial stiffness.

Patients with HTN(+)HFpEF had a significant increase in passive myocardial stiffness; collagen-

dependent and titin-dependent stiffness were increased. These data suggest that the development

of HFpEF is dependent on changes in both collagen and titin homeostasis.

Key words: heart failure, diastole, hypertension, hypertrophy, collagen, Titin

differential extraction assay), collagen assays (biochemistry or histology), or tittininn iiisosoofofoformrmrm aaandndnd

phosphorylation assays. Compared with controls, patients with HTN(-)HFpEF hahad dd nnono ccchahahangngngee iniin

LV end diastolic pressure (LVEDP), myocardial passive stiffness, collagen, or titin

phhosososphphphororylylylaatatiionn bububut t had an increase in biomarkeeersrr oof inflammatiionono (CRCRCRPPP, sST2, TIMP-1).

CCCommpmpared wwitiith h h bobobothh cccononntrtrololo aaandndnd HHHTNTNTN((-(-)H)H)HFpFpFpEFEFE , papapatienenntstss wwwititith h HTHHTN(N(N +)+)+)HFHFH pEpEEFFF hahahaddd ininnccrcreaeaasesesed d d

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phosphorylatatioioion nn ononon NNN2B2B2B SSS41414185855(S(S(S464669)9), , anannd d d ininncrcrcreaeae sesesed d d bibiiomomomarararkekekersrsr ooof f f inininflflflamamammamamatititiononon..


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Introduction

Patients with heart failure and a preserved ejection fraction (HFpEF) have been shown to have

abnormalities of left ventricular (LV) diastolic function including slowed/incomplete relaxation,

decreased suction/recoil, and increased passive chamber stiffness 1-5. However, the myocardial

basis for these changes remains incompletely understood. Some recent clinical studies have

suggested that development of HFpEF is accompanied by significant changes in the composition

and structure of the extracellular matrix (ECM), especially fibrillar collagen 6-8. Other studies

and editorials have emphasized the contribution of changes in titin (the giant molecular spring

protein that is one important factor responsible for cardiomyocyte passive stiffness) 9-20, 21;

specifically, changes in the phosphorylation of titin 22-32. However, in previous studies of patients

with HFpEF, the role of titin was examined in isolated, chemically demembranated

cardiomyocytes obtained from endocardial myocardial biopsies; these preparations did not

include surrounding ECM structures. The aforementioned changes in collagen and/or titin are

expected to increase passive myocardial stiffness; however, myocardial stiffness has never been

measured directly in myocardium from HFpEF patients. Thus, the magnitude of the assumed

increase in myocardial passive stiffness and therefore its importance as a determinant of diastolic

dysfunction are unknown. In addition, the role of increased passive stiffness in the development

and clinical course of patients with HFpEF and the relative contribution of changes in the ECM

versus titin to passive stiffness in HFpEF are also unknown. In the present study, we

hypothesized that changes in both collagen and titin occur during the development of HFpEF and

that these changes combine to cause a major increase in myocardial passive stiffness that

contributes to the development of HFpEF.

To determine the extent to which ECM collagen and titin contribute to changes in

pecifically, changes in the phosphorylation of titin 22-32. However, in previous ssttutudididiess oof ff papapattitiene ts

with HFpEF, the role of titin was examined in isolated, chemically demembranated

caardrddioioiommymyocococytytytes ooobbtbtaia ned from endocardial myoocacac rrddial biopsies; ttthehh see prprprepe arations did not

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myocardial passive stiffness in patients with HFpEF certain experimental conditions must be

met. First, since several potential determinants of passive stiffness could change concurrently

during the development of HFpEF, the contribution of each must be determined in samples of

myocardium which include an integrated, intact composite structure. To satisfy this condition,

methods of differential extraction have been developed to determine the separate contributions of

collagen and titin to stiffness in demembranated myocardial strips 9, 10. In the current studies

these methods were applied to strips prepared from LV myocardial biopsies obtained during

coronary artery bypass grafting (CABG). Second, the effects of co-morbidities and antecedent

diseases must be distinguished from changes associated with the clinical syndrome of HFpEF.

Two of the most common antecedent disease processes that lead to HFpEF are arterial

hypertension (HTN) and diabetes mellitus (DM) 33-37. In the current study, patients with HTN (or

DM combined with HTN) without HFpEF [HTN(-)HFpEF] were compared to patients with HTN

(or DM combined with HTN) with HFpEF [HTN(+)HFpEF]. Third, an appropriate referent

control group must be studied. For this purpose, CABG patients with no history of HTN or DM

were chosen. There are limitations in using these patients as referent controls in that CAD could

have uncertain effects on myocardial stiffness that cannot be distinguished from those related to

hypertension. However, CAD is present in a majority of patients with HFpEF (38); thus, a CAD

“background” is quite representative of the HFpEF population. Using these methods, the purpose

of this study was to determine whether patients with HFpEF and an antecedent history of HTN or

HTN/DM have an increase in passive myocardial stiffness and whether changes in stiffness are

dependent on changes in collagen and/or titin. Additionally, we sought to determine the

relationship between changes in myocardial passive stiffness and echocardiographic measures of

LV structure and function and selected plasma biomarkers.

Two of the most common antecedent disease processes that lead to HFpEF are aarrrterrriaial l l

hypertension (HTN) and diabetes mellitus (DM) 33-37. In the current study, patients with HTN (or

DMDMM ccomomombibibinenen d d wiwiwiththth HTN) without HFpEF [HTN(N(N(-))HFpEF] weree coc mpmppt ararared to patients with HTN

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Methods

Study Population

Recruitment

The study cohort consisted of 70 males and females recruited to undergo intraoperative LV

myocardial biopsy from amongst those scheduled for CABG at 1) Fletcher Allen Health Care in

Burlington, Vermont, the clinical facility of the University of Vermont College of Medicine

(UVM), 2) the Ralph H. Johnson Department of Veterans Administration Medical Center and the

Medical University of South Carolina Hospital Authority (MUSC) in Charleston, South Carolina,

and 3) selected NHLBI Heart Failure Research Network (HFRN) centers (University of Alberta

[Alberta, Canada], Intermountain Medical Center [Murray, UT], the Mayo Clinic [Rochester,

MN], Minnesota Heart Institute [Minneapolis, MN], University of Utah [Salt Lake City, UT] and

the Utah VA Medical Center [Salt Lake City, UT]) between October 1, 2008 and August 6, 2012

who satisfied the inclusion and exclusion criteria specified below. All patients signed consent

forms approved by their respective Institutional Review Boards.

Experimental Measurements

Demographic, medication and laboratory data and cardiac catheterization results (coronary

anatomy, LV end-diastolic pressure) were tabulated. The severity of coronary artery disease

(CAD) was graded based on the number of major vessels (left anterior descending, left circumflex,

right coronary arteries) with a stenosis >70%, with left main coronary stenosis considered as two

vessels. Patients recruited at UVM and MUSC underwent an echocardiographic-Doppler

examination to assess LV chamber structure and function. In addition, in these patients a 10 cc

plasma sample was obtained for measurement of biomarkers. Intra-operative LV anterior wall

epicardial biopsies were obtained as previously described (39, 40). Patients recruited to this

Alberta, Canada], Intermountain Medical Center [Murray, UT], the Mayo Clinic [[[RoRoRochchheseesteteter,r,r, r

MN], Minnesota Heart Institute [Minneapolis, MN], University of Utah [Salt Lake City, UT] and

hhe e UtUtUtahahah VVVA AA MeMedididiccacal Center [Salt Lake City, UTTT]])]) bbbetween Octobberee 1,, 222000008 and August 6, 2012

wwhoo o satisfied ththhee innnclclluususioioon nn ananand d d exexclclc uususioonnn crititterrria ssppeeecifffieieied d bbbellooww.. AAAllll ppaata iieentnttss ssisigngnnededed ccoonnsesesentntt

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protocol were part of an NIH grant (RO1HL089944) with multiple specific aims; therefore, biopsy

samples were allocated to several protocols, data from some of which have been published 39, 40.

Given the size of each biopsy, all protocols could not be performed on each biopsy. For the current

study, 25 biopsies were used to assess myocardial passive stiffness; 30 were used to measure tissue

collagen content; and 14 were used for titin phosphorylation studies (Table 1).

General Inclusion Criteria

Patients scheduled to undergo CABG over 21 years of age, with a preserved LVEF ( 50%),

normal wall motion and end-diastolic volume index (EDVi, < 75mL/m2), and without evidence of

previous myocardial infarction were eligible. Patients were categorized into three groups: control,

HTN(-)HFpEF and HTN(+)HFpEF.

Specific Patient Group Inclusion Criteria

Control patients fulfilled the general inclusion criteria, but did not have a history of HTN or DM.

HTN(-)HFpEF patients fulfilled the general inclusion criteria, and had a history of HTN

documented in their records and/or had been told of this diagnosis by a physician, and were

receiving medications for its treatment. These patients had no evidence of heart failure as defined

below.

HTN(+)HFpEF patients fulfilled the general inclusion criteria and had HTN and HFpEF as

specified by the European Society of Cardiology and Heart Failure Society of America criteria41,42.

These criteria require: 1) signs and/or symptoms of heart failure (Framingham or Boston criteria,

exercise testing, quality of life questionnaire), 2) LVEF 50 %, 3) LVEDVi < 75 mL/m2, 4)

evidence of diastolic LV dysfunction obtained invasively (cardiac catheterization) or non-

invasively (transmitral or tissue Doppler or left atrial size) and 5) exclusion of non-cardiac diseases

that could cause symptoms commonly present in patients with heart failure.

HTN(-)HFpEF and HTN(+)HFpEF.

Specific Patient Group Inclusion Criteria u

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Exclusion Criteria

Patients were excluded if they had a previous transmural myocardial infarction, LVEF < 50%,

LVEDVi > 75 mL/m2, significant valvular or other non-coronary heart disease, severe chronic

pulmonary disease requiring oral steroids and/or oxygen therapy, any non- cardiac disease or

condition known to affect myocardial function, anemia (Hgb < 13.0 g/dl), serum creatinine > 2.0

mg/dL, poorly controlled hypertension (blood pressure > 140/90 mmHg), off-pump or

emergency CABG, morbid obesity, history of substance abuse, inability to provide informed

consent, poorly controlled diabetes (HbA1c >8.5% within the past 6 months), active malignancy,

severe connective tissue disease , severe liver disease, hypertrophic cardiomyopathy, restrictive

cardiomyopathy or constrictive pericarditis, HIV or active infection.

Myocardial Biopsy Procedure

Anterior LV free wall epicardial biopsies weighing ~25-50 mg were obtained during CABG

soon after the patient was placed on cardiopulmonary bypass, as previously described 39,40. The

biopsy was placed in oxygenated HEPES-based Krebs buffer containing 30 mmol/L 2,3-

butanedione monoximine (BDM) at room temperature 39, 40. Small samples (< 5 mg) were

removed and frozen for collagen and titin studies or placed in formalin for histology. The

remainder of the tissue was processed for stiffness studies. From the section of the biopsy that

remained in buffer, tissue was dissected into pieces < 2 mm in length and placed in skinning

solution containing Triton-X100 at 4 C. For samples obtained at MUSC and the HFRN centers,

the skinning period coincided with overnight transit to UVM at 4 C. After 18-24 hour skinning,

strips were dissected to 150- 200 μm diameter and 800-1200 μm length and then underwent

measurements of myocardial stiffness as described below.

All patients were followed until discharge, with particular attention to ventricular

cardiomyopathy or constrictive pericarditis, HIV or active infection.

Myocardial Biopsy Procedure

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arrhythmias and bleeding complications. No adverse effects or post-operative complications

ascribable to the biopsy procedure were detected and all patients were discharged alive.

Measurement of Passive Myocardial Stiffness

At time of study, aluminum T-clips were attached to the ends of each strip. The strip was

mounted between a piezoelectric motor (Physik Instrumente, Auburn, MA) and a strain gauge

(Kronex Technologies, Oakland, CA) and initially lowered into a 30 μL droplet of relaxing

solution maintained at 37 C. The composition of relaxing solution is specified in previous

reports 39, 40. Sarcomere length (SL) was measured by Fourier Transform of digital images

(IonOptix Corp, Milton, MA) 9. Measurements of total, collagen-dependent, and titin-dependent

stiffness were made using a previously published differential extraction protocol 9, 10. The

extraction method removes the anchors of titin within the myofilament, leaving only ECM-based

stiffness. This method, originally developed and validated by Granzier and colleagues 9, 10, has

been successfully used in other studies 43. In previous control studies in which titin’s stiffness

was eliminated by protease treatment and the muscle strip was then treated with KCL/KI 12,

ECM stiffness was shown to be unaffected by the KCL/KI treatment. Thus, while the differential

extraction protocol causes irreversible myocardial damage it does not affect the measurements

that are central to the questions addressed in this study.

Echocardiography

Echocardiographic studies performed at UVM and MUSC were interpreted by a core laboratory

at MUSC. Studies were de-identified, coded, and interpreted in a blinded fashion. Measurements

were made using American Society of Echocardiography criteria 44.

Collagen

Collagen was assessed using both biochemical and histologic methods. Soluble, insoluble, and

tiffness were made using a previously published differential extraction protocoll 99, 10101 . ThThThe e

extraction method removes the anchors of titin within the myofilament, leaving only ECM-based

ttififffnfnfneesess. TTThihhiss memeethththodo , originally developed and d d vavav llidated by Graaanznn ieer r r aanand colleagues 9, 10, has

beeenn successfufullllyyy uususededd iin n ototheheher ststudududieiesss 4433. Innn ppprevviioouus cococontntrrroll l ststuududieies ininn wwwhihihichchh ttititininin’ss’s ssttiffffffnenessss

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total collagen content were determined using tissue samples sequentially extracted and assayed

directly using the microplate picrosirius red assay 45-47. Collagen volume fraction (CVF) was

measured using light microscopy with samples stained with picrosirius red (PSR) to detect

collagen and viewed with polarized light under dark field optics to detect birefringence of the

fibers 45.

Titin Studies

Titin isoform analysis was performed with 1% agarose gels using a vertical SDS-agarose gel

system as previously described 23, 48. LV myocardium co-expresses compliant N2BA titin and

stiffer N2B titin isoforms 9; their expression ratio was determined. We also measured titin

degradation as the ratio of T2 (~ 2 MDa degradation product of titin) to T1 (full length titin).

Titin phosphorylation levels were quantified via Western blotting 9, 25. Blots were stained

with Ponceau S (Sigma) to visualize the total protein transferred and then probed with phospho-

specific rabbit polyclonal antibodies against phosphorylated S11878(S26) and S12022(S170) of

the PEVK element 25. These sites are known to be phosphorylated by PKC. In addition, blots

were probed with phospho-specific rabbit polyclonal antibodies against phosphorylated

S4185(S469) of titin’s N2B element. This site is known to be phosphorylated by PKA and PKG

32. Membranes were labeled with secondary antibodies conjugated with fluorescent dyes with

infrared excitation spectra (CF680, goat anti-rabbit, Biotium Company, Hayward CA). Blots

were scanned using an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln NE) and

images analyzed using Li-Cor software. Ponceau S scans were analyzed in One-D scan to

normalize phosphorylation signal to protein loading.

Plasma Biomarkers

Biomarkers were chosen that reflect changes in ECM homeostasis, specifically matrix

degradation as the ratio of T2 (~ 2 MDa degradation product of titin) to T1 (full l llelengngngthhh tttitititininin).).).

Titin phosphorylation levels were quantified via Western blotting 9, 25. Blots were stained

wiwiththh PPPoononcececeauauau S (((SSiSiggma) to visualize the total prrotooteiin transferred anana d ththhenenen probed with phospho-r

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metalloproteinases [MMPs] and their tissue inhibitors [TIMPs]. Four classes of MMPs,

gelatinases (MMP-2 and MMP-9), collagenase (MMP-1 and 8), stromelysin (MMP-3), and

matrilysin (MMP-7), and all 4 tissue inhibitors of MMPs (TIMP-1, -2, -3, -4) were assayed. In

addition, N-terminal propeptide of brain naturetic peptide (NT-proBNP) was measured. Finally,

biomarkers that reflect a proinflammatory and/or profibrotic state, specifically CRP, IL-6, IL-8,

TNF- , and sST2 were examined.

Statistical analysis

Data are reported as mean ± SD in tables and text and mean ± SE in figures. A t-test was used to

detect differences in continuous variables, Pearson’s Chi-square test was used to detect

differences in categorical variables for demographics, collagen-dependent and titin-dependent

tension, collagen content, and titin phosphorylation and isoforms and plasma biomarkers

amongst referent controls, HTN(-)HFpEF and HTN(+)HFpEF groups. A general linear mixed

model (GLMM) was also employed to compare the relationship between stress and sarcomere

length in the three groups (Figure 1); this type of model is ideal for handling repeated measures

obtained within the same study subjects 49. Within the GLMM, linear and quadratic relationships

between stress and sarcomere length were considered, and an unstructured covariance structure

was selected after comparing the model’s AIC value to those from models incorporating other

covariance structures (e.g. autoregressive, compound symmetry) 50. In addition, stress was

compared between the 3 groups at selected common values of SL using ANOVA. Linear

correlations were used to examine the relationship between echocardiographic measurements and

stiffness measurements and between echocardiographic measurements and plasma biomarkers

using a least squares best fit model and a Pearson’s correlation coefficient.

differences in categorical variables for demographics, collagen-dependent and tititiniin-d-d- epepepennendededentntn t

ension, collagen content, and titin phosphorylation and isoforms and plasma biomarkers ff

ammononongsgsgsttt rererefefeferrrentntt cccoontrols, HTN(-)HFpEF and HTHTHTNNN(+)HFpEF grrououo pss.. AA A general linear mixed

mmoddedel (GLMM)M)) wwwass aaalsssoo emememplplp oyoyededed ttooo ccompppaaare thhhee rellalattitiononnsshhipip bbetetweweenenn sstrtrresesss anannd dd sasarrrcoomomeeereee

eengngngththth iin n ththhe e e ththrereee grgrg oououppsps ((FiFiigugugurerere 111);); ttthihiisss tytytypeee ooff f mmmoddedelll isisis iidededealal ffororor hhaaanddldlininng g g rerer pepepeatatateded mmmeaeasusurrees s

obtained witthihiinnn ththhe e e sasasamemem ssstututudydyd sssububu jejejectttsss 494949. WiWiWithththininin ttheheh GGGLMLMLMM,M,M, lllinii eaeaear r r ananandd d ququq adadadrararatititicc c rerer lationshipsss


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Results

Demographic and Echocardiographic Data

The mean age of the study group was 65 years; most subjects were male (Table 2). This age and

sex distribution is typical of CABG populations. By definition none of the controls had DM or

HTN. The prevalence of DM was comparable in both HTN groups [48% in HTN(-)HFpEF and

59% in HTN(+)HFpEF]. The extent of CAD was similar in the three groups (number of arteries

with a > 70% obstruction 2.5 ± 0.7). Compared with controls, blood pressure was higher in the two

HTN groups but not different from each other and within national guidelines. The mean creatinine

values were not statistically different between the 3 groups. There were significant differences in

the medications taken by each group (Table 2). As expected, both HTN groups were receiving

antihypertensive medications ( -blockers, ACE-I/ARBs and diuretics) more often than the

controls. Patients with HTN(+)HFpEF were taking diuretics and nitrates more often than the other

groups.

The structural and functional data obtained from the echocardiographic studies also

confirm the category definitions for each group (Table 3). By definition, LV volumes and EF were

normal in each group. Both HTN groups had increased LV mass and RWT. The number in each

HTN group with concentric LVH was comparable (39% in HTN(-)HFpEF and 41% in

HTN(+)HFpEF); the number with concentric remodeling was also comparable (32% in HTN(-

)HFpEF and 30% in HTN(+)HFpEF). Thus, the percentage with either concentric LVH or

concentric remodeling (i.e., increased RWT without increased LV mass) was ~70% in both groups.

Measurements that reflect diastolic function and filling pressure (E, E’, PCWP, LVEDP, LA

volume) were increased in HTN(+)HFpEF. With the exception of a small increase in LA volume,

these measurements were normal in HTN(-)HFpEF patients. LA enlargement was also more

he medications taken by each group (Table 2). As expected, both HTN groups wweeere e rer cecec ivivivinining g g

antihypertensive medications ( -blockers, ACE-I/ARBs and diuretics) more often than the

coontntntrororolslsls. PaPaPatititieeentstss wwwiti h HTN(+)HFpEF were takinnng gg dddiuretics and niniitrtt atesess mmmore often than the other

ggrouuupsp .

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confirm the cacaatetetegogogoryryry dddefeffinitititioioionsnsn fffororo eeeacacch hh grgrgrouououp p p (((TaTaTablblblee 333).).). BBBy y y dededefififininitititiononon, , LVLVLV vovoolululumememes s ana d EF weree


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frequent in the HTN(+)HFpEF group (41%) vs. HTN(-)HFpEF (20%).

Myocardial Stiffness

Relationships between myocardial stress and SL between 2.0-2.6 m for the three groups, which

reflect total passive myocardial stiffness, are shown in Figure 1. Results of the general linear

mixed model (GLMM) indicated that a model assuming a quadratic relationship between stress

and SL provided a superior fit compared to a linear model. The GLMM indicated that as SL

increased, the slope increased most rapidly in the HTN(+)HFpEF group (p<0.0001 compared with

both control and HTN(-)HFpEF), and that the control and HTN(-)HFpEF curves were not

significantly different from one another. Significant (p<0.01) differences were noted between the

HTN(+)HFpEF group and the other groups at 2.1 m and at each SL assessed up to and including

2.6 m. Passive myocardial tension was also examined at SL 2.6μm before and after differential

extraction to estimate collagen-dependent and titin-dependent stiffness (Figure 2). There were no

significant differences in collagen- and titin-dependent tension between control and HTN(-

)HFpEF. However, both collagen- and titin-dependent tension was significantly increased in

HTN(+)HFpEF compared with the other groups. Collagen-dependent stiffness was increased by

220% and titin-dependent stiffness was increased by 92% in the HTN(+)HFpEF group.

There were significant correlations between in vitro measurements of passive myocardial

tension and in vivo echocardiographic measurements of diastolic function (Figure 3). Thus, there

was a statistically significant direct relationship between collagen-dependent tension and left atrial

diameter (r2 = 0.42, p = 0.006) and PCWP (r2 = 0.46, p = 0.002) and between titin-dependent

tension and left atrial diameter (r2 = 0.43, p = 0.006) but not PCWP (r2 = 0.16, p = 0.11).

Collagen

Myocardial collagen content was measured using both biochemical and histologic methods.

HTN(+)HFpEF group and the other groups at 2.1 m and at each SL assessed up totoo andndn iincncnclululudididingng

2.6 m. Passive myocardial tension was also examined at SL 2.6μm before and after differential

exxtrtrracacactititiononn ttto oo eeestiimamamatet collagen-dependent and tittininin-dddependent stifffnenn ss (((FiFiFigug re 2). There were no

iiignnnifi icant diffffererenenenceess innn cccoolollalalaggegen-n-- aaandndn tiittin-d-ddeeppenndedeent ttteennsisi nonon bbeeetwweweenen coonontrtrrololol aandndd HHHTNTNTN(-(--

HHHFpFpFpEFEFEF.. HoHoHowwewevveer,r,, bbootth h cocolllagagagenenn--- aaanndnd tttitttininin-d-d-depppenene ddedennt tttennnsisisioonon wwwaaass sisisigngng iiificccanantltlt y y y ininccrcreeeaseeed inin

HTN(+)HFpEpEpEF F F cocoompmpmparara edee wwwititith h thththeee otototheheerr r grgrgrouououpspsps. . CoCoColllllagagenenen-d-ddepepepenenendedd ntntnt ssstititiffffffnenenesssss wwwasasas iiincncncrer ased by


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Biochemical studies assessed soluble, insoluble and total collagen (Figure 4A). CVF was

estimated by light microscopy of PSR stained sections (Figure 4B-F). There were no differences

in soluble collagen across the three groups. There was a significant increase in insoluble and total

collagen (by~100%) and CVF (by ~130%) in the HTN(+)HFpEF group compared with the

controls and HTN(-)HFpEF groups.

Titin

Titin N2B and N2BA isoforms and phosphorylation were examined in the 3 patient groups.

Three phosphorylation sites in titin’s spring region were examined: S11878(S26)and

S12022(S170) of the PEVK element, sites phosphorylated by protein kinase C (PKC) and

calcium/calmodulin dependent protein kinase II (CaMKII), and S4185(S469) of the N2B

element, a site phosphorylated by protein kinase A (PKA) and protein kinase G (PKG) (Figure

5). There were no differences in the N2BA/N2B titin ratio between the three groups (0.53±0.14

in control, 0.50±0.13 in HTN(-)HFpEF and 0.59±0.14 in HTN(+)HFpEF) and no changes in T2

(titin degradation product): T1 (full length titin) ratio amongst the groups. Patients with

HTN(+)HFpEF had a 31% higher phosphorylation value at the S11878(S26) PKC/CaMKII site,

no change at the S12022(S170) PKC CaMKII site and a 28% lower value at the S4185(S469)

PKA/PKG site compared with HTN(-)HFpEF. There were no significant differences between

HTN(-)HFpEF and control patients at any of the phosphorylation sites.

Plasma Biomarkers

Of the 16 biomarkers measured, four were significantly altered in the HTN patients compared

with the controls (Table 4). CRP, sST2, TIMP-1 and NT-proBNP were higher in the

HTN(-)HFpEF patients compared with the controls. Levels of sST2, TIMP-1 and NT-proBNP

increased further in the HTN(+)HFpEF group. There were no differences in MMP-1, 2, 3, 7, 9 or

calcium/calmodulin dependent protein kinase II (CaMKII), and S4185(S469) off ttthehehe NNN2B2B2B

element, a site phosphorylated by protein kinase A (PKA) and protein kinase G (PKG) (Figure

5).). TTTheheherere wwwererre nonoo ddifi ferences in the N2BA/N2B BB ttit ttiin ratio betweeenene thhe ee thththree groups (0.53±0.14

nn cccono trol, 0.5050±±0± ..1333 inin HHHTNTNTN(-(-(-)H)HFpFpFpEFEFF aandd 0..59±±±00..144 iinnn HHTHTN(N(N +++)HHFHFpEpEpEFFF) aaandndnd nno o chchhananngeeess innin TTT2

ttitittininin dddegegegraraadadadatitiononn ppprorodduductctc ):) TTT11 (f(f(fululull l l lelengngngththth titititiin)n)n) rratatioioi aaamomomonngngststst tthhehe gggroror uupupss.s PPPatata ieieientnttsss wwwiththth

HTN(+)HFpEpEpEF F F hahaad d d a a 313131% % % hihihighghhererer ppphohh spspsphohohoryryrylalalatitit ononon vvvala ueueue aaat t t thththee e S1SS 181818787878(S(S(S262626) ) PKPKPKC/C/C/CaCaCaMKII site,


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TIMP-2, 3, 4 or IL-6, 8 or TNF- between groups.

There were significant correlations between plasma biomarkers and echocardiographic

measurements of diastolic function (Figure 6); there was a statistically significant direct

relationship between PCWP and NT-proBNP (r2 = 0.32, p = 0.001) sST2 (r2 = 0.26, p = 0.005)

and TIMP-1 (r2 = 0.36, p < 0.001).

Correlations between NTpro-BNP and titin phosphorylation at S4185(S469),

S11878(S26) and S12022 (S170) and NTpro-BNP and titin-dependent stiffness were tested.

While the sample size was limited, there was no clear relationship between NTpro-BNP and

S4185(S469) (r2=0.009) or S12022 (S170) phosphorylation (r2= 0.04). However, there was a

trend with S11878(S26) (r2 = 0.32, p=0.2). There was also a trend between NTpro-BNP and titin-

dependent stiffness, r2 =0.22, p=0.08.

Finally, we also tested for correlations between sST2 and CVF and collagen-dependent

stiffness. There were trends for both relationships, with sST2 increasing with increased CVF,

r2=0.58, p=0.047 and increased collagen-dependent stiffness, r2=0.40, p=0.011.

Effects of Diabetes Mellitus

In the two HTN groups, the presence of DM did not affect echocardiographic parameters of

hypertrophy or diastolic function, in vitro measures of stiffness, collagen or titin assays or

biomarkers. DM also did not alter the differences in any of these parameters between HTN

without or with HFpEF.

Discussion

The results of this study support several novel conclusions. The current study showed for the first

time that patients with HTN(+)HFpEF have a significant increase in passive myocardial stiffness

rend with S11878(S26) (r2 = 0.32, p=0.2). There was also a trend between NTprprooo-BNBNBNPPP ananand d d titittin

dependent stiffness, r2 =0.22, p=0.08.

FFFinininalalalllyly, wewewe aalso tested for correlations beeetwtwtweeen sST2 andd CCCVFF aaannnd collagen-dependent

ttiffffnfness. Therere wwweeeree ttrt enenendsdsds fffooorr bobooththh reeelaaationnnshhhipss, wwiththh ssSTSTT222 ininccrcreeaeasisiinnng wwititthh h inincrcrreaeaasesed dd CVCVCVF,FF

22=0=0=0.5558,8,8, ppp=0=0=0.0.047477 aaandnd iincnccrereasssededed cccololollalalaggenenen-d-d-depepepenenendededentntt sttiifffffnenenesss, rrr22==0=0.44.40,0,0 pp==0=0.0.0011111..

Effects of DDiaiaabebeb tetetesss MeMeMelll itttususus


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measured directly in left ventricular myocardial strips. Although consistent with previous studies

in animal models of HFpEF 43, 45, 51, 52, this has never previously been documented in patients

with HFpEF. Previous studies in HFpEF patients have examined the passive properties of the

composite LV chamber 4 or the passive properties of isolated cardiomyocytes 19, 20, but not the

passive properties of the intact myocardium. Making direct measurements in intact myocardium

was a pivotal step for several reasons, including defining the relative mechanistic contributions

of changes in titin and collagen to the increased passive myocardial stiffness seen in patients

with HFpEF and the ability to differentiate the effects of co-morbid conditions on myocardial

properties from the effects of HFpEF itself. The techniques used to make direct measurements of

passive myocardial stiffness in left ventricular myocardial strips were developed using samples

from patients with heart failure and a reduced ejection fraction (HFrEF) but these techniques

have not been previously applied to patients with HFpEF.

Previous studies in patients with HFpEF examined the contribution of changes in titin to

the resting tension (passive stiffness) of isolated cardiomyocytes obtained from LV

endomyocardial biopsies; these studies also showed that collagen volume fraction was increased

19, 20. However, no previous study in patients with HFpEF has shown the relative contributions of

changes in both collagen and titin to passive myocardial stiffness. The current study showed for

the first time that the increase in myocardial stiffness in HFpEF patients was caused by changes

in both ECM fibrillar collagen and cardiomyocyte titin. Specifically, HFpEF patients had

increased collagen-dependent stiffness in association with increased fibrillar collagen content.

HFpEF patients also had increased titin-dependent stiffness in association with significant

changes in the phosphorylation state of titin, with decreased phosphorylation of a PKA/PKG site

in the N2B element and increased phosphorylation of one of the PKC sites in the PEVK element.

passive myocardial stiffness in left ventricular myocardial strips were developedd uusisiinggg ssamamamplplplese

from patients with heart failure and a reduced ejection fraction (HFrEFd ) but these techniques

haaveveve nnnototot bbbeeeee nnn prrrevevevioiously applied to patients witthh h HHHFpEF.

Previououss stttudddieiei s ininin ppatatatiieientnttss s wiwiw thth HFpFppEEEF eexaamiinineded ttthhee ccoonontrtribibbuuutiioon n ofofof cchahaangngngeses in n n tititititin n to

hhe e e rererestststiningg g tetetensnsioioon n (p(paaasssiiveve sstitit ffffffnenenessssss)) oofof iiisososollalateteed d d ccacarrdrdioioomymymyooocytytytesess ooobtbtbtaiainnened d frfrfromomom LLLVVVf

endomyocarrdidiialalal bbbioioiopspspsieieies;s ttthehehesese ssstutut dididiesss aaalslslsooo shshshowowowedede tthahaatt t cococolllllagagagenenen vvvolololumumumee e frfrfracacctititiononon wwwasaa increaseddd


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While all of the HFpEF patients examined in the current study had antecedent HTN and ~

50% had DM, the presence of these co-morbid antecedent disease processes alone, in the absence

of heart failure, did not alter total myocardial stiffness or its titin and collagen-dependent

components. This is the first clinical study in patients with HFpEF to examine and differentiate

the effects of a major antecedent disease substrate, such as hypertension, on myocardial structure

and function before the clinical syndrome of HFpEF has actually developed. There has been

significant controversy regarding the ability to differentiate or distinguish patients with

hypertension from those with HFpEF. It has been proposed that HFpEF is simply the aggregate

of a number of co-morbid factors such as hypertension, diabetes, CAD, obesity, etc. The current

study clearly demonstrates that there are specific structural and functional differences between

patients with hypertension without and with HFpEF. Similar conclusions are likely to be

applicable to other co-morbid or antecedent disease processes.

The current study also shows for the first time that the development of collagen and titin

based changes in diastolic function in patients with HFpEF occur in association with

proinflammatory and profibrotic stimuli as measured by plasma biomarkers. These data provide

additional support for the overall schematic hypotheses proposed by Paulus and Tschope (i.e.,

that proinflammatory and profibrotic signaling play a significant mechanistic role in the

development of HFpEF) and constitute new mechanistic insights into the pathophysiology

underlying HFpEF 13.

Hypertensive Heart Disease

HTN results in LV pressure-overload (PO) and causes a spectrum of structural, functional and

clinical outcomes that have collectively been called hypertensive heart disease (HHD). Data

from animal models of PO suggest that the myocardium responds to increased load by

tudy clearly demonstrates that there are specific structural and functional differereencnccesee bbbetetetweweweenen

patients with hypertension without and with HFpEF. Similar conclusions are likely to be

appplplplicicicababablelele tttoo otheheherrr cco-morbid or antecedent diseeeasasa eee processes. r

The cururrereenntt sstutuudydyy aaalslslso o shshowowowss ffoorr thee fifiirst tttimmme ttthhahatt ththt ee e dedeevveelolopmpmmenent t ofofof ccololllalalagegeg nnn ananandd tit tttin

baasesesed dd chchc ananngegeges s ininn dddiaiaststtolllicic fununnctctctioioion n n iinin pppatatatieieiennnts ss wiwiw tthth HHFpFpFpEFEFEF ooccccccuurur iiinn n asasssosociciatatatioioon n n wwiwitththff

proinflammatatororory y y ananand dd prprprofofibibibrororotiiccc ststs imimmulululi ii asasas mmmeaeaeasusuurrrededd by y y plplplasasasmamama bbbiooomamamarkrkrkererers.s.s TTThehehesesese dddata a provide


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undergoing cardiomyocyte hypertrophy which results in an increase in myofibrillar content 9, 10,

45, 51. In addition to the myofibrils, other structures and processes within both the cardiomyocyte

and the ECM are altered in response to increased load. In animal models these events have a

temporal sequence in which the additional changes in the cardiomyocyte and ECM occur only

after myofibrillar hypertrophy is well under way or even complete 45, 51. For example, in murine

and feline models PO during the first 1-2 weeks of PO, there are no significant changes in CVF

or diastolic filling pressures and no evidence of heart failure; however, after 4-8 weeks CVF and

filling pressures are increased and evidence of heart failure develops. Data from the current study

appear to parallel these temporal changes. Patients with HHD comprise a spectrum: HTN

without LVH, HTN with LVH/concentric remodeling but no heart failure [HTN(-)HFpEF], and

HTN with heart failure [HTN(+)HFpEF]. In the current study HHD patients were not followed in

a serial manner; data were obtained by cross-sectional analysis. However, it was only in the

patients with HTN(+)HFpEF that changes in collagen and titin were detected. Although in the

absence of sequential data in the same patients we cannot prove the concept that sequential

temporal changes occur in the cellular and molecular mechanisms that underlie HHD, our data

clearly support the idea that changes in collagen and titin constitute mechanisms are associated

with the transition from HTN to HFpEF.

Overall, there is good concordance between the current study and previous clinical

studies in pressure-overload and HFpEF 16, 19, 20, 26, 28. Van Heerebeck et al 19 and Borbely et al 20

showed that patients with pressure-overload induced HFpEF had increased cardiomyocyte

stiffness; however, because these mechanical studies were performed in isolated cardiomyocytes

with the ECM removed, the relative contribution of these changes in collagen and titin to

myocardial stiffness could not be determined.

without LVH, HTN with LVH/concentric remodeling but no heart failure [HTN(N(-)--)HFHFHFpEpEpEF]F]F],,, anana d

HTN with heart failure [HTN(+)HFpEF]. In the current study HHD patients were not followed in

a seseeriririalalal mmananannneer; dddaatataa were obtained by cross-sectctctioionnal analysis. HoHoH weeveveverrr, it was only in the

patiiiene ts with HTHTTNNN(+)+))HFHFFpEpEpEFFF thththatat cchhahanngngees innn cccollaaaggen anannd d ttit ttiin n wewwerere dddeete eececteteed.d.d. AAltltthohohouguggh ininin ttthehehe

abbbsesesencncncee ofoff ssseeqequeueentnttiaial l dadaatata inn ththheee sasasammeme ppatatatieieiennntstss wweee ccacannnnnototot ppproroovevev tthehehe cccoononcceceptptt tthahahat t seseseqququeeenttitialal

emporal chaangngngesese oooccccc ururur in n thththeee cececelllll ulululara aaandndnd mmmololo ecececulululararar mmmececechahahanininismsmsms thththatatat uuundndnderere liiie e e HHHHHHD,D,D our data


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Our results indicate that at SL 2.6 m increases in ECM collagen account for more than

2/3 of the increase in resting tension in HFpEF. However, the relative contributions of titin and

collagen to resting tension are SL-dependent; collagen accounts for a larger proportion at longer

SLs and titin for a larger proportion at short SLs. Their relative contributions are therefore

ultimately determined by the actual operating SL range in our patients, which is unknown. Based

on studies performed in pigs 53, we do know that in large mammals the upper end of the

operating range extends to 2.5-2.6 m. To the extent that the actual operating range is lower, the

increase in total resting tension in HFpEF will be more evenly divided between the two.

Data from the current study significantly advance our understanding of how these

mechanisms contribute to changes in myocardial stiffness by examining intact myocardial

samples that allowed integrated physiologic studies to selectively examine the individual

contributions of collagen and titin. The current study showed hypophosphorylation of PKG/PKA

sites on titin in HFpEF patients, consistent with the reduction in passive tension detected when

cardiomyocytes from HFpEF patients are treated with PKA/PKG 19, 20 and consistent with

phosphorylation studies in animal models 43, 54. In contrast to Van Heerebeck et al 19 and Borbely

et al 20, we did not detect changes in the N2BA/N2B ratio in HFpEF patients. In some but not all

of the studies from these investigators, the N2BA/N2B ratio was increased in HFpEF, which

would be expected to result in a potentially compensatory decrease in titin stiffness 16-20, 26, 28. It

is worth noting that the HFpEF patients studied by these investigators had overtly

decompensated heart failure and were therefore very likely at a more advanced stage of the

syndrome. Perhaps more advanced disease is required to elicit such a compensatory change. The

current study also showed for the first time that patients with HFpEF have hyperphosphorylation

of one of the PKC/CaMKII sites on titin. Previous animal studies have demonstrated that

mechanisms contribute to changes in myocardial stiffness by examining intact mmyoyoyocacc rdrdrdiaiaal ll

amples that allowed integrated physiologic studies to selectively examine the individual

coontntntririribububutititiononons off cccoolollal gen and titin. The current stststudududy showed hypppopoo hoospspsphhorylation of PKG/PKA

iiiteess on titin inn HFHFHFpEEFFF ppapatititienenntststs,, cocoonnsnsisssteeent wwwitth thhehe reddduucuctitiononon iin n paassssivivive tetennsnsioioon n dededetetetectcttededd wwhheh nnn

caardrdrdioioiomymymyococcytytyteses ffrooom m HFHFFpEp FF F papaatititieeentntn s s ararare ee trtrtreaeae teteted d wiwiththh PPKAKAKA/PPPKGKGKG 19,19, 2020 anndnd ccconononsisiststtenenent wwwitthth

phosphorylatatioioion n n stststudududieiees s s inn aaaninin mamamall l momom dededelslss 434343, 545454. InInn ccconono trrrasasa tt t totoo VVVanana HHHeeeeeerererebebebeckckc eeet t t alalal 191919 aand Borbelyyy


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hyperphosphorylation at these sites results in increased titin stiffness 23; however, the current

study is the first clinical study to show that this contributes to increased myocardial stiffness in

patients with HFpEF. In addition, while not yet studied in HFpEF, studies in HFrEF have

suggested that CaMKII-dependent phosphorylation of S12022 (S170) may reduce passive

stiffness 54.

The finding that both changes in collagen and titin may play a pivotal role in the

development of HFpEF in HHD suggests the possibility that there may be common upstream

mechanisms resulting in both changes 13. The presence of a HTN induced proinflammatory,

profibrotic, and/or redox stress state is supported by the plasma biomarker profiles found in the

current study. The increase in CRP, sST2 and TIMP-1 in the HFpEF patients supports this

hypothesis. Clearly, more investigation is needed in this area.

Diabetes Mellitus

Diabetes mellitus has been shown in both animal studies and clinical disease to cause abnormal

diastolic function that may contribute to the development of HFpEF 14, 16, 18. DM is reported to

cause changes in collagen homeostasis (by altering cross-linking) and titin phosphorylation.

Approximately 50% of the patients in the current study had DM as well as HTN. The presence of

DM in addition to HTN did not appear to alter myocardial stiffness, collagen or titin to an extent

greater than that of HTN alone. Because of their rarity in the CABG population, we were unable

to identify sufficient numbers of patients with DM without HTN for analysis. This lack of effect

differs from previous studies of pressure-overload (caused by aortic stenosis) in which the

presence of DM caused a significant increase in cardiomyocyte stiffness over and above that

caused by pressure-overload alone. This may be explained by patient selection differences. Thus,

previous studies did not specifically identify whether patients with pressure-overload or DM had

current study. The increase in CRP, sST2 and TIMP-1 in the HFpEF patients suppppopoorrtss thththisisis

hypothesis. Clearly, more investigation is needed in this area.

DiDiabababeteteteses MMMelele litutuusss

DDiaababetes mellilitutuss hhahas s bebeeenenn sshohohownwnn iinn n bbottth annnimmmal sttuudiieiesss ananndd clcliinniiccalal dddisseeasassee ttoto ccaauauseses aaabnbnnorormammal

didiasasastotoolililicc fufuuncncnctitiononn ttthahatt mmamayy cocoontntntririribububutete tttoo thththe ee dedeevevev llolopmpmpmenennttt ofoof HHHFpFppEFEFEF 1414,, 1666, 1, 1888. DMDMDM iiis s rreppporrtrtededd too o

cause changegesss ininn cccololollalaagegeen hohohomememeososostataasiss s s s (b(b(by yy alalaltetet ririr ngngng cccror ssssss-l-llinininkikikingngng)) ananand d d titititititin n n phphp osososphphphorororylyy ation.


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HFpEF or selectively examine patients with LVH without HFpEF 14, 16, 18. Additionally, the

current study may not have been powered sufficiently to examine the separate effects of DM,

there may be differences between pressure-overload caused by HTN versus aortic stenosis, and

the duration, severity and management of DM may differ between studies. The current study

does not necessarily bring into question the importance of DM in HFpEF, but it does raise the

question of whether HTN and DM together have a combinatorial effect on passive stiffness. In

addition, we did not examine the effects of DM on other determinants of diastolic function that

could affect passive stiffness such as calcium homeostasis and contractile proteins, key

determinants of relaxation rate and extent.

Effects of changes in collagen and titin homeostasis on diastolic function

Changes in collagen content, geometry, and composition are associated with abnormal diastolic

function in HHD and other comorbidities common in patients with HFpEF 55. Collagen content

is the product of the balance between collagen synthesis, post-synthetic processing, post-

translational modification and degradation. The current study was not designed to examine the

determinants of collagen homeostasis or mechanisms that alter it. However, the changes in

plasma biomarkers detected in the HFpEF patients may provide some insights 56. Increases in

sST-2 and TIMP-1 suggest that a profibrotic stimulus was present that would be expected to

increase collagen synthesis and decrease collagen degradation. TIMP-1 has been shown to

inhibit MMPs, the major degradation enzymes present in the ECM. ST2 is a member of the

interleukin 1 receptor family. Its functional ligand is interleukin 33 (IL-33), a cardiac fibroblast

protein. Binding of IL-33 to membrane ST2 elicits an antihypertrophic and antifibrotic response.

This cardioprotective effect is negated by soluble ST2 which prevents binding of IL-33 to

membrane bound ST2. Thus, increased TIMP-1 and sST-2 provide two potential mechanisms for

Effects of changes in collagen and titin homeostasis on diastolic function

Changes in collagen content, geometry, and composition are associated with abnormal diastolic

fuuncncctititioonon iiinnn HHHHH DDD aanand other comorbidities commmmonono iin patients witith hh HFFFpEpEpEF 55. Collagen content

ss thhehe product oof f f thhhe babab lalaancncnce e bebebetwtweeeeeenn cocollaggennn synntnthhessisiss,s, ppoosost-t-sysyynnththeteticici ppprorocececessssininng,g,g ppoost-t--

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the profibrotic state in HHD.

Titin’s I-band segment serves as a molecular spring that develops passive force when

extended57. Alternative splicing results in either the shorter and stiffer N2B or the longer and

more compliant N2BA isoforms, which are co-expressed within the sarcomere. When the

N2BA/N2B isoform ratio increases, cardiomyocyte and myocardial stiffness decrease as reported

in heart failure with reduced EF 58, 59. In contrast, Borbely et al reported an increase in the

N2BA/N2B isoform ratio in HFpEF patients but cardiomyocyte stiffness was increased 28,

implying that changes in phosphorylation outweigh the isoform shift. The N2BA/N2B ratio did

not change in the current study. As discussed above, it is possible that the isoform switch occurs

at a later stage of HFpEF. In addition, the N2BA:N2B ratio did not change in at least one study

of human HCM and DCM 60.

The phosphorylation state of 2 elements within titin’s spring segment also modulates

cardiomyocyte stiffness. Sites within the N2B element (e.g., S4185[S469]) are phosphorylated

by PKA and PKG, which decreases passive force 25, 32. Sites within the PEVK element

(S11878[S26] and S12022 [S170]) are phosphorylated by PKC (other PKC isoforms have not

yet been studied), which increases passive force. Additionally our data and the data of other

investigators suggest that these sites on titin are also a target of CaMKII 54, 61, 62. In both the

current and previous studies in HFpEF patients 28, hypophosphorylation of the N2B element has

been observed. Moreover, treatment of skinned cardiomyocytes from HFpEF patients with PKG

decreased cardiomyocyte stiffness 28, indicating that hypophosphorylation of PKA/PKG sites

contributes to elevated passive force. Importantly, PKG treatment did not lower stiffness to the

level present in control cardiomyocytes. It has been speculated 22 that this residual elevation in

tension is due to hyperphosphorylation of PKC sites. In the current study we demonstrate that

at a later stage of HFpEF. In addition, the N2BA:N2B ratio did not change in at t leleleasasstt onono e ee stststuduudy y

of human HCM and DCM 60.

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caardddioi myocyttee stsstiffffnneesss... SSSitititeseses wwititthihihinn n tthheee N222BBB elememmentt t ((e(e.g.gg.., SS414118855[S[SS4644 99]9])) arararee phphhosossphphphororrylyylatatateddd

byby PPPKAKAKA aandndnd PPPKGKGG,, whwhhicch h dedecrcrc eaeaaseseses ss papaasssssiviviveee fooorcrcr ee 255,5, 3222.. SiSiSitetetes s wiwiw ththhininn ttthehee PPPEVEVVK K K elelemememennttt

S11878[S26]6]6] aaandndnd SSS121220200 22 [S[S[S171770]0]0]) ) arara e e phphphosososphphphororo ylylylatatatedede bbby y y PKPKPKCCC (otototheheher r r PKPKPKCCC isisisofofofororormsmsm have not


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hyperphosphorylation of the S11878(S26) PKC/CaMKII site in the PEVK segment is associated

with increased myocardial stiffness in HFpEF patients. This constitutes a novel mechanism of

increased stiffness in HFpEF.

The increased PKC activity that is suggested by the increased S26 phosphorylation did

not result in phosphorylation of S12022 (S170). Previous studies indicate that PKC is

preferably active at sites with N-terminal and C-terminal basic residues 63. These studies also

indicate that PKC preferentially phosphorylates serines with basic residues within three amino

acids of both the C-and N-termini. Based on these results, PKC should have a stronger affinity

for S11878(S26) than S170 because of the closer proximity of the neighboring basic amino acids

(lysine [K] and arginine [R]). Finally, increased PKC activity has been shown to directly

increase protein phosphatase inhibitor-1 (PP1) activity 64, an effect which should facilitate

hypophosphorylation of PKA/PKG sites in the N2B element (its effect on PKC sites might be

negated by the increased PKC activity). Thus, increased PKC levels might increase stiffness

directly through phosphorylation of PEVK S11878(S26) and indirectly through PP1 activation

causing hypophosphorylation of PKA/PKG sites.

Limitations

The demographic characteristics of the current study population were typical of a CABG

population. However, our HFpEF patients were slightly younger and more often male than

typical patients in epidemiologic studies or randomized clinical trials. From the viewpoint of LV

structure and function, however, they were typical of previous HFpEF studies.

What constitutes an appropriate control group is a challenging issue in all research using

human myocardium. The degree of angiographic CAD was similar in all three study groups;

therefore, CAD was not likely to be a confounding variable across groups. Alternative choices

lysine [K] and arginine [R]). Finally, increased PKC activity has been shown ttooo didid reectctctlylyly

ncrease protein phosphatase inhibitor-1 (PP1) activity 64, an effect which should facilitate

hyhypopopophphphososphphphoooryllatatatiioion of PKA/PKG sites in the NN2N2BBB element (its eeefff ecct tt oonon PKC sites might be

nnegagated by thee iiinncncrereasasseddd PPPKCKCKC aacctititivivivityty)). Thuuus,,, inccreeeasededed PPKCKCKC llevvvelels mimim ghght t t ininncrcreaeaeasesee sstitt ffffffnenesssss

didirereectcttlylyly tthrhrrouououghgh phhohospspphohooryrylaaatitt ononon ofofof PPEVEVEVK KK SS1S118181 77878(S(S( 22626))) ananand d ininindddirereectctc lylyy tthrhrh ouououghghgh PPPP1P1P1 aaccctiivvatatiiionnn

causing hypopophphphosossphphphororrylyly attioioion n n ofoff PPPKAKAKA/P/PPKGKGKG sssititi esese ..


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for controls are limited and less than ideal. Unused explanted donor hearts are usually from

young subjects who have been subjected to high levels of stress for varying durations. Open

heart procedures in patients without CAD or LV remodeling (e.g., ASD closure) are now rare

and usually performed in younger patients. Samples from endomyocardial biopsies cannot be

used for mechanical tissue studies because of tissue trauma. On the other hand, CAD, much of

which is likely sub-clinical, is very common in HHD and HFpEF patients. Indeed, a recent report

indicates that a majority of HFpEF patients have epicardial coronary stenosis 38. Thus, the

presence of CAD can be considered a “real world” background in HTN patients and controls.

The limitation imposed by the presence of CAD, however, is that we cannot exclude possible

CAD effects on myocardial stiffness and/or distinguish them from the effects of hypertension or

diabetes.

The current study focused specifically on the contribution of collagen and titin to the

development of HFpEF. As in heart failure with a reduced ejection fraction (HFrEF), in HFpEF

other mechanisms also contribute to the development of heart failure. Thus, changes in calcium

homeostasis, energetics, and actin-myosin cross-bridge dynamics, which govern the speed and

completeness of relaxation, all may play a role in HFpEF in addition to changes in passive

stiffness.

Titin is a large and complex molecule with many phosphorylation sites outside the

mechanically relevant spring region; however, assessing all of them may not be required in the

context of the current study. The current study focused on three sites in the N2B and PEVK

elements that have been documented in several studies for which we have made and

characterized phospho-site specific antibodies. Two additional PKA/PKG sites in the N2B

element were published recently by Kotter et al (S4010 and S4099).In their recent study Kotter et

CAD effects on myocardial stiffness and/or distinguish them from the effects off hhhypypperrrteteensnsnsioioion n or

diabetes.

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ddedevvevelopment ofoff HHHFFpFpEFEE .. AsAsAs iiin nn hheaeaartrtt ffaaailuure wwwiiith aaa rreeduuucceed d ejejecectitiionnn ffraacctiioion n (H(H(HFrFrEFEFEF),),), iiinn HFHFHFpEpEpEF

ottheheher r memem chchhanananisismmms aalslsooo ccocontn ririribubuutetete tttooo ththhe e dededevvveloloopmpmpmeennt ofoff hhheeaeartrtt ffaaiailuuurerere. ThThThusus, chchhanangegegess innn cccalalciciiummm

homeostasis,s,, eeenenen rgrgrgetete iciccs,s,, andndnd aaactcttininin-m-mmyoyoosisis nn n crcrcrososo s-s-s brbrbrididdgegeg dddynynynamamamicicics,s wwwhihihichchch gggovovo ererern n n thththe e e sps eed and


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al showed that all three sites on the N2B element are hyophosphorylated in heart failure,

indicating that they change in concert 60. Thus, assessing the established Ser4185/485 site is

likely to reveal the general phosphorylation status of the N2B element.

It is important to acknowledge other limitations of the current study. We did not

systematically study all titin phosphorylation sites. The protein has at least 70 phosphorylation

sites, as shown by in-vivo phosphoproteomics using a SILAC approach 54. Moreover, recent

work has suggested that the N2B domain contains several more conserved serines which are

differentially phosphorylated in human HFrEF and in animal models of HFpEF 32, 43, 52.

Phosphospecific antibodies against the respective N2Bus sites were used in human HCM and

DCM hearts 60, 54. These studies underscore the complexity of titin phosphorylation schemes and

the need for further research in patients with HFpEF as well as other forms of heart failure that

will allow a more complete understanding of the contribution of titin to passive stiffness.

The current study also did not focus on upstream regulatory mechanisms effecting titin

phosphorylation such as changes in the expression, abundance and activity of relevant protein

kinases (e.g., PKA, PKG, CaMKII, PKC) and phosphatases (PP1, PP2a). Changes in kinase and

phosphatase activity have been shown to be important in other forms of heart failure 26, 43;

changes in HFpEF may represent important targets for novel therapies and important areas for

future research.

Several other important mechanisms influencing the ECM were not examined in the

current study, e.g., collagen isoform expression and content, troponin I and T, and galectin-3.

Unfortunately, the size of the LV biopsy limited the total number of experimental measurements

obtainable from each sample. Each of these additional mechanisms represents important

directions for future research.

DCM hearts 60, 54. These studies underscore the complexity of titin phosphorylattioioon n scchehehemememesss anand

he need for further research in patients with HFpEF as well as other forms of heart failure that

wiwilllll aallllllowoww aaa mmmorre ee ccocomplete understanding of theee ccooontribution of ttititi in ttooo pppassive stiffness. f

The cururrereenntt sstutuudydyy aaalslslso o didid d nononot t fofoccus onnn upppsttrreaaammm rereguguulalattoorryy mmeeechhahanininissmsmss efefeffefefectcttinnngg g tiititit nnn

phphhososo phphphororylylylatatatioionn suuuchch aass s chchannngegegesss ininin tthehee eeexpxpxprrer ssssssioionn,n, aabbubundndndaanancecece aandndnd acaca titiiviiityty ooof f rerer leleevavavantt prroroteteeinin

kinases (e.gg.,.,, PPPKAKAKA, , PKPKPKG,GG CCCaMaMaMKIKIKII,I, PPPKCKCKC) ) ) ananand d d phphphosososphphphatttasasaseseses (((PPPPPP1,11 PPPP2P2P2a)a)a .. ChChChananngegeges s s ininin kinase and


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Finally, the effects of medications taken by the patients prior to surgery on titin

phosphorylation and ECM homeostasis could not be assessed.

Clinical Implications

HTN is commonly a progressive process that leads to adverse cardiovascular remodeling,

abnormal diastolic function, and the development of heart failure, particularly HFpEF 65-67. Once

HFpEF has developed, subsequent morbidity and mortality rates are very high 67-76. Treatment of

HHD is thus a critical unmet challenge. One clear answer is prevention of HHD by early

treatment of high blood pressure, but prevention alone is insufficient. LV hypertrophy,

concentric remodeling, and diastolic dysfunction commonly develop without concomitant

symptoms, often before HTN has been detected. Furthermore, once LV remodeling and/or

HFpEF develop, even guideline prescribed blood pressure control does not reduce morbidity and

mortality. Randomized clinical trials in HFpEF of beta-blockers, angiotension converting

enzyme inhibitors, angiotension II receptor blockers, aldosterone blockers and PDE-5 inhibitors,

in which >90% of the patients had hypertension, have failed to show improvement in morbidity

or mortality 70-76. Therefore, there is a need for novel treatments beyond blood pressure control.

The development of novel therapies must overcome several critical barriers. Most

importantly, the cellular, extracellular, and molecular mechanisms that cause the progression

from HHD to HFpEF must be defined in man. Although animal models are useful, they do not

capture all of the key elements of complex and chronic human disease processes. Once proven

operative in patients, each mechanism can serve as a target for successful therapy. The current

study identifies two of these mechanisms; changes in collagen content and titin phosphorylation,

each of which is a potential target. In addition, plasma biomarkers that reflect these changes in

collagen, titin and the profibrotic milieu could be used to improve diagnostic criteria for HFpEF

ymptoms, often before HTN has been detected. Furthermore, once LV remodellininnggg annndd/d/ororor

HFpEF develop, even guideline prescribed blood pressure control does not reduce morbidity and

momortrtrtalalalititity.y. RRRaanndooommimizzed clinical trials in HFpEF oofof bbbeta-blockers, aaangioiootetetennsion converting

ennzyyzyme inhibititororrs,, aangnggioooteetensnsnsioioion n IIIII rrrececepeptor blbloockkeerrss, aalalddodoststterrrononee bblblocockekek rsrs aaandndnd PPDEDEDE-5-5-5 iinhnhhibibibitii ooors,

nn wwwhihihichchc >>909090% % oofof tthehe paatatieientntts s hahahaddd hyhyh ppepertrtrtenenenssiononon, , hahahaveve ffaiaiaileleledd tototo ssshohoow w w imimmpprprovovvememmenennt t iiin mmmoororbibididitytyty

or mortalityy 707070-767676. ThThThererrefefforre,e,e, tttheheererere iis s s a a nenen ededed fffororor nnovovoveeell l trtreaeaeatmtmtmenenentststs bbeyeyyononond d d blblblooooo d d d prprpresesessususurer control.


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and prognostic assessment. Thus, changes in biomarkers such as TIMP-1 and sST-2 may detect

the earliest transition from HTN to HFpEF. Finally, changes in these biomarkers could possibly

be used to monitor treatment efficacy before changes in myocardial structure and function or

clinical status are evident.

Conclusions

HTN in the absence of HFpEF was not associated with increased passive myocardial stiffness.

However, we show for the first time that patients with hypertension and HFpEF have markedly

increased passive myocardial stiffness due to increases in the contribution of both collagen and

titin. These results suggest that the development of HFpEF is linked to a major increase in

passive stiffness caused by changes in both collagen and titin homeostasis.

Funding Sources: This work was supported by National Institutes of Health grants

R56HL123478 (Zile), RO1HL089944 (LeWinter), U10 HL110342 (LeWinter), R01HL06288

(Granzier), and UL1TR000062 (Nietert), Merit Awards from the Veterans’ Affairs Health

Administration and the NHLBI Heart Failure Research Network. Dr. Michael Zile is supported

by the Research Service of the Department of Veterans Affairs (5101CX000415-02 and

5101BX000487-04).

Conflict of Interest Disclosures: None

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75. Pitt B, Pfeffer MA, Assmann SF, Boineau R, Anand IS, Claggett B, Clausell NN, , , DeDeD sasaii ASAS,,,Diaz R, Fleg JL, Gordeev I, Harty B, Heitner JF, Kenwood CT, Lewis EF, O'Meeaaara aa EE,E, Probstfield JL, Shaburishvili T, Shah SJ, Solomon SD, Sweitzer NK, Yang S, MMcKcKKinininlalaayy SMSMSM, TOPCAT Investigators. Spironolactone for heart failure with preserved ejection fraction. h N Engl J Med. 2014;3;3700:1383-1392

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Table 1. Sample Size for Each Endpoint

Referent Hypertension Control (-) HFpEF (+) HFpEF TotalEchocardiogram 17 31 22 70 Plasma Biomarker 12 22 18 52 Myocardial Stiffness* 9 10 6 25 Myocardial Collagen* 7 4 4 15 Collagen Volume Fraction* 5 5 5 15 Titin* 4 4 6 14 Abbreviations: * Biopsy size limited number of studies / patient, HFpEF = heart failure with a preserved ejection fraction.

Table 2. Patient Demographics Referent Hypertension Control (-) HFpEF (+) HFpEFAge (years) 65 ± 8 62 ± 9 66 ± 9 BSA (Kg/m2) 2.0 ± 0.2 2.1 ± 0.3 2.2 ± 0.3 Sex (% Male) 88% 90% 82% Diabetes Mellitus (%) 0 48% * 59% * Creatinine (mg/dL) 0.97 ± 0.21 0.94 ± 0.20 1.2 ± 0.5 Heart Rate (bpm) 65 ± 15 67 ± 10 68 ± 10Systolic BP (mmHg) 122 ± 7 132 ± 11 * 136 ± 14 *Diastolic BP (mmHg) 73 ± 7 70 ± 10 70 ± 11LV EDP (mmHg) 11 ± 4 13 ± 4 17 ± 5Medications (%)

-Blocker 65 81 82ACE-I/ARB 0 77 * 77 *CCB 0 26 * 27 *Diuretic 0 16 * 68 *#Lipid Therapy 65 87 86Nitrates 12 13 55 *#

CAD (# Vessels) 2.5 ± 0.7 2.5 ± 0.7 2.6 ± 0.6Abbreviations: Data = Mean ± SD, * = p < 0.05 vs Referent Control, # = p < 0.05 vs Hypertension (-) HFpEF, HFpEF = heart failure with a preserved ejection fraction, BSA = Body surface area, HR = heart rate, BP = blood pressure, -blocker = beta adrenergic receptor blocker, CCB = calcium channel blocker. ACE-I = angiotensin converting enzyme inhibitor, ARB = angiotensin receptor blocker, CAD = coronary artery disease, LVEDP = left ventricular end diastolic pressure.

Referent Hypertensionn Control (-) HFpEF (++)) HHFHFpEpEFF

Age (years) 65 ± 8 62 ± 9 66 ± 9 BSA A (K(K(Kg/g/g/mmm22)) 2.0 ± 0.2 2.1 ± 0.3 2.2 ± 0.3 SeSeex ((%(% MMalalleee) 8888% % 900% % % 8282% % DDiaababetes Melllilitutuuss (%%%) 00 4888% % * 595959% % *CrCrCreaeaatinine (((mgmgm //d/dL)L) 0.0.0 97977 ±±± 0.22211 0.0.949494 ± 00.2220 1..22 ±± 000.555HeHeararrttt RaRaRatetete ((bpbppm)m)m) 65655 ±±± 11155 5 676767 ±±± 111000 686868 ±±± 111000SSySy tstst lololiicic BBBPPP (m(m(m HmHmHg)g)g) 121212222 ±±± 777 131313222 ±±± 111111 *** 111363636 ±±± 111444 ***DiDiasastotolilicc BPBP ((mmmmHgHg)) 7373 ±± 77 7700 ±± 1010 7700 ±± 1111


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Table 3. Echocardiography

Referent Hypertension Control (-) HFpEF (+) HFpEFLV EDVi (mL/m2) 58 ± 11 60 ± 10 58 ± 14 LV ESVi (mL/m2) 20 ± 7 21 ± 4 21 ± 11 LV EF (%) 65 ± 7 65 ± 6 65 ± 14 LV Massi (g/m2) 88 ± 15 107 ± 20* 116 ± 20* LV Wall Thickness, cm 0.9 ± 0.1 1.1 ± 0.1* 1.2 ± 0.1* RWT 0.38 ± 0.04 0.45 ± 0.06* 0.48 ± 0.10*E (cm/s) 80.1 ± 16.9 76.7 ± 19.8 83.5 ± 22.2E’ (cm/s) 10.8 ± 1.8 10.1 ± 3.3 7.3 ± 2.0*#

PCWP (mmHg) 12.1 ± 2.8 12.5 ± 3.3 17.6 ± 5.4*# LA Voli (mL/m2) 20.8 ± 3.6 25.7 ± 8.0* 37.0 ± 11.9*# Abbreviations: Data = Mean ± SD, * = p < 0.05 vs Referent Control, # = p < 0.05 vs Hypertension (-) HFpEF, HFpEF = heart failure with a preserved ejection fraction (EF), LV = Left ventricle, BSA = body surface area, EDVi = end diastolic volume indexed (normalized) to BSA, ESVi = end systolic volume indexed (normalized) to BSA, LV Massi = LV mass indexed (normalized) to BSA, PCWP = pulmonary capillary wedge pressure as estimated from E/E’ ratio, LA Voli=left atrial volume indexed (normalized) to BSA, RWT = relative wall thickness, E = transmitral early peak filling velocity, E’ = Tissue Doppler mitral annular early peak diastolic velocity.

Table 4. Biomarkers

Referent Hypertension Control (-) HFpEF (+) HFpEFCRP, g/mL 2.4 ± 1.8 4.8 ± 2.9* 4.2 ± 2.1* IL-6, pg/mL 3.7 ± 3.5 3.3 ± 1.9 4.1 ± 4.5IL-8, pg/mL 8.3 ± 6.4 8.4 ± 7.3 12.9 ± 15.3 TNF- , pg/mL 3.4 ± 2.1 4.4 ± 2.1 4.3 ± 2.6sST2, ng/mL 25.6 ± 19.8 82.0 ± 35.7* 105.5 ± 31.4*# NT-proBNP, pg/mL 897 ± 629 1,507 ± 976* 2,355 ± 1,394*# MMP-1, pg/mL 383 ± 180 551 ± 403 372 ±362 MMP-2, ng/mL 326 ± 106 318 ± 69 353 ± 115 MMP-3, ng/mL 9.4 ±7.3 7.8 ± 2.9 8.1 ± 5.6 MMP-7, pg/mL 228 ± 132 468 ± 288* 212 ± 210# MMP-8, pg/mL 1110 ± 656 1257 ± 716 1852 ± 2198MMP-9, ng/mL 120 ± 73 114 ± 82 152 ± 160 TIMP-1, ng/mL 64 ± 27 101 ± 27* 128 ± 34*# TIMP-2, ng/mL 71 ± 15 79 ± 11 78 ± 13 TIMP-3, ng/mL 2.6 ± 1.1 3.5 ± 2.6 3.5 ± 1.6 TIMP-4, ng/mL 1.7 ± 0.8 1.7 ± 0.8 2.1 ±0.7 Abbreviations: HFpEF = heart failure with a preserved ejection fraction, MMP = matrix metalloproteinase, TIMP = tissue inhibitor of MMP, CRP = C-reactive protein, Il = Interleukin, TNF = tissue necrosis factor, sST2 = soluble ST2, NT-proBNP = N-terminal propeptide of brain natriuretic peptide, Data = Mean ± SD, * = p < 0.05 vs Referent Control, # = p < 0.05 vs Hypertension (-) HFpEF.

ndexed (normalized) to BSA, ESVi end systolic volume indexed (normalized) to BSA, LV Massi LV maasss iindndexexed normalized) to BSA, PCWP = pulmonary capillary wedge pressure as estimated from E/E’ ratio, LA Volii=llefefeftt t atatatririr alalal vvololo umumume endexed (normalized) to BSA, RWT = relative wall thickness, E = transmitral early peak filling velocity, EE’’ = TiTT sssueueue DDopoppplplp erer k

mitral annular early peak diastolic velocity.

Tablble e 4.4.4. BiBiomomomararrkekekersr

RRRefefefererenennt tt HyHyHyppeperrtenennsisisiononon CCCononnttrrol (-(-(-))) HHHFppEEFF (+))) HHFHFpEEEFF

CRCRRP,P,P, g/g/mLmLmL 2.2.2.44 ±±± 1.1.88 4.4.888 ±±± 2.2.9*9** 4.4..22 ±± 222.11*1* L-6-6, , pgpg//m/mLL 3.3 7 ±± 33.3.555 3.3.333 ±± 1.999 444.1 ±± 444.555L-8, pg/mLL 8.8..33 ±±± 6.6.6 444 8.8.8.4 4 4 ±±± 7.7.7.3 121212.9.99 ±± 15.3

TNTNFF pg//mLL 33 44 ±± 22 11 44 44 ±± 22 11 44 33 ±± 22 66


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Figure Legends:

Figure 1. Total myocardial stiffness expressed as the relationship between myocardial stress

(mN/mm2) versus cardiomyocyte sarcomere length ( m) for referent control patients (open

circle, solid line), patients with hypertension but without heart failure and a preserved ejection

fraction (HTN(-)HFpEF, closed circle, dashed line), and patients with hypertension and HFpEF

(HTN(+)HFpEF closed squares, dotted line). As sarcomere length increases, the slope increases

most rapidly in the HTN(+)HFpEF group (p<0.0001 when compared to both the control and

HTN(-)HFpEF groups). Overall, the curves for the control and HTN(-)HFpEF groups were not

significantly different from one another. Patients with HTN(+)HFpEF had an increase in total

myocardial stiffness as indicated by a leftward shift in the stress vs. sarcomere length

relationship; for any given sarcomere length 2.1 m, stress was higher in the HTN(+)HFpEF

vs. HTN(-)HFpEF or referent control patients. There were no significant differences between

HTN(-)HFpEF vs. referent control patients. * = p < 0.01 vs. referent control, # = p < 0.01 vs.

HTN(-)HFpEF.

Figure 2. Collagen-dependent and titin-dependent myocardial stress at a sarcomere length of 2.6

m for referent control patients (white column), patients with hypertension but without heart

failure and a preserved ejection fraction (HTN(-)HFpEF, cross-hatched column), and patients

with hypertension and HFpEF (HTN(+)HFpEF black column). Total stress is the numerical sum

of collagen and titin specific data. Patients with HTN(+)HFpEF had an increase in collagen-

dependent and titin-dependent myocardial stress. There were no significant differences between

HTN(-)HFpEF or referent control patients. * = p < 0.01 vs. referent control, # = p < 0.01 vs.

HTN(-)HFpEF.

ignificantly different from one another. Patients with HTN(+)HFpEF had an increreasasasee ininin tttotototalala

myocardial stiffness as indicated by a leftward shift in the stress vs. sarcomere leengnggththth

elaatiiononshshipip;; fof r r anana y y given sarcomere length 22.1. m, stress was highherere in the HTN(+)HFpEF

vsvss. HHHTN(-)HFHFFpEpEpEF F F ororr rrrefefeferererenene t t cococontntntroror l l l papapatitienenentststs.. ThThherrre wewewererere nnno oo sisisigngngnififificicicananant t t diffffferererenenencececes s bebebetwtwtweeeeeenn n

HTHTHTNNN(-)HFpEpEEFFF vvs.. reffeerreentt ccconnntrtrol pppatatieiennts. * = pp << 0.0.010101 vvsss. rrrefeeereeent cooonntntrorool, ## = ppp <<< 00.0011 vsss.

HTHTN(N((-)-))HFHFpEpEp F.F.


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Figure 3. Relationship between in vivo echocardiographic derived assessment of LV diastolic

dysfunction (left atrial diameter and echocardiographically estimated pulmonary capillary wedge

pressure [PCWP]) and in vitro measures of myocardial diastolic dysfunction (collagen-dependent

and titin-dependent myocardial stiffness) for all patients studied with both measures available.

There was a statistically significant direct relationship between collagen-dependent stiffness and

left atrial diameter (r2 = 0.42, p = 0.006) and PCWP (r2 = 0.46, p = 0.002) and between titin-

dependent stiffness and left atrial diameter (r2 = 0.43, p = 0.006) but not PCWP (r2 = 0.16, p =

0.11).

Figure 4. Myocardial collagen content in patients with hypertension (HTN) with(+) and

without(-) heart failure with a preserved ejection fraction (HFpEF). Referent control patients

(white column), patients with HTN(-)HFpEF (cross-hatched column), and patients with

HTN(+)HFpEF (black column). * = p < 0.01 vs. referent control, # = p < 0.01 vs.

HTN(-)HFpEF. Panel A: Soluble, insoluble, and total collagen measured biochemically. Patients

with HTN(+)HFpEF had an increase in insoluble and total collagen. There were no significant

differences between HTN(-)HFpEF or referent control patients. Panel B: Examples of picrosirius

stained myocardial sections. Patients with HTN(+)HFpEF had an increase in collagen. There

were no significant differences between HTN(-)HFpEF or referent control patients. Panel C:

Collagen volume fraction (CVF) measured from histologic sections. Patients with

HTN(+)HFpEF had an increase in CVF. There were no significant differences between

HTN(-)HFpEF or referent control patients..

Figure 5. Titin phosphorylation state in referent control patients (white column), patients with

Figure 4. Myocardial collagen content in patients with hypertension (HTN) witth(h((+)+)) aandndnd

without(-) heart failure with a preserved ejection fraction (HFpEF). Referent control patients

wwhihihitetete ccolololumumumnn), papapatitients with HTN(-)HFpEF (crororossss-hatched columummn)),, ananand d patients with

HHTNNN(+)HFpEEF F (b(b( lalackckck cccolololumumumn)nn). *** == p p << 0.0011 vvvs. rrrefferenenent t cocoonnttrorolll, ### == ppp <<< 00.0.00111 vsvs.

HTHTTN(N(N(-)-))HFHFFpEpEpEF.F. PPPaananelel AAA:: SoSolululublblbleee, iiinnsnsoololububublelele,, ananand d ttototatat l cocoollllllagagagenenen mmmeaeaasusus rrredd d bibiiococo heheh mmimicccalllly.y. PPatattieentntn s

with HTN(++)H)H)HFpFpFpEFEFEF hhhadadad aaan n n ininincrcrreaeaeasesese in n n ininnsososoluluublblb ee e ananand d d toootatatall l cococollllllagagagennn.. ThThThererere e e wewewererere nnnooo sisis gnificant


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hypertension but without heart failure and a preserved ejection fraction (HTN(-)HFpEF, cross-

hatched column), and patients with hypertension and HFpEF (HTN(+)HFpEF black column).

Three titin sites were examined: S11878(S26) and S12022(S170), sites known to be

phosphorylated by protein kinase c (PKC) and S4185(S469), a site known to be phosphorylated

by protein kinase a (PKA). Patients with HTN(+)HFpEF had an increase in S11878(S26), no

change in S12022(S170) and a decrease in S4185(S469). There were no significant differences

between HTN(-)HFpEF or referent control patients at any of the three sites. Insert: examples of

the phospho-blot (top) and total protein (bottom) for each of the patient groups. There are two

bands, the top band represents N2BA and the bottom band represents N2B. Data from both N2B

and N2BA bands were summed. * = p = 0.05.

Figure 6. Relationship between echocardiographically derived assessment of LV diastolic

dysfunction (estimated pulmonary capillary wedge pressure [PCWP]) and plasma biomarkers of

filling pressure (NT-proBNP, N-terminal propeptide of brain natriuretic peptide) and profibrotic

factors (soluble ST2 [sST2], tissue inhibitor of matrix metalloproteinase 1 [TIMP-1]). There was

a statistically significant direct relationship between PCWP and NT-proBNP (r2 = 0.32, p =

0.001), sST2 (r2 = 0.26, p = 0.005), and TIMP-1 (r2 = 0.36, p < 0.001).

and N2BA bands were summed. * = p = 0.05.

Fiigugugurerere 666. RRRelelelatioioonnsnshih p between echocardiograpphhihicaally derived asssssessmsmmeenent of LV diastolic

ddydysfsffunu ction (eeststimimimattedede ppp lululmomom nnanaryryy ccacappillllary wwwedggge preesesssusureree [[[PCPCCWPWWP]]))) ananand d plplplaasasmamaa bbbioioiomamamarkrkrkererrs s of

fiilllllinining gg prprpresesssususurere (((NTNTT-p-pproroBNBNP,P,P NNN-t-t-tererermmiminananalll prprpropoppepepe ttitiddee ooof f brbrbraiaain n nanan tttriuiuurerer titiic pepep ptptptididde)e)) aaanndnd ppprooofifibrbrrottticic

factors (solubublelele SST2T2T2 [[sSsSsST222],],] tttisissususuee inininhihiibibibitototorr r ofofo mamamatrtrt ixixi mmmetetetalalallololoprprprotoo eieieinananasesese 111 [[[TITIIMPMPMP-1-11])])]).. There wasss


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David Bull, Henk Granzier and Martin M. LeWinterBradshaw, Rebecca Slater, Bradley M. Palmer, Peter Van Buren, Markus Meyer, Margaret Redfield,

Michael R. Zile, Catalin F. Baicu, John Ikonomidis, Robert E. Stroud, Paul J. Nietert, Amy D.Contributions of Collagen and Titin

Myocardial Stiffness in Patients with Heart Failure and a Preserved Ejection Fraction:

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2015 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation published online January 30, 2015;Circulation.

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129129

초록

배경

본 연구의 목적은 좌심실 수축기능이 보존된 심부전(heart

failure with preserved ejection fraction, HFpEF) 환자에서 수

동 심근 경직도가 증가하는지 여부와 이러한 현상이 세포외기질

의 섬유성 콜라겐과 심근세포 내 티틴의 변화와 상관관계가 있는

지 알아보고자 하였다.

방법 및 결과

관상동맥 우회술을 시행 받는 70명의 환자에서 심초음파, 혈

중 바이오마커 분석 및 수술 중 좌심실 심외막 전벽에서 생검

을 시행하였다. 환자들을 고혈압이나 당뇨병이 없는 대조군(17

명), HFpEF가 없는 고혈압군[HTN(-)HFpEF, 31명], HFpEF

가 있는 고혈압군[HTN(+)HFpEF, 22명]의 세 개 군으로 분류하

고, 검체를 이용하여 수동 경직도 측정(differential extraction

assay), 콜라겐 분석(biochemistry or histology) 및 티틴의 아형

및 인산화 분석 등을 시행하였다. 대조군과 비교했을 때 HTN(-)

HFpEF군의 좌심실 확장기말 압력, 심근의 수동 경직도, 콜라겐

이나 티틴의 인산화 분석에서는 차이가 없었으나, 염증과 관련

된 바이오마커(C-reactive protein, soluble ST2, tissue inhibitor

of metalloproteinase 1)는 증가되어 있었다. HTN(+)HFpEF군

에서는 대조군이나 HTN(-)HFpEF군과 비교하여, 좌심실 확장

기말 압력, 좌심방 용적, NTproBNP(N-terminal propeptide of

brain natriuretic peptide), 수동 경직도, 불용성 콜라겐 등이 증

가되었으며, 티틴의 아형분석에서 PEVK S11878(S26)의 인산

화 증가와 N2B S4185(S469) 인산화 감소 및 염증과 관련된 바이

오마커의 증가가 관찰되었다.

결론

HTN(-)HFpEF는 수동 심근 경직도를 변화시키지 않았으나,

HTN(+)HFpEF 환자에서는 콜라겐과 티틴에 의존적으로 수동

심근 경직도가 유의하게 증가한다. 이는 HFpEF의 발생 과정에

서 콜라겐과 티틴의 항상성 변화가 연관이 있음을 시사한다.

좌심실 수축기능이 보존된 심부전 환자에서 보이는 심근 경직도는 콜라겐과 티틴의 항상성 변화 때문이다

박 훈 준 교수 가톨릭대학교 서울성모병원 순환기내과

Heart Failure

Documents

Myocardial Stiffness in Patients with Heart Failure and a ...circ.ahajournals.org/content/circulationaha/early/2015/01/30/... · Myocardial Stiffness in Patients with Heart Failure