Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin

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 depend on changes in extracellular matrix fibrillar collagen and cardiomyocyte titin.

Methods and results: Seventy patients undergoing coronary artery bypass grafting underwent an echocardiogram, plasma biomarker determination, and intraoperative left ventricular epicardial anterior wall biopsy. Patients were divided into 3 groups: referent control (n=17, no hypertension or diabetes mellitus), 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. In comparison with controls, patients with HTN(-)HFpEF had no change in left ventricular end-diastolic pressure, myocardial passive stiffness, collagen, or titin phosphorylation but had an increase in biomarkers of inflammation (C-reactive protein, soluble ST2, tissue inhibitor of metalloproteinase 1). In comparison with both control and HTN(-)HFpEF, patients with HTN(+)HFpEF had increased left ventricular end-diastolic pressure, left atrial volume, N-terminal propeptide of brain natriuretic peptide, 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 depends on changes in both collagen and titin homeostasis.

Keywords: collagen; diastole; heart failure; hypertension; hypertrophy.

Figure 1

Total myocardial stiffness expressed as the relationship between myocardial stress (mN/mm²) versus cardiomyocyte sarcomere length (μm) 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 μm, 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 μm 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.

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 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).