Passive Stiffness of Myocardium From Congenital Heart Disease and Implications for Diastole
Background— In ventricular dilatation or hypertrophy, an elevated end-diastolic pressure is often assumed to be secondary to increased myocardial stiffness, but stiffness is rarely measured in vivo because of difficulty. We measured in vitro passive stiffness of volume- or pressure-overloaded myocardium mainly from congenital heart disease.
Methods and Results— Endocardial ventricular biopsies were obtained at open heart surgery (n=61; pressure overload, 36; volume-overload, 19; dilated cardiomyopathy, 4; normal donors, 2). In vitro passive force-extension curves and the stiffness modulus were measured in skinned tissue: muscle strips, strips with myofilaments extracted (mainly extracellular matrix), and myocytes. Collagen content (n=38) and titin isoforms (n=16) were determined. End-diastolic pressure was measured at cardiac catheterization (n=14). Pressure-overloaded tissue (strips, extracellular matrix, myocytes) had a 2.6- to 7.0-fold greater force and stiffness modulus than volume-overloaded tissue. Myocyte force and stiffness modulus at short stretches (0.05 resting length, L0) was pressure-overloaded >normal≈volume-overloaded>dilated cardiomyopathy. Titin N2B:N2BA isoform ratio varied little between conditions. The extracellular matrix contributed more to force at 0.05 L0 in pressure-overloaded (35.1%) and volume-overloaded (17.4%) strips than normal myocardium. Stiffness modulus increased with collagen content in pressure-overloaded but not volume-overloaded strips. In vitro stiffness modulus at 0.05 L0 was a good predictor of in vivo end-diastolic pressure for pressure-overloaded but not volume-overloaded ventricles and estimated normal end-diastolic pressure as 5 to 7 mm Hg.
Conclusions— An elevated end-diastolic pressure in pressure-overloaded, but not volume-overloaded, ventricles was related to increased myocardial stiffness. The greater stiffness of pressure-overloaded compared with volume-overloaded myocardium was due to the higher stiffness of both the extracellular matrix and myocytes. The transition from normal to very-low stiffness myocytes may mark irreversible dilatation.
Received May 30, 2009; accepted November 13, 2009.
Abnormal cardiac filling caused by altered diastolic chamber stiffness is often proposed as a contributor to the pathophysiology of a range of cardiac diseases, especially ventricular hypertrophy and dilatation.1 However, quantifying diastolic chamber stiffness in vivo is extremely difficult and requires invasive measurement of the end-diastolic pressure-volume relationship (EDPVR).2 This is particularly problematic in ventricles with complex geometry (eg, the right ventricle, congenital heart disease).
Clinical Perspective on p 988
Myocardial stiffness is usually a major determinant of ventricular chamber stiffness, although forces exerted by tissues and cavities surrounding the heart (eg, pericardium, pleura, intrathoracic pressure) can affect and even, in some clinical syndromes, dominate chamber stiffness.3 An alternative to in vivo quantification of myocardial stiffness is direct in vitro measurement by stretching myocardial samples to different lengths and recording the force response.
This technique has established that, in normal rat cardiac muscle, titin is responsible for ≈90% of passive force at physiological sarcomere lengths.4,5 Titin is a huge elastic protein that extends across each half-sarcomere and is stretched in diastole when the sarcomere relaxes. In the heart, 2 isoforms of titin, N2B and N2BA, may be expressed. They differ in length in the I-band portion and influence tissue stiffness; myocardium expressing mainly the stiffer N2B isoform has a higher stiffness than myocardium in which N2BA is coexpressed.6 At long sarcomere lengths, additional elastic components increasingly contribute to stiffness (eg, collagen in the extracellular matrix [ECM]). However, extrapolation from the behavior of myocardium from normal rodents to that of human diseases is problematic. Although muscles with less stiff titin isoforms are in general less stiff,7–9 a survey of 37 different rabbit striated muscles found that the contribution of titin to total passive force and stiffness varied between muscles.9 Thus, the relative contribution of titin and the ECM to myocardial stiffness requires assessment for each family of diseases. Heart muscle from only a few conditions has been examined: ischemic and dilated cardiomyopathy (DCM),10–13 diastolic heart failure,14,15 and diabetes mellitus.16
Congenital heart disease offers many examples of ventricular hypertrophy or dilatation. We used the right ventricle of tetralogy of Fallot (TOF), double-chambered right ventricle (DCRV), stenotic right-ventricle-to-pulmonary-artery conduits, and the left ventricle of aortic stenosis (AS) as our models of hypertrophy caused by pressure overload. Our models of ventricular dilatation were the right ventricle of atrial septal defects (ASD), pulmonary regurgitation (PR), and the left ventricle of aortic regurgitation. Included for comparison was left ventricular tissue from normal donor hearts and severely dilated ventricles with DCM and end-stage heart failure that have been reported previously.10–13 This range of tissue enabled the characterization of the effect of pressure or volume overload on myocardial passive force and stiffness.
Detailed methods and patient information are provided in the online-only Data Supplement. In brief, endocardial ventricular biopsies were obtained with full consent and ethics approval (Royal Brompton, Harefield, and NHLI ethics protocols 01-006 and 01-194) and without any complications. Consent for donor hearts was obtained from the recipient (protocol 01-194). Biopsies were obtained within 10 minutes of cardioplegic arrest of the heart, except for donor hearts in which the ischemic time under cardioplegic arrest was 0.7 to 4 hours. Tissue was rapidly dissected into thin strips and either snap-frozen in liquid nitrogen or placed in glycerol/relaxing solution at 0°C. All of the solutions contained protease inhibitors. Collagen was quantified by hydroxyproline content. Titin isoforms were separated in a vertical 1% agarose gel electrophoresis system. Titin bands were confirmed in some samples by in-gel tryptic digestion and liquid chromatography–tandem mass spectrometry.
The force response to passive stretch was evaluated only in skinned (permeabilized) preparations to determine the contribution of different structural components: skinned muscle strips, the most physiological preparation with both ECM and myocytes; skinned muscle strips with thick and thin filaments and titin extracted, a predominantly ECM preparation; and skinned myocytes. The force response to passive stretch of striated muscle has a viscous component (increases with stretch speed) and an elastic component (increases with the size of the stretch). Although stress relaxation is due to the viscous component, the peak force was predominantly an elastic response.17,18
Collagenous regions were removed from muscle strips; they were skinned in relaxing solution containing 1% Triton X-100 and dissected to 0.1 to 0.25 mm wide and 1 to 2 mm long. Each muscle strip was mounted to a force transducer and servomotor with aluminum T clips. The strip was immersed in relaxing solution,19 and a low resting force of 5 μN was applied. A rapid ramp stretch (10 μm/ms) was followed by a length clamp of 1 second and ramp down (10 μm/ms) to baseline. Stretches were separated by 10 minutes to allow muscle recovery. Each strip underwent only 1 stretch of >0.05 L0 at the end of the experiment.
A subset of muscle strips were immersed in 0.6 mol/L KCl and then 1.0 mol/L KI while mounted between the force transducer and servomotor. This immersion removed virtually all of the myofilaments but left collagen intact and allowed evaluation of the ECM.5 Some extracted and nonextracted muscles were fixed in 2% glutaraldehyde while still attached to the transducers and processed for electron microscopy.
Myocytes were prepared by homogenizing the frozen tissue in relaxing solution and skinning in 1% Triton X-100.19 Myocyte fragments 120 μm in length and 20 to 30 μm in diameter were used. Skinned myocytes were attached to a sensitive force transducer and a servomotor.19 The mechanical protocol was similar to that for muscle strips except that mean sarcomere length was recorded with a 240-Hz charge-coupled device camera and commercial software. Cardiac catheterization was performed at the discretion of the responsible cardiologist.
A nonlinear stiffness can be estimated by the slope of the force-extension curve (Δ force/Δ extension in N/m) of the material. In this study, force was normalized to cross-sectional area (force/area=stress in N/m2), and stretch was expressed as a dimensionless fractional extension: (L−L0)/L0=Lagrangian strain, where L0 is the resting length and L is the stretched length.20 The slope of our force-extension (stress-strain) curves has units of Newtons per square meter and is the elastic modulus, elasticity or Young’s modulus of elasticity (in N/m2) of the material.20 To maintain consistency with the use of stiffness, this slope is called the stiffness modulus.
Hence, passive stiffness, in the sense of the resistance of a material to stretch, is evaluated here by the peak passive force in response to a stretch and the slope (stiffness modulus in N/m2) of the force-extension curves. Curves were fitted to force-extension data using splines,21 and the slope was calculated from the fitted curves using a finite-difference method. Note that compliance (used in the text) is the reciprocal of stiffness.
The mechanical data for strips in the Table were obtained from the median, minimum, and maximum of all of the subjects with a particular type of load (pressure overload, volume overload). Mechanical data for the strips for each subject were calculated from the force-extension curve that was fitted to the pooled data points from all of the strips from that subject.
Yield force of muscle strips was measured only from force-extension curves that had a clear inflection point and a plateau in the force-extension curve with the stiffness modulus dropping to ≈0 (Figure IA in the online-only Data Supplement). The force at this inflection point/plateau was taken as the yield force. In the filament-extraction experiments, the postextraction force-extension data reflect mainly the ECM mechanics and are labeled force and elastic modulus at extension 0.05 (ECM) and extension 0.2 (ECM) in the Table and VOecm and POecm in Figure 5.
Paired comparisons (before versus after extraction) were made by the Wilcoxon signed-rank test. The Mann-Whitney test was used for nonpaired comparisons of 2 groups. To compare the 4 cell types, a Kruskal-Wallis test was performed. R and the mgcv package21 were used for analysis.
Ventricular endocardial biopsies were obtained from 61 patients (35 male, 26 female patients), and the pressure-overloaded tissue (age range, 0.4 to 72.6 years) came from younger patients than volume-overloaded tissue (age range, 1.4 to 65.6 years) in all experiments The summary data from all of the experiments are given in the Table, and subject details are provided in the online-only Data Supplement.
Ninety-one strips were studied from 10 pressure-overloaded (9 right ventricles, 1 left ventricle) and 13 volume-overloaded (9 right ventricles, 4 left ventricles) hearts. There was insufficient tissue from DCM or donor hearts for muscle strip experiments. For most patients, several strips were studied, and the pooled force-extension data for that patient were fitted with a single curve that covered the full extension range.
Small rapid stretch of a strip produced a force response with a largely elastic upstroke followed by stress relaxation (Figure 1A and 1B). The force-extension relationship, derived from a series of stretches, showed a steep nonlinear rise in force (≤0.05 L0) and after a maximum or an inflection transitioned (0.05 to 0.1 L0) to a shallower curve, which rose more steeply at >0.2 L0. The data were more sparse for large than for short stretches. A striking feature is the clustering by load (Figure 1) rather than clinical diagnosis (Figure II in the online-only Data Supplement) or ventricle of origin (ie, the pressure-overloaded curves [AS, DCRV, TOF] lie to the left of and above the volume-overloaded curves [ASD, PR, aortic regurgitation]). The pressure-overloaded strips were stiffer than volume-overloaded strips (Table and Figure 1) at both 0.05 L0 (force, P<0.001; stiffness modulus, P<0.001) and 0.2 L0 (force, P=0.003; stiffness modulus P=0.03) extension. The inflection and plateau in the force-extension relationship, if present, were taken as the yield force. This yield force was lower in volume-overloaded (n=7) than pressure-overloaded (n=3) strips (Table), and the estimated force on titin molecules was 3.1 to 4.1 pN and 9.3 to 16.2 pN per titin, respectively (see the online-only Data Supplement).
Sarcomere length in the strips was not measureable by laser diffraction or confocal fluorescence microscopy. Thus, the relative disposition of the curves with respect to sarcomere length was unknown. To remove any sarcomere length–dependent lateral displacement of force-extension plots, the data were compared in stiffness modulus–force plots (Figure 1D, and in the online-only Data Supplement). Pressure-overloaded and volume-overloaded curve slopes were similar at low forces, although the pressure-overloaded curves reached higher stiffness moduli, confirming the differences between the passive mechanics of pressure-overloaded and volume-overloaded strips.
Contribution of the ECM
The contribution of collagen to passive mechanics of muscle strips was evaluated by measuring collagen content and assessing the effect of myofilament and titin extraction on passive mechanics. Stiffness modulus at 0.05 L0 increased with collagen content in pressure-overloaded strips but not in volume-overloaded strips (Figure 2). However, there was no difference in overall collagen content between the pressure-overloaded and volume-overloaded groups (Table).
Immersion in KCl and KI resulted in massive depletion of thick and thin filaments and titin5 with failure of activation with calcium (pCa2+, 4.5; not shown). Compared with preextraction values, resting force fell (Figure 3b), force (P=0.03) and stiffness modulus (P=0.05) decreased at short stretches (0.05 L0; Table and Figure 3), but larger stretches of 0.1 to 0.2 L0 were little affected (P=0.16; Figure 3C). Similarly, the stiffness modulus-force relationship was affected only at low forces (Table I in the online-only Data Supplement). After filament extraction, the stiffness modulus of the pressure-overloaded strips was still 2 to 4 times that of the volume-overloaded strips over the range of stretches used, demonstrating that the ECM was also stiffer in pressure-overloaded than volume-overloaded tissue.
The contribution of the ECM to the passive force of muscle strips was calculated as the ratio of the median of all of the postextraction strips/median of all of the nonextracted force at 0.05 L0 stretch. The ECM contribution to force at 0.05 L0 stretch was 35.1% for pressure-overloaded and 17.4% for volume-overloaded strips.
The myocyte force-extension curves were similar to those for muscle strips (compare Figure 4C and 4D with 1⇑C); curves for pressure-overloaded myocytes lay at shorter sarcomere lengths than those for the donor, volume-overloaded, and DCM myocytes (Figure 4C and 4D), and the maximum force reached was at least 2 times higher. The volume-overloaded and donor myocyte curves overlapped (Figure 4D). In contrast, DCM myocytes were shifted to the right relative to the donor and volume-overloaded cells. There were important differences between the force (P=0.006) and stiffness modulus (P=0.004) of the groups at 0.05 L0, and the pressure-overloaded cells had a higher force and stiffness modulus than the donor (P=0.016), volume-overloaded (P=0.008), and DCM (P=0.036) cells. A comparison of the resting sarcomere length of the myocytes was made by calculating for each cell the sarcomere length required to reach a given low force (0.75 kN/m2; Table): pressure overloaded< donor≈volume overloaded<DCM.
Myocytes were largely responsible for the passive force and stiffness modulus of muscle strips at short stretches (<0.05 L0). Strips and myocytes had similar forces and stiffness moduli at 0.05 L0 stretch (Table and Figure 5), and filament extraction led to a fall in resting force, and a loss of the early component of the force-extension curve resulting in a monophasic rather than biphasic curve (Figure 3).
To examine whether titin isoform variation accounted for differences in myocyte stiffness modulus, the titin N2B:N2BA ratio was determined for 16 patients (online-only Data Supplement). The relative amounts of the N2B and N2BA isoforms did not vary consistently between the pressure-overloaded, volume-overloaded, donor, and DCM tissue and are unlikely to account for differences in myocyte passive mechanics (Table).
Comparison With In Vivo Hemodynamics
In vivo EDP, measured by cardiac catheterization, was available in 14 patients. In these pressure-overloaded ventricles, in vivo EDP increased with myocardial stiffness modulus at 0.05 L0 stretch (spline fit to the pressure-overloaded points; Figure 6). In contrast, in the volume-overloaded ventricles, myocardial stiffness modulus was within the normal range and did not explain an elevated EDP. Note that our fit gives an excellent estimate for normal in vivo EDP of 5 to 7 mm Hg for ventricles with normal myocardial stiffness modulus.
Myocardium is a composite material, simplified in this study to myocytes and the ECM. The dominant stiffness of myocardium shifts from myocytes to the ECM as stretch size increases,5,22 and hemodynamic load affects both components. Within the range of tissue examined, the load on the ventricle (pressure-overload, volume-overload) was a dominant factor in the passive mechanical behavior, beyond that exerted by ventricle of origin and anatomic lesion.
Pressure-overloaded tissue (muscle strips, ECM, myocytes) had a 2.6- to 7.0-fold higher force and stiffness modulus than the comparable volume-overloaded tissue (Table). The ECM made a larger contribution to the force response at short stretches (pressure overload, 35.1%; volume overload, 17.4% at 0.05 L0 stretch) of our muscle strips than in normal rat myocardium (≈10%5). Our DCM cells had low stiffness, similar to DCM myofibrils10,11,13 and strips.12 In contrast, myocytes from moderately dilated ventricles (ASDs) had forces and stiffness moduli comparable to those of normal donors. This finding of normal passive mechanics with moderate ventricular dilatation in contrast to the extremely low stiffness tissue of severely dilated cardiomyopathic hearts is striking.
Mechanisms of Variation of Stiffness
The similar slopes of the stiffness modulus-force plots of volume-overloaded and pressure-overloaded strips at low forces suggest that similar elastic units are responsible in both tissues at short stretches/low forces (see the online-only Data Supplement). An increased number of elastic units in parallel may underlie the higher stiffness modulus of pressure-overloaded strips.23 We found that an increase in at least 1 component, collagen, was associated with an increase in stiffness modulus of pressure-overloaded muscle strips at short stretches (Figure 2). Although collagen levels were similar in the pressure-overloaded and volume-overloaded muscle strip groups as a whole, comparable to other diseases,24 the higher stiffness modulus of the ECM in pressure-overloaded muscle strips (Figure 3 and Table) suggests that changes in the collagen microarchitecture within strips (eg, increased cross-linking),25 may have occurred. Collagen cross-linking by advanced glycation end products can occur independently of hyperglycemia and may contribute to the increased stiffness modulus of the ECM.16,25
The yielding of our myocardial strips occurred at forces similar to rabbit skeletal split fibers (0.9 to 25 pN per titin7). This transition may occur in vivo because the estimated end-diastolic wall stress in dilated ventricles is 5 to 30 kN/m223,26 or 4 to 25 pN per titin molecule. Two potential mechanisms for muscle strip yielding are titin immunoglobulin unfolding (150 to 250 pN27,28) and bond rupture between myocyte surface receptors and their ECM ligands (α2β1 integrin-collagen, 65 pN; α5β1 integrin-fibronectin, 35 to 60 pN)29. Hence, myocytes are likely to uncouple from the ECM first.
The large differences in myocyte stiffness, especially the high stiffness of pressure-overloaded cells, remain unexplained. There was no systematic variation in titin isoforms across disease states (Table). Further studies are required to explore posttranslational modification of titin. Phosphorylation of titin in the N2B region by protein kinase A and G or the PEVK region by protein kinase Cα can decrease or increase the stiffness of titin, respectively,30–33 Additional studies should examine the stiffness of single pressure-overloaded myofibrils to determine whether a high stiffness modulus is a property of pressure-overloaded sarcomeres and should investigate the increased stiffness resulting from nonmyofibrillar structures such as an increase in the network density of the extrasarcomeric cytoskeleton,34 changes in microtubules,35 or an increase in desmin.
Several mechanisms are pertinent to the low stiffness of DCM tissue. First, because compliances in series are additive, longer myocytes (more sarcomeres in series36) or longer protein isoforms (more amino acids in series [eg, titin N2BA11]) will increase compliance (and decrease stiffness). Second, force-induced bond breakage can disrupt networks of structural proteins or uncouple myocytes from the ECM. Third, the force required to break molecular bonds increases with the speed of stretch.28 The slow stretch over years in chronic volume-overload or DCM may drop bond rupture forces to that attainable in vivo. Fourth, unfolding of a titin immunoglobulin domain increases its contour length, introducing an additional compliance in series, and refolding occurs only if the applied force drops to nearly 0.28 Because end-diastolic wall stress is never 0, unfolded domains will be trapped in the unfolded state and progressively accumulate. Unfolded immunoglobulin/fibronectin domains may also accumulate in other mechanically important proteins (eg, fibronectin, tenascin).
Implications for In Vivo Cardiac Function
These direct measurements of in vitro passive mechanics of myocardial strips and myocytes may extrapolate to in vivo ventricular performance. Figure 6 suggests that ventricular EDP increases with myocardial stiffness modulus in pressure-overloaded ventricles (ie, they have a steeper EDPVR). However, an elevated EDP in moderately dilated volume-overloaded ventricles can occur independently of an increased myocardial stiffness modulus by a raised end-diastolic volume shifting the ventricle to a steeper part of a normal EDPVR.37,38 In contrast, the myocytes of end-stage heart failure have an extremely low stiffness modulus, resulting in a shallow EDPVR.37,38 These severely dilated ventricles operate at the extreme right of their EDPVR at high end–diastolic volumes and, we hypothesize, at long sarcomere lengths (see the online-only Data Supplement). The transition from moderate volume-overload with normal stiffness myocytes to the very-low stiffness myocytes of end-stage heart failure may be a marker of irreversible dilatation.
Volume-overloaded hearts have elevated end-diastolic wall stress uncompensated for by increased wall thickness.23 We hypothesize that a persistently elevated end-diastolic wall stress may result in a less stiff myocardium and drive cardiac dilatation. As the heart dilates and myocytes operate at longer sarcomere lengths, a higher end-systolic wall stress will be required to generate the same systolic ventricular pressure (law of Laplace). If the required end-systolic wall stress is not achieved, then end-diastolic volume and wall stress may increase further and progressively drive dilatation.
This was an observational study from the perspective of clinical diagnosis, ventricular load, sex, and age at surgery. Stiffness of myocytes39 or ventricles40 is usually found to increase with age if all else is kept constant. However, our pressure-overload group had stiffer tissue and was younger than the volume-overload group. Although age is likely to have an impact within each clinical diagnostic category,41 across the categories in this study, load appeared to have the dominant effect.
A consequence of the large number of conditions evaluated is that the sample sizes are small, resulting in low statistical power. The Table shows how this applies to the myocytes (n=17 from 11 subjects), titin isoforms (16 subjects), and filament extraction experiments (10 subjects). However, although our data are more sparse in the myocyte and titin isoform studies, they are supported by previous reports.10–15,31,32
Sarcomere length was not measureable in muscle strips, and this was tackled with 3 methods. First, we set each strip to the same low resting force rather than the same resting sarcomere length. When muscles with different passive properties are compared, only one of these can be set constant at a time. Higher force in the pressure-overloaded than the volume-overloaded strips did not arise from a longer initial sarcomere length because the stiffer pressure-overloaded strips reached the same force at shorter sarcomere lengths than volume-overloaded strips, as found in the myocyte experiments. Second, there were large differences between the volume-overloaded and pressure-overloaded strips throughout the extension range, which should overcome any small variation in initial sarcomere length. Third, the stiffness modulus-force plots (Figure 1D, and in the online-only Data Supplement), which exclude sarcomere length dependence, also demonstrated systematic differences between the pressure-overloaded and volume-overloaded strips.
Human myocytes or myofibrils are usually prepared by homogenization of biopsies. Hence, it is difficult to exclude a small amount of ECM on the cells or between the bundles of 2 to 3 myofibrils that are typically used. However, any ECM component in our myocytes was small. The numeric values of force-extension curves of the donor and the DCM myocytes in this study are similar to previously published values for myofibrils10,11,13 and myocytes.12 In addition, the mechanical behavior of the myocytes was extremely different from that of the strips. Stretching the strips by 0.06 to 0.1 L0 led to irreversible changes, whereas the myocytes showed complete recovery.
Ideally, the relationship between wall stress and stiffness modulus would have been explored. However, the majority of our catheterization data are from right ventricles with congenital heart disease for which quantitative models of fiber architecture do not exist.
Either an increase (pressure-overload) or a decrease (severe- volume overload/DCM) in passive myocardial stiffness can be detrimental. Systolic pressure-overload results in increased stiffness of myocytes and ECM, with the diastolic consequence a stiffer ventricle and potential for impaired filling. The elevated end-diastolic wall stress of severe volume- overload results in a less stiff and more easily stretched myocardium and ultimately ventricular dilatation with systolic consequences. In the most common form of heart failure with a dilated ventricle, rather than the heart dilating as it fails, it may be more precise to say that it fails as it dilates.
Source of Funding
This study was funded by the British Heart Foundation.
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Changes in myocardial stiffness may contribute to the pathophysiology of cardiac disease. However, myocardial stiffness is difficult to measure in vivo, especially in the right ventricle or in congenital heart disease. Myocardial stiffness was measured in vitro from biopsies obtained from pressure-overloaded, volume-overloaded, donor, and dilated cardiomyopathy hearts. Pressure-overloaded tissue was stiff, suggesting that a raised end-diastolic pressure in these ventricles is due to a steeper-than-normal end-diastolic pressure–volume relationship. In contrast, the tissue from moderately dilated ventricles had relatively normal stiffness, implying that a raised end-diastolic pressure would be secondary to the ventricle operating at higher end-diastolic volumes in the steeper part of a normal end-diastolic pressure-volume curve. The dilated cardiomyopathy tissue had low stiffness, suggesting that the transition from normal to low stiffness may be a marker of irreversible dilatation.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.850677/DC1.