Cardiomyocyte Stiffness in Diastolic Heart Failure
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循环学杂志 2005年第2期
the Laboratory for Physiology (A.B., J.v.d.V., J.G.F.B., G.J.M.S., W.J.P.), Institute for Cardiovascular Research, VUMC, Amsterdam, the Netherlands
the Division of Clinical Physiology (A.B., Z.P., I.E.), Institute of Cardiology, UDMHSC, Debrecen, Hungary.
Abstract
Background— Heart failure with preserved left ventricular (LV) ejection fraction (EF) is increasingly recognized and usually referred to as diastolic heart failure (DHF). Its pathogenetic mechanism remains unclear, partly because of a lack of myocardial biopsy material. Endomyocardial biopsy samples obtained from DHF patients were therefore analyzed for collagen volume fraction (CVF) and sarcomeric protein composition and compared with control samples. Single cardiomyocytes were isolated from these biopsy samples to assess cellular contractile performance.
Methods and Results— DHF patients (n=12) had an LVEF of 71±11%, an LV end-diastolic pressure (LVEDP) of 28±4 mm Hg, and no significant coronary artery stenoses. DHF patients had higher CVFs (7.5±4.0%, P<0.05) than did controls (n=8, 3.8±2.0%), and no conspicuous changes in sarcomeric protein composition were detected. Cardiomyocytes, mechanically isolated and treated with Triton X-100 to remove all membranes, were stretched to a sarcomere length of 2.2 μm and activated with solutions containing varying [Ca2+]. Compared with cardiomyocytes of controls, cardiomyocytes of DHF patients developed a similar total isometric force at maximal [Ca2+], but their resting tension (Fpassive) in the absence of Ca2+ was almost twice as high (6.6±3.0 versus 3.5±1.7 kN/m2, P<0.001). Fpassive and CVF combined yielded stronger correlations with LVEDP than did either alone. Administration of protein kinase A (PKA) to DHF cardiomyocytes lowered Fpassive to control values.
Conclusions— DHF patients had stiffer cardiomyocytes, as evident from a higher Fpassive at the same sarcomere length. Together with CVF, Fpassive determined in vivo diastolic LV dysfunction. Correction of this high Fpassive by PKA suggests that reduced phosphorylation of sarcomeric proteins is involved in DHF.
Key Words: diastole ; heart failure ; myocytes ; contractility ; collagen
Introduction
Heart failure with preserved left ventricular (LV) ejection fraction (EF) is frequently referred to as diastolic heart failure (DHF) in contrast to systolic heart failure, which is characterized by HF with reduced LVEF.1–4 DHF is currently diagnosed in as much as 49% of HF patients.5 Diastolic LV dysfunction is also increasingly recognized,6 as evident from a recent population-based survey in which diastolic LV dysfunction was observed 5 times more often than systolic LV dysfunction.7
Despite the increased recognition of both DHF and diastolic LV dysfunction, their pathophysiology remains incompletely understood. Whether HF with preserved LVEF results from diastolic LV dysfunction8 or from subtle systolic LV dysfunction, unappreciated by a routine LVEF measurement9 and possibly exacerbated by high arterial impedance,10 is still a matter of debate. Furthermore, explanations proposed for diastolic LV dysfunction are divergent, ranging from high LV myocardial stiffness1,8 to pericardial or right ventricular constraint.11,12 Moreover, the relative importance of myocardial fibrosis and of high cardiomyocyte resting tension for LV myocardial stiffness remains undefined.13
Failure to resolve these controversies about DHF and diastolic LV dysfunction could arise from a lack of myocardial biopsy or necropsy material,4,13 which would allow clinical and hemodynamic features to be matched with cellular and molecular myocardial properties. We therefore studied endomyocardial biopsy samples from patients with DHF in whom sample procurement was requested because of clinical suspicion of restrictive cardiomyopathy or, in the case of transplant recipients, because of clinical suspicion of rejection and in whom subsequent histological examination revealed no signs of infiltrative myocardial disease or rejection. Apart from routine histological examination, biopsies were used for determination of collagen volume fraction (CVF), sarcomeric protein expression, and isolation of single cardiomyocytes, whose developed and resting tensions were measured to detect systolic and/or diastolic dysfunction at the cellular level.14,15 To evaluate the relative contributions to diastolic LV dysfunction of myocardial fibrosis and of cardiomyocyte resting tension, in vitro data were subsequently correlated with in vivo hemodynamic indices of diastolic LV function.
Methods
Patients
DHF patients were referred for cardiac catheterization and endomyocardial biopsy procurement because of clinical suspicion of restrictive cardiomyopathy (n=7) or cardiac allograft rejection (n=5). They had all been admitted to hospital because of worsening HF. LV endomyocardial biopsy samples were obtained from patients suspected of restrictive cardiomyopathy, and RV endomyocardial samples were obtained from the transplant recipients. Subsequent histological examination ruled out infiltrative myocardial disease or rejection in all patients. Coronary angiography showed the absence of significant coronary artery stenoses or graft vasculopathy. All patients satisfied the criteria as proposed by the European study group on DHF,16 ie, signs and symptoms of congestive HF, LVEF >45%, and LVEDP >16 mm Hg. They all had 1 or more predisposing risk factors for diastolic LV dysfunction (Table 1). 7 LV muscle mass (119±16 g/m2) was larger than normal (92±10 g/m2, P<0.05), and 5 patients had significant LV hypertrophy (>125 g/m2).
The control group consisted of 6 transplant recipients undergoing routine annual coronary angiography and biopsy procurement and of 2 patients referred for cardiac catheterization and endomyocardial biopsy procurement because of clinical suspicion of myocarditis. LV endomyocardial biopsy samples were obtained from the patients suspected of myocarditis, and RV endomyocardial samples were obtained from the transplant recipients. Histological examination ruled out the presence of myocarditis or rejection in all patients. They had no signs or symptoms of HF, an LVEF 50%, and an LVEDP 16 mm Hg.
Mean hemodynamic data of DHF patients and controls are listed in Table 2; individual hemodynamic data can be found in the Data Supplement Table. Hemodynamic data were derived from LV angiograms, 2D echocardiograms, and high-fidelity LV catheter pressure measurements. The local ethics committee approved the study protocol. Written, informed consent was obtained from all patients, and there were no complications related to catheterization or biopsy sample procurement.
Force Measurements in Isolated Cardiomyocytes
Force measurements were performed in mechanically isolated, single cardiomyocytes at 15°C as described previously.14 In brief, frozen biopsy samples (5 mg wet weight) were defrosted in cold relaxing solution (in mmol/L: free Mg 1, KCl 100, EGTA 2, Mg-ATP 4, and imidazole 10; pH 7.0), mechanically disrupted, and incubated for 5 minutes in relaxing solution supplemented with 0.2% Triton X-100 to remove all membranes. Thereafter, cells were washed twice in relaxing solution, and a single cardiomyocyte was attached with silicone adhesive between a force transducer and a piezoelectric motor (Figure 1A).
Resting sarcomere length in isolated cardiomyocytes was adjusted to 2.2 μm. Mean length between the attachments, width, and depth of control cardiomyocytes were 51±19, 24±8, and 18±5 μm, respectively. Corresponding values of DHF cardiomyocytes were 69±25, 28±10, and 21±6 μm. The composition of the relaxing and activating solutions was described previously.17 Their pCa (–10log[Ca2+]) ranged from 9.0 (relaxing) to 4.5 (maximal activation). Maximal activation at pCa 4.5 was used to calculate maximal calcium-activated isometric force (Fmax). Subsequently, exposure to a series of solutions with intermediate pCa yielded the force-pCa relation. All force values were normalized for myocyte cross-sectional area. A typical contraction-relaxation sequence is shown in Figure 1B. On transfer of the myocyte from relaxing to activating solution, isometric force started to develop. Once a steady-state force level was reached, the cell was shortened within 1 ms to 80% of its original length (slack test) to determine the baseline of the force transducer. The distance between the baseline and the steady force level is the total force (Ftotal). After 20 ms, the cell was restretched and returned to the relaxing solution, in which a second slack test of 10-second duration was performed to determine resting or passive force (Fpassive).
After establishing the baseline force-pCa relation, some of the myocytes were incubated for 40 minutes at 22°C in relaxing solution containing the catalytic subunit of protein kinase A (PKA, 100 U/mL; Sigma, batch 12K7495) and 6 mmol/L dithiothreitol (MP Biochemicals). Subsequently, a second force-pCa relation was determined.
Myocardial Tissue Properties
The extent of interstitial fibrosis was determined on elastica–von Gieson’s–stained sections of tissue placed in 5% formalin with an automated image analyzer (Prodit) and was expressed as low (0% to 5%, class I), intermediate (5% to 10%, class II), or high (10% to 15%, class III) CVF.18
Expression of myosin heavy chain, desmin, actin, troponin T (TnT), tropomyosin, troponin I (TnI), and myosin light-chain 1 and -2 (MLC-1 and MLC-2) was analyzed by 1D sodium dodecyl sulfate–polyacrylamide gel electrophoresis as described previously.15 To detect degradation products of myofilament proteins, Western immunoblot analysis was performed with specific monoclonal antibodies against desmin (clone DE-U-10, Sigma), TnT (clone 2G3, Spectral Diagnostics), TnI (clone 8I-7, Spectral Diagnostics), and MLC-1 and MLC-2 (clones F109.16A12 and F109.3E1, Alexis). Signals were visualized with a secondary horseradish peroxidase–labeled goat-anti-mouse antibody and enhanced chemiluminescence (Amersham Biosciences).
An ELISA was used to determine the phosphorylation status of TnI with specific monoclonal antibodies against whole TnI (clone 16A11) and dephosphorylated TnI (clone 22B11).19 Biopsy samples were treated with 10% trichloroacetic acid to preserve the phosphorylation status of proteins.
Data Analysis
Circumferential LV end-diastolic wall stress () was computed by using a thick-wall ellipsoid model of the LV: =PD/2hx[1– (h/D)–(D2/2L2)], where P is LVEDP, h is echocardiographically determined LV wall thickness, and D and L are LV short-axis diameter and long-axis length at the midwall, respectively.20
The radial myocardial stiffness modulus (E) was calculated to assess myocardial material properties from the formula: E=R/R=P/(h/h)=–P/ln h and assuming the increment in radial stress (R) to be equal but opposite in sign to the increment in P at the endocardium and the increment in radial strain (R) to be equal to the increment in wall thickness (h) relative to the instantaneous wall thickness. Because h/h=ln h, E equals the slope of a P versus ln h plot.18,21 Agreement between E and diastolic LV stiffness indices derived from multiple beat analyses during caval occlusion has previously been reported in patients with dilated cardiomyopathy.22
The Hill constant (nHill), a measure of the steepness of the sigmoidal force-pCa relation, was derived from a modified Hill equation fitted to the individual force-pCa data points of each myocyte: Ftotal=Fmaxx[Ca2+]nHill/(Ca50nHill+[Ca2+]nHill)+Fpassive, where Ca50 (or pCa50) corresponds to the [Ca2+] at which Ftotal–Fpassive=Fmax/2. pCa50 is a measure of Ca2+ sensitivity of the contractile apparatus.
Values are given as mean±SD. Statistical significance was set at P<0.05 and was obtained for multiple comparisons between groups by ANOVA followed by a Bonferroni test and for single comparisons by an unpaired Student t test. Monovariate and bivariate linear regression analyses were performed with SPSS (version 9.0).
Results
Hemodynamic Characteristics of the DHF Patients
Force Measurements in Single Cardiomyocytes
The average force-pCa relations obtained for 6 control and 9 DHF patients are shown in Figure 2A. Ftotal at pCa 4.5 did not significantly differ between the DHF (20.3±7.5 kN/m2; number of myocytes, n=23) and the control (24.2±12.4 kN/m2, n=15) groups. However, Fpassive was significantly higher in the DHF (6.6±3.0 kN/m2) than in the control (3.5±1.7 kN/m2, P<0.001) group. A higher Fpassive than in the control group was observed in both types of DHF patients: those who were transplant recipients (5.4±1.1 kN/m2, P<0.05) and those who were not (7.2±2.9 kN/m2, P<0.01). The Fpassive of the control group was comparable to previously reported values of cardiomyocytes isolated from nonfailing donor hearts.14,15 No significant differences were found between the 2 groups in terms of Ca2+ sensitivity of the contractile apparatus (pCa50) and the steepness of the force-pCa curves (ie, nHill; Table 3).
Myocardial Tissue Properties
DHF patients had a higher CVF than did controls (7.5±4.0% versus 3.8±2.0%, P<0.05). CVF of the DHF patients who were transplant recipients (7.5±3.0%) was similar to the CVF of the other DHF patients (7.5±3.0%). DHF patients were equally distributed over the 3 classes of CVF (Figure 3), and one third of patients therefore had low interstitial fibrosis. The higher values of LVEDP, , and E in these patients compared with those of controls with low interstitial fibrosis indicates that CVF is not the sole contributor to diastolic LV dysfunction. No conspicuous differences in expression of myosin heavy chain, desmin, actin, TnT, tropomyosin, TnI, or MLC-1 and MLC-2 were found between DHF and control myocardium. Western immunoblot analysis did not reveal any degradation product for desmin, TnT, TnI, MLC-1, and MLC-2 in either group. Moreover, the MLC-1–MLC-2 ratio did not differ between DHF patients (0.39±0.15) and controls (0.44±0.11). Phosphorylation status of TnI was determined in endomyocardial biopsy samples retrieved from 7 DHF patients and 7 controls. The ratio of dephosphorylated to total TnI was comparable in both groups (0.58±0.17 versus 0.53±0.17). Furthermore, there was no correlation between this ratio and Fpassive.
Correlation Between In Vivo Hemodynamics and In Vitro Force
When the DHF and control groups were combined, a monovariate linear regression analysis revealed significant correlations between the average Fpassive of all cardiomyocytes of each individual and the LVEDP, , or E measured in the same individual at the time of cardiac catheterization and biopsy sample retrieval (Figure 4). Note the quantitative agreement between the individual values of in vivo circumferential and Fpassive obtained in the isolated cardiomyocytes. These correlations were especially evident for values of Fpassive up to 5.0 kN/m2 and seemed to level off at higher values. A monovariate linear regression analysis also revealed significant correlations between CVF and LVEDP (R=0.63, P=0.009) or (R=0.68, P=0.004). In a bivariate linear regression analysis, the combination of Fpassive and CVF yielded stronger correlations with LVEDP (R=0.80, P=0.001) or (R=0.78, P=0.002) than Fpassive and CVF alone in monovariate analysis. Fpassive and CVF were unrelated (P=0.26).
PKA and Cardiomyocyte Force Development
After PKA treatment, a second force-pCa relation could be constructed for 11 cardiomyocytes isolated from biopsy samples of 4 control patients and for 16 cardiomyocytes isolated from biopsy samples of 7 DHF patients. Figure 1 (bottom) shows representative force recordings of a single cardiomyocyte retrieved from a DHF patient before and after PKA treatment at maximal (pCa 4.5) and intermediate (pCa 5.8) activation. At pCa 4.5, Ftotal remained the same, whereas at pCa 5.8, Ftotal was reduced after PKA. In addition, Figure 1 illustrates the reduced Fpassive (pCa 9.0) after PKA. For control and DHF groups, Ftotal at pCa 4.5 was similar before and after PKA (Table 3; Figure 2B and 2C). At intermediate pCa (eg, pCa 5.8), Ftotal was reduced after PKA because of PKA-induced myofilament desensitization (Figure 2B and 2C). The latter was also evident from the reduced pCa50 value observed in both DHF and control groups (Table 3). After PKA treatment, Fpassive of cardiomyocytes from DHF patients dropped to values observed in the control group both at baseline and after PKA treatment (Figures 2B, 2C, and 5A). In addition, in DHF patients, the PKA-induced fall in Fpassive was larger when baseline Fpassive was higher (Figure 5B).
Discussion
The present study analyzed endomyocardial biopsy samples obtained from patients with DHF and yielded the following information: (1) When cardiomyocytes isolated from these samples were stretched to a sarcomere length of 2.2 μm, Ftotal at maximal [Ca2+] was comparable to that of control cardiomyocytes, but their Fpassive was twice as high. (2) The increase in Fpassive was reversible because administration of PKA lowered Fpassive to a level observed in control cardiomyocytes. (3) In vivo hemodynamic measures of diastolic LV function such as LVEDP, , and E were correlated with in vitro measurements of both Fpassive and CVF.
High Fpassive
Because the mechanical isolation procedure removed endomysial collagen structures, the high Fpassive of cardiomyocytes retrieved from DHF patients can only result from deranged diastolic stiffness of the cardiomyocytes themselves. Because cardiomyocytes were incubated in solution supplemented with 0.2% Triton X-100 before the experiments, the integrity of sarcolemmal and sarcoplasmic membranes was disrupted, and the cardiomyocytes became dependent on externally supplied calcium for active force development. Under these conditions, disturbed calcium handling because of modified expression and/or phosphorylation of sarcoplasmic reticular Ca2+-ATPase,23 phospholamban,24,25 sarcoplasmic calcium release channel,26 and sodium/calcium exchanger27 is effectively ruled out as a cause of the observed elevation of Fpassive, which therefore needs to be attributed to alterations of myofilament or cytoskeletal proteins.
The present study revealed no difference between the DHF and control groups in the expression of cardiac sarcomeric proteins such as myosin heavy chain, actin, TnT, TnI, desmin, and tropomyosin. Protein composition may also change as a result of enhanced proteolysis. This is especially evident for TnI, whose calpain-mediated breakdown is accelerated by a high LVEDP.28 Western immunoblot analysis ruled out degradation of several contractile proteins, including TnI, in both the control and DHF groups. Therefore, it is unlikely that a change in isoform composition or protein degradation accounts for the high Fpassive of cardiomyocytes observed in the DHF group.
Moreover, the correction by PKA treatment provides evidence that the high Fpassive resulted from phosphorylation of its sarcomeric target proteins: TnI, myosin binding protein-C, and/or titin. Treatment with PKA induced a large drop in Fpassive in cardiomyocytes of DHF patients, whereas neither Fpassive in control cardiomyocytes nor Ftotal at pCa 4.5 in cardiomyocytes from both groups was altered (Table 3 and Figure 2). Such an isolated drop in Fpassive, unaccompanied by a fall in Ftotal, is more easily reconciled with an action of PKA on a myofilament rather than on a cytoskeletal phosphorylation site because the parallel alignment of the cytoskeleton with the myofilaments would predict a fall in Fpassive generated by the cytoskeleton to also lower Ftotal.
The present study also determined the phosphorylation status of TnI but found no difference in the ratio of dephosphorylated to total TnI between control and DHF groups. It has recently been demonstrated in animal studies that phosphorylation of myosin binding protein-C and titin modifies diastolic properties. Phosphorylation29 or expression of a mutant isoform30 releases the "braking" action of myosin binding protein-C on cross-bridge cycling, thereby decreasing Fpassive in skinned mouse myocardial strips. Similarly, PKA-mediated phosphorylation of the elastic N2B spring element of titin reduces diastolic stiffness in isolated rat cardiomyocytes.31 Because of limited procurement of myocardial tissue by endomyocardial biopsy techniques, phosphorylation of both proteins could not be assessed in the present study. Future studies on myocardial tissue from DHF patients should focus on the phosphorylation level of both proteins to detect the sarcomeric protein responsible for the high Fpassive of cardiomyocytes isolated from DHF patients.
In Vitro Versus In Vivo
When Fpassive values of control and DHF cardiomyocytes were pooled, in vitro measurement of Fpassive was correlated with in vivo indices of diastolic LV dysfunction, such as LVEDP, , and E (Figure 4). The quantitative agreement between and Fpassive indicates that diastolic LV dysfunction is determined to an important extent by the rise in Fpassive of the cardiomyocytes. The relations between Fpassive and indices of diastolic LV function all leveled off at higher values of LVEDP, , and E. This could have resulted from diuretic therapy to recompensate the patients before catheterization or from more intense interstitial fibrosis at the top end of diastolic LV dysfunction. In the in vitro setting, all cardiomyocytes were stretched to the same sarcomere length of 2.2 μm. In the in vivo setting, LV preload was uncontrolled, and especially the DHF patients, who underwent more intense diuretic therapy in the interval between admission for pulmonary edema and diagnostic cardiac catheterization, could have been operating at LV filling pressures lower than needed to achieve optimal sarcomere length. More intense interstitial fibrosis also provides an explanation for the relations between Fpassive and indices of diastolic LV function to level off at higher values of LVEDP, , and E. Endomyocardial biopsy samples from patients with DHF had higher CVF than did controls, and in a bivariate linear regression analysis, both Fpassive and CVF were significantly correlated with LVEDP and . A predominant effect of interstitial fibrosis at the top end of diastolic LV dysfunction is in line with previous experimental studies, which showed diastolic muscle stiffness to originate from structures within the sarcomere for sarcomere lengths <2.2 μm32 and from perimysial fibers once filling pressures exceeded 30 mm Hg.33
Degradation of collagen in pressure-overloaded, hypertrophied papillary muscles with plasmin did not reduce muscle stiffness to levels observed in normal muscles.34 Similarly, in the present study, patients with DHF and low CVFs still had higher LVEDP, , and E than did controls (Figure 3). Therefore, our data support the concept that diastolic LV dysfunction in the presence of a low CVF is explained by the higher Fpassive of the cardiomyocytes. However, because half of the DHF patients suffered from diabetes mellitus, collagen cross-links formed by advanced glycation end products could also explain the impairment of diastolic LV function at low CVF.35
The present study observed low CVF and high Fpassive values in some patients with DHF but failed to detect DHF patients with high CVF and low Fpassive values. This suggests diastolic LV dysfunction to result from a sequence of events, which start with a rise in cardiomyocyte Fpassive followed by the development of interstitial fibrosis. A similar sequence of events has also been reported in experimental tachypacing-induced HF models. In those models, elevation of diastolic LV muscle stiffness was paralleled by expression of the shorter and stiffer N2B isoform of titin36 and not by interstitial fibrosis, which only developed when an angiotensin II infusion was superimposed on the pacing stress.37 Similar coordination between titin isoform shift and extracellular matrix deposition has also been reported in other experimental models.38 The evidence provided by the present study that DHF patients can have low CVF also explains why angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors, which reduce interstitial fibrosis,39,40 have not been uniformly successful in large clinical trials in DHF patients.41
Study Limitations
Five of the 12 DHF patients and 6 of the 8 controls had undergone cardiac transplantation. Transplant recipients were included in the study because they frequently suffer DHF16 and because their myocardial biopsy material is readily available. The pathogenetic mechanisms responsible for their DHF could differ from that of other DHF patients because of ongoing rejection and use of immunosuppressant drugs. Force recordings of isolated cardiomyocytes and the extent of interstitial fibrosis of the transplanted subgroup of DHF patients were, however, similar to the measurements obtained in the other DHF patients, and both data sets were therefore merged into a single DHF group. The same also applied to the control group.
Isolation of cardiomyocytes and assessment of myocardial tissue properties was performed on a limited number of RV or LV samples and could potentially have overlooked tissue heterogeneity. The extent of tissue heterogeneity was addressed in previous studies on explanted hearts14,15 or surgically procured biopsy samples.42 In those studies, the variability of force measurements in cardiomyocytes isolated from different portions of the heart was always <5%. To validate the use of defrosted biopsy samples, force recordings of cardiomyocytes isolated from a biopsy sample immediately after procurement were compared with force recordings of cardiomyocytes isolated from a defrosted biopsy sample from the same patient. These force recordings yielded identical results.
Conclusions
Cardiomyocytes isolated from endomyocardial biopsy samples of DHF patients had elevated Fpassive, which together with CVF, determined in vivo diastolic LV dysfunction. Administration of PKA to these cardiomyocytes normalized Fpassive. Because the integrity of sarcolemmal and sarcoplasmic membranes was disrupted by prior Triton incubation and because expression of sarcomeric proteins and the phosphorylation level of TnI were unaltered, the PKA-induced fall in Fpassive probably resulted from correction of a phosphorylation deficit of myosin binding protein-C or titin. This hypophosphorylated sarcomeric protein could, together with extracellular matrix modification, be a specific myocardial target for drug therapy of DHF.
Acknowledgments
This study was supported by a Marie Curie host fellowship (contract number HPMT-GH-00-00114-12), a European Committee Research Training Network project (contract number HPRN-CT-2000-00091), and the Netherlands Organization for Scientific Research (VENI grant, 2002).
Footnotes
The online-only Data Supplement, which contains an additional table, can be found with this article at http://www.circulationaha.org.
References
Grossman W. Diastolic dysfunction in congestive heart failure. N Engl J Med. 1991; 325: 1557–1564.
Kitzman DW, Little WC, Brubaker PH, Anderson RT, Hundley WG, Marburger CT, Brosnihan B, Morgan TM, Stewart KP. Pathophysiological characterization of isolated diastolic heart failure in comparison to systolic heart failure. JAMA. 2002; 288: 2144–2150.
Zile MR, Brutsaert DL. New concepts in diastolic dysfunction and diastolic heart failure, part I: diagnosis, prognosis, and measurements of diastolic function. Circulation. 2002; 105: 1387–1393.
Redfield MM. Understanding ‘diastolic’ heart failure. N Engl J Med. 2004; 350: 1930–1931.
Cleland JG, Cohen-Solal A, Aguilar JC, Dietz R, Eastaugh J, Follath F, Freemantle N, Gavazzi A, van Gilst WH, Hobbs FD, Korewicki J, Madeira HC, Preda I, Swedberg K, Widimsky J. Management of heart failure in primary care (the IMPROVEMENT of Heart Failure Programme): an international survey. Lancet. 2002; 360: 1631–1639.
Redfield MM, Jacobsen SJ, Burnett JC Jr, Mahoney DW, Bailey KR, Rodeheffer RJ. Burden of systolic and diastolic ventricular dysfunction in the community: appreciating the scope of the heart failure epidemic. JAMA. 2003; 289: 194–202.
Fischer M, Baessler A, Hense HW, Hengstenberg C, Muscholl M, Holmer S, Doring A, Broeckel U, Riegger G, Schunkert H. Prevalence of left ventricular diastolic dysfunction in the community: results from a Doppler echocardiographic-based survey of a population sample. Eur Heart J. 2003; 24: 320–328.
Zile MR, Baicu CF, Gaasch WH. Diastolic heart failure—abnormalities in active relaxation and passive stiffness of the left ventricle. N Engl J Med. 2004; 350: 1953–1959.
Yu CM, Lin H, Yang H, Kong SL, Zhang Q, Lee SW. Progression of systolic abnormalities in patients with ‘isolated’ diastolic heart failure and diastolic dysfunction. Circulation. 2002; 105: 1195–1201.
Kawaguchi M, Hay I, Fetics B, Kass DA. Combined ventricular systolic and arterial stiffening in patients with heart failure and preserved ejection fraction: implications for systolic and diastolic reserve limitations. Circulation. 2003; 107: 714–720.
Pak PH, Maughan L, Baughman KL, Kass DA. Marked discordance between dynamic and passive diastolic pressure-volume relations in idiopathic hypertrophic cardiomyopathy. Circulation. 1996; 94: 52–60.
Morris-Thurgood JA, Frenneaux MP. Diastolic ventricular interaction and ventricular diastolic filling. Heart Fail Rev. 2000; 5: 307–323.
Kass DA, Bronzwaer JG, Paulus WJ. What mechanisms underlie diastolic dysfunction in heart failure; Circ Res. 2004; 94: 1533–1542.
van der Velden J, Klein LJ, Zaremba R, Boontje NM, Huybregts MA, Stooker W, Eijsman L, de Jong JW, Visser CA, Visser FC, Stienen GJM. Effects of calcium, inorganic phosphate, and pH on isometric force in single skinned cardiomyocytes from donor and failing human hearts. Circulation. 2001; 104: 1140–1146.
van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, Burton PB, Goldmann P, Jaquet K, Stienen GJM. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res. 2003; 57: 37–47.
How to diagnose diastolic heart failure. European Study Group on diastolic heart failure. Eur Heart J. 1998; 19: 990–1003.
Papp Z, Szabo A, Barends JP, Stienen GJM. The mechanism of the force enhancement by MgADP under simulated ischaemic conditions in rat cardiac myocytes. J Physiol. 2002; 543: 177–189.
Bronzwaer JG, Heymes C, Visser CA, Paulus WJ. Myocardial fibrosis blunts nitric oxide synthase–related preload reserve in human dilated cardiomyopathy. Am J Physiol. 2003; 284: H10–H16.
van der Velden J, Moorman AF, Stienen GJM. Age-dependent changes in myosin composition correlate with enhanced economy of contraction in guinea-pig hearts. J Physiol. 1998; 507: 497–510.
Paulus WJ, Heyndrickx GR, Buyl P, Goethals MA, Andries E. Wide-range load shift of combined aortic valvuloplasty-arterial vasodilation slows isovolumic relaxation of the hypertrophied left ventricle. Circulation. 1990; 81: 886–898.
Bronzwaer JG, de Bruyne B, Ascoop CA, Paulus WJ. Comparative effects of pacing-induced and balloon coronary occlusion ischemia on left ventricular diastolic function in man. Circulation. 1991; 84: 211–222.
Bronzwaer JG, Zeitz C, Visser CA, Paulus WJ. Endomyocardial nitric oxide synthase and the hemodynamic phenotypes of human dilated cardiomyopathy and of athlete’s heart. Cardiovasc Res. 2002; 55: 270–278.
Frank KF, Bolck B, Brixius K, Kranias EG, Schwinger RH. Modulation of SERCA: implications for the failing human heart. Basic Res Cardiol. 2002; 97 (suppl 1): I-72–I-78.
MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol. 2003; 4: 566–577.
Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002; 34: 951–969.
Marks AR, Reiken S, Marx SO. Progression of heart failure: is protein kinase A hyperphosphorylation of the ryanodine receptor a contributing factor; Circulation. 2002; 105: 272–275.
Weber CR, Piacentino V III, Houser SR, Bers DM. Dynamic regulation of sodium/calcium exchange function in human heart failure. Circulation. 2003; 108: 2224–2229.
Feng J, Schaus BJ, Fallavollita JA, Lee TC, Canty JM Jr. Preload induces troponin I degradation independently of myocardial ischemia. Circulation. 2001; 103: 2035–2037.
Flashman E, Redwood C, Moolman-Smook J, Watkins H. Cardiac myosin binding protein C: its role in physiology and disease. Circ Res. 2004; 94: 1279–1289.
Palmer BM, Georgakopoulos D, Janssen PM, Wang Y, Alpert NR, Belardi DF, Harris SP, Moss RL, Burgon PG, Seidman CE, Seidman JG, Maughan DW, Kass DA. Role of cardiac myosin binding protein C in sustaining left ventricular systolic stiffening. Circ Res. 2004; 94: 1249–1255.
Yamasaki R, Wu Y, McNabb M, Greaser M, Labeit S, Granzier H. Protein kinase A phosphorylates titin’s cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes. Circ Res. 2002; 90: 1181–1188.
Linke WA, Popov VI, Pollack GH. Passive and active tension in single cardiac myofibrils. Biophys J. 1994; 67: 782–792.
Factor SM, Flomenbaum M, Zhao MJ, Eng C, Robinson TF. The effects of acutely increased ventricular cavity pressure on intrinsic myocardial connective tissue. J Am Coll Cardiol. 1988; 12: 1582–1589.
Stroud JD, Baicu CF, Barnes MA, Spinale FG, Zile MR. Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment. Am J Physiol. 2002; 282: H2324–H2335.
Herrmann KL, McCulloch AD, Omens JH. Glycated collagen cross-linking alters cardiac mechanics in volume-overload hypertrophy. Am J Physiol. 2003; 284: H1277–H1284.
Wu Y, Bell SP, Trombitas K, Witt CC, Labeit S, LeWinter MM, Granzier H. Changes in titin isoform expression in pacing-induced cardiac failure give rise to increased passive muscle stiffness. Circulation. 2002; 106(: 1384–1389.
Senzaki H, Paolocci N, Gluzband YA, Lindsey ML, Janicki JS, Crow MT, Kass DA. ;-Blockade prevents sustained metalloproteinase activation and diastolic stiffening induced by angiotensin II combined with evolving cardiac dysfunction. Circ Res. 2000; 86: 807–815.
Wu Y, Cazorla O, Labeit D, Labeit S, Granzier H. Changes in titin and collagen underlie diastolic stiffness diversity of cardiac muscle. J Mol Cell Cardiol. 2000; 32: 2151–2162.
Brilla CG, Funck RC, Rupp H. Lisinopril-mediated regression of myocardial fibrosis in patients with hypertensive heart disease. Circulation. 2000; 102: 1388–1393.
Yamamoto K, Masuyama T, Sakata Y, Nishikawa N, Mano T, Yoshida J, Miwa T, Sugawara M, Yamaguchi Y, Ookawara T, Suzuki K, Hori M. Myocardial stiffness is determined by ventricular fibrosis, but not by compensatory or excessive hypertrophy in hypertensive heart. Cardiovasc Res. 2002; 55: 76–82.
Yusuf S, Pfeffer MA, Swedberg K, Granger CB, Held P, McMurray JJ, Michelson EL, Olofsson B, Ostergren J. Effects of candesartan in patients with chronic heart failure and preserved left-ventricular ejection fraction: the CHARM-Preserved Trial. Lancet. 2003; 362: 777–781.
van der Velden J, Klein LJ, van der Bijl M, Huybregts MA, Stooker W, Witkop J, Eijsman L, Visser CA, Visser FC, Stienen GJ. Isometric tension development and its calcium sensitivity in skinned myocyte-sized preparations from different regions of the human heart. Cardiovasc Res. 1999; 42: 706–719.(Attila Borbely, MD; Jolan)
the Division of Clinical Physiology (A.B., Z.P., I.E.), Institute of Cardiology, UDMHSC, Debrecen, Hungary.
Abstract
Background— Heart failure with preserved left ventricular (LV) ejection fraction (EF) is increasingly recognized and usually referred to as diastolic heart failure (DHF). Its pathogenetic mechanism remains unclear, partly because of a lack of myocardial biopsy material. Endomyocardial biopsy samples obtained from DHF patients were therefore analyzed for collagen volume fraction (CVF) and sarcomeric protein composition and compared with control samples. Single cardiomyocytes were isolated from these biopsy samples to assess cellular contractile performance.
Methods and Results— DHF patients (n=12) had an LVEF of 71±11%, an LV end-diastolic pressure (LVEDP) of 28±4 mm Hg, and no significant coronary artery stenoses. DHF patients had higher CVFs (7.5±4.0%, P<0.05) than did controls (n=8, 3.8±2.0%), and no conspicuous changes in sarcomeric protein composition were detected. Cardiomyocytes, mechanically isolated and treated with Triton X-100 to remove all membranes, were stretched to a sarcomere length of 2.2 μm and activated with solutions containing varying [Ca2+]. Compared with cardiomyocytes of controls, cardiomyocytes of DHF patients developed a similar total isometric force at maximal [Ca2+], but their resting tension (Fpassive) in the absence of Ca2+ was almost twice as high (6.6±3.0 versus 3.5±1.7 kN/m2, P<0.001). Fpassive and CVF combined yielded stronger correlations with LVEDP than did either alone. Administration of protein kinase A (PKA) to DHF cardiomyocytes lowered Fpassive to control values.
Conclusions— DHF patients had stiffer cardiomyocytes, as evident from a higher Fpassive at the same sarcomere length. Together with CVF, Fpassive determined in vivo diastolic LV dysfunction. Correction of this high Fpassive by PKA suggests that reduced phosphorylation of sarcomeric proteins is involved in DHF.
Key Words: diastole ; heart failure ; myocytes ; contractility ; collagen
Introduction
Heart failure with preserved left ventricular (LV) ejection fraction (EF) is frequently referred to as diastolic heart failure (DHF) in contrast to systolic heart failure, which is characterized by HF with reduced LVEF.1–4 DHF is currently diagnosed in as much as 49% of HF patients.5 Diastolic LV dysfunction is also increasingly recognized,6 as evident from a recent population-based survey in which diastolic LV dysfunction was observed 5 times more often than systolic LV dysfunction.7
Despite the increased recognition of both DHF and diastolic LV dysfunction, their pathophysiology remains incompletely understood. Whether HF with preserved LVEF results from diastolic LV dysfunction8 or from subtle systolic LV dysfunction, unappreciated by a routine LVEF measurement9 and possibly exacerbated by high arterial impedance,10 is still a matter of debate. Furthermore, explanations proposed for diastolic LV dysfunction are divergent, ranging from high LV myocardial stiffness1,8 to pericardial or right ventricular constraint.11,12 Moreover, the relative importance of myocardial fibrosis and of high cardiomyocyte resting tension for LV myocardial stiffness remains undefined.13
Failure to resolve these controversies about DHF and diastolic LV dysfunction could arise from a lack of myocardial biopsy or necropsy material,4,13 which would allow clinical and hemodynamic features to be matched with cellular and molecular myocardial properties. We therefore studied endomyocardial biopsy samples from patients with DHF in whom sample procurement was requested because of clinical suspicion of restrictive cardiomyopathy or, in the case of transplant recipients, because of clinical suspicion of rejection and in whom subsequent histological examination revealed no signs of infiltrative myocardial disease or rejection. Apart from routine histological examination, biopsies were used for determination of collagen volume fraction (CVF), sarcomeric protein expression, and isolation of single cardiomyocytes, whose developed and resting tensions were measured to detect systolic and/or diastolic dysfunction at the cellular level.14,15 To evaluate the relative contributions to diastolic LV dysfunction of myocardial fibrosis and of cardiomyocyte resting tension, in vitro data were subsequently correlated with in vivo hemodynamic indices of diastolic LV function.
Methods
Patients
DHF patients were referred for cardiac catheterization and endomyocardial biopsy procurement because of clinical suspicion of restrictive cardiomyopathy (n=7) or cardiac allograft rejection (n=5). They had all been admitted to hospital because of worsening HF. LV endomyocardial biopsy samples were obtained from patients suspected of restrictive cardiomyopathy, and RV endomyocardial samples were obtained from the transplant recipients. Subsequent histological examination ruled out infiltrative myocardial disease or rejection in all patients. Coronary angiography showed the absence of significant coronary artery stenoses or graft vasculopathy. All patients satisfied the criteria as proposed by the European study group on DHF,16 ie, signs and symptoms of congestive HF, LVEF >45%, and LVEDP >16 mm Hg. They all had 1 or more predisposing risk factors for diastolic LV dysfunction (Table 1). 7 LV muscle mass (119±16 g/m2) was larger than normal (92±10 g/m2, P<0.05), and 5 patients had significant LV hypertrophy (>125 g/m2).
The control group consisted of 6 transplant recipients undergoing routine annual coronary angiography and biopsy procurement and of 2 patients referred for cardiac catheterization and endomyocardial biopsy procurement because of clinical suspicion of myocarditis. LV endomyocardial biopsy samples were obtained from the patients suspected of myocarditis, and RV endomyocardial samples were obtained from the transplant recipients. Histological examination ruled out the presence of myocarditis or rejection in all patients. They had no signs or symptoms of HF, an LVEF 50%, and an LVEDP 16 mm Hg.
Mean hemodynamic data of DHF patients and controls are listed in Table 2; individual hemodynamic data can be found in the Data Supplement Table. Hemodynamic data were derived from LV angiograms, 2D echocardiograms, and high-fidelity LV catheter pressure measurements. The local ethics committee approved the study protocol. Written, informed consent was obtained from all patients, and there were no complications related to catheterization or biopsy sample procurement.
Force Measurements in Isolated Cardiomyocytes
Force measurements were performed in mechanically isolated, single cardiomyocytes at 15°C as described previously.14 In brief, frozen biopsy samples (5 mg wet weight) were defrosted in cold relaxing solution (in mmol/L: free Mg 1, KCl 100, EGTA 2, Mg-ATP 4, and imidazole 10; pH 7.0), mechanically disrupted, and incubated for 5 minutes in relaxing solution supplemented with 0.2% Triton X-100 to remove all membranes. Thereafter, cells were washed twice in relaxing solution, and a single cardiomyocyte was attached with silicone adhesive between a force transducer and a piezoelectric motor (Figure 1A).
Resting sarcomere length in isolated cardiomyocytes was adjusted to 2.2 μm. Mean length between the attachments, width, and depth of control cardiomyocytes were 51±19, 24±8, and 18±5 μm, respectively. Corresponding values of DHF cardiomyocytes were 69±25, 28±10, and 21±6 μm. The composition of the relaxing and activating solutions was described previously.17 Their pCa (–10log[Ca2+]) ranged from 9.0 (relaxing) to 4.5 (maximal activation). Maximal activation at pCa 4.5 was used to calculate maximal calcium-activated isometric force (Fmax). Subsequently, exposure to a series of solutions with intermediate pCa yielded the force-pCa relation. All force values were normalized for myocyte cross-sectional area. A typical contraction-relaxation sequence is shown in Figure 1B. On transfer of the myocyte from relaxing to activating solution, isometric force started to develop. Once a steady-state force level was reached, the cell was shortened within 1 ms to 80% of its original length (slack test) to determine the baseline of the force transducer. The distance between the baseline and the steady force level is the total force (Ftotal). After 20 ms, the cell was restretched and returned to the relaxing solution, in which a second slack test of 10-second duration was performed to determine resting or passive force (Fpassive).
After establishing the baseline force-pCa relation, some of the myocytes were incubated for 40 minutes at 22°C in relaxing solution containing the catalytic subunit of protein kinase A (PKA, 100 U/mL; Sigma, batch 12K7495) and 6 mmol/L dithiothreitol (MP Biochemicals). Subsequently, a second force-pCa relation was determined.
Myocardial Tissue Properties
The extent of interstitial fibrosis was determined on elastica–von Gieson’s–stained sections of tissue placed in 5% formalin with an automated image analyzer (Prodit) and was expressed as low (0% to 5%, class I), intermediate (5% to 10%, class II), or high (10% to 15%, class III) CVF.18
Expression of myosin heavy chain, desmin, actin, troponin T (TnT), tropomyosin, troponin I (TnI), and myosin light-chain 1 and -2 (MLC-1 and MLC-2) was analyzed by 1D sodium dodecyl sulfate–polyacrylamide gel electrophoresis as described previously.15 To detect degradation products of myofilament proteins, Western immunoblot analysis was performed with specific monoclonal antibodies against desmin (clone DE-U-10, Sigma), TnT (clone 2G3, Spectral Diagnostics), TnI (clone 8I-7, Spectral Diagnostics), and MLC-1 and MLC-2 (clones F109.16A12 and F109.3E1, Alexis). Signals were visualized with a secondary horseradish peroxidase–labeled goat-anti-mouse antibody and enhanced chemiluminescence (Amersham Biosciences).
An ELISA was used to determine the phosphorylation status of TnI with specific monoclonal antibodies against whole TnI (clone 16A11) and dephosphorylated TnI (clone 22B11).19 Biopsy samples were treated with 10% trichloroacetic acid to preserve the phosphorylation status of proteins.
Data Analysis
Circumferential LV end-diastolic wall stress () was computed by using a thick-wall ellipsoid model of the LV: =PD/2hx[1– (h/D)–(D2/2L2)], where P is LVEDP, h is echocardiographically determined LV wall thickness, and D and L are LV short-axis diameter and long-axis length at the midwall, respectively.20
The radial myocardial stiffness modulus (E) was calculated to assess myocardial material properties from the formula: E=R/R=P/(h/h)=–P/ln h and assuming the increment in radial stress (R) to be equal but opposite in sign to the increment in P at the endocardium and the increment in radial strain (R) to be equal to the increment in wall thickness (h) relative to the instantaneous wall thickness. Because h/h=ln h, E equals the slope of a P versus ln h plot.18,21 Agreement between E and diastolic LV stiffness indices derived from multiple beat analyses during caval occlusion has previously been reported in patients with dilated cardiomyopathy.22
The Hill constant (nHill), a measure of the steepness of the sigmoidal force-pCa relation, was derived from a modified Hill equation fitted to the individual force-pCa data points of each myocyte: Ftotal=Fmaxx[Ca2+]nHill/(Ca50nHill+[Ca2+]nHill)+Fpassive, where Ca50 (or pCa50) corresponds to the [Ca2+] at which Ftotal–Fpassive=Fmax/2. pCa50 is a measure of Ca2+ sensitivity of the contractile apparatus.
Values are given as mean±SD. Statistical significance was set at P<0.05 and was obtained for multiple comparisons between groups by ANOVA followed by a Bonferroni test and for single comparisons by an unpaired Student t test. Monovariate and bivariate linear regression analyses were performed with SPSS (version 9.0).
Results
Hemodynamic Characteristics of the DHF Patients
Force Measurements in Single Cardiomyocytes
The average force-pCa relations obtained for 6 control and 9 DHF patients are shown in Figure 2A. Ftotal at pCa 4.5 did not significantly differ between the DHF (20.3±7.5 kN/m2; number of myocytes, n=23) and the control (24.2±12.4 kN/m2, n=15) groups. However, Fpassive was significantly higher in the DHF (6.6±3.0 kN/m2) than in the control (3.5±1.7 kN/m2, P<0.001) group. A higher Fpassive than in the control group was observed in both types of DHF patients: those who were transplant recipients (5.4±1.1 kN/m2, P<0.05) and those who were not (7.2±2.9 kN/m2, P<0.01). The Fpassive of the control group was comparable to previously reported values of cardiomyocytes isolated from nonfailing donor hearts.14,15 No significant differences were found between the 2 groups in terms of Ca2+ sensitivity of the contractile apparatus (pCa50) and the steepness of the force-pCa curves (ie, nHill; Table 3).
Myocardial Tissue Properties
DHF patients had a higher CVF than did controls (7.5±4.0% versus 3.8±2.0%, P<0.05). CVF of the DHF patients who were transplant recipients (7.5±3.0%) was similar to the CVF of the other DHF patients (7.5±3.0%). DHF patients were equally distributed over the 3 classes of CVF (Figure 3), and one third of patients therefore had low interstitial fibrosis. The higher values of LVEDP, , and E in these patients compared with those of controls with low interstitial fibrosis indicates that CVF is not the sole contributor to diastolic LV dysfunction. No conspicuous differences in expression of myosin heavy chain, desmin, actin, TnT, tropomyosin, TnI, or MLC-1 and MLC-2 were found between DHF and control myocardium. Western immunoblot analysis did not reveal any degradation product for desmin, TnT, TnI, MLC-1, and MLC-2 in either group. Moreover, the MLC-1–MLC-2 ratio did not differ between DHF patients (0.39±0.15) and controls (0.44±0.11). Phosphorylation status of TnI was determined in endomyocardial biopsy samples retrieved from 7 DHF patients and 7 controls. The ratio of dephosphorylated to total TnI was comparable in both groups (0.58±0.17 versus 0.53±0.17). Furthermore, there was no correlation between this ratio and Fpassive.
Correlation Between In Vivo Hemodynamics and In Vitro Force
When the DHF and control groups were combined, a monovariate linear regression analysis revealed significant correlations between the average Fpassive of all cardiomyocytes of each individual and the LVEDP, , or E measured in the same individual at the time of cardiac catheterization and biopsy sample retrieval (Figure 4). Note the quantitative agreement between the individual values of in vivo circumferential and Fpassive obtained in the isolated cardiomyocytes. These correlations were especially evident for values of Fpassive up to 5.0 kN/m2 and seemed to level off at higher values. A monovariate linear regression analysis also revealed significant correlations between CVF and LVEDP (R=0.63, P=0.009) or (R=0.68, P=0.004). In a bivariate linear regression analysis, the combination of Fpassive and CVF yielded stronger correlations with LVEDP (R=0.80, P=0.001) or (R=0.78, P=0.002) than Fpassive and CVF alone in monovariate analysis. Fpassive and CVF were unrelated (P=0.26).
PKA and Cardiomyocyte Force Development
After PKA treatment, a second force-pCa relation could be constructed for 11 cardiomyocytes isolated from biopsy samples of 4 control patients and for 16 cardiomyocytes isolated from biopsy samples of 7 DHF patients. Figure 1 (bottom) shows representative force recordings of a single cardiomyocyte retrieved from a DHF patient before and after PKA treatment at maximal (pCa 4.5) and intermediate (pCa 5.8) activation. At pCa 4.5, Ftotal remained the same, whereas at pCa 5.8, Ftotal was reduced after PKA. In addition, Figure 1 illustrates the reduced Fpassive (pCa 9.0) after PKA. For control and DHF groups, Ftotal at pCa 4.5 was similar before and after PKA (Table 3; Figure 2B and 2C). At intermediate pCa (eg, pCa 5.8), Ftotal was reduced after PKA because of PKA-induced myofilament desensitization (Figure 2B and 2C). The latter was also evident from the reduced pCa50 value observed in both DHF and control groups (Table 3). After PKA treatment, Fpassive of cardiomyocytes from DHF patients dropped to values observed in the control group both at baseline and after PKA treatment (Figures 2B, 2C, and 5A). In addition, in DHF patients, the PKA-induced fall in Fpassive was larger when baseline Fpassive was higher (Figure 5B).
Discussion
The present study analyzed endomyocardial biopsy samples obtained from patients with DHF and yielded the following information: (1) When cardiomyocytes isolated from these samples were stretched to a sarcomere length of 2.2 μm, Ftotal at maximal [Ca2+] was comparable to that of control cardiomyocytes, but their Fpassive was twice as high. (2) The increase in Fpassive was reversible because administration of PKA lowered Fpassive to a level observed in control cardiomyocytes. (3) In vivo hemodynamic measures of diastolic LV function such as LVEDP, , and E were correlated with in vitro measurements of both Fpassive and CVF.
High Fpassive
Because the mechanical isolation procedure removed endomysial collagen structures, the high Fpassive of cardiomyocytes retrieved from DHF patients can only result from deranged diastolic stiffness of the cardiomyocytes themselves. Because cardiomyocytes were incubated in solution supplemented with 0.2% Triton X-100 before the experiments, the integrity of sarcolemmal and sarcoplasmic membranes was disrupted, and the cardiomyocytes became dependent on externally supplied calcium for active force development. Under these conditions, disturbed calcium handling because of modified expression and/or phosphorylation of sarcoplasmic reticular Ca2+-ATPase,23 phospholamban,24,25 sarcoplasmic calcium release channel,26 and sodium/calcium exchanger27 is effectively ruled out as a cause of the observed elevation of Fpassive, which therefore needs to be attributed to alterations of myofilament or cytoskeletal proteins.
The present study revealed no difference between the DHF and control groups in the expression of cardiac sarcomeric proteins such as myosin heavy chain, actin, TnT, TnI, desmin, and tropomyosin. Protein composition may also change as a result of enhanced proteolysis. This is especially evident for TnI, whose calpain-mediated breakdown is accelerated by a high LVEDP.28 Western immunoblot analysis ruled out degradation of several contractile proteins, including TnI, in both the control and DHF groups. Therefore, it is unlikely that a change in isoform composition or protein degradation accounts for the high Fpassive of cardiomyocytes observed in the DHF group.
Moreover, the correction by PKA treatment provides evidence that the high Fpassive resulted from phosphorylation of its sarcomeric target proteins: TnI, myosin binding protein-C, and/or titin. Treatment with PKA induced a large drop in Fpassive in cardiomyocytes of DHF patients, whereas neither Fpassive in control cardiomyocytes nor Ftotal at pCa 4.5 in cardiomyocytes from both groups was altered (Table 3 and Figure 2). Such an isolated drop in Fpassive, unaccompanied by a fall in Ftotal, is more easily reconciled with an action of PKA on a myofilament rather than on a cytoskeletal phosphorylation site because the parallel alignment of the cytoskeleton with the myofilaments would predict a fall in Fpassive generated by the cytoskeleton to also lower Ftotal.
The present study also determined the phosphorylation status of TnI but found no difference in the ratio of dephosphorylated to total TnI between control and DHF groups. It has recently been demonstrated in animal studies that phosphorylation of myosin binding protein-C and titin modifies diastolic properties. Phosphorylation29 or expression of a mutant isoform30 releases the "braking" action of myosin binding protein-C on cross-bridge cycling, thereby decreasing Fpassive in skinned mouse myocardial strips. Similarly, PKA-mediated phosphorylation of the elastic N2B spring element of titin reduces diastolic stiffness in isolated rat cardiomyocytes.31 Because of limited procurement of myocardial tissue by endomyocardial biopsy techniques, phosphorylation of both proteins could not be assessed in the present study. Future studies on myocardial tissue from DHF patients should focus on the phosphorylation level of both proteins to detect the sarcomeric protein responsible for the high Fpassive of cardiomyocytes isolated from DHF patients.
In Vitro Versus In Vivo
When Fpassive values of control and DHF cardiomyocytes were pooled, in vitro measurement of Fpassive was correlated with in vivo indices of diastolic LV dysfunction, such as LVEDP, , and E (Figure 4). The quantitative agreement between and Fpassive indicates that diastolic LV dysfunction is determined to an important extent by the rise in Fpassive of the cardiomyocytes. The relations between Fpassive and indices of diastolic LV function all leveled off at higher values of LVEDP, , and E. This could have resulted from diuretic therapy to recompensate the patients before catheterization or from more intense interstitial fibrosis at the top end of diastolic LV dysfunction. In the in vitro setting, all cardiomyocytes were stretched to the same sarcomere length of 2.2 μm. In the in vivo setting, LV preload was uncontrolled, and especially the DHF patients, who underwent more intense diuretic therapy in the interval between admission for pulmonary edema and diagnostic cardiac catheterization, could have been operating at LV filling pressures lower than needed to achieve optimal sarcomere length. More intense interstitial fibrosis also provides an explanation for the relations between Fpassive and indices of diastolic LV function to level off at higher values of LVEDP, , and E. Endomyocardial biopsy samples from patients with DHF had higher CVF than did controls, and in a bivariate linear regression analysis, both Fpassive and CVF were significantly correlated with LVEDP and . A predominant effect of interstitial fibrosis at the top end of diastolic LV dysfunction is in line with previous experimental studies, which showed diastolic muscle stiffness to originate from structures within the sarcomere for sarcomere lengths <2.2 μm32 and from perimysial fibers once filling pressures exceeded 30 mm Hg.33
Degradation of collagen in pressure-overloaded, hypertrophied papillary muscles with plasmin did not reduce muscle stiffness to levels observed in normal muscles.34 Similarly, in the present study, patients with DHF and low CVFs still had higher LVEDP, , and E than did controls (Figure 3). Therefore, our data support the concept that diastolic LV dysfunction in the presence of a low CVF is explained by the higher Fpassive of the cardiomyocytes. However, because half of the DHF patients suffered from diabetes mellitus, collagen cross-links formed by advanced glycation end products could also explain the impairment of diastolic LV function at low CVF.35
The present study observed low CVF and high Fpassive values in some patients with DHF but failed to detect DHF patients with high CVF and low Fpassive values. This suggests diastolic LV dysfunction to result from a sequence of events, which start with a rise in cardiomyocyte Fpassive followed by the development of interstitial fibrosis. A similar sequence of events has also been reported in experimental tachypacing-induced HF models. In those models, elevation of diastolic LV muscle stiffness was paralleled by expression of the shorter and stiffer N2B isoform of titin36 and not by interstitial fibrosis, which only developed when an angiotensin II infusion was superimposed on the pacing stress.37 Similar coordination between titin isoform shift and extracellular matrix deposition has also been reported in other experimental models.38 The evidence provided by the present study that DHF patients can have low CVF also explains why angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors, which reduce interstitial fibrosis,39,40 have not been uniformly successful in large clinical trials in DHF patients.41
Study Limitations
Five of the 12 DHF patients and 6 of the 8 controls had undergone cardiac transplantation. Transplant recipients were included in the study because they frequently suffer DHF16 and because their myocardial biopsy material is readily available. The pathogenetic mechanisms responsible for their DHF could differ from that of other DHF patients because of ongoing rejection and use of immunosuppressant drugs. Force recordings of isolated cardiomyocytes and the extent of interstitial fibrosis of the transplanted subgroup of DHF patients were, however, similar to the measurements obtained in the other DHF patients, and both data sets were therefore merged into a single DHF group. The same also applied to the control group.
Isolation of cardiomyocytes and assessment of myocardial tissue properties was performed on a limited number of RV or LV samples and could potentially have overlooked tissue heterogeneity. The extent of tissue heterogeneity was addressed in previous studies on explanted hearts14,15 or surgically procured biopsy samples.42 In those studies, the variability of force measurements in cardiomyocytes isolated from different portions of the heart was always <5%. To validate the use of defrosted biopsy samples, force recordings of cardiomyocytes isolated from a biopsy sample immediately after procurement were compared with force recordings of cardiomyocytes isolated from a defrosted biopsy sample from the same patient. These force recordings yielded identical results.
Conclusions
Cardiomyocytes isolated from endomyocardial biopsy samples of DHF patients had elevated Fpassive, which together with CVF, determined in vivo diastolic LV dysfunction. Administration of PKA to these cardiomyocytes normalized Fpassive. Because the integrity of sarcolemmal and sarcoplasmic membranes was disrupted by prior Triton incubation and because expression of sarcomeric proteins and the phosphorylation level of TnI were unaltered, the PKA-induced fall in Fpassive probably resulted from correction of a phosphorylation deficit of myosin binding protein-C or titin. This hypophosphorylated sarcomeric protein could, together with extracellular matrix modification, be a specific myocardial target for drug therapy of DHF.
Acknowledgments
This study was supported by a Marie Curie host fellowship (contract number HPMT-GH-00-00114-12), a European Committee Research Training Network project (contract number HPRN-CT-2000-00091), and the Netherlands Organization for Scientific Research (VENI grant, 2002).
Footnotes
The online-only Data Supplement, which contains an additional table, can be found with this article at http://www.circulationaha.org.
References
Grossman W. Diastolic dysfunction in congestive heart failure. N Engl J Med. 1991; 325: 1557–1564.
Kitzman DW, Little WC, Brubaker PH, Anderson RT, Hundley WG, Marburger CT, Brosnihan B, Morgan TM, Stewart KP. Pathophysiological characterization of isolated diastolic heart failure in comparison to systolic heart failure. JAMA. 2002; 288: 2144–2150.
Zile MR, Brutsaert DL. New concepts in diastolic dysfunction and diastolic heart failure, part I: diagnosis, prognosis, and measurements of diastolic function. Circulation. 2002; 105: 1387–1393.
Redfield MM. Understanding ‘diastolic’ heart failure. N Engl J Med. 2004; 350: 1930–1931.
Cleland JG, Cohen-Solal A, Aguilar JC, Dietz R, Eastaugh J, Follath F, Freemantle N, Gavazzi A, van Gilst WH, Hobbs FD, Korewicki J, Madeira HC, Preda I, Swedberg K, Widimsky J. Management of heart failure in primary care (the IMPROVEMENT of Heart Failure Programme): an international survey. Lancet. 2002; 360: 1631–1639.
Redfield MM, Jacobsen SJ, Burnett JC Jr, Mahoney DW, Bailey KR, Rodeheffer RJ. Burden of systolic and diastolic ventricular dysfunction in the community: appreciating the scope of the heart failure epidemic. JAMA. 2003; 289: 194–202.
Fischer M, Baessler A, Hense HW, Hengstenberg C, Muscholl M, Holmer S, Doring A, Broeckel U, Riegger G, Schunkert H. Prevalence of left ventricular diastolic dysfunction in the community: results from a Doppler echocardiographic-based survey of a population sample. Eur Heart J. 2003; 24: 320–328.
Zile MR, Baicu CF, Gaasch WH. Diastolic heart failure—abnormalities in active relaxation and passive stiffness of the left ventricle. N Engl J Med. 2004; 350: 1953–1959.
Yu CM, Lin H, Yang H, Kong SL, Zhang Q, Lee SW. Progression of systolic abnormalities in patients with ‘isolated’ diastolic heart failure and diastolic dysfunction. Circulation. 2002; 105: 1195–1201.
Kawaguchi M, Hay I, Fetics B, Kass DA. Combined ventricular systolic and arterial stiffening in patients with heart failure and preserved ejection fraction: implications for systolic and diastolic reserve limitations. Circulation. 2003; 107: 714–720.
Pak PH, Maughan L, Baughman KL, Kass DA. Marked discordance between dynamic and passive diastolic pressure-volume relations in idiopathic hypertrophic cardiomyopathy. Circulation. 1996; 94: 52–60.
Morris-Thurgood JA, Frenneaux MP. Diastolic ventricular interaction and ventricular diastolic filling. Heart Fail Rev. 2000; 5: 307–323.
Kass DA, Bronzwaer JG, Paulus WJ. What mechanisms underlie diastolic dysfunction in heart failure; Circ Res. 2004; 94: 1533–1542.
van der Velden J, Klein LJ, Zaremba R, Boontje NM, Huybregts MA, Stooker W, Eijsman L, de Jong JW, Visser CA, Visser FC, Stienen GJM. Effects of calcium, inorganic phosphate, and pH on isometric force in single skinned cardiomyocytes from donor and failing human hearts. Circulation. 2001; 104: 1140–1146.
van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, Burton PB, Goldmann P, Jaquet K, Stienen GJM. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res. 2003; 57: 37–47.
How to diagnose diastolic heart failure. European Study Group on diastolic heart failure. Eur Heart J. 1998; 19: 990–1003.
Papp Z, Szabo A, Barends JP, Stienen GJM. The mechanism of the force enhancement by MgADP under simulated ischaemic conditions in rat cardiac myocytes. J Physiol. 2002; 543: 177–189.
Bronzwaer JG, Heymes C, Visser CA, Paulus WJ. Myocardial fibrosis blunts nitric oxide synthase–related preload reserve in human dilated cardiomyopathy. Am J Physiol. 2003; 284: H10–H16.
van der Velden J, Moorman AF, Stienen GJM. Age-dependent changes in myosin composition correlate with enhanced economy of contraction in guinea-pig hearts. J Physiol. 1998; 507: 497–510.
Paulus WJ, Heyndrickx GR, Buyl P, Goethals MA, Andries E. Wide-range load shift of combined aortic valvuloplasty-arterial vasodilation slows isovolumic relaxation of the hypertrophied left ventricle. Circulation. 1990; 81: 886–898.
Bronzwaer JG, de Bruyne B, Ascoop CA, Paulus WJ. Comparative effects of pacing-induced and balloon coronary occlusion ischemia on left ventricular diastolic function in man. Circulation. 1991; 84: 211–222.
Bronzwaer JG, Zeitz C, Visser CA, Paulus WJ. Endomyocardial nitric oxide synthase and the hemodynamic phenotypes of human dilated cardiomyopathy and of athlete’s heart. Cardiovasc Res. 2002; 55: 270–278.
Frank KF, Bolck B, Brixius K, Kranias EG, Schwinger RH. Modulation of SERCA: implications for the failing human heart. Basic Res Cardiol. 2002; 97 (suppl 1): I-72–I-78.
MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol. 2003; 4: 566–577.
Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002; 34: 951–969.
Marks AR, Reiken S, Marx SO. Progression of heart failure: is protein kinase A hyperphosphorylation of the ryanodine receptor a contributing factor; Circulation. 2002; 105: 272–275.
Weber CR, Piacentino V III, Houser SR, Bers DM. Dynamic regulation of sodium/calcium exchange function in human heart failure. Circulation. 2003; 108: 2224–2229.
Feng J, Schaus BJ, Fallavollita JA, Lee TC, Canty JM Jr. Preload induces troponin I degradation independently of myocardial ischemia. Circulation. 2001; 103: 2035–2037.
Flashman E, Redwood C, Moolman-Smook J, Watkins H. Cardiac myosin binding protein C: its role in physiology and disease. Circ Res. 2004; 94: 1279–1289.
Palmer BM, Georgakopoulos D, Janssen PM, Wang Y, Alpert NR, Belardi DF, Harris SP, Moss RL, Burgon PG, Seidman CE, Seidman JG, Maughan DW, Kass DA. Role of cardiac myosin binding protein C in sustaining left ventricular systolic stiffening. Circ Res. 2004; 94: 1249–1255.
Yamasaki R, Wu Y, McNabb M, Greaser M, Labeit S, Granzier H. Protein kinase A phosphorylates titin’s cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes. Circ Res. 2002; 90: 1181–1188.
Linke WA, Popov VI, Pollack GH. Passive and active tension in single cardiac myofibrils. Biophys J. 1994; 67: 782–792.
Factor SM, Flomenbaum M, Zhao MJ, Eng C, Robinson TF. The effects of acutely increased ventricular cavity pressure on intrinsic myocardial connective tissue. J Am Coll Cardiol. 1988; 12: 1582–1589.
Stroud JD, Baicu CF, Barnes MA, Spinale FG, Zile MR. Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment. Am J Physiol. 2002; 282: H2324–H2335.
Herrmann KL, McCulloch AD, Omens JH. Glycated collagen cross-linking alters cardiac mechanics in volume-overload hypertrophy. Am J Physiol. 2003; 284: H1277–H1284.
Wu Y, Bell SP, Trombitas K, Witt CC, Labeit S, LeWinter MM, Granzier H. Changes in titin isoform expression in pacing-induced cardiac failure give rise to increased passive muscle stiffness. Circulation. 2002; 106(: 1384–1389.
Senzaki H, Paolocci N, Gluzband YA, Lindsey ML, Janicki JS, Crow MT, Kass DA. ;-Blockade prevents sustained metalloproteinase activation and diastolic stiffening induced by angiotensin II combined with evolving cardiac dysfunction. Circ Res. 2000; 86: 807–815.
Wu Y, Cazorla O, Labeit D, Labeit S, Granzier H. Changes in titin and collagen underlie diastolic stiffness diversity of cardiac muscle. J Mol Cell Cardiol. 2000; 32: 2151–2162.
Brilla CG, Funck RC, Rupp H. Lisinopril-mediated regression of myocardial fibrosis in patients with hypertensive heart disease. Circulation. 2000; 102: 1388–1393.
Yamamoto K, Masuyama T, Sakata Y, Nishikawa N, Mano T, Yoshida J, Miwa T, Sugawara M, Yamaguchi Y, Ookawara T, Suzuki K, Hori M. Myocardial stiffness is determined by ventricular fibrosis, but not by compensatory or excessive hypertrophy in hypertensive heart. Cardiovasc Res. 2002; 55: 76–82.
Yusuf S, Pfeffer MA, Swedberg K, Granger CB, Held P, McMurray JJ, Michelson EL, Olofsson B, Ostergren J. Effects of candesartan in patients with chronic heart failure and preserved left-ventricular ejection fraction: the CHARM-Preserved Trial. Lancet. 2003; 362: 777–781.
van der Velden J, Klein LJ, van der Bijl M, Huybregts MA, Stooker W, Witkop J, Eijsman L, Visser CA, Visser FC, Stienen GJ. Isometric tension development and its calcium sensitivity in skinned myocyte-sized preparations from different regions of the human heart. Cardiovasc Res. 1999; 42: 706–719.(Attila Borbely, MD; Jolan)