Impact of Treatment on Myocardial Lysyl Oxidase Expression and Collagen Cross-Linking in Patients With Heart Failure
The aim of this study was to investigate whether torasemide modifies collagen cross-linking in the failing human heart. We analyzed the degree of cross-linking and the expression of the enzyme lysyl oxidase, which regulates cross-linking, in the myocardium of patients with chronic heart failure at baseline and after 8 months of treatment with either torasemide or furosemide in addition to their standard heart failure therapy. Whereas lysyl oxidase protein expression was very scarce in normal hearts, it was highly expressed in failing hearts. Cross-linking was increased (P<0.001) in heart failure patients compared with normal hearts. These 2 parameters decreased (P=0.021 and P=0.034) in torasemide-treated patients and remained unchanged in furosemide-treated patients. In addition, more (P=0.009) patients showed normalization of left ventricular chamber stiffness in the torasemide subgroup than in the furosemide subgroup after treatment. Lysyl oxidase expression correlated with cross-linking (r=0.661; P<0.001), and cross-linking correlated with left ventricular chamber stiffness (r=0.452; P=0.002) in all patients. These findings show for the first time that lysyl oxidase overexpression is associated with enhanced collagen cross-linking in the failing human heart. In addition, we report that the ability of torasemide to correct both lysyl oxidase overexpression and enhanced collagen cross-linking results in normalization of left ventricular chamber stiffness in patients with heart failure. Lysyl oxidase may thus represent a target for reduction of stiff collagen and improvement of left ventricular mechanical properties in heart failure patients.
- clinical science
- extracellular matrix
- left ventricular chamber stiffness lysyl oxidase
Myocardial fibrosis may contribute to the increased risk of chronic heart failure (HF) in patients with cardiac diseases.1 A linkage between fibrosis and left ventricular (LV) dysfunction/failure may be established through different pathways, including increased passive stiffness that impairs diastolic function.2 Several studies using experimental models of pressure overload have demonstrated that LV chamber stiffness is affected by changes in both myocardial collagen quantity and quality, with the effect of changes in collagen concentration being modified by the degree of cross-linking.3–6
Several steps are involved in the process leading to exaggerated collagen deposition and fibrosis. Collagen is synthesized and secreted by fibroblasts and myofibroblasts as a procollagen precursor having amino-terminal and carboxy-terminal propeptides that are cleaved to yield the triple helical monomers of collagen by specific procollagen proteinases.7 After these proteolytic reactions, the collagen molecules are rapidly and spontaneously assembled into collagen fibrils. Chemical reactions slowly take place within existing collagen fibrils in tissues, leading to formation of covalent bonds between adjacent polypeptide chains and thus making the final collagen fibers less soluble in any solvent and more resistant against proteolytic enzymes. The first step in this reaction sequence is enzymatic: the copper (Cu)-dependent amine oxidase lysyl oxidase (LOX) catalyzes the oxidation of the ε-amino groups in lysine or hydroxylysine residues, resulting in the formation of corresponding aldehydes. Two such aldehydes can then spontaneously react with each other, or one aldehyde can bind to another ε–amino group; both mechanisms produce cross-links connecting 2 polypeptide chains.8,9
Previous studies in rats10,11 and patients12,13 with HF have shown that whereas treatment with torasemide was associated with a reduction in the amount of histologically proven myocardial fibrosis (as assessed from the measurement of the fraction of myocardial volume occupied by collagen tissue or CVF), treatment with furosemide did not. Thus, we hypothesized that torasemide, but not furosemide, may also modify the quality of collagen (ie, reduction of insoluble collagen and collagen cross-linking) in the myocardium of HF patients. In accordance with this hypothesis, the following end points were defined: (1) to test the effects of torasemide and furosemide, on top of the recommended treatment for HF, on insoluble collagen and collagen cross-linking in patients with HF, (2) to test the effects of torasemide and furosemide on myocardial LOX in these patients, and (3) to test the effects of torasemide and furosemide on LV chamber stiffness in the same patients.
An expanded version of the methods can be found in an online supplement available at http://www.hypertensionaha.org.
Patients and Study Design
This was a prospective, randomized, parallel group study. Twenty-four white patients with a previous diagnosis of chronic HF were included. After randomization, 12 patients were assigned to 10 to 20 mg torasemide daily (torasemide subgroup) and 12 patients to 20 to 40 mg furosemide daily (furosemide subgroup) for 8 months. Ten patients from each subgroup completed the study. Studies were performed on each patient at enrollment (baseline) and 8 months after randomization. Septal endomyocardial biopsies were obtained from autopsies of healthy subjects to assess control reference values.
Two-dimensional echocardiographic imaging, targeted M-mode recordings, and Doppler ultrasound measurements were obtained from each patient.
Amino-terminal probrain natriuretic peptide (NT-proBNP) was measured in serum samples by an enzyme-linked immunosorbent assay (Roche Diagnostics).
Histomorphologic and Immunohistochemical Studies
Three transvenous endomyocardial biopsies were taken from the middle area of the interventricular septum. The CVF was determined by quantitative morphometry in sections stained with collagen-specific picro-sirius red, as reported previously.14
To distinguish between cross-linked (insoluble) and noncross-linked (soluble) collagen, a colorimetric procedure was used.15,16 The degree of cross-linking was calculated as the ratio between the insoluble and the soluble forms of collagen. Immunohistochemical analysis for LOX was performed using a mouse monoclonal antibody against LOX (R&D Systems).
Western Blot Studies
Western blot studies were performed as described recently17 using a specific rabbit polyclonal antibody against LOX (R&D Systems).
Differences between different groups were tested using a Student t test for unpaired data or Mann–Whitney U test. Differences in parameters before and after treatment within each subgroup of patients were tested by a Student t test for paired data or Wilcoxon signed-rank test. Categorical variables were analyzed by the χ2 Fisher exact test. The correlation between continuously distributed variables was tested by univariate regression analysis and bivariate association. Data are expressed as means±SD and number of patients. A P value <0.05 was considered statistically significant. Analyses were performed with the SPSS 15.0 statistical package.
Expression of Myocardial LOX in Controls and the Whole Group of HF Patients
Figure 1 shows that whereas LOX was almost absent in the myocardium of a control subject, it was highly expressed in fibroblasts, areas of interstitial and perivascular fibrosis, and cardiomyocytes in the myocardium of an HF patient. As shown in Figure 2, one 32-kDa band corresponding to the active form of LOX was identified in myocardial samples from all HF patients. In contrast, the active form of LOX was undetectable in myocardial samples from control subjects.
Types of Collagen and Collagen Cross-Linking in Controls and the Whole Group of HF Patients
As shown in Table 1, the amounts of both insoluble and soluble collagens were significantly higher in HF patients than in control subjects. Whereas insoluble collagen corresponded to 58.7±2.43% of total collagen in control hearts, it represented 76.4±5.88% in failing hearts, a significant difference (P<0.001). The degree of collagen cross-linking was significantly increased in the myocardium of HF patients compared with control subjects (Table 1).
Effects of Treatment in the 2 Subgroups of HF Patients
Clinical, Echocardiographic, and Biochemical Data
Clinical and echocardiographic characteristics of the 2 subgroups of patients at baseline are presented in Table 2. No significant differences were observed between the subgroups in the etiologies of HF. A depressed ejection fraction (EF; below the normal cutoff value of 0.40) was observed in 70% and 50% of patients from the furosemide subgroup and the torasemide subgroup, respectively. Doppler criteria of diastolic dysfunction were present in 50% and 70% of patients from the furosemide subgroup and the torasemide subgroup, respectively. An abnormally high LV chamber stiffness constant (KLV) value was observed in 60% and 50% of patients from the furosemide subgroup and the torasemide subgroup, respectively. None of these differences reached statistical significance.
Eight months after randomization, patients in the torasemide subgroup and the furosemide subgroup received mean daily dosages of 11.1±0.8 mg and 33.1±3.0 mg of these agents, respectively. Baseline medications other than loop diuretics were maintained unchanged during the treatment period in the 2 subgroups of patients. No adverse effects occurred during the study in either subgroup. The frequency of complications (including hospitalizations and exacerbations of HF) was similar in the 2 subgroups.
Although nonsignificant differences in the values of EF were found after treatment in the 2 subgroups of treatment, the frequency of patients showing normalization of this parameter after treatment was higher (P=0.025) in the torasemide subgroup than in the furosemide subgroup (80% versus 40%). In addition, although the final values of KLV were similar in the 2 subgroups of patients, the frequency of patients showing normalization of KLV after treatment was greater (P=0.009) in the torasemide subgroup than in the furosemide subgroup (80% versus 0%). As shown in Table 2, the levels of NT-proBNP decreased in the torasemide subgroup but remained unchanged in the furosemide subgroup. After treatment, NT-proBNP levels were lower in torasemide-treated patients than in furosemide-treated patients. Finally, the New York Heart Association functional class decreased in the torasemide subgroup and remained unchanged in the furosemide subgroup (Table 2).
Expression of Myocardial LOX
No significant differences in the expression of LOX protein were found at baseline between the 2 subgroups of patients (Figure 2). Whereas the expression of LOX protein remained unchanged in furosemide-treated patients (5.06±2.57 arbitrary densitometric units [A.D.U.] versus 5.08±2.61 A.D.U.), it decreased (P=0.034) in torasemide-treated patients (4.86±1.78 versus 3.34±1.85 A.D.U.; Figure 2).
Total Collagen, Types of Collagen, and Collagen Cross-Linking
No differences in the baseline values of CVF were observed between the 2 subgroups. Whereas CVF decreased in the torasemide subgroup after treatment (8.29±1.65% versus 4.24±0.74%; P<0.001), it remained unchanged in the furosemide subgroup (7.93±3.05% versus 7.04±3.48%; P=0.549). Final values of CVF were lower (P<0.001) in the torasemide subgroup than in the furosemide subgroup.
The amounts of insoluble and soluble collagen and the degree of cross-linking of the 2 subgroups of patients are presented in Table 3. No significant differences between the 2 subgroups of patients were found in these parameters at baseline. Whereas insoluble collagen increased in furosemide-treated patients (P=0.012), it decreased in torasemide-treated patients (P=0.045). In addition, soluble collagen increased (P=0.046) in torasemide-treated patients but remained unchanged in furosemide-treated patients. Thus, whereas the degree of collagen cross-linking did not change in furosemide-treated patients, it decreased (P=0.021) in torasemide-treated patients.
A positive correlation was found between the expression of LOX protein and the amount of insoluble collagen (r=0.603; P=0.004) and the degree of cross-linking (r=0.661; P<0.000) in all patients at baseline and after treatment (Figure 3). These correlations remained significant when we excluded the influence of a number of potential confounding factors (ie, age, body weight, systolic blood pressure, diastolic blood pressure, LV mass index, LV diastolic volume, KLV, and EF) in partial correlation analysis.
The amount of insoluble collagen was positively correlated with KLV (r=0.507; P=0.007) in all patients at baseline and after treatment (Figure 4). Similarly, the degree of cross-linking was positively correlated with KLV (r=0.452; P=0.002) in all patients at baseline and after treatment (Figure 4). The 2 correlations remained significant when we excluded the influence of a number of potential confounding factors (ie, age, body weight, systolic blood pressure, diastolic blood pressure, LV mass index, LV diastolic volume, and EF) in partial correlation analysis.
Finally, negative correlations were found between LOX and the deceleration time (r=−0.579; P=0.001) and between cross-linking and the deceleration time (r=−0.336; P=0.037) in all patients at baseline and after treatment. These correlations also remained significant after excluding the influence of the above potential confounding factors in partial correlation analysis.
The main findings of this study are as follows: (1) the expression of LOX and the degree of collagen cross-linking are abnormally increased in the myocardium of chronic HF patients, (2) both LOX expression and collagen cross-linking decrease in torasemide-treated chronic HF patients but remain unchanged in furosemide-treated chronic HF patients, and (3) direct correlations exist between LOX and collagen cross-linking and between collagen cross-linking and LV stiffness in chronic HF patients. Collectively, these findings show for the first time that LOX plays a critical role in collagen alterations in the failing human heart.
The Cu-dependent enzyme LOX critically controls the process whereby collagen molecules are assembled and covalently cross-linked to one another, resulting in fibers with increased material stiffness and greater resistance to degradation. Whereas LOX mRNA upregulation has been reported previously in explanted hearts of patients with idiopathic dilated cardiomyopathy,18 low levels of collagen cross-linking have been reported in the hearts of patients with the same cardiac disease.19 However, none of these studies explored whether the changes in LOX were simultaneously associated with variations in the formation of insoluble collagen (cross-linked). Here we show that an increase of LOX was associated with an increase of insoluble collagen in the human failing heart, suggesting that upregulation of the enzyme is responsible for excessive collagen cross-linking present in patients from this study. In support of this possibility is our finding that in torasemide-treated patients, the decrease in LOX was associated with the reduction in both collagen cross-linking and the amount of insoluble collagen.
Dyshomeostasis of micronutrients such as Cu and zinc (Zn) is part of the systemic illness that accompanies HF and is simultaneously operative in promoting myocardial remodeling.20 In particular, the role of Cu in regulating the activity of LOX and Cu/Zn-superoxide dismutase as well as the role of Zn essential to the activity of angiotensin-converting enzyme and matrix metalloproteinases is thought to be of relevance for the regulation of collagen matrix. In this conceptual framework, it is tempting to speculate that because of the different pharmacokinetic properties and pharmacodynamic actions of torasemide and furosemide,21 these 2 compounds may differentially influence Cu and Zn homeostasis, and this, in turn, may have a different impact on the activity of LOX and other metalloenzymes in the heart.
Of interest, we found that in addition to its localization in the interstitial space and fibroblasts, LOX was also expressed in cardiomyocytes in patients with chronic HF. We have shown recently that cardiomyocytes from chronic HF patients express procollagen type I carboxy-terminal proteinase (PCP) that regulates the synthesis of mature collagen type I,13 as well as matrix metalloproteinase-1 that regulates the degradation of collagen fibers.17 Collectively, these observations suggest that in addition to fibroblasts, cardiomyocytes play a major role in regulating collagen turnover in the failing human heart.
LOX can be regulated at 3 levels: synthesis of LOX precursor by fibroblasts and other fibrogenic cells,8 extracellular conversion of the precursor into the active enzyme because of the action of PCP,22,23 and direct stimulation of the activity of the enzyme by cytokines such as transforming growth factor-β.24 We reported recently that PCP activation was abnormally increased in the myocardium of chronic HF patients and that torasemide, but not furosemide, decreased myocardial PCP activation in these patients.13 In addition, it has been reported that the expression of transforming growth factor-β is reduced in the myocardium of torasemide-treated rats compared with control rats.10,11 It is thus tempting to speculate that the reduction in LOX observed after treatment in patients receiving torasemide can be related to the ability of this compound to deactivate PCP or downregulate transforming growth factor-β.
Quantitative assessment of fibrosis in various murine transgenic models, larger animal failure models, and even in humans has not always found correlations between stiffness and collagen content. In other studies, it has been demonstrated that the relative ratio of insoluble to soluble collagen (ie, the degree of cross-linking) is more important. For example, in rats exposed to aortic binding, hypertrophy was accompanied by increased total collagen, yet decreases in insoluble/soluble collagen ratio, and no change in myocardial stiffness.6 In contrast, spontaneously hypertensive rats had elevated total collagen and higher insoluble/soluble ratio, which correlated with chamber stiffening.6 Our findings that LV stiffness is correlated with the amount of insoluble collagen and the degree of cross-linking are consistent with the notion that in the failing human heart, the quality of collagen (specifically cross-linking) plays a key role in translating quantity into mechanical stiffness and functional performance of the left ventricle.
This hypothesis receives some support from changes in clinical and biochemical parameters observed in treated patients. In fact, a greater number of patients showed normalization in KLV and EF in the torasemide subgroup than in the furosemide subgroup. In addition, levels of NT-proBNP decreased in torasemide-treated patients but not in furosemide-treated patients. In this regard, it is interesting to remark that myocardial stiffness has been shown to be the most important determinant of the plasma BNP production in patients with HF.25 Therefore, the ability of torasemide to reduce LOX and collagen cross-linking may contribute to its beneficial clinical impact on HF patients.
Several limitations of the current study must be recognized. First, this was an individually randomized, parallel-group study involving a small number of patients. In addition, it must be recognized that therapy with β-blockers, angiotensin-converting enzyme inhibitors, or angiotensin-receptor antagonists may have influenced the findings. Second, although we did not assess LOX precursor (ie, zymogen) or LOX activity (ie, zymography), the associations found between LOX expression and insoluble collagen and collagen cross-linking suggest that the activity of the enzyme may parallel its expression in the failing human heart. Third, KLV is difficult to measure in clinical practice, even with invasive techniques. However, studies in animals26 and humans27 with different hemodynamic conditions have demonstrated that the deceleration time provides an accurate estimate of LV operating stiffness. Finally, it is important to consider the possibility that the different pharmacology of furosemide and torasemide, even in HF patients,21 may also contribute to the differential effects of the 2 compounds on the cardiac parameters tested in this study.
Findings from this study suggest a role for LOX upregulation in the excess of collagen cross-linking present in patients with chronic HF. In addition, our data suggest that the ability of torasemide to correct both LOX overexpression and enhanced collagen cross-linking may be involved in amelioration of KLV in HF patients treated with this compound. These results support the notion that therapeutic strategies directed not just at reducing the amount of collagen but also at reducing collagen cross-linking should be implemented in the prevention of the adverse impact of myocardial fibrosis on cardiac function in chronic HF patients.
The authors thank Sonia Martínez for her valuable technical assistance.
Sources of Funding
This work was funded through the agreement between the Foundation for Applied Medical Research (FIMA) and UTE project CIMA, the Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares (RECAVA) from the Instituto de Salud Carlos III, Ministry of Health, Spain (grant RD06/0014/0008), and the European Union (InGenious HyperCare, grant LSHM-CT-2006-037093).
- Received October 16, 2008.
- Revision received October 26, 2008.
- Accepted November 17, 2008.
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