Identification of a Potential Cardiac Antifibrotic Mechanism of Torasemide in Patients With Chronic Heart Failure

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1 Journal of the American College of Cardiology Vol. 50, No. 9, by the American College of Cardiology Foundation ISSN /07/$32.00 Published by Elsevier Inc. doi: /j.jacc Heart Failure Identification of a Potential Cardiac Antifibrotic Mechanism of Torasemide in Patients With Chronic Heart Failure Begoña López, PHD,* Arantxa González, PHD,* Javier Beaumont, PHD,* Ramón Querejeta, MD, PHD, Mariano Larman, MD, Javier Díez, MD, PHD* Pamplona and San Sebastián, Spain Objectives Background Methods Results Conclusions This study sought to investigate whether torasemide inhibits the enzyme involved in the myocardial extracellular generation of collagen type I molecules (i.e., procollagen type I carboxy-terminal proteinase [PCP]). Torasemide has been reported to reduce myocardial fibrosis in patients with chronic heart failure (HF). Chronic HF patients received either 10 to 20 mg/day oral torasemide (n 11) or 20 to 40 mg/day oral furosemide (n 11) in addition to their standard HF therapy. At baseline and after 8 months from randomization, right septal endomyocardial biopsies were obtained to analyze the expression of PCP by Western blot and the deposition of collagen fibers (collagen volume fraction [CVF]) with an automated image analysis system. The carboxy-terminal propeptide of procollagen type I (PICP) released as a result of the action of PCP on procollagen type I was measured in serum by radioimmunoassay. The ratio of PCP active form to PCP zymogen, an index of PCP activation, decreased (p 0.05) in torasemidetreated patients and remained unchanged in furosemide-treated patients. A reduction (p 0.01) in both CVF and PICP was observed in torasemide-treated but not in furosemide-treated patients. Changes in PCP activation were positively correlated (p 0.001) with changes in CVF and changes in PICP in patients receiving torasemide. These findings suggest the hypothesis that the ability of torasemide to reduce myocardial fibrosis in chronic HF patients is related to a decreased PCP activation. Further studies are required to ascertain whether PCP may represent a new target for antifibrotic strategies in chronic HF. (J Am Coll Cardiol 2007;50:859 67) 2007 by the American College of Cardiology Foundation Myocardial fibrosis has been proposed as a major determinant of altered left ventricular function leading to chronic heart failure (HF) in patients with cardiac diseases (1,2). It has been proposed that myocardial fibrosis is primarily the result of the uncoupling between increased synthesis and unchanged or decreased degradation of collagen type I fibers (3,4). Collagen type I is synthesized and secreted by fibroblasts as procollagen type I precursor having amino-terminal and carboxy-terminal propeptides that are cleaved to yield the triple helical monomers (Fig. 1) (5). The enzyme procollagen type I carboxy-terminal proteinase (PCP) is a neutral, From the *Division of Cardiovascular Sciences, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; Division of Cardiology, Donostia University Hospital, San Sebastián, Spain; Division of Hemodynamics, Guipuzcoa Polyclinics, San Sebastián, Spain; and the Department of Cardiology and Cardiovascular Surgery, University Clinic, University of Navarra, Pamplona, Spain. Partially funded by the Red Temática de Investigación Cardiovascular (RECAVA), Ministry of Health, Spain, and by the arrangement between the FIMA and UTE-CIMA project. Manuscript received February 20, 2007; revised manuscript received April 25, 2007, accepted April 30, Ca 2 -dependent proteinase responsible for the cleavage of the carboxy-terminal propeptide of procollagen type I (PICP) that is further released to the blood stream (Fig. 1) (6,7). The hydrolytic activity of PCP is enhanced up to 10-fold by a 55-kDa glycoprotein enhancer (PCPE) and by its 36- and 34-kDa PCPE proteolytic fragments (Fig. 1) (8,9). In addition, PCP stimulates extracellular activation of the enzyme lysyl oxidase that controls the formation of covalent cross-links between collagen type I molecules to form collagen type I fibrils (Fig. 1). In fibrogenic cells, the enzyme is primarily secreted as a 112-kDa inactive zymogen with a proregion that is removed to yield the 96-kDa active form (10). We reported recently that long-term treatment with different loop diuretics may have a variable impact on myocardial fibrosis and collagen type I biosynthesis in chronic HF patients (11). In fact, although torasemide-treated patients showed decreased myocardial collagen accumulation and serum PICP, furosemide-treated patients did not. In addition, a greater improvement of New York Heart Association functional

2 860 López et al. JACC Vol. 50, No. 9, 2007 Fibrosis and Heart Failure August 28, 2007: Abbreviations and Acronyms CVF collagen volume fraction HF heart failure MMP matrix metalloproteinase mrna messenger ribonucleic acid PCP procollagen type I carboxy-terminal proteinase PCPE procollagen type I carboxy-terminal proteinase enhancer PICP carboxy-terminal propeptide of procollagen type I TIMP tissue inhibitor of matrix metalloproteinase class and of parameters assessing cardiac function was observed in torasemide-treated patients than in those treated with furosemide. We thus have explored whether torasemide alters the expression of the PCP/PCPE system and this, in turn, results in reduced PCP activity and decreased collagen deposition in the myocardium of newly recruited chronic HF patients. To evaluate this possibility we studied changes in myocardial PCP, PCPE, and collagen volume fraction (CVF), and serum PICP in HF patients receiving either torasemide or furosemide. The effects of these drugs on the synthesis of procollagen type I precursor (i.e., 1 chain of procollagen type I messenger ribonucleic acid [mrna]) and the degradation of collagen type I fibers (i.e., matrix metalloproteinase [MMP]-1 and tissue inhibitor matrix metalloproteinase [TIMP]-1) were also analyzed. Methods Patients and study protocols. This was an individually randomized, open-label, parallel-group pilot study. All subjects gave written, informed consent before participating in the study. The investigation conformed to the principles outlined in the Declaration of Helsinki. The study protocol Figure 1 Generation of Collagen Type I Fibers Schematic representation of the steps involved in the synthesis and processing of collagen type I molecules. DNA deoxyribonucleic acid; PCP procollagen type I carboxy-terminal proteinase; PCPE procollagen type I carboxy-terminal proteinase enhancer; PICP carboxy-terminal propeptide of procollagen type I. was approved by the Institutional Ethics Committee of the Donostia University Hospital. The study population consisted of 22 white patients. All patients were required to have a previous diagnosis of chronic HF by the presence of a least 1 major and 2 minor criteria of the Framingham study (12) during the last 6 months. Whereas 80% of the patients enrolled had hypertensive heart disease, the remaining 20% showed ischemic heart disease. None of the patients had suffered from previous myocardial infarction. A depressed ejection fraction ( 0.40) was observed in 55% of patients. Although all patients were clinically stable, they were randomly assigned to receive either torasemide or furosemide in accordance with recent data (13). After randomization, 11 patients were assigned to torasemide 10 to 20 mg daily (torasemide group) and 11 patients to furosemide 20 to 40 mg daily (furosemide group) for 8 months. The doses were used in accordance with current guidelines for patients with chronic HF (14). Existing recommended salt intake restriction (4 g/day) and concomitant HF medications (i.e., an angiotensin-converting enzyme inhibitor, or an angiotensin-receptor antagonist, and a betaadrenergic blocker) were continued during the study (14). None of the patients were treated with aldosterone antagonists. A number of studies, including endomyocardial biopsy, were performed in each patient at enrollment (baseline) and 8 months after randomization. A group of 12 normotensive subjects (7 men and 5 women, mean age 56 years, range 39 to 72 years) were used as control subjects for histomorphologic and biochemical studies. They were subjects with clinically presumed coronary artery disease who were found to lack the disease at a coronary angiography. Echocardiographic assessment. Two-dimensional echocardiographic imaging, targeted M-mode recordings, and Doppler ultrasound measurements were obtained in each patient as recommended (15). Left ventricular mass was measured, and left ventricular mass index was calculated by dividing left ventricular mass by body surface area. The following pulsed Doppler measurements were obtained: maximum early transmitral velocity in diastole, maximum late transmitral velocity in diastole, the deceleration time of the early mitral filling wave, and isovolumic relaxation time. Ejection fraction was calculated according to Quinones et al. (16). Biochemical determinations. Venous blood samples were drawn at 09:00 h in an upright position. Plasma aldosterone was measured by radioimmunoassay using a commercial kit. Serum PICP was determined by radioimmunoassay according to a method previously described (17). Histomorphologic and immunohistochemical studies. Three transvenous endomyocardial biopsies were taken from the middle area of the interventricular septum with a bioptome Cordis 96 cm (7-F) under fluoroscopic guidance after angiographic examination. The CVF was determined by quantitative morphometry with an automated image

3 JACC Vol. 50, No. 9, 2007 August 28, 2007: López et al. Fibrosis and Heart Failure 861 analysis system in sections stained with collagen-specific picrosirius red, as previously reported (18). Immunohistochemical analysis for PCP and PCPE was performed on formalin-fixed and paraffin-embedded sections. Immunohistochemical staining was performed by the avidin peroxidase-labeled dextran polymer method. Positive staining was visualized with DAB Plus (Boehringer Mannheim Corp., Indianapolis, Indiana), and tissues were counterstained with Harris hematoxylin (Sigma, St. Louis, Missouri). A mouse monoclonal antibody against PCP (dilution 1:100; R&D Systems, Abingdom, England) and PCPE (dilution 1:100; R&D Systems) was used as the primary antibody. Western blot studies. A 5- g sample of total protein obtained from transvenous endomyocardial biopsies was processed for Western blot as recently described (19). Specific rabbit polyclonal antibodies against PCP (R&D Systems, specificity 95%), PCPE (R&D Systems; specificity 100%), MMP-1 (Oncogene, Cambridge, Massachusetts; specificity 100%) and TIMP-1 (Chemicon, Hofheim, Germany; specificity 95%) were incubated at dilutions of 1:500, 1:100, 1:2,000, and 1:200, respectively. Bands were detected by peroxidase-conjugated secondary antibodies (Amersham Biosciences, Barcelona, Spain) and visualized with the ECL-Plus chemiluminescence system (Amersham Biosciences). Autoradiograms were analyzed using an automatic densitometer (Molecular Imager FX, Bio-Rad, Barcelona, Spain). The blots were also probed with a monoclonal beta-actin antibody (Sigma) as a control for loading. Data are expressed as arbitrary densitometric units relative to beta-actin expression. Reverse transcriptase-polymerase chain reaction (RT- PCR) study. The mrna levels of the 1 chain of procollagen type I were analyzed by real-time quantitative RT-PCR as recently described (20). Reverse transcription was performed with 200 g of total RNA by using Superscript III reverse transcriptase (Invitrogen, Eugene, Oregon). Real-time PCR was performed with an ABI PRISM 7000 Sequence Detection System according to the manufacturer s recommendations (Applied Biosystems, Madrid, Spain) by using specific TaqMan MGB fluorescent probes for human mrna of the 1 chain of procollagen type I (Hs ), and a specific TaqMan MGB fluorescent probe for human constitutive 18S ribosomal RNA as endogenous control. Data are expressed as arbitrary units relative to 18S ribosomal RNA. Statistical analysis. Differences in parameters between normotensive controls and the whole group of HF patients at baseline and between the 2 groups of HF patients at baseline and after treatment (absolute values and deltas) were tested using a Student t test for unpaired data once normality was shown (Shapiro-Wilks test); otherwise, a nonparametric test (Mann-Whitney U test) was used. Differences in parameters before and after treatment within each group of patients were tested by a Student t test for paired data once normality was shown (Shapiro-Wilks test); otherwise, a nonparametric test (Wilcoxon test) was used. The correlation between continuously distributed variables was tested by univariate regression analysis and bivariate association (Spearman coefficient). Data are expressed as mean value SEM. A value of p 0.05 was considered statistically significant. Results Baseline characteristics. Baseline clinical and echocardiographic characteristics of the 2 groups of patients are presented in the Table 1. No significant differences were observed between the groups in the parameters tested. As shown in Figure 2, 2 PCP bands of 112 and 96 kda corresponding to zymogen and active form, respectively, were identified in myocardial samples from all patients. In addition, the 55-kDa full-length and the 36-kDa proteolytic fragment of PCPE were also detected in all samples (Fig. 3). Figure 4 shows that although PCP and PCPE were mostly expressed in fibroblasts and areas of interstitial and perivascular fibrosis, these molecules also were present in cardiomyocytes. Myocardial parameters evaluated in the normotensive group and the whole group of HF patients are presented in Table 2. As expected, values related to collagen expression and deposition, as well as PICP values, were higher in patients than in normotensive subjects. In addition, PCP zymogen was decreased and PCP activation, assessed as the ratio between its active form and its zymogen, and 36-kDa PCPE were increased in HF patients compared with normotensive subjects. No significant differences were found between the 2 groups of patients in the baseline values of parameters assessing myocardial fibrosis and collagen type I synthesis and degradation (Table 3). The PCP activation did tend to be higher in the torasemide group ( ) than in the furosemide group ( ), but the difference did not reach statistical significance. A positive correlation (r 0.687, p 0.001) was found between plasma aldosterone and PCP activation in all patients (Fig. 5). Effects of treatment. GENERAL ASPECTS. Eight months after randomization, patients in the torasemide group (n 11) and the furosemide group (n 11) received mean daily dosages of mg and mg of these agents, respectively. Baseline medications other than loop diuretics were maintained unchanged during the treatment period in the 2 groups of patients. No adverse effects occurred during the study in either group. The frequency of complications (including hospitalizations and exacerbations of HF) was similar in the 2 groups (data not shown). CLINICAL AND ECHOCARDIOGRAPHIC DATA. As shown in Table 1, the changes in body weight and blood pressure were similar in the 2 groups of patients. The values of left ventricular end-diastolic volume showed a nonsignificant trend toward a decrease in torasemide-treated but not in furosemide-treated patients (Table 1). The values of left ventricular ejection fraction showed a nonsignificant trend

4 862 López et al. JACC Vol. 50, No. 9, 2007 Fibrosis and Heart Failure August 28, 2007: Effects in the 2of Groups Treatment of Patients on Clinical WithParameters Chronic Heart Assessed Failure Table 1 Effects of Treatment on Clinical Parameters Assessed in the 2 Groups of Patients With Chronic Heart Failure Furosemide Group Torasemide Group Parameters Baseline Treatment Baseline Treatment Age, yrs Gender, M/F 9/2 8/3 Body weight, kg Causes of HF, n HHD 9 8 IHD 2 3 Types of HF, n EF EF Medications, n ACEI or ARA Beta-blocker Digoxin Diuretic Furosemide Torasemide Hct amiloride SBP, mm Hg * * DBP, mm Hg * * LVMI, g/m RWT LVEDD, mm V E /V A IVRT, ms DT, ms EF NYHA functional class, n I 0 0 II II IV Aldosterone, pg/ml Data are expressed as the mean value SEM or number of patients. *p 0.01 versus values at baseline in the same group. p 0.01 for changes in NYHA functional class 8 months after randomization. ACEI angiotensin-converting enzyme inhibitor; ARA angiotensin type I receptor antagonist; DBP diastolic blood pressure; DT deceleration time; EF ejection fraction; Hct hydrochlorothiazide; HF heart failure; HHD hypertensive heart disease; IHD ischemic heart disease; IVRT isovolumic relaxation time; LVEDD left ventricular end-diastolic diameter; LVMI left ventricular mass index; NYHA New York Heart Association; RWT relative wall thickness; SBP systolic blood pressure; V A maximum late transmitral velocity in diastole; V E maximum early transmitral velocity in diastole. toward an increase in torasemide-treated but not furosemide-treated patients (Table 1). The number of patients showing improvement of at least 1 grade in New York Heart Association functional class was greater (p 0.01) in the torasemide group than in the furosemide group. MYOCARDIAL FIBROSIS. Although CVF decreased (p 0.01) in the torasemide-treated patients, it remained unchanged in furosemide-treated patients (Table 3). In addition, CVF was lower (p 0.01) in torasemide-treated patients than in furosemide-treated patients. Furthermore, the delta for this parameter was also different between the 2 groups (torasemidetreated patients %, furosemide-treated patients %, p 0.05). PARAMETERS RELATED TO COLLAGEN TYPE I SYNTHESIS AND DEGRADATION. As shown in Table 3, the expression of 1(I) mrna decreased (p 0.05) to a similar extent in the 2 groups of treated patients. Whereas administration of torasemide was associated with a reduction (p 0.01) in serum PICP, this parameter did not change in the furosemide group (Table 3). In addition, serum PICP measured 8 months after randomization was lower (p 0.01) in the torasemide group than in the furosemide group. The delta for this parameter was different between the 2 groups (torasemide-treated patients %, furosemide-treated patients %, p 0.05). A positive correlation was found between changes in serum PICP and changes in CVF (r 0.692, p 0.01) in

5 JACC Vol. 50, No. 9, 2007 August 28, 2007: López et al. Fibrosis and Heart Failure 863 Figure 2 Western Blot Autoradiograms of PCP Representative Western blot autoradiograms of myocardial PCP from 1 patient with chronic heart failure at baseline and 8 months after randomization to furosemide (left), and 1 chronic heart failure patient at baseline and 8 months after randomization to torasemide (right). Abbreviations as in Figure 1. torasemide-treated patients (Fig. 6). No significant changes in either MMP-1 or TIMP-1 were observed in the 2 groups with treatment (Table 3). PCP AND PCPE EXPRESSION. The expression of both PCP zymogen and PCP active form increased (p 0.05) in the furosemide group, but remained unchanged in the torasemide group (Table 3, Fig. 2). Whereas PCP activation remained unchanged in furosemide-treated patients ( ), it decreased (p 0.05) in torasemide-treated patients ( ). The expression of full-length PCPE did not change with treatment in either group of patients (Table 3, Fig. 3). The 36-kDa PCPE fragment decreased (p 0.05) in torasemide-treated patients and remained unchanged in furosemide-treated patients (Table 3, Fig. 3). Changes in PCP activation were positively correlated with both changes in CVF (r 0.876, p 0.001) (Fig. 7A) and changes in serum PICP (r 0.897, p 0.001) (Fig. 7B) in torasemide-treated patients. In addition, a positive correlation (r 0.588, p 0.05) was found between final values of plasma aldosterone and the inhibition of PCP activation in torasemide-treated patients. These correlations were not found in furosemide-treated patients. No other correlations were found among the remaining parameters tested in this study. Discussion The main findings of this study are as follows: 1) 1(I) mrna levels (an index of the synthesis of procollagen type I precursor) decreased to a similar extent in torasemide- Figure 3 Western Blot Autoradiograms of PCPE Representative Western blot autoradiograms of myocardial PCPE from 1 patient with chronic heart failure at baseline and 8 months after randomization to furosemide (left), and 1 chronic heart failure patient at baseline and 8 months after randomization to torasemide (right). Abbreviations as in Figure 1.

6 864 López et al. JACC Vol. 50, No. 9, 2007 Fibrosis and Heart Failure August 28, 2007: Figure 4 PCP and PCPE Expression Histological sections of myocardial biopsy specimens from 1 patient with chronic heart failure. (A, C) Immunostaining for PCP and PCPE, respectively. These molecules were identified in brown and located mostly in areas of interstitial and perivascular fibrosis (arrows). Positive staining also was seen in cardiomyocytes (arrowheads). (B, D) Negative controls for the correspondent primary antibody omission. Magnification 100. Abbreviations as in Figure 1. treated patients with chronic HF as in furosemide-treated patients; 2) although the expression of both PCP zymogen and active form increased in chronic HF patients treated with furosemide, they did not change after treatment with torasemide; 3) the activation of PCP decreased in torasemide-treated patients, but remained unchanged in furosemide-treated patients; 4) the expression of 36-kDa PCPE fragment was reduced with treatment in chronic HF patients receiving torasemide, whereas it remained unchanged in patients receiving furosemide; and 5) no changes in the expression of full-length PCPE and the MMP-1/TIMP-1 balance were found in either group of chronic HF patients with treatment. Collectively, these findings generate the working hypothesis that the ability of torasemide to interfere with the myocardial PCP/PCPE system may be one contributing factor to its antifibrotic effect. Nevertheless, other mechanisms can also be involved in the antifibrotic effect of torasemide (e.g., its ability to interfere with the direct trophic actions of the profibrotic factor angiotensin II) (21). Although initially isolated from the medium of cultured fibroblasts (22), PCP has been further characterized in a number of cell types (6,7). In this regard, we found that in addition to its localization in the interstitial space and fibroblasts, PCP and PCPE are expressed in cardiomyocytes as found in endomyocardial biopsy tissue obtained from patients with chronic HF. We have shown recently that cardiomyocytes from chronic HF patients express MMP-1 Clinical Normotensive of Patients andwith Cardiac Subjects Chronic Parameters and Heart in the Failure Assessed Whole Group in Table 2 Clinical and Cardiac Parameters Assessed in Normotensive Subjects and in the Whole Group of Patients With Chronic Heart Failure Parameters Normotensive Patients HF Patients CVF, % * 1(I) mrna, AU * PCP zymogen, ADU 15, , * PCP active form, ADU 22, , PCP activation Full-length PCPE, ADU kDa fragment PCPE, ADU * PICP, g/l * MMP-1, ADU TIMP-1, ADU Data are expressed as the mean value SEM. *p 0.01 versus values in normotensive patients. 1(I) mrna 1 chain of procollagen type I messenger ribonucleic acid; ADU arbitrary densitometric units; AU arbitrary units; CVF collagen volume fraction; MMP matrix metalloproteinase; PCP procollagen type I carboxy-terminal proteinase; PCPE procollagen type I carboxy-terminal proteinase enhancer; PICP carboxy-terminal propeptide of procollagen type I; TIMP tissue inhibitor of matrix metalloproteinase.

7 JACC Vol. 50, No. 9, 2007 August 28, 2007: López et al. Fibrosis and Heart Failure 865 Effects Type HeartIFailure Synthesis of Treatment and Degradation Parameters Assessed Relatedin tothe Myocardial 2 GroupsFibrosis of Patients and Collagen With Chronic Table 3 Effects of Treatment on Parameters Related to Myocardial Fibrosis and Collagen Type I Synthesis and Degradation Assessed in the 2 Groups of Patients With Chronic Heart Failure Furosemide Group Torasemide Group Parameters Baseline Treatment Baseline Treatment CVF, % * 1(I) mrna, AU PCP zymogen, ADU 10, , , , PCP active form, ADU 21,616 2,106 25, , , Full-length PCPE, ADU kDa fragment PCPE, ADU PICP, g/l * MMP-1, ADU TIMP-1, ADU Data are expressed as the mean value SEM. *p 0.01 versus values at baseline in the same group. p 0.01 versus values after 8 months of randomization in the furosemide group. p Abbreviations as in Tables 1 and 2. and TIMP-1 that regulate collagen degradation (19). Therefore, because of the critical role that the PCP/PCPE system plays in collagen type I synthesis and deposition, it is likely that in addition to fibroblasts, cardiomyocytes regulate collagen turnover in the failing human heart. Activity of PCP can be regulated at 3 levels: 1) PCP zymogen synthesis; 2) activation of PCP zymogen into PCP active form; and 3) PCPE availability (8 10). In this regard, changes reported here in furosemide-treated patients at the level of PCP zymogen and active form would suggest that the activity of the enzyme increased in these patients. In support of this possibility, we found that despite a decrease in the availability of procollagen type I precursor, PICP and CVF did not change in furosemide-treated patients. On the other hand, changes found in patients receiving torasemide at the level of PCP activation and 36-kDa PCPE fragment would suggest that PCP activity diminished with treatment in these patients. This change in combination with a diminished availability of procollagen type I precursor can explain the associated decrease in PICP and CVF observed in torasemide-treated patients. Because transforming growth factor-beta-1 has been shown to stimulate the synthesis of both PCP zymogen and active form in fibrogenic cell cultures (23,24), it is tempting to speculate that this cytokine mediates up-regulation of myocardial PCP associated with furosemide treatment. However, it has been reported that myocardial transforming growth factor-beta-1 decreases in Dahl high-salt HF rats treated with furosemide (25). Thus, further studies are necessary to clarify the effects of this compound on myocardial PCP. Figure 5 Association Between Aldosterone and PCP Activation Positive correlation (y 0.004x 1.907) between plasma aldosterone and activation of myocardial PCP (as assessed by the ratio of PCP active form to PCP zymogen) in all patients with chronic heart failure. Abbreviations as in Figure 1. Figure 6 Association Between PICP and CVF Positive correlation (y 1.338x 18.73) between the reduction in serum PICP and the reduction in myocardial collagen volume fraction (CVF) in patients with chronic heart failure treated with torasemide. Abbreviations as in Figure 1.

8 866 López et al. JACC Vol. 50, No. 9, 2007 Fibrosis and Heart Failure August 28, 2007: Figure 7 Associations Among PCP Activation and CVF and PICP (A) Positive correlation (y 0.012x 27.48) between the reduction in PCP activation (as assessed by the ratio of PCP active form to PCP zymogen) and the reduction in myocardial CVF in patients with chronic heart failure treated with torasemide. (B) Positive correlation (y 0.483x 9.979) between the reduction in PCP activation (as assessed by the ratio of PCP active form to PCP zymogen) and the reduction in serum PICP in patients with chronic HF treated with torasemide. Abbreviations as in Figure 6. Aldosterone has been found to increase PCP activity and PCPE expression in cultured rat heart fibroblasts (26). In addition, it has been reported that aldosterone is involved in enhanced PCPE mrna expression seen in the remodeling rat myocardium after infarction (27). Interestingly, we found that a direct correlation exists between plasma aldosterone and PCP activation in chronic HF patients at baseline, suggesting that the mineralocorticoid may regulate PCP activity in humans. In this regard, it is important to remark that transcardiac extraction of aldosterone is reduced in chronic HF patients treated with torasemide (28) and that torasemide blocks the binding of the hormone to its mineralocorticoid receptor (29). Our finding that a direct correlation exists between final values of aldosterone and inhibition of PCP activation in torasemide-treated patients would suggest that the effects of torasemide on the myocardial PCP/PCPE system in chronic HF patients are related to this hormone. Chemical inhibitors of PCP activity that are nontoxic to cells in culture have been developed. These inhibitors are hydroxamic acid derivatives that bind specifically the active site of the zinc (Zn) atom of PCP and therefore inhibit enzyme activity (30). Of interest, whereas torasemide possess a Zn ligand residue, furosemide does not (31). Thus, this chemical difference may add another mechanism through which torasemide, but not furosemide, interferes with the PCP/PCPE system. Study limitations. This was a study involving a relatively small number of patients in whom diastolic function was not extensively investigated, but because of the nature of the goals under investigation, this design is appropriate. In addition, it must be recognized that therapy with angiotensinconverting enzyme inhibitors or angiotensin-receptor antagonists that inhibit collagen synthesis and reduce fibrosis may have influenced the findings. Nevertheless, because there is no available evidence showing that angiotensin II modifies the PCP/PCPE system, it is reasonable to assume that the effect of torasemide on collagen type I synthesis is exerted on top of the potential effects of angiotensin-converting enzyme inhibitors and angiotensin-receptor antagonists. Although no direct assessment of tissue PCP activity (i.e., zymography) was performed, serum PICP has been proposed as a reliable index of collagen type I synthesis within the human myocardium (32). Furthermore, the association reported here between PCP activation and serum PICP suggests that this peptide is a specific biomarker of the activity of myocardial PCP in patients with chronic HF. We have not studied the procollagen amino-proteinase that cleaves the amino-terminal propeptide of procollagen type I. However, impairment of amino-terminal propetide is not incompatible with in vivo fibrillogenesis (33). In addition, in vitro studies have shown that collagen monomers that retain the amino-propeptide are readily incorporated into growing fibrils along with normal monomers (34). In contrast, collagen type I monomers that retain PICP are not incorporated into growing fibrils (34). Because an excess of collagen type III deposition occurs in the myocardium of HF patients, and picrosirius red binds to collagen molecules other than type I, such as type III, we cannot exclude the possibility that the changes in myocardial collagen found in our patients also may be caused by changes in the deposition of fibril-forming collagen type III molecules not investigated here. In addition, changes in collagen cross-linking and in other components of the extracellular matrix (e.g., glycoproteins and proteoglycans) that have not been studied here also may be altered in the failing heart.

9 JACC Vol. 50, No. 9, 2007 August 28, 2007: López et al. Fibrosis and Heart Failure 867 Conclusions Our findings led us to hypothesize that the inhibition of the PCP/PCPE system may be one of the potential mechanisms involved in the ability of torasemide to repair myocardial fibrosis in chronic HF patients. Further studies must be conducted to identify the precise molecular pathways through which torasemide inhibits the system in these patients. Findings reported here set the stage for the development of antifibrotic cardiac therapies targeting the PCP/PCPE system. Acknowledgment The authors thank Sonia Martínez for her valuable technical assistance. Reprint requests and correspondence: Dr. Javier Díez, Área de Ciencias Cardiovasculares, CIMA, Avenida Pío XII 55, Pamplona, Spain. REFERENCES 1. Burlew BS, Weber KT. Connective tissue and the heart. Functional significance and regulatory mechanisms. Cardiol Clin 2000;18: Brower GL, Gardner JD, Forman MF, et al. 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