Heart 2001;86:701–708
`
`701
`
`1
`
`Oslo, Norway). The cells were incubated at
`37(cid:176)C in a humidified 5% carbon dioxide
`atmosphere, in the presence of phytohaemag-
`glutinin (10 µg/ml) and Phorbol 12-myristate
`13 acetate (1 ng/ml). The cells were har-
`vested at time 0 and 24 hours after stimula-
`tion for total RNA extraction. After RNA
`extraction TGF-♢
`1 gene expression was
`quantified by the PCR MIMIC method
`(Clontech, Palo Alto, California, USA).
`Clontech, primers sequences are: 5’ GCC
`CTG GAC ACC AAC TAT TGC T 3’ and 5’
`AG GCT CCA AAT GTA GGG GCA GG
`3’. The PCR product generated from
`MIMIC fragment has 270 bp while those
`from target cDNA has 161 bp, and were
`separated by 1.7–2% agarose gels and stained
`with ethidium bromide. Densitometry was
`performed and a standard curve was estab-
`lished by plotting the log value of the band
`intensity ratio of the MIMIC fragment and
`the target fragment with log MIMIC mol-
`ecule number. The initial target molecule
`number can be calculated and is equal to the
`MIMIC molecule number when the log band
`intensity ratio is zero. The value was normal-
`ised as cDNA molecule per cell or gram of
`tissue.
`The results showed that serum TGF-♢
`concentrations were higher in the dilated car-
`diomyopathy
`group
`(mean (SE)
`28.4
`(2.2) ng/ml) compared to normals (14.7
`(1.6) ng/ml; p = 0.0001, Mann Whitney
`test). The individual values are shown in fig 1.
`There was a significant increase in macro-
`phage TGF-♢
`1 mRNA in dilated cardio-
`myopathy patients compared to controls
`(18.9 (6.0) v 1.54 (0.5) · 10−3 molecules/cell;
`p = 0.006). Individual values are also shown
`in fig 1. There was no relation between
`TGF-♢
`and ejection fraction
`1 mRNA,
`(r = 0.027; p = 0.5), clinical course over the
`previous 6–24 months as assessed by the
`change
`in ejection fraction (r = 0.005;
`p = 0.77), or left ventricular diastolic func-
`tion (TGF-♢
`1 mRNA expression in those
`with a restrictive left ventricular filling
`pattern on echocardiography was the same as
`those without: 2.02 (0.29) and 2.05 (0.26),
`respectively).
`the
`In response to injury or disease,
`production of TGF-♢
`1 increases cell prolif-
`eration and ECM production is stimulated to
`repair and heal the tissue1. This is achieved
`through simultaneously stimulating the syn-
`thesis of ECM proteins, inhibiting the actions
`of proteases that degrade ECM, and increas-
`ing receptors on cell surfaces. Increased con-
`centrations of TGF-♢
`1 produced by injury,
`injecting TGF-♢
`1 or transferring the TGF-♢
`gene invariably leads to tissue fibrosis.
`TGF-♢
`1 may be especially important in
`dilated cardiomyopathy in which there is an
`overall
`increase in intramyocardial fibrillar
`collagen. Recently, Pauschinger presented
`results in 18 patients with dilated cardio-
`myopathy suggesting that the gene expression
`of collagen type III correlated with the gene
`expression of TGF-♢
`1 in myocardial biopsy
`specimens, although they did not identify the
`cell type responsible.5 However, ours is the
`first published report
`to demonstrate in
`patients with idiopathic dilated cardiomyopa-
`thy increased macrophage gene expression
`for TGF-♢
`1 associated with increased circu-
`lating concentrations.
`It is well documented in patients with idio-
`pathic dilated cardiomyopathy that cytotoxic
`lymphocytes
`and macrophages may be
`present in significant numbers within the
`myocardium, which is not apparent using
`
`1
`
`SCIENTIFIC
`LETTERS
`
`1
`
`Transforming growth factor-♢
`expression in dilated cardiomyopathy
`1 (TGF-♢
`Transforming growth factor-♢
`1) is a
`multifunctional cytokine that has an impor-
`tant role in the regulation of cell growth, dif-
`ferentiation, and repair in a variety of tissues.1
`In addition to its role in the cell cycle and
`apoptosis, TGF-♢
`1 induces the synthesis of
`extracellular matrix (ECM) and is upregu-
`lated by angiotensin II.1 2 Dilated cardio-
`myopathy is characterised by myocyte loss,
`hypertrophy of residual myocytes, increased
`interstitial fibrosis, and abnormalities of the
`cytoskeleton.3 Cytotoxic lymphocytes and
`macrophages are also present in the myocar-
`dium in increased numbers which may
`provide a source of TGF-♢
`1.4 Therefore in
`this study we have investigated whether
`patients with chronic heart failure caused by
`idiopathic dilated cardiomyopathy have in-
`creased plasma concentrations of TGF-♢
`1,
`and whether this is associated with increased
`
`macrophage gene expression for TGF-♢
`1
`compared to those with normal left ventricu-
`lar
`function. Patients with ischaemic or
`hypertensive heart disease were excluded
`because of the potential confounding eVects
`on TGF-♢
`1 concentrations of atherosclerosis
`and pressure overload with left ventricular
`hypertrophy.
`Twenty patients who presented with symp-
`toms and signs of chronic heart failure with
`no obvious cause and with the clinical
`diagnosis of dilated cardiomyopathy (by the
`World Health Organization criteria) were
`studied. All patients had right and left cardiac
`catheterisation with standard haemodynamic
`measurements, and coronary angiography
`was performed to exclude significant valvar,
`coronary artery disease, hypertensive heart
`failure, as well as constrictive pericardial dis-
`ease or restrictive cardiomyopathy before
`proceeding
`to right
`ventricular biopsy.
`Twenty age and sex matched healthy controls
`with no evidence of cardiac disease (all with
`normal echocardiograms) were also studied.
`Serum TGF-♢1 was assayed with an enzyme
`linked immunosorbent assay (ELISA) (R &
`D System, Minneapolis, USA) The intra- and
`interassay coeYcient of variation was < 15%.
`Peripheral blood mononuclear cells were iso-
`lated and were resuspended in medium for
`total cell counting. CD14 cells were isolated
`using dynabeads M-450 CD14 (Dynal, AS,
`
`DCM
`
`Normals
`
`p < 0.0001
`
`p = 0.0006
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`100
`
`Serum TGF-b1 (ng/ml)
`
`75
`
`50
`
`25
`
`Macrophage TGF-b1 mRNA
`
`(molecules/cell · 10–3)
`
`0
`
`DCM
`Figure 1 Scattergram showing individual values for serum TGF-♢
`1 concentrations (upper) and
`macrophage mRNA concentrations (lower). DCM, idiopathic dilated cardiomyopathy.
`
`Normals
`
`www.heartjnl.com
`
`Colibri Heart Valve LLC, Exhibit 2024, Page 1 of 8
`
`

`

`702
`
`Heart 2001;86:701–708
`
`light microscopy and requires electron micro-
`scopy or
`immunohistologic
`techniques.4
`Using special
`immunohistologic staining
`methods we have found that approximately
`40% of myocardial biopsy specimens have
`increased lymphocytes and macrophages,
`similar to the published results.4 However, we
`have not been able to detect any enteroviral
`genome in right ventricular endomyocardial
`biopsy specimens from this group of patients
`(unpublished data). Although macrophages
`are recognised to be important mediators of
`cardiac injury, repair, and TGF-♢
`1, there may
`be other cellular sources for the raised plasma
`TGF-♢
`1 from within the myocardium, and in
`situ hybridisation PCR studies are required
`to define precisely which particular cells are
`the source.
`In summary we have shown, for the first
`time, increased gene expression for TGF-♢
`1
`in macrophages from some patients with
`idiopathic dilated cardiomyopathy associated
`with increased plasma concentrations. Prob-
`ably this reflects the role of macrophages in
`tissue repair, remodelling and healing. But
`excessive production of TGF-♢
`1 may account
`for the increased collagen deposition found in
`dilated cardiomyopathy, which impairs ven-
`tricular compliance and diastolic function,
`and worsens heart failure.
`
`J E SANDERSON
`K B LAI
`I O L SHUM
`S WEI
`L T C CHOW*
`Division of Cardiology,
`Department of Medicine & Therapeutics,
`*Department of Anatomical & Cellular Pathology,
`The Chinese University of Hong Kong,
`Prince of Wales Hospital,
`Hong Kong SAR, China
`Correspondence to: Professor JE Sanderson, De-
`partment of Medicine & Therapeutics, The Chinese
`University of Hong Kong, 9/F Clinical Sciences
`Bldg, Prince of Wales Hospital, Shatin, NT, Hong
`Kong SAR, China; jesanderson@cuhk.edu.hk
`
`This study was supported by a grant from the Uni-
`versity Grants Committee, Hong Kong (no CUHK
`292/96M). We thank Ms Skiva Chan RN and Ms
`Leata Yeung RN for their help.
`
`1 Blobe GC, Schiemann WP, Lodish HF. Role of
`transforming growth factor ♢ in human disease.
`N Engl J Med 2000;342:1350–8.
`2 Kawano H, Do YS, Kawano Y, et al. Angio-
`tensin II has multiple profibrotic eVects in
`human cardiac fibroblasts. Circulation 2000;
`101:1130–7.
`3 Somma S Di, Marotta M, Salvatore G, et al.
`Changes in myocardial cytoskeletal intermedi-
`ated filaments and myocyte contractile dys-
`function in dilated cardiomyopathy: an in vivo
`study in humans. Heart 2000;84:659–67.
`4 Kuhl U, Seeberg B, Schultheiss H-P, et al.
`Immunohistological characterization of infil-
`trating lymphocytes in biopsies of patients with
`clinically suspected dilated cardiomyopathy.
`Eur Heart J 1994;15(suppl C):62–7.
`5 Pauschinger M, Knopf D, Petschauer S, et al.
`Dilated cardiomyopathy is associated with
`significant changes in collagen type I/III ratio.
`Circulation 1999;99:2750–6.
`
`Idiopathic dilated cardiomyopathy: lack
`of association with haemochromatosis
`gene in the CARDIGENE study
`
`gene
`The hereditary haemochromatosis
`(HFE) has recently been proposed as a
`disease modifying gene.1 The rationale is that
`two common mutations of the HFE gene
`(C282Y and H63D) are found in a majority
`of patients with genetic haemochromatosis
`who are either homozygotes (C282Y/C282Y)
`or compound heterozygotes (C282Y/H63D).
`
`Table 1 Distribution of genotypes for each polymorphism in cases and in control subjects
`
`Genotype
`
`+/+
`
`C282Y/+
`
`H63D/+
`
`C282Y/H63D
`
`Cases (n=418)
`Controls (n=372)
`
`246 (58.9 %)
`228 (61.3 %)
`
`36 (8.6 %)
`30 (8.1 %)
`
`127 (30.4 %)
`106 (28.5 %)
`
`9 (2.2 %)
`8 (2.2 %)
`
`Frequencies of genotypes are indicated in absolute numbers, and percentages in parenthesis.
`
`These mutations have been shown to con-
`tribute to more subtle modifications of iron
`homeostasis at the heterozygous state.2 In
`turn,
`iron may predispose to myocardial
`damage through the production of activated
`oxygen species. Recently, Mahon and col-
`leagues have reported an association be-
`tween the H63D mutation and idiopathic
`dilated cardiomyopathy (IDCM).3 In this
`study, 207 unrelated white patients with
`dilated cardiomyopathy and 200 controls
`were tested for HFE C282Y and H63D
`mutations. An increased proportion of
`H63D heterozygotes was
`found among
`patients (36%) as compared to the control
`group (27%). No association was found with
`C282Y mutation and as the H63D mutation
`had a relatively minor eVect on iron status,
`these authors proposed that this association
`may be unrelated to iron metabolism. Surber
`and colleagues reported a study with 161
`patients diagnosed with suspicion of myocar-
`ditis or IDCM where C282Y heterozygotes
`IDCM patients were significantly more
`frequent compared to controls.4
`We have determined the frequency of both
`HFE mutations in the CARDIGENE study,
`a case–control study of IDCM enrolled in 10
`diVerent hospitals in France (for details on
`the population see Tesson and colleagues5).
`There were 426 patients with IDCM (339
`men, 87 women). The mean (SD) age at
`diagnosis was 53 (10) years, and 229 patients
`had undergone cardiac transplantation. Pa-
`tients with chronic excess alcohol consump-
`tion were not excluded. The study control
`group consisted of 401 subjects free of
`cardiovascular disease selected from the
`MONICA (monitoring trends and determi-
`nants
`in cardiovascular disease) project
`in France and matched for sex and age
`(329 men and 72 women, mean age 46 (8)
`years).
`HFE C282Y and H63D mutations were
`tested by polymerase chain reaction (PCR)
`and allele-specific detection.
`Amplification was performed in a 96 well
`microtitration plate as previously described6;
`200 ng of genomic DNA were amplified in a
`total volume of 25 µl containing 20 pmol of
`5’ and 3’ primers, 0.2 mM dNTPs, 6 mM
`MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH
`8.3), 5 pmol of each allele-specific molecular
`Beacon, and 0.75 U of AmpliTaq Gold DNA
`polymerase (ABI, France). The enzyme was
`heat activated at 96(cid:176)C for 10 minutes
`followed by 40 cycles of denaturation at
`95(cid:176)C for 20 seconds, annealing at 55 (cid:176)C for
`20 seconds and extension at 72(cid:176)C for 20
`seconds in UNO-Thermoblock (Biometra,
`Göttingen, Germany). After a final denatura-
`tion at 95(cid:176)C for two minutes, hybridisation
`with the probes was carried out at 55(cid:176)C for
`five minutes. The emission of fluorescence
`was recorded in a plate fluorometer fluostar
`(BMG, Germany) in two wavelength sys-
`tems: 480–520 nm for fluorescein (FAM)
`and
`520–590
`for
`tetramethylrhodamine
`(TAMRA).
`Primers and allele-specific probes were
`synthesised
`by Eurogentech
`(Seraing,
`Belgium):
`
`www.heartjnl.com
`
`C282Y:
`forward primer: CTGTACCCCCTGGG-
`GAAGAGCAGAG
`reverse primer: CCCAGATCACAATGAG-
`GGGCTGATC
`mutated probe: FAM- gcgac CCACCTGG-
`TACGTATAT gtcgc –DABC YL
`normal probe: TAMRA- gcgac CCACCT-
`GGCACGTATAT gtcgc –DABCYL
`H63D:
`forward primer: GCTTTGGGCTACGT-
`GGATGACCAGC
`reverse primer: CCATGGAGTTCGGGG-
`CTCCACAC
`mutated probe: FAM- gcgac TTCT ATGAT-
`GATGAGAGTC gtcgc –DABCYL
`normal probe: TAMRA- gcgac TTCTAT-
`GATCATGAGAGTC gtcgc –DABCYL
`DABCYL:
`4-4’[dimethylaminophenylazo]
`benzoid acid.
`Data were analysed using the SAS statisti-
`cal software (SAS Institute Inc, Cary, North
`Carolina, USA). Hardy-Weinberg equilib-
`rium was tested by a ♻2 test with 1 degree of
`freedom (df). Allele frequency was calculated
`by gene counting. The association of each
`polymorphism with the disease was tested by
`a ♻2 test comparing cases and controls with 2
`df for H63D and 1 df for C282Y since, owing
`to low numbers, we pooled homozygotes and
`heterozygotes for the rare allele.
`The association with variables characteris-
`ing the severity of disease was tested similarly
`by comparing cases below and above the
`median of ejection fraction (median = 24)
`and left ventricular dilation (median = 40),
`and cases with and without cardiac transplan-
`tation.
`Genotypes of both H63D and C282Y
`mutations could be determined in 418 cases
`and 372 controls. There was no significant
`deviation from Hardy-Weinberg equilibrium
`in controls as well as in cases. We did not find
`any association between the disease and any
`of the two HFE mutations (table 1). The
`odds ratio (95% confidence interval (CI)) for
`Y282 and D63 carrying were 0.94 (95% CI
`0.58 to 1.52) and 0.90 (95% CI 0.66 to
`1.23), respectively. Allele frequencies were
`similar in controls and patients (Y282: 0.051
`and 0.055, D63: 0.17 and 0.18, respectively).
`Within the patient group,
`there was no
`relation between genotype and the severity of
`the disease, assessed by ejection fraction, left
`ventricular dilation, or cardiac transplanta-
`tion.
`Our results on a large and well character-
`ised population do not confirm the implica-
`tion of these two common mutations in the
`HFE gene as genetically predisposing factors
`in IDCM. These contradictory results may
`result from diVerent factors including sample
`size, diVerent genetic background, selection
`criteria including degree of cardiac dilatation
`or ejection fraction reduction, or bias in the
`recruitment of the control population. These
`conflicting results point out the need for large
`populations in the search for genes suscepti-
`ble to IDCM. Future studies should therefore
`be undertaken with clearly defined subgroups
`
`Colibri Heart Valve LLC, Exhibit 2024, Page 2 of 8
`
`

`

`Heart 2001;86:701–708
`
`in order to determine the modulating influ-
`ence of HFE mutations in dilated cardiomy-
`opathy.
`
`G HETET1
`B GRANDCHAMP1
`C BOUCHIER2 3
`V NICAUD3 4
`L TIRET3 4
`G ROIZÈS5
`M DESNOS6
`K SCHWARTZ7
`R DORENT8
`M KOMAJDA2 3 9
`FOR THE CARDIGENE GROUP
`1INSERM U409, Association Claude Bernard,
`Université Paris VII, Paris
`2Laboratoire Génétique et InsuYsance Cardiaque,
`Association Claude Bernard/Université Paris VI,
`Groupe hospitalier Pitié-Salpêtrière, Paris
`3IFR 14 “Coeur, Muscles et Vaisseaux”, Paris
`4INSERM U525, Paris
`5INSERM U249, Montpellier
`6Service de Cardiologie,
`Hôpital Européen G Pompidou, Paris
`7INSERM U523, Paris
`8Service de Chirurgie Cardiaque,
`9Service de Cardiologie,
`Groupe hospitalier Pitié-Salpêtrière, Paris,
`France
`Correspondence to: Dr C Bouchier, Laboratoire
`Génétique et
`InsuYsance Cardiaque, Pavillon
`Rambuteau, Groupe hospitalier Pitié-Salpêtrière,
`47 bd de l’Hôpital, 75651 Paris cedex 13, France;
`bouchier@chups.jussieu.fr
`
`1 Feder JN, Gnirke A, Thomas W, et al. A novel
`MHC class I-like gene is mutated in patients
`with hereditary haemochromatosis. Nat Genet
`1996;13:399–408.
`2 Whitfield JB, Cullen LM, Jazwinska EC, et al.
`EVects of HFE C282Y and H63D polymor-
`phisms and polygenic background on iron
`stores in a large community sample of twins.
`Am J Hum Genet 2000;66:1246–58.
`3 Mahon NG, Coonar AS,
`JeVery S, et al.
`Haemochromatosis gene mutations in idio-
`pathic dilated cardiomyopathy. Heart. 2000;84:
`541–7.
`4 Surber R, Sigusch HH, HoVmann A, et al. Idio-
`pathic dilated cardiomyopathy: association
`with the hereditary haemochromatosis gene.
`Eur Heart J 2000;21:147.
`5 Tesson F, Charron P, Peuchmaurd M, et al.
`Characterization of a unique genetic variant in
`the beta1-adrenoceptor gene and evaluation of
`its role in idiopathic dilated cardiomyopathy.
`CARDIGENE Group. J Mol Cell Cardiol.
`1999;31:1025–32.
`6 Hetet G, Elbaz A, Gariepy J, et al. Association
`studies between hemochromatosis gene muta-
`tions and the risk of cardiovascular diseases.
`Eur J Clin Invest 2001;30:1–8.
`
`EVects of cardiac resynchronisation on
`maximal and submaximal exercise
`performance in advanced heart failure
`patients with conduction abnormality
`
`Atriobiventricular pacing (cardiac resynchro-
`nisation) is a promising treatment for patients
`with advanced heart failure and conduction
`abnormality.1 These patients have severely
`impaired exercise tolerance and this has been
`shown to be multifactorial. The present study
`examined,
`in detail, the eVect of cardiac
`resynchronisation on maximal and submaxi-
`mal exercise capacity as evaluated by meta-
`bolic exercise testing.
`
`A consecutive series of patients from a sin-
`gle centre with cardiac resynchronisation
`therapy, able to perform baseline and follow
`up treadmill tests, were enrolled in the study.
`Inclusion criteria were New York Heart
`Association (NYHA) functional class III/IV
`heart failure on maximal medical treatment,
`left ventricular ejection fraction < 35%, left
`ventricular
`end
`diastolic
`dimension
`> 60 mm, and QRS duration > 130 ms.
`Patients with chronic atrial fibrillation were
`excluded. The protocol was approved by the
`hospital research and ethics committee and
`all subjects gave written informed consent.
`Left ventricular
`(LV)
`stimulation was
`accomplished by a lead inserted into an LV
`branch of
`the coronary sinus. Specially
`designed leads were used (Medtronic 2187 or
`2188, Medtronic Inc, Minneapolis, Minne-
`sota, USA). Right atrial and ventricular leads
`were positioned in the right atrial appendage
`and right ventricular apex, respectively. All
`patients received cardiac resynchronisation
`devices (InSync, Medtronic Inc) with one
`atrial and two ventricular ports. Optimal
`atrioventricular delays were
`individually
`determined by analysing transmitral flow
`using Doppler echocardiography.
`Exercise testing was performed before and
`1–3 months after
`implantation. Patients
`underwent symptom limited maximal breath-
`by-breath cardiopulmonary exercise testing
`at baseline and follow up using the Modified
`Naughton protocol with continuous electro-
`cardiographic recording. Oxygen consump-
`tion (V~ O2, ml/min), carbon dioxide produc-
`tion (V~ CO2, ml/min) and minute ventilation
`(V~E,
`l/min) were continuously measured
`using an automated breath by breath system
`(Med Graphics CPX-D, Medical Graphics
`Co, St Paul, Minnesota, USA). Anaerobic
`threshold was determined by the V slope
`method and confirmed by the ventilatory
`equivalence method. The patients were all
`encouraged to exercise until the respiratory
`exchange ratio (that is,V~ CO2/V~ O2) was greater
`than 1.0 which is a good indication that near
`maximal eVort has been reached. Maximal
`exercise capacity was assessed by exercise
`duration and peak oxygen consumption
`(peak V~ O2). Submaximal exercise capacity
`was assessed in three ways: by measuring
`oxygen consumption at anaerobic threshold
`(V~ O2@AT)2; by examining continuous oxygen
`uptake using the oxygen uptake eYciency
`slope (OUES)3; and by measuring the
`respiratory response to exercise (V~E/V~ CO2
`slope).4
`We studied 15 patients (13 males and two
`females); 14 had NYHA class III and one had
`class IV heart failure. The aetiology was
`ischaemic in 10 patients, valvar in two, and
`idiopathic in three. Mean (SD) age was 66.2
`(9.6) years. Baseline ejection fraction was
`23.6 (7.5)%, left ventricular end diastolic
`dimension was 71.9 (9.5) mm, QRS duration
`was 169.3 (27.6) ms, and PR interval was
`226.1 (44.8) ms. Fourteen patients had left
`bundle branch block and one had a pre-
`existing standard dual chamber pacemaker.
`
`Table 1 Comparison of exercise parameters before and after cardiac resynchronisation (n=15)
`
`Baseline
`
`With CR
`
`% Change
`
`p Value
`
`382.27 (170.90)
`467.87 (177.25)
`+22.4
`0.04
`Exercise duration (sec)
`Peak V~ O2 (ml/min/kg)
`14.31 (3.41)
`16.02 (4.01)
`+11.9
`0.03
`V~ O2@AT (ml/min/kg)
`10.0 (1.8)
`10.8 (1.6)
`+8.0
`0.13
`517.59 (148.15)
`611.97 (210.25)
`+18.2
`0.02
`OUES
`V~E/V~ CO2 slope
`40.53 (4.26)
`37.13 (5.08)
`−8.4
`0.02
`CR, cardiac resynchronisation; OUES, oxygen uptake eYciency slope; V~ O2, oxygen consumption; V~ CO2, car-
`bon dioxide production; V~E, minute ventilation.
`
`www.heartjnl.com
`
`703
`
`reduced to 148.1
`QRS duration was
`(13.5) ms after atriobiventricular pacing. The
`reduction in QRS duration was 21.1
`(22.4) ms.
`All 15 patients completed the metabolic
`exercise test at baseline and at follow up.
`Anaerobic threshold was identifiable in all
`exercise tests. All patients reached a respira-
`tory exchange ratio of > 1.0 with a mean
`(SD) of 1.17 (0.09) at baseline. At follow up,
`the respiratory exchange ratio was 1.17
`(0.12); only one patient had a respiratory
`exchange ratio of < 1.0.
`The exercise results are listed in table 1.
`Both maximal parameters and two out of
`three submaximal parameters improved after
`pacing. There was no significant correlation
`between baseline QRS or QRS width reduc-
`tion and any of the parameters.
`Maximal exercise capacity as assessed by
`peak V~ O2 is a strong independent predictor in
`a broad range of heart failure patients. Indeed
`peak V~ O2 is widely accepted as the best single
`measure of prognosis in ambulatory patients
`with severe heart failure.5 However, peak V~ O2
`is poorly correlated with everyday physical
`activity which is largely a series of submaxi-
`mal exercises. This may explain why there is a
`poor correlation between self reported exer-
`tional symptoms and maximal exercise ca-
`pacity. In this regard, submaximal exercise
`parameters may provide better estimation of
`functional capacity of daily living.
`Oxygen consumption at anaerobic thresh-
`old (V~ O2@AT) has been used as an estimate
`of
`submaximal capacity in heart
`failure
`patients.2 There was a small increase in this
`variable from 10.0 (1.8) ml/min/kg before to
`10.8 (1.6) ml/min/kg with cardiac resynchro-
`nisation, although this did not reach signifi-
`cance (p = 0.13). However V~ O2@AT has rec-
`ognised limitations.2
`The kinetics of oxygen consumption at the
`start and end of exercise are delayed in
`patients with heart
`failure. Therefore an
`index of oxygen kinetics before anaerobic
`threshold might better reflect submaximal
`exercise capacity and thus patient’s exertional
`symptoms. The OUES is one such index and
`was first proposed and validated as a new
`index of submaximal cardiorespiratory re-
`serve by Baba and colleagues in 1996.3 Also,
`OUES is eVort independent and thus less
`likely to be aVected by subjective factors
`including training eVects or intra-observer
`variability. For these reasons, OUES may be
`the most valid assessment of submaximal
`exercise capacity. In this study OUES showed
`a major improvement from 517.6 (148.2) at
`baseline to 612.0 (210.2) with cardiac resyn-
`chronisation (p = 0.02).
`Respiratory eYciency (V~E/V~ CO2 slope) is
`also a measure of
`submaximal exercise
`capacity. Heart failure patients often have an
`excessive ventilatory response to exercise4
`and this can be quantified by analysing the
`relation between carbon dioxide output
`(V~ CO2) and minute ventilation (V~E). In our
`study there was a clear and significant
`improvement in this variable with cardiac
`resynchronisation (41.8 (7.1) before v 38.1
`(9.0) after, p = 0.04).
`In conclusion, this is the first detailed
`analysis of the eVects of cardiac resynchroni-
`sation on exercise performance and our
`results show that atriobiventricular pacing
`significantly improved maximal and submaxi-
`mal exercise capacity. The study has signifi-
`cant limitations in being small and uncon-
`trolled; however, larger randomised studies
`are underway to investigate whether these
`
`Colibri Heart Valve LLC, Exhibit 2024, Page 3 of 8
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`

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`704
`
`Heart 2001;86:701–708
`
`12 24 36 48 60 72 84 96 108
`
`120
`
`12 24 36 48 60 72 84 96 108
`
`120
`
`120
`12 24 36 48 60 72 84 96 108
`Hours after onset of chest pain
`
`A
`
`B
`
`20
`
`10
`
`0
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`C
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`IL-10 concentration (pg/ml)
`
`IL-6 concentration (pg/ml)
`
`IL-12 concentration (pg/ml)
`
`Figure 1 Time course of interleukin (IL)-10
`(A), IL-6 (B), and IL-12 (C) in 20 patients
`with acute myocardial infarction. Data are
`mean (SD). *p < 0.01 compared with mean
`value at 12 hours after onset of chest pain.
`
`IL-10 decrease. No correlation between
`IL-10 and IL-12 was identified.
`In conclusion, our study shows reciprocal
`elevation of circulating IL-10 and IL-6
`following AMI. Further study is required to
`reveal
`the physiological role of IL-10 in
`patients with AMI.
`
`NOBUO KOTAJIMA*
`TAKAO KIMURA*†
`TSUGIYASU KANDA‡
`ATSUSHI KUWABARA*†
`YUKIHITO FUKUMURA*
`MASAMI MURAKAMI*†
`ISAO KOBAYASHI*†
`*Clinical Laboratory Center,
`†Department of Laboratory Medicine,
`‡Department of General Medicine,
`Gunma University School of Medicine,
`3-39-15 Showa-machi, Maebashi,
`Gunma 371-8511, Japan
`Correspondence to: Takao Kimura;
`tkimura@med.gunma-u.ac.jp
`
`1 Shibata M, Endo S, Inada K, et al. Elevated
`plasma levels of interleukin-1 receptor antago-
`nist and interleukin-10 in patients with acute
`myocardial infarction. J Interferon Cytokine Res
`1997;17:145–50.
`2 Yang Z, Zingarelli B, Szabo C. Crucial role of
`endogenous interleukin-10 production in myo-
`cardial
`inschemia/reperfusion injury. Circula-
`tion 2000;101:1019–26.
`3 Frangogiannis NG, Mendoza LH, Lindsey ML,
`et al. IL-10 is induced in the reperfused
`myocardium and may modulate the reaction to
`injury. J Immunol 2000;165:2798–808.
`4 Kanda T, Inoue M, Kotajima N, et al. Circulat-
`ing interleukin-6 and interleukin-6 receptors in
`patients with acute and recent myocardial
`infarction. Cardiology 2000;93:191–6.
`5 Kaneko K, Kanda T, Yokoyama T, et al. Expres-
`sion of
`interleukin-6 in the ventricles and
`coronary arteries of patients with myocardial
`infarction. Res Commun Mol Pathol Pharmacol
`1997;97:3–12.
`
`positive eVects of cardiac resynchronisation
`on exercise performance are real and can be
`translated into an impact on mortality.
`D BIRNIE
`L P SOUCIE
`S SMITH
`A S L TANG
`Ottawa Heart Institute,
`40 Ruskin Road,
`Ottawa, Ontario K1Y 4W7
`Canada
`
`Correspondence to: Dr Birnie;
`davidbirnie@yahoo.ca
`
`We are grateful for the assistance of our research
`nurses Patricia Theorot-Patrick and Clare Carey.
`
`1 Gras D, Mabo P, Tang T et al. Multisite pacing
`as a supplemental treatment of congestive heart
`failure: preliminary results of the Medtronic
`InSync study. Pacing Clin Electrophysiol 1998;
`21:2249–55.
`2 De Groote P, Millaire A, Decoulx E, et al. Kinetics
`of oxygen consumption during and after exercise
`in patinets with dilated cardiomyopathy. New
`markers of exercise intolerance with clinical
`implications. J Am Coll Cardiol 1996;28:168–75.
`3 Baba R, Nagashima M, Masahike G, et al. Oxy-
`gen uptake eYciency slope: a new index of car-
`diorespiratory functional reserve derived from
`the relation between oxygen uptake and minute
`ventilation during incremental exercise. J Am
`Coll Cardiol 1996;28:1567–72.
`4 Francis DP, Shamim W, Davies LC, et al.
`Cardiopulmonary exercise testing for prognosis
`in chronic heart failure: continuous and inde-
`pendent prognostic value from VE/VCO2 slope
`and peak VO2. Eur Heart J 2000;21:154–61
`5 Mancini D, LeJemtel T, Aaronson K. Peak VO2:
`a simple yet enduring standard. Circ 2000;101:
`1080–2.
`
`Reciprocal increase of circulating
`interleukin-10 and interleukin-6 in
`patients with acute myocardial
`infarction
`
`Increased circulating interleukin (IL)-10 is
`reported in patients with acute myocardial inf-
`arction (AMI)1 and in mice with myocardial
`ischaemia/reperfusion2;
`induction of
`IL-10
`mRNA in ischaemic/reperfused myocardium3
`was
`also
`recently
`reported.
`Ischaemic/
`reperfused myocardium is protected by IL-10
`through inhibition of tumour necrosis factor ♡
`production2 and down regulation of
`IL-6
`mRNA in a canine model.3 We have already
`shown induction of circulating IL-6, and a
`positive relation between natriuretic peptides in
`AMI.4 We also showed IL-6 expression in
`ischaemic myocardium5; however, no study has
`investigated circulating concentrations of IL-6
`and IL-10 at the same time. Increased IL-10
`inhibited IL-12 induction in an animal model,3
`while circulating IL-12 has not been investi-
`gated in patients with AMI. In this study we
`show a reciprocal increase in circulating IL-10
`and IL-6, but not IL-12, in patients with AMI.
`Twenty patients admitted to Fujioka Hospi-
`tal within six hours of the onset of AMI symp-
`toms, and 20 healthy controls, were enrolled
`after
`informed consent was obtained. In
`patients with AMI,
`informed consent was
`obtained at admission; all patients immediately
`underwent percutaneous transluminal coron-
`ary angioplasty (PTCA). The 20 patients
`comprised 16 men and 4 women aged 45–69
`years, and the 20 controls were 16 men and 4
`women aged 45–65 years. No subjects had
`collagen diseases, liver disease, renal failure,
`malignancy, infection, autoimmune disease, or
`thyroid disease. The diagnosis of AMI was
`based on chest pain resistant
`to glyceryl
`trinitrate, electrocardiographic ST segment
`elevations in more than two leads with or with-
`out Q wave formation, and significant in-
`creases in plasma creatine phosphokinase
`
`(CK) and lactate dehydrogenase (LDH). The
`MB isoenzyme of CK, a relatively specific
`indicator of myocardial damage, was elevated
`by > 5% in all 20 patients. Blood specimens
`were drawn from the femoral vein on admis-
`sion and every 12 hours until 120 hours after
`admission. Plasma was centrifuged for 15
`minutes at 3000 rpm at 4ºC and stored at
`−80ºC until assayed. IL-6 was measured with
`a chemiluminescent enzyme immunoassay kit
`(Fujirebio, Tokyo, Japan). IL-10 and IL-12
`were measured by another chemiluminescent
`enzyme immunoassay kit (BioSource Inter-
`national Inc, Camarillo, California, USA). CK
`and LDH were measured by autoanalyser.
`Atrial natriuretic peptide (ANP) and brain
`natriuretic peptide (BNP) were measured by
`radioimmunoassay. Data are presented as
`mean (SD). Statistical analysis was performed
`using unpaired t test, analysis of variance
`(ANOVA), or linear regression. A probability
`level of p < 0.05 was considered significant.
`Mean (SD) concentrations of IL-6, IL-10,
`and IL-12 in controls were 0.7 (0.5), 3.4
`(1.3), and 53.9 (16.1) pg/ml, respectively,
`and in patients with AMI were 18.5 (8.5),
`14.9 (6.2), and 55.3 (14.9) pg/ml, respec-
`tively. Mean concentrations of plasma IL-6
`and IL-10 were significantly increased in
`patients with AMI compared with controls,
`and IL-10 reached a peak concentration
`within 24 hours after onset of chest pain (fig
`1A). A prolonged increase in IL-6 concentra-
`tion was detected, showing two peaks after
`IL-10 decreased (fig 1B). Circulating IL-12
`did not diVer from controls throughout the
`study (fig 1C). Peak concentrations of IL-10
`were positively correlated with both ANP and
`BNP
`(p < 0.01,
`p < 0.01,
`R2 = 0.41;
`R2 = 0.36, respectively, data not shown). The
`first peak of IL-6 in AMI was correlated with
`ANP and BNP as previously reported (data
`not shown).4 Mean concentrations of IL-10
`were significantly higher in the 11 patients
`with severe AMI (16.8 (6.8) pg/ml, Killip
`class II, III, or IV, group B) than in the nine
`patients with uncomplicated AMI
`(7.4
`(3.3) pg/ml, Killip class I, group A). Neither
`IL-10 nor IL-12 correlated with CK or LDH,
`while IL-6 positively correlated with CK and
`LDH (data not
`shown). No significant
`relation was detected between IL-6 and
`IL-10, between IL-6 and IL-12, and between
`IL-10 and IL-12 in patients with AMI.
`In this study we have shown reciprocal
`elevation of IL-10 and IL-6 in patients with
`AMI. A recent study showed that IL-10
`induction and down regulation of IL-6 in
`myocardium required reperfusion after is-
`chaemia, while ischaemia without reperfusion
`induced prolonged IL-6 mRNA expression.3
`In our subjects both IL-1