MD Pietro Di Pasquale

MD Sergio Cannizzaro

MD Sergio Fasullo

MD Filippo Ganci

MD Giorgio Marenghini

MD Sebastiano Scalzo

MD Francesco Giambanco

MD Giuseppe Vitale

MD Giovanni Polizzi

MD Salvatore Paterna

Paolo Borsellino

Combination of indomethacin and statin compared with indomethacin and placebo in patients with first episode of acute pericarditis: preliminary findings

Keywords: Indomethacin, Acute Pericarditis, Rosuvastatine. Clinical Science Immediate

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Acute pericarditis is a common and frequent disease to be considered in the differential diagnosis of chest pain. Most patients with acute pericarditis have either viral or idiopathic pericarditis. In many cases, acute pericarditis has a brief and benign course after empirical treatment with non-steroidal anti-inflammatory drugs (NSAIDs). A viral aetiology is often presumed but evidence for this is not often sought due to the expense involved and the time required before the results of laboratory tests are available. The aims of treatment are to relieve symptoms, and most patients are hospitalized for complete diagnosis and observation of complications, particularly pericardial effusion and cardiac tamponade [START_REF]Heart Disease: A Textbook of Cardiovascular Medicine[END_REF][START_REF] Spodick | Acute pericarditis: current concepts and practice[END_REF]. Although as many as 90% of isolated cases of acute pericarditis are idiopathic or viral, the list of other potential causes is extensive. The mainstay of therapy is NSAIDs and, recently, colchicine has also shown promise in the treatment of recurrent pericarditis [START_REF] Adler | Colchicine treatment for recurrent pericarditis: a decade of experience[END_REF]. Indomethacin and other NSAIDs can inhibit the activity of COX, a property which accounts for their shared therapeutic effects [START_REF] Vane | Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs[END_REF]. Recent studies have shown that it is possible to modulate Creactive protein (CRP) levels with pharmacological intervention, including HMG-CoA reductase inhibitor drugs like statins [START_REF] Ridker | Long-term effects of pravastatin on plasma concentration of C-reactive protein: the Cholesterol and Recurrent Events (CARE) Investigators[END_REF][START_REF] Ridker | Rapid reduction in C-reactive protein with cerivastatin among 785 patients with primary hypercholesterolemia[END_REF][START_REF] Strandberg | Effect of statins on C-reactive protein in patients with coronary artery disease[END_REF]). The latter reduce cardiovascular risk also by diminishing arterial inflammation, as suggested by a reduction in serum CRP levels; this appears to be independent of a reduction in LDL cholesterol levels [START_REF] Ridker | Measurement of C-reactive protein for the targeting of statin therapy in the primary prevention of acute coronary events[END_REF][START_REF] Jialal | Effect of hydroxymethyl glutaryl CoA reductase inhibitor therapy on high sensitive C-reactive protein levels[END_REF]. On the basis of these data a pilot study was carried out to evaluate the safety and feasibility of the indomethacin/statin combination, and if this should prove to be more effective than indomethacin plus placebo in patients with a first episode of acute pericarditis. Our endpoints were: 1) feasibility and safety; and 2) effects on clinical inflammation markers, pericardial effusion and ST-segment evolution.

MATERIALS AND METHODS

The present study was performed in accordance with the declaration of Helsinki, and written informed consent was obtained from each patient before starting the study. The protocol was approved by the local ethical committee.

Population. 55 consecutive patients, hospitalized for a first episode of acute pericarditis at G.F. Ingrassia Hospital, Palermo, Italy, were studied prospectively between January 2005 and December 2006.

Inclusion criteria: age > 18 years, prodrome of fever, malaise, and myalgia, retrosternal or left precordial chest pain and shortness of breath, rapid and regular heart rate, pericardial friction rub, elevated white-cell count > 10,0 x 10 L, elevated erythrocyte sedimentation rate (ESR) >35 mm/h, high values of serum CRP (normal value <05 mg/dl), plasma troponin I (TNI) > 1.0 pg/ml), serum creatin kinase (CK) > 80 IU/l) and its MB fraction concentration [START_REF]Heart Disease: A Textbook of Cardiovascular Medicine[END_REF][START_REF] Spodick | Acute pericarditis: current concepts and practice[END_REF][START_REF] Permanyer-Miralda | Primary acute pericardial disease: a prospective series of 231 consecutive patients[END_REF][START_REF] Imazio | Cardiac troponin I in acute pericarditis[END_REF], classic ECG alterations (widespread upward concave ST-segment elevation and PR-segment depression), and echocardiographic evidence of a small pericardial effusion on admission (an echo-free space, anterior plus posterior, of <10 mm during diastole) [START_REF] Weitzman | The incidence and natural history of pericardial effusion after cardiac surgery: an echocardiographic study[END_REF]. As this was a pilot study, only patients with small pericardial effusion were included. Acute pericarditis was diagnosed when at least 4 of the following criteria were present: pericarditic chest pain, pericardial friction rub, widespread ST- Exclusion criteria: patients with infections, kidney failure, metastatic disease, those receiving medications, and radiation therapy, history of recent heart attack (myocardial infarction), rheumatoid arthritis and lupus, tuberculosis and AIDS, recent chest trauma, and receiving antiflammatory drugs before hospitalization, statin etc. Considering that most cases probably had a viral or idiopathic aetiology, with a possible brief and benign course after NSAIDs, we intentionally avoided performing an initial full diagnostic evaluation in all cases.

Study protocol and follow up: Just after admission, the suitable patients were double-blind randomized into two groups: one group received indomethacin 150 mg daily plus rosuvastatin 10 mg daily; the other group received indomethacin 150 mg daily plus placebo. Both groups received the treatments (rosuvastatin plus indomethacin or placebo plus indomethacin) up to the normalization of inflammation markers (CRP <0.5 pg/ml, WC <7.0 x 10 L, ESR < 20 mm/h) and for the whole following week. After baseline clinical and laboratory assessments, the patients were evaluated daily with a complete physical examination, a careful symptom check , blood pressure (BP) and heart rate (HR); blood samples were drawn daily to determine WCc, ESR, CRP, TNI, CK/MB (Dade Behring Diagnostics), pro-NT-BNP plasma levels (Roche), serum urea and creatinine. All laboratory measurements were made in a core laboratory and a validated assay for high-sensitive CRP was used (Dade Behring Co., Marburg, Germany). Chest-x ray was performed on admission and at discharge, while ECG and echocardiogram were performed daily up to discharge in order to control pericardial effusion. After discharge, the patients were followed as outpatients every week for the 1st month and every two weeks for the subsequent two months to evaluate a recurrence of pericarditis. Clinical evaluation, ECG, echocardiographic and laboratory assessments were performed at every follow up by two blinded physicians to evaluate if recurrence of pericarditis had occurred. No other anti-inflammatory drug was added or used during the study period. The side effects of indomethacin were recorded and gastroprotection with omeprazole (20 mg/day) was also administered..

Statistical Analysis

Continuous data are reported as the mean value ± SD and compared, using the unpaired t test and variance analysis (ANOVA) for repeated measures with Bonferroni correction for intragroup data. Categorical variables are expressed as proportions or percentages and compared using chi-square analysis. A value of p < 0.05 was considered to show statistical significance. Pericardial friction rub was present in 45 cases and small pericardial effusion was present in 47 patients. The treatment was effective in all the 55 cases and no patient displayed initial treatment failure. The treatment was well tolerated without serious drug side effects. We recorded only one minor side effect, including dyspepsia, abdominal pain, and gastritis (from the statin group). After a mean follow-up of 3 months, no recurrence of pericarditis and any rebound effect after discontinuing the statin therapy was observed. The two groups were similar by clinical characteristics, age, sex and laboratory parameters (Table 1). The statin group showed a significant faster reduction/normalization in CRP values (5.0+1 vs 6.0+2 days, p=0.022); in ST segment normalization (3.5+1 vs 4.5+1 days, p=0.001); pericardial effusion (4.5+1 vs 5.5+1 days, p=0.001); ESR (5+1vs 6+2 days; p=0.022), while WCc (3.0+2 vs 4.0+2 days) and hospitalization time (5.5+2 versus 6.5+2 days) showed a not significant but favourable trend, p=0.069 (Table 2). These data were calculated when the patients were completely free of symptoms and, in the same time period ESR, WCc fell in the normal range (<20 mm/h and <7.0 x 10 L respectively). This moment was also chosen to analyze the CRP values. TNI and CK/MB in patients presenting with increased levels at entry did not show any differences in normalization time and plasma levels during the study period (Table 2). The absence of effects of the combined treatment on TNI and CK/MB was probably due to the kinetic of myocardial enzymes, which usually depends on baseline values and which represents myocardial damage already present at entry. Pro-NT-BNP (Roche) values showed normal values (< 125 pg/ml) either on admission or during the study period. Table 3 shows the daily variations in plasma inflammation markers (WCc, ERS, CRP) during hospitalization. The statin group displayed earlier changes in CRP, WCc and ESR in comparison with the placebo group. These results are in agreement with the faster favourable clinical evolution, as reported by an analysis of the ST-segment and pericardial effusion resolution.

RESULTS

All

DISCUSSION

This study points to the safety of a combined indomethacin/statin therapy. Moreover, this combination, determining an earlier normalization of clinical and laboratory inflammatory markers in patients with a first episode of acute pericarditis, was also observed. To date, this is the first time that a combination of NSAID and statin has been used in the setting of such patients. This treatment took into consideration the results of recent reports, in which the important anti-inflammatory effects of statins were discussed [START_REF] Waehre | Hydroxymethylglutaryl coenzyme a reductase inhibitors down-regulate chemokines and chemokine receptors in patients with coronary artery disease[END_REF][START_REF] Nissen | 20070092 © 2007 The Authors Journal compilation © 2007 Biochemical Society Lipid Lowering (REVERSAL) Investigators[END_REF][START_REF] Kinlay | High-Dose Atorvastatin Enhances the Decline in Inflammatory Markers in Patients With Acute Coronary Syndromes in the MIRACL Study Circulation[END_REF]. Statins can interfere with the proinflammatory pathway of adhesion and migration at the level of protein expression and function. For example, statins diminish the expression of the integrin dimer CD11b (Mac-1, the cognate ligand of ICAM-1 on the endothelium and other cells) on monocytes and they also inhibit the adhesion of leukocytes to endothelial cells [START_REF] Weber | HMG-CoA reductase inhibitors decrease CD11b expression and CD11b-dependent adhesion of monocytes to endothelium and reduce increased adhesiveness of monocytes isolated from patients with hypercholesterolemia[END_REF][START_REF] Kimura | Effects of fluvastatin on leukocyte-endothelial cell adhesion in hypercholesterolemic rats[END_REF][START_REF] Rasmussen | Diverse effects of inhibition of 3-hydroxy-3methylglutaryl-CoA reductase on the expression of VCAM-1 and E-selectin in endothelial cells[END_REF] Statins can selectively inhibit leukocyte adhesion by direct interaction with the leukocyte-function antigen-1 (LFA-1), thereby providing definitive evidence for functions which are independent of lipid lowering. [START_REF] Weitz-Schmidt | Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site[END_REF] By binding to a new allosteric site within this ß 2 integrin, rather than targeting HMG-CoA reductase, statins block the adhesion of lymphocytes to the endothelium to such a degree so as to suppress the inflammatory response in peritonitis in mice. [START_REF] Weitz-Schmidt | Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site[END_REF]. Statins are also reported to ameliorate inflammatory diseases in murine autoimmune models, by inhibiting Th1 responses [START_REF] Leung | A novel anti-inflammatory role for simvastatin in inflammatory arthritis[END_REF], and to influence cell activation through the inhibition of NFκB [START_REF] Hilgendorff | Statins differ in their ability to block NF-kappaB activation in human blood monocytes[END_REF][START_REF] Dechend | Modulating angiotensin II-induced inflammation by HMG Co-A reductase inhibition[END_REF] [START_REF] Li | Immunoregulatory effects of atorvastatin on experimental autoimmune myocarditis in lewis rats[END_REF][START_REF] Aktas | Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1 cells through atorvastatin[END_REF]. The type of immune response supported may also depend on the ability of statins to induce the release of the T H 2-promoting cytokines (eg, IL-4, IL-5, IL-10, or transforming growth factor-ß), and diminish the secretion of the T H 1 subtype (eg, IL-2, IL-12, or interferon. [START_REF] Youssef | Biochemical Society a Th2 bias and reverses paralysis in central nervous system autoimmune disease[END_REF]. Statins may also repress the activation of T lymphocytes by inhibiting major histocompatibility complex class II antigen expression. [START_REF] Kwak | Statins as a newly recognized type of immunomodulator[END_REF]. Notably, HMG-CoA reductase inhibitors also regulate the expression and function of these mediators. In addition, statins determined the prevention of atrial fibrillation (AF) in an experimental model of pericarditis with elevated CRP, and prevented the promotion of atrial electrophysiological and structural changes resulting from inflammation. [START_REF] Kumagai | The HMG-CoA reductase inhibitor atorvastatin prevents atrial fibrillation by inhibiting inflammation in a canine sterile pericarditis model[END_REF]. Similarly, a recent clinical study has shown that the use statins in patients with lone AF was associated with a decrease in the recurrence of AF after successful cardioversion [START_REF] Siu | Prevention of atrial fibrillation recurrence by statin therapy in patients with lone atrial fibrillation after successful cardioversion[END_REF]. These data show that statins can mediate modest but clinically apparent anti-inflammatory effects by modifying vascular risk factors in the context of high-grade autoimmune inflammation [START_REF] Mccarey | Trial of Atorvastatin in Rheumatoid Arthritis (TARA): double-blind, randomised placebo-controlled trial[END_REF]. Indomethacin is a widely used NSAID and it is generally known to exhibit multiple biological functions by inhibiting cyclooxygenases. Indomethacin inhibits the activity of the enzyme cyclooxygenase by decreasing the formation of the precursors of prostaglandins and thromboxanes from arachidonic acid or by activating peroxisome proliferator-activated receptors [START_REF] Brooks | Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle[END_REF] However, recent studies have revealed that a number of the actions of indomethacin are independent of COXs. [START_REF] Zhang | Malignant transformation and antineoplastic actions of nonsteroidal anti-inflammatory drugs (NSAIDs) on cyclooxygenase-null embryo fibroblasts[END_REF]. In addition, it was shown that the novel PGD 2 receptor chemoattractant receptor-homologous molecule, expressed on Th2 cells (CRTH2), is another functional target for indomethacin [START_REF] Lehmann | Peroxisome proliferatoractivated receptors and are activated by indomethacin and other non-steroidal anti-inflammatory drugs[END_REF][START_REF] Hirai | Cutting Edge: Agonistic Effect of Indomethacin on a Prostaglandin D 2 Receptor,CRTH2[END_REF].In addition, indomethacin was able to reduce virus infection at a postbinding step early in the infection cycle, thereby inhibiting virus protein synthesis, but it did not seem to affect viral RNA synthesis [START_REF] Rossen | Inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step[END_REF]. We hypothesized that the combination of both drugs, which displays different actions in the inflammatory process, could determine an enhancing of antiflammatory effects and it could also explain the observed effects on CRP plasma levels from day 1 of treatment. We studied patients with acute pericarditis because these patients are young and with no other diseases which could interfere with the inflammatory state determined by pericarditis. The randomized patient sample comprised patients exclusively at low risk because this was the first time that this combination had been used. Indeed, the primary endpoint was safety and feasibility. The effects observed on clinical and laboratory inflammatory markers were of great interest but the low number of patients and brief observation period were insufficient in permitting definitive conclusions. We believe that the favourable trend in reducing hospitalization time (not significant), the significant earlier normalization in ST-segment elevation and pericardial effusion were obtained due to a more rapid cessation of the inflammatory process. Our data are insufficient to suggest additive or synergistic effects of the indomethacin/statin combination and should, therefore, be considered as a preliminary stage and to be interpreted with caution; further investigation, however, is warranted.

Clinical

Clinical

  Science Immediate Publication. Published on 11 Jul 2007 as manuscript CS20070092 © 2007 The Authors Journal compilation © 2007 Biochemical Society segment elevation on the electrocardiogram (ECG), echocardiographic evidence of a small pericardial effusion, elevated white-cell count (WCc), elevated erythrocyte sedimentation rate (ESR), high values of serum CRP, and increased values of TNI and CK and its MB fraction concentration.

  the 55 enrolled patients (mean age 29.3 ± 5.2 years, range 20-38 years, 34 men and 21 women) showed an acute onset, pericardial chest pain and ECG ST-segment elevation with a typical ECG evolution at entry. Clinical Science Immediate Publication. Published on 11 Jul 2007 as manuscript CS20070092 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  and diminished T-cell proliferation, probably Clinical Science Immediate Publication. Published on 11 Jul 2007 as manuscript CS20070092 © 2007 The Authors Journal compilation © 2007 Biochemical Society via the direct engagement of the T-cell receptor.

  Science Immediate Publication. Published on 11 Jul 2007 as manuscript CS20070092 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1 : Clinical characteristics and baseline values of the enrolled patients. Data are expressed as mean+ SD.

 1

	_________________________________________________________________________
		Statin	Placebo	P<
	_________________________________________________________________________
	Patients No	28	27	ns
	Age (years)	29.5+5.7	29.2+4.8	ns
	Sex (M/F)	18/10	16/11	ns
	Systolic Blood Pressure (mmHg) 118+8.1	117.7+7.3	ns
	Diastolic Blood Pressure (mmHg) 72.8+6.4	73.3+7.0	ns
	Heart Rate (b/min)	101+2+8.2	100.8+7.7	ns
	BUN (mg/dl)	29.56+5.4	28.7+5.2	ns
	Creatinine (mg/dl)	0.82+0.22	0.84+0.24	ns
	Ejection Fraction%	60.1+3.7	59.8+3.9	ns
	C-Reactive Protein (mg/dl)	9.36+3.3	9.25+3.0	ns
	Erythrocyte sediment. rate (mm/h) 40.1+6.41	39.96+6.

74 ns Troponin I (pg/ml) 1.21+0.7 1.18+0.6 ns White Cell count (x 10 L) 11,21+2.3 11,11+2.6 ns Creatin Kinase (IU/l) 193.8+50.43 189.5+45.35 ns ___________________________________________________________________________ White cell count values are x10/L. Clinical Science Immediate Publication. Published on 11 Jul 2007 as manuscript CS20070092

Table : 2: Effects of the combination of statin and indomethacin versus indomethacin and placebo on the normalization time of inflammatory markers, ST-segment normalization and pericardial effusion disappearance (days). The time (days) was calculated from admission (baseline values) to normalization of inflammation markers and when the patients were symptom-free and in the same time ESR, WCc resulted in normal range (<20 mm/h and <7.0 X 10 L respectively). This moment was also chosen to analyze the CRP values. Data are expressed as mean+SD.

 :

	________________________________________________________________________________
		Statin	Placebo	p<
	Time of hospitalization (days)	5.5+2	6.5+2	0.069
	ST segment normalization (days)	3.5+1	4.5+1	0.001
	Pericardial effusion (days)	4.5+1	5.5+1	0.001
	ESR (mm/h <20) (days)	5.0+1	6.0+2.	0.022
	WCc (<7.0 X 10 L)(days)	3.0+2	4.0.+2	0.069
	CRP (< 0.5 mg/dl)(days)	5.0+1	6.0.+2	0.022
	TNI (<0.5 pg/ml)(days)	4.5+1	4.5+1	ns
	CK (<80 IU/L)(days)	3.5+1	3.5+1

ns _______________________________________________________________________________ WCc=White Cells count; ESR= Erythrocyte sedimentation rate; CRP= C-reactive protein; TNI= Troponin I; CK=creatin Kinase. Clinical Science Immediate Publication. Published on 11 Jul 2007 as manuscript CS20070092 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table : 3: Changes in C-reactive protein, erythrocyte sedimentation rate, and white cell count during hospitalization period. Data are expressed as mean + SD _____________________________________________________________________________________

 :

	Days	Statin	Placebo P<	Statin	Placebo P<	Statin	Placebo	P<
		C-Reactive--Protein	erythrocyte sedimentation rate	White Cells count
	Entry	9.36+3.3 9.25+3.0 0.89	40,1+5,5 39,9+5.7 ns	11210+2590	1110+2355	ns
	Days 1	6.3+2.3	8.5+2.5 0.001	35+4,5	37+5,4	0.141	10125+1855	10.600+1950 0.359
	Days 2	3.1+1.1	4.9+1.5 0.0001	3 0,5+4,1 34+4,7	0.005	9185+1445	9850+1650	0.117
	Days 3	1.2+0.5	2.5+0.8 0.0001	30,1+3,8 32+3,8	0.069	8750+1235	9.200+1475	0.225
	Days 4	0.82+0.4 1.5+0.7 0.0001	26,5+3,4 26+3,7	n s	7350+1110	8250+1325	0.008
	Days 5	0.71+0.2 0.92+0.3 0.003	19,9+2,8 26+3.2	0.0001	6150+855	7500+650	0.0001
	Days 6	0.5+0.2	0.65+0.2 0.007	18,1+2,1 20.1+1,9 0.0001	5965+525	6285+755	0.073
	*P<	0.0001	0.0001	0.0001	0.0001		0.0001	0.0001

________________________________________________________________________________________________________ *p= intragroup differences (ANOVA).

Clinical Science

Immediate Publication. Published on 11 Jul 2007 as manuscript CS20070092 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society


Faidon Magkos

Yannis Tsekouras

Stavros A Kavouras

Bettina Mittendorfer

Ph.D Labros S Sidossis
email: lsidossis@hua.gr

Improved insulin sensitivity after a single bout of exercise is curvilinearly related to exercise energy expenditure

Keywords: acute exercise, insulin resistance, dose-dependent, threshold

A single bout of moderate-intensity exercise increases whole-body insulin sensitivity for 12-48 hours post-exercise. However, the relationship between exercise energy expenditure and the improvement in insulin sensitivity is not known. We hypothesized that the exercise-induced increase in whole-body insulin sensitivity, assessed with the homeostasis model assessment of insulin resistance (HOMA IR ), is directly related to the energy expended during exercise. We studied 30 recreationally active, non-obese men (age: 27 ± 5 years, body mass index: 24 ± 2 kg/m 2 ) in the post-absorptive state on two separate occasions: once after exercising at 60% of peak oxygen consumption for 30-120 min on the preceding afternoon (expending a total of 1.28-5.76 MJ) and once after an equivalent period of rest; blood samples were obtained the following morning. Exercise-induced changes in HOMA IR were curvilinearly related to exercise energy expenditure (r = -0.666, P = 0.001) with a threshold of ~3.77 MJ (900 kcal) for improvements in HOMA IR to manifest. In particular, HOMA IR was reduced by 32 ± 24% (P = 0.003) in subjects who expended more than 3.77 MJ during exercise, but did not change for those who expended fewer than 3.77 MJ (-2 ± 21%, P = 0.301). Furthermore, the magnitude of change in HOMA IR after exercise was directly associated with baseline (i.e., resting) HOMA IR (r = -0.508, P = 0.004); this relationship persisted in multivariate analysis. We conclude that improved wholebody insulin resistance after a single bout of exercise is curvilinearly related to exercise energy expenditure, and requires unfeasible amounts of exercise for most sedentary individuals.

Introduction

Regular, moderate-intensity, endurance-type physical activity is associated with significantly reduced risk for type 2 diabetes and cardiovascular disease, in part due to enhanced insulin action [START_REF] Bassuk | Epidemiological evidence for the role of physical activity in reducing risk of type 2 diabetes and cardiovascular disease[END_REF]. Whole-body insulin sensitivity is higher in trained athletes than in untrained subjects, and improves considerably with exercise training in previously sedentary individuals [START_REF] Borghouts | Exercise and insulin sensitivity: a review[END_REF]. Most of the enhancement in insulin action associated with exercise training is attributed to the last bout of exercise, and is lost after 3-6 days of inactivity [START_REF] Borghouts | Exercise and insulin sensitivity: a review[END_REF][START_REF] Henriksen | Effects of acute exercise and exercise training on insulin resistance[END_REF][START_REF] Henriksson | Influence of exercise on insulin sensitivity[END_REF][START_REF] Van Baak | Relationships with physical activity[END_REF]. In fact, insulin-mediated whole-body glucose uptake is increased 12-48 hours after a single exercise session in both healthy [START_REF] Mikines | Effect of physical exercise on sensitivity and responsiveness to insulin in humans[END_REF][START_REF] Bogardus | Effect of muscle glycogen depletion on in vivo insulin action in man[END_REF][START_REF] Perseghin | Increased glucose transportphosphorylation and muscle glycogen synthesis after exercise training in insulin-resistant subjects[END_REF][START_REF] Annuzzi | Increased insulinstimulated glucose uptake by exercised human muscles one day after prolonged physical exercise[END_REF] and insulin-resistant subjects [START_REF] Perseghin | Increased glucose transportphosphorylation and muscle glycogen synthesis after exercise training in insulin-resistant subjects[END_REF]10,[START_REF] Devlin | Enhanced peripheral and splanchnic insulin sensitivity in NIDDM men after single bout of exercise[END_REF] but returns to baseline values thereafter [START_REF] Borghouts | Exercise and insulin sensitivity: a review[END_REF][START_REF] Henriksen | Effects of acute exercise and exercise training on insulin resistance[END_REF][START_REF] Henriksson | Influence of exercise on insulin sensitivity[END_REF][START_REF] Van Baak | Relationships with physical activity[END_REF].

The amount of exercise required to elicit an enhancement in insulin sensitivity remains uncertain.

Furthermore, there is considerable inter-individual variability in the metabolic response to acute exercise and the concomitant changes in glucose and insulin dynamics [12], some of which may be related to baseline insulin resistance [10,[START_REF] Devlin | Enhanced peripheral and splanchnic insulin sensitivity in NIDDM men after single bout of exercise[END_REF]13]. Understanding the dose-response nature of these relationships will have important physiological and public health implications. Current public guidelines advocate 30-60 minutes of moderate-intensity exercise on most days of the week [START_REF] Bassuk | Epidemiological evidence for the role of physical activity in reducing risk of type 2 diabetes and cardiovascular disease[END_REF]; however, ~70% of the adult population fails to meet the recommended 30-min goal of regular exercise and ~40% does not engage in any kind of physical activity [14]. It was therefore our objective to examine the relationship between the energy expended during exercise and basal whole-body insulin sensitivity, assessed by using the homeostasis model assessment of insulin resistance (HOMA IR ), in healthy untrained men.

Methods

Subjects and preliminary testing

Thirty non-obese, recreationally active but untrained men participated in the study (Table 1). All subjects engaged in moderate-intensity physical activities ≤ 2 times per week, and were considered to be in good health after completing a medical evaluation, which included a history and physical examination and standard blood tests. None were smoking or taking medications.

Body composition (fat mass and fat-free mass) was determined by dual-energy X-ray absorptiometry (Delphi-W, Hologic, Waltham, MA / DPX-MD+, Lunar, Madison, WI), and peak oxygen consumption (VO 2peak ) was measured with an incremental exercise test on a treadmill (SensorMedics, Vmax229, Yorba Linda, CA) or a cycloergometer (TrueOne 2400, ParvoMedics, Salt Lake City, UT), depending on whether subjects were assigned to perform running or cycling exercise (see Experimental protocol). Written informed consent was obtained from all subjects before participation in the study, which was approved by the Human Studies Committee and the General Clinical Research Center Advisory Committee at Washington University School of Medicine in St. Louis, MO, and the Bioethics Committee of Harokopio University, Athens, Greece.

Experimental protocol

Each subject completed two studies within three weeks, in randomized order: once after resting and once after exercising on the preceding afternoon. Subjects were instructed to adhere to their regular diet and to refrain from exercise for a minimum of 3 days before the start of each study (rest and exercise). For the exercise study, they came in the laboratory and cycled on a semirecumbent cycloergometer (Cateye Fitness, Dallas, TX) or ran on a motor-driven treadmill (Technogym Runrace, Italy) at 60% of their VO 2peak for 30 to 120 min between 1730-1930 h (7 subjects exercised for 30 min; 7 for 60 min; 9 for 90 min, and 7 for 120 min). There were no significant differences in VO 2peak between individuals who exercised for different amounts of time (3.3 ± 0.8, 3.2 ± 0.6, 3.3 ± 0.7 and 3.2 ± 0.5 l/min, respectively; P = 0.883 by one-way analysis of variance). Oxygen consumption was measured at regular intervals during exercise (every 5, 15, or 30 min, depending on the duration of exercise) to calculate total energy expenditure of the bout (range: 1.28-5.76 MJ, i.e., 306-1376 kcal) [15] and adjust the workload, if necessary, to maintain the desired VO 2 within ± 5%. The different modes and durations of exercise were chosen in order to cover the range from the minimum recommended amount of exercise (i.e., 30 min of moderate-intensity) [START_REF] Bassuk | Epidemiological evidence for the role of physical activity in reducing risk of type 2 diabetes and cardiovascular disease[END_REF] to an exercise session that most untrained individuals would find it difficult to complete (i.e., 2 h in duration) [16], and ensured that the experimental conditions would closely reflect a realistic situation, in which unaccustomed individuals engage in various activities for variable amounts of time. Subjects were not allowed access to any kind of food or beverage during exercise, other than water. For the resting study, subjects lied in bed or sat in a chair. After completion of the exercise or the equivalent period of rest, they consumed the same standardized meal (12 kcal per kg body weight; 50-55% carbohydrate, 30-35% fat and 15-20% protein) at ~2000 h, in order to eliminate between-trial differences in energy balance other than the energy deficit incurred by exercise, and then fasted (expect for water) until completion of the study the next day.

At 0800 h the following morning, an arterialized blood sample was obtained from a heated forearm vein for the determination of basal plasma glucose and insulin concentrations. Blood was collected in chilled tubes containing heparin (for glucose) or sodium EDTA plus aprotinin (for insulin) and placed immediately on ice. Plasma was separated by centrifugation within 30 min of collection, and samples were stored at -80ºC until analysis. Plasma glucose concentration was determined by the glucose oxidase method on an automated glucose analyzer (YSI 2300 STAT+, Yellow Spring Instruments, Yellow Springs, OH). Plasma insulin concentration was measured by radioimmunoassay (Linco Research, St. Louis, MO). The HOMA IR score, which reflects whole-body insulin resistance, was calculated as the product of fasting plasma insulin (in mU/l) and glucose (in mmol/l) concentrations divided by 22. 5 [17].

Statistical analysis

Data were analyzed with SPSS 13 for Windows (SPSS Inc, Chicago, IL). All data sets were normally distributed according to the Kolmogorov-Smirnov procedure. Data are presented as means ± standard deviations (SD). Results after rest and after exercise were compared with Student's paired, two-tailed t test. Relationships between variables of interest were examined with correlation and regression analyses. P < 0.05 was considered statistically significant.

Results

Fasting plasma glucose and insulin concentrations, and therefore the HOMA IR score, were significantly lower the morning after exercise than rest (Table 2). There responses varied considerably between individuals (Table 2), but were not affected by the mode of exercise (running or cycling) in any consistent manner (not shown).

Total energy expenditure during exercise correlated negatively with exercise-induced changes in fasting plasma glucose (r = -0.482, P = 0.007) and insulin (r = -0.521, P = 0.003) concentrations.

There was a negative, curvilinear relationship between total energy expenditure during exercise and changes in HOMA IR score, with no apparent effect of exercise below a caloric expenditure of ~3.77 MJ (i.e., 900 kcal) (r = -0.666, P = 0.001; Figure 1). Hence HOMA IR was reduced by ~30% in subjects who expended more than 3.77 MJ during exercise (n = 12, P = 0.003), but did not change for those who expended fewer than 3.77 MJ (n = 18, P = 0.301; Figure 1). There were no significant differences in subject characteristics between these two groups (Table 1).

The relationship between changes in HOMA IR score and exercise energy expenditure was linear when the latter was expressed relative to body weight (r = -0.577, P = 0.001). However, the percent of variance explained by this linear relationship (R 2 = 33.3%) was less than that explained by the curvilinear relationship with total energy expenditure of exercise (R 2 = 44.4%).

The magnitude of change in HOMA IR after exercise was inversely correlated with baseline (i.e., resting) HOMA IR score (r = -0.508, P = 0.004; Figure 2).

In multivariate linear regression analysis, including exercise energy expenditure, age, body mass index, body weight, fat mass and fat-free mass, VO 2peak , and baseline (i.e., resting) plasma glucose and insulin concentrations and HOMA IR score, total energy expenditure during exercise (standardized β = -0.494, P = 0.001) and baseline HOMA IR score (β = -0.420, P = 0.005) were the only significant independent predictors of the exercise-induced change in HOMA IR , accounting for 49.4% of the total variance (F = 13.2, P < 0.001).

Discussion

We investigated the relationship between the energy expended during a single bout of moderateintensity endurance exercise, performed in the evening, and fasting plasma glucose and insulin concentrations and insulin resistance (HOMA IR ) the following morning in healthy, non-obese, untrained men. Our findings suggest that improvements in whole-body insulin sensitivity, reflected by decreased HOMA IR score, are curvilinearly related to exercise energy expenditure with a threshold of ~3.77 MJ (900 kcal) for a beneficial effect to manifest. However, exercisemediated changes in whole-body insulin resistance are inversely related to baseline HOMA IR score, even within this group of normoglycaemic and normoinsulinaemic subjects with normal HOMA IR [18], implying that insulin-resistant individuals may benefit more from exercise than insulin-sensitive ones. Nonetheless, single sessions of typical recreational activities are unlikely to have a significant impact on whole-body insulin sensitivity in healthy sedentary individuals.

It is well established that a single session of strenuous exercise leads to increased insulinmediated whole-body glucose uptake for some 12-48 hours post-exercise in healthy and insulinresistant subjects [START_REF] Mikines | Effect of physical exercise on sensitivity and responsiveness to insulin in humans[END_REF][START_REF] Bogardus | Effect of muscle glycogen depletion on in vivo insulin action in man[END_REF][START_REF] Perseghin | Increased glucose transportphosphorylation and muscle glycogen synthesis after exercise training in insulin-resistant subjects[END_REF]10,[START_REF] Devlin | Enhanced peripheral and splanchnic insulin sensitivity in NIDDM men after single bout of exercise[END_REF]. In our study, whole-body insulin sensitivity improved proportionally with the energy expended during exercise when this exceeded ~3.77 MJ, whereas no changes in HOMA IR score occurred after a total energy cost of exercise less than 3.77 MJ (900 kcal). This energy expenditure threshold is equivalent to ≥60-90 min of exercise at 60% of VO 2peak in our recreationally active men, which by far exceeds current public recommendations for physical activity [START_REF] Bassuk | Epidemiological evidence for the role of physical activity in reducing risk of type 2 diabetes and cardiovascular disease[END_REF]. In fact, most sedentary individuals will probably not be able to exercise for more than 1 hour at this intensity [16]. The existence of an energy expenditure threshold in the insulin-sensitizing effect of exercise is in line with findings on other physiological outcomes of acute exercise, e.g., the lowering of plasma triglyceride concentrations [19,20]. Our findings also help explain the lack of a consistent effect of exercise on insulin sensitivity in some previous studies, because too little exercise was performed (corresponding to total caloric expenditures ≤2.1 MJ or 500 kcal) [13,21]. These results collectively suggest that improvements in basal whole-body insulin sensitivity after a single bout of exercise require unfeasible amounts of exercise for most untrained individuals.

It is possible that regular exercise training lowers the threshold of exercise required to improve whole-body insulin sensitivity; the effects of acute exercise on insulin action appear to be more pronounced in the trained state [START_REF] Borghouts | Exercise and insulin sensitivity: a review[END_REF] and every-day, leisure-time physical activity is associated with dose-dependent reductions in diabetes risk in epidemiological surveys [22][23][24][25]. However, whether exercise training or the intensity of exercise modify in any way the dose-response relationship between energy expenditure and insulin sensitivity remains to be determined.

Furthermore, the inverse association between exercise-induced changes in HOMA IR and baseline HOMA IR implies that less exercise may be required to improve insulin sensitivity in insulinresistant subjects than those with good insulin sensitivity at baseline, i.e., before engaging in exercise. For instance, some of our subjects with higher baseline HOMA IR scores, albeit within the normal range [18], exhibited large improvements in insulin resistance (i.e., ~20-40% reduction) after expending only during exercise. Likewise, insulin-mediated whole-body glucose disposal increased to a greater extent in insulin-resistant obese than in insulin-sensitive lean subjects ~12 hours after a strenuous session of exercise [10],

and HOMA IR decreased the morning after a single, 90-min exercise bout at 60% of VO 2peak in men but not in women, whose baseline HOMA IR was half that of men [26]. Regular exercise, even of low energy cost, should therefore not be rejected as a means to improve insulin sensitivity in insulin-resistant subjects.

The exact cellular mechanisms responsible for the increase in insulin sensitivity late into recovery from a single bout of exercise are not well understood. Exercise-induced changes in insulin sensitivity have been linked to the depletion of skeletal muscle glycogen and/or triglyceride stores [27]. Depletion of muscle glycogen [START_REF] Mikines | Effect of physical exercise on sensitivity and responsiveness to insulin in humans[END_REF][START_REF] Bogardus | Effect of muscle glycogen depletion on in vivo insulin action in man[END_REF][START_REF] Perseghin | Increased glucose transportphosphorylation and muscle glycogen synthesis after exercise training in insulin-resistant subjects[END_REF]10,[START_REF] Devlin | Enhanced peripheral and splanchnic insulin sensitivity in NIDDM men after single bout of exercise[END_REF] leads to enhanced postexercise uptake of glucose to facilitate glycogen replenishment [START_REF] Borghouts | Exercise and insulin sensitivity: a review[END_REF][START_REF] Henriksen | Effects of acute exercise and exercise training on insulin resistance[END_REF]28]. The major cellular event underlying this phenomenon is increased translocation of the GLUT4 isoform of glucose transporter from its intracellular storage sites to the cell surface [29]; however, the mechanism(s) responsible for mediating this event, the signals involved, and the amount of exercise required to elicit these signals remain poorly defined [28]. Glycogen repletion in the recovery from exercise occurs in two distinct phases: an early, insulin-independent period of rapid glycogen resynthesis (lasting ~1 hour after cessation of exercise), and a subsequent period (up to 1-2 days postexercise) of slow glycogen resynthesis which is insulin-dependent [30]. This mechanism fits well with the results from our study, in which muscle glycogen stores were probably not depleted to any significant extent, except for those subjects who exercised for ≥60-90 min and expended more than ~3.77 MJ (900 kcal), as skeletal muscle glycogen is reduced dose-dependently with the total energy cost and hence the duration of exercise [30,31]. We are not aware of any studies examining the possible dose-dependency of exercise-induced changes in relevant signaling pathways. Furthermore, glycogen resynthesis rates are substantially slower in insulin-resistant than insulin-sensitive subjects in the late post-exercise period [32], which could help explain our observation that relatively insulin-resistant subjects (as indicated by higher baseline HOMA IR scores) enjoyed greater decreases in HOMA IR after exercise than relatively insulin-sensitive ones, because persistence of low glycogen stores augments the exercise-induced enhancement in muscle insulin action [28]. Intramuscular triglyceride content is also closely associated with insulin sensitivity [33]. Diminution of skeletal muscle lipid stores and/or enhanced lipid oxidation after exercise could therefore also facilitate muscle insulin action [34]. The relative contribution of changes in skeletal muscle glycogen and triglyceride metabolism to the exerciseinduced improvements in insulin sensitivity and the role, if any, of the energy expenditure of exercise are currently not known.

In summary, we investigated the relationship between the total energy expended during a single bout of moderate-intensity evening exercise and changes in fasting plasma glucose and insulin concentrations and HOMA IR in healthy, non-obese, untrained men. Our results indicate that more than 1 hour of moderate-intensity exercise is required to improve basal whole-body insulin sensitivity, assessed with the HOMA IR score. Whether the same applies for repeated exercise sessions (i.e., training) remains to be studied. Although interpretation of our findings is limited by the use of HOMA IR as a surrogate index of whole-body insulin resistance, there is a good correlation (r = 0.7-0.9) between HOMA IR and estimates of insulin sensitivity derived from hyperinsulinaemic-euglycemic clamp and minimal model analysis [35]. These findings may therefore be useful for the development of appropriate exercise protocols targeted at ameliorating insulin resistance.

Figure 1

 1 Figure 1Exercise-induced changes in homeostasis model assessment of insulin resistance (HOMA IR ) as a function of total energy expenditure during exercise (a), and for subjects who expended fewer or more than 3.8 MJ (900 kcal) during the exercise bout (b; values are means ± SD; * P < 0.05 versus rest).

Figure 2

 2 Figure 2Exercise-induced changes in homeostasis model assessment of insulin resistance (HOMA IR ) as a function of baseline (i.e., resting) HOMA IR score.

FIGURE 1 Exercise

 1 FIGURE 1

Clinical Science Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1

 1 Subject characteristics

	All subjects	Exercise energy expenditure
		<3.77 MJ (900 kcal) >3.77 MJ (900 kcal)

Table 2

 2 Fasting plasma glucose and insulin concentrations and HOMA IR score after a single evening exercise bout or an equivalent period of rest

			Rest	Exercise	Exercise-induced change (%)	P value
	Glucose	(mmol/l)	5.3 ± 0.4	5.1 ± 0.4	-3 ± 6	0.005
	Insulin	(mU/l)	6.5 ± 2.6	5.4 ± 2.1	-12 ± 26	0.004
	HOMA IR score		1.56 ± 0.67	1.25 ± 0.52	-14 ± 26	0.003
	Values are means ± SD.

Clinical Science Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070134 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We wish to thank Amalia Yanni and Maria Maraki for technical support, and the study subjects for their participation. This study was supported by grants from the American Heart Association (0365436Z and 0510015Z), the Hellenic Heart Foundation, and National Institutes of Health grants AR 49869, DK 56341 (Clinical Nutrition Research Unit), and RR 00036 (General Clinical Research Center). Bruce, C. R. and Hawley, J. A. (2004) Improvements in insulin resistance with aerobic exercise training: a lipocentric approach. Med Sci Sports Exerc 36, 1196-1201


Rory B Mcgoldrick

Martyn Kingsbury

Mark A Turner

Desmond J Sheridan

Alun D Hughes
email: a.hughes@imperial.ac.uk

Mcgoldrick Rb

MA Turner

Sheridan Dj

Hughes Ad

Left ventricular hypertrophy

Keywords: left ventricular hypertrophy, vasodilation, coronary artery. Clinical Science Immediate

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Left ventricular hypertrophy (LVH) is an adaptive increase in myocardial mass in response to work overload and over time confers a major increase in morbidity and mortality [START_REF] Gordon | Premature mortality from coronary heart disease. The Framingham study[END_REF]. In particular, LVH is a major precursor for heart failure [START_REF] Cooper | Left ventricular hypertrophy is associated with worse survival independent of ventricular function and number of coronary arteries severely narrowed[END_REF], although the mechanisms for progression to heart failure are poorly understood. Previous studies have demonstrated reduced coronary vascular reserve [14;17] in LVH and this is likely to be a major contributing factor to development of failure [START_REF] Vatner | Coronary vascular mechanisms involved in decompensation from hypertrophy to heart failure[END_REF].

Several mechanisms have been suggested to account for the adverse effect of LVH on cardiac function and impaired coronary flow reserve. These include a reduced density of arterioles in hypertrophied hearts [3;11;14;19]; increased perivascular compression due to changes in myocardial structure [START_REF] O'gorman | Investigation of impaired coronary vasodilator reserve in the guinea pig heart with pressure induced hypertrophy[END_REF], and a greater compressive effect of systole [START_REF] O'gorman | Investigation of impaired coronary vasodilator reserve in the guinea pig heart with pressure induced hypertrophy[END_REF]. However there is also evidence suggesting a reduction in active coronary artery vasodilator function in hypertrophied hearts [START_REF] Kingsbury | Endogenous and exogenous coronary vasodilatation are attenuated in cardiac hypertrophy: a morphological defect?[END_REF]. However, these studies have been conducted in intact hearts and therefore do not allow a distinction to be made between increases in vascular impedance arising from passive or structural changes or impairment of active vasodilator function.

The object of these studies therefore was to examine vasodilator function directly in isolated coronary arteries using a guinea-pig pressure-overload model of LVH, and to compare them with mesenteric small arteries in order to understand to what extent a primary defect in active vasodilation may contribute to the impairment of coronary flow reserve.

METHODS & MATERIALS

Proximal aortic stenosis was induced by banding the aorta of male Dunkin-Hartley guinea pigs (600-800 g) as previously described [START_REF] Kingsbury | Investigation of distal aortic compliance and vasodilator responsiveness in heart failure due to proximal aortic stenosis in the guinea pig[END_REF]. In brief, animals were anaesthetized and a small high-density plastic clip with an internal diameter of 1.99 mm placed on the upper portion of the ascending aorta.

Haemodynamic measurements, morphology, myography

After 168±3 days animals were anaesthetized with a bolus intra-peritoneal dose (60 mg/kg) of pentobarbitone (Animal Care Ltd., York, UK). Electrocardiograms (ECG), left atrial (LA), right ventricular (RV) and left ventricular (LV) pressures and aortic flow were measured [START_REF] Kingsbury | Investigation of distal aortic compliance and vasodilator responsiveness in heart failure due to proximal aortic stenosis in the guinea pig[END_REF]. Subsequently, animals were killed by cervical dislocation and the heart, lung and mesentery removed. Small branches of the left epicardial coronary artery and 3 rd order branches of the superior mesenteric artery were dissected and mounted as ring segments in an isometric microvascular myograph [START_REF] Mulvany | Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats[END_REF]. The myograph contained 12 mL physiological saline solution (PSS) containing NaCl 118 mmol/L, KCl 4.69mmol/L, MgSO 4 .7H 2 O 1.18 mmol/L, KH 2 PO 4 1.18 mmol/L, NaHCO 3 25 mmol/L, Glucose 5.5 mmol/L, CaCl 2 .6H 2 O 2.5 mmol/L, Na 2 EDTA 0.03 mmol/L aerated with 95% O 2 and 5% CO 2 . Arteries were allowed to equilibrate for 1 hour and then were set at a "normalized" internal circumference of 0.9.L 100 , estimated to be 90% of the circumference they would maintain if relaxed and exposed to 100 mm Hg transmural pressure. This was calculated for each individual artery on the basis of the passive lengthtension characteristics of the artery and the LaPlace relationship [START_REF] Mulvany | Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats[END_REF]. Before the start of the studies, vessels were tested for viability with a depolarizing potassium solution (KPSS: PSS with equimolar substitution of 118 mmol/L KCl for NaCl), all vessels studied produced a tension equivalent to ≥10kPa and were considered viable. Relaxation was examined by precontracting vessels to near maximal tone using the thromboxane mimetic, U46619 (1μmol/L), and adding the relaxant cumulatively once stable tone was achieved. Responses were calculated as % inhibition of precontracted tone and the degree of pre-contraction (measured as active media stress) did not differ between banded and sham animals (data not shown). At the end of each experiment, arterial wall morphology was assessed at six positions with the artery mounted in the myograph under minimal tension using a optical microscope as previously described [START_REF] Mulvany | Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats[END_REF]. After removal of the coronary artery the heart was cleaned, dried at 70°C for 72 hours and weighed.

Statistical analysis

All data are means ±SEM. Concentration response data were fitted to a logistic function using Prism 2.01 (GraphPad Software Inc, San Diego, USA) and compared using analysis of variance (ANOVA).

In addition -log(EC 50 ) values were calculated and maximum responses (E max ) were determined from measured values. Since inhibition of relaxation may be manifested either as an reduction in -log(EC 50 ) or E max depending on efficacy and receptor reserve, statistical comparisons between these parameters were also made using a two way unpaired Student's t-test if ANOVA was significant. p < 0.05 was considered significant.

Drugs and reagents

Reagents were prepared immediately prior to use. Acetylcholine (ACH), forskolin (FSK) haemoglobin (Hb), indomethacin, isoprenaline (ISO), NG-monomethyl-L-arginine (LNMMA), noradrenaline, sodium nitroprusside (SNP), U46619 and all other reagents were purchased from Sigma-Aldrich (Dorset UK).

RESULTS

Effect of banding on Cardiac Morphology

Changes in gross heart morphology following aortic banding are illustrated in Table 1. Aortic banding for 168±3 days increased heart weight:body weight ratio by 79% (p < 0.01) compared with sham controls. This reflected marked increases in the LV (52%), RV (100%) and atrial (170%) mass (p < 0.01 in each case). In addition, lung weight:body weight ratio increased by 96% (p < 0.01) suggesting a degree of heart failure in association with the hypertrophy. These changes represent an increase in organ weight as the body weight of banded and sham control groups was similar (Table 1).

Effect of banding on systemic haemodynamics

Haemodynamic data in sham operated and aortic banded animals are shown in Table 2. The data are similar to those reported previously in this model [12;13], and although blood pressure is low in both banded and sham animals these values are typical for the anaesthetized guinea pig [START_REF] Hauser | Cardiovascular parameters in anaesthetized guinea pigs: a safety pharmacology screening model[END_REF] and only slightly lower than measurements in conscious animals [START_REF] Provan | Development of a surgical approach for telemetering guinea pigs as a model for screening QT interval effects[END_REF]. As expected aortic banding resulted in an increased systolic pressure gradient between the LV and the carotid artery. This was accompanied by a 37% (p < 0.001) reduction in aortic flow and a 47% (p < 0.05) increase in systemic vascular resistance. ECG-derived heart rate was unchanged, but banding increased QRS R wave voltage by 94% (p < 0.01), QRS duration by 32% (p < 0.05) and QTc interval duration by 15% (p < 0.05).

Effect of banding on arterial structure

The effects of aortic banding on coronary and mesenteric artery morphology are shown in Table 3.

Aortic banding resulted in significant increases in adventitial (52%; p<0.05), medial (59%; p<0.01) and total wall (52%; p<0.001) thickness of coronary arteries, but mesenteric arterial structure was unaffected (Table 3). The internal diameters of coronary and mesenteric arteries were not significantly changed by banding.

Effect of banding on vasodilator responses of coronary and mesenteric arteries

Relaxation in response to ACH was reduced in coronary arteries from banded animals (figure 1 & Table 4), but relaxation to ACH in mesenteric arteries was similar in banded and sham animals (figure 1 &table 4). In view of the inhibition of ACH-induced relaxation in the coronary artery, the role of cyclooxygenase products and NO was investigated in this tissue. Preincubation with the cyclooxygenase inhibitor, indomethacin (3µmol/L) did not alter responses to ACH, and responses to ACH remained inhibited in coronary arteries from banded animals exposed to indomethacin (table 4).

In contrast, preincubation with a combination of the NO inhibitors, LNMMA (10µmol/L) + oxyhaemoglobin (10µmol/L), reduced responses to ACH and there was no longer a significant difference in ACH-induced relaxation in coronary arteries from banded and sham animals following inhibition of NO (table 4). Relaxation to ISO was reduced in banded animals in both coronary and mesenteric arteries (figure 2), while responses to FSK (figure 3) and SNP (figure 4) were reduced in coronary arteries from banded animals, but not in mesenteric arteries (figures 3 & 4).

Clinical Science Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070136

DISCUSSION

We have shown that cardiac hypertrophy due to aortic banding is associated with impaired relaxation of coronary arteries in response to endothelium-dependent and endothelium-independent agents.

Previous studies have demonstrated impaired coronary flow reserve in cardiac hypertrophy [7;10;12] and the present studies show that this is due, at least in part, to impaired vasodilation of coronary arterial smooth muscle. Aortic banding-induced also caused impaired β-adrenoceptor mediated relaxation in both coronary and mesenteric resistance arteries. This is likely to be due to downregulation of β-adrenoceptors secondary to the sympathetic overactivity and enhanced levels of plasma catecholamines, previously observed in this model [START_REF] Kingsbury | Investigation of distal aortic compliance and vasodilator responsiveness in heart failure due to proximal aortic stenosis in the guinea pig[END_REF]. In addition as has been previously described [START_REF] Kingsbury | Endogenous and exogenous coronary vasodilatation are attenuated in cardiac hypertrophy: a morphological defect?[END_REF], there was an increased adventitial and medial thickness of coronary arteries probably as adaptive response to the elevated coronary arterial pressures resulting from aortic banding. Whether the changes in relaxation responses are a direct consequence of coronary artery smooth muscle hypertrophy is not known, but Boonen et al., [START_REF] Boonen | Mesenteric small artery changes after vasoconstrictor infusion in young rats[END_REF] found no difference in vasodilation in response to ISO and SNP in hypertrophied mesenteric small arteries from spontaneously hypertensive rats and Wistar Kyoto rats infused chronically with angiotensin II compared with arteries from normotensive Wistar Kyoto rats, suggesting that hypertrophy per se may not affect vasodilator responsiveness.

Aortic banding causes a number of neuroendocrine changes including elevated levels of catecholamines [START_REF] Kingsbury | Investigation of distal aortic compliance and vasodilator responsiveness in heart failure due to proximal aortic stenosis in the guinea pig[END_REF], natriuretic peptides [START_REF] Kingsbury | Investigation of distal aortic compliance and vasodilator responsiveness in heart failure due to proximal aortic stenosis in the guinea pig[END_REF], endothelin-1 [START_REF] Bell | Vitamin C and quinapril abrogate LVH and endothelial dysfunction in aortic-banded guinea pigs[END_REF], tumor necrosis factor-α [START_REF] Bell | Vitamin C and quinapril abrogate LVH and endothelial dysfunction in aortic-banded guinea pigs[END_REF] and, inconsistently, activation of the renin-angiotensin system [1;12]. It is also associated with increased coronary endothelial cell NAD(P)H-dependent superoxide production [START_REF] Bell | Vitamin C and quinapril abrogate LVH and endothelial dysfunction in aortic-banded guinea pigs[END_REF]. The extent to which these changes are responsible for the effects seen is unknown, but the reduction in both endotheliumdependent and endothelium-independent relaxation clearly indicates that the vasodilator responsiveness of coronary arterial smooth muscle is abnormal. Our observations do not exclude concomitant abnormalities in coronary endothelial function, since changes in endothelial function and / or NO release could be obscured by the alteration in vasorelaxant properties of the smooth muscle in our studies. Nevertheless, a previous study in this animal model [START_REF] Kingsbury | Endogenous and exogenous coronary vasodilatation are attenuated in cardiac hypertrophy: a morphological defect?[END_REF] did not observe differences in coronary artery NO release under basal conditions or during reactive hyperaemia in an isolated perfused Langendorf preparation. While the mechanism responsible for the impaired vasorelaxant function following aortic banding is uncertain it is notable that ACH, FSK, and SNP all cause vasodilation via increases in cyclic nucleotides. Additional studies investigating levels of cyclic nucleotides, activity of phosphodiesterases and activity of myosin light chain phosphatase (the major mediator of the relaxant action of cyclic nucleotides) would therefore be of interest. After inhibition of NO synthase, responses to ACH were reduced, but did not differ significantly between sham and banded animals. This observation suggests that relaxation to other factors released by ACH, such as EDHF [START_REF] Busse | EDHF: bringing the concepts together[END_REF], may be unaffected by banding, but in view of the reduced responses to ACH after inhibition of NO synthase, further studies are required to confirm this interpretation.

Until recently the importance of the vascular tone of coronary arteries in the maintenance of coronary resistance was largely ignored, as it was believed that ischaemia caused maximal vasodilatation of the coronary microvasculature. However, more recent studies have demonstrated that even during ischaemia, coronary resistance vessels retain vasomotor tone and can respond to vasoconstrictor stimuli [START_REF] Duncker | Regulation of coronary vasomotor tone under normal conditions and during acute myocardial hypoperfusion[END_REF]. Consequently the impairment of relaxation seen in coronary arteries in this study is likely to contribute to reduced cardiac perfusion and progressive deterioration of ventricular function as hypertrophy evolves. However, since the current studies were performed on hypertrophic hearts with concurrent evidence of heart failure, it is not possible to determine whether the vascular dysfunction precedes or merely potentiates cardiac dysfunction.

In conclusion, left ventricular hypertrophy secondary to aortic banding causes reduced relaxation of systemic resistance arteries to β-adrenoceptor activation and an impaired relaxation of coronary small arteries to endothelium-dependent and endothelium-independent vasodilators.

TABLES

  Sham-operated animals underwent identical operative procedures but the plastic clip was not placed on the aorta. All animal work and surgery was performed in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986, HMSO, London and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Figure 1 .

 1 Figure 1. Effect of aortic banding on acetylcholine (ACH) relaxation of A) coronary resistance arteries,

Figure 2 .

 2 Figure 2. Effect of aortic banding on isoprenaline (ISO) relaxation of A) coronary resistance arteries,

Figure 3 .

 3 Figure 3. Effect of aortic banding on forskolin (FSK) relaxation of A) coronary resistance arteries, B)

Figure 4 .

 4 Figure 4. Effect of aortic banding on sodium nitroprusside (SNP) relaxation of A) coronary resistance

Clinical Science Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070136

Table 1 .

 1 Effect of aortic banding on cardiac structure. BW = body weight, HW = heart weight, LV = left ventricular weight, RV = right ventricular weight, A = atrial weight. Data are means ±SEM. ** = p <0.01 compared with sham.

	Clinical
		Sham	Banded
	BW (g)	1358±35	1313±28
	HW:BW (%)	0.049±0.001	0.088±0.010**
	LV:BW (%)	0.038±0.002	0.058±0.005**
	RV:BW (%)	0.006±0.001	0.012±0.002**
	A:BW (%)	0.007±0.001	0.019±0.004**

Science Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070136

Table 2 .

 2 Effect of aortic banding on cardiac and systemic haemodynamics.

	Sham	Banded

Clinical Science

Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070136

Table 3 .

 3 Effect of aortic banding on coronary and mesenteric resistance artery structure.

		Coronary	Mesenteric
		Sham	Banded	Sham	Banded
	Diameter (µm)	292±31	321±21	331±25	310±31
	Adventitia (µm)	17±3	26±4*	21±4	19±2
	Media (µm)	22±7	35±2**	28±3	30±5
	Wall (µm)	40±8	61±5**	49±5	49±3
	Data are means ±SEM of 6-7 observations. * = p < 0.05 and ** = p < 0.01 compared with sham.

Table 4 .

 4 Effect of aortic banding on relaxation of coronary and mesenteric arteries.

		Sham		Banded
	Coronary arteries	-logEC 50 (M) E max (%)	-logEC 50 (M) E max (%)
	ACH †	7.4±0.1	95±3	6.8±0.1	76±5
	ACH + indomethacin †	7.2±0.1	92±5	6.8±0.2	77±4
	ACH + Hb + LNMMA	5.7±0.2	36±6	5.1±0.4	43±13
	Mesenteric arteries
	ACH	6.7±0.2	95±4	6.4±0.2	91±4
	ACH + indomethacin	6.7±0.1	82±4	6.8±0.1	87±3
	ACH + Hb + LNMMA	6.0±0.3	28±6	5.7±0.1	25±4
	Acetylcholine (ACH), haemoglobin (Hb), NG-monomethyl-L-arginine (LNMMA), Data are means
	±SEM of 6-7 observations, † = p <0.01 compared with sham by ANOVA.

Clinical Science

Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070136

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070136 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGMENTS

We thank Dr R Garcha for technical assistance with these studies.

Clinical Science Immediate Publication. Published on 18 Jul 2007 as manuscript CS20070136


Heart rate variability measures: a fresh look at reliability

Pinna G, Maestri R, Torunski A, Danilowicz-Szymanowicz L, Szwoch D, La Rovere MT, and Raczak G, Heart Rate Variability Measures: A fresh look at reliability. Clin Sci (Lond), 2007. By Dr. Gavin Sandercock.

Abstract.

Heart rate variability (HRV) is a non-invasive maker of cardiac autonomic modulation utilised in many hundreds of scientific studies each year. The reliability of heart rate variability has been frequently investigated yet remains poorly quantified.

Assessing the reliability of a measure that assesses dynamic physiological processes and shows large between and within-subject variation is a complex task. In this issue of Clinical Science Pinna et al. [START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF] provide excellent insight into the test-retest reliability of commonly used HRV indices and put the values obtained into context by comparing them with levels of between-subject variation and by producing sample size estimates.

Commentary.

Scientific publications utilising heart rate variability (HRV) as an index of cardiac autonomic modulation are increasing year on year [START_REF] Parati | Point: cardiovascular variability is/is not an index of autonomic control of circulation[END_REF]. It would therefore seem logical to conclude that the thousands of authors who publish scholarly articles using this measure are fully aware of its concurrent validity and retest reliability, two qualities that should be easily identifiable in any clinical measurement. Recent discussions [START_REF] Parati | Point: cardiovascular variability is/is not an index of autonomic control of circulation[END_REF] and reviews of the literature [START_REF] Sandercock | The reliability of short-term measurements of heart rate variability[END_REF] demonstrate that an adequate consensus on either of these qualities has not been fully reached.

In this issue of Clinical Science, Pinna et al. [START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF] assess the reliability of time and frequency domain measures of HRV, in a well designed and statistically sound investigation. Their study [START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF] provides much needed information, enabling the reader to determine the value of HRV in terms of retest reliability. Pinna et al. [START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF] promote an interesting discussion regarding absolute and relative reliability of HRV indices. Their paper evaluates the reliability of selected measures and suggests potential, theoretical sample size requirements needed during future empirical investigations. The authors conclude that reliability of HRV may appear low, or even unsatisfactory, insofar that even the better-performing indices often double or halve in value from test to retest. When this relatively poor absolute reliability is put into context, the authors are able to conclude that: 'for most indexes…random variation represents a limited portion of the between-subject variability'.

Assessing the reliability of HRV measures in this way and the creation of sample size estimates is original, innovative and provides the reader with useable information regarding application and use of HRV. What this innovative approach demonstrates is that many of the existing studies which use HRV as a dependent variable lack statistical power. When we reviewed the literature examining reliability of short-term HRV analysis we found that high quality studies were rare [START_REF] Sandercock | The reliability of short-term measurements of heart rate variability[END_REF]. In fact, nearly half the studies identified in our initial search were rejected due to poor design and in particular, poor choice of statistical analysis. One previous study of high methodological quality [START_REF] Sinnreich | Five minute recordings of heart rate variability for population studies: repeatability and age-sex characteristics[END_REF] provided findings highly consistent with those of Pinna et al. [START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF], both in terms of reliability and of absolute values for HRV indices obtained. Sinnreich et al. [START_REF] Sinnreich | Five minute recordings of heart rate variability for population studies: repeatability and age-sex characteristics[END_REF] produced, what was then, probably the best quality study in relation to HRV reliability and the present study [START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF] adds greatly to this body of literature.

The similarity in HRV index values between Sinnreich et al. [START_REF] Sinnreich | Five minute recordings of heart rate variability for population studies: repeatability and age-sex characteristics[END_REF] and Pinna et al. Time domain values show reasonable homogeneity but spectral measures appear particularly heterogeneous. Although some of the variation will be due to individual differences and methodological inconsistencies, the range of values appears unsatisfactory. To illustrate this point using the high frequency spectral power (HF), I cite values from two papers examining HRV in sportive populations, which used broadly similar data collection methods. In the first, HF was reported as (mean ± S.D.) 5839 ± 1839 ms 2 [START_REF] Sacknoff | Effect of athletic training on heart rate variability[END_REF] whilst in the second, HF was reported as 3.4 ± 1.8 ms 2 [START_REF] Scott | Enhanced cardiac vagal efferent activity does not explain training-induced bradycardia[END_REF]. Both these values are somewhat disparate from the 1996 Taskforce report [START_REF] Taskforce | Heart rate variability: standards of measurement, physiological interpretation and clinical use[END_REF] which provides normal values for HF as 975 ± 203 ms 2 . The problem here lies not in which value is 'correct', this is merely an illustration, but in the fact that such values can be published without reference to the fact that they vary so widely from 'expected' values.

Large-scale, sex and age-determined normative data for short-term HRV do exist [START_REF] Sinnreich | Five minute recordings of heart rate variability for population studies: repeatability and age-sex characteristics[END_REF][START_REF] Kuo | Effect of aging on gender differences in neural control of heart rate[END_REF][START_REF] Hemingway | Does autonomic function link social position to coronary risk? The Whitehall II study[END_REF] but reference to such values rarely is made. Authors and reviewers alike should be cautious when values for spectral measures are radically different from those considered normal and authors in particular should seek to provide reasoning for such discrepancies.

Authors should be encouraged to report spectral measures as raw (ms 2 ) units and provide appropriate measures of central tendency and dispersion. Logarithmic transformation is often necessary prior to parametric analysis, but reporting only the mean (± S.D) of the natural logarithm does not adequately describe the data. The simple addition of appropriate descriptive statistics would greatly enhance the potential for the reader to evaluate the values for spectral HRV measures reported.

Perhaps when a recognised range of values for spectral HRV exists, research into expected magnitudes of change can begin in earnest. Finally, when such values are established, HRV studies with a priori power calculations will be more commonplace and HRV may be able to occupy a role as a clinical tool in risk stratification and therapeutic target for intervention. peer-reviewed publications annually it surely cannot be unreasonable to assume that some consensus toward a normative range for HRV indices can be reached. Perhaps then, sample size calculations in HRV will be as common place as they are in the assessment of other physiological measurements.

Clinical

  Science Immediate Publication. Published on 23 Apr 2007 as manuscript CS20070137 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  [START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF] leads to a second noteworthy point. Pinna et al.[START_REF] Pinna | Heart Rate Variability Measures: A fresh look at reliability[END_REF] raise an important point by using simulated data for sample size estimates because: 'reference values as to what change in HRV parameters would be clinically relevant are lacking'. It has long been known that depressed levels of selected HRV indices are excellent predictors of mortality in some populations[START_REF] Kleiger | Decreased heart rate variability and its association with increased mortality after acute myocardial infarction[END_REF] but what represents a normal value for HRV on which to base sample size estimates proves elusive.Measures of central tendency and dispersion show us that there appears to be a genuine and large inter-subject variation in many HRV indices, particularly spectral measures. Clinical Science Immediate Publication. Published on 23 Apr 2007 as manuscript CS20070137 © 2007 The Authors Journal compilation © 2007 Biochemical Society Similarly, some studies show very large changes (and therefore large effect sizes) in HRV indices due to a variety of interventions. The reliability of HRV must, therefore, be interpreted both in terms of expected values at baseline and predicted magnitude of change. Such interpretation is, however, problematic due in part to the heterogeneity in values reported in the literature.

Clinical

  figures for required sample sizes in future intervention studies in which HRV indices are independent variables. Accuracy of future sample size calculations would, however, be much greater if reference values for HRV indices are agreed and cited. With over 500

Clinical Science Immediate Publication. Published on 23 Apr 2007 as manuscript CS20070137 © 2007 The Authors Journal compilation © 2007 Biochemical Society