Cardiorespiratory responses, nitric oxide production and inflammatory factors in patients with myocardial infarction after rehabilitation

Accepted Manuscript Cardiorespiratory responses, nitric oxide production and inflammatory factors in patients with myocardial infarction after rehabilitation José V. Subiela, Sonia H. Torres, Juan B. De Sanctis, Noelina Hernández PII:

S1089-8603(17)30258-6

DOI:

10.1016/j.niox.2018.03.006

Reference:

YNIOX 1759

To appear in:

Nitric Oxide

Received Date: 25 September 2017 Revised Date:

3 March 2018

Accepted Date: 5 March 2018

Please cite this article as: José.V. Subiela, S.H. Torres, J.B. De Sanctis, N. Hernández, Cardiorespiratory responses, nitric oxide production and inflammatory factors in patients with myocardial infarction after rehabilitation, Nitric Oxide (2018), doi: 10.1016/j.niox.2018.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

CARDIORESPIRATORY RESPONSES, NITRIC OXIDE PRODUCTION AND INFLAMMATORY

RI PT

FACTORS IN PATIENTS WITH MYOCARDIAL INFARCTION AFTER REHABILITATION.

Running title: Skeletal muscle inflammation in rehabilitated myocardial infarction patients. José V Subiela, MD, PhDa, Sonia H Torres, MD, PhDa, Juan B De Sanctis, PhDb, Noelina Hernández,

M AN U

SC

PhDa.

a. Institute of Experimental Medicine, Section of Muscle Adaptation (SEAM), Medical Faculty, Central University of Venezuela, Caracas, Venezuela.

TE D

b. Institute of Immunology, Medical Faculty, Central University of Venezuela, Caracas, Venezuela

EP

Corresponding author: Dr. Sonia H. Torres. Instituto de Medicina Experimental. Sección para el Estudio de la Adaptabilidad Muscular (SEAM). Facultad de Medicina. Universidad Central de Venezuela. Ciudad Universitaria. Caracas. Venezuela. Postal Code: 1050 E.mail: [email protected] Work telephone: 58- 212- 6053395 Home telephone: 58-212.-7307503 Cell phone: 58 04143116551. WhatsApp +58 4143116551

AC C

Emails of authors José V Subiela: [email protected] Juan B De Sanctis: [email protected] Noelina Hernández: [email protected]

FUNDING This work was funded by “Consejo de Desarrollo Científico y Humanístico”. Universidad Central de Venezuela. 09-00-6717 CONFLICTS OF INTEREST: none.

CONTRIBUTORS: All authors have materially participated in the research and article preparation and have approved the final article. 1

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

2

ABSTRACT

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

There is evidence that myocardial infarction (MI) patients have an inflammatory process that includes skeletal muscles, and exercise has been reported to reduce some inflammatory markers. The aim of this work was to study NO and some inflammatory markers in quadriceps muscle of MI patients before and after cardiac rehabilitation. Muscle biopsy was obtained in17 MI patients before and after CR and only once in 11 healthy subjects. Several cardiorespiratory and metabolic parameters were evaluated and skeletal muscle levels of nitric oxide synthases, nitrate, nitrite, nitrotyrosine, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukin- 6 (IL-6) and CD154. After CR there was an increase in maximal oxygen consumption (21.2±1.4 vs 25.7±2.5 mL/kg/min, P<0.0001); work load (116.2±14.9 vs 140±17 W, P<0.0001); pulmonary ventilation (59.8±7,5 vs 73.8±11.6 L/min, P<0.0001); anaerobic threshold (53.8%±3.5% vs 60.2%±3.3% of maximal VO2, P<0.0001), maximal lactatemia (8.1±1.4 vs 9.3±1.5 mmol/L, P<0.0001), and oxygen pulse (11.7±1.6 vs 14.0±1.9 mL/pulse, P<0.0001). CSA of type I fibers increased (4380±1868 vs 5237±1530 µm2, P=0.02), and nitrate (18.6±3.04 vs 20.7±2.0 ng/mg, P<0.001). There was a negative correlation between BMI, fat%, waist and hip circumferences and NO synthase, nitrite and nitrate after CR. The inflammatory mediators were higher in patients than in control subjects and did not change with CR. TGF-β correlated directly with nitrite and nitrate and inversely to other inflammatory factors. In conclusion, there is an increase of nitrate post CR, indicating a more effective NO production. TGF-β was related to anti-inflammatory processes even before CR.

Key Words: Cardiac rehabilitation, cardiorespiratory indices, nitric oxide, inflammation factors Abbreviations:

Highlights:

AC C

EP

TE D

AT = aerobic threshold BMI= body mass index CAD= coronary artery disease CD154 = CD40 ligand. Protein in macrophage membrane, CD4 + activated T cell, endothelial and other cells. CVD=cardiovascular disease CR = cardiac rehabilitation CSA = cross sectional area eNOS = constitutive endothelial nitric oxide synthase iNOS = inducible nitric oxide synthase nNOS = constitutive neuronal nitric oxide synthase TNF-α = Tumor necrosis factor alfa TGF-β = Transforming growth factor beta IL-6 = interleukin 6

After cardiac rehabilitation nitrate levels increased in skeletal muscle. Body fat correlated inversely with higher NO production in rehabilitated patients. Transforming growth factor β was related with decreased inflammation and higher NO.

3

ACCEPTED MANUSCRIPT 1.INTRODUCTION. Cardiovascular diseases (CVD) are the leading cause of death in the world. According to WHO in 2012, 17.5 million people died from CVD, of which 7.5 million deaths were from coronary artery disease and 6.7 million from cerebrovascular accidents [1]. Exercise based CR programs were developed for patients after

RI PT

acute myocardial infarction (MI) to help them regain their previous exercise capacity and facilitate reintegration into working life. It has been demonstrated that CR is a highly efficient, innocuous and economic therapeutic intervention [2]. There is ample evidence that diet and medical treatment of risk

SC

factors play a role in prevention and control of CVD [3]; moreover, CR including its 6 fundamental aspects (patient education, psychological aid, occupational therapy, dietetic orientation, medical treatment and

M AN U

exercise) is crucial in secondary prevention of the disease. Some studies indicate that the modification of risk factors have a very positive impact on morbidity due to cardiac events, both recent and recurrent. After an acute coronary syndrome, the effects seem to be even more pronounced leading to a 40% decline in 6month mortality. [3, 4]. Physical training improves exercise capacity more than conventional medical

TE D

therapy alone [5]. A year program of regular physical exercise in selected patients with stable coronary artery disease (CAD) resulted in higher event-free survival and exercise capacity compared with standard percutaneous coronary intervention [6]. Since 1995, CR participation after acute coronary syndrome and

treatment [7].

EP

coronary artery bypass grafting is associated with reduced mortality even in the modern era of CAD

AC C

Training induces in both healthy subjects and CAD patients an increase in maximal oxygen consumption, which is the product of the capacity of the cardiovascular system to supply oxygen (i.e. cardiac output) and the capacity of skeletal muscles to use oxygen (i.e. arterial-venous oxygen difference).

In healthy

individuals this increase is aproximately produced in equal proportion by central and peripheral adaptations while in CAD subjects impaired cardiac function would place a higher contribution on adaptive changes in the periphery, and consequently demand less effort on cardiac function [8]. Regular physical exercise as part of a multifactorial intervention improves symptom free exercise tolerance and myocardial perfusion. Because no net regression of epicardial coronary stenosis was observed in the majority of patients, and many 4

studies of collateralization in CAD patients have reported that exercise did not improve angiographically

ACCEPTED MANUSCRIPT

detectable collateralization, the current evidence does not firmly support the hypothesis that these beneficial effects of exercise result from a blunted progression and/or an increased regression of coronary lesions, instead it indicates that exercise increases endothelium dependent dilation in the conduit arteries and larger resistance arteries [9], as a result of increased nitric oxide (NO) production and bioavailability [10].

RI PT

Exercise also produces higher myogenic control, increased metabolic vasodilatation in small resistance arteries, and direct cardiac effects as improved calcium handling, increased P13K activity, reduction of fibrosis and cardiomyocyte apoptosis [4].

SC

Adaptive changes to endurance training in peripheral muscle are: increased oxygen extraction by correcting endothelial dysfunction and increasing basal NO formation [11]; improvement of oxidative metabolism,

M AN U

showed by increase in activity of oxidative enzymes [12]; increased levels of phosphocreatine/inorganic phosphates [13]; changes in mitochondrial ultrastructure [14]; increase of radical scavenger enzyme activity [15]; increase of COX-activity inversely correlated with iNOS expression /iNOS protein content [16]. In addition, there is a reduction of sarcopenia related to attenuation of MuRF-1 expression, a component of the

TE D

ubiquitin-proteasome system involved in muscle proteolysis [17].

There are several reports of muscle inflammation in general diseases as type II diabetes mellitus [18] and chronic obstructive pulmonary disease (COPD) [19]. Patients with acute coronary syndrome, stable CAD, or

EP

chronic heart failure (CHF) showed elevated levels of inflammatory markers in peripheral blood mononuclear cells [20], plasma [21, 22] or skeletal muscle [23].

AC C

Exercise has been reported to reduce some inflammatory markers in skeletal muscle of CHF patients [23], but not in plasma [22]. Generation of NO is related to inflammatory markers, as demonstrated by the induction of iNOS expression in skeletal muscle by IL-1b and NFkB activation [24]. Most of the work on peripheral effects of exercise on skeletal muscle in patients with CVD has been done ln patients with CHF, however, there is scarce information in MI patients. The hypothesis of the present work is that the improvement in oxygen consumption that is obtained with the rehabilitation program currently used in our University Hospital may be related to an increase in skeletal muscle NO production and to reduction in the muscle inflammatory state. For that purpose, nitric oxide synthases (NOS), nitrate, nitrite, 5

nitrotyrosine, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukin-6

ACCEPTED MANUSCRIPT

(IL-6) and CD154 were determined, along with several cardiorespiratory and metabolic parameters.

2. METHODS 2.1. Study subjects

RI PT

Seventeen male patients (54±8 years old) who entered a 12 weeks program of CR in the Unit of Cardiac Rehabilitation of the “Hospital Clínico Universitario” in Caracas (Venezuela), 8 weeks after been released from hospitalization due to a myocardial infarction (MI) of low or medium risk profile [25]. Each patient

SC

was examined before and after the CR program to be his own control. Eleven healthy male subjects (56±1 years old) were also examined to compare with them the results of the MI patients before and after CR

M AN U

(Table 1). All patients were treated with β-blockers, 16 had anti-aggregation therapy, 9 received ARAII, 4 received calcium antagonists, 3 were taking IECA and one, anticoagulant therapy; treatment was maintained during rehabilitation and for the performance of the ergometric tests. The Bioethical Committee of the hospital approved the study and patients and healthy subjects signed the informed consent.

TE D

2.2. Body composition

Body fat percentage was estimated by skinfold thickness measurements using a caliper in four body regions; and equations to calculate body density and fat % [26]. Waist and hips circumference [27], and body mass

EP

index were assessed by standard procedures. 2.3. Ergometric test

AC C

An exercise test was performed before and after the CR program, in an electrically braked cycle ergometer (Lode Excalibur Sport, USA), frequency of 60 cycles per min. After 3 min of unloaded pedaling, 25 watts´ resistance increases were added every 3 min until exhaustion, or when blood pressure or heart rate increased or decreased disproportionally, or ECG changes showed signs of complex arrhythmias or myocardial ischemia. ECG was monitored during the whole test, and the 15 final seconds (s) of each stage were recorded. Blood pressure (BP) was measured in the final 30 s of each stage and at 1, 2, 3, 5, 7 and 10 min post-test. Physiological variables were registered in an automatic equipment (ULTIMA 20 MedGraphics,

6

USA), with a metabolic and respiratory analyzer, following the established protocol. All patients finished

ACCEPTED MANUSCRIPT

the test despite of minor ECG and cardiac rhythm changes in some subjects,

2.4. Physical training The CR program used is that currently practiced in the University Hospital of Caracas. Training program

RI PT

consisted in three weekly sessions of approximately 60 min of exercise, during 12 weeks for a total of 36 sessions during the study period. Sessions consisted of three parts: 1) warming and neuromuscular conditioning including loosening, stretching, articular mobility and flexibility, during 15 min. 2) aerobic

SC

exercise during 20 min the first two weeks and then 30 min the rest of the sessions, bicycling or walking on a thread-mill in the firsts sessions, that allowed a better control of HR and BP, and walking on a track

M AN U

in the lasts sessions. The intensity was tailored for each patient according to the result of the prerehabilitation ergometric test (60-80% of VO2 max). 3) final recovery and relaxation exercises for 10-15 min. First week started with 60% of maximum heart rate obtained in the previous effort test, if well tolerated, it was increased to 70% in the second week, to 80% in the third week and kept at this level until

TE D

the end of the 12 weeks. Walk speed/pedaling rate were adjusted accordingly to maintain the intensity at 80%. Al training sessions were supervised by physiotherapists and one cardiologist.

EP

2.5. Lactatemia

Blood lactate was measured in the ear lobe, before the test and 3 min after the end. Blood was analysed with

AC C

a mini-photometric equipment (Miniphotometer Dr Lange Plus 20, Berlin, Germany). 2.6. Echocardiogram

A trans-thoracic echocardiogram (TTE) was performed with a Philips Sonos 4500 equipment, before and after CR. The axes determined were; long left parasternal, short left parasternal, sub-costal, and 4 chambers, with a transducer of 2.5 MHz. The inter-ventricular septum width, the diameters of the posterior wall of the left ventricle and the left atrium, and the ejection fraction were measured. 2.7. Skeletal muscle determinations

7

Patients underwent needle muscle biopsies [28] before and after CR. Healthy subjects were biopsied only

ACCEPTED MANUSCRIPT

once. The sample of approximately 50 mg was immediately blotted and divided in three parts; the first part was embedded in OCT compound (Tissue TEK; Sakura Finetek Torrance, CA) and dipped in isopentane frozen in liquid nitrogen; the two other parts were directly frozen in isopentane, all samples were wrapped in identified aluminum paper and placed in liquid nitrogen to transport them to a freezer to be stored at -80ο

RI PT

until processing. The procedure from taking the sample to be frozen lasted less than 1 min. 2.7.1. Histochemical Procedures

Fiber type classification and capillary measurements were done in 10 µm transverse serial sections cut in a

SC

cryostat at – 20°C, with the adenosine triphosphatase reaction after alkaline (pH 10.3) and acid (pH 4.37 and pH 4.6) preincubation [29]. Capillaries were visualized by the α-amylase periodic acid Schiff reaction [30].

M AN U

Photomicrographs at a final magnification X 200 were made, and fibers were identified by comparison with the adenosine triphosphatase sections. An area of the photograph was delimited, measured with a planimeter, and fibers and capillaries were counted to calculate the mean fiber CSA, capillaries per square millimeter, and capillaries per fiber ratio. Capillaries around each fiber type were counted and all the fibers

TE D

of one type were drawn together to measure area by planimetry to calculate the mean area of each fiber type. 2.7.2. Determinations of muscle metabolism enzymes. Muscle samples for enzymes activities were weighted at -20οC, homogenized in ice cooled potassium

EP

phosphate buffer. The activities of citrate synthase (CS) 3-31hydroxy-acyl-CoA-dehydrogenase (HAD) and lactate dehydrogenase (LDH) were assayed using fluorometric techniques [31].

AC C

2.7.3. Assessment of nitrate, nitrite, eNos, iNOs, nNOs and nitrotyrosine levels. The muscle sample was weighed and homogenized with a glass pestle in 1 mL of Tris-HCl buffer, 10 mmol/L, pH 7.5; NaCl, 150 mmol/L; ethylene-diamine tetra-acetic acid, 5 mmol/L; triton x-100, 1% (volume/volume); leupeptin, 10 µg/mL; aprotinin,10 g/mL; and pepstatin, 2.5 g/mL. The homogenate was centrifuged at 90g for 5 min. Supernatant was used for the assays. Protein concentration was assessed by the BCA protein assay kit (Pierce Biochemicals; Rockfort IL). NO levels were determined indirectly by quantification of their oxidized products of degradation, using nitrate reductase and the Griess reagent. A standard curve was obtained with sodium nitrate dissolved in water or in a pool of normal sera. Nitrite 8

concentration was determined at 540 nm using enzyme-linked immunosorbent assay (ELISA) plate reader

ACCEPTED MANUSCRIPT

(Multiscan MCC/340; Labsystems; Helsinki, Finland). All three NO synthases were assessed using sandwich ELISA assays. Constitutive eNOS was detected using a commercial kit (R&D Systems; Minneapolis, MN). Monoclonal and polyclonal antibodies for nNOS were from BD Biosciences (San Diego CA), and the recombinant enzyme for the standard curve was from Calbiochem (San Diego, CA).

RI PT

iNOS was assessed using a pair of antibodies (Serotec Corporation; Kidlington, Oxford, UK). The sensitivity of all assays was 25 pg/mL. Total amount of nitrotyrosine was determined by ELISA. The mouse immunoglobulin (Ig)-G monoclonal antibody, the polyclonal antibody against nitrotyrosine and the

SC

polyclonal goat anti-rabbit IgG-peroxidase antibody were purchased from Upstate Biotechnology (LakePlacid, NY, USA). The quantification of nitrotyrosine was performed using a standard curve with

the assay was 50 pg/mL [19]. 2.7.4. Assessment of inflammatory markers.

M AN U

known concentrations of nitrotyrosine from chemically modified bovine serum albumin. The sensitivity of

CD154 levels were detected by a commercial sandwich immunoenzymatic assay purchased from Chemicon

TE D

Corporation (Temécula, CA, USA). The ELISA plates were already coated with the capturing monoclonal. The samples were diluted as specified by the manufacturer in the assay buffer, and the only modification was that samples were incubated for 18 h at 4º C. Quantitative analysis was performed with a standard

EP

curve. The detection limit was 1 ng CD154 per mg of protein. TGF-β and IL-6 were measured with ELISA sandwich in solid phase following the protocol recommended by the manufacturers of the reactive (R&D

AC C

Systems, Minneapolis, USA).

TNF-a was assessed by a third generation TNF-a quantikine assay (R & D systems) following the manufacturer’s instructions, except that samples were diluted as suggested for serum samples and incubated for 18 h instead of 3 h. The sensitivity of the assay was 0.5–3 pg/mL. 2.8. Statistical Analysis Data in tables is expressed as mean ± SD. The results of each variable were evaluated in patients before and after CR with Student paired “t” test. The comparison of control subjects with patients before and after CR was done with non-paired “t” test. The Pearson product-moment correlation coefficient for grouped data and 9

Spearman for ungrouped data were used to establish correlations between the different variables of the

ACCEPTED MANUSCRIPT

study. The statistical program StatSoft Statistica was used for the analysis. A “P” value of less than 0.05 was considered statistically significant.

3. RESULTS

RI PT

3.1. Anthropometric characteristics Table 2 shows the bio-anthropometric characteristics of the patients. Although there was not a significant difference in weight and BMI, fat percentage was reduced after rehabilitation, indicating an increase in fat

SC

free mass and body density. The anthropometric characteristics measured in the healthy subjects group were not different compared to those of the patients before rehabilitation: weight (71.9±14.8 vs 75.5± 10.2 kg),

M AN U

height (167±8 vs165±17 cm), and BMI (26.43±3.0 vs 26.27±17.0), all P>0.05.

3.2. Exercise capacity

After CR, a highly significant increase in maximal oxygen consumption was found (P< 0.001), all patients

TE D

passed the first category of Weber and Janicki [32]. Work load and work time increased; pulmonary ventilation, anaerobic threshold, maximal lactatemia and oxygen pulse also showed significant increments (p<0.001), (Table 3).

EP

3.3. Skeletal muscle characteristics

3.3.1. Cross sectional area and capillary indexes

AC C

Patients showed an increase in the CSA of type I fibres after CR, P< 0.02 (Table 1). Before CR the patients, compared to healthy subjects, had a lower mean CSA of fibers (P<0.02), however, after CR there were no differences between the CSA of muscle fibers of patients and the healthy subjects (Table 1). Capillary density did not change after CR and did not differ between the healthy subject and the patient groups. (Table 1). 3.3.2. Muscle metabolism enzymes There were no changes after CR in muscle oxidative enzymes: (Table 1). CS activity was found higher in patients than in healthy subjects, both before and after rehabilitation (P<0.05). HAD activity was similar in 10

healthy subjects and patients; LDH was lower in patients before CR than in healthy subjects (P<0.05);

ACCEPTED MANUSCRIPT

however, significance of the difference disappeared after CR (Table 1). 3.3.3. NO production in muscle After CR, nitrate level was found highly significantly increased (P<0.001) (Table 1). No modification in the activity of NOS isoforms was seen after CR. Nitrate and nitrite concentrations were lower in patients as

RI PT

compared to healthy subjects (p<0.0001 and p<0.005 respectively); on the contrary, nitrotyrosine was higher (p<0.001) (Table 1). Although the value of nitrate increased after CR, it did not reach that of the healthy subjects. The constitutive isoforms of NOS, nNOS and eNOS, were lower in patients compared to healthy

SC

subjects (p<0.0001 and 0.05 respectively), and the inducible isoform iNOS was higher (p<0.005) (Table 1). 3.3.4. Inflammatory markers in muscle

M AN U

In Table 1 it is shown that there were not significant differences in the levels of the inflammatory markers post CR in comparison with those before rehabilitation. The measured inflammatory markers were significantly higher in patients as compared to healthy subjects, both before and after CR (TNF-α P<0.05, TGF-β and IL-6 P<0.005, and CD154 P<0.002). In all the healthy subjects, the levels of TNF-α were under

TE D

the sensitivity of the method (Table 1).

3.4. Correlations between the studied variables

Positive and negative correlations between anthropometric characteristics versus NO products, enzymes and

EP

inflammatory markers are shown in Table 4. In Table 5 are reported the correlations between aerobic (CS and HAD) and anaerobic (LDH) metabolism enzymes; NO production enzymes (NOS), NO products

AC C

(nitrite, nitrate and nitrotyrosine) and the inflammatory mediators (TNF-α, TGF-β, IL-6 and CD154). There was a negative correlation (see Table 4 for P values) between weight, BMI, fat%, waist and hip circumferences and eNOS, nitrite or nitrate after CR (Table 4). TGF-β correlated directly with nitrite and nitrate before CR, with all NOS after CR, and inversely to other inflammatory factors (see Table 5 for P values.). Correlation between the differences of the values after and before CE were significant for nitrite and maximal oxygen consumption expressed as VO2 mL/Kg.min (P<0.0001), and between nitrite and nitrate (P<0.002).

11

4. DISCUSSION

ACCEPTED MANUSCRIPT

4.1. Main findings. There is ample information on the effects of regularly practiced exercise in skeletal muscle of healthy subjects. However, the morphological and functional changes in skeletal muscles in disease, and their modification by CR, is less known. The present study was done in patients with MI. In agreement with our

RI PT

hypothesis it was found a significant increase of nitrate levels in muscle after CR (P<0.001); the increase of nitrate and/or nitrite is considered a physiological increase of NO production. In addition, the differences of nitrite levels and maximal oxygen consumption (VO2 mL/Kg.min) after and before CE showed a direct

SC

correlation (P<0.0001). Also, a direct correlation was found between the differences pre- and postrehabilitation values of nitrite and nitrate (P<0.002). Another relevant finding was the inverse correlation

M AN U

between weight, BMI, percentage of body fat and circumference of waist and hip, and muscle eNOS values and, except percentage of body fat, also to nitrite; moreover, percentage of body fat was inversely related to nitrate levels (Fig. 1). The decrease of body fat percentage was not accompanied by weight change or by decrease of body mass index, which means an increase of lean body mass and body density. These results

TE D

could be interpreted as a higher capacity of subjects with less fat to produce NO. On the other hand, the second part of our hypothesis, the decrease in the levels of inflammatory markers after CR, could not be demonstrated (Table 1). An important finding regarding inflammatory markers was the positive correlation

EP

of TGF-β with nitrites and nitrates before CR, and post CR with nitrite and all the three NOS (Table 4, Fig. 2). Fig. 3 illustrates the negative relationship of TGF-β with nitrotyrosine (post CR), with TNF-α (pre- and

AC C

post- CR) and also with IL-6 (before CR). These results are in favor to a role of TGF-β as an antiinflammatory factor in patients with MI. 4.2. Cardiorespiratory effects The results obtained in cardiorespitatory functions with the present program of CR are likethose found by others:19.1% and 21.2 % for absolute and relative maximal oxygen consumption [33,34]. The changes in work load (watts) and exercise time were 12.6% and 20.3%, without change in mechanical efficiency. Maximal pulmonary ventilation increased 23.4%. There was a highly significant increase in oxygen pulse, which is a better indicator than VO2 for the appreciation of changes in maximal cardiac output [35]. In the 12

present study the increment of AT after CR was 11.8% and that of lactatemia was 13.6% [36]. Accordingly,

ACCEPTED MANUSCRIPT

AT, work load and pulmonary ventilation correlated directly to VO2 max. 4.3. Muscular changes 4.3.1. Fiber types and CSA In the present work no differences was found in the distribution of fiber type after CR, although Hambrecht

RI PT

et al [37] have reported a marginal increase in type I fibres proportion. This difference may be due to the longer duration of their program (6 months compared to 3 months). CSA of type I fibers augmented, and this caused that the differences in mean CSA disappeared after CR between patients and healthy subjects; this

SC

result differs from that of only 4 weeks training study [17]. 4.3.2. Muscle metabolism enzymes

M AN U

A group of MI patients has been studied in a previous work of the same laboratory (12); that group showed low levels of CS, 9.0 ± 2.9 µmol/gr.min before CR that increased to 16.0 ± 4.8, P<0.01 after only 6 weeks of a similar program of CR, they also showed an increase in HAD and a decrease in LDH after rehabilitation in contrast with the group of the present study that did not show any change in the enzyme levels with

TE D

rehabilitation. In the present work the level of CS was higher in patients before CR than in healthy subjects and the difference was maintained after CR. These results may be explained because our healthy subjects were sedentary and the present group of patients was heterogeneous in their activity level. It is to be noticed

EP

that patients joined the CR program 8 weeks after leaving hospital with a recommendation to initiate a program of moderate exercise before joining formal CR; most of the patients confirmed to have done it.

AC C

However, it was observed that the three patients that showed the higher increment in in max VO2 (25%, 26% and 27%) did have an increase in CS and HAD levels. 4.3.3. NO generation

The patients studied showed endothelial dysfunction, with low levels of nitrite and nitrate, eNOS and nNOS, and increased nitrotyrosine and iNOS in skeletal muscle. This indicates a low production of “physiological” NO with high production of “pathological” NO that reacted with superoxide to form peroxynitrite [15, 38] as shown by the increase of nitrotyrosine, the end-product of the nitrosylation of tyrosine by peroxynitrite. In addition, iNOS showed a negative correlation with both constitutive isozymes nNOS and eNOS levels. 13

Other authors have found increased iNOS expression and nitrotyrosine levels in skeletal muscle of patients

ACCEPTED MANUSCRIPT

with chronic heart failure [39]. It is well known that regular physical exercise, personally tailored and controlled, has important effects on cardiovascular health and survival [40,41], attenuates disease progression [9], reduces hypertension [42] and produces changes in the lipoprotein subclass profile [43]. Reactive oxygen species (ROS), inflammatory

RI PT

factors and NO are involved in the development of these responses [15,4,10]. Unexpectedly eNOS was not increased in muscle after CR in the current work. However, the direct correlation between nNOS and nitrite present before CE reached a higher signification post-rehabilitation; moreover, a high significance direct

SC

correlation appeared between eNOS and nitrite. This may be interpreted as an improvement in vasodilator

M AN U

capacity in muscle after CR.

4.3.4. Inflammatory markers

There is evidence of the role of inflammatory processes in cardiovascular diseases: it has been demonstrated in the development of atherosclerosis [21]; expression of TNF-α and IL-6, together with other interleukins

TE D

(IL-10, IL-23A, IL-27 and IL-37), was significantly higher in peripheral blood mononuclear cells of patients with acute coronary syndrome [20]; patients with cardiac hearth failure showed increased serum levels of TNF-alpha, IL-6, and IL-1-beta [22]. In skeletal muscle of patients with cardiac heart failure it has been

EP

found increased levels of TNF-α, IL-1-beta, IL-6 with normal values in serum, [23]. In the present work

AC C

patients showed increased levels of TNF-α, TGF-β and IL-6, and CD154 in skeletal muscle. It has been demonstrated that training for 6 months significantly reduced the local expression of TNF-α, IL1-beta, IL-6 and iNOS in skeletal muscle of patients with cardiac hearth failure [23]. Other authors did not find reduction of inflammatory markers in blood after 4 months of high intensity training in patients with cardiac heart failure and arterial hypertension, but they did in patients with idiopathic dilated cardiomyopathy [22]. Differences in results may be related to duration of training and to the cardiovascular status of patients. It is to note that the standard deviation of the values of inflammatory markers in our patients was high, and some of them showed reduction after CR. It also may be possible that these 14

inflammatory markers have a modulatory effect in different moments of the rehabilitation process and in a

ACCEPTED MANUSCRIPT

different metabolic milieu.For example TGF-β is a pleiotropic peptide released by many cell types and involved in the regulation of tissue growth and homeostasis in many processes including inflammation and its resolution [44]. It represses skeletal muscle specific gene expression and has also been reported to modulate proliferation in satellite cells [45]. The relations between different inflammatory markers may also

RI PT

change during the rehabilitation process; for instance, in the present study TGF-β levels showed an inverse correlation with TNF-α both before and after CR; with IL-6 only before, and with nitrotyrosine after CR. Some markers have been demonstrated to modify NO production, TGF-β suppressed IL-1-induced iNOS

SC

expression and NO production in chondrocytes [46]. In our results, after CR, TGF-β correlated directly with all the tree isoforms of NOS, but also post CR iNOS negatively correlated to nitrotyrosine, suggesting that

M AN U

NO produced by iNOS was not reacting with ROS, but joining the vasodilator pool of NO. All this suggests that in our patients TGF-β was acting as an anti-inflammatory factor and this effect was more important after CR. Other change in relationship between the different inflammatory markers was seen with CD154, before CR CD154 levels directly correlated with IL-6 and TNF-α and the two last mentioned cytokines were also

TE D

directly related between them. However, after rehabilitation the significance of the direct correlation between CD154 and IL-6 was reduced, and the direct correlation between TNF-α and CD154 disappeared.

exercise. 4.4. Limitations

EP

These results suggest that the modulation effect between pro-inflammatory markers may be affected by

AC C

Due to the invasive nature of procedure used to obtain the skeletal muscle samples, a relatively small number of subjects was included in the study; this probably limits the direct extrapolation of the present findings to the entire population of patients with myocardial infarction. The diversity of the level of physical activity in the patients provided a heterogeneous group that made difficult to obtain significant statistical results in some of the variables studied. The method applied to measure % body fat may be not sufficiently accurate, however it was performed by personnel that evaluates athletes and the results are coherent with the data of BMI and waist and hip circumferences. Greiss method is not the most sensitive assay to measure

15

nitrite, but the results obtained with nitrate were highly significant, and we could not find other references of

ACCEPTED MANUSCRIPT

direct NO measurements in skeletal muscle in MI patients to compare.

5. CONCLUSION The present work reinforces the knowledge of the positive effects of CR in MI patients on oxygen

RI PT

consumption, physical work capacity, cardiac function, lactate production, anaerobic threshold and anthropometric characteristics. Skeletal muscle showed increase in NO production, as shown by the increase in the level of nitrate. The change in nitrite levels in muscle after rehabilitation was directly correlated with

SC

the increase in maximal oxygen consumption. CSA of the muscle fibers increased. The enhancement of the correlations of NOS isoforms with nitrite and nitrate suggests a more efficient production of NO. There was

M AN U

found an inverse relationship between NO production and BMI, body fat percentage, and waist and hip circumferences, indicating a better NO production in subjects with less body fat. The direct association of TGF-β with NO, nitrite, nitrate and NOS, and inverse relationship with inflammatory markers TNF-α and IL-6 in skeletal muscle strongly suggest an anti-inflammatory role. The direct correlation between pro-

TE D

inflammatory markers was less significant or disappeared after CR. All these results may be interpreted as a change of the modulatory effects of inflammatory markers after the rehabilitation treatment, concurrent with

AC C

EP

the improvement on exercise capacity of the patients.

16

6. ACKNOWLEDGEMENTS.

Our special thanks to Professor Stephen Tillett for correction of the English language and to

EP

TE D

M AN U

SC

RI PT

Assistant Professor Luis Vázquez (Public Health Department) for his help with statistics.

AC C

•

ACCEPTED MANUSCRIPT

17

ACCEPTED MANUSCRIPT 7. REFERENCES [1] World Health Organization. (2013, March) Cardiovascular diseases (CVDs) fact sheet No. 317. Retrieved from http://www.who.int/mediacentre/factsheets/ fs317/en/index.html.

RI PT

[2] Al Quait A, Doherty P. Does cardiac rehabilitation favor the young over the old? Open Heart, 2016; 3: doi: 10.1136/openhrt-2016- 000450.

[3] Franklin B, Cushman M. Recent advances in preventive cardiology and lifestyle medicine. Circulation

SC

2011; 123: 2274-83.

[4] Gielen S, Schuler G, Adams V. Cardiovascular effects of exercise training: molecular mechanisms.

M AN U

Circulation. 2010; 122:1221-38.

[5] Akashi Y, Koike A, Osada N, Omiya K, Itoh H. Short-Term Physical Training Improves Vasodilatory Capacity in Cardiac Patients. Jpn Heart J 2002; 43: 13-24.

[6] Hambrecht R, Walther C, Möbius-Winkler S, Gielen S, Linke A, Conradi K, et al. Percutaneous

TE D

Coronary Angioplasty Compared With Exercise Training in Patients With Stable Coronary Artery Disease. A Randomized Trial. Circulation. 2004; 109:1371-78.

[7] Rauch B, Davos CH, Doherty P, Saure D, Metzendorf MI, Salzwedel A, et al. The prognostic effect

EP

of cardiac rehabilitation in the era of acute revascularisation and statin therapy: A systematic review and meta-analysis of randomized and non-randomized studies - The Cardiac Rehabilitation Outcome Study

AC C

(CROS). Eur J Prev Cardiol. 2016; 23:1914-1939). [8] Hagberg JM. Central and peripheral adaptations to training in patients with coronary artery disease. In Saltin B (ed). Biochemistry of Exercise VI. Champaign. Human Kinetics Publishers. 1986. 267-77. [9] Gielen S, Laughlin MH, O’Conner C, Duncker DJ. Exercise Training in Patients with Heart Disease: Review of Beneficial Effects and Clinical Recommendations. Prog Cardiovasc Dis.2015; 57: 3 4 7 – 3 5 5. [10] Hambrecht R, V. Adams V, Erbs S, Linke, A, Kränkel N, Shu Y, et al. Regular physical activity improves endothelial function in patients with Coronary Artery Disease by increasing phosphorylation of endothelial nitric oxide synthase. Circulation. 2003; 107:3152-58. 18

[11] Hambrecht R, Fiehn E, Weigl C, Gielen S, Hamann C, Kaiser R, et al. Regular Physical Exercise

ACCEPTED MANUSCRIPT

Corrects Endothelial Dysfunction and Improves Exercise Capacity in Patients With Chronic Heart Failure. Circulation. 1998; 98:2709-15. [12} Torres SH, Almeida D, Rosenthal J, Lozada Fernandez Y, Hernandez N. Skeletal muscle changes with training in patients with coronary artery disease. J Cardiopulm Rehabil .1990; 10: 271-7.

RI PT

[13] Cottin Y, Walker P, Rouhier-Marcer I, Cohen M, Louis P, Didier JP, et al. Relationship between increased peak oxygen uptake and modifications in skeletal muscle metabolism following rehabilitation after myocardial infarction. J Cardiopulm Rehabil. 1996;16:169-74.

SC

[14] Hambrecht R, Fiehn E, Yu J, Niebauer J, Weigl C, Hilbrich L, et al. Effects of endurance training on mitochondrial ultrastructure and fiber type distribution in skeletal muscle of patients with stable chronic

M AN U

heart failure. J Am Coll Cardiol 1997; 29:1067–73.

[15] Linke A, Adams V, Schulze PC, Erbs S, Gielen S, Fiehn E, et al. Antioxidative effects of exercise training in patients with chronic heart failure: increase in radical scavenger enzyme activity in skeletal muscle. Circulation. 2005; 11: 1763-70.

TE D

[16] Gielen S, Adams V, Linke A, Erbs S, Möbius-Winkler S, Schubert. Exercise training in chronic heart failure: correlation between reduced local inflammation and improved oxidative capacity in the skeletal muscle. Eur J. Cardiovasc Prev Rehabil. 2005;12:393-400.

EP

[17] Gielen S, Sandri M, Kozarez I, Kratzsch J, Teupser D, Thiery J, et al. Exercise training attenuates MuRF-1 expression in the skeletal muscle of patients with chronic heart failure independent of age: the

AC C

randomized Leipzig Exercise Intervention in Chronic Heart Failure and Aging catabolism study. Circulation. 2012;125:2716-27.

[18] Torres SH, De Sanctis JB, Briceño M de L, Hernández N, Finol HJ. Inflammation and nitric oxide production in skeletal muscle in type 2 diabetic patients. J Endocrinol 2004; 181: 419-27. [19] Montes de Oca M, Torres SH, De Sanctis JB, Mata A, Hernández N, Tálamo C. Skeletal muscle inflammation and nitric oxide in patients with COPD. Eur Respir J 2005; 26: 390-9

19

[20] Al Shahi H, Shimada K, Miyauchi K, Yoshihara T, Sai E, Shiozawa T, et al. Elevated circulating levels

ACCEPTED MANUSCRIPT

of inflammatory markers in patients with acute coronary syndrome. Int J Vasc Med 2015;2015:805375. doi: 10.1155/2015/805375 Epub 2015 Oct 4. [21] Zakynthinos E, Pappa N. Inflammatory biomarkers in coronary artery disease. J Cardiol. 2009; 53: 31733.

RI PT

[22] Byrkjeland R, Nilsson BB, Westheim AS, Arnesen H, Seljeflot I. Inflammatory markers as related to disease severity in patients with chronic heart failure: limited effects of exercise training. Scand J Clin Lab Invest. 2011; 71: 598-605.

SC

[23] Gielen S, Adams V, Möbius-Winkler S, Linke A, Erbs S, Yu J. Anti-inflammatory effects of exercise training in the skeletal muscle of patients with chronic heart failure. J Am Coll Cardiol. 2003 Sep

M AN U

3;42(5):861-8.

[24] Adams V, Nehrhoff B, Späte U, Linke A, Schulze PC, Baur A, et al. Induction of iNOS expression in skeletal muscle by IL-1beta and NF kappa B activation: an in vitro and in vivo study. Cardiovasc Res. 2002 Apr;54(1):95-104.

TE D

[25] D'Agostino RB Sr, Vasan RS, Pencina MJ, Wolf PA, Cobain M, Massaro JM, et al. General cardiovascular risk profile for use in primary care: the Framingham heart study. Circulation. 2008; 117: 74353.

EP

[26] Durnin JVGA, Womersley J. Body fat assessed from total body density and its estimation from skinfold

AC C

thickness measurements on 481 men and women age 16 to 72 years. Br J Nutr 1974; 32: 77-97. [27] National Institutes of Health. Clinical guidelines for the identification, evaluation and treatment of overweight and obesity in adults. Bethesda, MD: National Institutes of Health, 1998. [28] Bergstrom J. Muscle electrolytes in man determined by neutron activation analysis on needle biopsy specimens: A study on normal subjects, kidney patients with chronic diarrhea. Scand J Clin Lab Invest 1962; 14 (Suppl 68):1-110. [29] Brooke MH, Kaiser KK. Muscle fibers types: how many and what kind? Arch Neurol 1970; 23:369–79.

[30] Andersen P. Capillary density in skeletal muscles of man. Acta Physiol Scand 1975; 95:203-5. 20

ACCEPTED MANUSCRIPT [31] Lowry OH, Passonneau JV. A flexible system of enzyme analysis. New York: Academic Press; 1972 [32] Weber KT, Janicki JS. Cardiopulmonary exercise testing for evaluation of chronic cardiac failure. Am J Cardiol 1985; 55: 22A-31A.

with coronary artery disease. Ann Rehabil Med. 2011 Jun; 35: 381–87.

RI PT

[33] Kim C, Youn JE, Choi HE. The effect of a self exercise program in cardiac rehabilitation for patients

[34] Moore SA, Jakovljevic DG, Ford GA, Rochester L, Trenell MI. Exercise induces peripheral muscle but not cardiac adaptations after stroke: A randomized controlled pilot trial. Arch Phys Med Rehabil. 2016; 97:

SC

596-603.

2010; 25: 17-27.

M AN U

[35] Allison T, Burdiat G. Pruebas de esfuerzo cardiopulmonar en la práctica clínica. Rev Urug Cardiol

[36] Iellamo F, Manzi V, Caminiti G, Vitale C, Castagna C, Massaro M, et al. Matched dose interval and continuous exercise training induce similar cardiorespiratory and metabolic adaptations in patients with heart failure. Int J Cardiol. 2013; 167:2561-65.

TE D

[37] Hambrecht R, Fiehn E, Yu J, Niebauer J, Weigl C, Hilbrich L, et al. Effects of endurance training on mitochondrial ultrastructure and fiber type distribution in skeletal muscle of patients with stable chronic heart failure. J Am Coll Cardiol 1997; 29:1067–73.

EP

[38] Panth N, Paudel KR, Parajuli K. Reactive Oxygen Species: A Key Hallmark of

Cardiovascular

AC C

Disease. Adv Med. 2016; 2016:9152732. doi:10.1155/2016/9152732. [39] Hambrecht R, Adams V, Gielen S, Linke A, Möbius-Winkler S, Yu J, et al. Exercise intolerance in patients with chronic heart failure and increased expression of inducible nitric oxide synthase in the skeletal muscle. J Am Coll Cardiol. 1999; 33:174-9. [40] Christle JW, Schlumberger A, Haller B, Gloeckl R, Halle M, Pressler A. Individualized vs. group exercise in improving quality of life and physical activity in patients with cardiac disease and low exercise capacity: results from the DOPPELHERZ trial. Disabil Rehabil 2016; 28:1-6.

21

[41] Kureshi F, Kennedy KF, Jones PG, Thomas RJ Arnold SV, Sharma P, et al. Association between

ACCEPTED MANUSCRIPT

cardiac rehabilitation participation and health status outcomes after acute myocardial infarction. JAMA Cardiol. 2016 JAMA Cardiol. 2016; 1:980-8. [42] Neves VJ, Fernandes T, Roque FR, Soci UP, Melo SF, de Oliveira EM. Exercise training in hypertension: Role of microRNAs. World J Cardiol. 2014; 6: 713-27.

RI PT

[43] Sarzynski MA, Burton J, Rankinen T, Blair SN, Church TS, Després JP, et al. The effects of exercise on the lipoprotein subclass profile: a meta-analysis of 10 interventions. Atherosclerosis. 2015; 243:364–72. [44] Amiram A, Timor O. Hanging in the balance: endogenous anti-inflammatory mechanisms in tissue

SC

repair and fibrosis. J Pathol 2013; 229: 250–63.

[45] Kollias HD, McDermott JC. Transforming growth factor-β and myostatin signaling in skeletal muscle. J

M AN U

Appl Physiol 2008 ;104: 579-87.

[46] Vuolteenaho K, Moilanen T, Jalonen U, Lahti A, Nieminen R, van Beuningen HM, et al. TGFβ inhibits IL-1 -induced iNOS expression and NO production in immortalized chondrocytes. Inflamm res. 2005; 54: 420-27.

TE D

[47] Hannan AL, Hing W, Simas V, Climstein M, Coombes JS, Jayasinghe R, et al. High-intensity interval training versus moderate intensity continuous training within cardiac rehabilitation: a systematic review and

AC C

EP

meta-analysis. Open Access Journal of Sports Medicine. 2018;9: 1–17.

22

ACCEPTED MANUSCRIPT TEXTS OF FIGURES FIGURE 1 Correlations between body mass index (BMI) and body fat percentage (% fat) after cardiac rehabilitation.

RI PT

eNOS: endothelial nitric oxide synthase. A: p<0.005. B: p<0.005. C: p<0.05. D: p<0.02.

FIGURE 2

Correlations between transforming growth factor beta (TGF-β) and products and enzymes of nitric oxide.

SC

eNOS: endothelial nitric oxide synthase, nNOS: neuronal nitric oxide synthase. A: Before cardiac rehabilitation p<0.0005. B: After cardiac rehabilitation p<0.05, before cardiac rehabilitation the correlation

M AN U

between these variables was present with similar characteristics (not shown). C: After cardiac rehabilitation, p<0.01. D: After cardiac rehabilitation, p<0.005.

FIGURE 3

TE D

Correlations between transforming growth factor beta (TGF-β) and inflammatory factors. TNFα: tumor necrosis factor alfa, IL-6: interleukin 6. A: Before cardiac rehabilitation p<0.05. B: After cardiac

AC C

EP

rehabilitation p<0.05. C: Before cardiac rehabilitation p<0.01. D: After cardiac rehabilitation p<0.005.

23

ACCEPTED MANUSCRIPT TABLE 1. Comparison between healthy subjects and patients before (BCR) and after (ACR) cardiac rehabilitation, and comparison between patient values BCR and ACR. VARIABLES

Healthy

PBCR

subjects

H vs PBCR

PACR

P

H vs. PACR

PBCR vs.

P

PACR p

45±12

43±13

NS

42±9

NS

NS

% type IIA fibers

39±13

40±9

NS

43±10

NS

NS

% type IIX fibers

16±9

16±8

NS

16±9

NS

NS

Mean CSA (µm2)

5618±1273

3789±1104

<0.001

4399±1534

NS

NS

CSA type I fib(µm2)

6447±1441

4380±1868

<0.02

5237±1530

NS

<0.02

CSA type II fib(µm2)

5568±1331

4193±945

<0.05

5523±1830

NS

NS

CSA type IIX fib (µm2)

5166±1514

4218±1490

NS

5237±2452

NS

NS

Cap Adj. I (n)

4.6±0.8

4.8±0.8

NS

4.7±0.5

NS

Ns

Cap Adj. IIA (n)

4.2±0.8

4.2±0.3

Cap Adj. IIX (n)

3.6±1.0

3.0±0.8

DC (cap x mm2)

308±83

410±98

C x F (n)

1.65±0.29

1.57±0.34

CS (ng/mg-prot)

11.14±2.10

14.32±3.0

HAD (ng/mg-prot)

10.04±1.90

LDH (ng/mg-prot)

196±53.00

Nitrite (ng/mg)

17.44±4.47

Nitrate (ng/mg)

24.04±3.85

NO2- + NO3- (ng/mg)

4 1.83±6.49

Nitrotyrosine(ng/mg)

7.93±3.86

SC

M AN U 4.4±0.3

NS

NS

NS

3.7±0.9

NS

NS

<0.05

411±128

<0.05

NS

NS

1.63±1.59

NS

NS

<0.05

15.90±5.08

<0.05

NS

TE D

NS

10.15±2.6

NS

10.86±3.69

NS

NS

151.1±45.92

<0.05

150.8±58.20

NS

NS

5.64±0.56

<0.000000

5.79±0.70

<0.000000

NS

18.59±3.04

<0.005

20.71±2.00

<0.005

<0.001

24.23±3.21

<0.000000

26.61±2.29

<0.000000

NS

13.70±3.95

<0.001

14.90±5.04

<0.001

NS

EP

AC C

RI PT

% type I fibers

nNOS (ng/mg-prot)

120.00±31.36

12.19±3.99

<0.000000

13.33±3.52

<0.000000

NS

iNOS (ng/mg-prot)

8.05±4.21

14.05±3.60

<0.005

14.31±3.72

<0.005

NS

eNOS (ng/mg-prot)

43.65±31.35

21.92±5.27

<0.05

23.79±6.22

<0.05

NS

TNF-α (pg/mL)

< 5.00

63.67±59.50

<0.05

53.50±46.81

<0.05

NS

TGF-β (pg/mL)

19.66±4.64

162.67±97.61

<0.005

172.20±102.0

<0.005

NS

IL-6(pg/mL)

18.64±4.79

182.17±108.25

<0.005

165.9±106.70

<0.005

NS

CD 154 (pg/mL)

24.32±6.47

130.50±79.09

<0.002

108.4±50.14

<0.002

NS

H: Healthy. PBCR: patients before cardiac rehabilitation. PACR: patients after cardiac rehabilitation.CSA: Cross sectional area. Cap adj..I: Mean number of capillaries around type one fiber. C x F: Capillary per fiber index. DC: Capillary density. NS: no significant.

24

ACCEPTED MANUSCRIPT TABLE 2. Bio-anthropometric data and body composition. Before RC

After RC

P

Age (years)

53.9±8.03

-

-

Weight (kg)

75.6±10.24

74.4±8.62

NS

Height (cm)

168.4±5

-

BMI (kg/m2)

26.3±3.02

26.0±2.58

Fat %

16.8±4.60

15.0±2.58

Waist (cm)

93.3±6.58

92.3±6.30

Hip (cm)

96.9±5.09

95.7±4.73

<0.05

Waist/Hip

0.96±0.06

0.96±0.05

NS

M AN U

SC

RI PT

Variables

NS

<0.001 <0.01

AC C

EP

TE D

BMI: Body mass index.

-

25

TABLE 3. Cardio-circulatory and respiratory variables in exercise test before and after

ACCEPTED MANUSCRIPT rehabilitation. Before RC

After RC

P

Watts

116±15

140±17

<0.001

Exercise time (sec)

836±107

1006±125

<0.001

VE (L/min)

59.8±7.5

73.8±11.6

<0.001

VO2 max (mL/min)

1596±216

1901±223

<0.001

VO2 max(mL/kg/min)

21.2±1.4

25.7±2.5

<0.001

VE/VO2

37.2±5.1

VE/VCO2

31.9±4.5

La max (mM/L)

SC

RI PT

Variables

NS

33.2±3.8

NS

8.1±1.4

9.3±1.5

<0.001

AT (% VO2 max)

53.8±3.5

60.2±3.3

<0.001

Oxymetry (SpO2%)

96.1±1.3

96.0±1.1

NS

HR max (bpm)

135±13

136±15

NS

183±11

186±12

NS

92±5

91±4

NS

DP (SBP x HR)

24673±3085

25286±3708

NS

VO2/HR (mL/beat)

11.7±1.6

14.0±1.9

<0.001

AC C

EP

DBP max (mmHg)

TE D

SBP max (mmHg)

M AN U

38.7±4.0

AT: anaerobic threshold. DP: double product. La max: maximal lactate.

VP: pulmonary ventilation. Max HR: maximal heart rate. VP: pulmonary ventilation. VO2/HR (mL/beat): Oxygen pulse

26

ACCEPTED MANUSCRIPT

TABLE 4.- CORRELATIONS BETWEEN ANTHROPOMETRIC CHARACTERISTICS vs. NO PRODUCTS, ENZYMES AND INFLAMMATORY MARKERS. PRE-REHABILITATION eNOS

NO2-

nNOS

NO3-

NO2-+NO3-

IL-6

RI PT

Weight 0.65*e

0.58*e

NO3-

NO2-+NO3-

-0.58*f

-0.61*f

M AN U

SC

Height BMI FFMII % fat Waist Hip Waist/hip

POST-REHABILITATION nNOS

-0.63*f -0.76**f -0.70*f

IL-6

-0.66*f

TE D

-0.74**f

NO2-0.57**f

-0.56*f

-0.56*f -0.71**f -0.74**f

-0.55*f

EP

Weight Height BMI FFMI % fat Waist Hip Waist/hip

eNOS -0.59*f

AC C

p < 0.05; ** p< 0.01; *** p < 0.001. Negative correlations are preceded by a negative sign. Letters are used to point that signification was: a= similar before and after CR, b= higher after CR, c=lower after CR, d= changed from + to – after CR, e=present only before CR, f= present only after CR

27

ACCEPTED MANUSCRIPT

TABLE 5 CORRELATIONS BETWEEN OXIDATIVE AND GLYCOLITIC ENZYMES, ISOFORMS OF NOS, NO PRODUCTS AND INFLAMMATORY MARKERS PRE-REHABILITATION AND POST-REHABILITATION

NO2-

NO2-+NO3-

PRE-REHABILITATION nNOS iNOS

NITROT.

0.58*d 0.62*b

NO2-+NO3-

TGF-β -0.62*e

IL-6

-0.75**a

0.64*a 0.87**e 0.89***e

-0.76**e

-0.60*a

0.79**e -0.72**e

CD 154

0.52*f -0.57*d 0.59*f 0.96***a

0.58*f -0.70**f

-0.56*a 0.56*b

-0.59*a

0.56*e 0.77**c

eNOS

TNF-α

TGF-β

IL-6

CD 154 0.62*

TE D

0.78***b

NO2-

POST-REHABILITATION NITROT. nNOS iNOS

EP

CS HAD LDH NO2NO3NO2-+NO3NITROT. nNOS iNOS eNOS TNF-α TGF-β IL-6

LDH

M AN U

-0.56*b

HAD

TNF-α

0.64*e

0.99***a

POST.R

eNOS

RI PT

LDH

SC

HAD 0.55*b

AC C

PRE-R CS HAD LDH NO2NO3NO2-+NO3NITROT. nNOS iNOS eNOS TNF-α TGF-β IL-6

0.83***b

0.87***b

0.68**f -0.74**a

-0.84***b

0.65* -0.58*a 0.71**b

0.63*f -0.62*a

-0.56*f

-0.65*

0.65*a

-0.75**f 0.75**f 0.57*f 0.77*f -0.65*a 0.59*c

p < 0.05; ** p< 0.01; *** p < 0.001. Negative correlations are preceded by a negative sign. Letters are used to point that signification was: a= similar before and after CR, b= higher after CR, c=lower after CR, d= changed from + to – after CR, e=present only before CR, f= present only after CR 28

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

29

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

30

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

31

ACCEPTED MANUSCRIPT

Highlights:

After cardiac rehabilitation nitrate levels increased in skeletal muscle. Body fat correlated inversely with higher NO production in rehabilitated patients.

AC C

EP

TE D

M AN U

SC

RI PT

Transforming growth factor β was related with decreased inflammation and higher NO.