RICERCA ITALIANA     

Molecular and cellular mechanisms underlying exercise-induced protection

Università degli Studi di Milano

Abstract

1. This project originates from the observation that chronic moderate aerobic exercise (training) reduces the incidence of the major cardiovascular and degenerative diseases. Although training has became an integrative part of preventive and rehabilitative medicine in the most advanced countries, there is still considerable space for targeted basic studies:
1.1 The molecular mechanisms underlying training-induced protection are not yet well understood. For example, for what concerns myocardial protection, it is unclear which factor(s) play(s) the most important roles among preconditioning (i.e., the development of transitory resistance to severe insults as a result of prior exposure to moderate insults), development of collateral coronary arteries, induction of heat shock proteins, improved anti-oxidant capacity and blood coagulation control.
1.2 It is unclear whether training-induced protection is attributable to training per se or to the associated lifestyle changes. For example, abstinence from smoking, moderate food and beverage intake and decreased psychological stress are variables that, although concurring in eliciting protection, tend to confound the cause-effect mechanisms.
1.3 The mechanisms underlying loss of protection after training cessation (detraining) are poorly elucidated. This issue is relevant not only for practical purposes, but it is also useful in order to understand to a larger extent the molecular mechanisms mediating protection: determining if the disappearance of the effects elicited by training is associated with the higher or lower expression of key proteins might help to clarify the most critical proteins that induce protection.

2. With the aim to better understand these issues, we will use as an animal model a homogenous population of rats exposed to moderate training at an intensity and duration comparable to that of humans regularly practising physical exercise at an amateur level. Training in rats will be suitably characterised with physiologic and morphologic parameters. At the end of training (3 months), a group of animals will be sacrified, whereas a second group will be evaluated one month after the cessation of training (detraining). In both groups, as well as in a control group, the following parameters will be monitored:
2.1 Development of cardioprotective mechanisms against ischemia-reperfusion injury.
2.2 Alterations in capillary vessel formation and morphological alterations in skeletal and cardiac muscles.
2.3 Gene expression (intracellular mRNA accumulation and protein quantitative assessment) relative to stress response, angiogenesis, antioxidant capacity, thrombosis and hemostasis.
2.4 Evaluation of the events occurring in response to damage, such as apoptosis, the modulation of which might provide information on cytoprotection in muscles and other tissues.

3. The work will be divided among four Units, which boast a range of complementary scientific and technological skills in various biomedical fields. Animal training and biological samples preparation will be performed by the same Unit to ensure uniformity. The other Units will perform the various assays and analyses using previously acquired skills, techniques and equipment. The project is expected to expand our knowledge of the molecular and cellular mechanisms involved in training-induced protection not only in muscles, but also in other organs and tissues. On a long-term perspective, the identification of the molecules that are primarily involved in training is expected to suggest strategies to induce cytoprotection in people unable to perform adequate training (obese patients, and patients with neurological damage, osteo-articular conditions and uncompensated heart failure). <<<

Principal Investigator

Arsenio VEICSTEINAS Università degli Studi di MILANO

Research Objectives

1. Analysis of the problem. The epidemiological findings, as well as the basic hypotheses raised from studies in human, animal and in vitro models, are reviewed in the "Background" section. These findings support the idea that moderate, regular aerobic physical exercise (training) reduces the weight of a number of risk factors for cardiovascular (coronary heart disease, ictus, hypertension) and degenerative (atherosclerosis, type II diabetes) diseases. Training, therefore, should trigger protective mechanisms for several tissues, especially the myocardium. Despite the considerable epidemiological evidence, however, the molecular mechanisms underlying this phenomenon have not yet been fully clarified.

2. Aim of the project. To emphasise the role of training in eliciting cytoprotection, all the potential confounding variables not related to training must be either eliminated or minimised. To this aim, we propose an animal model where male rats from a homogeneous population are subjected to training of intensity and duration comparable to that of humans who take physical regular exercise. At the end of the training and detraining periods, the rats will be sacrificed and various tissues will be harvested for a number of biochemical, molecular, morphologic and physiologic analyses.

3. Primary objectives:
3.1 To confirm that training is protective in terms of myocardial resistance to ischemia-reperfusion injury independently of the associated life style factors.
3.2 To assess how the gene expression of some proteins mediate the body response to stress, improve the antioxidant activity, ameliorate muscle morphology (new capillary formation) and optimize the control of blood coagulation.
3.3 To determine the loss of the protective effects elicited by training after its cessation (detraining).

4. Limits of the project. In this project, we will not investigate the correlation between the training intensity and the extent of protection or resistance to stress, the effects of the diet (antioxidants or food with risk factors) and the time course changes of the effects of training and detraining. These aspects will be investigated in future projects, which will be designed accordingly to the results obtained in this project and might include the effect of factors such as gender and age.

5. Specific aims. Given that we have already determined the best protocol to train experimental animals for 12 weeks, in order to attain the primary objectives above described, we plan an extensive investigation into the following parameters:
5.1 Metabolic and cardiovascular adaptation to training.
5.2 Morphology (histology, histochemistry and immunohistochemistry) of trained muscle.
5.3 Assessment of the damage to myocardium and skeletal muscle cells, both involved (to assess direct effects) and not involved (to assess remote effects, if any) in training, as well as in organs as liver and blood components.
5.4 Induction of protection against ischemia-reperfusion injury in the myocardium using the isolated perfused rat heart model.
5.5 Intracellular accumulation of mRNAs encoding stress proteins, transcription factors, growth factors, pro and anti-apoptotic proteins in tissues directly and non-directly involved in exercise.
5.6 Development of angiogenesis and new capillary formation.
5.7 Control of thrombosis and hemostasis.

6. Perspectives. Attaining the above objectives has a number of long-term consequences. First, we expect to get new information related to a phenomenon that is not yet fully supported by scientific data. Second, we will assess how molecular and cellular mechanisms are involved in training-induced protection and in cytoprotection. We expect that assessing to a greater accuracy the role of the molecules involved in training-induced cytoprotection might suggest strategies aimed at inducing cytoprotection in persons unable to perform adequate exercise, e.g., patients affected by neurological or osteo-articular pathologies, severe physical disabilities, chronic heart failure and obesity.

7. Strategy. A single Unit will be responsible of the training protocols in a single animal species, thereby reducing to a reasonable minimum the individual variability. This Unit will distribute the material to the other Units. All Units will concentrate on specific analyses in which they are expert, using already operative equipment and techniques. Four Units characterised by complementary competence and experience will carry out the project. This will lead to an integrated interpretation of the results in terms of cell paths involved in the generation of training-induced cytoprotection. The project will be split into two phases. In phase 1, we plan to assess the role of training. In phase 2, we will assess the role of detraining. Both phases will use adequate control groups and will be based on a statistically significant number of animals. <<<

First Results

The end of this Phase will match two primary objectives of this project:
- Confirming the hypothesis that aerobic training is protective irrespectively of life style.

- Investigating the expression of key proteins and genes that potentially mediate the training-induced response, change the antioxidant response and alter muscle morphology.Results expected from Phase 2
The end of Phase 3 will match the last of the primary objectives:
- Determining to what extent the protective effects of training persist after training cessation for one month.
At the end of this Phase we will also evaluate possible further development of this project. Though rooted to knowledge already acquired by the involved Units, the present project is innovative in many aspects and is likely to open new lines of study. For example, if exercise results to be protective, further studies will be needed to determine the relationships between training intensity or frequency and the extent of protection. Other questions may concern the effects on the exercise-induced protection of dietary components, such as antioxidants or caloric uptake, age, gender and common disease risk factors (e.g., hyperglycemia, hypertriglyceridemia, hypercholesterolemia). The present project makes an effort to accelerate the transfer to human applications and may constitute the starting point for the identification of specific markers that will enable to develop kits or microarrays for the assay of such markers in blood samples from humans. The results of this project will also help to understand the molecular and cellular mechanisms underlying the body response to exercise, and could have several practical and technological implications. The knowledge of the metabolic pathways involved in exercise-induced cytoprotection may provide suggestions for the development of drugs for the pharmacological activation or inhibition of those pathways, leading to cytoprotection in subjects who cannot take regular exercise (e.g., bedridden, paraplegic, and obese patients), thereby contributing to improving human health. <<<

Timescale

24 months

National and international background

1 Epidemiology of moderate physical exercise. Notwithstanding increasing evidence that moderate physical exercise prevents the development and progression of chronic degenerative diseases such as atherosclerosis, coronary heart disease and type II diabetes mellitus (Haennel and Lemire, 2002; Marliss and Vranic, 2002), most people in industrialised countries customarily lead sedentary lives. The beneficial effect of moderate regular exercise on the main risk factors for cardiovascular disease was documented by a number of epidemiological studies, including the Multiple Risk Factor Intervention Trial (Leon et al., 1987), the Harvard Alumni Study (Paffenbarger et al., 1993), the Lipid Research Clinic Mortality Follow-up Study (Ekelund et al., 1988) and several large scale prospective studies (Blair et al., 1989). These studies clearly indicate that physically active people have lower incidence of myocardial infarction and greater survival from heart attack compared with sedentary counterparts (Berlin and Colditz, 1990). Exercise programs in sedentary people must be carefully tailored, and the recommendations contained in the 2001 revision of the 1995 guidelines proposed by the American College of Cardiology and the American Heart Association (Fletcher et al., 2001) suggest that a three times a week volume of exercise of 1400 Kcal/week is necessary and sufficient to produce the expected benefits (American College of Sports Medicine, Resource Manual for guidelines for exercise testing and prescription 1998). However, it is likely that under a certain threshold there is little effect, whereas above another threshold the cardiovascular risk might outweigh the benefits.

2 Experimental models to study exercise as a protective factor. Recommending exercise to improve protection against degenerative diseases requires full clarification of the mechanisms whereby exercise produces favourable effects on the cardiovascular system. Factors as age, sex, race, habits, smoking, stress, cultural background, work, diet, and individual motivation, unavoidable variables in most human studies, make it difficult to design trials that focus solely on the protective mechanisms of exercise. Animal studies have the advantages that the cellular, metabolic and molecular mechanisms can be investigated easily, the genetic makeup is uniform and life-style as well as anthropomorphic and individual choices can be ruled out.

3. Hypotheses. Although it is clear that exercise is capable of reducing risk factors associated with disease, the mechanisms responsible for exercise-induced protection continues to be debated, and the major hypotheses that explain, at least in part, the resistance to stress conferred by exercise are now discussed.

3.1 Hypertension. Exercise clearly reduces the risk of developing hypertension (Shephard and Balady, 1999) and lowers blood pressure in mildly hypertensive men (Pescatello et al., 1991). It is thus likely that exercise is responsible for altering the central command of local and global blood flow. It is known that alterations in baroreceptor sensitivity during exercise contribute to decrease post-exercise resting blood pressure. The transient nature of the baroreceptor setpoint ultimately emphasizes the importance of exercise to improve blood pressure control. Related to blood pressure, local control of blood flow is also a function of mechanistic alterations in vascular endothelial tissue as discussed below. Collectively, the literature consensus indicates that exercise is beneficial in reducing the risk of hypertension as a cardiovascular risk factor through these and other mechanisms.

3.2 Glucose metabolism and obesity. Exercise has beneficial effects on glucose metabolism and insulin sensitivity (Shephard and Balady, 1999). Although the underlying mechanism is not fully clarified, exercise improves glucose transport across muscle cell membranes by up-regulating glucose transporters and decreasing liver glucose release. Exercise also improves substrate utilization thereby reducing the dependence on blood and liver glucose stores. Furthermore, clear links are established between exercise and obesity, adult-onset diabetes and weight loss. Exercise-induced weight loss often results in lower blood fatty acids concentration, reduced Langerhans beta-cell stimulation and lowered insulin secretion. Nonetheless, most studies show only modest body fat loss with exercise alone, but when caloric restriction is added to exercise, the average weight loss is 3-4 times greater than with exercise alone. Loss of body fat via diet and exercise is significant in reducing cardiovascular risk because body composition and fat distribution are linked to cardiovascular mortality (Blair, 1993).

3.3 Ischemia-reperfusion (I/R) injury. Cardioprotection is a major hallmark among the general beneficial effects induced by exercise. The debated ischemic preconditioning hypothesis (Yellon and Downey, 2003) attributes protection to the development of transitory resistance to severe insults elicited by prior exposure to one or more insults of moderate duration or severity. Initially developed for the heart, today the preconditioning hypothesis has been confirmed in several organs and tissues, including liver (Carini and Albano, 2003), brain (Dirnagl et al., 2003), skeletal muscle (Quan et al., 2004), intestine (Wu et al., 2004) and others, with essentially the same basic features. By increasing resistance to I/R, exercise may also be considered a form of preconditioning (Bolli, 2000). Importantly, the protection elicited by preconditioning may be effective with a 24-48 h delay after trigger to exploit what is called the "second window of protection" (Kis et al., 2003).

3.3.1 Oxidative injury. Free radicals are highly reactive molecules with an unpaired electron in their outer orbital. The superoxide anion, produced by molecular oxygen univalent reduction, is a well known oxygen-derived radical that promotes formation of other dangerous reactive oxygen species (ROS) as peroxides and hydroxyl radicals. ROS cause several types of oxidative injury targeted at lipids and proteins (Demirel et al., 1998; Powers et al., 1998). Lipids are damaged via reaction with polyunsaturated fatty acids, which results into radical propagation with formation of carbon-based radicals, which alters membrane fluidity and increase their permeability. Damage to proteins, marked by formation of carbonyls groups on the aminoacid side chains, leads to structural and functional alterations that impair several cell functions. It is now widely accepted that ROS are produced in great amounts early during I/R and contribute to cytotoxicity and tissue damage. However, ROS may also activate important metabolic signalling pathways. Low concentrations of ROS and reactive nitrogen-based species are indeed critically implicated in the control of a number of pathological processes (Carmody and Cotter, 2001; Hancock et al., 2001). ROS burst during I/R, and possibly during exercise, may therefore result into increased expression and activity of mitochondrial complexes (Sammut et al., 2001), antioxidants and stress proteins (Fehrenbach and Northoff, 2001; Radak et al., 2000; Sen, 2001). The up-regulation of these target genes and deployment of their products would allow cells, tissues and organs to withstand stress successfully (Bolli, 2000; Maulik and Das, 2000). Under basal conditions, 2-3% of the oxygen consumed is not completely reduced (Halliwell and Gutteridge, 1998) and plasma hydrogen peroxide level may be up to 5 micromoles/L (Frei et al., 1988). When the system is overloaded, as it may happen during exercise, it is likely that ROS generation increases, thereby increasing the protection against successive stressful conditions.

3.3.2 Heat shock proteins. The temporary increase in body temperature during exercise may induce the synthesis of heat shock proteins (HSPs), molecular chaperones that protect other proteins against denaturation by any agent that alters their native folding (Marini et al., 1996; Yu and Chung, 2001), which may include ROS. An ancestral mechanism of protection, this path is well conserved in all cell lines, thereby potentially explaining why the response to exercise involves various organs. Among the several HSPs, HSP70 is a recognised cytoprotective agent during myocardial I/R, but now it appears dubious that HSPs expression alone is sufficient to induce protection because skeletal muscle fibres in which HSP70 is constitutionally overexpressed following gene transfer are not resistant to I/R (Lepore et al., 2001) and I/R resistance is not associated to HSP70 overepxression (Ronchi et al., 2004). Furthermore, if exercise is performed in the absence of any body temperature increase, HSP70 is not increased despite exercise-induced cardioprotection, indicating that cardioprotection does not depend on HSP70 expression alone (Taylor et al., 1999). Nevertheless, hearts isolated from trained animals, when exposed to I/R, recover better than those from sedentary animals (Milano et al., 2001). Another protein thought to potentially play an important role in mediating exercise-induced protection, e.g., GRP94 (Gorza and Vitadello, 2000), was found to be unaffected by exercise (Gorza et al., unpublished data, see figure below. E=exercise, C=control, GRP78 is the reference protein).


3.3.3 Angiogenesis. Exercise increases the number of small blood vessels by stimulating angiogenesis (Laufs et al., 2004), a recognised phenomenon during embryonic development, placenta formation, wound healing and tumour growth. Exercise also improves the regulation of arterial tone and platelet aggregation, most likely by increasing the release of vasoactive factors, especially nitric oxide (NO) (Bowles and Wamhoff, 2003). NO inhibits vascular contraction, leukocyte adhesion, vascular smooth muscle cell growth, and platelet aggregation (Rubio and Morales-Segura, 2004). Therefore, besides its positive effect on the fibrinolytic system, by decreasing the likelihood of thrombotic events (Shephard and Balady, 1999), exercise is predicted to reduce the risk of coronary thrombosis by improving the intrinsic control of coronary vascular resistance. Early studies reported that 6 months of regular endurance exercise in old patients is associated with significant improvement in haemostatic parameters, e.g., reduced fibrinogen, increased tissue plasminogen activator and reduced plasminogen inhibitor-1 (Stratton et al., 1991). Another study reported that 12 weeks moderate intensity exercise in middle-aged men reduced platelet aggregation by 52% (Rauramaa et al., 1986). Collectively, epidemiological studies indicate that exercise reduces the risk of thrombosis, but again the underlying molecular mechanisms are not yet fully clarified.

3.3.4 Vascular endothelial growth factor (VEGF). One of the most important angiogenic factors, VEGF increases vascular permeability, promotes endothelial cells proliferation and new vessels growth, thereby increasing local blood flow (Ferrara et al., 2003), particularly in skeletal and heart muscles, by binding to its cognate receptor VEGFR-1 (LeCouter et al., 2003). Whereas for some Authors exercise clearly increases the expression of angiogenesis-promoting genes (Laufs et al., 2004), others were unable to demonstrate changes in rat skeletal muscle capillary density after 8 weeks training, whereas the same training protocol could increase capillarisation when carried out under hypoxia (Olfert et al., 2001). The angiogenic response to exercise is important as it occurs in patients with chronic heart failure, where the utility of exercise is still matter of controversy. VEGF and VEGF mRNA levels in muscle biopsies almost doubled after 8 weeks of training in patients with chronic NYHA class II-III heart failure, while the capacity for exercise was also increased by 36% (Gustafsson et al., 2001).

4 Reversibility of protection (detraining). Although the persistence of the protection induced by training has not been determined with precision, the dynamics of the reversal of the cardiovascular and protective effects induced by training is primarily important to assess the efficacy of exercise-induced protection. When elderly master athletes were subjected to three months detraining, clinically silent signs of ischemia were revealed by thallium scintigraphy in 3 out of 10 subjects after three months inactivity (Begum and Katzel, 2000). These signs were considered predictive of the appearance of cardiovascular events over a 5-year follow-up. The ongoing large-scale STRRIDE (Studies of a Targeted Risk Reduction Intervention through Defined Exercise) study is seeking to determine both the optimal volume and intensity of training in humans so as to minimise the cardiovascular risk, and the effects of 2-week detraining on the reversibility of the protective effects (Kraus et al., 2001). Besides its evident practical importance, studying detraining has important basic implications, because one can correlate the loss of protection with the corresponding alteration in some of the investigated markers, each of which is expected to have its own time-course. For example, endothelin-1 and nitrites/nitrates return to pre-training levels after eight-week detraining (Maeda et al., 2001), while the lipid profile related to reduced risk of cardiovascular events (as increased HDL-cholesterol) returns to normal after two-week inactivity (Mankowitz et al., 1992). By contrast, the training-induced increase in maximal oxygen consumption completely disappears after four-week detraining, while the exercise-induced changes in carbohydrate metabolism, insulin sensitivity and glucose transporters return to baseline in about 10 days (Mujika and Padilla, 2000). Also, capillary density and the activities of oxidative enzymes return to pre-training levels after about one-month detraining (Mujika and Padilla, 2001). Finally, lack of correlation between HSP72 and catalase, and loss of exercise-induced cardioprotection after 18 days detraining might lead to reconsider the role of these factors in exercise-induced protection (Lennon et al., 2003) <<<