Identification of hemostatic genes expressed in human and rat leg muscles and a novel gene (LPP1/PAP2A) suppressed during prolonged physical inactivity (sitting)

Theodore W Zderic 0 Marc T Hamilton 0 0 Inactivity Physiology Department, Pennington Biomedical Research Center , LA, Baton Rouge 70808 , USA Background: Partly because of functional genomics, there has been a major paradigm shift from solely thinking of skeletal muscle as contractile machinery to an understanding that it can have roles in paracrine and endocrine functions. Physical inactivity is an established risk factor for some blood clotting disorders. The effects of inactivity during sitting are most alarming when a person develops the enigmatic condition in the legs called deep venous thrombosis (DVT) or coach syndrome, caused in part by muscular inactivity. The goal of this study was to determine if skeletal muscle expresses genes with roles in hemostasis and if their expression level was responsive to muscular inactivity such as occurs in prolonged sitting. Methods: Microarray analyses were performed on skeletal muscle samples from rats and humans to identify genes associated with hemostatic function that were significantly expressed above background based on multiple probe sets with perfect and mismatch sequences. Furthermore, we determined if any of these genes were responsive to models of physical inactivity. Multiple criteria were used to determine differential expression including significant expression above background, fold change, and non-parametric statistical tests. Results: These studies demonstrate skeletal muscle tissue expresses at least 17 genes involved in hemostasis. These include the fibrinolytic factors tetranectin, annexin A2, and tPA; the anti-coagulant factors TFPI, protein C receptor, PAF acetylhydrolase; coagulation factors, and genes necessary for the posttranslational modification of these coagulation factors such as vitamin K epoxide reductase. Of special interest, lipid phosphate phosphatase-1 (LPP1/ PAP2A), a key gene for degrading prothrombotic and proinflammatory lysophospholipids, was suppressed locally in muscle tissue within hours after sitting in humans; this was also observed after acute and chronic physical inactivity conditions in rats, and exercise was relatively ineffective at counteracting this effect in both species. Conclusions: These findings suggest that skeletal muscle may play an important role in hemostasis and that muscular inactivity may contribute to hemostatic disorders not only because of the slowing of blood flow per se, but also potentially because of the contribution from genes expressed locally in muscles, such as LPP1. - Introduction Low muscular activity has been associated with cardiovascular diseases [1,2]. In contrast to the more established literature on the effects of formal types of exercise, there is an emerging interest in the underlying molecular responses to physical inactivity and the more common non-exercise activities such as standing and low-intensity physical activity [3]. Deep venous thrombosis (DVT) can occur from an acute lack of normal ambulatory activity [4-6], and we are unaware of any evidence that it can be prevented by exercise [7,8]. For example, it was first reported over 60 years ago that sitting in London air raid shelters was associated with a 6-fold increased incidence of DVT [6]. Then, Homans noted in 1954 that the spontaneous thrombosis which almost exclusively occurs in the legs is due to a state imposed by airplane flights, automobile trips, and even attendance at the theater [4]. There was a report in that same decade of a patient with thrombosis occurring as a result of prolonged sitting while the patient was watching television [5] and the insidious risks of typical amounts of sitting or other sedentary behavior has continually been observed [9,10]. Currently, there are ~2 million cases of DVT reported each year in the United States, leading to 250,000 deaths due largely to pulmonary embolism [11]. What distinguishes DVT from other forms of thrombosis is that it occurs deep in the muscle of the legs. Because the hypercoagulable state develops locally within the legs, there has been a long held supposition that dysregulation of cellular processes locally in the leg muscle tissue may somehow contribute to the risk of DVT in the legs. Support for this proposition would be buttressed by the detection of significant hemostatic gene expression (mRNA) in human skeletal muscle. The first inkling for this came from a recent report that there is a pool of tPA protein (tissue plasminogen activator) localized within the skeletal muscle tissue [12], consistent with the muscle producing tPA via local gene expression, but not ruling out the possibility that the protein was delivered to muscle from the circulation. In addition, a more recent study reported that skeletal muscle expressed not only tPA but also 3 additional hemostasis related genes (CTAP III, tetranectin, and PAI-1) in overweight men with metabolic syndrome [13]. Giving a further hint of a role for skeletal muscle in hemostasis, the expression of these genes was altered by 9 months of exercise training to result in a pattern that would be consistent with less risk of thrombosis [13]. As little is known about the role of skeletal muscle in hemostasis at this point, determining if skeletal muscle expresses a number of hemostasis related genes may open the door toward a new insight for the role of this tissue in hemostasis. Therefore, the first aim of this study was to comprehensively examine what hemostasis genes are expressed in skeletal muscle through the use of microarray technology. Furthermore, we sought to determine if any of these expressed genes would be altered by commonly used models of reduced contractile activity in the legs (hindlimb unloading in rats and sitting in humans). A translational study of rodent and human skeletal muscle was performed given the eventual need for physiological studies inducing thrombosis in experimental animal models while manipulating expression of select hemostatic genes. The identification of a hemostatic gene(s) impacted by prolonged inactivity like sitting may be important to investigate as a candidate for a role in the pathogenesis of DVT. Methodology Rat experiments Animal procedures were approved by an Institutional Animal Care and Use Committee and performed in respect of the American Physiology Society principles for research on animals. Female Sprague Dawley rats (Harlan) weighing approximately 200 g were housed individually (450 cm x 241 cm) in a temperature- and lightcontrolled environment (12:12 hrs lightdark cycle). The soleus muscle (deepest skeletal muscle in the rat hindlimb) was obtained under anesthesia (16.2 mg ketamine, 0.66 mg xylazine, 1.05 mg acepromazine) from all rats and frozen in liquid nitrogen after the completion of the following described physical activity/inactivity treatments. Altogether, 89 rats were used in these studies. In the first set of experiments, the effect of the acute (12 hrs) removal of normal standing/ambulatory activity on (...truncated)