Ingeniería inversa 

Exercise-induced muscle damage from contact related sport can occur in two ways. Firstly the eccentric loading of the muscle causes sarcomere disruption in myofibrils and damage to the excitation-coupling system (Proske and Morgan 2001). Secondly blunt force trauma causes structural integrity loss of the muscle cell membrane which results in the release of intramuscular constituents into the plasma. Muscle damage is followed by regeneration, repair and adaptation, and is associated with primary inflammation, cell proliferation, apoptosis and the formation of scar tissue (Huard et al., 2002). This subsequently reduces muscle force production (Connolly et al., 2003; Howell et al., 1993) and motor neuron activation and sensitivity (Prasartwuth et al., 2005). Significant concern arises when blunt force trauma occurs from a high force impact common in contact sport that can result in catastrophic loss of muscle cell integrity resulting in significant health concerns including rhabdomyolysis (Brown 2004).

Quantification of skeletal muscle damage severity following exercise is one of the more common measurements of stress. Markers generally include myoglobin, CK, carbonic anhydrase III (CAIII), skeletal troponin I (sTnI), lactate dehydrogenase (LDH), aspartate aminotransferase and aldolase. Careful consideration following intense exercise has to be given to the effect of oxidative stress on muscle damage which will be discussed in a later section. For the purpose of this section, only direct intramuscular constituents will be discussed.

Serum concentrations of CK have been significantly increased following running of varying durations and intensities (Kanter et al., 1988; Ostrowski et al., 2000; Rahimi et al., 2010a), eccentric loading (Nosaka and Clarkson 1996; Nosaka and Newton 2002) and used as a marker of overload in professional soccer players (Lazarim et al., 2009). They all seem to indicate its release is intensity and duration dependent (Kanter et al., 1988; Strachan et al., 1984). Specifically, its concentrations tend to become more significantly elevated with physical impact (Ehlers et al., 2002; Hoffman et al., 2002) while identifying rugby and rugby league as sports that induce severe muscle damage (Cunniffe et al., 2010; McLellan et al., 2010, 2011a; Smart et al., 2008). Meanwhile, LDH, CAIII and sTnI have all been shown to increase following a marathon or eccentric loading exercise (Fu et al., 2009; Kobayashi et al., 2005; Nosaka et al., 1992; Sorichter et al., 1997). Whilst these markers provide an effective means of understanding the level of muscle damage associated with exercise, they are predominantly quantified in serum or plasma and have comparatively slow kinetics which makes them obsolete for the acute and immediate nature of this research.

1.5.2 Myoglobin

Myoglobin is a 17 kDa single-chain oxygen-carrying hemoprotein restricted to cardiomyocytes and oxidative skeletal myofibers. The detection of myoglobin in the bloodstream is a diagnostically relevant measurement indicative of muscle damage (Nelson and Cox 2000). It facilitates O2 delivery from the intra-capillary erythrocyte to the

mitochondria in order to maintain oxidative phosphorylation for myocardial contractility. Myoglobin deficient mice have been found to be viable with preserved cardiac function due to their ability to mount a complex compensatory response involving increased vascularization, induction of the hypoxia gene program, reduced cell width, elevated

hematocrit and increased coronary flow and coronary flow reserve (Grange et al., 2001; Mammen et al., 2003; Meeson et al., 2001). Myoglobin’s other potential roles also include acting as an O2 reservoir, a cytoprotective protein against reactive oxygen species (ROS), and

as a modulator of nitric oxide (NO) (Flögel et al., 2001; Kojda and Kottenberg 1999; Trochu et al., 2000).

Myoglobin’s use in the clinical field is critical for diagnosis of patients with rhabdomyolysis (Feinfeld et al., 1992) or acute kidney injury (Zager and Burkhart 1997). It’s extremely fast elimination kinetics (Suzuki et al., 1999) and relatively fast time to peak in comparison to CK (Mikkelsen and Toft 2005) provides a sharper response and immediate opportunity to gauge muscle damage severity. It is a sensitive and credible muscle damage marker of choice in rugby union due to the number of high force impacts (Smart et al., 2008). It is rapidly filtered by the glomeruli and reabsorbed by the proximal tubules where it is catabolized as a result of its small globular size (Bagley et al., 2007; Hamilton et al., 1989). When the filtered load exceeds the re-absorptive capacity of the tubule, myoglobin spills over into the urine, colouring it red (Beetham 2000; Don et al., 1997). However it is only when concentrations exceed 100 mg/dL that urine becomes discoloured by myoglobin (Gabow et al., 1982). This is corroborated by a study on naval officers who demonstrated concentrations from 3.2 – 410 mg/L following training exercises where coffee-brown discolouration was noted (Smith 1968).

Furthermore, urinary myoglobin is notorious for its instability. Temperature, pH, unidentified urinary compounds smaller than 10kDa, short half-life, metabolism to bilirubin, dissociation at acidic pH and time to analysis have all been identified as causes for myoglobin instability (Alterman et al., 2007; Chen-Levy et al., 2005; Gabow et al., 1982; King et al., 2010; Naka et al., 2005; Wu et al., 1994). It has been recommended that urine samples be adjusted to an alkaline pH (8 - 9.5), analysed immediately with avoidance of multiple freeze-thaw cycles to eliminate potential loss (Chen-Levy et al., 2005).

1.5.2.3 Exercise Effect

Similar to other muscle damage markers, myoglobin release is intensity and duration dependent. A study investigating the effects of 25 triathletes in competition, noticed those

with the fastest finishing time had the highest myoglobin concentration (Thomas and Motley 1984). In comparison, during a 48 hour adventure race, myoglobin was observed to continually increase throughout the duration (Wichardt et al., 2011). Myoglobin has also been shown to increase in response to intense resistance exercise resulting in black discolouration of the urine and eight days in hospital (Moeckel-Cole and Clarkson 2009), downhill treadmill and outdoor running (Peake et al., 2005; Sorichter et al., 1999), as well as duathlon, ironman and cycling (Neubauer et al., 2008; Sugama et al., 2012; Suzuki et al., 1999). Relevant to this research, Takarada (2003) observed a significant increase in plasma myoglobin concentration following two games of elite amateur rugby union that correlated with the number of impacts each player experienced. This implies that the blunt force trauma of the impacts, whether they are tackles, ball carries or ruck related, seem to cause ultra- structural damage to the muscle cells resulting in myolysis rather than damage through eccentric loading.

The elimination kinetics of myoglobin are unique characteristics that provide an advantage over other markers. Cycling for 90 minutes at 90 watts (W) saw myoglobin concentrations peak at both one and three hours post (Suzuki et al., 1999). Running on a treadmill at a gradient of - 10 % also resulted in myoglobin peaking between one and three hours (Peake et al., 2005), while myoglobin also peaked 45 minutes following the cessation of rugby play (Takarada 2003). In contrast, sustained myoglobin increases have been observed up to 24 hours after an ironman race that also showed signs of increase in certain individuals as much as 19 days later; a potential sign of impaired recovery (Neubauer et al., 2008). This elevation has been similarly observed 48 - 72 hours following six sets of 85 % maximum voluntary contractions (Tseng et al., 2012). A cooling aid was applied in the latter study that may have delayed recovery. Based on the literature, myoglobin may provide an instantaneous assessment of muscle damage severity that is superior to the delayed rise in other conventional markers (Cunniffe et al., 2010).

Exercise induced myoglobin release can also contribute to the syndrome known as exertional rhabdomyolysis. This is a potentially life-threatening syndrome characterised by the breakdown and necrosis of striated skeletal muscle resulting in the subsequent release of potentially toxic intracellular contents into the systemic circulation (Giannoglou et al., 2007; Khan 2009; Sauret et al., 2002). This can occur directly through blunt force trauma that

directly disrupts the cell membrane resulting in release of its constituents, or through an increase in intracellular free ionized calcium to a level much higher than normal in the cytoplasm and mitochondria (Giannoglou et al., 2007). It has been observed in several sports including American Football, swimming, body-building and running (Clarkson 2007; Do et al., 2007; Galvez et al., 2008; Kraemer et al., 2013), with myoglobin often contributing to the detrimental effects. Myoglobinuria however does not occur without rhabdomyolysis, but rhabdomyolysis does not necessarily lead to visible myoglobinuria (Khan 2009).

Acute renal failure (ARF) is a complication of rhabdomyolysis that is dependent on the severity and duration of renal dysfunction. It can lead to chronic renal failure, damage to the heart or nervous system, and death (Bagley et al., 2007; Hamilton et al., 1989; Huerta- Alardín et al., 2005). Myoglobin has been associated with renal dysfunction through several possible mechanisms. If levels exceed the protein-binding capacity of the plasma, myoglobin can precipitate in the glomerular filtrate. In conjunction with uric acid, this can lead to intraluminal casts, increased intra-tubular pressure, and subsequently decreased glomerular filtration rate (Don et al., 1997; Huerta-Alardín et al., 2005; Vanholder et al., 2000; Zutt et al., 2014). It also has nephrotoxic effects at acidic pH where it dissociates into ferrihaemate and globin which potentiates acute tubular necrosis (Khan 2009; Naka et al., 2005), alongside the heme group enhancing renal vasoconstriction and ischemia through activation of the cytokine cascade (Beetham 2000; Huerta-Alardín et al., 2005). Together with the large increases in myoglobin reported in contact related sports, these mechanisms may contribute to ARF in professional athletes.