Mitochondrial and sarcoplasmic reticulum abnormalities in cancer cachexia: Altered energetic efficiency?

Background: Cachexia is a wasting condition that manifests in several types of cancer, and the main characteristic is the profound loss of muscle mass. Methods: The Yoshida AH-130 tumour model has been used and the samples have been analyzed using transmission electronic microscopy, real-time PCR and Western blot techniques. Results: Using in vivo cancer cachectic model in rats, here we show that skeletal muscle loss is accompanied by fiber morphologic alterations such as mitochondrial disruption, dilatation of sarcoplasmic reticulum and apoptotic nucleus. Analyzing the expression of some factors related to proteolytic and thermogenic processes, we observed in tumor-bearing animals an increased expression of genes involved in proteolysis such as ubiquitin ligases Muscle ring finger 1 ( MuRF-1) and Muscle Atrophy F-box protein (MAFBx ) . Moreover, an overexpression of both sarco/endoplasmic Ca 2+ -ATPase (SERCA1) and adenine nucleotide translocator (ANT1) (factors related to cellular energetic efficiency) was observed. Tumor burden also leads to a marked decreased in muscle ATP content. Conclusions: In addition to muscle proteolysis, other ATP-related pathways may have a key role in muscle wasting, both directly by increasing energetic inefficiency, and indirectly, by affecting the sarcoplasmic reticulum-mitochondrial assembly that is essential for muscle function and homeostasis. General Significance: The present study reports, for the first time?, profound morphological changes in cancer cachectic muscle, which are visualized mainly in alterations in sarcoplasmic reticulum and mitochondria. These alterations are linked to pathways that can account for some of energy inefficiency associated with cancer cachexia.

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INTRODUCTION
In advanced malignant diseases, cachexia appears to be one of the most common systemic manifestations. The presence of cachexia always implies a poor prognosis, having a great impact on the patients' quality of life and survival [1]. The skeletal muscle loss is the main characteristic of cancer cachexia and the principal cause of function impairment, fatigue and respiratory complications [2]. Several important molecular mechanisms have been shown to be involved in the increased muscle catabolism observed in cancer-induced cachexia, such as greater ubiquitin-proteasome-dependent proteolysis, apoptosis, and activation of uncoupling proteins [3]. Interaction of these mechanisms leads to muscle-mass loss by promoting protein and DNA breakdown and energy inefficiency.
Skeletal muscle is a very heterogeneous tissue and is used to respond to a broad range of functional demands in each animal species. It represents approximately 50% of the whole total weight and plays a central role in the whole-body metabolism [4]. Consequently, the loss of muscle in catabolic syndromes, such as cancer cachexia, represents a devastating condition not only for patient's quality of life, but also as a surgical risk and also decreasing the response to chemotherapy [3,5]. It is important to emphasize that approximately 30% of body weight loss represents a 75% of muscle loss. This factor leads to patient death, and it is the most prominent phenotypic feature in cancer cachexia [6]. In many years, the experts are challenging to understand what mechanisms are involved in the maintenance of muscle mass for the development of strategies to attenuate the wasting and improve muscle function [7].
It is known that mitochondria and sarcoplasmic reticulum (SR) have a key role in the muscular function. This advantageous assembly reflects the Ca 2+ releasing from SR, stimulating mitochondrial ATP production helping to meet increased energy demand during muscle contraction, a process called excitation-contraction (EC) coupling [8]. On the other hand, functionally intact mitochondria inhibit undesired localized SR Ca 2+ release by controlling the local redox environment of the calcium release units (for a review, see ref. [8]). Thus, bidirectional SR-mitochondrial communication provides a powerful local control mechanism for integrating Ca 2+ release/reuptake and ATP utilization during muscle contraction with ATP production and skeletal muscle bioenergetics [9].
Reduced thermodynamic efficiency will result in an increased weight loss. The laws of thermodynamics are silent on the existence of variable thermodynamic efficiency in metabolic processes.
Therefore such variability can be related to differences in weight loss. An alteration of energy balance is the immediate cause of cachexia [10]. Although alterations of energy intake are often associated with cachexia, it has lately become clear that increased energy expenditure is the main cause of wasting associated with different types of pathological conditions, such as cancer, infections and chronic heart failure. Different types of molecular mechanisms contribute to involuntary body weight loss [11].
Taking into consideration that skeletal muscle loss is the most prominent characteristic of cancer cachexia, and that the mitochondria and SR have an important role in the muscle function and energetic metabolism [2], the purpose of this work has been to examine if a dysregulation of mitochondria and SR functions could be involved in the development of cancer cachexia in animals bearing cachectic tumors, analyzing the putative pathways involved in energy efficiency and homeostasis.

Animals
5 weeks old male Wistar rats (Interfauna, Barcelona, Spain) were maintained at 22 ± 2 °C with a regular light-dark cycle (light on from 08:00 a.m. to 08:00 p.m.) and had free access to food and water.
The diet (Panlab, Barcelona, Spain) consists of 63.9% carbohydrate, 14.5% protein, and 4% fat (the residue was non-digestible material). Food intake was measured daily. All animal manipulations were conducted in accordance with the European Community guidelines for the use of laboratory animals.

Tumor inoculation
Rats were divided into two groups, namely controls (n=6) and tumor hosts (n=7). The latter received an intraperitoneal inoculum of 10 8 AH-130 Yoshida ascites hepatoma cells obtained from exponential tumors [12]. 8 days after tumor transplantation, the animals were weighed and anesthetized with an i.p. injection of ketamine/xylazine mixture (3:1) (Imalgene and Rompun respectively). The tumor was harvested from the peritoneal cavity, and its volume and cellularity were evaluated. Several tissues were rapidly excised, weighed, and frozen in liquid nitrogen except for the muscles processed for electron microscopy and histology (see below).

Histology, SDH staining and total activity
During rat sacrifice, the EDL muscles were rapidly excised and divided in two parts in the mid-belly region, half directly frozen in liquid nitrogen for the enzymatic activity, half mounted in OCT and frozen in melting isopentane for histology. Ten micrometers of transverse sections were cut on a cryostat and later stained for SDH (succinate dehydrogenase) incubating for 30 min at 37°C with 1 mg/mL NTB (nitrotetrazolium blue chloride) and 27 mg/mL Na-succinate in PBS. Afterwards the slides were washed three times in PBS, mounted with glycerol and photographed at different magnifications. Fiber crosssectional area (CSA) was determined on randomly chosen 100 individual fibers (for both oxidative and glycolytic ones) by the Image J software.
As for the total SDH activity, the muscles were homogenized (5% wt/vol) in ice-cold 150 mM NaCl, 10 mM KH 2 PO 4 , 0,1 mM EGTA, 2 times x 30 sec. using a turrax device and centrifuged 5 min. at 800 x g.
The supernant was collected and total protein content measured using the BCA protein assay (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). 50µL of protein homogenate were incubated with 200 µL reaction buffer containing 10 mM Na-succinate, DCPIP 50 µg/mL, 10 mM phosphate buffer (pH 7,4), 2 mM KCN, 10 mM CaCl 2 , 0,05% BSA. The absorbance at 600 nm was measured at t0, 3 min and 20 min after the addition of proteins. The rate of disappearance of the absorbance between 3 and 20 min was corrected for the total protein loaded and used to calculate the SDH content.

Transmission Electronic Microscopy
Muscle pieces of 1 mm 2 were removed under a stereomicroscope and transferred to glass vials filled with 2% parafomaldehyde and 2.5% glutaraldehyde in phosphate buffer. They kept in the fixative during 24 h at 4ºC. Then, they were washed with the same buffer and potsfixed with 1% osmium tetroxide in the same buffer containing 0.8% potassium ferricyanide at 4ºC. Then the samples were dehydrated in acetone, infiltrated with Epon resin during 2 days, embedded in the same resin orientated for longitudinal sectioning and polymerised at 60ºC during 48 hour. Semithin sections were made in order to look for muscle fibers at light microscope. When they were found, ultrathin sections were obtained using a Leica Ultracut UC6 ultramicrotome and mounting on Formvar-coated copper grids. They were staining with 2% uranyl acetate in water and lead citrate. Then, sections were observed in a JEM-1010 electron microscope (Jeol, Japan) equipped with a CCD camera SIS Megaview III and the AnalySIS software. Intermyofibrillar mitochondrial morphology was classified into unchanged and altered (swelling-related ultraestructural changes). Mitochondrial counting was performed from 25-30 micrographs, which were fields randomly taken at 20000x magnification, from three different areas of one grid.

ATP measurement
The determination of ATP using bioluminescence was performed using the commercially available were confirmed in each correspondent group. Primer sequences were designed through Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
The Operon tool (https://www.operon.com/oligos/toolkit.php) was used to check the putative primer-dimer formation. All reactions were performed with Power SyBR ® Green Master Mix (Applied Biosystems) according to manufacturer's instructions. The relative amount of transcripts was calculated using comparative C T method and Acidic Ribosomal Phosphoprotein P0 mRNA was used for normalization [13].

Western Blot
Protein isolations from EDL and GSN muscles were performed using the Mitochondria Isolation

Statistical analysis
Statistical analysis of the data was performed by means of one-way analysis of variance (ANOVA).

RESULTS AND DISCUSSION
The implantation of the Yoshida AH-130 ascites hepatoma caused a marked decrease in body weight, together with an important decrease in skeletal muscle mass represented through 27% of extensor digitorius longus (EDL) and 25% of gastrocnemius (GSN) and 24% of tibialis when compared with control animals ( Figure 1A; Table S1). These changes were accompanied by a significant decrease in food intake (18%). These data agree with previous results from our own laboratory [12].
A thorough analysis of muscle morphology in EDL cross sections of controls and AH-130 bearing rats shows that muscle atrophy occurs in both fast and slow twitch muscle fibers. In fact, the cross section area is dramatically reduced in both oxidative and glycolytic fibers (SDH positive and negative, respectively; Figure  Fiber cross-sectional area (CSA) was determined on oxidative and glycolytic fyber types from EDL muscles indicating that in both fiber types atrophy is present ( Figure 1C and 1D). The results are indicated as percentage of fiber number of different areas and it could be observed that in tumour-bearing animals there are an increased number of fibers with small area. It is also observed that total SDH activity is lower in EDL from tumour-bearing animals pointing out that there was a damage in the mitochondria of this group.
Tumor burden induced important morphological changes at the level of skeletal muscle. In  Figure 3J, 3K, 3L). Cristae were also often whorled ( Figure 3J).
Muscle morphology in the tumor-bearing animals also included sarcomere disorganization, nucleus with an irregular membrane and chromatin compaction ( Figure 3H, 3I), features that characterize the apoptotic process. Indeed, apoptosis is linked to muscle wasting during cancer, both in animals [14] and human subjects [15]. As can be observed in Figure 2, in addition to a clear mitochondrial damage, the SR is dilated, with profound alterations in the triad structure ( Figure 2L). It is interesting to observe that satellite cells show nucleus with apoptotic morphology similar to what is observed in GSN muscle fibers ( Figure 3G and I). Previous studies have shown mitochondrial defects associated with the development of myopathies [16,17]. The results presented here agree with those that link apoptosis with higher mutation rate in mitochondrial genes and an increased mitochondrial mass in human myopathies that are associated with intolerance to exercise, defects in the energy production and muscle weakness [18].
New experiment PAZ: Additionally to the quatification of area and number of mitochondrias, the percentatge of normal and altered mitochondria have been also measured. As can be seen in figure 4, EDL and GSN from tumour-bearing animals showed an increased number of altered mitochondria compared with the control muscles. This result is in accordance to the decreased ATP content (in GSN) and to the decreased SDH activity (in EDL) in tumour-bearing muscles. Table 1 shows the consequences of tumor burden on pattern of gene expression in both GSN and EDL muscles. It can be seen that tumor-bearing rats manifested a marked increase in expression for genes related to muscle proteolysis such as ubiquitin (UB) (by 5.4-fold and 3.4-fold for EDL and GSN, respectively), muscle specific ubiquitin ligases MuRF-1 (by 3-fold and 5.5-fold for EDL and GSN, respectively) and MAFBx (by 6.2-fold and 5.4-fold for EDL and GSN, respectively), and also calpain-2 (2.2fold for both muscles). These results agree with previous results from our own group, where it showed that muscle proteolysis is a common mechanism linked to cachexia present in cancer. [12,19], Recent studies have shown that MAFBx ligase seems to be involved in the direct degradation of MyoD protein and myofibrillar proteins, whereas MuRF-1 seems to be involved in the degradation of thick filaments [20,21]. Calcium-dependent proteases (calpains) also seem to play a role in the muscle wasting process [22].
SR and mitochondria have an important inter-relationship and the orchestral regulation of share pathways between these organelles is critical for Ca 2+ signaling in EC coupling, for the maintenance of cellular homeostasis, for energy supply and for cellular fate under stress conditions [8,9]. Based on the morphological alterations in these organelles found in cancer cachexia, we decide to analyze if the disruption of SR-mitochondrial communication could be an important factor to explain muscle dysfunction and posterior muscle loss in cancer cachexia. By means of real time PCR and Western Blot techniques, the expression of some targets connected to this hypothesis in EDL and GSN muscles was analyzed.
An increased expression of ryanodine receptor 1 (RyR1) gene was observed, as shown in the homeostasis, leading to a rise in body temperature and muscle rigidity upon exposure to a fluorinated inhalation anesthetic such as halothane [23]. Moreover, the increase of RyR1 and its dysregulation has been shown in muscle weakness and fatigue in aging [24,25]. Therefore, it can be speculated that overexpression of RyR1 can increase the Ca 2+ leak from SR in cachexia and can be involved in the stress responses, that triggers proteolytic and apoptotic processes [23,25].
It is particularly interesting to remark on the significant increased expression in both SERCA1 and 2 genes in EDL muscles in the tumor-bearing animals, as shown in Table 1. In GSN muscles, tumor burden also resulted in an increased SERCA1 gene expression by 3.6-fold ( exchange with a high thermodynamic efficiency across the SR membrane [26,27]. In skeletal muscle, SERCA1 has the capacity to interconvert different forms of energy. Thus, SERCA1 activity may be coupled to Ca 2+ translocation from the cytosol to the SR, a process that requires a considerable amount of energy, since it represents a dynamic process against a concentration gradient. Some of the energy associated with the activity of the pump is released as heat [28]. Interestingly, the pump may also function in an uncoupled way, releasing just heat without translocating Ca 2+ [26,29]. In fact, the rate of uncoupled ATPase activity is much higher than the coupled one. It is for this reason that SERCA1, in addition to being involved in Ca 2+ translocation, has also been related to non-shivering thermogenesis [30]. Conversely, in SERCA2 most of the hydrolytic ATP-derived energy is coupled to Ca 2+ transport to the lumen of the SR [28]. It is clear that in adult mammals, despite a small heat production in brown adipose tissue, the major contributor for thermogenesis is the skeletal muscle since it represents about 50% body weight. The increased gene expression of SERCA1 was accompanied by increased protein content both in EDL (by 4fold, Figure 3A) and GSN (by 3.6-fold, Figure 3A) muscles from tumor animals. SERCA2 protein content was also increased in EDL muscles ( Figure 3C). It may thus be suggested that SERCA, in particular SERCA1, could be involved in the energetic inefficiency that characterizes the cachectic condition [10]. In fact, upregulation of SERCA1 in skeletal muscle has been described in disuse atrophy [31]. It is also very interesting to remark that ATP consumption by SERCA pumps accounts for 50% of resting metabolic rate in mice [32]. If we take into consideration the fact that cancer patients have an increased energy expenditure [33], which contributes to weight loss, it becomes clear the importance that SERCA pumps may have in cancer cachexia. In addition, previous studies have shown that an increased cytosolic ADP concentration, due to an increased SERCA activity, leads to Ca 2+ leakage from the SR in fast-twitch muscles, effectively reducing the capacity of SERCA to maintain an adequate Ca 2+ intracellular gradient, and thus leading to increased energetic inefficiency [34].
It is interesting to note that, in addition to the energetic implications of SERCA, it may play a key role in apoptosis. From this point of view, it was characterized a truncated variant of the SERCA1, S1T, that amplifies ER stress, determines endoplasmic reticulum Ca 2+ depletion due to increased Ca 2+ leak, an increased number of endoplasmic reticulum-mitochondria contact sites, and inhibition of mitochondria movements that leads to trigger apoptotic processes [35]. Considering the present morphological results that show the apoptotic processes in cachectic muscles and also previous studies [14], the altered SRmitochondria interactions could be involved in muscle dysfunction and cell death observed in cachexia.
In the mitochondrial internal membrane, the adenine nucleotide translocator (ANT) carries out the ATP/ADP exchange between cytoplasm and mitochondrial matrix. Interestingly, there are four different ANT isoforms (ANT1, ANT2, ANT3 and ANT4) but ANT1 is the predominant isoform present in skeletal muscle and, although not normally included as a part of the OXPHOS system, it is a key protein regulating the mitochondrial ATP/ADP flux [36]. It seems that the activity of the transporter may be intimately linked to uncoupling of the respiratory chain, basically due to proton leakage across the inner mitochondrial membrane and, therefore, disruption of the electrochemical proton gradient that drives ATP synthesis in the mitochondria [37]. Interestingly, our results show an increased ANT1 gene expression in both GSN and EDL muscles during tumor growth (2.2 and 2-fold respectively), as shown in Table 1. These increases in mRNA content are accompanied by significant elevations in protein content ( Figure 3D, E). These alterations may be an indication of uncoupling in mitochondria from tumor-bearing animals. ANT1 also seems to play a key role in the pathogenesis of fascioscapulohumeral muscular dystrophy, one of the most common hereditary muscle diseases, where ANT1 is significantly elevated in muscle resulting in mitochondrial dysfunction associated with oxidative stress, leading to progressive weakness involving the face, shoulders, hips and feet [38]. In addition, [39] have reported an increased expression of ANT1 in soleus muscles undergoing immobilization-induced atrophy. Based on above evidences, the high expression of ANT1 in cachexia can be linked to an improved proton leak that leads to a decrease of ATP production, maybe at the beginning of the cachectic stage. In addition to its role in wasting, there is a connection between ANT1 and apoptosis. ANT1 has been known to be a major component of the permeability transition pore complex (PTP), a high conductance channel that cause an abrupt increase in the permeability of small solutes across the inner mitochondrial membrane, contributing to mitochondria-mediated apoptosis [40]. In any case, ANT1 could therefore have an important role in muscle apoptosis during tumor burden [14,15].
The consequences of the abnormal SR-mitochondrial morphology and molecular profile of expression observed in cachectic muscle were reflected in total muscle ATP content. Indeed, as can be seen in Figure 3F, it was significantly reduced by 17% in the tumor-bearing animals. This could be explained taking into consideration an impairment of oxidative capacity of mitochondria, leading to PTP formation and apoptotic signals. Similar observations have previously been reported [41]. In fact, very recently, a defect in ATP synthesis during cancer has been described by our group, directly linked to mitochondrial dysfunction [42].
It is worth mentioning the increased gene expression of mitofusin-2 (Mfn2) and proliferatoractivated receptor gamma coactivator (PGC-1α) in EDL muscles of tumor-bearing rats (Table 1). Mfn2 is a mitochondrial protein involved in the regulation of mitochondrial morphology and distribution [43].
Moreover, Mfn2 is related to the interactions between endoplasmic reticulum and mitochondria which govern interorganellar Ca 2+ signaling, having a key role in endoplasmic reticulum morphology [44].
Moreover, the SR-mitochondrial apposition performed by Mfn2 predisposes mitochondria to high Ca 2+ microdomains and to the consequent overloading, leading eventually to apoptosis by excessive Ca 2+ transfer [45,46]. Furthermore, it is known that PGC-1α participates in the stimulation of Mfn2 expression under a variety of conditions characterized by enhanced energy expenditure (for review, see ref. [47]).
Hence, in cachexia the overexpression of PGC-1α can activate Mfn2 expression, this leading to a Ca 2+ dysregulation and formation of permeability transition pore (PTP), which is involved in apoptotic processes. It is known that Bax, a proapoptotic gene, is overexpressed in cachectic muscles [48]; therefore the apoptotic signals in cachexia could also be started by Mfn2 and Bax.
Finally, considering that SERCA and ANT1 are related to energy dissipation in SR and mitochondria, it may be suggest that they could have a role in energy inefficiency associated with cachexia. This process could induce SR stress by high and constant citoplasmic Ca 2+ levels, having dramatic consequences for the muscle cell, thus leading to a self-amplifying circuitry between SR and mitochondria by Mfn2 that eventually results in muscle death. The increased Ca 2+ levels induce a decrease in ATP content not only by OXPHOS dysfunction, but also through thermogenic processes, such as those performed through ANT1. Recent evidences describes SERCA not only as a heat pump, functioning like a thermogenic device in the skeletal muscle, but also as a directly regulator of Ca 2+ signaling in mitochondria [49,50]. These facts lead to suggest that the main energy dissipation observed in cachexia could be related to an increased expression of SERCA, more precisely by implication of SERCA reactions that produce heat and also lead to Ca 2+ leak. The proton leak activity of UCP3 [51] and ANT1 into mitochondria can worsen the catabolic condition in cachexia, potentiate the ADP accumulation [34], Ca 2+ leak and heat production as represented in the Figure 4. The constant Ca 2+ leakage from SR and disruption of mitochondrial functions amplify subsequently the degradation signals, triggering proteolytic processes through calpain, cathepsins and Ub-Proteasome systems, and apoptotic signals performed by PTP formation through ANT1 and Bax [48].

CONCLUSIONS
The present study reports, for the first time, profound morphological changes in cancer cachectic muscle, which are visualized mainly in alterations in SR and mitochondria. posar alguna cosa més de morfologia de ME !!!! These alterations are linked to pathways that can account for some of energy inefficiency associated with cancer cachexia. It is known that SR-mitochondrial assembly is crucial for muscle activity, where SR Ca 2+ release stimulates the mitochondrial ATP production to supply the increased energy demand during EC coupling [9]. EC uncoupling is a term used to describe a highly reproducible and specific phenomenon, in which the normal EC process in a skeletal muscle fiber is disrupted [52]. This phenomenon is thought to be implicated in situations such as ageing, muscle fatigue and some muscle diseases [52,53]. Altogether, the present data suggests that EC uncoupling could be an event previous to activation of proteolytic processes in catabolic conditions. Indeed, the factors implicated in EC uncoupling are strictly related to SR and mitochondrial disruption. This condition can activate proteolytic and apoptotic signals, leading to muscle atrophy in cachexia. The therapeutic implications of understanding the commented assembly and the energy dissipation pathways involved, such as SERCA and ANT1 are very obvious and deserve future research and could well lead to new approaches for the treatment of cancer cachexia.

Figure 1
A: EDL muscle weight from control (C) and tumor-bearing (AH-130) rats. B: graph showing the reduction of total SDH activity in AH-130 vs C rats. C: micrographs (4x and 20x magnification) of EDL cross sections stained for SDH activity. Scale bar: 200 µm. D: fiber CSA independently measured in oxidative and glycolytic fibers. Statistical significances were analyzed using one-way ANOVA (* p<0.05; *** p<0.001).

Figure 2
Electron micrographs depicting morphological mitochondrial differences between extensor digitorum longus (EDL) muscles from control and tumor-bearing rats.
A-F: EDL from control rats. G-L: EDL from tumor-bearing rats. Scale bar: indicated. Graphs are showing differences in intermyofibrilar mitochondria's area and number between control and tumor groups.
Mitochondrial counting was performed from 25-30 micrographs, which were fields randomly taken at 20000x magnification, from three different areas of one grid. Statistical significances were analyzed using one-way ANOVA (*** p<0.001).

Figure 3
Electron micrographs depicting morphological mitochondrial differences between gastrocnemius (GSN) muscles from control and tumor-bearing rats.
A-F: GSN from control rats. G-L: GSN from tumor-bearing rats. Scale bar: indicated. Graphs are showing differences in intermyofibrilar mitochondria´s area and number between control and tumor groups.
Mitochondrial counting was performed from 25-30 micrographs, which were fields randomly taken at 20000x magnification, from three different areas of one grid. Statistical significances were analyzed using one-way ANOVA (* p<0.05; *** p<0.001).

Figure 4
Ultraestructural changes in mitochondrial morphology in gastrocnemius and EDL muscles from control and tumor-bearing rats.
A. Pictures A-B: EDL muscle; and C-D: Gastrocnemius muscle. A,C: Control. B,D: Tumor. White arrows are pointing to swelling mitochondrias. Magnification 20000x.
B. Percentage of unchanged and altered (including electro-lucent and swelling alterations) mitochondrias. Statistical significance of the results was determined using one-way ANOVA (* p<0.05; ** p<0.01).

Figure 5
A, SERCA1 protein content in extensor digitorum longus (EDL) muscles. B, SERCA1 protein content in gastrocnemius (GSN) muscles from control and tumor-bearing rats. C, SERCA2 protein content in extensor digitorum longus (EDL) muscles. D, ANT1 protein content in EDL muscles. E, ANT1 protein content in GSN muscles from control and tumor-bearing rats. Na + , K + -ATPase alpha subunit was used as invariant control.
F, Differences of ATP content in GSN muscle between control and tumor-bearing rats. Results are expressed as µM ATP/g of GSN muscle. For further details, see Materials and Methods. Columns, mean of four animals in each group; bars, SE. The results are expressed as a percentage of controls. Statistical significances were analyzed using one-way ANOVA (* p<0.05; ** p<0.01).