4.1. Changes in the Abundance of Proteins Involved in Calcium Homeostasis in the HspB1-NullMouse
The proteomic analysis allowed us to detect two proteins involved in calcium homeostasis as differentially abundant in the mutant mouse, namely, sarcalumenin (Srl) with six spots, and calsequestrin (Casq1).
In skeletal muscle cells, fine regulation of Ca2+
storage, uptake and release is achieved through the concerted action of three major classes of Sarcoplasmic Reticulum (SR) calcium-regulatory proteins: calsequestrin, junctate and sarcalumenin (Srl) for calcium storage; SR calcium release channels such as ryanodine receptors (RyR) for calcium release; and SR Ca2+
–ATPase (SERCA) pumps for calcium reuptake [17
]. Srl are major luminal glycoproteins that codistribute with SERCA and play a role in Ca2+
transport and sequestration [18
]. It maintains muscle Ca2+
homeostasis and is involved in the sequestration of Ca2+
in the non-junctional region of the SR [18
]. The expression of Srl is similar in slow and fast-twitch muscle fibers. After the initiation of muscle contraction by increasing cytoplasmic Ca2+
is pumped back to the SR by SERCA, leading to relaxation. Srl-deficient mice were apparently normal in growth, health, and reproduction, indicating that Srl is not essential for fundamental muscle functions. Srl-deficient skeletal muscle carrying irregular SR ultrastructure retained normal force generation but showed slow relaxation phases after contractions. A weakened Ca2+
uptake activity was detected in the SR prepared from Srl mutant muscle, indicating that Srl contributes to Ca2+
buffering in the SR lumen, and also to the maintenance of Ca2+
pump proteins [18
]. There are two Srl isoforms (160-kDa and 53-kDa) that are generated by alternative splicing [18
]. In the present study, six spots of the 53-kDa isoform were found to be more abundant in the muscle of the HspB1
-null mouse, and this overexpression was confirmed by Western blotting (Table 4
). These spots could correspond to phosphorylation of Srl. In accordance with this hypothesis, Hadad et al.
] revealed the phosphorylation of Srl in cardiac and skeletal muscle by Casein kinase II (CkII). They demonstrated that while the phosphorylation of the purified Srl protein by exogenously added CK II is independent of Ca2+
, the phosphorylation of the SR membrane-associated proteins is absolutely Ca2+
could be involved in the transport of ATP into the SR lumen or in the protein-membrane association-dissociation processes. The phosphorylation of Srl induces modifications in the ryanodine receptor properties, and both the Ca2+
and the ryanodine binding affinities are modified. As there are no indications for a direct interaction between Srl and the RyR, it was suggested that this effect could be done via calsequestrin. Action potentials may elicit contractions by releasing Ca2+
from the SR via the RyR. This latter is modulated directly or indirectly by various ions, small molecules and proteins, including calsequestrin. In the present study, a proteomic analysis of Tibialis anterior
muscle of both groups of mice identified an up-regulation of calsequestrin-1 (Casq1, spot 902) in the HspB1
-null mouse [21
Casq1 is an important regulatory of Ca2+
. It is located in the supramolecular membrane assembly in the terminal cisternae region of muscle fibers. It represents a high-capacity, medium-affinity binding protein that is restricted to junctional SR. Casq1 plays a key role in both homeostasis and terminal cisternae structure and acts as the physiological mediator of the excitation-contraction relaxation cycle through the regulation of the ryanodine receptor complex (Ryr) [22
]. Casq1 isoform is expressed in fast-twitch II fibers at increasing levels from birth to adulthood [23
]. Interestingly, Casq1 knock-out does not alter contractile responses of muscle fibers but induces profound ultrastructure remodeling, reducing the content of the stored Ca2+
and the amplitude of Ca2+
]. This demonstrates the crucial role of the protein for the fine muscle architecture in agreement with the electronic microscopy observations in the m. Soleus
and m. Tibialis anterior
of the HspB1-null mouse [7
signaling mechanisms in skeletal muscle control a multitude of cellular processes (for review [23
ions are well known for their implication in muscle contraction. Under resting conditions, the cytosolic-free Ca2+
level is higher in type I than type II fibers, and this contributes to the structural differences in the composition of fiber types. Type II fibers require higher Ca2+
gradients for muscle contraction; this is consistent with a higher density of RyR channels in type II fibers comparatively to type I.
release is highly regulated by smaller molecules such as ATP and Mg2+
. A major metabolic pathway in skeletal muscle that provides a high amount of ATP generation per time is the anaerobic glycolysis [23
ions contribute to the regulation of glycolysis as they affect the enzymatic speed of several crucial enzymes of this pathway. This is consistent with our results showing modifications of abundances of the protein involved in Ca2+
storage and in glycolysis.
4.2. Changes in the Abundance of Proteins Involved in Energy Metabolism in the HspB1-Null Mouse
Overloaded sarcoplasmic concentrations of Ca2+
are responsible for the activation of muscle metabolism and acceleration of lactate production in postmortem
]. These high Ca2+
concentrations have also been implicated as initiators of apoptosis via some signaling pathways in skeletal muscle [26
]. In this study, seven proteins involved in energy metabolism were reported to be different between the two groups. Moreover, one major result of this study is the very low phosphofructokinase (Pfk) activity observed in the HspB1
-null mouse. Pfk is the main rate-controlling enzyme of glycolysis in various tissues which catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate to yield ADP and fructose-1,6-bisphosphate [25
]. Its activity is controlled by the concentrations of a large number of metabolites, including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate. The down-regulation of Pfk in the present study in HspB1-null mice, together with the finding that lactate accumulates in river prawn following hypoxia [26
], may suggest that this protein may be a key factor in accelerating glycolysis to stimulate anaerobic ATP production in postmortem
muscle. Three enzymes are candidates to be the major actors of this regulation because of their high negative free energy, namely, hexokinase, Pfk, and pyruvate kinase. Of the three, Pfk is considered to be the major regulatory enzyme for glycolysis in skeletal muscle. This energy barrier makes sense as pyruvate kinase catalyzes the final reaction and hexokinase is not involved in glycolysis at all when the process is begun from glycogen. Otherwise, the Pygm could catalyze the degradation of glycogen and lead to technological defects in meat (e.g., Pale, Soft, Exudative (PSE) meat) [27
In humans, a muscular metabolic disorder known as glycogen storage disease type VII, characterized by glycogen storage disease (excess glycogen accumulation), is induced by Pfk deficiency. The symptoms are similar to deficiencies of phosphoglycerate kinase, phosphoglycerate mutase, lactate dehydrogenase, β-enolase and aldolase A. From these data, we can speculate that HspB1
-null mice should be more fatigable and sensitive to physical exercise than their controls. This may be supported by the finding in the present study that the tripartite motif protein 72 protein (Trim72) was more abundant in the HspB1
-null animals compared to wild-type controls. Indeed, Trim72 is abundantly expressed in striated muscle tissues and has been shown to function as a critical component of the cell membrane repair [28
]. We also identified, as shown in the correlation network, interactions between Trim72 with Casq1 (a calcium-binding protein) and Pygm (involved in carbohydrate metabolic process). Moreover, alternative routes of anaerobic carbohydrate catabolism are less efficient for ATP production and probably do not provide enough energy to maintain aerobic consumption in the HspB1
-null mice. This hypothesis needs to be validated.
On another hand, Ca2+ ions are able to modulate Pfk activity by the Ca2+-dependent activation of Calmodulin (CaM) which interacts with Pfk by binding on two binding sites on Pfk monomers. This generates stable Pfk dimers, which exhibit increased catalytic activity of Pfk, in part preventing allosteric inhibition of the enzyme, e.g., by ATP, citrate and lactate. This regulation contributes to an increase of Pfk activity via increased abundance of Ca2+. In the present study, we could speculate that the higher storage of Ca2+ through modifications of Casq1 and Srl abundances could result in a decrease of Pfk activity.
Two mitochondrial proteins of energy metabolism (Mdh1 and Pdhb) were found to be more abundant in the HspB1
-null mouse. The Ca2+
influx into mitochondria increases the energy conversion potential, which is very important for the energetic homeostasis in contracting muscle. The activity of dehydrogenase enzymes such as Mdh1 and Pdhb are dependent on mitochondrial Ca2+
]. As a pluripotent organelle, the mitochondrion plays multiple roles in calcium homeostasis, apoptosis and physiology in postmortem
skeletal muscle [30
]. Under high calcium flux, the integrity of mitochondria would be destroyed, leading to the release of cytochrome C and other pro-apoptotic factors, which finally triggers apoptosis [17
]. This may happen in the HspB1
-null mouse as suggested in Cassar-Malek et al.
Our proteomic analyses therefore provided strong evidences of the enhancement in both glycolytic and mitochondrial energy metabolism in the skeletal muscle of the HspB1-null mouse. Changes in mitochondrial oxidative capacity may be the reason of more abundant metabolic proteins in the mutant mouse.
It has been proposed by several authors [23
] that Ca2+
ions influence the activity of Ca2+
-sensitive phosphatases and kinases. This could explain the differences observed in Ak1 and Ckm abundances in HspB1
-null mice, which could be the consequence of differences in Ca2+
in muscle fibers of the two genotypes. Other roles would be also assumed by these proteins. Ckm is an enzyme found in sarcolemma and in SR of muscle cells, where it is functionally involved in the calcium transport and ATPase activity [32
]. Ckm can also act as an antioxidant by scavenging free radicals [33
]. Antioxidants can minimize meat discoloration by decreasing lipid oxidation, thereby limiting metmyoglobin formation [34
]. The higher expression of Ckm observed in wild-type mouse could possibly be associated with higher demand for energy during hypoxia, but it is not the case for HspB1-null mouse, where numerous enzymes of energy metabolism are highly abundant. Moreover, the increase in Mdh1 in HspB1
-null mouse may therefore indicate a corresponding increase in oxidative phosphorylation capacity [30
Moreover, a down-regulation of one protein spot (Spot 2362) identified as mitochondrial Hsp70 (mtHsp70; also known as GRP75 or HspA9) was found in the HspB
-null mouse. This lower abundance in HspA9 in the HspB1
-null mouse may be linked to better mitochondrial functioning with less reactive oxygen species production. This suggests that the mitochondria (perhaps more intact and efficient) are attempting to maintain function despite the eventual decrease of pyruvate flux into the TCA cycle. HspA9 is the only chaperone described to be regulated by glucose privation, Ca2+
homeostasis and perturbation of glycolysis [35
]. This is consistent with the modifications of the abundance of this enzyme and with the correlations observed in the present study. It plays a key role in the folding of matrix-localized mitochondrial proteins and in the transport of proteins into the mitochondrion [36
]. In cattle meat, HspA9 was related to pH3h postmortem
, ultimate pH (pHu), and L*
color coordinates [37
]. It plays a role in Ca2+
trafficking from the SR to the sarcoplasm, which may also explain relationship between HspA9 and the enzymes of the glycolytic pathway described by Gagaoua et al.
] in cattle. These authors described correlations of this enzyme with proteins of glycolysis such as Mdh1 and Ldh-B, with structural proteins, proteolytic enzymes and anti-oxidant enzymes such as Park7, which was also modified in the HspB1
-null mouse (Table 4
4.3. Changes in the Abundance of Proteins Involved in Other Functions
The bioinformatic network revealed three proteins outside our dataset (Ywhae, Kcnma1, and Hsd3b4) which were highly connected to nine proteins of our dataset mainly protein of energy metabolism.
Among these three proteins, Kcnma1 proteins (Potassium Channel, Calcium Activated Large Conductance Subfamily M Alpha, Member 1), also called MaxiK channels, are composed of two subunits. The physical association between these two units is regulated by intracellular calcium and the activity is activated by both membrane depolarization, cytosolic Mg2 + b
or an increase in cytosolic Ca2+
. GO annotations related to this gene include actin binding and calcium-activated potassium channel activity. These channels selectively transport K+ ions across biological membranes and play a key role in controlling the membrane potential in a number of systems [38
]. Mice with an invalidation of the Kcnma1
gene are characterized by cerebellar ataxia in the form of an abnormal conditioned eye-blink reflex, abnormal locomotion and a pronounced deficiency in motor coordination [39
]. The interaction of this protein with a large number of proteins of our dataset seems to be logical, as we have seen above that the invalidation of HspB1
had consequences for the abundances of several proteins involved in calcium metabolism.
Ywhae gene encodes tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, 14-3-3 epsilon. The 14-3-3 proteins (phospho-serine/-threonine binding proteins) belong to a large, highly conserved family functioning as a dimer in diverse biological processes such as signal transduction, metabolism, protein trafficking, signal transduction, apoptosis, cell cycle regulation and potassium channel regulation [40
]. The 14-3-3 proteins bind to a lot of target proteins, as observed in our bioinformatic network. A phosphoproteome study showed that 14-3-3 proteins could regulate glucose homeostasis in response to insulin or to energetic stress [41
]. They are also known to sequester phosphorylated Bad
in the cytoplasm in healthy cells and inhibit apoptosis. The 14-3-3 epsilon protein may also play a role in regulating microfilament stability during heat stress by maintaining their phosphorylation states [42
]. This protein was also reported to interact with heat shock proteins [42
Hsd3b4 (3beta-hydroxysteroid dehydrogenase type 4) belongs to the 3-β-HSD family. It plays a crucial role in the biosynthesis of all classes of hormonal steroids and catalyzes the conversion of dihydrotestosterone to 5alpha-androstanediol in the presence of the cofactor NADPH. In cattle, Guillemin et al.
] observed a higher abundance of HspB1 in steers (castrated animals) comparatively to young bulls (non-castrated animals). According to this data, we could hypothesize that the androgen pathway could be modified in HspB1
-null mice and that the androgen receptor (Ar) could be a target of HspB1
invalidation. In accordance with this hypothesis, Zoubeidi et al.
] showed that HspB1 in vitro
knock-down induced Ar degradation via the proteasome-mediated pathway. In their study, HspB1
knock-down inhibited the androgen-stimulated nuclear translocation of the Ar with subsequent suppression of the Ar-regulated gene expression. A recent report also indicates that Hsp27 is a mediator in the repression of Ar function [49
]. In cattle, Guillemin et al.
] also showed lower abundance of HspA8, HspA9, and Park7 in steers compared to young bulls. In accordance with the hypothesis of the modification of the androgen pathway in HspB1
-null mice, we observed lower abundance of HspA8 and Park7 (Table 4
), and of HspA8 in HspB1
-null mice, confirming the hypothesis of relationships between these proteins. Altogether, these data suggest that Hsp27/Ar interactions could contribute to modulate the abundance of these proteins as indicated in the present study.
Altogether, our results show that the invalidation of the HspB1 gene has direct or indirect consequences mainly on cellular homeostasis of calcium, energetic metabolism and apoptosis. These biological functions are also those that have an important role in beef sensory qualities such as tenderness.
4.4. Implications for Meat Quality
The results of the present study suggest relationships between Hsp27 and ATP production and, hence, the energy supply of contracting muscle regulated by the Ca2+
-dependent enhancement of glycolytic enzyme activity and mitochondrial respiration. A relation between Hsp27 abundance and glycolytic activity has been previously described. For example, Liu et al.
], in the skeletal muscle of Korean chickens, described an increase of glycolytic metabolism associated with the up-regulation of Hsp27. By proteomic analysis, these authors found also modifications of Srl abundance associated with modifications of Hsp27, and of enzymes of glycolytic metabolism, as in the present study. As a consequence, the difference in glycolytic metabolism can cause a differential rate of pH drop (due to the accumulation of lactate), which may affect directly protein degradation and meat quality traits (e.g., tenderness and water-holding capacity).
In cattle, we observed an inverse relationship between small Hsp, in particular Hsp27, and tenderness according to the type of muscle. In fast glycolytic muscle like the Tibialis anterior
, Hsp27 was negatively correlated with tenderness, while fast or glycolytic proteins such as MyHC IIx and Ldh were positively correlated with tenderness. In a slow oxidative muscle these relationships are reversed [51
]. This illustrates an opposite relationship between Hsp27 and proteins representative of fast glycolytic type in agreement with the present results showing higher abundance of some fast glycolytic proteins in the HspB1
-null mice. In the present study, we failed to find differences in the abundances of small Hsps, namely, Hsp20 and αB-crystallin, described in the m. Soleus
of the HspB1
-null mouse [6
]. This suggests a muscle-specific effect of the invalidation of HspB1
. Consistently, an inverse postmortem
evolution of Hsp27 according to muscle type has been described [52
The modifications in Ca2+
homeostasis and of energy metabolism observed consequently to the invalidation of HspB1
gene could be linked to postmortem
modifications of muscle with crucial role on the establishment of meat quality. The postmortem
degradation of ATP, and the increase of the activity of glycolytic enzymes are associated with pH drop [53
]. These events are controlled by muscle Ca2+
homeostasis. Therefore, it is well documented that proteins involved in Ca2+
regulation are important for meat quality [54
]. Aberrant calcium regulation in early postmortem
period was reported to be associated with inferior meat quality [55
]. For example, birds with PSE meat failed to respond to heat stress because of a delay in the up-regulation of the Ca2+
-regulating genes Ryr
. It is also known that fast glycolysis and a rapid buildup of lactate in early-stage postmortem
muscle may be a direct cause of the PSE defect [56
]. Thus, insight into the metabolism pathways will help us to understand and prevent the occurrence of PSE meat. Otherwise, the presence of elevated sarcoplasmic Ca2+
concentrations soon after animal bleeding in PSE meat has been well documented [57
]. The components of calcium channels located in the membrane of SR have been demonstrated to be correlated with the variation of calcium concentration in sarcoplasm of many tissues, including skeletal muscle [17
]. For example the expression variance of SERCA can induce skeletal muscle Ca2+
disorders and PSE meat appearance [58
]. Further, functional and morphological disorders of mitochondria were found in PSE meat [59
], which were considered to induce or represent the appearance of apoptosis in skeletal muscle [60
]. Another interesting link between apoptosis and muscle-to-meat conversion relies to the Ca2+
content of muscle cells. Earlier studies reported the acceleration of the tenderization process in meat administered with exogenous Ca2+
]. The protein 14.3.3 epsilon, for which a role in meat tenderness has never been described in the literature, was found with a higher abundance in group of Longissimus thoracis
muscles of high tenderness in the French Blonde d’Aquitaine beef breed [61
]. Consequently, a role in tenderness could be explained by its implication in the protection of cells from apoptosis.
On the other hand, in meat, it is well documented that skeletal muscle alterations induced by modification of Ca2+ flux in SR during ageing result in protein proteolysis involving ultra-structural modifications. α-actin is one of the myofibrillar proteins the most concerned by this proteolysis. As Hsp27 is known to protect α-actin, lower α-actin abundance in the muscle of HspB1-null animals could be expected. This was confirmed by Western blotting analysis in the present study.
Moreover, Pfk, whose activity was profoundly decreased in the HspB1
-null mutants, plays a role in coupling glycolysis to many metabolic pathways, and would affect the technological traits of meat. For example, Krischek et al.
] reported that Pfk activity was greater in pork with a faster pH drop.