MD Maria-Christina Zennaro
email: maria-christina.zennaro@inserm.fr

Primary aldosteronism Takes (KCNJ)Five!

Primary aldosteronism (PA) is the most common form of endocrine hypertension, due to autonomous aldosterone production from the adrenal cortex. Patients with PA typically present with hypertension, high plasma aldosterone levels associated with low plasma renin activity, and varying degrees of hypokalemia and metabolic alcalosis [START_REF] Funder | Case detection, diagnosis, and treatment of patients with primary aldosteronism: an endocrine society clinical practice guideline[END_REF]. The prevalence of PA increases with the severity of hypertension and is currently estimated around 10% in referred patients and 4% in primary care [(2) and references therein; [START_REF] Plouin | Trends in the prevalence of primary aldosteronism, aldosterone-producing adenomas, and surgically correctable aldosterone-dependent hypertension[END_REF]], but as high as 20% in patients with resistant hypertension [START_REF] Douma | Prevalence of primary hyperaldosteronism in resistant hypertension: a retrospective observational study[END_REF][START_REF] Calhoun | Hyperaldosteronism among black and white subjects with resistant hypertension[END_REF]. The two main causes of PA are aldosterone producing adenoma (APA) and bilateral adrenal hyperplasia (BAH), also called idiopathic hyperaldosteronism. Efficient and timely screening for PA is of major importance, given the severe cardiovascular outcome of aldosterone excess that is independent of blood pressure levels [START_REF] Rossi | Primary aldosteronism: cardiovascular, renal and metabolic implications[END_REF][START_REF] Milliez | Evidence for an increased rate of cardiovascular events in patients with primary aldosteronism[END_REF]. Yet, the pathogenic mechanisms leading to aldosterone hypersecretion and cell proliferation are largely unknown.

Recently, a few recurrent somatic mutations of the KCNJ5 gene, coding for the potassium channel Kir3.4 have been implicated as a cause of APA, while an inherited mutation was identified in a family with a Mendelian form of early severe hypertension which features massive adrenal hyperplasia and is referred to as familial hyperaldosteronism type 3 [FH3, [START_REF] Choi | K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension[END_REF][START_REF] Mulatero | A new form of hereditary primary aldosteronism: familial hyperaldosteronism type III[END_REF]]. These mutations all lie near or within the selectivity filter of the Kir3.4 channel; they result in a loss of channel selectivity, with increased sodium conductance leading to membrane depolarization. These changes are presumed to be responsible for constitutive aldosterone secretion and cell proliferation by promoting opening of membrane voltagedependent calcium channels which is followed by activation of the calcium signaling pathway, the main trigger for aldosterone production in adrenal zona glomerulosa cells. In this issue of Endocrinology, Oki et al now formally establish a causal relationship between KCNJ5 mutations and hyperaldosteronism. They demonstrate that the inherited KCNJ5 T158A mutation produces a marked stimulation in aldosterone biosynthesis which is dependent on membrane depolarization followed by calcium influx into adrenal cortical carcinoma cells (Oki et al, 2012).

The paper by Oki et al indeed tackles a central issue that had not previously been addressed, i.e. the causal link between KCNJ5 mutations, membrane depolarization and aldosterone overproduction and cell proliferation. By transiently infecting adrenocortical HAC15 cells with a lentivirus expressing wild type KCNJ5 or mutated KCNJ5-T158A, the authors show that expression of channels harboring the T158A mutation potentiated basal aldosterone production, which was further stimulated by angiotensin II and the protein kinase A activator, forskolin. Using different fluorescent dyes, they confirmed the enhanced sodium influx through the mutated Kir3.4 channel, leading to membrane depolarization and increased intracellular calcium concentrations. KCNJ5-T158A transduced cells presented significantly increased expression of CYP11B2, the gene coding for aldosterone-synthase which ensures the last three enzymatic steps of aldosterone biosynthesis. Expression of CYP11B1, coding for 11β-hyroxylase, was also increased, as was basal and stimulated cortisol production. KCNJ5-T158A also induced significant production of 18-oxocortisol, a hybrid steroid largely produced in affected members of the original family with FH3 [START_REF] Geller | A novel form of human mendelian hypertension featuring nonglucocorticoidremediable aldosteronism[END_REF]. CYP11B2 expression and aldosterone production were both inhibited by calcium channel antagonists and calmodulin inhibitors, confirming the mechanistic link to activation of the calcium signaling pathway.

While these results confirm the pathogenic role of the KCNJ5 T158A mutation in promoting aldosterone-overproduction, the same does not hold true for its effects on cell proliferation. Indeed, KCNJ5-T158A had an inhibitory effect (~30% reduction) on HAC15 cell proliferation, in stably infected cells, an effect which was not due to increased apoptosis and was unrelated to calcium signaling. Interestingly, a similar negative effect of KCNJ5 mutations on cell proliferation was recently described for two other mutants. The KCNJ5 G151R mutation is one of the recurrent somatic mutations found in APA and has recently been described as inherited mutation in two families with early onset, severe progressive hyperaldosteronism and adrenal cortex hyperplasia, requiring bilateral adrenalectomy in childhood to control blood pressure [START_REF] Scholl | Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5[END_REF]. Despite this adrenal phenotype, in vitro studies demonstrated that the G151R mutation significantly reduced cell survival when transfected into human embryonic kidney 293T cells. A second mutation affecting the same amino acid, G151E, was recently described in a family diagnosed with non-glucocorticoid remediable familial hyperaldosteronism (12). This mutation was associated with a much milder phenotype of the two affected family members, compared to classical FH3, with blood pressure levels and hypokalemia easily corrected by medical therapy. Remarkably, the adrenals appeared normal by CT scanning, and hybrid steroids were produced at a rate comparable to that of other patients with sporadic PA. Again, the KCNJ5-G151E mutant channel was similarly permeable for sodium and potassium, resulting in depolarization of the plasma membrane and a continuous sodium influx (12). The KCNJ5 G151E mutation was reported in two additional families with early onset hyperaldosteronism of unknown cause [START_REF] Scholl | Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5[END_REF]. These subjects also had easily controlled hypertension and no evidence of adrenal hyperplasia. Remarkably, enough, in this study KCNJ5-G151E channels produced a much larger sodium conductance as compared to KCNJ5-G151R, resulting in rapid sodiumdependent cell lethality [START_REF] Scholl | Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5[END_REF]. Although G151E carriers do not present adrenal cortex hyperplasia, indicating that the mutation may limit adrenocortical cell proliferation yet promoting aldosterone overproduction, it is difficult to conceive how the G151R mutation, which also leads to reduced cell survival in the same study, induces bilateral adrenal hyperplasia when inherited and APA formation when occurring somatically. Based on these studies and the work by Oki et al, it remains therefore unclear whether and how KCNJ5 mutations are responsible for increased cell proliferation. Alternatively, the observed effects raise the possibility that KCNJ5 mutations are responsible for aldosterone hypersecretion, while increased cell proliferation is triggered by other, yet to be identified, mechanisms.

Consistent with this hypothesis is the observation that inactivation of several relevant adrenal potassium channels in mice leads to increased aldosterone production, but not to adrenal cortex hyperplasia or APA formation (Table 1). Somatic KCNJ5 mutations have been demonstrated to be present in a large proportion of patients with APA, with an estimated prevalence in unselected patients of ~34% (13), and even higher frequencies described depending on the sample size, the screening procedures for selecting patients for adrenalectomy and genetic background (14-15). However, although germinal KCNJ5 mutations are responsible for inherited forms of familial hyperaldosteronism, they are not similarly causative for sporadic BAH (13). Even though unilateral adrenalectomy represents the treatment of choice for PA in specialized departments, the fact that only 30% of operated patients are cured and that a subset of patients don't even go into surgery because of the multi-tiered process for subtype identification, sets medical treatment as an attractive therapeutic option even in lateralized forms of PA. Kir3.4 potassium channels may thus represent interesting new drug targets for a subset of APA not eligible for surgery. Cell as well as animal models expressing mutant Kir3.4 channels may provide valuable tools to address the functional consequences of KCNJ5 mutations, allowing to further dissect the mechanistic determinants of aldosterone overproduction and increased cell proliferation in PA.

Table 1 .

 1 Mouse models inactivated for potassium channels featuring adrenal aldosterone hypersecretion.

	Inactivated channel	PA Phenotype	Adrenal cortex	Hyperplasia/	Gender difference	Ref
	(gene)		expression	APA	described
	maxiK	KCNMA1 KO:	KCNMA1:	NO	NO	(16-17)
	(KCNMA1/KCNMB1)	Hyperaldosteronism,	ZG>>ZF, ZR
		hypokalemia, normal renin levels;	KCNMB1: adrenal
		KCNMB1 KO:	gland
		hyperaldosteronism linked to
		renal K retention and
		hyperkalemia
	TASK1 (KCNK3)	Glucocorticoid remediable	ZG, ZF, ZR	NO	Females only	(18)
		hyperaldosteronism, decreased
		plasma renin, hypokalemia,
	TASK1/TASK3	Hyperaldosteronism, reduced	TASK3: ZG	NO	NO	(19)
	(KCNK3/KCNK9)	plasma renin,
	KvLQT1/IsK	Hyperaldosteronism in KCNE1	ZG (KCNQ1, KCNE1)	NO	NO	(20)
	(KCNQ1/KCNE1)	KO mice, normal renin, fecal
		sodium and potassium loss


S Eadmusik

C Molette

X Fernandez

H Rémignon
email: remignon@ensat.fr

Prof. Hervé Rémignon

EARLY MUSCLE PH AND TEMPERATURE MEASUREMENTS SUFFICIENT TO DETECT PSE BREAST MEAT IN TURKEYS?

Keywords: Meat, Turkeys, Protein

Within a large flock of turkey toms (2000 BUT9 conventionally reared and 26 slaughtered), early muscle pH measurements were randomly done to distinguish two groups 27 of birds presenting low (fast glycolysing, GR) or normal (normal glycolysing, GN) values. 28 2. Subsequently, ultimate pH values and meat quality parameters were also recorded. Meat 29 quality parameters from GR or GN samples differ more or less indicating more or less severe 30 PSE conditions. Proteins extracted from the samples at 20 min post mortem were quite similar 31 while they greatly differ at 24 h post mortem. Moreover, among the GR birds, a subgroup of 32 animals (called AB) presented SDS-PAGE profiles largely different from other GR or GN 33 birds. 34 3. All the subsequent analysis developed on meat quality parameters as well as for protein 35 extractabilities also differ between AB and other animals indicating that they must be 36 considered differently in term of PSE syndrome development. 37 4. Western blots against Myosin Heavy Chain and actin at 24 h post mortem indicate that 38 myofibrillar proteins alterations are different in AB and GR or GN samples. 39 5. At 20 min post mortem, glycogen content was lowest in AB samples while the glycolytic 40 potential was similar in all samples at the time of death. Measurements of PFK enzyme 41 specific activity did not indicate a different regulation of post mortem glycolysis in AB 42 samples. 43 6. Our results suggest that a unique pH measurement at 20 min post is insufficient to detect 44 animals more prone to developing a severe PSE syndrome in turkeys. In consequence, it is 45 suggested that a more precise evaluation of the kinetics of pH and temperature decrease has to 46 be conducted to understand the aetiology of meat quality parameters alterations in poultry. 47 INTRODUCTION 48

3 adverse effects on poultry meat quality. In pigs, the genetic origin of the syndrome is well 51 established (e.g., [START_REF] Barbut | Progress in reducing 454 the pale, soft and Exudative (PSE) problem in pork and poultry meat[END_REF], but it remains obscure in poultry (Rémignon et al., 52 2007). Nevertheless, there is a large consensus in mammals, as well as in poultry, to consider 53 that the combination of a low pH and a high temperature in post-mortem muscle is at the 54 origin of protein alterations which lead to meat quality modifications (Alvarado and Sams, 55 2004). These alterations concern, to a greater or lesser extent, the colour of meat, its water-56 holding capacity, its texture and also its ability to succeed in meat-product enhancements like 57 marination, curing-cooking processing or sausage elaboration [START_REF] Mckee | Rigor mortis development at elevated temperature 502 induces pale exudative turkey meat characteristics[END_REF]58 Wynveen et al., 1999;[START_REF] Alvarado | Turkey carcass chilling and protein denaturation 449 in the development of Pale, Soft and Exudative meat[END_REF]. 59

Several facets of the production of live birds and/or management of carcases have been 60 investigated, mainly in turkeys and chickens (for review, [START_REF] Barbut | Progress in reducing 454 the pale, soft and Exudative (PSE) problem in pork and poultry meat[END_REF], to describe the 61 factors which trigger the PSE syndrome. A lot of attention has also been given to the speed of 62 post-mortem glycolysis, which is thought to be more important than the animal itself (Choi et 63 al., 2007;[START_REF] Gratacos-Cubarsi | Determination of changes in protein 480 conformation caused by pH and temperature[END_REF][START_REF] Molette | Modification of glycolyzing 506 enzymes lowers meat quality of turkey[END_REF][START_REF] Hahn | The relationship between pH 482 decline post-mortem and biochemical and physico-chemical properties of turkey breast meat[END_REF]64 Rathgeber et al., 1999a). Indeed, an acceleration of muscle glycolysis is required to produce 65 low pH values at any time soon after slaughter, when the temperature of muscles is still high 66 [START_REF] Petracci | The effect of holding 516 temperature on live shrink, processing yield, and breast meat quality of broiler chickens[END_REF]. Several experiments have then been conducted to maintain poultry 67 muscle at high post-mortem temperatures and these have shown that such conditions lead to 68 poor meat quality if the rate of pH fall is not concomitantly reduced [START_REF] Molette | Effect of rate of pH fall on turkey 504 breast meat quality[END_REF]69 Alvarado and Sams, 2002;Rathgeber et al., 1999b). Studies have also been conducted to sort 70 animals according to their early muscle pH value and have demonstrated that fast glycolysing 71 muscles (i.e. with a low early muscle pH value) are generally associated with poor meat 72 qualities similar to those described when PSE or PSE-like syndrome develops (Warris and 73 Brown, 1987;[START_REF] Pietrzak | Effect of rapid rigor mortis 519 processes on protein functionality in pectoralis major muscle of domestic turkeys[END_REF]Rathgeber et al., 1999a;[START_REF] Wynveen | Effects of muscle pH and chilling on development of PSE-like turkey breast meat[END_REF]James et 74 al., 2006). Nevertheless, all these studies have observed the consequence of the presence of 75 The measurement of early pH values cannot be generalised in processing plants because 78 it is destructive and not easy to perform. Soon after death, plasma membranes are still in their 79 native form and most of the water present in the muscle is retained in the myofibrillar 80 structure. Therefore, to have a good estimation of early pH values, it is absolutely necessary 81 to grind muscle samples in a buffer and to be able immediately to stop the anaerobic 82 glycolysis (iodoacetate is generally used as recommended by [START_REF] Jeacocke | The temperature dependence of anaerobic glycolysis in beef muscle 489 held in a linear temperature gradient[END_REF]. On the 83 contrary, after the setup of rigor mortis, a large part of the water have been expelled from the 84 myofibrillar structure and released into the extracellular spaces after plasma membrane 85 disruptions. Thus, a direct measurement of proton concentration is possible with an adapted 86 (meat-type) pH electrode. 87

In consequence, although numerous studies are available on the variability of ultimate 88 pH (pHu) values within a given flock (Fernandez et al., 2002;[START_REF] Hahn | The relationship between pH 482 decline post-mortem and biochemical and physico-chemical properties of turkey breast meat[END_REF]Fraqueza et 89 al., 2006), few have paid attention to this variability in pH measurements made soon after the 90 birds have been slaughtered. Because muscle pH monitoring is not easily performed on a 91 slaughter line, authors generally report only one early pH value (recorded at 15 or 20 min 92 post-mortem) to define characteristics of fast or normal glycolysing muscles. Nevertheless, 93 we must consider that post-mortem glycolysis is a dynamic phenomenon which cannot be 94 described by a single measurement. The complexity of these measurements is increased if the 95 monitoring of internal muscle temperature has also to be taken into account. 96 Besides this, conclusions coming from the comparison of fast and normal glycolysing 97 muscles are not clear because some workers have reported alterations of sarcoplasmic 98 [START_REF] Warner | Muscle protein changes 546 post-mortem in relation to pork quality traits[END_REF][START_REF] Joo | The relationship of 492 sarcoplasmic and myofibrillar protein solubility to colour and water-holding capacity in 493 porcine longissimus muscle[END_REF]Rathgeber et al., 1999a) or myofibrillar proteins 99 [START_REF] Mckee | Rigor mortis development at elevated temperature 502 induces pale exudative turkey meat characteristics[END_REF]Rathgeber et al., 1999a;[START_REF] Kuo | Quality characteristics of Chinese sausages made fromPSE 495 pork[END_REF]. These protein 100 alterations may concern phosphorylase [START_REF] Pietrzak | Effect of rapid rigor mortis 519 processes on protein functionality in pectoralis major muscle of domestic turkeys[END_REF]Rathgeber et al., 1999a,b;101 Alvarado and Sams, 2004) or creatine kinase (Rathgeber et al., 1999b) or, for example, 102 myokinase or triose phosphate isomerase and myosine (Rathgeber et al., 1999b) or 103 cytoskeletic proteins such as titin [START_REF] Warner | Muscle protein changes 546 post-mortem in relation to pork quality traits[END_REF]. 104

In the present study, we investigated whether all turkeys which exhibit a low early pH 105 value (20 min post-mortem) must be considered as a whole or if some of them could exhibit 106 different protein alterations which could make them more or less typical of the PSE 107 syndrome. To be sure that all the finally observed meat quality alterations were only due to 108 different glycolytic rates, all the birds were initially randomly selected from a commercial 109 flock with special attention being given to ultimate pH value (pHu) and glycogen reserves to 110 ensure that the amplitude of the decline in pH did not interact with the rate of pH fall. 111

MATERIALS AND METHODS 112

Animals 113 A flock of 2000 turkey toms (line BUT9, BUT Ltd., Chester, UK), was reared in a 114 commercial farm to 19 weeks of age (average live weight = 15 kg). All the birds were fasted 115 for 10 h before slaughter and then conventionally processed in a slaughter-house (Gastronome 116 SA,Moncoutant,France). 117

Sampling 118

At the end of the slaughtering process, the pH-values of 458 breast muscles (Pectoralis major, 119 Pm) were randomly measured (pH 20min ) according to [START_REF] Jeacocke | The temperature dependence of anaerobic glycolysis in beef muscle 489 held in a linear temperature gradient[END_REF]. Two grams of breast 120 muscle were homogenised in 18 ml of 5 mM iodoacetate using an Ultra Turrax (IKA-121 WERKE, Staufen, Germany). The pH 20min was then measured by using a portable pH-meter 122 combined with a glass electrode. The internal breast muscle temperature was also measured at 123 20 min post-mortem and 20 g of muscle were collected and immediately frozen in liquid 124 nitrogen. 125 animals: the first one (Normal Glycolysis, GN, n = 20) was made up with animals showing 127 pH 20min values similar to the mean value (6.21 + 0.01) of the whole population, while the 128 second one (Rapid Glycolysis, GR, n = 20) was made up with birds presenting the lowest 129 pH 20min values (5.80 + 0.07). Following storage overnight at 4°C, pHu was then directly 130 measured by inserting a glass pH-probe into the Pm muscle in GN and GR samples. At 24 h 131 post-mortem, other Pm samples were harvested as described above and kept at -80°C until 132 analysed. 133

Meat quality measurements 134

Colour 135

Meat colour was measured at 24 h post-mortem on the belly part of the breast meat with a 136 Minolta CR-300 chromameter (Osaka, Japan) The instrument was set to measure CIE L*, a*, 137 b* values using illuminant D and 65° standard observer. 138

Water-holding capacity 139

Drip loss was evaluated on scallops (around 100 g) of breast muscle collected 1 d post-140 mortem. The scallops were then stored for 9 d at 4°C in a polystyrene tray covered with a 141 scattering film (air permeable). After final weighing, drip loss was expressed as a percentage 142 of the initial weight at 1 d [START_REF] Honikel | Reference methods for the assessment of physical characteristics of 485 meat[END_REF]. After 9 d of storage, the scallops were weighed 143 (pre-thaw weight) and frozen at -20°C for 2 weeks. They were allowed to thaw overnight at 144 4°C and were then reweighed (precook weight). Thaw loss was expressed as a percentage of 145 the pre-thawing weight. The thawed meat was further used to evaluate cook loss and sausage 146 yield. The scallops were vacuum-packaged and cooked in an 80°C water bath for 15 min. 147

After cooking, samples were then cooled down to room temperature, rapidly wiped and 148 weighed. Cooking loss was expressed as a percentage of the precooking weight. 149 Sausages were produced following the method described by [START_REF] Shand | Kappa-carrageenan, sodium chloride 532 and temperature affect yield and texture of structured beef rolls[END_REF]. Thawed 151 meat (52.5g) was trimmed, chopped and mixed with iced water (21g, 0°C) and salt (1.5g) for 152 25 s using a Moulinex ILLICO (Lyon, France) blender. The meat batter was then transferred 153 to a plastic bag and vacuum packaged (residual pressure < 10mb) to remove air bubbles. 154 Twenty grams of meat batter were poured into a polypropylene tube and centrifuged at 700 g 155 at 4°C for 10 min. The tubes were then placed in a hot water bath until the internal 156 temperature of meat paste reached 80°C. After cooling down, the tubes were removed and the 157 sausages were weighed. The yield was expressed as a percentage of initial weight. 158

Meat batter yield 150

Warner-Bratzler shear test 159

The texture of cooked meat was evaluated according to [START_REF] Honikel | Reference methods for the assessment of physical characteristics of 485 meat[END_REF]. A 1-cm-wide strip of 160 cooked meat was obtained by cutting parallel to the longitudinal axis of the scallop myofibres. 161 Shear force was measured using a MTS Synergie 200 apparatus (MTS, Eden Prairie, MN, 162 USA). The maximum force needed to shear the sample represented the meat tenderness and 163 was expressed in Newtons. 164

Protein extractability 165

Muscular proteins of Pm samples collected at 20 min and 24 h post-mortem were extracted 166 following the methods of Rathgeber et al. (1999a) and [START_REF] Boles | Effect of 463 porcine stress syndrome on the solubility and degradation of myofibrillar/cytoskeletal 464 proteins[END_REF]. One gram of 167 sample was homogenised in 20 ml of a low ionic strength (LIS) buffer, containing 0.05 M 168 potassium phosphate buffer, 1 mM NaN 3 and 2 mM EDTA/pH 7.3 at 2°C for 10 sec. The 169 homogenised sample was then agitated for 30 min at 4°C and centrifuged at 17500 g at 2°C 170 for 15 min. Ten millilitres of the supernatant (LIS fraction) were collected at 2 cm above the 171 bottom of the tube while the remaining supernatant was discarded. A supplemental 20 ml LIS 172 buffer was added to resuspend the pellet which was then homogenised, agitated and 173 centrifuged as previously describe. The supernatant was discarded and the procedure was 174 repeated with high ionic strength (HIS) buffer, containing 0.55 M KCl, 0.05 M potassium 175 phosphate buffer, 1 mM NaN 3 and 2 mM EDTA/pH 7.3 at 2°C. Following centrifugation, 10 176 ml of supernatant (HIS fraction) was collected as described above and the excess supernatant 177 removed. The pellet was re-suspended in 40 ml of potassium phosphate buffer containing 75 178 mM KCl, 10 mM KH 2 PO 4 , 2 mM MgCl 2 and 2 mM EGTA/pH 7 and sonicated (Sonificateur,179 Bioblock Scientific,Illkirch,France) for 1 min to solubilise the fraction (pellet fraction). 180

Protein content in LIS, HIS and pellet fractions were determined by the BCA protein assay kit 181 (PIERCE, Rockford, IL, USA). Results are expressed as a percentage of total proteins. 182

Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) 183

SDS-PAGE was performed in order to separate proteins in each buffer-extracted fraction from 184 samples collected at 20 min and 24 h post-mortem. Samples containing 2% (w/v) SDS, 5% β-185 mercaptoethanol, 10% glycerol and 62 mM Tris-HCl, pH 6.8 in a ratio 1:1 v/v (Laemmli, 186 1970) were heated at 95°C for 5 min. SDS-PAGE was performed following the method 187 described by [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of 497 bacteriophage T4[END_REF] of co-substrate (NADH) at 340 nm and 25°C for 2 min [START_REF] Beutler | (Ed) Red cell metabolism. A 461 manual of biochemical methods[END_REF]. The reaction was 230 assayed by mixing 1 µl of enzyme extract with 1 ml of solution containing 0.07 M Tris-HCl 231 (pH 8), 1.4 mM MgCl 2 , 4.5 mM KCL, 0.71 mM phosphophenol pyruvate, 0.65 mM fructose-232 1,6-diphosphate, 0.4 mM NADH, 1 mM ATP, 1.8 mM fructose-6-phosphate, 0.08 U PK and 233 0.96 U LDH. The PFK activity was expressed as µmol of NADH disappeared per min and per 234 g of muscle (µmol/min/g). 235

The preparation of enzyme extract for COX activity was performed as described above 236 except that, after centrifugation, the supernatant was frozen and defrosted 4 times in liquid 237 nitrogen to break cell wall and to release the enzyme contained in the mitochondrial matrix. 238

The enzyme extract was kept at -20°C until used. Twenty µl of enzyme extract were mixed 239 with 1 ml of solution (pH 7.4) containing 90 µM Cytochrome c (reduced form) and 50 mM 240 potassium phosphate (Smith and Conrad, 1956). The activity was assayed at 550 nm and 241 25°C. The COX activity was expressed as micromoles of Cytochrome c (reduced form) 242 disappeared per min and per g of muscle . 243

Glycogen, glucose, G-6-P, lactate and glycolytic potential determination 244

Amounts of glycogen, glucose, glucose-6-phosphate (G-6-P) and lactate of Pm muscle 245 collected at 20 min and 24 h post-mortem were measured by enzymatic procedures according 246 to [START_REF] Dalrymple | A method for the extraction of glycogen and 468 metabolits from a single muscle sample[END_REF] and [START_REF] Bergmeyer | D-Glucose. 457 Determination with hexokinase and glucose-6-phosphate dehydrogenase[END_REF]. About 200 mg of Pm muscle 247 was homogenised in 10 ml of 0.5 M perchloric acid. Aliquots (0.5 ml) of homogenate were 248 taken for the enzymatic determination of glycogen and G-6-P after glycogen hydrolysis with 249 amyloglucosidase. The rest of the homogenate was centrifuged and the supernatant was used 250 for analysis of glucose, G-6-P and lactate. Glycogen content was calculated as the difference 251 between the results of the 2 sets. Lactate was determined in the supernatant resulting from the 252 centrifugation of the homogenate (20 min at 2500 g). Results were expressed in µmol/g of 253 fresh tissue. Glycolytic potential was then calculated according to [START_REF] Monin | Pork of low technological quality with a normal rate of 508 muscle pH fall in the immediate post mortem period: The case of the Hampshire breed[END_REF] From these observations, it clearly appears that not all GR samples can be considered as 278 equivalent, at least for their proteome extracted with high or very high ionic strength buffers. 279

Results presented in Figure 2 failed to show differences in the quantity of MHC and 280 actin extracted at 20 min post-mortem. However, at 24 h post-mortem, WB performed with 281 antibody against MHC and actin clearly distinguished AB samples from others ones. A larger 282 quantity of MHC was detected in the proteins extracted from GN samples. AB and GR-AB 283 samples could also be significantly distinguished. Nevertheless, results from the quantity of 284 actin extracted at that later time cannot be used to distinguish GN from GR samples or GR-285 AB from GN ones, but AB samples exhibited significantly lower amount of actin extracted 286 than in all other samples. 287

Twenty min post-mortem, there were few differences between the groups of birds in 288 the quantity of muscular proteins extracted by the different buffers, thus making the 289 consequences of an acceleration of the glycolysis post-mortem difficult to interpret (Table 1). 290

Nevertheless, it was noticed that higher quantities of proteins were present in HIS and pellet 291 extracts coming from GN samples. This indicated that the lowest soluble muscular proteins 292 were affected by an acceleration in the rate of glycolysis which made them less extractible. 293 This tended to be more evident if GR samples are distinguished as AB and GR-AB samples 294 because AB samples presented lower (HIS extracts) or similar values to those found in After the onset of rigor mortis (24 h post-mortem), LIS and HIS protein extracts 297 exhibited higher amounts in GN than in GR samples. This difference between fast and normal 298 glycolysing samples was also more evident when AB samples were distinguished from GR-299 In that case, all the AB samples were significantly different from the GN ones. This 300 indicated that AB samples were more distinct to GN than GR-AB ones. 301 Meat quality parameters reported in Table 2 show that initial pH values were similar 302 in AB and GR-AB samples but lower in GR than in GN ones. In contrast, pHu values were 303 similar in all samples and within a normal range. Considering muscle temperature at 20 min 304 post-mortem, results indicated that values for GN, GR and GR-AB samples were similar 305 (around 42 °C), while values from AB samples were slightly but significantly higher (around 306 43°C). Considering different water loss values, GR samples presented similar (thawing or 307 cook loss) or higher (drip losses) values than GN samples. Contrastingly, AB samples had 308 higher drip and cook loss than GR-AB or GN samples, while thawing ones were similar in all 309 groups. The processing yields obtained from meat batter preparation were not significantly 310 different for GN and GR samples, but it was significantly lower for AB than for GR-AB or 311 GN samples (82.11, 88.95 and 90.40 respectively). After cooking, GN meat samples 312 presented lower shear force values (F max ) than GR ones, but AB and GR-AB samples were 313 not found to be significantly different. Nevertheless, AB and GR-AB samples were still 314 significantly tougher than GN ones. Higher L* values (53.03 ± 2.87) recorded for raw meat 315 samples also permitted to easily distinguish AB samples from other ones. However, L* values 316 were similar in GN and GR samples (49.06 ± 2.92 and 50.14 ± 2.51 respectively). When a* 317 values were significantly higher in GR samples than in GN ones, but not in GR-AB ones, they 318 were also significantly increased in samples coming from the AB group. 319 Glycolytic potential (GP) values were similar in all samples (Table 3) suggesting that 320 muscular reserves in carbohydrates at the time of death were not different between all the 321 animals. Nevertheless, soon after death, we can observe that glycogen content is higher in GN 322 than in all other samples. On the contrary, glucose content was lower in GN samples than in 323 GR ones and was similar in AB and GR-AB samples. Lactate was higher in GR than in GN 324 samples but failed to distinguish AB to GR-AB samples. No significant differences among all 325 the animals were recorded for G6P. At the end of the rigor mortis onset (24 h post-mortem), 326 similar quantities of all the considered metabolites were measured in all samples. 327 If small significant differences in COX enzyme specific activity (Figure 3) were 328 recorded in the different samples, a much higher value was reported for the PFK enzyme 329 specific activity: in GN samples, this value was approximately 6 fold higher than in GR, Soon after death (20 min post-mortem), SDS-Page profiles were similar between all the 333 groups of birds. This demonstrates that the analysed samples were very close in term of 334 proteome composition, as already shown by [START_REF] Pietrzak | Effect of rapid rigor mortis 519 processes on protein functionality in pectoralis major muscle of domestic turkeys[END_REF] and Rathgeber et al. 335 (1999b), who reported that normal or fast glycosing muscles were not differently organised in 336 terms of constitutive or functional proteins. On the contrary, at the end of the rigor mortis 337 onset, SDS-Page profiles were similar for all extracted fractions in GN animals while they 338 allowed us to make a clear difference between the GR ones. This indicates that, despite the 339 fact that all the GR birds were initially identified as being homogenous for the characteristic 340 of the rate of glycolysis (same low initial pH 20min values), a clear distinction could be found 341 among them, considering the consequences of this parameter on the solubility of muscular 342 proteins. This distinguishes AB from GR-AB birds. The relevance of the AB group was 343 initially based on differences of SDS-Page profiles for HIS and pellet extracts, mainly 344 because of different intensities for two bands located at 45 and 200 kDa. The identification of 345 the modified bands was made by mass spectrometry analysis and revealed the presence of 346 actin and myosin heavy chain (MHC) at 45 and 200 kDa respectively. Alterations of 347 myofibrillar proteins due to the acceleration of post-mortem glycolysis had previously been 348 reported by Rathgeber et al. (1999b). Modifications of muscular proteins extractabilities were 349 Table 3 andFigure partly visible at 20 min post-mortem because AB samples always differed from GN, but not 350 completely from GR-AB ones (same value for pellet extract). At 24 h post-mortem, AB 351 samples differed widely not only from GN ones but also from GR-AB, mainly for HIS 352 extracts which contained most of the extractable myofibrillar proteins. By using values of 353 extractabilities, AB samples could always be distinguishable from GN ones whatever the time 354 post-mortem or the considered fraction. In contrast and for all the considered fractions, GR-355 AB samples were not always found to be different from the GN ones. Quantitative analysis of 356 the presence of actin and myosin within the different HIS fractions by WB confirmed that the 357 level of considered proteins was similar for all the samples at 20 min post-mortem. At 24 h 358 post-mortem, there were always significantly smaller amounts of actin and MHC present in 359 AB samples than in other ones, indicating a different degree of those proteins alteration 360 between the different samples. Whereas these alterations of actin and MHC in fast 361 glycolysing muscles have previously been reported by Rathgeber et al. (1999b), Alvarado and 362 Sams (2004) reported no effect of post-mortem glycolysis acceleration on the extraction of 363 myofibrillar proteins, as observed here, if AB samples are excluded from the analysis by 364 comparing GR-AB with GN ones. This indicates that, depending on the presence or otherwise 365 of AB samples among GR ones, the occurrence of myofibrillar protein alterations is in 366 response to accelerated glycolysis post-mortem. The presence of an associated increase in 367 muscle temperature only in AB samples, together with a lack of any difference in pHu values, 368 suggests that AB samples must be considered as true PSE ones while GR-AB are probably 369 less affected. The percentage of AB samples among all the GR ones could then explain why 370 some authors found large effects on meat quality parameters due to accelerated post-mortem 371 glycolysis, while others concluded that the combination low early post-mortem pH and high 372 temperature values had a much smaller effect. It is generally accepted that the combination of 373 low pH and high temperature values, within muscle, leads to protein alterations (Joo et Wilson;[START_REF] Wilson | Sarcoplasmic proteins influence water-555 holding capacity of pork myofibrils[END_REF] and consequently modifies protein extractabilities as well as 375 meat quality [START_REF] Alvarado | Turkey carcass chilling and protein denaturation 449 in the development of Pale, Soft and Exudative meat[END_REF][START_REF] Gratacos-Cubarsi | Determination of changes in protein 480 conformation caused by pH and temperature[END_REF]. To our 376 knowledge, but with the exception of a report by [START_REF] Offer | Modelling of the formation of Pale, Soft and Exudative meat: Effect of 511 chilling regime and rate and extend of glycolysis[END_REF] who estimated the part of 377 myosin degraded in several pH and temperature conditions, neither the conditions nor the 378 duration of pH and temperature limit values have precisely been described. As a consequence, 379

Figures 1 & 2 and
we must ask what are the incorrect conditions for meat quality. For example, is a condition 380 described by a pH value lower than 6.0 and a temperature lower than 40°C during 10 min 381 worst than one described by a pH value of 6.2 and a temperature higher than 40°C during a 382 shorter or a longer time? As muscle to meat transformation is a dynamic phenomenon 383 depending on time, temperature and pH values, we could take into account these three 384 parameters to better predict consequences on the future of proteins and on associated meat 385

qualities. 386

All data collected to determine the energetic status of sampled muscles confirmed that 387 AB as well as GR-AB had rapidly started post-mortem glycolysis. Indeed, GP level was the 388 same in all samples, which is in good accordance with observed similar pHu values because 389 GP directly reflects the level of stock energy at the time of death [START_REF] Monin | Pork of low technological quality with a normal rate of 508 muscle pH fall in the immediate post mortem period: The case of the Hampshire breed[END_REF]Sellier, 1985, Le 390 Bihan-Duval et al., 2008). At the end of the onset of rigor mortis, all the recorded values for 391 energetic compounds were at the same level, indicating that the extent of post-mortem 392 glycolysis was at the same level in all samples, [START_REF] Warris | Depletion of glycogen 550 reserves in fasting broiler chickens[END_REF]El Rammouz et al., 393 2004). This was in good agreement with final pHu values. However, whereas glycogen and 394 glucose levels were higher in GR (including AB and GR-AB samples) than in GN at 20 min 395 post-mortem, the lactate content was the reverse. This demonstrated that glycolysis began 396 more quickly in GR than in GN muscles, as could be deduced from pH 20min values (Shen et 397 al., 2006). In our study, lactate levels, which are mainly responsible for pH fall in post-398 mortem muscles, were similar in AB and GR-AB samples as were pH 20min values. PFK 399 enzyme is considered to be a key enzyme in anaerobic glycolysis because it can act as a 400 regulator of its efficiency. When the level of production of energy (i.e. ATP) is high within 401 the cell, the specific activity of PFK enzyme is reduced to economise the stock of glycogen 402 [START_REF] Wegener | Different modes of activating phosphofructokinase, a 552 key regulatory enzyme of glycolysis, in working vertebrate muscle[END_REF]. This is probably the case here at 20 min post-mortem because, 403 in GR samples, the mean pH value reported was low, indicating a high level of anaerobic 404 glycolysis. In contrast, the level of anaerobic glycolysis in GN samples stayed low (as 405 demonstrated by low levels of lactate and high pH 20min values) while the PFK specific activity 406 remained high. In all samples, low values for specific activities of COX enzyme indicated that 407 all the samples were glycolytic muscles as previously observed in turkey PM (Opalka et al., 408 2004). No differences in the specific activity of the PFK enzyme were recorded between AB 409 and GR-AB samples, demonstrating that a difference in glycolysis regulation should exist 410 between all the GR and GN samples independently from PFK specific activity modulations. It 411 is likely that, at the beginning of the slaughtering process, some birds would have enhanced 412 their muscular glycolysis rate, as did GR, but only few (AB ones) were able to maintain it for 413 a longer time than the majority of the fast glycolysing ones (here the GR-AB samples). This 414 would probably lead to the worst combination of pH and temperature values in the muscle 415 and could explain the poorest final quality of meat (here for AB samples). However, the 416 origins of this deep disorder in post-mortem glycolysis in the muscle is still unknown. 417

When we consider meat quality parameters such as drip loss, cooking l, colour values 418 (mainly L* and a*) and processing yield, our conclusions on the effect of the acceleration of 419 glycolysis post-mortem are completely different if we compare AB, GR-AB or GR samples 420 with GN. With AB samples, most of the recorded parameters differ from GN, while the final 421 conclusions are much less evident with GR or GR-AB. For example, higher L* values of meat 422 in fast-glycolysing samples have been widely discussed, but some workers found this 423 parameter could be used to sort PSE samples [START_REF] Barbut | Problem of pale soft exudative meat in broiler chickens[END_REF] (Molette et al., 2002;[START_REF] Fraqueza | 477 Incidence of pectoralis major turkey muscles with light and dark colour in a Portuguese 478 slaughterhouse[END_REF]. In this study, we demonstrated 425 that, depending on the origin of samples, AB or GR-AB group, one can find equal or higher 426 L* values for meat samples with a low initial pH value than in normal ones. If we take as a 427 reference the cut-off value of L* > 52, as proposed by [START_REF] Barbut | Problem of pale soft exudative meat in broiler chickens[END_REF], we would sort all the 428 AB samples as being PSE ones, while all the GR-AB would be considered as the GN ones, 429 despite that they present very different early pH values. As a consequence, it seems clear that 430 only measuring one early pH value is not sufficient to accurately detect samples liable to 431 develop the meat quality alterations generally associated with PSE syndrome in poultry. 432

Conclusion 433

This paper demonstrates that a simple measurement of one early muscle pH value is probably 434 insufficient to satisfactorily distinguish turkeys prone to develop PSE syndrome and all the 435 associated consequences for the quality of meat. Indeed, if the presence of a high muscle 436

  and high temperature in poultry muscles at early times post-mortem, but no one 76 has yet proposed a clear origin of those conditions. 77

  then sorted according to their pH 20min values from all the measured 126

  using a Mini-Protean II electrophoresis unit or a Protean Xi 188 Unit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Samples were loaded at 40 µg protein 189 per lane for LIS, HIS and pellet fractions, 5 and 10 µg for western blots of actin and Myosin 190 Heavy Chain (MHC), respectively. Resolving gel of 12% polyacrylamide was used for LIS 191 fraction and 8% polyacrylamide was applied for HIS and pellet fractions and western blots of 192 actin and MHC. Gels were run at 17 mA or 35 mA/gel (for Mini-Protean II and Protean Xi 193 Units, respectively), constant current, until the dye front reached the bottom of the gel. Gels 194 were stained overnight in Coomassie brilliant blue R-250 containing 5% (v/v) glacial acetic 195 acid, and 45% (v/v) ethanol. Gels were then destained twice in the same solution excluding 196 Coomassie blue. 197 Western blots of Actin and Myosin Heavy Chain 198 HIS fraction was used to perform western blots of actin and MHC. Following SDS-PAGE, gel 199 was steeped into transfer buffer containing 25 mM Tris, 192 mM glycine and 20% v/v 200 The enzyme extract was obtained from centrifugation at 10000 g for 5 min at 4°C. The 226 supernatant was collected and stored at 0°C. Activity of PFK was spectophotometrically 227 measured in the presence of pyruvate kinase (PK) and lactate dehydrogenase (LDH) by using 228 a UVIKON 943 spectrometer (Kontron Instruments, Watford, UK) to follow a disappearance 229

  as 254 follows: GP = 2[(glycogen) + (glucose) + (glucose-6-phosphate)] + (lactate). It was 255 expressed as µmol of lactate equivalent per g of fresh meat. 256 Statistics 257 Experimental data were analysed using the GLM-general factorial ANOVA procedure using 258 the SAS software system for Windows statistical package, version 9.1 (SAS Institute Inc., 259 Cary, NC, USA). The GLM model was Y ij = µ + G i + e ij , where Y ij = dependent observation; µ 260 = overall mean; G i = effect of Group (i = AB, GR-AB and GN in a model or GR and GN in 261 another one) and e i = the residual error. Statistical significant effects were further analysed and 262 means compared using Student-Newman-Keuls multiple range test. Statistical significance 263 was determined at P <0.05. 264 RESULTS 265 SDS-Page profiles are reported in Figure 1. For the different proteins extracted at 20 min 266post-mortem, gel patterns were very similar indicating that samples from the different groups 267 of birds probably contained the same proteins. In contrast, at 24 h post-mortem, if 268 sarcoplasmic proteins (LIS buffer extracted) presented the same profile, noticeable differences 269 were present for HIS buffer and pellet protein extracts. Those differences concerned mainly 270 two bands, located at 200 and 45 kDa respectively, which were identified by mass 271 spectrometry analysis (data not shown) as myosin heavy chain (MHC) and actin respectively. 272 Whereas all the GN samples presented similar SDS-Page profiles, GR samples could easily be 273 divided in two groups. The GR-AB (Fast Glycolysing animals not ABnormals) samples were 274 very similar to the GN ones while the AB (ABnormals Fast Glycolysing) ones exhibited 275 the bands corresponding to MHC and actin proteins. Four AB samples 276 were easily identified creating a new subgroup representing 20% of the whole GR samples. 277

Figure 1 .Figure 2 .Figure 3 .

 123 temperature and a low pH value in muscle are required to develop PSE syndrome, the 437 duration of the conditions encountered post-mortem are also fundamental. Further 438 experiments describing the precise kinetics of pH and temperature evolution post mortem are 439 necessary to adequatelyl understand the etiology of the syndrome. The different possible 440 combinations of (pH, temperature and duration) could have large consequences on muscle 441 proteins and variably affect meat quality. Other investigations are also required to understand 442 why, which and how some turkeys are able to develop such conditions of muscle to meat 443 transformation leading to the poorest final quality of poultry products. Examples of SDS-Page gels from muscular proteins extracted with different buffers 590 (LIS=Low Ionic Strength, HIS=High Ionic Strength, Pellet= residue after Lis and His buffers 591 extractions) at 24 h post mortem from GR (Fast Glycolysing) and GN (Normal Glycolysing) 592 turkeys. One different animal is present per lane and arrows indicate major variation in band 593 staining. Western blot values with antibody against Myosin Heavy Chain (top) and actin 598 (bottom) at 20 min and 24 h post mortem for the different groups of turkeys. Values are means 599 ± SD and are expressed as percentages of reference protein. GN = Normal Glycolysing, GR 600 = Fast Glycolysing, AB = Abnormal GR, GR-AB = GR animals not AB. a,b AB, GR-AB and 601 GN means with different superscripts differ significantly at P <0.05, A,B GN and GR means 602 with different superscripts differ significantly at P <0.05. Specific activities (in UI) of the PFK (PhosphoFructoKinase) and cytochrome 606 oxydase c (COX) enzymes in the different groups of turkeys. Values are means ± SD. GN = 607 Normal Glycolysing, GR = Fast Glycolysing, AB = Abnormal GR, GR-AB = GR birds not 608 AB. a,b AB, GR-AB and GN means with different superscripts differ significantly at P <0.05, 609A,B GN and GR means with a different superscript differ significantly at P <0.05.

Table 1

 1

	near here

Table 2

 2

	near here

Table 1 .

 1 Percentage of proteins extracted by buffer with increasing ionic strength at 20 min 611or 24 h post-mortem in different groups of turkeys. Values are means ± SD and expressed as AB, GR-AB and GN means with different superscripts differ significantly at P <0.05. 614A,B GN and GR means with different superscripts differ significantly at P <0.05. 615 GN = Normal Glycolysing, GR = Fast Glycolysing, AB = Abnormal GR, GR-AB = GR 616 turkeys not AB. LIS = Low Ionic Strength buffer, His = High Ionic Strength buffer, Pellet = 617 residue after Lis and His buffers extractions.

	612
	613		percentages of total proteins
	Time post mortem	%	AB	GR-AB	GN	GR
	20 min	LIS	22.7±2.38 a	25.0±0.92 ab	25.0±1.03 b	24.8±0.93
		HIS	5.9±1.03 a	7.0±0.78 b	7.6±1.17 b,A	6.8±1.00 B
		Pellet	23.2±5.10 a	26.3±3.33 a	30.7±2.87 b,A	26.3±3.09 B
	24 h	LIS	23.6±0.69 a	27.0±1.67 b	29.3±1.13 c,A	26.3±2.05 B
		HIS	5.4±0.95 a	16.6±4.45 b	18.2±4.67 b,A	14.4±6.00 B
		Pellet	32.5±5.46 a	27.8±6.01 ab	27.0±4.36 b	28.8±6.07
	a,b 618
	619
	620

Table 2 .

 2 Meat quality parameters in different groups of turkeys. Values are means ± SD AB, GR-AB and GN means with different superscripts significantly differ at P <0.05. 623 A,B GN and GR means with different superscripts significantly differ at P <0.05. 624 GN = Normal Glycolysing, GR = Fast Glycolysing, AB = Abnormal GR, GR-AB = GR 625 turkeys not AB. 626

	621
		AB	GR-AB	GN	GR
	pH 20	5. 8±0.12 a	5.8±0.39 a	6.2±0.01 b,A	5.8±0.07 B
	pH u	5.8±0.03	5.8±0.09	5.8±0.09	5.8±0.08
	T 20 (°C)	43.1±2.22 a	41.7±0.91 ab	41.7±0.66 b	42.1±1.37
	Drip losses (%)	6.2±1.47 a	3.7±1.06 b	3.2±0.08 b,A	4.2±1.45 B
	Thawing losses (%)	4.8±0.33	4.5±2.35	4.0±1.79	4.5±2.07
	Cooking losses (%)	17.7±1.24 a	14.4±2.05 b	14.2±2.17 b	15.1±2.34
	F max (N)	43.5±14.83 a	30.8±11.04 a	25.0±4.25 b,A	33.4±12.57 B
	L*	53.0±2.87 a	49.4±1.89 b	49.1±2.92 b	50.1±2.51
	a*	7.3±1.04 a	5.5±1.147 b	5.1±0.60 b,A	5.9±1.34 B
	b*	0.76±1.31	0.14±0.85	0.19±0.64	0.26±0.92
	Processing yield (%) 82.1±1.34 a	89.0±4.17 b	90.4±.14 b,A	87.6±4.68 B
	622
	a,b

E-mail: br.poultsci@bbsrc.ac.uk URL: http://mc.manuscriptcentral.com/cbps British Poultry Science