# Cells shrinkage and phosphatidylserine externalization in post mortem muscle by fluorescence microscopy

S. Becila, Y. Boudida, Mohammed Gagaoua, K. Hafid, H. Boudchicha, H.

Smili, R. Belachehabe, C. H. Herrera-Mendez, M. A. Sentandreu, Roland Labas, et al.

<figure><img src="image_1.png" /></figure>

► To cite this version:

S. Becila, Y. Boudida, Mohammed Gagaoua, K. Hafid, H. Boudchicha, et al. Cells shrinkage and phosphatidylserine externalization in post mortem muscle by fluorescence microscopy. 3. International Multidisciplinary Microscopy and Microanalysis Congress (InterM), Oct 2015, Oludeniz, Turkey. 10.1007/978-3-319-46601-9_7. hal-01606895

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# Cells Shrinkage and Phosphatidylserine Externalization in Post Mortem Muscle by Fluorescence Microscopy

S. Becila, Y. Boudida, M. Gagaoua, K. Hafid, H. Boudchicha, H. Smili, R. Belachehabe, C.H. Herrera-Mendez, M.A. Sentandreu, R. Labas, T. Astruc, A. Boudjellal, B. Picard and A. Ouali

**Abstract** *Postmortem* (PM) meat tenderization is a complex biochemical process that involves multiple endogenous proteolytic enzymes and that is not completely understood. Cell death by apoptosis is recently proposed as a novel mechanism in this process. The signification of the caspases implication in muscle proteins degradation is that the cells are dying through apoptosis, a major cell death programme. Our purpose was therefore to verify this statement using different well known hallmarks of the apoptotic process including cell shrinkage, phosphatidylserine (PS) externalization. Cells start to shrink few minutes after animal death and reach a maximum shrinkage about 24 h postmortem while they showed the typical rounding appearance of apoptotic cells. Externalization of PS is detectable within 1 h postmortem and increased gradually with time. The present work is therefore the first to provide direct evidence supporting the onset of apoptosis in post-mortem muscle. The present findings should lead us to reconsider as a whole, the mechanisms contributing of the development of meat qualities (tenderness, flavor, juiciness ...) by a detailed analysis of the apoptosis associated biochemical and physiological modifications taking place within the cells.

<footnote>S. Becila (✉) · Y. Boudida · M. Gagaoua · K. Hafid · H. Boudchicha · H. Smili · R. Belachehabe · A. Boudjellal
INATAA, Bothers Mentouri Constantine University, Aïn El Bey Road, 25000 Constantine, Algeria
e-mail: becila2002@yahoo.fr</footnote>

<footnote>R. Labas · T. Astruc · B. Picard · A. Ouali
Theix, QuaPA, INRA de Clermont Ferrand, 63122 St Genes Champanelle, France</footnote>

<footnote>C.H. Herrera-Mendez
Departamento de Ingeniería Agroindustrial, Universidad de Guanajuato, 38900 Salvatierra, Mexico</footnote>

<footnote>M.A. Sentandreu
Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Avenida Agustín 7, 46980 Valencia, Spain</footnote>
## Abbreviations

FITC Fluorescein isothiocyanate

IgG Immunoglobulin

PS Phosphatidylserine

# 1 Introduction

Over the last years, several reports suggested a contribution of caspases, especially caspase 3, to the overall proteolysis taking place in *postmortem* muscle. After perfusion with the caspase inhibitor Z-VAD-fmk, a reduction in tilapia muscle toughness was thus reported by Ishida et al. [1]. Similarly, a significant reduction of proteolysis in chicken skeletal muscle was observed upon treatment with a specific caspase 3 inhibitor whereas calpain activity was unchanged over the whole treatment length [2]. On the other hand, Kemp and Parr [3] demonstrated the high sensitivity of myofibrils to the action of caspase 3. These findings are in good agreement with the conclusion of Camou et al. [4] who reconsidered the role of calpains in *postmortem* muscle and concluded their investigations by stressing that other proteolytic systems than calpains are involved in *postmortem* proteolysis and pore specially caspases. Hence, caspases contribute very likely to *postmortem* proteolysis. The activation of effector caspases requires the preliminary initiation of apoptotic process by specific stimulus. Upon initiation of the apoptotic process, one or several initiator caspases are activated and these will in turn activate, through limited proteolysis, the effector caspases. The effector caspases will then degrade cellular proteins up to total cell dismantling [5].

After animal bleeding, the dynamic regulation of the biological processes existing in the living cells will not be efficient anymore and if the apoptotic process is set up *postmortem*, it will be very probably never fully completed. There is two major reasons supporting this assumption: (a) the physicochemical conditions (pH, osmotic pressure, ...) which will change unfavorably and (b) because of the lower efficiency of the supplying energy pathways, the energy needs for a full completion of the process will probably not be fully covered.

Although several sets of evidence suggested the contribution of caspases to *postmortem* proteolysis, especially of caspase 3, the onset of the apoptotic process in *postmortem* muscle has never been clearly established. The purpose of the present work is to fill in this gap and provide experimental data demonstrating that apoptosis is set up in *postmortem* muscle. To achieve this goal two major changes characterizing the apoptotic process (reviewed in [6]). Have been selected and tested in *postmortem* rat muscles: (a) cell shrinkage and rounding; (b) phosphatidylserine externalization and. The whole set of findings reported in this paper led us to conclude that the apoptotic process starts to be set up in the first few minutes postmortem and progresses then during time.
# 2 Experimental

## 2.1 Animals and Muscle Sampling

Five 10–12 weeks old Wistar rats provided by the research center rat breeding group were used in the present work. Animals were anesthetized by intra-peritoneal injection of sodium pentobarbital (0.1 ml/100 g of rat weight) and then killed by decapitation. For each animal, *Gastrocnemius* (red muscle) and *Plantaris* (white muscle) muscles were excised from the left hind leg within 1 h postexsanguination. The muscle from the right hind leg was maintained on place and excised 24 h postmortem after storage of the carcass at 10 °C for 5 h and then at 4 °C up to 1 day postmortem. These two muscles were used to study the extent of cell shrinkage and rounding together with the phosphatidylserine externalization. Because more samples were needed for DNA fragmentation studies, this experiment was performed on a larger muscle, namely rat *Longissimus* muscle, from which samples were taken at each predefined times postmortem from the left side and then from the right side of the carcass.

## 2.2 Muscle Cells Shrinkage

Muscle strips from *Gastrocnemius* and *Plantaris* muscles were immersed in 4 % paraformaldehyde and 0.1 % E were cut and observed without any additional staining or treatment with an Axioplan 2E Zeiss light microscope (Zeiss, Lyon, France).

## 2.3 Estimation of the Extent of Shrinkage

The estimation was carried out on samples excised just after bleeding from rat *Longissimus* muscle, a larger muscle enabling analysis of a greater number of samples. Samples were prepared for microscopy examination as described above for cells shrinkage. The contrast of the image was adjusted in order to identify clearly the cell limits using Corel Paint Shop Pro X application from Macromedia (Paris). After printing the image presented in Fig. 1, cells were cut out with or without the associated extracellular space and pieces of paper corresponding to the cell alone (grey (G) area in Fig. 1) and to the cell plus the extracellular space (grey (G) + black (B) area in Fig. 1) were weighted. The extent of shrinkage was calculated using the following equation:

$$ \text{Shrink} = (1 - (G/(G+B))) * 100 $$

where “Shrink” is the extent of shrinkage (%), “G”, the weight of the cell alone and (G + B), the weight of the cell plus the associated extracellular space.
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Estimation of the extent of shrinkage on samples excised just after bleeding from rat *Longissimus* muscle</figcaption></figure>

Measurements were carried out on 1–3 cells per cut and 5 cuts were analyzed for each animal. A total of 52 fibers were analyzed on samples prepared within the first 10 min after animal decapitation. As a reference we used the cell volume for a negligible extracellular space (B = 0 and cell weight = B + G).

## 2.4 Phosphatidylserine Externalization and Laminin Labelling

Phosphatidylserine externalization was investigated using 2.5 × 6 mm strips from Gastrocnemius and Plantaris muscles. These samples were stretched and fixed with needles on cork plates before immersion in 1 % (v/v) paraformaldehyde, 0.1 % (v/v) glutaraldehyde, 0.1 M sucrose in 0.1 M sodium phosphate buffer, pH 7.2, for 15 min at room temperature. FITC-Annexin V Conjugate (Sigma-Aldrich A9210, Saint-Quentin Fallavier, France) was used for Phosphatidylserine detection. After three washes with PBS buffer for 5 min, muscle strips were incubated in PBS buffer containing FITC labeled Annexin V (1.8 µg/ml) for 24 h at 4 °C and kept overnight in the dark upon mild stirring. One strip was used as a control and similarly incubated in the same buffer but in the absence of Annexin V. After three washes with PBS buffer for 5 min, muscles were cut into small pieces (1 × 3 mm) parallel to the fibre axis. These small cuts were frozen at −160 °C in isopentane immersed in liquid nitrogen and subsequently kept at −80 °C until use. Transversal sections (10 µm) were then cut using a Reichert Frigocut 2800 (Leika) and dried in the dark at room temperature before fixation in PBS buffer containing 2 % formol. Thin Annexin V labeled cuts were then treated for Laminin labeling as previously described [7], using a rabbit anti-Laminin (Sigma-Aldrich) primary antibody (dilution: 1/100) followed by the addition of a cyanine 3 conjugated goat anti-rabbit IgG (dilution: 1/150) as the secondary antibody (Santa Cruz SC-5879, Santa Cruz Biotechnology Inc., Heidelberg, Germany). Finally, sections were mounted on glass slides in Mowiol and observed by using an Axioplan 2E Zeiss light microscope. FITC-Annexin V conjugate was revealed using λexc 488 nm and λemm 530 nm whereas for cyanine.
## 2.5 Preparation of Samples for Electron Microscopy

For routine ultrastructural studies, rat muscle samples were treated as previously described [8].

# 3 Results and Discussion

## 3.1 Muscle Cells Shrinkage

Cell shrinkage which is one of the first steps of the cell death program [9], has been investigated on both rat *Gastrocnemius* and *Plantaris* muscles. Similar findings were obtained for both muscles. Thus only those corresponding to *Gastrocnemius* muscle are presented hereafter. As shown in Fig. 2a, after 1 h postmortem, some cells are shrunk (left part of the white line) whereas some other are not (right part of the white line). It is worthy to remember here that each cell can decide by itself,

**Fig. 2** Cell shrinkage and related structural damage of the membrane. a Transversal section of rat *Gastrocnemius* muscle excised 1 h *postmortem* (×1000). The *white line* delimited the left region of the section where shrinkage is more important than in the *right* one.

b Membrane disruption at the costamere level observed by electron microscopy examination of a 2 h *postmortem* sample (magnification: ×100,000). The *black arrow* indicate the position of the hole in the membrane and the *white open arrowheads* shows the released portion of the membrane still bound to the costamere/Z-line (Z) transversal filaments.

c Transversal section of rat *Gastrocnemius* muscle excised 24 h (×1000). For more details see the materials and methods section

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
independently from the others, to commit suicide by engaging into the cell death program at the right time. This explains why, within the same cut, the behaviour of each cell can be different. The extra-cellular space is thus minimal and more regular in the right part of the plate as compared to the left part, where this space is abnormally large and variable from one area to another.

An estimation of the extent of shrinkage has been performed as described in the Materials and Methods section on rat Longissimus samples excised just after bleeding. For each sell examined, the reference is the same cell before shrinkage meaning that shrink = 0 and total cell weight = G + B. On average, cell volume decreased by about 20 % (20 ± 6 %). age This shrinkage results very likely from the cytoskeletal induced traction forces on the cell membrane with a concomitant water out flux towards the extracellular space.

The intensity of the forces concerned at the time of the cellular retraction is sufficiently important to cause wrenching of anchorage structures (costameres, integrins, ...) from the membrane. They also cause a retraction of all intracellular organelles contributing, at least partly, to their dysfunctions. This feature is illustrated in Fig. 2b for a 2 h postmortem sample from Gastrocnemius muscle showing a clear release (black arrow) of part of the membrane corresponding to the costamere region, the membrane fragment being still anchored to the transversal intermediate filaments emanating from the Z-line (open arrowhead).

Figure 2c presents a cut from the same Gastrocnemius muscle at time 24 h postmortem. After 24 h, all cells are shrunk and extra-cellular space increased significantly, suggesting a time-dependent progress of the cell death process. Another interesting feature is that, in the 24 h postmortem sample, almost all fibres showed the typical rounding appearance of cells dying by apoptosis [9].

## 3.2 Phosphatidylserine Externalisation

Analysis of phosphatidylserine externalisation was performed on both rat Gastrocnemius and Plantaris muscles and, as no difference was observed, only results of the former muscle are presented herein. Besides cell shrinkage, another characteristic feature of apoptosis is the externalization of phosphatidylserine (PS), a class of acidic phospholipids normally located on the internal leaflet of the lipid bilayer. However, in some physiologic conditions, the internal PS is exposed to the external surface especially in dying cells [10, 11]. Phosphatidylserine (PS) externalization is thus regarded as one of the earliest hallmarks of cells undergoing programmed cell death [12]. In living organisms, macrophages recognize the PS externalized cells through a specific PS receptor, resulting in a possible contribution of macrophages to the target cell dismantling [13].

To comfort the idea that the onset of the cell death program takes place immediately after bleeding, an assumption supported by the intense cell shrinkage observed as soon as 1 h postmortem, redistribution of PS has been investigated on small muscle fibers bundles using a FITC labeled specific PS-binding protein,
Annexin V, as a specific probe. To attest the extracellular location of PS, we referred to Laminin, a major component of the basal lamina. In living cells, the primary function of the Laminin protein family is the anchorage of the plasma membrane to the extracellular matrix. Hence, these proteins are located in the vicinity of the external leaflet of the plasma membrane [14] and constitute an interesting internal control to verify that the PS labeling is extracellular and not intracellular.

Small muscle fiber bundles taken at 1 h (Fig. 3a) and 24 h (Fig. 3b) postmortem were incubated with FITC-labeled annexin V for 24 h and then frozen. Frozen cuts obtained from the Annexin V labeled samples (Fig. 3a1) were then treated with a primary rabbit Laminin antibody, revealed with a FITC-labeled goat anti-rabbit secondary antibody (Fig. 3a2) and examined under the light microscope for Annexin V and Laminin fluorescence. Note that serial thin cuts were obtained from the central part of the frozen sample avoiding the peripheral fibers which can be damaged during preparation of the sample.

As depicted in Fig. 3a1 (1 h postmortem sample) and Fig. 3b1 (24 h postmortem sample) analysis of the Annexin V distribution in transversal muscle thin cuts led to a specific green fluorescent labeling of the periphery of the fibers. In addition, the labeling intensity appeared to be greater in the 24 h postmortem sample suggesting a time course progress in the PS externalization during the first 24 h post-exsanguination. No fluorescence was detected in fibers treated with the same buffer in which Annexin V was omitted (Fig. 3a4). Immunostaining of rat muscle fibers prepared 1 h (Fig. 3a2) or 24 h (Fig. 3b2) postmortem with a Laminin rabbit polyclonal antibody revealed with a cyanin 3 conjugated goat anti-rabbit IgG led to specific fluorescent red labeling of the periphery of the fibers. When the first antibody was omitted, no fluorescence was detected (Fig. 3b4).

Merging images corresponding Laminin and PS labeling led to a yellow color, a result indicative of a co-localization of Laminin and PS-bound Annexin V. Such color change was observed both at 1 h (Fig. 3a3) and 24 h (Fig. 3b3) postmortem samples. The co-localization of both antigens confirms that Annexin V is bound to extracellular PS and not to intracellular acidic phospholipids. It is noteworthy that the yellow color is more intense in the 24 h sample, suggesting an increase in the number of externalized PS between 1 and 24 h postmortem. The present results clearly indicate that PS groups are externalized in postmortem muscle as soon as 1 h postmortem and that the extent of PS externalization increased between 1 and 24 h postmortem. By contrast, Laminin labeling tends to decrease between 1 h (Fig. 3a2) and 24 h (Fig. 3b2), a feature indicative of a probable degradation of this protein soon after death.

Since the beginning of the present decade, several reports suggested a contribution of caspases, especially caspase 3, to the postmortem proteolysis [1, 2, 4]. On the other hand, caspases are the driven motor of the programmed cell death by apoptosis development. The present work intended therefore to obtain evidence supporting the onset of the apoptotic process in postmortem muscle.

The concept of apoptosis was introduced by Kerr et al. 4 [15] to indicate a form of cellular death totally different from necrosis, from both the morphological and
<figure><img src="image_7.png" /><figcaption>**Fig. 3** Phosphatidylserine externalization and Laminin labeling. **a** Samples from rat *Gastrocnemius* muscle excised 1 h *postmortem*. **b** Samples from rat *Gastrocnemius* muscle excised from the second leg 24 h *postmortem*. **1** Samples labeled with a FITC-Annexin V conjugate at 1 h (a) and 24 h (b) postmortem. Note the greater fluorescent labeling of the 24 h sample as compared to 1 h (λexc 488 nm; λemm 530 nm). **2** Samples labeled with a rabbit specific Laminin antibody revealed with a cyanine 3 goat anti-rabbit IgG conjugate at 1 h (a) and 24 h postmortem. Note the lower fluorescent labeling of the 24 h sample as compared to 1 h probably indicative of the degradation of the Laminin (λexc 554 nm; λemm 568 nm). **3** Merged image from 1 and 2. **4** Control samples for annexin V (a4) and Laminin (b4) labeling in which the FITC Annexin V conjugate and the primary anti-Laminin antibody respectively were omitted</figcaption></figure>
biochemical points of view. In muscle tissue, chronological events conducting to apoptosis are the same than in mononucleated cells which are usual models for such studies [16]. As previously reviewed [6], in contrast to necrosis often considered as a "disordered" cellular death, apoptosis is regarded as an "ordered" cellular death proceeding through a series of chronological and strictly regulated events: Dying cells lose their contact with neighboring cells and signal their status by externalization of phosphatidylserine which are normally located on the inner leaflet of the membrane. Concomitantly, the general condensation affecting the dying cells and their organelles led to an intense shrinkage and a significant reduction in the cell volume together with an increase in the extracellular space. Mitochondria undergo several major modifications leading to a release of pro-apoptotic proteins including cytochrome c. After condensation of the nucleus, chromatin is cleaved in regular fragments of approximately 180 pairs of bases.

According to Green programmed cell death is a rapid process taking place within few minutes to few hours. Hence, if apoptosis [17]. Set up in postmortem muscle, the first characteristic changes induced by process would be detectable soon after animal death. Four of the above hallmarks of apoptosis, including cell shrinkage, phosphatidylserine externalization, DNA fragmentation and actin degradation, were therefore investigated.

It was shown that cell shrinkage is detectable few minutes after exsanguination and that this change progress over time to reach its maximum about 24 h post-mortem. For samples excised within 1 h postmortem, the cell volume decreased by about 20 % but all cells are not concerned at the same time, comforting the idea that each cell decides when it will commit to suicide and engages into the cell death programme. After 24 h, all cells are shrunk and all have the rounding shape characterizing apoptosis. Cell shrinkage led to an important increase in the extra-cellular space. This increase in the extracellular space was generally ascribed to a decrease in the capacity of myofibrillar protein to bind water during the acidification of muscle and the consequent pH drop [18, 19]. The causes of the large raise of the extracellular space in postmortem muscle would have to be reconsidered in the light of the present findings.

One of the major consequences of cell shrinkage is the strong constraints supported by the anchorage structures associated to the plasma membrane (costameres, integrins, ...) which can be strongly altered. As illustrated in the present work, costameres are sometimes wrenched from the membrane while they are still attached to the transversal intermediate filaments emanating from the Z-line. This limited opening of the membrane will facilitate water flux towards the extracellular space.

The second hallmark of apoptosis considered was the externalization of phosphatidylserine (PS) which move from the inner to the outer leaflet of the plasma membrane. Using FITC-labeled Annexin V as a specific probe of these acidic phospholipids, it was shown that a strong green labeling was noted in the vicinity of the membrane. To attest the extracellular location of PS, we referred to Laminin, a major component of the basal lamina located in the vicinity of the external membrane leaflet, which ensures the connection of the extracellular matrix to the cell membrane. Laminin was labeled with a specific antiserum and revealed using a
cyan 3-conjugate secondary antibody (red color). When the fluorescent images were merged the color changed to yellow indicative of a co-localization of the proteins, i.e. Annexin V and Laminin. In addition, we noted a decrease in the Laminin labeling intensity probably suggesting a probable alteration of this protein over the first 24 h postmortem. In contrast, intensity of PS labeling seemed to increase over this time laps suggesting a time dependent raise in the number of PS group transferred on the outer leaflet.

Recently Huang et al. [20] demonstration thereby strengthened the hypothesis that in post mortem muscle, oxygen deprivation-induced apoptosis is dependent on the mitochondrial pathway. On the other hand, proteins involved in the regulation of the apoptotic process whether released from mitochondria (AIF, Endo G, Cytochrome c, ...) or expressed in muscle cells (IAPs, caspase inhibiting serpin, ...) have never been revealed by 2D gel electrophoresis proteome analysis and thus they would merit to be selectively targeted using alternative more specific approaches [21].

## 4 Conclusion

In conclusion, the present work provides, for the first time, indisputable evidence supporting the set up of apoptosis in muscle immediately after animal death. It has been further proven that this process affects cell metabolism, oxidative processes affecting proteins, lipids and meat color, cytoskeletal network degradation. These different events contributing the development of different meat qualities (tenderness, color, flavor and juiciness) must be reconsidered in relation with the complex apoptotic induced modifications of cell metabolism and physiology.

**Acknowledgments** Many thanks to Christine Barboiron, Arnaud Delavaud and Jean-Luc Montel (URH unit, C2M team) for their invaluable assistance in light microscopy studies. This work is part of one of the QUAPA transversal project funded by INRA. The visit of S. Becila in our institute was funded by a grant from the Algerian Ministry of Education. Many thanks for this support.

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