Effects of carnosine on contractile apparatus Ca²⁺ sensitivity and sarcoplasmic reticulum Ca²⁺ release in human skeletal muscle fibers

There is considerable interest in potential ergogenic and therapeutic effects of increasing skeletal muscle carnosine content, although its effects on excitation-contraction (EC) coupling in human muscle have not been defined. Consequently, we sought to characterize what effects carnosine, at levels attained by supplementation, has on human muscle fiber function, using a preparation with all key EC coupling proteins in their in situ positions. Fiber segments, obtained from vastus lateralis muscle of human subjects by needle biopsy, were mechanically skinned, and their Ca(2+) release and contractile apparatus properties were characterized. Ca(2+) sensitivity of the contractile apparatus was significantly increased by 8 and 16 mM carnosine (increase in pCa(50) of 0.073 ± 0.007 and 0.116 ± 0.006 pCa units, respectively, in six type I fibers, and 0.063 ± 0.018 and 0.103 ± 0.013 pCa units, respectively, in five type II fibers). Caffeine-induced force responses were potentiated by 8 mM carnosine in both type I and II fibers, with the potentiation in type II fibers being entirely explicable by the increase in Ca(2+) sensitivity of the contractile apparatus caused by carnosine. However, the potentiation of caffeine-induced responses caused by carnosine in type I fibers was beyond that expected from the associated increase in Ca(2+) sensitivity of the contractile apparatus and suggestive of increased Ca(2+)-induced Ca(2+) release. Thus increasing muscle carnosine content likely confers benefits to muscle performance in both fiber types by increasing the Ca(2+) sensitivity of the contractile apparatus and possibly also by aiding Ca(2+) release in type I fibers, helping to lessen or slow the decline in muscle performance during fatiguing stimulation.     

Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca2 + -ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca^2?+? release, and the light fraction enriched in Ca^2?+?-ATPase (SERCA), an enzyme responsible for Ca^2?+? transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca^2?+?- ATPase activity and Ca^2?+?-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca^2?+?-uptake and Ca^2?+?-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.     

Molecular topography of the Ca2+ — ATPase of sarcoplasmic reticulum

Summary The Ca²⁺ binding site region of the Ca²⁺ — ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca²⁺ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca²⁺ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I^– indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca²⁺ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca²⁺ binding is shown to be near the membrane surface.     

Calcium transport by rabbit skeletal muscle microsomes (“fragmented sarcoplasmic reticulum”)

Ca²⁺ binding by skeletal muscle microsomes in 5 mM ATP exhibited saturation kinetics in the range of Ca²⁺ concentrations between 3 · 10^?8 and 10^?5 M. Approximately 140 nmoles binding sites per mg protein were found. These had a Ca²⁺ binding constant of approximately 4.5 · 10⁶ M^?1 with half saturation at 2.2 · 10^?7 M Ca²⁺. In the presence of oxalate, much larger amounts of Ca²⁺ (approx. 6 μmoles/mg protein) were taken up by the microsomes (Ca²⁺ uptake), but the rate of Ca²⁺ uptake increased linearly with [Ca²⁺] when ionized Ca²⁺ concentrations were below 3 · 10^?6 M. At Ca²⁺ concentrations above 3 · 10^?6, Ca²⁺ uptake was inhibited. Double reciprocal plots of the Ca²⁺ dependence of the initial rates of Ca²⁺ uptake in the concentration range between 3 · 10^?7 M and 10^?5 M, unlike those of Ca²⁺ binding, did not demonstrate saturation kinetics, but could be interpreted as representing a non-saturable system with inhibition at higher Ca²⁺ concentrations. In view of these differences, and because Ca²⁺-binding sites were almost fully saturated at 10^?6 M Ca²⁺, whereas Ca²⁺ uptake rate increased linearly with increasing [Ca²⁺] to approximately 3 · 10^?6 M, the Ca²⁺-binding sites are not shown kinetically to participate in oxalate-dependent Ca²⁺ uptake.     

Further characterization of light and heavy sarcoplasmic reticulum vesicles. Identification of the ‘sarcoplasmic reticulum feet’ associated with heavy sarcoplasmic reticulum vesicles

Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle.Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca²⁺/μl of water space. After KCl washing both contained less than 4 nmol Ca²⁺/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca²⁺ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca²⁺ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca²⁺-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca²⁺-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’.     

Does intrinsic fluorescence reflect conformational changes in the Ca2+-ATPase of sarcoplasmic reticulum?

We have investigated the kinetics of the intrinsic fluorescence drop observed when ATP is added to purified sarcoplasmic reticulum ATPase in a potassium-free medium containing magnesium and calcium, at pH 6 and 20°C. Under these conditions, analysis of the fluorescence drop is complex. Several events contributed to the rate of the fluorescence drop initiated by turnover, including phosphorylation, conformational transition of the phosphorylated complex, and dephosphorylation. On the other hand, when 75% of total fluorescence was quenched by energy transfer to the membrane-bound ionophore A23187, the observed turnoverdependent drop in residual fluorescence mainly reflected the conformational transition of the phosphorylated ATPase. Combination of fast kinetics with the quenching of selected tryptophan residues is suggested to be a promising tool for the study of proteins containing many of these residues.     

Synchronization of stochastic Ca²(+) release units creates a rhythmic Ca²(+) clock in cardiac pacemaker cells

In sinoatrial node cells of the heart, beating rate is controlled, in part, by local Ca2(+) releases (LCRs) from the sarcoplasmic reticulum, which couple to the action potential via electrogenic Na(+)/Ca2(+) exchange. We observed persisting, roughly periodic LCRs in depolarized rabbit sinoatrial node cells (SANCs). The features of these LCRs were reproduced by a numerical model consisting of a two-dimensional array of stochastic, diffusively coupled Ca2(+) release units (CRUs) with fixed refractory period. Because previous experimental studies showed that β-adrenergic receptor stimulation increases the rate of Ca2(+) release through each CRU (dubbed I(spark)), we explored the link between LCRs and I(spark) in our model. Increasing the CRU release current I(spark) facilitated Ca2(+)-induced-Ca2(+) release and local recruitment of neighboring CRUs to fire more synchronously. This resulted in a progression in simulated LCR size (from sparks to wavelets to global waves), LCR rhythmicity, and decrease of LCR period that parallels the changes observed experimentally with β-adrenergic receptor stimulation. The transition in LCR characteristics was steeply nonlinear over a narrow range of I(spark), resembling a phase transition. We conclude that the (partial) periodicity and rate regulation of the "Calcium clock" in SANCs are emergent properties of the diffusive coupling of an ensemble of interacting stochastic CRUs. The variation in LCR period and size with I(spark) is sufficient to account for β-adrenergic regulation of SANC beating rate.     

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca²⁺ in the Ca²⁺-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca²⁺-uptake, and heat up during ATP synthesis and Ca²⁺-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca²⁺-ATPases, not only act as Ca²⁺-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Temperature dependence of cardiac sarcoplasmic reticulum Ca²⁺-ATPase from rainbow trout Oncorhynchus mykiss

In this work, the temperature dependence of the sarco-endoplasmic reticulum Ca(2+) -ATPase (SERCA2) activity from rainbow trout Oncorhynchus mykiss cardiac ventricles was measured and compared with the mammalian SERCA2 isoform. The rate of ATP-dependent Ca(2+) transport catalysed by O. mykiss vesicles was totally abolished by thapsigargin and the Ca(2+) ionophore A(23187) . At warm temperatures (25 and 30° C), the SERCA2 from O. mykiss ventricles displayed the same rate of Ca(2+) uptake. At 35° C, the activity of the O. mykiss enzyme decreased after 20 min of reaction time. The rate of Ca(2+) uptake catalysed by the mammalian SERCA2 was temperature dependent exhibiting its maximal activity at 35° C. In contrast to the rate of Ca(2+) uptake, the rate of ATP hydrolysis catalysed by O. mykiss SERCA2 was not significantly different at 25 and 35° C, but the rate of ATP hydrolysis catalysed by the rat Rattus norvegicus SERCA2 isoform at 35° C was two-fold higher than at 25° C. At low temperatures (5 to 20° C), the rate of Ca(2+) uptake from O. mykiss SR was less temperature dependent than the R. norvegicus isoform, being able to sustain a high activity even at 5° C. The mean ±s.e. Q(10) values calculated from 25 to 35° C for ATP hydrolysis were 1·112 ± 0·026 (n = 3) and 2·759 ± 0·240 (n = 5) for O. mykiss and R. norvegicus, respectively. Taken together, the results show that the O. mykiss SERCA2 was not temperature dependent over the 10 to 25° C temperature interval commonly experienced by the animal in vivo. The Q(10) value of SERCA2 was significantly lower in O. mykiss than R. norvegicus which may be key for cardiac function over the wide environmental temperatures experienced in this eurythermal fish.     

Improving cardiac Ca⁺² transport into the sarcoplasmic reticulum in heart failure: lessons from the ubiquitous SERCA2b Ca⁺² pump

As a major Ca2+ pump in the sarcoplasmic reticulum of the cardiomyocyte, SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) controls the relaxation and contraction of the cardiomyocyte. It is meticulously regulated by adapting its expression levels and affinity for Ca2+ ions to the physiological demand of the heart. Dysregulation of the SERCA2a activity entails poor cardiomyocyte contractility, resulting in heart failure. Conversely, improving cardiac SERCA2a activity, e.g. by boosting its expression level or by increasing its affinity for Ca2+, is a promising strategy to rescue contractile dysfunction of the failing heart. The structures of the related SERCA1a Ca2+ pump and the Na+/K+-ATPase of the plasma membrane exposed the pumping mechanism and conserved domain architecture of these ion pumps. However, how the Ca2+ affinity of SERCA2a is regulated at the molecular level remained unclear. A structural and functional analysis of the closely related SERCA2b Ca2+ pump, i.e. the housekeeping Ca2+ pump found in the endoplasmic reticulum and the only SERCA isoform characterized by a high Ca2+ affinity, aimed to fill this gap. We demonstrated the existence of a novel and highly conserved site on the SERCA2 pump mediating Ca2+ affinity regulation by the unique C-terminus of SERCA2b (2b-tail). It differs from the earlier-described target site of the affinity regulator phospholamban. Targeting this novel site may provide a new approach to improve SERCA2a function in the failing heart. Strikingly, the intramembrane interaction site of the 2b-tail in SERCA2b shares sequence and structural homology with the binding site of the β-subunit on the α Na+/K+-ATPase. Thus P-type ATPases seem to have developed related mechanisms of regulation, and it is a future challenge for us to discover these general principles of P-type regulation.     

Does muscle activation occur by direct mechanical coupling of transverse tubules to sarcoplasmic reticulum?

Our knowledge of the physiological and biochemical constituents of skeletal muscle excitation has increased greatly during the last few years but this has not led to a consensus of the physiological mode of muscle activation. Three hypotheses of transmission, involving either transmitter-receptor interaction or direct mechanical coupling, are still under active consideration. The hypothesis of direct mechanical coupling currently being evaluated proposes that the dihydropyridine receptor in the transverse tubules serves as a voltage sensor that communicates directly with the junctional foot protein/Ca2+ channel of sarcoplasmic reticulum to initiate opening of the channel.     

The role of PGC-1α in the regulation of skeletal muscle metabolism

The purpose of this review was to provide an understanding of the role of PGC-1α in the regulation of skeletal muscle metabolism and to describe the results of studies on the association of the polymorphism gene PPARGC1A with human muscle performance.     

Neurotrophic control of the transmembrane Cl− pump in mammalian muscle fibers

Resting membrane potential of both innervated and denervated rat diaphragm muscle fibers was investigated when the concentration of potential-producing ions was changed in the external fluid and following treatment with furosemide. It was found that equilibrium potential ( ) equalled resting potential level in innervated muscle for Cl^–, but shifts to more positive values compared with resting membrane potential following section of the nerve. Furosemide retards development of post-denervation depolarization of the muscle membrane. It is deduced that trophic influences originating from the motor nerve activates the furosemide-sensitive Cl^– influx system, leading to raised intracellular concentration of Cl^–, a shift in (E_Cl) and depolarization of the muscle membrane.S. V. Kurashov Medical Institute, Minsitry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 766–771, November–December, 1987.     

Interaction between the sarcoplasmic reticulum and mitochondria in the control of contractile calcium in guinea-pig atria--does it exist?

The inhibitory action of caffeine on calcium (Ca2+) release from the sarcoplasmic reticulum (SR) and interference with mitochondrial (Ca2+) fluxes by a mitochondrial uncoupler protonfore CCCP were utilized to define a calcium pool responsible for potentiation of post-rest twitch tension in guinea-pig atria. The Ca2+ fluxes were assessed by means of 45Ca2+. Caffeine and CCCP when applied separately did not affect post-rat 45Ca2+ content. Yet, when they were applied together it was markedly reduced to the resting level. It is concluded that a possible source of contractile Ca2+ may be located in mitochondria and an eventual shift of Ca2+ between mitochondria and the SR seems to be a plausible assumption.     

Photoaffinity labeling of the (Ca+Mg)ATPase of skeletal and cardiac sarcoplasmic reticulum with [γ-32P]-8-azido-ATP

8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca²⁺. The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca²⁺ and Mg²⁺ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.     

Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.     

How does ryanodine modify ion handling in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel?

Under appropriate conditions, the interaction of the plant alkaloid ryanodine with a single cardiac sarcoplasmic reticulum Ca(2+)-release channel results in a profound modification of both channel gating and conduction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this paper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group 1a monovalent cations, alkaline earth divalent cations, and organic monovalent cations. We have quantified the change in single-channel conductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the nature of the permeant cation. The group 1a monovalent cations (K+, Na+, Cs+, Li+) have values of fractional conductance in a narrow range (0.60- 0.66). With divalent cations fractional conductance is considerably lower (Ba2+, 0.22 and Sr2+, 0.28), whereas values of fractional conductance vary considerably with the organic monovalent cations (ammonia 0.66, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylamine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the relative permeability of, representative cations in the ryanodine-modified channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-ion, four-barrier three-well rate theory model of conduction in the unmodified channel. Our findings indicate that the high affinity, essentially irreversible, interaction of ryanodine with the cardiac sarcoplasmic reticulum Ca(2+)-release channel produces a conformational alteration of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group 1a monovalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the conduction pathway for the alkaline earth divalent cations is also increased, however the relative permeability of this class of cations is reduced compared to the unmodified channel. The influence of modification on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunological detection of m- and μ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca²⁺-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15^th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy.