The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post‐natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3−/− mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation–contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).
Ryanodine receptors (RyRs) are intracellular homotetrameric Ca2+ release channels whose subunits are encoded by three different genes indicated as RyR1, RyR2 and RyR3 (Meissner, 1994; Sorrentino, 1995; Sutko and Airey, 1996). RyR1 channels are located in the terminal cisternae of the sarcoplasmic reticulum of skeletal muscle fibres, while RyR2 channels are found in the corresponding structures of cardiac myocytes (Meissner, 1994; Sutko and Airey, 1996). Both RyR1 and RyR2 are also found, although at much lower levels of expression than in striated muscles, in several other tissues and type of cells (Giannini et al., 1995; Sorrentino, 1995). The third isoform, RyR3, is characterized by a wide pattern of expression but lack of a preferential association with one tissue (Giannini et al., 1992, 1995). At variance with RyR1 and RyR2, for which a major functional role is to release Ca2+ in order to regulate skeletal and cardiac contraction, there is no evidence of a physiological role for RyR3. Based on its wide expression, it has been proposed that RyR3 may participate in intracellular Ca2+ release in conjunction with other Ca2+ release channels, including the inositol trisphosphate (InsP3) receptors and other RyR isoforms, via a mechanism of activation mediated by Ca2+ itself, named Ca2+‐induced Ca2+ release (CICR) (Giannini et al., 1992, 1995; Berridge, 1993).
Among other tissues, RyR3 was also detected in some mammalian skeletal muscle preparations (Giannini et al., 1992; Conti et al., 1996) and in cultured myocytes (Giannini et al., 1995; Takeshima et al., 1995; Tarroni et al., 1997), although its expression in skeletal muscles is still debated (Buck et al., 1997). In vertebrate skeletal muscles, RyR1 is essential for releasing Ca2+ from the sarcoplasmic reticulum following electrical depolarization of the plasma membrane, a process referred to as excitation–contraction coupling (Fleischer and Inui, 1989; Schneider, 1994; Takeshima et al., 1994; Nakai et al., 1996; Sutko and Airey, 1996). The finding that RyR3 is present, even though at lower levels than RyR1, in mammalian skeletal muscles echoes the observation made in non‐mammalian vertebrate skeletal muscles where equal amounts of two isoforms, α and β, are expressed (Sutko and Airey, 1996). These two isoforms have been shown, at least in frogs and chicken, to correspond to mammalian RyR1 and RyR3, respectively (Oyamada et al., 1994; Ottini et al., 1996). Although both α‐ and β‐RyR isoforms are expressed in most muscles of birds, frogs and fishes, differential expression of RyR isoforms in specialized muscles of non‐mammalian vertebrates correlates with particular contractile properties of those muscles (Sutko and Airey, 1996). Expression of the α‐RyR isoform alone has been observed in the extraocular muscles of chicks and fishes, and in the swimbladder muscle of toadfish (O‘Brien et al., 1993). Since these muscles are among the fastest contracting muscles in vertebrates, it has been suggested that the β‐RyR isoform is not compatible with fast contracting muscles (O'Brien et al., 1995).
In addition to the biochemical data on the heterogeneity of RyR isoforms in vertebrate skeletal muscles, other lines of evidence suggest that two functionally distinct Ca2+ release channels may be involved in the regulation of skeletal muscle contraction. Morphological analysis of fractured faces of sarcoplasmic and of T‐tubule membranes have revealed that both RyRs and the voltage sensor, the di‐hydropyridine receptors (DHPRs), are organized in regular arrays of particles (Block et al., 1988). Accurate measurements of the distance between DHPRs on the plasma membrane and between RyRs on the sarcoplasmic reticulum have shown that the distance between DHPRs is such that only one out of two RyRs may be in close contact with the DHPRs (Block et al., 1988; Franzini‐Armstrong and Kish, 1995; Flucher and Franzini‐Armstrong, 1996). Since in skeletal muscle excitation–contraction coupling a direct contact between RyRs and DHPRs is important for activation of Ca2+ release from the sarcoplasmic reticulum, it would appear that one out of two RyRs is not activated directly by this mechanism.
A second observation in favour of a two‐release channel system comes from physiological studies of Ca2+ release from the sarcoplasmic reticulum, where the existence of two components, a fast activating and inactivating component of large amplitude and a smaller more steady component, has been reported (Fleischer and Inui, 1989; Rios et al., 1992; Meissner, 1994; Schneider, 1994). It has been proposed that the former represents a CICR component that is activated by the initial Ca2+ released by the latter smaller component that is regulated directly by voltage (Rios et al., 1992). The relationship between coupled and uncoupled RyRs and the proposed two components of Ca2+ release in skeletal muscles has not yet been resolved. It should be noted that the structural organization with coupled and uncoupled RyRs has also been observed in muscles of non‐mammalian and mammalian vertebrates that contain only one isoform of RyR (Block et al., 1988; O'Brien et al., 1993; Franzini‐Armstrong and Kish, 1995). Thus, that particular structural organization seems to be a general motif in vertebrate skeletal muscles, but the presence of two RyR isoforms is not always required.
The reason for the seemingly redundant expression of RyR1 and RyR3 in skeletal muscle is therefore not clear. The central role of RyR1 in skeletal muscle excitation–contraction coupling has been confirmed by studies of RyR1 knockout mice (Takeshima et al., 1994, 1995; Nakai et al., 1996), in which skeletal muscle excitation–contraction coupling is abolished. RyR1 is also crucial for skeletal muscle excitation–contraction coupling in non‐mammalian vertebrates, as exemplified by the cn/cn mutant, a naturally occurring chicken mutant that does not express RyR1 and in which skeletal excitation–contraction coupling is abolished although RyR3 expression levels remain high (Ivanenko et al., 1995; Sutko and Airey, 1996). Thus the RyR3 isoform cannot substitute for RyR1 function, and whether RyR3 participates at all in the regulation of skeletal muscle contraction remains to be clarified (Sutko and Airey, 1996; Takeshima et al., 1996).
In this study, we provide the first evidence that (i) RyR3 is widely expressed in murine skeletal muscles selectively during the neonatal phase of muscle development, while in adult muscles its expression is restricted mainly to the diaphragm and soleus muscles; (ii) skeletal muscle contraction in RyR3 knockout is impaired during the first weeks after birth, indicating that the RyR3 isoform has a functional role in optimizing muscle contractility during the neonatal phase of development of skeletal muscles; and (iii) the contribution of RyR3 to excitation–contraction coupling is apparently devoted to the Ca2+ release amplification.
RyR3 is widely expressed in neonatal but not in adult skeletal muscles
The process of skeletal muscle development requires fine regulation of the expression of the contractile proteins and of proteins that accumulate and release Ca2+ in the sarcoplasmic reticulum. Most muscle‐specific proteins start to be expressed during embryonic development, yet a significant increase in the synthesis of these proteins occurs around birth and in the neonatal period of muscle development. Several muscle proteins undergo isoform transition during this period (Schiaffino and Reggiani, 1996). The developmental expression pattern of the RyR3 isoform of Ca2+ release channel was investigated by Western blot analysis of microsomal fractions prepared from total hindlimb muscles isolated from mice at day 18 of fetal development, at day 2, 5, 15 and 25 after birth and from adult (i.e. 60‐day‐old) mice. Expression of RyR1 and RyR2 isoforms and of the sarcoplasmic reticulum Ca2+‐ATPase (SERCA) was analysed in parallel. An increase in RyR1 and Ca2+‐ATPase was observed in the early days after birth, with high levels maintained in adult mice. The RyR3 isoform was also detected in the hindlimb muscles of mice, where its expression attained the highest levels 15 days after birth (Figure 1A). Later, however, the RyR3 content was reduced in hindlimb muscles and became undetectable in adult mice.
The RyR3 content in post‐natal skeletal muscles was evaluated further in individual skeletal muscles including biceps and quadriceps femoris, extensor digitorum longus, soleus, tibialis anterior, diaphragm and abdominal muscles dissected from 15‐day‐old and from adult mice. As shown in Figure 1B, the RyR3 protein was expressed in all skeletal muscles of 15‐day‐old mice. It was, however, not detectable in the same muscles of adult mice, with the exception of the diaphragm and soleus muscles, where it remained present (Conti et al., 1996) at lower levels than in younger mice. A similar pattern of expression for RyR3 was also found in skeletal muscles of rats (not shown). In both mouse and rat skeletal muscles, the cardiac isoform (RyR2) was never detected. Altogether, these results reveal a unique expression pattern for RyR3 in neonatal skeletal muscles. The wider pattern of expression of RyR3 in neonatal skeletal muscles compared with the restricted pattern observed in adult animals suggests that RyR3 could participate specifically in either Ca2+ release in relation to metabolic processes or in excitation–contraction coupling, during that period of development.
Generation of RyR3 knockout mice
To test whether RyR3 has a functional role in neonatal skeletal muscles, we took advantage of the homologous recombination technique. To disrupt the RyR3 gene, a replacement vector was constructed were the neomycin resistance gene was inserted into the sixth exon (counting from the last one). This eliminates the carboxy‐terminus of the protein which includes the transmembrane segments involved in formation of the pore region of the Ca2+ channel, thus generating a non‐functional RyR3 allele (see Figure 2A). E14 embryonic stem (ES) cells were electroporated and two of the selected clones containing the mutated gene were used to generate chimeric mice. Homozygous mice were obtained by intercrossing heterozygous RyR3−/+ mice, and the progeny were genotyped by Southern blot (Figure 2B). RyR3 knockout mice (RyR3−/−) appeared normal and were detected at a normal frequency in the progeny, as also observed in an independent RyR3 knockout mouse line (Takeshima et al., 1996). Western blot analysis confirmed the absence of the RyR3 protein in the microsomal fraction from diaphragm muscle (Figure 2C) and other organs (not shown). Expression levels of the RyR1 isoform in skeletal muscles were similar in RyR3−/− and RyR3+/+ mice (Figure 2D). RyR3−/− mice were indistinguishable in appearance from wild‐type controls. Histological examination of skeletal muscles of RyR3 knockout mice at the level of both the light and the electron microscope failed to reveal gross abnormalities in sarcomere and triad organization (not shown).
Impaired response to electrical stimulation in neonatal skeletal muscles of RyR3 knockout mice
The contractile properties of skeletal muscles of RyR3 knockout mice were studied on muscle strips dissected from the diaphragm of neonatal (15 days) and adult (60–90 days) RyR3−/− and RyR3+/+ mice. Although the preparations from neonatal animals developed less tension during fused tetani (50 Hz) than the preparations from adult mice, there were no significant differences between RyR3−/− and RyR3+/+ mice (see Figure 3A). However, in 15‐day‐old mice, tension developed in a twitch at low stimulation frequency (0.1 Hz) was significantly lower in RyR3−/− than in RyR3+/+ mice: expressed as a fraction of tetanic tension (twitch/tetanus ratio) it represented 0.23 ± 0.03 in RyR3+/+ (n = 10) and 0.15 ± 0.01 in RyR3−/−(n = 10, P<0.05) (Figure 3B). The same was true for tension developed in unfused tetani (5 and 10 Hz). At higher stimulation rates, the difference was cancelled by fusion and summation of the responses. As can be seen in Figure 3C, the force–frequency curve appeared shifted downward and rightward in RyR3−/− compared with RyR3+/+ mice. In contrast, in adult mice preparations, the differences in twitch/tetanus ratio and in the force–frequency curve disappeared (Figure 3D). The time course of the twitch showed a significant age‐dependent change. Time to peak tension (Figure 3E) and time to half‐relaxation (not shown) became shorter in adult muscles, but no difference was detectable between RyR3−/− and RyR3+/+ mice. Other contractile properties, including response to the fatiguing stimulation protocol (not shown) and depression of the contractile response produced by removing extracellular Ca2+ (Figure 3F), were not different in RyR3−/−versus RyR3+/+ mice, although, as expected (Dangain and Neering, 1991), the effect of lowering Ca2+ concentration in the perfusing medium was greater in neonatal than in adult muscles.
The large contracture induced by caffeine in neonatal mice is strongly reduced in RyR3 knockout mice
The most striking difference between muscles of RyR3−/− and RyR3+/+ mice was found when tension development was induced by exposure to millimolar caffeine concentrations. Although mammalian muscles are less responsive to caffeine than amphibian muscles (Luttgau and Oetliker, 1968), release of calcium from sarcoplasmic reticulum and tension development without electrical stimulation can be induced by caffeine at concentrations >5 mM (Fryer and Neering, 1989). In RyR3+/+ mice, neonatal diaphragm strips developed more tension in response to caffeine than adult diaphragm strips. At caffeine concentrations >10 mM, the difference increased abruptly: 30 mM caffeine induced a contracture four times greater in neonatal than in adult diaphragm strips (see Figure 4). The kinetics of tension development were markedly different, as tension peaked in ∼10 s in adult and in 400–600 s in neonatal strips.
Remarkably, the large response to caffeine observed in RyR3+/+ neonatal diaphragm strips was abolished in knockout RyR3−/− preparations. The kinetics of tension development were, however, not altered, as tension development remained slower in neonatal than in adult strips (see Figure 4). The response to caffeine was also tested in extensor digitorum longus and soleus muscles with comparable results. The large contracture evoked by 30 mM caffeine in extensor digitorum longus (51% of tetanic tension n = 3) and soleus (38% of tetanic tension n = 3) of 15‐day‐old RyR3+/+ mice was markedly reduced in extensor digitorum longus and soleus muscles of RyR3−/− mice of the same age (7.4 and 8.6% of tetanic tension respectively, n = 3). In adult mice, the caffeine contracture was barely detectable in extensor digitorum longus, and ranged between 5 and 10% of tetanic tension in soleus without significant differences between RyR3+/+ and RyR3−/− preparations (not shown).
We have found that, in mammalian skeletal muscles, RyR3 is already expressed in the late stages of fetal development and that it attains its higher levels of expression between 2 and 3 weeks after birth. The RyR3 levels then decrease and this isoform is no longer detected in adult muscles, with the notable exception of the diaphragm and the soleus muscles. While the RyR3 levels start to decrease at 3 weeks after birth, the levels of other sarcoplasmic proteins, including the predominant RyR1 isoform of Ca2+ release channel and the Ca2+‐ATPase, increase steadily. The contrast between the wide pattern of expression of RyR3 in skeletal muscles of neonatal mice and the restricted expression observed in adult skeletal muscles suggests a specific requirement for RyR3, in addition to RyR1, at this stage of muscle development. Why neonatal skeletal muscles require the expression of RyR3 is not clear, but is worth remembering that usage of isoforms different from those present in adult fibres is observed commonly in neonatal skeletal muscles for some proteins of the contractile apparatus and sarcoplasmic reticulum (Schiaffino and Reggiani, 1996). Expression of RyR3 in the first 2–3 weeks after birth corresponds to a period where skeletal muscles undergo a series of major morphological and biochemical changes which precede the formation of adult skeletal muscles (Flucher and Franzini‐Armstrong, 1996; Schiaffino and Reggiani, 1996).
The functional relevance of the RyR3 isoform during this period of muscle development is supported by results obtained with RyR3 knockout mice. These results indicate that, in agreement with the preferential expression of the RyR3 isoform in the post‐natal period of mammalian skeletal muscle development, neonatal muscles lacking RyR3 are ∼35% less efficient in translating electrical stimulation into force generation than muscles from control mice. Impairment of force generation in RyR3−/− mice following electrical stimulation represents the first physiological evidence that the RyR3 isoform participates in excitation–contraction coupling in skeletal muscles. Although the force deficit observed in skeletal muscles of RyR3−/− mice can be compensated easily by increasing the rate of motorneuron discharge, in agreement with preservation of mice motility in the absence of RyR3 (Takeshima et al., 1996), an impairment of muscle performance in RyR3−/− mice under extreme conditions of muscle activity cannot be ruled out definitively.
The relevance of RyR3 in the response of skeletal muscles to contraction‐inducing stimuli was even more evident when muscles from neonatal RyR3 knockout mice were exposed to high concentrations of caffeine, a known activator of Ca2+ release through RyRs (Fryer and Neering, 1989; Meissner, 1994). In neonatal RyR3−/− mice, caffeine‐induced contracture was in fact reduced to ∼20% of that of control mice. Although several factors, including a disproportion between ability to release and to take up Ca2+ into the sarcoplasmic reticulum (Luttgau and Oetliker, 1968; Makabe et al., 1995), might also participate in setting the stage for the large caffeine contracture observed in neonatal mice, the depression of the caffeine response observed in RyR3−/− mice demonstrates that the RyR3 isoform is necessary for this large response to caffeine. This observation is in agreement with results obtained in muscles from RyR1 knockout mice which, although lacking excitation–contraction coupling, retained an unexpected high responsiveness to caffeine that tentatively was attributed to RyR3 (Takeshima et al., 1994; Takeshima et al., 1995; Nakai et al., 1996). The functional consequences of the RyR3 knockout in neonatal skeletal muscle contractility reported here are even more surprising when we consider that available data indicate that RyR3 represents only 1/20–1/100 of the total RyRs in mammalian skeletal muscles (Conti et al., 1996; Murayama and Ogawa, 1997; A.Conti and V.Sorrentino, unpublished data). Although we must await more work to establish the relative abundance of RyR3 and the structural and molecular organization of excitation–contraction coupling in developing muscles, these results strongly suggest that RyR3 could contribute to a secondary component of excitation–contraction coupling which creates an amplification mechanism for regulation of skeletal muscle contraction through a CICR mechanism (Fleischer and Inui, 1989; Rios et al., 1992; Schneider, 1994; Takeshima et al., 1995; Sutko and Airey, 1996).
It is of interest to discuss our results within the framework of the excitation–contraction coupling model for skeletal muscles (see Figure 5), where, at triads, only one of every two Ca2+ release channels on the sarcoplasmic reticulum faces the voltage sensor on the plasma membrane (Block et al., 1988; Flucher and Franzini‐Armstrong, 1996). It is accepted that the RyR1 isoform transduces, probably by mechanical coupling with the DHPR (Fleischer and Inui, 1989; Rios et al., 1992; Schneider, 1994; Sutko and Airey, 1996), the signal between the plasma membrane and the sarcoplasmic reticulum (Takeshima et al., 1994, 1995; Fleig et al., 1996; Nakai et al., 1996). This specialized function cannot be performed by the RyR3 isoform in either chicken or mice, as shown by lack of excitation–contraction coupling in natural chicken mutant and experimental mice lacking RyR1 (Takeshima et al., 1994; Ivanenko et al., 1995; Nakai et al., 1996). On these bases, the RyR1 isoform is a logical candidate to build the coupled channels that face the voltage sensor. In most non‐mammalian skeletal muscles, the RyR1 and RyR3 isoforms, given their equal ratio, could be organized in the working model shown in Figure 5A, with an alternating pattern where only RyR1 channels are opposed to DHPRs. In mammalian muscles that contain only the RyR1 isoform, as well as in those muscles of chicks and fishes that express only the α‐RyR1 isoform, it has to be accepted that RyR1, in addition to contributing to the coupled channels, must also contribute to the channels that are not coupled with the voltage sensor, as shown in Figure 5B. In agreement with the expectation that contraction following electrical stimulation is controlled essentially by activation of Ca2+ release channels containing the RyR1 isoform, RyR3 knockout mice possess a functional excitation–contraction coupling. However, in neonatal skeletal muscles of RyR3−/− mice, a strong reduction in the response to both electrical and caffeine stimulation was observed. We propose, therefore, that the RyR3 isoform, when expressed as in the neonatal stage of muscle development, forms uncoupled receptors which provide the CICR‐based amplificatory component of excitation–contraction coupling (Fleischer and Inui, 1989; Rios et al., 1992; Schneider, 1994; Sutko and Airey, 1996). Given the evidence that in mammalian skeletal muscles RyR3 is not in an equimolar ratio with RyR1, RyR3 could contribute to only a fraction of the uncoupled channels, as shown in Figure 5C. However, we should mention that, because of the current agreement that RyRs are homotetramers, the possibility of heterotetrameric channels has not been considered in Figure 5, although it cannot be excluded.
An obvious question arises from the results reported here. How can RyR3 channels, which represent only a small fraction of the total Ca2+ release channels present in skeletal muscles, be responsible for the effects that we observe to be present in neonatal RyR3 knockout mice? Several models have been proposed to explain the functional significance of the relationship between DHPRs and RyRs in skeletal muscles, and probably the data reported here need to be incorporated into such models (Sutko and Airey, 1996). The use of the RyR3 isoform for the uncoupled release channels is likely to reflect specific regulatory properties of RyR3 (Percival et al., 1994; O‘Brien et al., 1995; Takeshima et al., 1995) that might make it more effective than RyR1 in a CICR mechanism. Specific activation/inactivation kinetics or differential sensitivity to Ca2+ or other agonists and antagonists of channel activity might be the determinants of these properties (Percival et al., 1994; O'Brien et al., 1995; Sitsapesan et al., 1995; Takeshima et al., 1995; Rakovic et al., 1996; Sutko and Airey, 1996). Thus, qualitative diversity between isoforms might account for the apparent discrepancy (see above) between the small amount of RyR3 detected in mammalian muscles and the large functional impact of its deletion described in this study. We can speculate that the presence of a more sensitive system for a CICR‐mediated amplification might be of particular relevance in neonatal muscles in view of the as yet incomplete development of the triad structure (Luff and Atwood, 1971; Flucher and Franzini‐Armstrong, 1996).
As a concluding remark, it should be considered that co‐expression of various isoforms of Ca2+ release channels, originating from both alternative splicing and expression of different genes from both the InsP3‐R and the RyR families (Berridge, 1993; Furuichi et al., 1994; Marziali, 1996; Striggow and Ehrlich, 1996; Sutko and Airey, 1996), has been observed with increasing frequency in numerous cell types. The involvement of multiple channels with different regulatory properties has been proposed to be a means of providing a more refined regulation of specific cellular functions which are dependent on Ca2+ release (Berridge, 1993; Furuichi et al., 1994; Kasai and Petersen, 1994; Clapham, 1995; Striggow and Ehrlich, 1996). Our finding that, in neonatal muscles, activation of the RyR1 isoform to achieve an optimal force development requires the action of the RyR3 isoform provides the first experimental evidence of such a case.
Materials and methods
Microsomal vesicle preparations
Skeletal muscles isolated from mice at the indicated ages were used to prepare the microsomal fractions. Muscle tissue was frozen quickly in liquid nitrogen, and microsomes were prepared as previously described (Conti et al., 1996). Briefly, tissue samples were homogenized in ice‐cold buffer A [320 mM sucrose, 5 mM Na‐HEPES pH 7.4 and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] using a Dounce homogenizer. Homogenates were centrifuged at 7000 g for 5 min at 4°C. The supernatant obtained was centrifuged at 100 000 g for 1 h at 4°C. The microsomes were resuspended in buffer A and stored at −80°C. The protein concentration of the microsomal fraction was quantified using the Bradford protein assay kit (BioRad).
Western blot analysis
Microsomal proteins were separated by SDS–PAGE, as described (Giannini et al., 1995; Conti et al., 1996). Proteins were then transferred to a nitrocellulose membrane (Schleicher & Schüll) by blotting gels for 5 h at 350 mA at 4°C in a transfer buffer containing 192 mM glycine, 25 mM Tris, 0.01% SDS and 10% methanol. Membranes were incubated for 3 h in a blocking buffer containing 150 mM NaCl, 50 mM Tris–HCl pH 7.4, 0.2% Tween‐20, 5% non‐fat milk. Primary antibodies (diluted 1:3000) were incubated with membranes overnight at room temperature. Polyclonal rabbit antisera able to distinguish between the three RyRs were developed against purified GST fusion proteins corresponding to the region of low homology situated between the transmembrane domains 4 and 5 (divergent region 1, or D1) of the RyR1, RyR2 and RyR3 proteins, as previously described (Giannini et al., 1995), and have been shown not to cross‐react with each other. The Y1F4 monoclonal antibody against the Ca2+‐ATPase SERCA1 isoform (SERCA) was kindly provided by Dr J.M.East. Antigen detection was performed using the alkaline phosphatase detection method.
RyR3 gene targeting
A library of 129/Sv strain mouse genomic DNA was screened with a murine RyR3 probe corresponding to the sequence encoding the pore region of the channel. Isolated clones were characterized by sequence analysis, and the exon–intron structure of the region encoding the last seven exons of the murine RyR3 gene was characterized (G.Giannini, A.Nori and V.Sorrentino, unpublished). An insertion targeting vector containing 1.0 and 6.0 kb fragments surrounding the neomycin resistance gene, and the herpes virus thymidine kinase gene, was constructed. The linearized vector was electroporated into the E14 ES cell line and colonies were isolated following selection in G418 and gancyclovir, by standard methods. Double resistant cell lines were grown and characterized for homologous recombination by Southern blot after SpeI digestion and hybridization with a 0.9 kb EcoRI–HindIII probe. Four clones containing the targeted allele of 9.3 kb were isolated and injected into C57Bl/6 blastocysts. Transmission of the mutated allele to the progeny was obtained in four out of five chimeric mice obtained with two different ES clones. These mice were mated to C57Bl/6 mice to generate heterozygous mutant mice that were then cross‐bred to generate homozygous RyR3‐deficient (RyR3−/−) mice.
Analysis of muscle contractile properties
Diaphragm strips including the insertions on the rib and on the central tendon were dissected and mounted between a force transducer (AE 801Sensonor Horten, Norway) and a movable hook in a perfusion bath (volume 2 ml) containing bicarbonate Krebs solution. The solution was bubbled with O2–CO2 mixture and the temperature was kept constant at 22°C. The perfusion solution could be exchanged quickly (5–10 s) by flushing ∼20 ml of the new solution through the chamber. The preparations were stretched just above slack length (resting tension ∼5% of twitch tension) and supramaximally activated with field stimulation. The output of the tension transducer was stored in a PC after A/D conversion and recalled for analysis. CEA 1401 A/D converter and CEA Spike2 (CEA Cambridge UK) software were used.
After stabilization at 0.1 Hz, twitch and tetani (0.5 s duration) at frequencies of 5, 10, 20 and 50 Hz were recorded. Fatigue was induced with a protocol based on a 1:4 duty ratio with tetani of 0.5 s duration and 50 Hz stimulation frequency.
The depressing effect of external Ca2+ removal was studied on twitches produced at the rate of 0.1 Hz. Contractures with increasing doses of caffeine (range 10–30 mM) in the absence of electrical stimulation were produced sequentially. Each contracture was followed by washing with Krebs solution without caffeine to produce a complete relaxation of the preparation.
We acknowledge the contribution of G.Giannini and A.Nori to the initial stages of this project and L.Wrabetz and J.Meldolesi for comments on the manuscript. F.B. was supported by a short‐term EMBO fellowship during part of this project. This work was supported by a DFG grant Scho340/2 to H.R.S. and by European Community Grant BMH4‐CT96‐0656 to V.S. The financial support of Telethon Italy, grant No. 653 to V.S. and No. 1017 to C.R., is gratefully acknowledged.
- Copyright © 1997 European Molecular Biology Organization