Introduction

Top Abstract Introduction Materials and methods Results Discussion References

Nitric oxide (NO), a second messenger with numerous biologic functions, is synthesized by a group of flavoproteins known as nitric oxide synthases (NOS). Three NOS isoforms, endothelial cell (ecNOS), neuronal (nNOS), and inducible NOS, have been cloned so far. In normal skeletal muscle fibers, NO modulates a wide range of processes, including glucose uptake, excitation-contraction coupling, Ca²⁺ homeostasis, sarcolemmal ionic pumps, mitochondrial respiration, and neuromuscular transmission (1). NO also plays a significant role in the maturation of skeletal muscle in vivo (5) and regulates the fusion of cultured myoblasts in vitro (6). nNOS, the main NOS isoform responsible for NO synthesis in normal muscle fibers, is localized beneath the sarcolemma of mainly fast-twitch fibers (4). The association of nNOS with the sarcolemma is mediated through its interaction with the dystrophin complex. Disruption of the dystrophin complex in Duchenne and Becker muscular dystrophies results in downregulation of nNOS expression (7, 8) as well as displacement of nNOS from the sarcolemma to the cytoplasm.

Jaffrey and Snyder (9) used yeast two-hybrid screening of the rat hippocampal cDNA library to identify a small (89-amino acid) protein designated protein inhibitor of nNOS (PIN). This protein selectively associates with residues 163 to 245 of nNOS and destabilizes the nNOS dimer, thereby inhibiting nNOS activity. Recent studies indicate that PIN mRNA and protein are highly expressed in the brain and testes of normal humans (10). Amino acid sequencing of PIN suggests that it is one of the most conserved proteins in nature, with a high homology to the light-chain component of dynein in Drosophila (10), C. elegans (11), and Chlamydomonas (12). Dyneins are highly complex molecular microtubule-based motors responsible for retrograde translocation of membranous vesicles along microtubules. The critical importance of PIN as a component of cytoplasmic dynein was demonstrated recently in Drosophila in which partial loss-of-function mutations in PIN protein were associated with morphogenetic defects in bristle and wing development (10).

Despite the recent discovery of PIN as an endogenous regulator of nNOS activity, little is known about its expression and regulation in normal muscle fibers. The main aim of this study was therefore to investigate whether PIN is expressed in various ventilatory and limb muscle fibers and whether its expression is regulated in a fashion similar to that of nNOS. We also assessed whether PIN associates with the dynein complex inside skeletal muscle fibers.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Reagents

Western blotting apparatus and gels were obtained from Novex Inc. (San Diego, CA). Monoclonal anti-nNOS antibody was supplied by Transduction Laboratories (Lexington, KY). Affinity-purified polyclonal anti-PIN antibody was raised in rabbits by injecting histidine-tagged recombinant PIN, and purification was achieved with the glutathione-S-transferase (GST)-PIN column (9). Enhanced chemiluminescence (ECL) detection kits and nylon membranes were purchased from Amersham Canada (Oakville, ON, Canada). One hundred base-pair DNA markers and reverse transcriptase were procured from Life Technologies, Inc. (Gaithersburg, MD). A TA cloning kit was provided by Invitrogen, Inc. (San Diego, CA). Cy3-labeled secondary antibody was obtained from Jackson ImmunoResearch, Inc. (West Grove, PA).

Tissue Preparation

Rats. Pathogen-free Sprague-Dawley rats (250-350 g) were killed by decapitation, and the cerebellum, diaphragm, intercostal, gastrocnemius, tibialis anterior, and soleus muscles were quickly excised and frozen in liquid nitrogen. For immunostaining, the tissues were first flash-frozen in cold isopentane (20 s), then immersed in liquid nitrogen and stored at 80°C. To assess the regulation of PIN expression during in vivo muscle development, six pathogen-free, time-dated, pregnant Sprague-Dawley rats obtained from Charles River Laboratories (St. Constant, PQ, Canada) were killed at the gestational age of 18 d (E-18), and diaphragms from their fetuses were quickly dissected and frozen in liquid nitrogen. Diaphragms from littermate fetuses were pooled.

Mice. Five-week-old C57/B6 mice (Charles River Laboratories) were killed by sodium pentobarbital overdose, and their diaphragms were excised, quickly frozen in liquid nitrogen, and stored at 80°C.

Human. Proper ethical approval was obtained for human experimentation. Tissue samples were obtained from the sternal region of the diaphragm of adult patients undergoing cardiac transplantation. Small portions of the diaphragm were quickly frozen and stored at 80°C.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction

Total RNA was isolated from different tissues by the method of Chomczynski and Sacchi (13). For cloning of the PIN coding sequence, total RNA (1 µg) from rat diaphragms was reverse transcribed by using random primers and Maloney murine leukemia virus reverse transcriptase. Polymerase chain reaction (PCR) amplification of the PIN coding sequence was accomplished by forward primer 5'-ATGTGCGACCGAAAGGCCGTAGATC-3' and reverse primer 5'-TTAACCAGATTTGAACAGAAGAATGGCC-3'. PCR was carried out at 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s for a total of 30 cycles. The resultant cDNA was subjected to electrophoresis in 2% agarose gel and visualized by ethidium bromide staining. The appropriate size DNA fragment was excised, ligated into cloning vector pCRII.1, and then sequenced (McGill University automated DNA sequence facility).

Northern Blotting

Total RNA (30 µg) was mixed with 1 µl of 10 µg/µl ethidium bromide and separated by electrophoresis on 1.5% agarose gels containing 2.2 M formaldehyde. The RNA was then transferred to nylon membranes and rRNA was visualized under ultraviolet light and photographed to verify that equal amounts of RNA were loaded on all lanes. The membranes were then challenged with randomly primed 270-bp ³²P-labeled PIN cDNA recently amplified from rat tissues (14). The blots were washed in 0.2× standard saline citrate and 0.2% sodium dodecyl sulfate (SDS) for 10 min at room temperature, then at 65°C for 1 h. The membranes were exposed to X-ray film using an intensifying screen at 70°C.

Immunoblotting

Frozen tissues were homogenized in 6 vol (wt/vol) of homogenization buffer (pH 7.4, 10 mM N-2-hydroxyethyl-piperazine-N'-ethanesulfonic acid buffer, 0.1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM dithiotreitol, 1 mg/ml phenylmethylsulfonyl fluoride [PMSF], 0.32 mM sucrose, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 1% Triton X-100). The crude homogenates were centrifuged at 4°C for 15 min at 8,000 × g. The supernatants (80 µg total protein) were heated for 15 min at 90°C and then loaded on 18% Tris-glycine SDS-polyacrylamide gels. Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk, and subsequently incubated with either affinity-purified anti-PIN antibody (1:500) or monoclonal anti-nNOS (1:1,000) antibody. Recombinant PIN protein expressed as GST-fusion protein in E. coli (9) and pituitary lysate were respectively used as positive controls. Specific proteins were detected with horseradish peroxidase-conjugated antimouse secondary antibody and ECL reagents. The blots were scanned with an imaging densitometer (Model GS700, 12-bit precision and 42-µm resolution; Bio-Rad Inc., Hercules, CA), and optical densities of the protein bands were quantified with SigmaGel software (Jandel Scientific, San Rafael, CA). Predetermined molecular weight standards were used as markers. Protein concentration was measured by the Bradford method with bovine serum albumin as the standard.

Immunoprecipitation

To investigate whether PIN associates with the cytoplasmic dynein complex, we immunoprecipitated cytoplasmic dynein using an antibody raised against the 74-kD intermediate chain subunit of mammalian dynein (IC74) (15). Adult rat gastrocnemius muscles were homogenized in Triton X-100 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN₃, 0.05% Triton X-100, 2 mM EDTA, 1 mg/ml PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin, pH 7.4). The homogenate was spun at 27,000 × g for 15 min and the supernatant was recovered, respun, and then incubated with monoclonal anti-IC74 antibody (10 µl/0.5 g tissue) (Chemicon Inc., Temecula, CA) for 2 h at 4°C. Protein A-agarose conjugate and secondary rabbit antimouse antibody were then added, and the samples were incubated for another hour. After centrifugation, the pellets were washed twice with a buffer containing 125 mM Tris-HCl, pH 8.1, 500 mM NaCl, 0.5% Triton X-100, 10 mM EDTA, and 0.02% NaN₃. The final wash was performed with water. Proteins were eluted with electrophoresis sample buffer, and immunoblotting of supernatant and eluted proteins was undertaken as described. Membranes were probed with anti-IC74 and anti-PIN antibodies. Proper negative controls included omission of primary antibody and omission of protein A-agarose conjugate and secondary antibodies.

Immunohistochemistry

Air-dried cryostat sections (10 µm) were rehydrated with phosphate-buffered saline (PBS; pH 7.4, 3-5 min) and blocked for 1 h with normal donkey serum. The sections were then incubated overnight with either affinity-purified anti-PIN antibody (see above) or polyclonal anti-nNOS antibody (16). After three washes with saline PBS, the sections were incubated with Cy3-labeled donkey antirabbit secondary antibody. Slides were visualized with a Nikon fluorescence microscope (Melville, NY) and a Molecular Dynamics (Sunnyvale, CA) confocal argon laser scanning system equipped with a Cy3 filter. A similar protocol was used for negative control sections except that anti-PIN antibody was preincubated with recombinant PIN protein (9).

	Results

Top Abstract Introduction Materials and methods Results Discussion References

Cloning of Rat PIN cDNA

Amplification of the mammalian PIN coding sequence from rat diaphragmatic RNA by reverse transcription- PCR produced a single 270-bp band. Sequencing of this band revealed an open reading frame of an 89-amino acid protein, which is 100% homologous to human dynein light chain 1 (10) (Figure 1).

View larger version (17K): [in this window] [in a new window]   Figure 1.   Nucleotide and deduced amino acid sequence of PIN cDNA cloned from rat diaphragmatic RNA.

Expression of PIN during Muscle Development

Figure 2 illustrates PIN mRNA (A) and protein (B) expression in embryonic and adult rat diaphragms. A major ~ 0.9-kb band was detected in both muscles, with embryonic expression more than 4-fold higher than in mature muscle (Figure 2A). In addition, a minor transcript of an equivalent size of 18 S ribosomal RNA was detected only in the embryonic diaphragm (filled triangle, Figure 2A). Probing the rat cerebellum with PIN cDNA revealed a major 0.9-kb transcript similar to that expressed in skeletal muscle and a minor 2.5-kb transcript that was not detected in skeletal muscle RNA (open triangle, Figure 2A). Immunoblotting of the rat cerebellum and embryonic and adult diaphragms with anti-PIN antibody revealed an ~ 8-kD band that was 2.5-fold more intense in the embryonic diaphragm than in mature muscle (Figure 2B). A similar protein band was detected in the cerebellum (Figure 2B).

View larger version (26K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 2.   (A) Northern blot analysis of total RNA samples obtained from the rat cerebellum, embryonic (E-18), and adult diaphragms. Hybridization with PIN cDNA revealed a major PIN transcript of about 0.9 kb in size. Minor transcripts (filled and empty arrows) were also detected in the cerebellum and embryonic diaphragms. Note the differences in PIN mRNA expression between embryonic and adult diaphragms. (B) Immunoblotting of protein samples obtained from the rat cerebellum, embryonic (E-18), and adult diaphragms with anti-PIN antibody. Also shown is pure PIN protein obtained by expressing GST-PIN fusion protein in E. coli. Note that PIN is expressed at a much higher level in the embryonic diaphragm than in adult muscle.

Intra- and Interspecies Differences in PIN Expression

Figure 3 illustrates the differences in PIN mRNA (A) and protein (B) expressions between various muscles of adult rats. A strong 0.9-kb band was detected in the soleus muscle, whereas weaker expression was evident in the tibialis and gastrocnemius muscles (Figure 3A). Unlike PIN mRNA, PIN protein was expressed to a similar degree in various muscles of adult rats (Figure 3B). In addition to the 8-kD protein band, the anti-PIN antibody detected two additional bands with apparent masses of 13- and 22-kD (open arrows, Figure 3B). These bands were undetectable when anti-PIN antibody was preincubated with pure PIN protein (data not shown). Figure 4 illustrates the relationship between the intensity of PIN mRNA and protein expression and the percentage of type I fibers in various mature rat muscles (17). Although PIN mRNA expression correlated significantly and positively with the percentage of type I fibers (r = 0.93, P < 0.001), no correlation was found between PIN protein and the percentage of type I fibers. Figure 5 shows interspecies differences in diaphragmatic PIN protein expression. PIN protein was expressed to a similar degree in human, mouse, and rat diaphragms. By comparison, nNOS protein expression was more prominent in the human diaphragm than in rat and mouse diaphragms (Figure 5).

View larger version (49K): [in this window] [in a new window]   View larger version (23K): [in this window] [in a new window]   Figure 3.   (A) Northern blot analysis of total RNA obtained from various muscles of adult rats. Hybridization with PIN cDNA revealed a significant level of PIN mRNA expression in the soleus, whereas much weaker expression was evident in other muscles. The bottom of this panel shows ethidium bromide staining of total RNA. (B) Immunoblotting of various adult rat muscle samples with anti-PIN antibody. Note that PIN protein is ubiquitously expressed in the tibialis (T), intercostal (I), gastrocnemius (G), soleus (S), and diaphragm (D) muscles. In addition to the main 8-kD protein band, anti-PIN antibody detected two other bands (open arrows). These bands, along with the main PIN protein band, were undetectable when anti-PIN antibody was preincubated with pure PIN protein.

View larger version (20K): [in this window] [in a new window]   Figure 4.   Relationships between the optical densities of PIN mRNA (filled circles) and protein (empty circles) bands and the percentage of type I fibers in various muscles of adult rats. The percentage of type I fibers correlated positively with the intensity of PIN mRNA. By comparison, PIN protein did not correlate with the percentage of type I fibers.

View larger version (28K): [in this window] [in a new window]   Figure 5.   Differences in PIN (top) and nNOS (bottom) protein expressions among diaphragms of different species. Note that, unlike nNOS expression, PIN protein abundance is similar among mouse, human, and rat diaphragms.

Localization of PIN Protein

Immunostaining of the adult rat diaphragm (Figure 6A) and soleus (Figure 6B) muscles with anti-PIN antibody revealed positive staining in close proximity to the sarcolemma and the nuclei. Faint cytoplasmic staining was also evident in a few muscle fibers (Figure 6). Strong, positive PIN staining was also detected in muscle spindles (Figure 7B) and in the axons of nerve fibers supplying skeletal muscles (Figure 7A). No staining was evident when PIN antibody was preadsorbed with pure PIN protein (data not shown).

View larger version (80K): [in this window] [in a new window]   Figure 6.   Confocal microscopy of adult rat diaphragm (A) and soleus (B) muscle sections stained with anti-PIN antibody. Note the strong positive staining in close proximity to the sarcolemma and nuclei. Faint cytoplasmic staining is also evident in a few muscle fibers.

View larger version (106K): [in this window] [in a new window]   Figure 7.   Immunostaining with anti-PIN antibody showing a strong positive reaction (white color) inside the axons of nerve fibers (A) and muscle spindles (B) of the adult rat soleus muscle.

Association of PIN with the Dynein Complex

Cytoplasmic dynein in gastrocnemius muscle homogenates was immunoprecipitated with anti-IC74 antibody. Figure 8 reveals that PIN protein was immunoprecipitated by anti-IC74 antibody, indicating that PIN associates with the dynein complex in skeletal muscles. It should also be emphasized that PIN protein was still detectable in the supernatant, suggesting that not all PIN protein molecules associate with IC74 protein.

View larger version (21K): [in this window] [in a new window]   Figure 8.   Immunoprecipitation of the dynein complex of rat gastrocnemius muscle by anti-IC74 antibody. Crude muscle samples, immunocomplexes (ppt), and supernatants (supt) were probed with anti-IC74 antibody (top) and anti-PIN antibody (bottom). In addition to complete precipitation of IC74 protein, anti-IC74 antibody also precipitated a significant portion of PIN protein.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

The main findings of this study are (1) PIN mRNA and protein are expressed in skeletal muscle fibers of various species; (2) significantly higher levels of PIN mRNA and protein are expressed in embryonic muscles than in adult muscles; (3) PIN protein is localized at several sites, including the sarcolemma and nuclei of muscle fibers, nerves supplying muscle fibers, and muscle spindles.

Discovery of PIN

Since the independent discovery of PIN in mammalian cells by several groups (9, 10, 18), diverse proteins have been found to interact and associate with PIN. These include flagellar dynein (12), cytosolic dynein (18), nNOS (9), myosin V (19), and IB (20). In addition, PIN appears to be essential for normal development in Drosophila as loss-of-function mutations of the drosophila PIN homolog are lethal (10). It is unclear whether this phenotype in Drosophila reflects an essential role for PIN in the normal function of dynein, myosin V, IB, nNOS, or some other, as yet unidentified, PIN-binding protein. In relation to NO production, it has been shown that PIN interacts specifically with the N-terminal part of nNOS and inhibits nNOS selectively by preventing the dimerization of nNOS monomers (9). Our group has confirmed recently that nNOS protein interacts directly with PIN (14). We also reported that PIN protein is expressed to differing degrees in various regions of the rat brain (14).

Significant progress has been made recently in elucidating the role of PIN as a component of dynein, which is a multisubunit enzyme complex responsible for retrograde axonal transport, nuclear migration, and movement of organelles such as lysosomes, chromosomes, and the Golgi apparatus. Many subunits of dynein have been cloned, including heavy, intermediate, light intermediate, and light chains (21). Unlike heavy and intermediate chains, the nature, localization, and functional significance of light intermediate and light chains in mammalian cells are not yet clear. An M_r 8,000 mammalian homolog of Drosophila and flagellar dynein light chains with an identical amino acid sequence to PIN have been cloned recently from rat and bovine brains (18). In oligodentrocytes, this protein is localized at the cell periphery and colocalizes with cytoplasmic dynein. This finding confirmed that PIN and M_r 8,000 are the same protein and that PIN is an important component of the dynein complex.

Little is known about the existence, functional significance, and localization of the dynein complex in skeletal muscle fibers. Yoshida and colleagues (22) described abundant heavy-chain dynein expression in muscle spindles with no evidence of positive immunostaining in muscle fibers. In a separate study, Paschal and coworkers (23) reported that various rat tissues, including skeletal muscles, express significant levels of the 74-kD intermediate chain of dynein. Our results indicate for the first time that PIN is ubiquitously expressed in skeletal muscle fibers of various species. More importantly, we found that PIN protein is localized in distinct compartments inside and outside skeletal muscle fibers, namely, the sarcolemma, muscle spindles, and nerves supplying muscle fibers. The fact that these sites also contain abundant nNOS expression suggests that PIN is likely to play a role in regulating nNOS activity in these sites. In addition, the association of PIN with IC74 in muscle homogenates (Figure 8) suggests that PIN may also exist as a component of the dynein complex. Taken together, the localization of PIN in different sites inside muscle fibers suggests the existence of more than one intracellular pool of PIN. In mammalian brains, King and colleagues (18) identified at least three biochemically distinct pools of PIN: (1) non-microtubule-associated; (2) microtubule-associated and eluted with adenosine triphosphate and salt (component of the dynein complex); and (3) microtubule-associated but not salt-extractable (18). The finding of a portion of PIN immunoprecipitates with the dynein complex (Figure 8) supports the notion that distinct pools of PIN exist in skeletal muscle fibers.

Little information is available regarding the changes in dynein complex expression during development. In Drosophila, only the 4.5-kb mRNA transcript of dynein light chain 1 is regulated during embryonic development, whereas the main 2.4-kb transcript remains ubiquitously expressed (10). In mammals, expression of the 74-kD intermediate chain of cytoplasmic dynein is relatively constant during brain development, suggesting that the cytoplasmic dynein level is not developmentally regulated. Our study shows for the first time that PIN mRNA and proteins undergo significant developmental regulation (Figure 2). Although we did not investigate the regulation of various intracellular pools of PIN during muscle development, it is likely that the higher level of PIN detected in embryonic muscle fibers is a reflection of a greater requirement for dynein activity in the developing muscle fiber. In addition to being a component of the cytoplasmic dynein complex, PIN expression in the embryonic diaphragm is likely to play an important role in regulating the augmented nNOS activity recently described by our group (5).

Our results also indicate that the distribution of PIN mRNA in various rat skeletal muscles does not relate to the profile of PIN protein expression (Figures 3 and 4). It is possible that post-transcriptional and/or post-translational mechanisms are involved in the disparity between PIN mRNA and protein expression. One of these possible mechanisms is mRNA stability. It is likely that the stability of PIN mRNA varies in different skeletal muscle fibers. Differences in the PIN mRNA transcription rate between various muscles are also likely. Finally, post-translational modifications such as phosphorylation are likely to influence PIN protein degradation and thus impact on PIN protein levels in different muscle fibers. However, we believe that, unlike IC74, which is phosphorylated in glial and cultured neurons (24), PIN protein does not undergo major post-translational changes in mammalian cells because pure bacterially produced PIN (obtained by cleaving purified GST-PIN fusion protein at the GST-PIN junction with Factor Xa protease) migrated with the same apparent molecular mass as the mammalian PIN found in skeletal muscles and the cerebellum (Figure 2). Clearly, more detailed studies are needed to establish the exact mechanisms responsible for the regulation of PIN protein expression in mammalian cells.

NO Production in Skeletal Muscles

It has been well established that NO is synthesized inside skeletal muscle fibers by the nNOS isoform (4, 25). This isoform is localized at the junctional and extrajunctional sarcolemma and muscle spindles. Localization of nNOS to these compartments is accomplished through direct interaction between nNOS and the -1 syntrophin component of the dystrophin complex (26). However, the dissociation between nNOS expression and that of dystrophin and -1 syntrophin after denervation or in patients with muscular dystrophies (8, 27) suggests the existence of as yet unidentified molecules that are involved in anchoring nNOS to subcellular compartments of skeletal muscle fibers. Our main finding of abundant PIN expression in close proximity to the sarcolemma raises an intriguing possibility that PIN may be involved not only in regulating nNOS activity but also in anchoring nNOS protein to the sarcolemma. If this is true, PIN would function in a fashion similar to caveolin-1 and caveolin-3 in endothelial cells and skeletal muscle fibers. Recent studies indicate that ecNOS protein is anchored to endothelial cell membranes through an inhibitory heteromeric complex with caveolin-1 (28). This complex serves not only to anchor ecNOS to the cell membranes but regulates ecNOS activity as well. Similarly, there is evidence that nNOS protein associates with caveolin-3 in skeletal muscle fibers (29). It is therefore possible that PIN is involved in anchoring nNOS to membrane-associated proteins and regulating nNOS activity by directly inhibiting the formation of nNOS dimers. Moreover, the existence of PIN inside muscle fibers signifies the possible involvement of this protein in the regulation of various processes that are normally modulated by endogenous nNOS activity. These processes include excitation-contraction coupling, glucose uptake, mitochondrial respiration, and neuromuscular transmission.

In summary, our study indicates for the first time that PIN, the endogenous regulator of nNOS activity, is abundantly expressed in skeletal muscle fibers of various species and that it is regulated during skeletal muscle development. We also found that PIN is localized in distinct compartments, which include the sarcolemma, nucleus, muscle spindles, and nerve fibers supplying skeletal muscles.