The immunolocalization of protein kinase C (PKC) isozymes a , pl, and pll, was investigated in human skeletal muscle. All three isozymes were present on the muscle fiber surface membrane and within the muscle fibers. The a-isozyme was most clearly delineated on the surface membrane of the muscle fiber and on small blood vessels in the connective tissue. The axons of myelinated intramuscular nerves stained intensely for the pl isozyme, whereas the endoneurial connective tissue reacted more strongly for the a- and pll-isozymes. PKC isozymes may regulate intracellular signal transduction in human skeletal muscle, as in the other tissues, but their exact role in muscle remains unknown. Key words: protein kinase C isozymes human skeletal muscle skeletal muscle MUSCLE & NERVE 15:496-499 1992
LOCALIZATION OF PROTEIN KINASE C IN HUMAN SKELETAL MUSCLE SATOSHI NAKANO, MD, PhD, SHUN SHIMOHAMA, MD, PhD, TSUNAO SAITOH, PhD, ICHIRO AKIGUCHI, MD, PhD, and JUN KIMURA, MD
Protein kinase C (PKC), a phospholipid- and calcium-dependent kinase, acts through cell-surface signal transduction mechanisms. Distributed throughout the body, it regulates many cellular functions such as secretion, proliferation, and differentiation.""" It is also present in the skeletal muscle, but its functional role in myofibers remains unclear. An understanding of the PKC regulatory mechanisms in muscle in vivo requires precise localization of the PKC enzymes. Molecular cloning techni ues have revealed cDNAs for 7 PKC transcripts,' whereas Northern blotting has detected mRNAs for the a- and P-isozymes in muscle."," We have previously characterized antibodies against PKC isozymes, a , PI, and PII, using specific amino acid sequences predicted from their c D N A s . " ' ~ ~Using '~ these antibodies, we now describe the immunolocalization
From the Department of Neurology, Faculty of Medicine, Kyoto University, Japan (Drs Nakano. Shimohama, Akiguchi, and Kimura). and Department of Neurosciences, School of Medicine, University of California, San Diego. California (Dr. Saitoh) Acknowledgment. We thank Dr A.G. Engel for reading the manuscript We also thank Dr M Tanaka for performing muscle biopsy. This work was supported by a grant-in-aid from the Tokyo Biochemical Research Foundation Address reprint requests to Shun Shimohama, MD, PhD, Department of Neurology, Kyoto University Hospital, 54 Shogoin Kawaharacho, Sakyoku. Kyoto 606. Japan. Accepted for publication April 9, 1991 CCC 0148-639X1921040496-04$04.00 0 1992 John Wiley & Sons, Inc.
496
PKC in Human Skeletal Muscle
of each isozyme in human skeletal muscle. The results provide a basis for further studies directed at understanding the role of PKC in skeletal muscle. MATERIALS AND METHODS
Yolyclonal antisera to PKC isozymes ( a , PI, and PII) were prepared as described Briefly, antisera were raised in rabbits against synthetic peptides predicted from the human cDNA sequence of the C-terminal portion. The amino acid sequences used were PQFVHPILQSAV [PKC ( a ) ,661-6721, SYTNPEFVINV [PKC (PI), 661-6711, and NSEFLKPEVKS [PKC (PII), 663-6731. All recognized their respective peptide antigen on dot blots at dilutions up to 1/2000 to 1/5000.
Antibody Preparation.
Skeletal Muscle. Seven human limbmuscle specimens from 4 men and 3 women, 43 to 75 years of age, were studied. The patients had neurogenic muscular atrophy (2), inflammatory myopathy (2), unspecified myopathy (l), and minimal nonspecific abnormality (2).
Human
The specimens, fixed in 4% formaldehyde in phosphate buffer (pH 7.4) for 1 hour at 4"C, were washed with 20% sucrose in phosphate-buffered saline (PBS), frozen in isopentane chilled by liquid nitrogen, and sectioned at 8-pm in a cryostat at -20°C. The sections attached to glass slides were immersed in PBS containing 0.3% H,O, for 30 minutes to block the endogenous peroxidase activity. The sections were then
Immunolocalization.
MUSCLE & NERVE
April 1992
incubated overnight at 4°C with anti-PKC(a), antiPKC(PI), and anti-PKC(PI1). Preimmune rabbit serum, or an antibody absorbed specifically with an excess of competing free peptide immunogen, were used as controls. The sections were treated with biotinylated antirabbit IgG (7.5 kg/mL; Vector Laboratories) and, after rinsing, with avidin D peroxidase (1 :800; Vector Laboratories). Each rinse consisted of 3 changes of PBS over 20 minutes. PKC-positive structures were visualized by incubating the section with 0.02% 3,3'diaminobenzidine tetrahydrochloride (DAB), 0.6% Ni ammonium sulfate, and 0.005% H,O, in 0.05 mol/L tris-HC1 buffer (pH 7.6). RESULTS
The immunoreactivity for each PKC isozyme varied in intensity and distribution in human skeletal muscle (Table 1). We recognized four levels of reaction: (+ +) intense, (+) intermediate, (+/-) weak, and (-) negative. Intense immunoreactivity for a-PKC was detected on surface membranes of muscle fibers, on endomysial capillaries, and on small perimysial vessels in all cases (Fig. la). Strong reactivity for a-PKC was also present on surface membranes of intrafusal fiber, capsules of muscle spindle (Fig. lb), and endoneurium of intramuscular nerves (Fig. lc). The distribution of immunoreactive sites for PI-PKC was less distinct on the surface membranes, sometimes forming interrupted lines (Fig. 2a). Small blood vessels either showed weaker immunoreactivity to PI-PKC than to a-PKC, or none at all. By contrast, the axons of intramuscular myelinated nerves reacted sharply for PI-PKC, but the endoneurial connective tissue elements showed no reactivity (Fig. 2b).
Table 1. Incidence and intensity of immunoreactivity of 3 PKC antibodies in human skeletal muscle. a
PI
PI1
++ +,++ ++
+,++
t,+
+,++
2
i
?
++
PKC isozyme Muscle fiber Surface membranes Sarcoplasm Small vessels Intramuscular nerve Myelinated axons Endoneurium Perimysial loose Connective tissue
++
LOCALIZATION OF PROTEIN KINASE C IN HUMAN SKELETAL MUSCLE SATOSHI NAKANO, MD, PhD, SHUN SHIMOHAMA, MD, PhD, TSUNAO SAITOH, PhD, ICHIRO AKIGUCHI, MD, PhD, and JUN KIMURA, MD
Protein kinase C (PKC), a phospholipid- and calcium-dependent kinase, acts through cell-surface signal transduction mechanisms. Distributed throughout the body, it regulates many cellular functions such as secretion, proliferation, and differentiation.""" It is also present in the skeletal muscle, but its functional role in myofibers remains unclear. An understanding of the PKC regulatory mechanisms in muscle in vivo requires precise localization of the PKC enzymes. Molecular cloning techni ues have revealed cDNAs for 7 PKC transcripts,' whereas Northern blotting has detected mRNAs for the a- and P-isozymes in muscle."," We have previously characterized antibodies against PKC isozymes, a , PI, and PII, using specific amino acid sequences predicted from their c D N A s . " ' ~ ~Using '~ these antibodies, we now describe the immunolocalization
From the Department of Neurology, Faculty of Medicine, Kyoto University, Japan (Drs Nakano. Shimohama, Akiguchi, and Kimura). and Department of Neurosciences, School of Medicine, University of California, San Diego. California (Dr. Saitoh) Acknowledgment. We thank Dr A.G. Engel for reading the manuscript We also thank Dr M Tanaka for performing muscle biopsy. This work was supported by a grant-in-aid from the Tokyo Biochemical Research Foundation Address reprint requests to Shun Shimohama, MD, PhD, Department of Neurology, Kyoto University Hospital, 54 Shogoin Kawaharacho, Sakyoku. Kyoto 606. Japan. Accepted for publication April 9, 1991 CCC 0148-639X1921040496-04$04.00 0 1992 John Wiley & Sons, Inc.
496
PKC in Human Skeletal Muscle
of each isozyme in human skeletal muscle. The results provide a basis for further studies directed at understanding the role of PKC in skeletal muscle. MATERIALS AND METHODS
Yolyclonal antisera to PKC isozymes ( a , PI, and PII) were prepared as described Briefly, antisera were raised in rabbits against synthetic peptides predicted from the human cDNA sequence of the C-terminal portion. The amino acid sequences used were PQFVHPILQSAV [PKC ( a ) ,661-6721, SYTNPEFVINV [PKC (PI), 661-6711, and NSEFLKPEVKS [PKC (PII), 663-6731. All recognized their respective peptide antigen on dot blots at dilutions up to 1/2000 to 1/5000.
Antibody Preparation.
Skeletal Muscle. Seven human limbmuscle specimens from 4 men and 3 women, 43 to 75 years of age, were studied. The patients had neurogenic muscular atrophy (2), inflammatory myopathy (2), unspecified myopathy (l), and minimal nonspecific abnormality (2).
Human
The specimens, fixed in 4% formaldehyde in phosphate buffer (pH 7.4) for 1 hour at 4"C, were washed with 20% sucrose in phosphate-buffered saline (PBS), frozen in isopentane chilled by liquid nitrogen, and sectioned at 8-pm in a cryostat at -20°C. The sections attached to glass slides were immersed in PBS containing 0.3% H,O, for 30 minutes to block the endogenous peroxidase activity. The sections were then
Immunolocalization.
MUSCLE & NERVE
April 1992
incubated overnight at 4°C with anti-PKC(a), antiPKC(PI), and anti-PKC(PI1). Preimmune rabbit serum, or an antibody absorbed specifically with an excess of competing free peptide immunogen, were used as controls. The sections were treated with biotinylated antirabbit IgG (7.5 kg/mL; Vector Laboratories) and, after rinsing, with avidin D peroxidase (1 :800; Vector Laboratories). Each rinse consisted of 3 changes of PBS over 20 minutes. PKC-positive structures were visualized by incubating the section with 0.02% 3,3'diaminobenzidine tetrahydrochloride (DAB), 0.6% Ni ammonium sulfate, and 0.005% H,O, in 0.05 mol/L tris-HC1 buffer (pH 7.6). RESULTS
The immunoreactivity for each PKC isozyme varied in intensity and distribution in human skeletal muscle (Table 1). We recognized four levels of reaction: (+ +) intense, (+) intermediate, (+/-) weak, and (-) negative. Intense immunoreactivity for a-PKC was detected on surface membranes of muscle fibers, on endomysial capillaries, and on small perimysial vessels in all cases (Fig. la). Strong reactivity for a-PKC was also present on surface membranes of intrafusal fiber, capsules of muscle spindle (Fig. lb), and endoneurium of intramuscular nerves (Fig. lc). The distribution of immunoreactive sites for PI-PKC was less distinct on the surface membranes, sometimes forming interrupted lines (Fig. 2a). Small blood vessels either showed weaker immunoreactivity to PI-PKC than to a-PKC, or none at all. By contrast, the axons of intramuscular myelinated nerves reacted sharply for PI-PKC, but the endoneurial connective tissue elements showed no reactivity (Fig. 2b).
Table 1. Incidence and intensity of immunoreactivity of 3 PKC antibodies in human skeletal muscle. a
PI
PI1
++ +,++ ++
+,++
t,+
+,++
2
i
?
++
PKC isozyme Muscle fiber Surface membranes Sarcoplasm Small vessels Intramuscular nerve Myelinated axons Endoneurium Perimysial loose Connective tissue
++
