THE ANATOMICAL RECORD 23052-56 (1991)
Expression of a Fast Myosin Heavy Chain mRNA in Individual Rabbit Skeletal Muscle Fibers With Intermediate Oxidative Capacity DAVID J. DIX AND BRENDA RUSSELL EISENBERG Department of Biochemistry, North Carolina State University, Box 7622, Raleigh, North Carolina 27695-7622 (D.J.D.); Department of Physiology and Biophysics, University of Illinois, Mail Code 901, Box 6998, Chicago, Illinois 60680 (B.R.E.)
ABSTRACT In situ hybridization (ISH) of myosin heavy chain (MHC) mRNA, immunofluorescent detection of MHC protein, and oxidative enzyme histochemistry were performed on the same fibers in serially sectioned rabbit skeletal muscle. By combining these three techniques quantitatively, on a fiber-by-fiber basis, fibers that expressed mRNA complementary to a fast MHC cDNA pMHC24-79 of unknown subtype (Maeda et al., 1987) were classified into fiber types with respect to slow myosin expression and oxidative capacity. As expected, slow fibers had low hybridization to pMHC24-79. Fast fibers were divided into three subtypes. mRNA from the low oxidative fibers (fast-glycolytic, IIB) did not hybridize with pMHC2479. Fast fibers whose mRNA hybridized best to pMHC24-79 were mainly in the intermediate range of oxidative capacity (probably IIX). The fast fibers with the highest oxidative capacity had low hybridization to this MHC mRNA (probably IIA). Thus, pMHC24-79 was identified as a clone of a fast isomyosin, tentatively designated as the fast IIX with intermediate oxidative capacity. The expression of more than a single species of fast and slow isomyosin mRNAs in classically defined fiber type was considered in interpreting these results. Seven distinct myosin heavy chain (MHC) genes have been cloned and classified in rat (Mahdavi et al., 1986).These include three adult skeletal isoforms: fastglycolytic (IIB); fast-oxidative (IIA); and slow-oxidative (I), the same gene as p-cardiac). A fourth adult skeletal muscle isoform has recently been reported, labelled IIX by one group (Schiaffino et al., 1986, 1988, 1989) and IId by another (Termin et al., 1989). This new discovery necessitates the re-evaluation of all the classical subdivisions of fast fiber-types. In rat fibers expressing IIX isomyosin the oxidative enzyme content is intermediate compared to fast-glycolytic (IIB) and fast-oxidative (IIA) fibers. However, in mouse the IIX fibers have a higher oxidative content than IIA fibers and IIA fibers are the rarest of the three fast isoforms (Schiaffino et al., 1991). Rabbit fibers are more similar to rat than mouse in their isomyosin expression and the relative oxidative enzyme content amongst these fibers. Previously we have used a n a-MHC cDNA as a hybridization probe for slow MHC mRNA in skeletal muscle fibers (Dix and Eisenberg, 1988). This was possible because of the high degree of similarity between the a-cardiac and p-cardiac isomyosins. p-cardiac MHC is identical to slow MHC in skeletal muscle in all species studied to date. In the present work a subclone of the rabbit fast skeletal MHC cDNA pMHC24-79 (Maeda et al., 1987) provides RNA probes for in situ hybridization (ISH). By matching ISH results to MHC immunofluorescence and oxidative enzyme histochemistry within the same fibers in serial sections, we were able to determine which skeletal muscle fiber type ex0 1991 WILEY-LISS. INC.
presses this fast MHC mRNA. Thus, identifying which fast isomyosin gene is represented by the pMHC24-79 cDNA. METHODS Animals and Tissue
Governmental and institutional guidelines for animal care and use were followed a t all times. Two female New Zealand (NZ) white rabbits, approximately 2.5 kg body weight, provided medial gastrocnemius muscles. Rabbits were anesthetized with a n intramuscular injection of Acepromazine maleate and ketamine HC1 (0.8 mg and 35 mg, respectively, per kg body weight) and muscles removed. Rabbits were euthanized by lethal injection. Blocks of tissue for ISH, immunofluorescence, and histochemistry were flashfrozen in isopentane cooled with liquid nitrogen and then stored at -80°C in isopentane until use. In Situ Hybridization
Hybridizations were performed as previously described (Dix and Eisenberg, 1988). RNA probes were transcribed from a subcloned 377-bp Sau 3A fragment of rabbit fast skeletal MHC cDNA pMHC24-79 (Maeda et al., 1987). This fragment from the 3'-end of the cod-
Received July 10, 1990; accepted October 15, 1990. Address reprint requests to Dr. Brenda Eisenberg, Department of Physiology and Biophysics, University of Illinois, Mail Code 901, Box 6998, Chicago, IL 60680.
FAST MYOSIN mRNA AND OXIDATIVE CAPACITY
53
ing region was ligated into transcription vector Bluescribe M13 + (Stratagene, San Diego, CA) such th a t T3 RNA polymerase transcribes complementary antisense RNA and T7 RNA polymerase transcribes sense RNA (the kind gift of Dr. K. Maeda, Max Planck Institut, Heidelberg, FRG). Biotin-1 1-UTP (Bethesda Research Laboratories, Gaithersburg, MD) substituted for UTP in transcription reactions. Hybridization with the fast MHC probe was 52°C for 3 h. After hybridization, sections were treated with RNase A, washed, and then the biotin label was detected with streptavidin-alkaline phosphatase (SAP, Clontech, Palo Alto, CA). Sections were viewed and photographed with a Nikon Microphot-FXA. Histochemistry
NADH tetrazolium-reductase histochemistry to assess oxidative enzyme content was done as published (Dubowitz and Brooke, 1973). lmmunofluorescence
Immunofluorescent detection of slow MHC protein was accomplished with the monoclonal antibody HPM7 at 1:2,000 dilution (Kennedy et al., 1986, a kind gift of Dr. R. Zak of the University of Chicago). Slow specificity of HPM-7 in rabbit was confirmed by comparison with ATPase-stained serial sections. Sections were viewed and photographed with a Nikon MicrophotFXA with indirect immunofluorescence. The internal microphotometer was used to record fluorescent intensities. Video Image Analysis
The optical density (OD) of stain in individual fibers following ISH and histochemistry were measured by a n IBM-AT based video image analysis system (TM540 CCD camera, Pulnix America, Sunnyvale, CA; PCVision plus frame-grabber, Imaging Technology, Woburn, MA; ImageMeasure software, Microscience, Federal Way, WA) added on to a Nikon Microphot-FXA microscope. This sytem’s response was calibrated with a Kodak photographic step tablet number 3 (OD range 0.05-3.05). Mean OD measurements were converted to a linear relative scale (0-1.0) for ISH and histochemistry values. All OD measurements were of individual fibers. This allowed subtraction of background from appropriate serial sections on a fiber-by-fiber basis to provide the OD of the signal due to the detection of the endogenous mRNA. Mean OD values were total OD signal divided by fiber cross-sectional area. RESULTS ISH with the pMHC24-79 RNA probe and subsequent histochemical detection resulted in a mosaic staining pattern of muscle fibers in medial gastrocnemius (Fig. 1A). Control sections hybridized with the sense RNA probe stained a t very low levels (not shown) in agreement with our previous results (Dix and Eisenberg, 1988, 1990, 1991). Immunofluorescent detection of slow MHC with the HPM-7 antibody identified slowoxidative fibers (Fig. 1B); this identification was confirmed by the ATPase reaction in serial sections. NADH-tetrazolium reductase histochemistry assayed oxidative enzyme content and helped differentiate the
Fig. 1. Serially sectioned medial gastrocnemius. A. ISH of fast skeletal MHC mRNA with biotinylated pMHC24-79 RNA probe. B. Immunofluorescence with antislow MHC HPM-7 identifies the slow-oxidative (SO) fibers. C. NADH-tetrazolium reductase histochemistry differentiates fast-glycolytic (FG) and fast-oxidative (FO) fibers. Note that fast-oxidative fibers vary in the ISH staining. Those fast-oxidative fibers that stain darkest following hybridization with pMHC2479 probe are mainly of intermediate oxidative capacity. Bar = 50 pm.
three fast fiber types. Fast-glycolytic fibers stain lightly because of their low mitochondria1 content. The moderate and darker stained fast fibers with greater oxidative enzyme content in Figure 1C were the two fast-oxidative fiber types (IIA and 11x1. After classifying individual fibers via the‘ results in Figs. 1B,C, it was possible to go back to Figure 1A and note that fibers staining the darkest with the pMHC24-79 probe were mainly in the intermediate range of oxidative capacity. The micrographs in Figure 1 are representative of hundreds of fibers observed. Video image analysis of 100 fibers provided relative OD of stain within individ-
54
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Fig. 2. Scattergram of relative ODs from ISH with the fast MHC mRNA probe vs. NADH-tetrazolium reductase histochemistry within single fibers. Triangles denote fibers which reacted with the HPM-7 antibody (slow-oxidative fibers). Open circles are fast-glycolytic fibers. Filled circles are intermediate and high oxidative fast fibers. The fibers hybridized with the greatest amounts of pMHC24-79 probe are fast fibers with intermediate oxidative capacity.
ual fibers. A scattergraph of relative OD for ISH versus NADH-histochemistry from these 100 fibers illustrates the clustering of data points into three major groups (Fig. 2). Fast-glycolytic fibers and slow-oxidative fibers which stained lightly following ISH with pMHC24-79 probe, and fast-oxidative fibers, some of which stained darker following ISH. Mean values of ISH and histochemistry relative OD for each of these fiber-types are presented in Table 1. Closer examination of the 48 fast-oxidative fibers revealed two subgroups within this classification (Table 1). The mean relative OD of slow-oxidative fibers following ISH with the fast MHC mRNA probe was considered as nonspecific staining of muscle fibers by the probe. Thus 0.29 plus 0.40 (2 SD) or 0.69 provided a reasonable lower confidence limit for legitimate mRNA hybridization. There were 27 fast-oxidative fibers with a n ISH relative OD over 0.69. The mean relative OD for NADH-histochemistry of these fibers was 0.26 (0.09 SD). Twenty-one fast-oxidative fibers had a n ISH OD of
Expression of a Fast Myosin Heavy Chain mRNA in Individual Rabbit Skeletal Muscle Fibers With Intermediate Oxidative Capacity DAVID J. DIX AND BRENDA RUSSELL EISENBERG Department of Biochemistry, North Carolina State University, Box 7622, Raleigh, North Carolina 27695-7622 (D.J.D.); Department of Physiology and Biophysics, University of Illinois, Mail Code 901, Box 6998, Chicago, Illinois 60680 (B.R.E.)
ABSTRACT In situ hybridization (ISH) of myosin heavy chain (MHC) mRNA, immunofluorescent detection of MHC protein, and oxidative enzyme histochemistry were performed on the same fibers in serially sectioned rabbit skeletal muscle. By combining these three techniques quantitatively, on a fiber-by-fiber basis, fibers that expressed mRNA complementary to a fast MHC cDNA pMHC24-79 of unknown subtype (Maeda et al., 1987) were classified into fiber types with respect to slow myosin expression and oxidative capacity. As expected, slow fibers had low hybridization to pMHC24-79. Fast fibers were divided into three subtypes. mRNA from the low oxidative fibers (fast-glycolytic, IIB) did not hybridize with pMHC2479. Fast fibers whose mRNA hybridized best to pMHC24-79 were mainly in the intermediate range of oxidative capacity (probably IIX). The fast fibers with the highest oxidative capacity had low hybridization to this MHC mRNA (probably IIA). Thus, pMHC24-79 was identified as a clone of a fast isomyosin, tentatively designated as the fast IIX with intermediate oxidative capacity. The expression of more than a single species of fast and slow isomyosin mRNAs in classically defined fiber type was considered in interpreting these results. Seven distinct myosin heavy chain (MHC) genes have been cloned and classified in rat (Mahdavi et al., 1986).These include three adult skeletal isoforms: fastglycolytic (IIB); fast-oxidative (IIA); and slow-oxidative (I), the same gene as p-cardiac). A fourth adult skeletal muscle isoform has recently been reported, labelled IIX by one group (Schiaffino et al., 1986, 1988, 1989) and IId by another (Termin et al., 1989). This new discovery necessitates the re-evaluation of all the classical subdivisions of fast fiber-types. In rat fibers expressing IIX isomyosin the oxidative enzyme content is intermediate compared to fast-glycolytic (IIB) and fast-oxidative (IIA) fibers. However, in mouse the IIX fibers have a higher oxidative content than IIA fibers and IIA fibers are the rarest of the three fast isoforms (Schiaffino et al., 1991). Rabbit fibers are more similar to rat than mouse in their isomyosin expression and the relative oxidative enzyme content amongst these fibers. Previously we have used a n a-MHC cDNA as a hybridization probe for slow MHC mRNA in skeletal muscle fibers (Dix and Eisenberg, 1988). This was possible because of the high degree of similarity between the a-cardiac and p-cardiac isomyosins. p-cardiac MHC is identical to slow MHC in skeletal muscle in all species studied to date. In the present work a subclone of the rabbit fast skeletal MHC cDNA pMHC24-79 (Maeda et al., 1987) provides RNA probes for in situ hybridization (ISH). By matching ISH results to MHC immunofluorescence and oxidative enzyme histochemistry within the same fibers in serial sections, we were able to determine which skeletal muscle fiber type ex0 1991 WILEY-LISS. INC.
presses this fast MHC mRNA. Thus, identifying which fast isomyosin gene is represented by the pMHC24-79 cDNA. METHODS Animals and Tissue
Governmental and institutional guidelines for animal care and use were followed a t all times. Two female New Zealand (NZ) white rabbits, approximately 2.5 kg body weight, provided medial gastrocnemius muscles. Rabbits were anesthetized with a n intramuscular injection of Acepromazine maleate and ketamine HC1 (0.8 mg and 35 mg, respectively, per kg body weight) and muscles removed. Rabbits were euthanized by lethal injection. Blocks of tissue for ISH, immunofluorescence, and histochemistry were flashfrozen in isopentane cooled with liquid nitrogen and then stored at -80°C in isopentane until use. In Situ Hybridization
Hybridizations were performed as previously described (Dix and Eisenberg, 1988). RNA probes were transcribed from a subcloned 377-bp Sau 3A fragment of rabbit fast skeletal MHC cDNA pMHC24-79 (Maeda et al., 1987). This fragment from the 3'-end of the cod-
Received July 10, 1990; accepted October 15, 1990. Address reprint requests to Dr. Brenda Eisenberg, Department of Physiology and Biophysics, University of Illinois, Mail Code 901, Box 6998, Chicago, IL 60680.
FAST MYOSIN mRNA AND OXIDATIVE CAPACITY
53
ing region was ligated into transcription vector Bluescribe M13 + (Stratagene, San Diego, CA) such th a t T3 RNA polymerase transcribes complementary antisense RNA and T7 RNA polymerase transcribes sense RNA (the kind gift of Dr. K. Maeda, Max Planck Institut, Heidelberg, FRG). Biotin-1 1-UTP (Bethesda Research Laboratories, Gaithersburg, MD) substituted for UTP in transcription reactions. Hybridization with the fast MHC probe was 52°C for 3 h. After hybridization, sections were treated with RNase A, washed, and then the biotin label was detected with streptavidin-alkaline phosphatase (SAP, Clontech, Palo Alto, CA). Sections were viewed and photographed with a Nikon Microphot-FXA. Histochemistry
NADH tetrazolium-reductase histochemistry to assess oxidative enzyme content was done as published (Dubowitz and Brooke, 1973). lmmunofluorescence
Immunofluorescent detection of slow MHC protein was accomplished with the monoclonal antibody HPM7 at 1:2,000 dilution (Kennedy et al., 1986, a kind gift of Dr. R. Zak of the University of Chicago). Slow specificity of HPM-7 in rabbit was confirmed by comparison with ATPase-stained serial sections. Sections were viewed and photographed with a Nikon MicrophotFXA with indirect immunofluorescence. The internal microphotometer was used to record fluorescent intensities. Video Image Analysis
The optical density (OD) of stain in individual fibers following ISH and histochemistry were measured by a n IBM-AT based video image analysis system (TM540 CCD camera, Pulnix America, Sunnyvale, CA; PCVision plus frame-grabber, Imaging Technology, Woburn, MA; ImageMeasure software, Microscience, Federal Way, WA) added on to a Nikon Microphot-FXA microscope. This sytem’s response was calibrated with a Kodak photographic step tablet number 3 (OD range 0.05-3.05). Mean OD measurements were converted to a linear relative scale (0-1.0) for ISH and histochemistry values. All OD measurements were of individual fibers. This allowed subtraction of background from appropriate serial sections on a fiber-by-fiber basis to provide the OD of the signal due to the detection of the endogenous mRNA. Mean OD values were total OD signal divided by fiber cross-sectional area. RESULTS ISH with the pMHC24-79 RNA probe and subsequent histochemical detection resulted in a mosaic staining pattern of muscle fibers in medial gastrocnemius (Fig. 1A). Control sections hybridized with the sense RNA probe stained a t very low levels (not shown) in agreement with our previous results (Dix and Eisenberg, 1988, 1990, 1991). Immunofluorescent detection of slow MHC with the HPM-7 antibody identified slowoxidative fibers (Fig. 1B); this identification was confirmed by the ATPase reaction in serial sections. NADH-tetrazolium reductase histochemistry assayed oxidative enzyme content and helped differentiate the
Fig. 1. Serially sectioned medial gastrocnemius. A. ISH of fast skeletal MHC mRNA with biotinylated pMHC24-79 RNA probe. B. Immunofluorescence with antislow MHC HPM-7 identifies the slow-oxidative (SO) fibers. C. NADH-tetrazolium reductase histochemistry differentiates fast-glycolytic (FG) and fast-oxidative (FO) fibers. Note that fast-oxidative fibers vary in the ISH staining. Those fast-oxidative fibers that stain darkest following hybridization with pMHC2479 probe are mainly of intermediate oxidative capacity. Bar = 50 pm.
three fast fiber types. Fast-glycolytic fibers stain lightly because of their low mitochondria1 content. The moderate and darker stained fast fibers with greater oxidative enzyme content in Figure 1C were the two fast-oxidative fiber types (IIA and 11x1. After classifying individual fibers via the‘ results in Figs. 1B,C, it was possible to go back to Figure 1A and note that fibers staining the darkest with the pMHC24-79 probe were mainly in the intermediate range of oxidative capacity. The micrographs in Figure 1 are representative of hundreds of fibers observed. Video image analysis of 100 fibers provided relative OD of stain within individ-
54
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Fig. 2. Scattergram of relative ODs from ISH with the fast MHC mRNA probe vs. NADH-tetrazolium reductase histochemistry within single fibers. Triangles denote fibers which reacted with the HPM-7 antibody (slow-oxidative fibers). Open circles are fast-glycolytic fibers. Filled circles are intermediate and high oxidative fast fibers. The fibers hybridized with the greatest amounts of pMHC24-79 probe are fast fibers with intermediate oxidative capacity.
ual fibers. A scattergraph of relative OD for ISH versus NADH-histochemistry from these 100 fibers illustrates the clustering of data points into three major groups (Fig. 2). Fast-glycolytic fibers and slow-oxidative fibers which stained lightly following ISH with pMHC24-79 probe, and fast-oxidative fibers, some of which stained darker following ISH. Mean values of ISH and histochemistry relative OD for each of these fiber-types are presented in Table 1. Closer examination of the 48 fast-oxidative fibers revealed two subgroups within this classification (Table 1). The mean relative OD of slow-oxidative fibers following ISH with the fast MHC mRNA probe was considered as nonspecific staining of muscle fibers by the probe. Thus 0.29 plus 0.40 (2 SD) or 0.69 provided a reasonable lower confidence limit for legitimate mRNA hybridization. There were 27 fast-oxidative fibers with a n ISH relative OD over 0.69. The mean relative OD for NADH-histochemistry of these fibers was 0.26 (0.09 SD). Twenty-one fast-oxidative fibers had a n ISH OD of
