ARTICLE |
Correspondence to: Susanne Sender, Vegetative Physiologie, 4220, Medizinische Hochschule Hannover, 30623 Hannover, Germany.
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Summary |
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We investigated carbonic anhydrase IV (CA IV) in rat and human heart with immunohistochemical methods by both light and electron microscopy. In cryosections that were incubated with anti-CA IV/FITC, the capillaries showed a strong reaction for CA IV. In paraffin and semithin sections treated with anti-CA IV/ABC (avidinbiotinperoxidase complex) blood vessels, capillaries, and sarcolemma (SL) were positively stained. By staining ultrathin sections with anti-CA IV/immunogold, CA IV could also be demonstrated at the latter two locations, including the specialized sarcolemmal structures intercalated discs, and T-tubules. In addition, by this method CA IV was seen to be associated with the sarcoplasmic reticulum (SR). The absence of immunostaining in SR and/or SL with some techniques probably indicates a problem of accessibility of the antigenic sites. In line with the immunohistochemical results, CA IV mRNA expression was visualized in both endothelial and muscle cells by in situ hybridization histochemistry. (J Histochem Cytochem 46:855861, 1998)
Key Words: carbonic anhydrase IV, capillaries, sarcolemma, sarcoplasmic reticulum, heart, skeletal muscle, immunohistochemistry, in situ hybridization
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Introduction |
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SEVERAL STUDIES have provided evidence for the existence of carbonic anhydrase (CA) in mammalian heart muscle.
A membrane-bound CA, CA IV, has been shown by immunohistochemistry to be present in capillary endothelium of rat and human skeletal muscles (
The purpose of this study was to investigate in heart muscle the localization of membrane-bound CA IV to different cells and subcellular structures. Antisera against CA IV were used in various techniques to immunostain sections of rat and human heart for visualization of CA IV at light microscopic and electron microscopic levels. Furthermore, in situ hybridization (ISH) histochemistry was applied to determine the presence of CA IV mRNA. This technique was carried out on human heart muscle sections and, in view of the immunohistochemical results for CA IV in skeletal muscle mentioned above, also on human skeletal muscle sections.
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Materials and Methods |
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Tissue Preparation
Pieces of rat and human heart muscle were dissected and frozen in liquid nitrogen. Human heart muscle samples were obtained from explanted hearts of patients undergoing transplantation. The samples were taken from unaffected tissue portions immediately after explantation. Cryosections were screened to exclude tissue specimens with visible degradation. Other sections 57 µm thick intended for further processing were fixed and stored as decribed by
In addition, pieces of heart muscle were fixed for 46 hr in one of the following fixatives: (a) 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate-HCl buffer, pH 7.3; (b) 0.1% glutaraldehyde/4% paraformaldehyde in the same buffer; (c) 3% glutaraldehyde in the same buffer; or (d) paraformaldehyde/lysine/periodate (PLP) in 0.1 M phosphate buffer as described by
Immunohistochemistry
Preparation and specificity of the applied antisera, anti-rat lung CA IV and anti-human lung CA IV, were previously described (
Light Microscopy
The cryosections were stained by immunofluorescence according to
Electron Microscopy
Ultrathin sections about 80 nm thick were etched with a saturated solution of sodium metaperiodate for 30 min. They were incubated overnight at 4C with a solution of 0.1% BSA/5% goat nonimmune serum/0.05 M Tris-HCl, pH 7.6, and anti-CA IV (diluted 1:2001:500). Sections were further treated according to the protocol given by
Generation of Single-stranded Riboprobes
Full-length single-stranded riboprobes (sense and anti-sense) were synthesized from full-length 1105-base pair (BP) CA IV cDNA (
In Situ Hybridization
Human skeletal muscle samples were frozen in liquid nitrogen. Cryosections 10 µm thick were mounted on TESPA-coated slides (Sigma). The sections were fixed in 4% paraformaldehyde in PBS for 20 min. They were then incubated with 25 µg/ml pronase E (Sigma) in 50 mM Tris/5 mM EDTA, pH 7.4, for 10 min at 37C. The sections were refixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed in PBS, and dehydrated in 70% ethanol. After prehybridization at 37C for 2 hr, the sections were hybridized overnight at 37C with the [35S]-UTP-labeled human CA IV riboprobes. Experimental tissue sections were incubated with the anti-sense CA IV riboprobe and control sections with the sense riboprobe. After stringent washing (
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Results |
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Light Microscopy
Immunofluorescence Method: CA IV in Capillary Endothelium.
Incubation of heart muscle cryosections with anti-CA IV/FITC resulted in positive staining of the capillary endothelium in rat (Figure 1A) and human heart (Figure 1B). The muscle cells showed no apparent staining. Control sections incubated with nonimmune serum/FITC showed only background fluorescence (Figure 1C). Therefore, the immunostaining indicates the association of CA IV with heart capillaries.
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AvidinBiotinPeroxidase Complex Method: CA IV in Vascular Endothelium and Sarcolemma. After incubation with anti-CA IV/ABC, paraffin sections of rat heart showed intense brown staining in the capillary endothelium and the endothelium of blood vessels (Figure 2A). A positive reaction was also seen at the SL, which exhibited a brown color of variable intensity. The cytoplasm in only some of the cardiac muscle cells showed yellowish staining, which was also seen in control sections (not shown). We conclude, therefore, that CA IV is present in the vascular endothelium and in the SL. This result is confirmed by semithin sections of rat cardiac muscle incubated with anti-CA IV/ABC or nonimmune serum/ABC, respectively. There was strong SL and capillary staining in parts of the antiserum-treated sections (Figure 2B). With this technique, the capillary staining appeared not only on the luminal but also on the external surface of the capillary wall. As in the cryosections (Figure 1A and Figure 1B) and in the ABC-treated paraffin sections (Figure 2A), the cytoplasm showed no specific staining. In the control sections, almost no immunoreactivity was visible throughout the sections (Figure 2C).
In Situ Hybridization to CA IV-specific mRNA. The hybridization of isotope-labeled riboprobe with tissue intrinsic CA IV mRNA transcripts was shown in heart (Figure 3A) and skeletal muscle (Figure 3C). In cardiac muscle sections, an intense hybridization signal was seen in many nuclei and, to a lesser extent in the cytoplasm. In control sections (Figure 3B) in which a sense RNA probe was used, the amount of silver grains was significantly reduced. On the basis of the number and distribution of the positive nuclei, one can conclude that both cardiomyocytes and capillary endothelial cells express CA IV. In addition, in both heart and skeletal muscle sections there was a prominent signal in larger blood vessels. This is shown in Figure 3C for skeletal muscle, together with a high signal intensity over nuclei and a lower signal intensity in the cytoplasm. Because the nuclei of skeletal muscle cells are often close to capillaries, it is sometimes difficult to unequivocally attribute the signal to one of these cell types. However, some capillaries showing accumulation of siver grains can definitely be identified. Therefore, we conclude that there may be an identical pattern of CA IV mRNA expression in heart and skeletal muscle.
Electron Microscopy
The applied fixatives and embedding media gave similar results regarding intensity and localization of the CA IV immunoreaction. Figure 4F shows a section from human heart; the other sections were taken from rat cardiac muscle.
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CA IV in Capillary Endothelium. After incubation with anti-CA IV/immunogold, capillary endothelial cells showed an accumulation of gold particles, predominantly in the proximity of the luminal membrane (Figure 4A and Figure 4B).
CA IV in the Sarcolemma. The SL, too, exhibited strong immunoreaction for CA IV (Figure 4C). Furthermore, CA IV appeared to be associated with specialized sarcolemmal structures, intercalated discs and T-tubules. As shown in Figure 4D and Figure 4F, patches of gold particles were found next to the intercalated discs. Very few particles were observed within the cells. An accumulation of gold particles could also be seen associated with membranous structures close to the Z discs (Figure 4E). We interpret this staining pattern as a positive reaction of T-tubules, which extend inward from the cell surface at this site, and/or as staining of junctional SR.
CA IV in Sarcoplasmic Reticulum. CA IV inside the muscle cells themselves is shown in Figure 4G and Figure 4H. Clusters of gold particles were associated with junctional complexes between sarcolemma and SR membrane, situated at the level of Z discs. In Figure 4G, both junctional SR and T-tubules are positively stained, whereas mitochondria remain unstained. Figure 4G shows an example of a triadic junctional complex, although the majority of junctional complexes in heart are diadic. What are called "terminal cisternae" in Figure 4G are much smaller in heart than in skeletal muscle and may not be wider than the L-system in general. In the longitudinal system, seen to be located between the myofibrils, we also found CA IV (Figure 4H). Control sections incubated with antigen-blocked antiserum/immunogold showed almost no background staining throughout the whole section (Figure 4I).
The ultrathin sections therefore support and extend the light microscopic results mentioned above. CA IV is located on the luminal membrane of capillary endothelial cells and is associated with SL and SR of cardiac muscle cells.
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Discussion |
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CA IV in Capillary Endothelium
The localization of CA IV in capillary endothelium of heart muscle is consistent with our earlier demonstration of CA IV in skeletal muscle capillaries (
Concerning the physiological role of this CA,
CA IV in the Sarcolemma
As in skeletal muscle (
CA IV in the Sarcoplasmic Reticulum
In heart muscle, SR CA IV could be demonstrated at the electron microscopic but not at the light microscopic level, a phenomenon that has also been observed in skeletal muscle sections (
It has been postulated that CA in skeletal muscle SR is necessary to rapidly produce and buffer H+ that move across the SR membrane in exchange for Ca++ (
CA IV mRNA Expression in Endothelium and Muscle Cells
The ISH histochemistry clearly shows that CA IV mRNA is present in heart muscle and skeletal muscle cells as well as in capillaries and other vessels. This is in accordance with the immunohistochemical results presented above, indicating the synthesis of CA IV in the same cell types that exhibit CA IV immunoreactivity.
The percentage similarities between human CA IV and other human CAs present in skeletal muscle cells and/or erythrocytes are 31% for CA I, 36% for CA II, and 30% for CA III at the amino acid level (
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Acknowledgments |
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Supported by the Deutsche Forschungsgemeinschaft, grant Gr 489/10.
The excellent technical assistance of Iris Albers and Elke Mallon and the expert photographic assistance of Angelika Hundt, are gratefully acknowledged.
Received for publication September 17, 1997; accepted March 11, 1998.
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