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/ . EmbryoL exp. Morph. Vol. 55, pp. 1-15, 1980
Printed in Great Britain © Company of Biologists Limited 1980
Immunofluorescence studies for myosin,
a-actinin and tropomyosin in developing hearts
of normal and cardiac lethal mutant Mexican
axolotls, Ambystoma mexicanum
By LARRY F. LEMANSKI, 1 REBECCA A. FULDNER
AND DANIEL J.PAULSON
From the Department of Anatomy and The Muscle Biology Laboratory,
University of Wisconsin-Madison
SUMMARY
Recessive mutant gene c in axolotl embryos results in an absence of normal heart function.
Immunofluorescence studies were done to determine the distributions of myosin, tropomyosin
and a-actinin in the hearts of normal and mutant siblings. Anti-myosin specifically stains the
A bands of myofibrils in normal hearts and reveals a progressive increase in myofibril organization with development. Mutant hearts display less staining for myosin than normal and
localization is mainly in amorphous collections. Anti-a-actinin stains the Z lines of myofibrils
in normal myocytes. Mutant cells also have significant staining for a-actinin but show no
striations. Antitropomyosin intensely stains the I bands of myofibrils in normal cells; however, there is very little staining for tropomyosin in mutant hearts. Thus, mutant myocardial
cells have reduced but significant amounts of actin (Lemanski, Mooseker, Peachey &
lyengar, 1976) and myosin, even though non-filamentous, and substantial amounts of
a-actinin. The cells appear to contain little tropomyosin.
INTRODUCTION
A recessive mutant gene, designated c for 'cardiac lethal', was discovered in
the Mexican axolotl, Ambystoma mexicanum (Humphrey, 1972). The gene
appears to exert its effect by abnormal inductive processes from the anterior
endoderm (Lemanski, Paulson & Hill, 1979), a potent heart inductor tissue in
salamander embryos (Jacobson & Duncan, 1968; Fullilove, 1970). Mutant
(c/c) embryos are obtained by mating heterozygous ( + /c) adults and can be
first distinguished from their normal ( + / + or +/c) siblings at stage 35
(Schreckenberg & Jacobson, 1975) when the normals develop vigorously contracting hearts. The hearts of mutant embryos at this stage appear normal upon
gross examination except they fail to beat properly in vivo and circulation is not
1
Author's address: Department of Anatomy, Bardeen Medical Laboratories, University of
Wisconsin, 1215 Linden Drive, Madison, WI 53706, U.S.A.
2
L. F. LEMANSKI, R. A. FULDNER AND D. J. PAULSON
established (Justus & Hollander, 1971; Humphrey, 1972; Justus, 1978; Kulikowski & Manasek, 1977, 1978; Lemanski, 1978). During later stages the
mutant hearts distend and remain thin-walled. In spite of an absence of circulation the mutant embryos survive through stage 41 (about 20 days beyond the
heart-beat stage), presumably by simple diffusion of oxygen directly into the
tissues. Skeletal muscle is not affected by gene c (Lemanski et al. 1976).
Morphological studies on mutant and normal embryonic hearts from stage 34
(normal heart-beat stage) to stage 41 reveal that normal hearts become trabeculated and the myocytes contain numerous well-organized myofibrils (Lemanski,
1913a, b). The mutant ventricular myocardium remains a single cell layer thick
and the cells contain relatively few thin (6 nm) and thick (15 nm) myofilaments;
there are no distinct sarcomeric myofibrils. The mutant cells contain instead
large amorphous proteinaceous collections in their peripheral sarcoplasm where
myofibrils organize initially in normal cells (Lemanski, 1973 c). These morphological data make it apparent that gene c results in abnormal heart cell differentiation. Most striking is the absence of well-organized muscle sarcomeres.
Biochemical experiments extend these basic morphological studies. Sodium
dodecylsulfate (SDS) polyacrylamide gel electrophoresis (Lemanski et al. 1976)
and radioimmunoassay studies (Lemanski, Joseph & Iyengar, 1975; Lemanski,
1976) suggest that mutant hearts contain approximately 50-75 % of the normal
quantity of myosin. Heavy-meromyosin (HMM)-binding studies in combination
with the gel electrophoresis data indicate that actin (43000 daltons) also is
present in somewhat reduced but substantial quantity in mutant hearts; however, most of the actin is not in a filamentous form (Lemanski et at. 1976).
Electrophoresis experiments indicate further that tropomyosin (34000) is very
significantly reduced in quantity when compared to normal controls. Thus, the
published morphological and biochemical data suggest that mutant hearts
contain myosin and actin in somewhat reduced, but significant amounts, even
though non-filamentous; however, tropomyosin appears to be substantially
reduced.
In the present study, we have determined the distributions of myosin, aactinin and tropomyosin in hearts of normal and mutant siblings at stages
35-41 by using immunohistochemical techniques. In addition, correlations were
made with hearts of stage-33 (pre-heart-beat) normal embryos.
MATERIALS AND METHODS
Embryos. The axolotl embryos were reared in 25 % Holtfreter's solution and
staged according to the system of Schreckenberg & Jacobson (1975). Hearts
from stage-33 known-wild-type normals ( + / + ) and from normal ( + / + or
+ /c) and mutant (c/c) siblings (obtained by + /ex + /c) matings) at stages 35,
39 and 41 were investigated.
Morphology. The embryonic hearts were prepared for histological and ultrastructural study by methods detailed in earlier papers (Lemanski, 1913a, b, c).
Contractile proteins in developing axolotl hearts
Fig. 1. Double immuncdiffusion plate to test antimyosin. AM, antimyosin antibodies; MY, purifted myosin from axolotl skeletal muscle.
Fig. 2. Double immuncdiffusion plate to test antitropomyosin. AT, antitropomyosin
antibodies; TR, purified tropomyosin from chicken skeletal muscle.
Fig. 3. Double immuncdiffusion plate to test anti-a-actinin. AA, anti-a-actinin antibodies; aA, purifbd a-actinin.
Antigens and antibodies. Antibodies raised against highly purified muscleprotein antigens, prepared by 'conventional' as well as by SDS-gel electrophoresis methods, were used in the immunohistochemical studies. Myosin was
extracted from fresh minced adult axolotl skeletal muscle and purified by
DEAE Sephadex column chromatography (Richards, Chung, Menzel & Olcott,
1967). Tropomyosin was purified from ether-dried chicken skeletal muscle
(Bailey, 1948). a-Actinin was prepared from porcine skeletal muscle (Goll,
Suzuki, Campbell & Holmes, 1972). In addition to the above 'conventional'
preparative methods, antibodies were prepared against chicken heart myosin
(heavy chain), tropomyosin and a-actinin that had been purified by slab SDS-gel
electrophoresis (Lazarides, 1975). Antibodies against the above proteins were
prepared in young rabbits. Preimmune rabbits were screened carefully to
eliminate non-specific staining on the tissue samples to be used in the experiments. Antibodies against the native (conventionally prepared) antigens were
obtained by giving initial subcutaneous injections of 4 mg of protein in Freund's
complete adjuvant followed by three subcutaneous booster injections of 2 mg
L. F. LEMANSKI, R. A. FULDNER AND D. J. PAULSON
Fig. 4. Light micrograph of a portion of the heart from a stage-41 normal embryo.
The myocardium is trabeculated (T) and is composed of cells containing organized
myofibril tracts (arrows), x 450.
Fig. 5. Light micrograph of a portion of the heart from a stage-41 cardiac lethal
mutant embryo. The myocardium is a single cell layer in thickness with no trabeculae.
Yolk platelets (Y) are present. There are no visible organized myofibrils. x 450.
of protein in Freund's incomplete adjuvant at 2-week intervals. The rabbits
were bled from the lateral ear veins 8 days after the final injection and the
y-globulins obtained by ammonium sulfate fractionation. The antibodies were
stored at — 70 °C until ready for use. Antibodies to the antigens purified by
SDS polyacrylamide gel electrophoresis were prepared as above except that the
relevant bands were cut from the gels after slight Coomassie blue staining,
homogenized in physiological saline and injected into the rabbits without
Freund's adjuvant. The purity of antibody preparations prepared from both
native and electrophoresed antigens was tested by double immunodiffusion and
immunoelectrophoresis against purified antigen and against crude heart muscle
homogenates from chicken and salamander. Each antibody gave a single precipitin line indicative of high degrees of purity (Figs. 1-3). For a given antigen
the tissue-staining pattern for the two different antibody preparations was
identical.
Immunofluorescence staining. Tissues to be examined immunohistochemically
were fixed 4 h at 0 °C in a periodate-lysine-paraformaldehyde solution (McLean
F I G U R E S 6 AND 7
Fig. 6. Electron micrograph of portions of heart cells from a stage-41 normal embryo.
Myofibrils are numerous and well organized. A, A band; I, I band; Z, Z line.
X28400.
Fig. 7. Electron micrograph of portions of heart cells from a stage-41 mutant embryo.
There are no organized myofibrils. Amorphous proteinaceous collections are present
in the peripheral areas of the cell. Sometimes dense bodies (d) and a few filaments
are visible within or associated with the amorphous collection (arrow), x 28400.
Contractile proteins in developing axolotl hearts
6
L. F. LEMANSKI, R. A. FULDNER AND D. J. PAULSON
& Nakane, 1974). Frozen sections, 2 jam thick, were air dried on glass slides
and stained by an indirect method. The primary antibodies in phosphatebuffered saline (0-9% NaCl in 0-05 M phosphate buffer, pH 7-0) plus 25%
glycerol (PBS-glycerol) were placed over the sections for 1 h at 37 °C. After
several rinses in PBS-glycerol totalling at least 1 h, the sections were stained for
1 h at 37 °C with FITC-labeled IgG fraction of antirabbit IgG prepared in goat
(Miles Laboratories, Kanakee, 111.). The controls for the antibody staining
included: (a) incubation with preimmune globulins from the same rabbit that
produced the specific antibodies, (b) staining with antibodies that had been
absorbed with excess purified antigen, and (c) staining with the second antibody
only. There was very little background staining on the control slides. The FITCstained sections were viewed with a Zeiss Universal light microscope equipped
with epifluorescence illumination and photographs were taken using 35 mm
Kodak Plus-X film at 60 sec exposure times. Light micrographs of the same
section areas were taken using phase optics for comparison.
RESULTS
Morphology. In spite of the functional difference, studies using the light
microscope show no discernible variance between normal and mutant hearts
FIGURES
8-15
Fig. 8. (a-b) Corresponding phase contrast (a) and immunofluorescence (b) light
micrographs of adult heart tissue stained with antimyosin. The antibody specifically
stains the A bands of organized myofibrils. Arrows illustrate corresponding areas.
x800.
Fig. 9. Immunofluorescence micrograph of antimyosin-stained stage-35 normal
heart. The A bands of organized myofibrils are stained. Also, there are a few
myosin positive amorphous areas, x 800.
Fig. 10. Immunofluorescence micrograph of antimyosin-stained stage-39 normal
heart. There is staining of the A bands in organized myofibrils as well as staining of
some amorphous areas, x 800.
Fig. 11. Immunofluorescence micrograph of antimyosin-stainedstage-41 normal heart.
A band staining is prominent in the myofibrils. Some of the 'amorphous-appearing'
areas are actually myofibrils in cross section and myofibrils out of the plane of focus.
x800.
Fig. 12. Immunofluorescence micrograph of stage-33 (pre-heart-beat) normal heart.
There is no sarcomere staining; however, several myosin-positive amorphous areas
are visible in the cell peripheries, x 800.
Fig. 13. Immunofluorescence micrograph of a stage-35 mutant heart. The staining is
limited to amorphous areas at the cell peripheries. There is no sarcomeric A band
staining as in stage-35 normal heart cells, x 800.
Fig. 14. Immunofluorescence micrograph of a stage-39 mutant heart. The myosin
containing amorphous collections are prominent, but there is no evidence of
sarcomeric myofibril organization, x 800.
Fig. 15. Immunofluorescence micrograph of a stage-41 mutant heart. The myosin
containing amorphous collections are prominent, but there is still no evidence of
sarcomeric myofibril organization, x 800.
Contractile proteins in developing axolotl hearts
8
L. F. LEMANSKI, R. A. FULDNER AND D. J. PAULSON
during early stages (Lemanski, 1973 c). By stage 41, however, histological
abnormalities in mutant hearts are obvious. Normal embryonic hearts at this
stage have well-developed trabeculae composed of myocytes containing
numerous organized myofibril tracts (Fig. 4). The mutant hearts fail to trabeculate and show no sign of sarcomeric myofibrils (Fig. 5). Yolk platelets,
virtually absent from normal hearts by stage 41, still remain in the cytoplasm
of some mutant cells.
Electron microscopy confirms the absence of sarcomeric myofibrils in mutant
heart ventricles. Normal myocytes contain organized myofibrils at stage 35
directly beneath and parallel to the plasma membrane; by stage 41, the normal
myocardium is composed of highly differentiated muscle cells containing
numerous well-organized myofibrils (Fig. 6). The mutant ventricular cells at
early stages contain amorphous proteinaceous collections along with a few 6 nm
(actin-like) and 15 nm (myosin-like) filaments, and what appear to be Z bodies;
however, even as late as stage 41, organized sarcomeres are absent (Fig. 7).
Immunohistochemistry. Immunofluorescence studies reveal that antimyosin
very specifically stains the A bands of organized myofibrils (Fig. 8 a, b). The
normal embryonic heart tissues at stage 34 show a few organized myofibrils
FIGURES
16-23
Fig. 16. (a, b) Corresponding phase contrast (a) and immunofluorescence (b) light
micrographs of adult heart tissue stained with anti-a-actinin. The antibody stains
the Z lines of organized myofibrils. Arrows illustrate corresponding areas, x 800.
Fig. 17. Immunofluorescence micrograph of anti-a-actinin-stained stage-35 normal
heart. The myofibrillar Z lines are stained and there is staining at the peripheries
of the cells, x 800.
Fig. 18. Immunofluorescence micrograph of anti-a-actinin-stained stage-39 normal
heart. There is staining of the myofibrillar Z lines along with some staining at the
cell peripheries.
Fig. 19. Immunofluorescence micrograph of anti-a-actinin-stained stage-41 normal
heart. Most of the a-actinin appears to be localized within the Z lines of organized
myofibrils. x 800.
Fig. 20. Immunofluorescence micrograph of stage-33 normal heart (pre-heart-beat
stage) stained with anti-a-actinin. The staining pattern is diffuse for the protein with
no obvious Z line staining, x 800.
Fig. 21. Immunofluorescence micrograph of stage-35 cardiac mutant heart stained
with anti-a-actinin. There seems to be localization of the protein at the peripheries
in the cells. Some cells appear to contain more of the protein than others. No Z line
staining can be seen, x 800.
Fig. 22. Immunofluorescence micrograph of anti-a-actinin-stained stage-39 cardiac
mutant heart. There is an increase in staining from stage 35, but the general distribution and pattern of staining remains basically the same (see Fig. 21). x 800.
Fig. 23. Immunofluorescence micrograph of anti-a-actinin-stained stage-41 cardiac
mutant heart. There is extensive staining of the tissue indicating rather substantial
quantities of a-actinin. There are no obvious sarcomeric Z lines stained, although
'spots' are visible.
Contractile proteins in developing axolotl hearts
10
L. F. LEMANSKI, R. A. FULDNER AND D. J. PAULSON
with distinct A band staining as well as myosin-containing amorphous areas in
some regions of the cells (Fig. 9). With progressing development there is an
increase in the organization of sarcomeres (Fig. 10) until by stage 41, most of
the myosin in the cells is localized in the A bands of the now numerous organized
myofibrils (Fig. 11). The mutant cells throughout development show no sarcomeric pattern of myosin localization. Most of the myosin-positive regions in
the mutant hearts take the form of amorphous collections in the cell peripheral
cytoplasm (Figs. 13-15), where myofibrils first organize in normal cells
(Lemanski, 1973 c). The pattern of staining in mutant cells is reminiscent of that
seen in stage-33 (pre-heartbeat) normal cells (Fig. 12). While the accumulated
myosin in mutant hearts increases with advancing development, there is clearly
less in mutant hearts than in normal control tissues at the same stage. Nevertheless the mutant hearts do show substantial staining for the protein, certainly
more than in any non-muscle tissues examined (i.e. gut, liver, brain).
Anti-a-actinin stains the Z lines of organized myofibrils (Fig. 16a, b). Sarcomeric Z line staining is present in stage-35 normal hearts (Fig. 17) and there is
an increase in Z line staining with development (Figs. 18-19) corresponding to
the increased numbers of organized myofibrils (Lemanski, 1973&). The a-actinin
staining is often localized along the sarcolemma at the cell peripheries. The
F I G U R E S 24-31
Fig. 24. (a, b) Corresponding phase contrast (a) and immunofluorescence (b) light
micrographs of adult heart tissue stained with antitropomyosin. The antibody
specifically stains the I bands of the organized myofibrils. Arrows illustrate corresponding areas, x 800.
Fig. 25. Immunofluorescence micrograph of antitropomyosin-stained stage-35
normal heart. Most of the protein is localized at the cell peripheries in amorphous
collections. There is some sarcomeric I band staining, x 800.
Fig. 26. Immunofluorescence micrographs of antitropomyosin-stained stage-39
normal heart. Organized myofibrils show I band staining. A few stained amorphous
areas are visible, x 800.
Fig. 27. Immunofluorescence micrograph of antitropomyosin-stained stage-41
normal heart. The myofibrillar I bands are stained as are some peripherally located
amorphous areas, x 800.
Fig. 28. Immunofluorescence micrograph of antitropomyosin-stained stage-33
normal heart (pre-heart-beat stage). While there is no sarcomeric pattern of
staining, substantial stain-positive areas are present as amorphous collections in
the peripheral cytoplasm of the cells, x 800.
Fig. 29. Immunofluorescence micrograph of antitropomyosin-stained stage-35
mutant heart. There is very little stain-positive material present x 800.
Fig. 30. Immunofluorescence micrograph of antitropomyosin-stained stage-39
mutant heart. A few lightly stained amorphous areas can be distinguished in the
cells. x800.
Fig. 31. Immunofluorescence micrograph of antitropomyosin-stained stage-41
mutant heart. There is very little staining for the protein at this stage. It appears to
be somewhat less than that seen in earlier mutant stages (see Figs. 29-30). x 800.
Contractile proteins in developing axolotl hearts
11
12
L. F. LEMANSKI, R. A. FULDNER AND D. J. PAULSON
mutant heart cells also show substantial staining for a-actinin (Figs. 23-23);
however, the pattern is more diffuse than in normal cells. While there is no
obvious Z line staining in the mutant cells even as late as stage 41, there are
little 'spots' of the protein which are similar in appearance to those seen in some
normal cells at earlier stages (e.g. compare Figs. 17 and 18 and 20 with Fig. 23).
We believe that these 'spots' in both normal and mutant cells represent collections of a-actinin which are closely associated with, or perhaps even plastered
against, the plasma membrane. Thus, these immunohistochemical studies
suggest that mutant cells have substantial staining for a-actinin but the protein
does not become organized into sarcomeric Z bands as in normal cells. Advanced
mutant hearts (stage 41) have certain staining characteristics of normal organs
at earlier stages (stages 35 and 39).
Anti-tropomyosin antibodies specifically stain the I bands of organized
myofibrils (Fig. 24a, b). The normal heart cells show significant staining for
tropomyosin at stage 35 and, like myosin and a-actinin, there is an increase in
sarcomere staining with advancing development (Figs. 25-27). At stage 41, the
bulk of the staining for tropomyosin in normal heart cells is in the I bands of
organized myofibrils. The mutant hearts throughout development show little
staining for tropomyosin (Figs. 29-31). While there does appear to be a slight
increase of tropomyosin-staining in mutant cells between stages 35 and 39, this
increase does not continue to stage 41. If anything, the individual cells seem to
show progressively less staining for the protein with advancing development
beyond stage 39. Stage-33 (pre-heartbeat) normal hearts show as much staining
for tropomyosin as any of the three stages of mutant hearts we have examined
(Fig. 28).
DISCUSSION
The present immunohistochemical studies were undertaken to determine the
distributions of myosin, a-actinin and tropomyosin in the normal and mutant
heart cells. Great care was taken to screen the rabbits and produce antigens of
the highest possible purity for our studies. Myosin in the normal hearts is
localized primarily in the A bands of organized myofibrils during the advanced
developmental stages (39-41). At early stages the normal hearts have a number
of amorphous myosin collections, which presumably represent areas in the
cells where myosin has accumulated but not yet been incorporated into myofibrils. The pre-heart-beat (stage 33) normal myocardial cells show no sarcomere
staining even though intensely stained amorphous collections are visible. The
mutant hearts throughout development do not display sarcomere staining,
although they do have rather extensive accumulations of amorphous myosinpositive areas which are reminiscent in distribution and appearance to the
myosin of pre-heart-beat normal cells. It appears that myosin has accumulated
in substantial quantity in mutant hearts, but fails to become incorporated into
sarcomeric myofibrils.
Contractile proteins in developing axolotl hearts
13
The immunohistochemical data suggests that a-actinin is also abundant in
mutant hearts, and like myosin and actin, is not incorporated into myofibrils
as is the case in normal cells. Rather the a-actinin appears to be generally
diffuse with a few concentrated collections of the protein at the cell peripheries.
Our immunohistochemical experiments corroborate the previously published
electrophoresis data (Lemanski et at. 1976) suggesting that muscle-type tropomyosin (34000 daltons) is quantitatively reduced in mutant hearts when compared to normal. In fact, pre-heart-beat normal cells (stage 33) show as much
staining for the protein as mutant hearts at stages 35-41. Furthermore, the
mutant hearts do not show an increase in this protein with advancing development beyond stage 39. If anything, there appears to be a decrease in tropomyosin staining within individual mutant heart cells. Whether this decline
represents a dilution effect from continued cell division, a breakdown of the
protein, or some other factor, remains moot. What is clear is that mutant hearts
have substantially lower than normal amounts of antigenically detectable
tropomyosin at all of the stages studied. These studies do not exclude the possibility that mutant heart cells contain antigenically altered or abnormal tropomyosin, such that the protein escapes recognition by the antibodies used.
Further, we cannot rule out the possibility that mutant heart cells contain
significant quantities of non-muscle-type tropomyosin (30000 daltons) (Cohen
& Cohen, 1972; Fine, Blitz, Hitchcock & Kamier, 1973). These questions
require further study.
Thus, although the mutant hearts studied here contain almost normal complements of the myofibrillar proteins, myosin, actin and a-actinin, tropomyosin
appears to be quantitatively reduced or, at least, biochemically altered. In any
event, normal myofibrillogenesis fails. In view of the failure of mutant heart
actin to form into filaments (Lemanski et al. 1976) and in view of the close
molecular association between actin and tropomyosin in organized myofibrils
of muscle (Gergeley, 1976), studies were undertaken recently in our laboratory
to determine whether there might be a casual relationship between the deficiency
of normal muscle tropomyosin in mutant hearts and their failure to form filamentous actin. The results of that study suggest that there is indeed a correlation (Lemanski, 1979). When glycerated mutant hearts are incubated in a
solution containing purified tropomyosin from chicken or rabbit skeletal
muscle, large numbers of 6 nm actin filaments form from the amorphous
collections in the cells; negative staining experiments confirm this result. Therefore, all available evidence suggests that insufficient or abnormal tropomyosin
in mutant heart cells may be a key in their failure to form normally organized
myofibrils. It is inviting to speculate that the presence of normal muscle-type
tropomyosin in developing embryonic hearts may be a necessary prerequisite
for actin to become filamentous and for myofibrillogeneis to succeed.
EMB 55
14
L. F. LEMANSKI, R. A. F U L D N E R AND D. J. PAULSON
This study was supported by NIH Grant HL 22550 and by a Grant-in-Aid from the
American Heart Association. The work was done during the tenure of an Established Investigatorship from the American Heart Association awarded to Dr Lemanski. Ms Fuldner
is supported by a Predoctoral Fellowship from the National Science Foundation. We thank
Ms Mikiye Nakanishi for excellent technical assistance and Dr Darrel Goll and Dr Judith.
Schollmeyer for providing us with porcine a-actinin. We are grateful to Ms Donna
Leonard for superb secretarial assistance.
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