CARDIAC MUSCLE Its Ultrastructure in the Finch and Hummingbird with Special Reference to the Sarcoplasmic Reticulum PAUL H . JEWETT, J . R . SOMMER, and E . A . JOHNSON From the Departments of Pediatrics, Pathology, and Physiology, Duke University Medical Center, and the Veterans Administration Hospital, Durham, North Carolina 27706 ABSTRACT Cardiac muscle fibers of the hummingbird and finch have no transverse tubules and are smaller in diameter than those of mammalian hearts . The fibers are connected by inter- calated discs which are composed of desmosomes and f. adherentes ; small nexuses are often interspersed . As in cardiac muscle of several other animals, the junctional SR of the couplings is highly structured in these two birds but, in addition, and after having lost sarcolemmal contact, the junctional SR continues beyond the coupling to extend deep into the interior of the cells and to form belts around the Z-I regions of the sarcomeres . This portion of the sarco- plasmic reticulum, which we have named "extended junctional SR," and which is so promi- nent and invariant a feature of cardiac cells of hummingbirds and finches, has not been ob- served in chicken cardiac cells. The morphological differences between these species of birds may be related to respective differences in heart rates characteristic for these birds . INTRODUCTION Comparison of the fiber structure of hearts of were primarily concerned with a description of different animals can provide a great deal of in- mitochondrial morphology (2). Some features of sight into the relationships between the structure of the anatomy of heart muscle from a variety of cardiac muscle and its function ; during phylogene- other birds have previously been reported (3-9). sis and ontogenesis essential features of structure, The present inquiry adds the description of a dis- and of function, survive . crete anatomical feature in cardiac muscle of the We have chosen to select the hearts of the hum- hummingbird and finch, which consists of a strik- mingbird and of the finch for a comparative look ingly prominent morphological differentiation of because of the very fast heart rates which dis- portions of the sarcoplasmic reticulum and which, tinguish these two species from the previously because of its extent and because of its specific studied chicken (1) . We have further chosen to set intracellular location, provokes intriguing specula- aside the myofilaments, for it appeared to be more tions as to its possible functional role. rewarding, at the moment, to say something about forms of cell apposition, and about the ultrastruc- Definitions, Criteria, and Dimensions ture and topography of the sarcoplasmic reticulum . The ultrastructure of the heart of the humming- For additional terminology the reader is re- bird has been investigated before, but these studies ferred to previous publications (10-14) . Our find- 50 THE JOURNAL OF CELL BIOLOGY . VOLUME 49, 1971 . pages 50-65 ings in the finch and hummingbird have necessi- MATERIALS AND METHODS tated the subdivision of the sarcoplasmic reticulum (SR) into the following components, although all Animals are continuous with one another . The subdivisions Eight adult zebra finches (Taeniopygia castanotis) of are (a) free, (b) junctional, and (c) extended junctional, either sex, one adult female hummingbird (rufous SR. The free SR is that portion of the SR which is tailed), and one adult female chicken (Leghorn) were composed of a network of tubules (20-80 mµ wide) anesthesized with ether . The beating hearts were re- that, electron optically, have usually a clear lumen moved, opened, and immersed in fixative for 2-4 hr . and that surround the A bands of the sarcomeres ; it Small samples of ventricular and atrial subendocar- .2 mm thick, were is that part of the SR that does not have structural dial myocardium, not more than 0 dissected out about 30 min after immersion in the fixa- features of the junctional, nor of the extended tive, and then left in the fixative for the remainder of junctional, SR . refers to The junctional SR (11) the time. that portion of the SR which contains electron- opaque granules (functional granules), and that Fixatives forms couplings (10) with the sarcolemma, either at the periphery of the cell (peripheral couplings ; Either 4% glutaraldehyde in 0 .2 M cacodylate mammals and birds), or at transverse tubules, buffer with 5 mm calcium chloride, or Karnovsky's i.e., in the interior of the cell (interior couplings ; fixative (18) was used at room temperature and at pH 7.2-7 .4. Tissue samples were rinsed for 30 min in mammals) (11, 12) . Sandwiched between the either distilled water or 0 .2 M cacodylate buffer, post- junctional SR and the sarcolemma, as part of the fixed in freshly prepared 2% osmium tetroxide in couplings, there are seen periodic densities (junc- Veronal-acetate buffer (19) for 2 hr . dehydrated tional processes) . In the finch and hummingbird a through graded alcohols, and embedded in either portion of the SR is intercalated between the Maraglas or Epon 812 . Some tissues were contrasted junctional SR and the free SR that has the mor- with uranyl acetate in 50% ethanol or water en bloc phology of junctional SR (i .e. SR tubule, func- for 60 min after osmification. All specimens were tional granules, junctional processes), but that is stained on the grid with lead citrate . not in contact with the sarcolemma. We have named this SR portion, tentatively, "extended Special Studies junctional SR" because, except for the absence of Finch and young chicken (3-5 days old) hearts actual contact with sarcolemma, its morphology were injected with 100 mg of horseradish peroxidase appears identical to that of the junctional SR of in 3 ml of Ringer solution (20) . After administration couplings . of ether anesthesia the thorax was opened, the aorta The nexuses noted in the two birds under study was clamped, and the peroxidase solution was injected are gap junctions. We have avoided the terms by hand syringe through a 22 gauge needle into the "macula" and "fascia occludens" in this paper, right and left ventricular cavities with sufficient force and refer to all of these structures simply as nexuses to moderately distend the ventricle. A heart of an adult hen was also injected with peroxidase in a simi- (15), because that indifferent term names a struc- lar manner (500 mg in 15 nil of Ringer solution) . turally discrete entity without qualification ; After injection of the exogenous peroxidase, the hearts "macula" and "fascia" signify, yet do not specify, were quickly removed, opened, and immersed for 4 different sizes of these junctional complexes. The hr in 4% glutaraldehyde in cacodylate buffer as above use of the word "occludens" has become inap- (see Fixatives). Samples about 0.2 mm thick were then propriate since a gap was discovered between the dissected out, rinsed briefly in buffer, and exposed for unit membranes of the nexus (16) . 15-30 min to a solution containing 20 ml Tris buffer, M fibers (working myocardium) only are de- 10 ml 3-3-diaminobenzidine tetrahydrochloride, and scribed in this report . Although we have seen ap- 1 nil 1 % hydrogen peroxide . These samples were then parent P fibers with the light microscope (cf . 17), postfixed in 2% osmium tetroxide in Veronal-acetate buffer for 1% hr. dehydrated through graded alcohols, there are surprisingly few of them as compared and embedded in Epon 812 without further contrast with the chicken . enhancement, and thin sections were cut for electron All magnifications are reasonable approxima- microscopy . tions within the limits of a routinely calibrated For measurement of fiber diameters, similar multi- electron microscope, and within the limits of ple small (0 .2 mm thick) individual ventricular myo- routine photographic processing. cardial samples from these hearts were separately PAUL H. JEWETT, J. R . SOMMER, AND E . A . JOHNSON Cardiac Muscle 51 FIGURE 1 Hummingbird . Ventricular fibers contain numerous profiles of extended junctional SR (arrows) in the interior of the fibers at the Z lines and away from contact with the sarcolemma . Transverse tubules are absent . Junctional SR forms peripheral couplings at Z lines (arrowheads) . Free SR (SR) fills the interfibrillary spaces around A bands. Many mitochondria and lipofuscin deposits (lip) are present. Extracellular space (E) . Open arrows mark endothelial cells . Scale, 1 µ. X 11,000. FIGURE 2 Finch, ventricular fibers . Exogenous peroxidase is seen in capillaries (large black arrow), in the wide (E) and narrow (open arrows) extracellular spaces, and in the intercalated discs (ID) . The interior of the fibers is free of reaction product, demonstrating the absence of transverse tubules. Nu- cleated red blood cell, Nuc; fixation artifacts, X. Scale, 2 µ. X 5000 . embedded in Epon 812, and 1 .s thick cross-sections used as a calibration standard (Budd Co., Instrument were cut, mounted on glass slides, and photographed Div., Phoenixville, Pa.) . No more than one section with a Leitz Orthomat (phase contrast) (E . Leitz Inc., from each ventricular sample was studied to preclude Rockleigh, N.J .). A Unitron slide micrometer was measuring any fiber more than once . 50 such fibers PAUL H. JEWETT, J. R . SOMMER, AND E. A . JOHNSON Cardiac Muscle 53 were measured from each bird, and the greatest cell and thin filaments forming A and I bands, but had diameter was recorded, without respect to its co- no apparent M lines . Mitochondria were nu- incidence with the nuclear region of the cell . Calcula- merous and showed complex cristae, though per- tions of the range and mean diameter of myofibers of haps less marked than is seen in the canary (3). In- finch, young chick, and adult hen were made . Areas dividual fibers were surrounded by a typical sar- of preparations where peroxidase did not completely fill the extracellular space were excluded from study, colemma (plasma-plus basement membrane) since fiber diameter measurements are subject to con- having numerous sarcolemmal vesicles and pits siderable error when all cell borders cannot be clearly along its surface .