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Aspects of the mammalian cardiac sarcotubular system revealed by freeze fracture electron microscopy
Journal of Molecular
and Cellular
Cardiology
(1981)
Aspects of the Mammalian System Revealed by Freeze Donald Laboratory
13, 3i3-380
Cardiac Sarcotubular Fracture Electron Microscopy J. Scales
of Phq’sioloQ and Biophj,sics, 1 ~rlir~er.$)~ of‘ the Pargk, San Francisco, California 94 1 1.5. I Y.S.;l.
(Keceioed 19 August 1980, accepted in wisedjorm
15 December 1980 j
1). J. %AI.ES. Aspects of the Mammalian Cardiac Sarcotubular Sy~tun Rcvcaled by I:rwz~ Fracture Elrc tron klicroscopy. Journal oj ~\foftwrfnr m/d Celhlar Cnrdiohgr (1981) 13, 373.380. Freeze frnc-turc methods wrre used to examine the sarcoplasmir rctic,ulum (SR) of dog cardiac muscle. The free SR consists of three rrgions: narrow tubules of tbv free SK, fcncstrated membranes of the 1LI retc, and llat cistrrnal SR. Cistcrnal SK is a new ultraacr~cctural feature of cardiac SR. The cisternal regions appear scattcrcd througllout the ccl1 alld frvqwntly arc found near mitochondria. Thcrc is littlc diffucnw brt~~~v tbr narrow tubules of frrc SR and tlw junctional SR which is connected to the T tubulrs. Somrtimrs the tub~llnl j~rnctionat SK broadrns and wraps around a ‘f’ tubule. Thra SK is also connrv~rd IO ttw sarcolemma at the intercalated disc and somclimc\ is found IIKU ,gap junctions. E:\,idcru c of SK-mitoc bondria connections is presented.
Introduction Reported studies of the morphology of cardiac muscle are sparse in comparison to structural studies of skeletal muscle. A few reviews of cardiac ultrastructurc are available [G, 7. 12-161 and they point out an anatomical diversity that exists in cardiac ultrastructure not only among various animal species, but also among diffcrent regions of a given heart. Avian hearts for example, have no T tubules nor do Purkinje fibers [g, 51. C:anine atria1 fibers and human atrial fibers haw a particularly prominent SK [I4]. Th e relative volumes of the myofibrils, mitochondria, SR. and T tubules in cardiac muscle vary greatly among animal spccics [IJ. 1/i]. In this paper \ve explore the distribution of the sarcoplasmic reticulum in the left ventricle of dog heart muscle. The SR is easily recognized in freeze fi.acturc by its high density of intramembranous particles on the P fracture faces. If one is lucky, the fracture planes can reveal large areas of SR membrane, so that the continuity of the netlvork can be followed more clearly than thin section views, \vhere the idcntification of the membrane may not alllays be so obvious. We present several frcczc: fracture views in this paper in order to describe the configuration of the SK, \\ith particular attention to its various connections to other membranes. Materials
and
Methods
Tissue from the left ventricle of Mongrel dogs \vas fixed by immersion at room temperature in 4” (, glutaraldchyde in 0.1 M cacodylate, pH 7.4. The tissue leas cut into pieces less than 0.5 mm in width with Yannas scissors while in the fixing solution and after 15 min the specimens were rinsed twice in 0.1 M cacodylate for 10 and 0022-2828:81;040373
. 08 $02.OOjO
tC 1981
Academic
Pres>
1~.
(London!
I.irnitld
374
D. J. Scales
15 min. They were transferred to 0.1 M cacodylate containing 5”,, glycerol for 15 mirl, lO()G glycerol for 20 min, and 200.;, glycerol for 1 h. The samples \vere frozen in liquid Freon 22, fractured and replicated on a Balzers 360 M at - 100°C: and 1 :.. lo-” torr. The replicas were cleaned in bleach, rinsed in double distilled water and mounted on uncoated 300 mesh copper grids. Electron microscopy was done with a Philips EM 200 operting at 80 kV.
Results
The T tubules of dog ventricular muscle range from 0.12 to 0.28 pm in diameter and occur at the level of the Z disc (Figure 1). In close proximity we find the membranes of the sarcoplasmic reticulum (SR). Generally a T tubule is accompanied by a single tubule of SR that may follow it for a distance as shown in Figure 2, but then may broaden and wrap around it (Figure 3). There are no neatly arranged cisternae of junctional SR (jSR) like those seen in skeletal muscle. Instead the jSR resembles the narrow 20 to 50 nm tubules of the longitudinal SR with occasional cisternal elements that cross over the T tubules. In addition to these there are other cistcrnal regions of the SR that are found in the myoplasm. Figure 4 shows several of these flat manifold-like cisternae (cSR) that are smoothly connected to tubules of SR. Figures 5, 6 and 7, show other examples of cisternal SR in dog ventricular muscle. A common feature of these cisternae is the presence of 20 nm round depressions on the E face and corresponding protuberances on the P face. In Figure 8 we see extended fracture faces of three outer mitochondrial membranes. At the top right is a fragment ofcisternal SR connected to free SK below it and subjacent to the mitochondrion. Similar fragments of E face cistcrnal SR, each demonstrating 20 nm round depressions, are subjacent to the middle and bottom mitochondria. T tubules run between the mitochondria and at the top a fragment of E face junctional SR fits between the T tubule and the mitochondria above it. Particles bridge the spaces on both sides of the jSR. SR is frequently found near mitochondria. In Figure 9 a tubule of SR is connected to a mitochondrion by a linear array of bridging particles. In Figure 4 cisternal SR ih connected to a mitochondrion (arrows) and in Figure 10 a transverse fracture reveals the circular profile of a cross-fractured mitochondrion with a tubular process of SR in contact with it. Junctions between the SR and the sarcolemma are called peripheral couplings. Figure 11 shows a case where SR makes contact with the intercalated disc. Of particular interest here is the presence of a gap junction on the same process of the intercalated disc. The arrow indicates an array of pits, typical of the E face of a gap junction.
Discussion
The sarcoplasmic reticulum of into two broad classes: junctional the network that is connected peripheral plasmalemma they the T system are called interior intensely in thin sections [3, 9,
mammalian ventricular myocardium can be divided SR and free SR. The junctional SR is that part of to the plasmalemma. If the connections occur at the are called peripheral couplings, while connections to couplings. The connections or junctional feet stain 101 and appear particulate in freeze fracture replicas
Cardiac
Sarcotubular
System
s7.5
FIGURE 1. Longitudinal fracture shows placrmrnt of T tubules (‘I‘\ and mitwlw~~drin (11 I. Y 154 000. FIGURE 2. The P face of a T tubule (T) shows numerous intramembranous particles. To thr Ictt s a fragment of the E face of junctional SR (jSR). Particlrs bridge thr spncc hctwern thcsc fractt~tx Bees (arrowsi and represent the junctional feet. Y 69 800. I:IC;URE 3. Here a narrow tubule of free SR broadens and wraps around a T tuhtk. !I7 liO0.
D. J. Scales
Cardiac
Sarcotubular
System
a-.1/i
378
D. J. Scales
FIGURE 9. Several par&&s can be sect between a tubule of SK (SR) and a mitochondrion (MI. x 69 100. FIGURE 10. The circular profile ol’ a cross-liracturcd mitocbondria can be swn on the Icfc (XII. A tubule of SR approaches from the right and makes contact with the mitochondrion. 57 HOO. FIGURE 11. Gap junctions (G) arr found Frequently on the junctional sarcnlrmma or intcrvalatrd disc. A broadened aspect of the SR is sern is contact with a process of’ the intercalated disc near a gap junction. i: 44 200.
Cardiac
Sarcotubular
379
System
[8]. Identical structures appear to bridge the space between SR and mitochondria as shown here. The free SR consists of 10 to 50 nm tubules that form a lacelvork around the myofibrils and Lvhich form the M rete midway between two Z discs. Smoothl) connected to the tubules of free SR are occasional flattened sacs that WC call cistcrnal SR (cSR). Similar regions were described recently in rabbit hearts [I], and an earlier report of thin-sectioned mouse myocardium described the appcarancc of occasional expanded regions of SR although these were attributed to en face views of junctional SR [Z]. These expanded regions resemble extended junctional SR (EjSR) found predominantly in fibers without T tubules. In these cases the EjSR is a thin sac smoothly connected to free SR as reported here, but is generally intercalated into the Z rctc. Cisternal SR described in this report appears randomly throughout the myoplasm and in fibers having T tub&s. The only cases reported of EjSR in mammalian ventricular fibers show it in a different form called lamellar junctional SR [II, I?]. For these reasons we feel that cisternal SR represents a new component of SR in mammalian myocardium. Conclusion
The SR compartment of cardiac cells is connected to the periphery of the cell and also makes interior couplings with T tubules and mitochondria. At least in dog and rabbit ventricular muscle the SR consists of numerous cistcrnal regions joined by the narrow tubules of free SR. It is not known if this is a common feature of mammalian myocardium. Acknowledgements
I gratefully acknowledge the assistance of Dr Michele Chiesi and the helpful discussions with Dr Parris Kidd. Freeze fracturing was done at the Electron Microscope Laboratory of the University of California at Berkeley. This work was supported by a grant from the USPHS (5 PO1 HL 16607).
REFERENCES 1. DOLBER, P. & SOMMER, J. Freeze fracture appearance of rabbit cardiac sarcoplasmic reticulum. In Proceedings of the Annual Meeting of the Electron i\licrosco/)y Society of America, 38th, G. Bailey, Ed., pp. 630-631. Baton Rouge, Lousiana: Claitor’s Publishing Division ( 1980). 2. FORBES, M. & SPERELAKIS, N. Myocardial couplings: their structural variations in the mouse. Journal of Ultrastructural Research 58, 50-65 ( 1977). 3. FRANZINI-ARMSTRONG, C. Studies of the triad. I. Structure of the junction in frog twitch fibers. Journal of Cell Biology 47, 488-499 (1970). 4. JEWETT, P., SOMMER, J. & JOHNSON, E. Cardiac muscle: its ultrastructure in the finch and hummingbird with special reference to the sarcoplasmic reticulum. Journal of Cell Biology
49,
50-65
(1971).
5. JOHNSON, E. & SOMMER, physiologic implications
J. A strand of cardiac muscle: its ultrastructure and the electroof its geometry. Journal of Cell Biology 33, 103-129 (1967).
6.
MCNUTT, (1975).
N.
7.
McNuTr,
N. & FAWCETT,
G. Langer
Ultrastructure
& A. Brady,
of the
myocardial
sarcolemma.
D. Myocardial ultrastructurc. Eds. pp. l-49. New York: John
Circulation
Research
In The Mammalian Wiley & Sons (1974).
37.
I-13
-hQocardium,
380 8.
9. 10. 11.
12. 13. 14.
15.
16.
17.
D. J. Scales RAYNS, D., DEVINE, C. & SCTHERLAND, C. Freeze fracture studies of membrane systems in vertebrate muscle. I. Striated muscle. Journal of Ultrastructure Research 50, 306-321 (1975). REVEL, J. The sarcoplasmic reticulum of the bat cricothyroid muscle. Journal of Cell Biology 12, 571-588 (1962). SOMLYO, A. V. Bridging structures spanning the junctional gap at the triad of skeletal muscle. j’ournal of Cell Biolop 80, 743-750 (1979). SOMMER, J. Sr JEWETT, I’. Cardiac muscle: a comparative ultrastructural, anatomical Ed.. pp. 89-106. New York: Academic Press view. In Cardiac Hyj,ertroj,hy, N. Alpert, (1971). SOMMER, J. & JOHNSON, E. A comparative study of Purkinje fibers and ventricular Gbers. Journal of Cell Biology 36, 497-526 (1968). SOMMER, J. & JOHNSON, E. Comparative ultrastructure of cardiac cell membrane specializations. American Journal of Cardiology 25, 184-194 (1970). SOMMER, J. & JOHNSON, E. Ultrastructure of cardiac muscle. In Handbook of Physiologv--The Cardioz~uscular System, R. Berne, N. Sperelakis & S. Geiger, Eds, pp. 113-186. Bethesda, Maryland: American Physiological Society (1979). SOMMER, J. & M'AUCH, R. The ultrastructure of the mammalian cardiac muscle cellwith special emphasis on the tubular membrane system. American j’ournal of PathologV 82, 192-232 (1976). SPERELAKIS, N.. FORBES, M. & RURIO, R. ‘The tubular system of myocardial cells: ultrastructure and possible function. In Recent Adr’ances in Studies on Cardiac Structure and ~letabolism. N. Dhalla, Ed., pp. 1633194. Baltimore, Maryland: University Park Press (1972). WAUGH, R. & SUMMER, J. Lamellar junctional sarcoplasmic reticulum, a specialization of cardiac sarcoplasmic reticulum. Journal of Cell Biology 63, 337-343 (1974).
and Cellular
Cardiology
(1981)
Aspects of the Mammalian System Revealed by Freeze Donald Laboratory
13, 3i3-380
Cardiac Sarcotubular Fracture Electron Microscopy J. Scales
of Phq’sioloQ and Biophj,sics, 1 ~rlir~er.$)~ of‘ the Pargk, San Francisco, California 94 1 1.5. I Y.S.;l.
(Keceioed 19 August 1980, accepted in wisedjorm
15 December 1980 j
1). J. %AI.ES. Aspects of the Mammalian Cardiac Sarcotubular Sy~tun Rcvcaled by I:rwz~ Fracture Elrc tron klicroscopy. Journal oj ~\foftwrfnr m/d Celhlar Cnrdiohgr (1981) 13, 373.380. Freeze frnc-turc methods wrre used to examine the sarcoplasmir rctic,ulum (SR) of dog cardiac muscle. The free SR consists of three rrgions: narrow tubules of tbv free SK, fcncstrated membranes of the 1LI retc, and llat cistrrnal SR. Cistcrnal SK is a new ultraacr~cctural feature of cardiac SR. The cisternal regions appear scattcrcd througllout the ccl1 alld frvqwntly arc found near mitochondria. Thcrc is littlc diffucnw brt~~~v tbr narrow tubules of frrc SR and tlw junctional SR which is connected to the T tubulrs. Somrtimrs the tub~llnl j~rnctionat SK broadrns and wraps around a ‘f’ tubule. Thra SK is also connrv~rd IO ttw sarcolemma at the intercalated disc and somclimc\ is found IIKU ,gap junctions. E:\,idcru c of SK-mitoc bondria connections is presented.
Introduction Reported studies of the morphology of cardiac muscle are sparse in comparison to structural studies of skeletal muscle. A few reviews of cardiac ultrastructurc are available [G, 7. 12-161 and they point out an anatomical diversity that exists in cardiac ultrastructure not only among various animal species, but also among diffcrent regions of a given heart. Avian hearts for example, have no T tubules nor do Purkinje fibers [g, 51. C:anine atria1 fibers and human atrial fibers haw a particularly prominent SK [I4]. Th e relative volumes of the myofibrils, mitochondria, SR. and T tubules in cardiac muscle vary greatly among animal spccics [IJ. 1/i]. In this paper \ve explore the distribution of the sarcoplasmic reticulum in the left ventricle of dog heart muscle. The SR is easily recognized in freeze fi.acturc by its high density of intramembranous particles on the P fracture faces. If one is lucky, the fracture planes can reveal large areas of SR membrane, so that the continuity of the netlvork can be followed more clearly than thin section views, \vhere the idcntification of the membrane may not alllays be so obvious. We present several frcczc: fracture views in this paper in order to describe the configuration of the SK, \\ith particular attention to its various connections to other membranes. Materials
and
Methods
Tissue from the left ventricle of Mongrel dogs \vas fixed by immersion at room temperature in 4” (, glutaraldchyde in 0.1 M cacodylate, pH 7.4. The tissue leas cut into pieces less than 0.5 mm in width with Yannas scissors while in the fixing solution and after 15 min the specimens were rinsed twice in 0.1 M cacodylate for 10 and 0022-2828:81;040373
. 08 $02.OOjO
tC 1981
Academic
Pres>
1~.
(London!
I.irnitld
374
D. J. Scales
15 min. They were transferred to 0.1 M cacodylate containing 5”,, glycerol for 15 mirl, lO()G glycerol for 20 min, and 200.;, glycerol for 1 h. The samples \vere frozen in liquid Freon 22, fractured and replicated on a Balzers 360 M at - 100°C: and 1 :.. lo-” torr. The replicas were cleaned in bleach, rinsed in double distilled water and mounted on uncoated 300 mesh copper grids. Electron microscopy was done with a Philips EM 200 operting at 80 kV.
Results
The T tubules of dog ventricular muscle range from 0.12 to 0.28 pm in diameter and occur at the level of the Z disc (Figure 1). In close proximity we find the membranes of the sarcoplasmic reticulum (SR). Generally a T tubule is accompanied by a single tubule of SR that may follow it for a distance as shown in Figure 2, but then may broaden and wrap around it (Figure 3). There are no neatly arranged cisternae of junctional SR (jSR) like those seen in skeletal muscle. Instead the jSR resembles the narrow 20 to 50 nm tubules of the longitudinal SR with occasional cisternal elements that cross over the T tubules. In addition to these there are other cistcrnal regions of the SR that are found in the myoplasm. Figure 4 shows several of these flat manifold-like cisternae (cSR) that are smoothly connected to tubules of SR. Figures 5, 6 and 7, show other examples of cisternal SR in dog ventricular muscle. A common feature of these cisternae is the presence of 20 nm round depressions on the E face and corresponding protuberances on the P face. In Figure 8 we see extended fracture faces of three outer mitochondrial membranes. At the top right is a fragment ofcisternal SR connected to free SK below it and subjacent to the mitochondrion. Similar fragments of E face cistcrnal SR, each demonstrating 20 nm round depressions, are subjacent to the middle and bottom mitochondria. T tubules run between the mitochondria and at the top a fragment of E face junctional SR fits between the T tubule and the mitochondria above it. Particles bridge the spaces on both sides of the jSR. SR is frequently found near mitochondria. In Figure 9 a tubule of SR is connected to a mitochondrion by a linear array of bridging particles. In Figure 4 cisternal SR ih connected to a mitochondrion (arrows) and in Figure 10 a transverse fracture reveals the circular profile of a cross-fractured mitochondrion with a tubular process of SR in contact with it. Junctions between the SR and the sarcolemma are called peripheral couplings. Figure 11 shows a case where SR makes contact with the intercalated disc. Of particular interest here is the presence of a gap junction on the same process of the intercalated disc. The arrow indicates an array of pits, typical of the E face of a gap junction.
Discussion
The sarcoplasmic reticulum of into two broad classes: junctional the network that is connected peripheral plasmalemma they the T system are called interior intensely in thin sections [3, 9,
mammalian ventricular myocardium can be divided SR and free SR. The junctional SR is that part of to the plasmalemma. If the connections occur at the are called peripheral couplings, while connections to couplings. The connections or junctional feet stain 101 and appear particulate in freeze fracture replicas
Cardiac
Sarcotubular
System
s7.5
FIGURE 1. Longitudinal fracture shows placrmrnt of T tubules (‘I‘\ and mitwlw~~drin (11 I. Y 154 000. FIGURE 2. The P face of a T tubule (T) shows numerous intramembranous particles. To thr Ictt s a fragment of the E face of junctional SR (jSR). Particlrs bridge thr spncc hctwern thcsc fractt~tx Bees (arrowsi and represent the junctional feet. Y 69 800. I:IC;URE 3. Here a narrow tubule of free SR broadens and wraps around a T tuhtk. !I7 liO0.
D. J. Scales
Cardiac
Sarcotubular
System
a-.1/i
378
D. J. Scales
FIGURE 9. Several par&&s can be sect between a tubule of SK (SR) and a mitochondrion (MI. x 69 100. FIGURE 10. The circular profile ol’ a cross-liracturcd mitocbondria can be swn on the Icfc (XII. A tubule of SR approaches from the right and makes contact with the mitochondrion. 57 HOO. FIGURE 11. Gap junctions (G) arr found Frequently on the junctional sarcnlrmma or intcrvalatrd disc. A broadened aspect of the SR is sern is contact with a process of’ the intercalated disc near a gap junction. i: 44 200.
Cardiac
Sarcotubular
379
System
[8]. Identical structures appear to bridge the space between SR and mitochondria as shown here. The free SR consists of 10 to 50 nm tubules that form a lacelvork around the myofibrils and Lvhich form the M rete midway between two Z discs. Smoothl) connected to the tubules of free SR are occasional flattened sacs that WC call cistcrnal SR (cSR). Similar regions were described recently in rabbit hearts [I], and an earlier report of thin-sectioned mouse myocardium described the appcarancc of occasional expanded regions of SR although these were attributed to en face views of junctional SR [Z]. These expanded regions resemble extended junctional SR (EjSR) found predominantly in fibers without T tubules. In these cases the EjSR is a thin sac smoothly connected to free SR as reported here, but is generally intercalated into the Z rctc. Cisternal SR described in this report appears randomly throughout the myoplasm and in fibers having T tub&s. The only cases reported of EjSR in mammalian ventricular fibers show it in a different form called lamellar junctional SR [II, I?]. For these reasons we feel that cisternal SR represents a new component of SR in mammalian myocardium. Conclusion
The SR compartment of cardiac cells is connected to the periphery of the cell and also makes interior couplings with T tubules and mitochondria. At least in dog and rabbit ventricular muscle the SR consists of numerous cistcrnal regions joined by the narrow tubules of free SR. It is not known if this is a common feature of mammalian myocardium. Acknowledgements
I gratefully acknowledge the assistance of Dr Michele Chiesi and the helpful discussions with Dr Parris Kidd. Freeze fracturing was done at the Electron Microscope Laboratory of the University of California at Berkeley. This work was supported by a grant from the USPHS (5 PO1 HL 16607).
REFERENCES 1. DOLBER, P. & SOMMER, J. Freeze fracture appearance of rabbit cardiac sarcoplasmic reticulum. In Proceedings of the Annual Meeting of the Electron i\licrosco/)y Society of America, 38th, G. Bailey, Ed., pp. 630-631. Baton Rouge, Lousiana: Claitor’s Publishing Division ( 1980). 2. FORBES, M. & SPERELAKIS, N. Myocardial couplings: their structural variations in the mouse. Journal of Ultrastructural Research 58, 50-65 ( 1977). 3. FRANZINI-ARMSTRONG, C. Studies of the triad. I. Structure of the junction in frog twitch fibers. Journal of Cell Biology 47, 488-499 (1970). 4. JEWETT, P., SOMMER, J. & JOHNSON, E. Cardiac muscle: its ultrastructure in the finch and hummingbird with special reference to the sarcoplasmic reticulum. Journal of Cell Biology
49,
50-65
(1971).
5. JOHNSON, E. & SOMMER, physiologic implications
J. A strand of cardiac muscle: its ultrastructure and the electroof its geometry. Journal of Cell Biology 33, 103-129 (1967).
6.
MCNUTT, (1975).
N.
7.
McNuTr,
N. & FAWCETT,
G. Langer
Ultrastructure
& A. Brady,
of the
myocardial
sarcolemma.
D. Myocardial ultrastructurc. Eds. pp. l-49. New York: John
Circulation
Research
In The Mammalian Wiley & Sons (1974).
37.
I-13
-hQocardium,
380 8.
9. 10. 11.
12. 13. 14.
15.
16.
17.
D. J. Scales RAYNS, D., DEVINE, C. & SCTHERLAND, C. Freeze fracture studies of membrane systems in vertebrate muscle. I. Striated muscle. Journal of Ultrastructure Research 50, 306-321 (1975). REVEL, J. The sarcoplasmic reticulum of the bat cricothyroid muscle. Journal of Cell Biology 12, 571-588 (1962). SOMLYO, A. V. Bridging structures spanning the junctional gap at the triad of skeletal muscle. j’ournal of Cell Biolop 80, 743-750 (1979). SOMMER, J. Sr JEWETT, I’. Cardiac muscle: a comparative ultrastructural, anatomical Ed.. pp. 89-106. New York: Academic Press view. In Cardiac Hyj,ertroj,hy, N. Alpert, (1971). SOMMER, J. & JOHNSON, E. A comparative study of Purkinje fibers and ventricular Gbers. Journal of Cell Biology 36, 497-526 (1968). SOMMER, J. & JOHNSON, E. Comparative ultrastructure of cardiac cell membrane specializations. American Journal of Cardiology 25, 184-194 (1970). SOMMER, J. & JOHNSON, E. Ultrastructure of cardiac muscle. In Handbook of Physiologv--The Cardioz~uscular System, R. Berne, N. Sperelakis & S. Geiger, Eds, pp. 113-186. Bethesda, Maryland: American Physiological Society (1979). SOMMER, J. & M'AUCH, R. The ultrastructure of the mammalian cardiac muscle cellwith special emphasis on the tubular membrane system. American j’ournal of PathologV 82, 192-232 (1976). SPERELAKIS, N.. FORBES, M. & RURIO, R. ‘The tubular system of myocardial cells: ultrastructure and possible function. In Recent Adr’ances in Studies on Cardiac Structure and ~letabolism. N. Dhalla, Ed., pp. 1633194. Baltimore, Maryland: University Park Press (1972). WAUGH, R. & SUMMER, J. Lamellar junctional sarcoplasmic reticulum, a specialization of cardiac sarcoplasmic reticulum. Journal of Cell Biology 63, 337-343 (1974).