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Left ventricular apical torsion and architecture are not inverted in situs inversus totalis
ARTICLE IN PRESS
Progress in Biophysics and Molecular Biology 97 (2008) 513–519 www.elsevier.com/locate/pbiomolbio
Left ventricular apical torsion and architecture are not inverted in situs inversus totalis Tammo Delhaasa,b,, Wilco Kroonb, Peter Bovendeerdd, Theo Artsc a
Department of Pediatrics, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, P.O. Box 5800, NL-6202 AZ Maastricht, The Netherlands b Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands c Department of Biophysics, Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands d Department of Biomedical Technology, Eindhoven University of Technology, P.O. Box 513, NL-5600 MB Eindhoven, The Netherlands Available online 9 February 2008
Abstract Occasionally, individuals have a complete, mirror-image reversal of their internal organ position, called situs inversus totalis (SIT). Whereas gross anatomy is mirror-imaged in SIT, this might not be the case for the internal architecture of organs, e.g. the myofiber pattern in the left cardiac ventricle. We performed a Magnetic Resonance Tagging study in nine controls and in eight subjects with SIT to assess the deformation pattern in the apical half of the LV wall. It appeared that both groups had the same LV apical deformation pattern. This implies that not only the superficial LV apical layers in SIT follow a normal, not inverted pattern, but the deeper layers as well. Apparently, the embryonic L/R controlling genetic pathway does determine situs-specific gross anatomy morphogenesis but it is not the only factor regulating fiber architecture within the apical part of the LV wall. We propose that mechanical forces generated in the not-inverted molecular structure of the basic right-handed helical contractile components of the sarcomere play a role in shaping the LV apex. r 2008 Published by Elsevier Ltd. Keywords: Cardiac development; Left/right asymmetry; Magnetic resonance imaging; Mechanics; Myocardium; Adaptation
Contents 1. 2.
3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Experimental subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Image acquisition and analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
514 514 514 514 515 516
Corresponding author at: Department of Pediatrics and Physiology, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, P.O. Box 5800, NL-6202 AZ Maastricht, The Netherlands. Tel.: +31 43 387 5239; fax: +31 43 387 5246. E-mail address: [email protected] (T. Delhaas).
0079-6107/$ - see front matter r 2008 Published by Elsevier Ltd. doi:10.1016/j.pbiomolbio.2008.02.004
ARTICLE IN PRESS T. Delhaas et al. / Progress in Biophysics and Molecular Biology 97 (2008) 513–519
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5.
Grants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518 Editor’s note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
1. Introduction The human body displays an almost perfect exterior bilateral symmetry. Internally, however, there are striking asymmetries. About 99.99% of the human population has developed identical thoraco-abdominal asymmetry with the cardiac apex, a bilobed lung, the stomach, and the spleen at the left, and the venae cavae, a trilobed lung, the appendix, the major liver lobe, and the gallbladder at the right. This normal arrangement of organs is called situs solitus. Occasionally, individuals have a complete, mirror-image reversal of this asymmetry, called situs inversus totalis (SIT). Whereas gross anatomy is mirror-imaged in SIT, this might not be the case for the internal architecture of organs, e.g. the myofiber pattern in the left cardiac ventricle. Among mammals, including humans, this myofiber pattern has an invariant nature in normal hearts (Grimm et al., 1976; Hsu et al., 2001; Streeter and Hanna, 1973; Streeter et al., 1969). Myofiber orientation varies gradually across the LV wall: subepicardial fibers follow the path of a left-handed helix around the cavity, fibers in the mid-wall are circumferentially oriented, and subendocardial fibers follow a right-handed helical path. Due to this specific pattern of fiber orientation, the LV exhibits torsion around its long axis during the ejection phase such that the apex rotates counter-clockwise with respect to the base, as viewed from the apex (Arts et al., 1984; Taber et al., 1996). Whereas to our knowledge no data are available on e.g. the orientation of the spiral fiber layers in the wall of the stomach or the arteries in subjects with SIT, for the heart some reports exist (Asami and Koizumi, 1989; Matsumura et al., 1990; Taussig, 1926). They all show that in SIT at least the superficial LV apical layers follow a normal, not inverted, pattern. We performed a Magnetic Resonance Tagging study to assess the deformation pattern in the apical half of the LV wall in controls and in subjects with SIT. It appeared that both groups had the same LV apical deformation pattern. This implies that not only the superficial LV apical layers in SIT follow a normal, not inverted pattern, but the deeper layers as well. Apparently, the embryonic L/R controlling genetic pathway does determine situs-specific gross anatomy morphogenesis but it is not the only factor regulating fiber architecture within the apical part of the LV wall. We propose that mechanical forces generated in the notinverted molecular structure of the basic right-handed helical contractile components of the sarcomere play a role in shaping the LV apex. 2. Methods 2.1. Experimental subjects Nine healthy controls (one female, eight males; age 10–56 years (median 30 years)) and eight persons with SIT (three females, five males; age 8–70 years (median 23 years)) were investigated. Six of the latter group had Kartagener syndrome, characterized by SIT, ciliary dyskinesia, bronchiectasis with chronic cough, sinusitis, and variable impairment of fertility. None of the SIT group had other structural (cardiac) defects or abnormalities. The Medical Ethics Committee of the University Hospital Maastricht approved the research protocol and informed consent was obtained from all participants. 2.2. Image acquisition and analysis MRI experiments were performed at 1.5 T (Gyroscan NT, Philips Medical Systems, Best, The Netherlands). Images were acquired in breathhold using ECG-triggering. Using spatial modulation of the magnetization (Axel and Dougherty, 1989), two series of line-tagged images (T1 FFE EPI) with lines in two orthogonal directions were obtained over a period of 80–90% of the cardiac cycle in two parallel short-axis slices of the LV apex. Additionally balanced FFE non-tagged reference cine images were made. The following parameter
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settings were used: phase interval 20 ms, slice distance 10–15 mm, slice thickness 8 mm, inter-tag distance 5 mm, field of view 200 mm for children and 250 mm for adults, image size 256 256 pixels. MR-images were analyzed off-line (Van Der Toorn et al., 2002). The LV wall was manually outlined for each slice in a mid-systolic reference cine-image. The horizontally tagged images were spatially band filtered around the line tag frequency (spatial frequency 0.14 mm1, ratio of bandwidth to center frequency 1.0). With the use of a correlation method previously applied for pulsed ultrasonic echo signals (de Jong et al., 1990), vertical displacement maps for each time interval were calculated from the successive images with horizontal tag lines. Similarly, horizontal displacement maps were obtained from the images with vertical tag lines. From these displacement maps the following time-dependent parameters for 2 LV short axis slices were determined: rotation (F), cross-sectional cavity area (Ac) and wall area (Aw). Torsion (T) in the section between the slices was calculated as the axial gradient in rotation angle multiplied by the average of the outer radii (ro) of the upper (u) and lower (l) slice: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Fu Fl ruo ruo þ r1o Aw þ Ac with ro ¼ T¼ , (1) d 2 p with d denoting the distance between the two cross-sections. Physically, this measure of torsion may be interpreted as the shear angle on the epicardial surface between both cross-sections. 3. Results Torsion pattern of the LV apex in controls and in subjects with SIT are shown in Fig. 1. Because ECGtriggering is used, the starting point within the cardiac cycle for all depicted torsion patterns is early systole. The duration of the systolic phase is indicated by the hatched bar and ranges between 250 and 350 ms, depending on the subject’s heart frequency. For all subjects studied, except for the two subjects with SIT indicated by the asterisk, torsion becomes steadily more negative during the systolic phase. Because torsion is proportional to the base-to-apex gradient in rotation angle (see Section 2), negative torsion indicates that, looking on the apex, the rotation in the apical cross section slice is counter clockwise relative to the more basal cross section slice. After the systolic phase, torsion returns towards zero. The influence of atrial systole on left ventricular filling and deformation is not clear since the use of ECG-triggering limits data acquisition to the initial 80–90% of the cardiac cycle. The magnitude of maximum torsion during systole is different for all subjects and ranges from 0.04 to 0.22 rad. Mean maximal systolic torsion is not significantly different between the controls (0.1770.02) and
situssolitus (=normal)
Torsion [rad]
0.10
situs inversus totalis
0.00
-0.10
-0.20 systole 0
200
400 Time (ms)
600
800
Fig. 1. Time course of torsion of the left ventricular short-axis slices in normal subjects and in those with situs inversus totalis. The asterisks indicate the two subjects with SIT that showed a different torsion trajectory.
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those with SIT (0.1370.06). The two subjects with SIT that showed a different systolic torsion trajectory (indicated by the asterisk) were an 8-years-old female with SIT and a 16-years-old female with Kartagener. Both subjects were not completely able to hold their breath during acquisition, making their data less reliable. 4. Discussion Our results clearly show that although gross anatomy is mirror-imaged in SIT, this is not the case for LV deformation. The negative torsion of the apical half of the LV in SIT is in six out of eight SIT subjects like normal LV torsion, both in sign and amplitude. This can only exist if the myocardial fiber orientation pattern in the LV apex is also as in normal subjects. Our results are in line with the only three anatomical/histological studies in SIT hearts that we could track after extensive literature search (Asami and Koizumi, 1989; Matsumura et al., 1990; Taussig, 1926). All three qualitative studies mentioned showed that in SIT apical fiber orientation follows a normal pattern. Whereas Taussig points out that the fiber direction in the superficial layers of the SIT heart is essentially the same as in the normal heart, she also mentions that there is no gradual transition in fiber orientation from the epicardium towards the endocardium: ‘‘y Immediately upon peeling off the thinnest possible superficial layer one came upon a deeper muscle layer which ran at right angles to the superficial layer’’ (Taussig, 1926). The study by Asami and Koizumi (1989) shows that the vortex cordis is never reversely directed even not in situs inversus or L-loop anomaly (see Fig. 2). They also show that the LV apex in SIT exhibits the same transmural gradient in fiber orientation as in the normal heart. The latter observation was also made by Matsamura et al. (1990). Life thrives on mirror asymmetry, a designation between left- and right-handedness in the basic structures of amino acids and organic molecules. Right-handed DNA is the rule for all life forms. The appearance of a symmetric exterior in vertebrates, including humans, disguises dramatic left/right asymmetries of the interior body plan. This left/right axis is a decision-type axis rather than a continuous gradient type like the anteroposterior and the dorso-ventral axes. The invariant nature of body situs within and across vertebrate species implies that a highly conserved pathway controls the specification of the left/right axis (Fujinaga, 1997;
Fig. 2. The vortex cordis has the same direction in situs solitus ( ¼ normal organ arrangement) and in situs inversus totalis (modified after Asami and Koizumi, 1989).
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Hamada et al., 2002). A disturbed initiation of the cascade of signals leading to situs solitus ( ¼ normal organ position) might give rise to a complete left-right reversal of the inner body. Perturbations within the pathway result in defects of laterality, called heterotaxia, which manifests as aberrant visceral organ position and/or asymmetry and an assortment of cardiac defects. Our data, as well as the one from the mentioned anatomical/histological studies of the SIT heart, provide developmental biologists with information that puts the L/R asymmetry establishment into a new perspective because it suggests that the embryonic L/R controlling genetic pathway does determine situs-specific gross anatomy morphogenesis but that it is not the only factor regulating fiber architecture within the LV wall. The apical part is then considered to have a myocardial fiber direction development that is situs-independent. Possible fiber orientation regulating factors, other than the L/R controlling genetic pathway, might be sought in the existence of a symmetry axis within the heart caused by different gene patterns present in specific parts of the myocardium (Christoffels et al., 2000), in different origin of myocardium e.g. from primary or secondary heart field (Waldo et al., 2001), in mechanical factors or in combinations of these factors. We propose the following possible role of mechanical factors in the development of the normal, i.e. noninverted fiber orientation pattern as found in the LV apex of subjects with SIT. As said before, life thrives on mirror asymmetry, a designation between left- and right-handedness in the basic structures of amino acids and organic molecules. The handedness of molecules in SIT is not different from the one in normal subjects. The major contractile filaments of the cardiac myocytes, actin and myosin, have both right-handed spirals. Single polypeptide globular molecules of G-actin form the double-strand right-handed helical polymer F-actin, also known as the thin filament. Myosin-II, the basic structure of the thick filament, is a dimer of myosin molecules with their long tails intertwined in a right-handed helix. Anti-parallel pairs of myosin-II dimers assemble to form a myosin thick filament. A basic property of a single helical structure is that it will unwind when force is applied to it. Bundles of right-handed helical structures will also exhibit torsional motion when they are subject to stress, either generated internally or caused by stretch, a fact illustrated in Fig. 3. The bundled black-white intertwined threads, all with a right-handed helix, are loaded by a heavy weight. Whereas each individual right-handed helix unwinds, the overall structure exhibits the formation of a left-handed helix. This illustrates that events on micro-level will have its impact on macro-level. We propose that torsional behavior of myocytes will be transmitted to the overall myocardial structure. To be important, such a tiny effect requires amplification. This might be found in the adaptation of myocyte size and orientation to mechanical load. Any tiny difference in myofiber orientation becomes amplified until one final form dominates. Because myocytes in SIT and situs solitus must have the same structure, this might explain that normal subjects as well as those with SIT have the same myofiber architecture of the LV apex. The fact that molecular signals are not the only forces that pattern and shape the developing embryo has also been shown by Scott and Stainier (2003) who demonstrated that mechanical stresses sensed by cells also seem to be involved in creating the body plan. Differences in the pattern of torsion between normal subjects and those with SIT cannot be explained by differences in heart rate variability or right and/or left ventricular loading conditions between the two groups. None of our subjects had conduction disturbances, myocardial infarct, or signs of (pulmonary) hypertension. In the normal heart, through slice motion will not affect observed torsion because the longitudinal gradient in rotation angle is about uniform. In SIT, some differences in torsion differences may be attributed to throughslice motion. The variation in measured patterns of torsion in SIT suggests that every single SIT patient has a unique fiber orientation pattern, especially in the more basal parts of the left ventricle. Deformation in these more basal parts of the left ventricle influences of course the torsion pattern in the apical part. Future more detailed studies on the deformation of both apical and basal slices might shed more light on the differences found for the apical torsion pattern within the SIT group. The fact that LV apical deformation in normal subjects and in those with SIT is not completely congruent might also be sought in the different, i.e. mirror-imaged, gross-anatomy at the basal part of the LV. In this region not only mechanical forces but also hemodynamical forces imposed by the gross anatomy will play a role. The direction of blood expelled from the LV is more or less concomitant with the long axis of the LV and, hence, will not induce any rotational movement of the LV. However, the RV outflow tract is directed oblique to the LV long axis. If blood is expelled from the RV, it will impose a rotational force on the LV in the direction opposite to the blood flow direction. This force will be maximal just below the equator of the heart and result in the elongation of subepicardial fibers during ejection, if these fibers are oriented in directions
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Fig. 3. The bundled black–white intertwined threads, all with a right-handed helix, are loaded by a heavy weight. Whereas each individual helix unwinds, the overall structure exhibits the formation of a left-handed helix.
other than a left-handed helix in the case of situs solitus and a right-handed one in SIT. Every single SIT patient has found a unique solution to deal with the mechanical and hemodynamical forces, and, hence, has a unique LV torsion pattern. The SIT-heart therefore provides a unique possibility to study regulatory mechanisms for cardiac fiber orientation, which is very important in understanding and predicting effects of cardiac remodeling in asynchronously activated hearts, after myocardial infarction, and after surgical interventions. Because myocardial structure is one of the determining factors for electrical conduction, the SIT heart might also be used to study cardiac mechano-electric interactions in more detail. In conclusion: (1) gross anatomy is mirror-imaged in SIT; (2) the LV apical deformation pattern as well as its helical myofiber pattern are not mirror-imaged in SIT; and (3) the LV apex might develop independently from the left-right controlling genetic pathway. In this developmental process mechanical forces imposed by the basic non-inverted right-handed helical structures of the contractile elements of the myocytes might play a major role. 5. Grants Supported by Grant 2000T036 from The Netherlands Heart Foundation and Grant PF155 from the University Hospital Maastricht. T.D. is a Clinical Fellow of The Netherlands Heart Foundation (Dr. E. Dekker Fund). Editor’s note See also related communications in this issue by Taggart and Lab (2008) and Cherubini et al. (2008). References Arts, T., Meerbaum, S., Reneman, R.S., Corday, E., 1984. Torsion of the left ventricle during the ejection phase in the intact dog. Cardiovasc. Res. 18, 183–193.
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Asami, I., Koizumi, K., 1989. The vortex cordis is never reversely directed, even in situs inversus and L-loop anomaly. Kaibogaku Zasshi 64, 36–45. Axel, L., Dougherty, L., 1989. MR imaging of motion with spatial modulation of magnetization. Radiology 171, 841–845. Cherubini, C., Filippi, S., Nardinocchi, P., Teresi, L., 2008. An electromechanical model of cardiac tissue: Constitutive issues and electrophysiological effects. Prog. Biophys. Mol. Biol. 97, 562–573. Christoffels, V.M., Habets, P.E., Franco, D., Campione, M., de Jong, F., Lamers, W.H., Bao, Z.Z., Palmer, S., Biben, C., Harvey, R.P., Moorman, A.F., 2000. Chamber formation and morphogenesis in the developing mammalian heart. Dev. Biol. 223, 266–278. de Jong, P.G.M., Arts, T., Hoeks, A.P.G., Reneman, R.S., 1990. Determination of tissue motion velocity by correlation interpolation of pulsed ultrasonic echo signals. Ultrason. Imaging 12, 84–98. Fujinaga, M., 1997. Development of sidedness of asymmetric body structures in vertebrates. Int. J. Dev. Biol. 41, 153–186. Grimm, A.F., Katele, K.V., Lin, H.L., 1976. Fiber bundle direction in the mammalian heart. An extension of the ‘‘nested shells’’ model. Basic Res. Cardiol. 71, 381–388. Hamada, H., Meno, C., Watanabe, D., Saijoh, Y., 2002. Establishment of vertebrate left-right asymmetry. Nat. Rev. Genet. 3, 103–113. Hsu, E.W., Buckley, D.L., Bui, J.D., Blackband, S.J., Forder, J.R., 2001. Two-component diffusion tensor MRI of isolated perfused hearts. Magn. Reson. Med. 45, 1039–1045. Matsumura, H., Aizawa, Y., Kumaki, K., 1990. Myocardial architecture in situs inversus viscerum totalis. In: Clark, E.B., Takao, A. (Eds.), Developmental Cardiology: Morphogenesis and Function. Futura Publishing Co., Inc., Mount Kisco, NY, pp. 605–624. Scott, I.C., Stainier, D.Y., 2003. Developmental biology: twisting the body into shape. Nature 425, 461–463. Streeter, D.D., Hanna, W.T., 1973. Engineering mechanics for successive states in canine left ventricular myocardium: II fiber angle and sarcomere length. Circ. Res. 33, 656–664. Streeter, D.D., Spotnitz, H.M., Patel, D.P., Ross Jr., J., Sonnenblick, E.H., 1969. Fiber orientation in the canine left ventricle during diastole and systole. Circ. Res. 24, 339–347. Taber, L.A., Yang, M., Podszus, W.W., 1996. Mechanics of ventricular torsion. J. Biomech. 29, 745–752. Taggart, P., Lab, M., 2008. Cardiac mechano-electric feedback and electrical restitution in humans. Prog. Biophys. Mol. Biol. 97, 452–460. Taussig, H.B., 1926. The anatomy of the heart in two cases of situs transversus. Bull. Johns Hopkins Hosp. 39, 199–202. Van Der Toorn, A., Barenbrug, P., Snoep, G., Van Der Veen, F.H., Delhaas, T., Prinzen, F.W., Maessen, J., Arts, T., 2002. Transmural gradients of cardiac myofiber shortening in aortic valve stenosis patients using MRI tagging. Am. J. Physiol. Heart Circ. Physiol. 283, H1609–H1615. Waldo, K.L., Kumiski, D.H., Wallis, K.T., Stadt, H.A., Hutson, M.R., Platt, D.H., Kirby, M.L., 2001. Conotruncal myocardium arises from a secondary heart field. Development 128, 3179–3188.
Progress in Biophysics and Molecular Biology 97 (2008) 513–519 www.elsevier.com/locate/pbiomolbio
Left ventricular apical torsion and architecture are not inverted in situs inversus totalis Tammo Delhaasa,b,, Wilco Kroonb, Peter Bovendeerdd, Theo Artsc a
Department of Pediatrics, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, P.O. Box 5800, NL-6202 AZ Maastricht, The Netherlands b Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands c Department of Biophysics, Cardiovascular Research Institute Maastricht, Maastricht University, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands d Department of Biomedical Technology, Eindhoven University of Technology, P.O. Box 513, NL-5600 MB Eindhoven, The Netherlands Available online 9 February 2008
Abstract Occasionally, individuals have a complete, mirror-image reversal of their internal organ position, called situs inversus totalis (SIT). Whereas gross anatomy is mirror-imaged in SIT, this might not be the case for the internal architecture of organs, e.g. the myofiber pattern in the left cardiac ventricle. We performed a Magnetic Resonance Tagging study in nine controls and in eight subjects with SIT to assess the deformation pattern in the apical half of the LV wall. It appeared that both groups had the same LV apical deformation pattern. This implies that not only the superficial LV apical layers in SIT follow a normal, not inverted pattern, but the deeper layers as well. Apparently, the embryonic L/R controlling genetic pathway does determine situs-specific gross anatomy morphogenesis but it is not the only factor regulating fiber architecture within the apical part of the LV wall. We propose that mechanical forces generated in the not-inverted molecular structure of the basic right-handed helical contractile components of the sarcomere play a role in shaping the LV apex. r 2008 Published by Elsevier Ltd. Keywords: Cardiac development; Left/right asymmetry; Magnetic resonance imaging; Mechanics; Myocardium; Adaptation
Contents 1. 2.
3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Experimental subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Image acquisition and analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
514 514 514 514 515 516
Corresponding author at: Department of Pediatrics and Physiology, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, P.O. Box 5800, NL-6202 AZ Maastricht, The Netherlands. Tel.: +31 43 387 5239; fax: +31 43 387 5246. E-mail address: [email protected] (T. Delhaas).
0079-6107/$ - see front matter r 2008 Published by Elsevier Ltd. doi:10.1016/j.pbiomolbio.2008.02.004
ARTICLE IN PRESS T. Delhaas et al. / Progress in Biophysics and Molecular Biology 97 (2008) 513–519
514
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1. Introduction The human body displays an almost perfect exterior bilateral symmetry. Internally, however, there are striking asymmetries. About 99.99% of the human population has developed identical thoraco-abdominal asymmetry with the cardiac apex, a bilobed lung, the stomach, and the spleen at the left, and the venae cavae, a trilobed lung, the appendix, the major liver lobe, and the gallbladder at the right. This normal arrangement of organs is called situs solitus. Occasionally, individuals have a complete, mirror-image reversal of this asymmetry, called situs inversus totalis (SIT). Whereas gross anatomy is mirror-imaged in SIT, this might not be the case for the internal architecture of organs, e.g. the myofiber pattern in the left cardiac ventricle. Among mammals, including humans, this myofiber pattern has an invariant nature in normal hearts (Grimm et al., 1976; Hsu et al., 2001; Streeter and Hanna, 1973; Streeter et al., 1969). Myofiber orientation varies gradually across the LV wall: subepicardial fibers follow the path of a left-handed helix around the cavity, fibers in the mid-wall are circumferentially oriented, and subendocardial fibers follow a right-handed helical path. Due to this specific pattern of fiber orientation, the LV exhibits torsion around its long axis during the ejection phase such that the apex rotates counter-clockwise with respect to the base, as viewed from the apex (Arts et al., 1984; Taber et al., 1996). Whereas to our knowledge no data are available on e.g. the orientation of the spiral fiber layers in the wall of the stomach or the arteries in subjects with SIT, for the heart some reports exist (Asami and Koizumi, 1989; Matsumura et al., 1990; Taussig, 1926). They all show that in SIT at least the superficial LV apical layers follow a normal, not inverted, pattern. We performed a Magnetic Resonance Tagging study to assess the deformation pattern in the apical half of the LV wall in controls and in subjects with SIT. It appeared that both groups had the same LV apical deformation pattern. This implies that not only the superficial LV apical layers in SIT follow a normal, not inverted pattern, but the deeper layers as well. Apparently, the embryonic L/R controlling genetic pathway does determine situs-specific gross anatomy morphogenesis but it is not the only factor regulating fiber architecture within the apical part of the LV wall. We propose that mechanical forces generated in the notinverted molecular structure of the basic right-handed helical contractile components of the sarcomere play a role in shaping the LV apex. 2. Methods 2.1. Experimental subjects Nine healthy controls (one female, eight males; age 10–56 years (median 30 years)) and eight persons with SIT (three females, five males; age 8–70 years (median 23 years)) were investigated. Six of the latter group had Kartagener syndrome, characterized by SIT, ciliary dyskinesia, bronchiectasis with chronic cough, sinusitis, and variable impairment of fertility. None of the SIT group had other structural (cardiac) defects or abnormalities. The Medical Ethics Committee of the University Hospital Maastricht approved the research protocol and informed consent was obtained from all participants. 2.2. Image acquisition and analysis MRI experiments were performed at 1.5 T (Gyroscan NT, Philips Medical Systems, Best, The Netherlands). Images were acquired in breathhold using ECG-triggering. Using spatial modulation of the magnetization (Axel and Dougherty, 1989), two series of line-tagged images (T1 FFE EPI) with lines in two orthogonal directions were obtained over a period of 80–90% of the cardiac cycle in two parallel short-axis slices of the LV apex. Additionally balanced FFE non-tagged reference cine images were made. The following parameter
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settings were used: phase interval 20 ms, slice distance 10–15 mm, slice thickness 8 mm, inter-tag distance 5 mm, field of view 200 mm for children and 250 mm for adults, image size 256 256 pixels. MR-images were analyzed off-line (Van Der Toorn et al., 2002). The LV wall was manually outlined for each slice in a mid-systolic reference cine-image. The horizontally tagged images were spatially band filtered around the line tag frequency (spatial frequency 0.14 mm1, ratio of bandwidth to center frequency 1.0). With the use of a correlation method previously applied for pulsed ultrasonic echo signals (de Jong et al., 1990), vertical displacement maps for each time interval were calculated from the successive images with horizontal tag lines. Similarly, horizontal displacement maps were obtained from the images with vertical tag lines. From these displacement maps the following time-dependent parameters for 2 LV short axis slices were determined: rotation (F), cross-sectional cavity area (Ac) and wall area (Aw). Torsion (T) in the section between the slices was calculated as the axial gradient in rotation angle multiplied by the average of the outer radii (ro) of the upper (u) and lower (l) slice: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Fu Fl ruo ruo þ r1o Aw þ Ac with ro ¼ T¼ , (1) d 2 p with d denoting the distance between the two cross-sections. Physically, this measure of torsion may be interpreted as the shear angle on the epicardial surface between both cross-sections. 3. Results Torsion pattern of the LV apex in controls and in subjects with SIT are shown in Fig. 1. Because ECGtriggering is used, the starting point within the cardiac cycle for all depicted torsion patterns is early systole. The duration of the systolic phase is indicated by the hatched bar and ranges between 250 and 350 ms, depending on the subject’s heart frequency. For all subjects studied, except for the two subjects with SIT indicated by the asterisk, torsion becomes steadily more negative during the systolic phase. Because torsion is proportional to the base-to-apex gradient in rotation angle (see Section 2), negative torsion indicates that, looking on the apex, the rotation in the apical cross section slice is counter clockwise relative to the more basal cross section slice. After the systolic phase, torsion returns towards zero. The influence of atrial systole on left ventricular filling and deformation is not clear since the use of ECG-triggering limits data acquisition to the initial 80–90% of the cardiac cycle. The magnitude of maximum torsion during systole is different for all subjects and ranges from 0.04 to 0.22 rad. Mean maximal systolic torsion is not significantly different between the controls (0.1770.02) and
situssolitus (=normal)
Torsion [rad]
0.10
situs inversus totalis
0.00
-0.10
-0.20 systole 0
200
400 Time (ms)
600
800
Fig. 1. Time course of torsion of the left ventricular short-axis slices in normal subjects and in those with situs inversus totalis. The asterisks indicate the two subjects with SIT that showed a different torsion trajectory.
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those with SIT (0.1370.06). The two subjects with SIT that showed a different systolic torsion trajectory (indicated by the asterisk) were an 8-years-old female with SIT and a 16-years-old female with Kartagener. Both subjects were not completely able to hold their breath during acquisition, making their data less reliable. 4. Discussion Our results clearly show that although gross anatomy is mirror-imaged in SIT, this is not the case for LV deformation. The negative torsion of the apical half of the LV in SIT is in six out of eight SIT subjects like normal LV torsion, both in sign and amplitude. This can only exist if the myocardial fiber orientation pattern in the LV apex is also as in normal subjects. Our results are in line with the only three anatomical/histological studies in SIT hearts that we could track after extensive literature search (Asami and Koizumi, 1989; Matsumura et al., 1990; Taussig, 1926). All three qualitative studies mentioned showed that in SIT apical fiber orientation follows a normal pattern. Whereas Taussig points out that the fiber direction in the superficial layers of the SIT heart is essentially the same as in the normal heart, she also mentions that there is no gradual transition in fiber orientation from the epicardium towards the endocardium: ‘‘y Immediately upon peeling off the thinnest possible superficial layer one came upon a deeper muscle layer which ran at right angles to the superficial layer’’ (Taussig, 1926). The study by Asami and Koizumi (1989) shows that the vortex cordis is never reversely directed even not in situs inversus or L-loop anomaly (see Fig. 2). They also show that the LV apex in SIT exhibits the same transmural gradient in fiber orientation as in the normal heart. The latter observation was also made by Matsamura et al. (1990). Life thrives on mirror asymmetry, a designation between left- and right-handedness in the basic structures of amino acids and organic molecules. Right-handed DNA is the rule for all life forms. The appearance of a symmetric exterior in vertebrates, including humans, disguises dramatic left/right asymmetries of the interior body plan. This left/right axis is a decision-type axis rather than a continuous gradient type like the anteroposterior and the dorso-ventral axes. The invariant nature of body situs within and across vertebrate species implies that a highly conserved pathway controls the specification of the left/right axis (Fujinaga, 1997;
Fig. 2. The vortex cordis has the same direction in situs solitus ( ¼ normal organ arrangement) and in situs inversus totalis (modified after Asami and Koizumi, 1989).
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Hamada et al., 2002). A disturbed initiation of the cascade of signals leading to situs solitus ( ¼ normal organ position) might give rise to a complete left-right reversal of the inner body. Perturbations within the pathway result in defects of laterality, called heterotaxia, which manifests as aberrant visceral organ position and/or asymmetry and an assortment of cardiac defects. Our data, as well as the one from the mentioned anatomical/histological studies of the SIT heart, provide developmental biologists with information that puts the L/R asymmetry establishment into a new perspective because it suggests that the embryonic L/R controlling genetic pathway does determine situs-specific gross anatomy morphogenesis but that it is not the only factor regulating fiber architecture within the LV wall. The apical part is then considered to have a myocardial fiber direction development that is situs-independent. Possible fiber orientation regulating factors, other than the L/R controlling genetic pathway, might be sought in the existence of a symmetry axis within the heart caused by different gene patterns present in specific parts of the myocardium (Christoffels et al., 2000), in different origin of myocardium e.g. from primary or secondary heart field (Waldo et al., 2001), in mechanical factors or in combinations of these factors. We propose the following possible role of mechanical factors in the development of the normal, i.e. noninverted fiber orientation pattern as found in the LV apex of subjects with SIT. As said before, life thrives on mirror asymmetry, a designation between left- and right-handedness in the basic structures of amino acids and organic molecules. The handedness of molecules in SIT is not different from the one in normal subjects. The major contractile filaments of the cardiac myocytes, actin and myosin, have both right-handed spirals. Single polypeptide globular molecules of G-actin form the double-strand right-handed helical polymer F-actin, also known as the thin filament. Myosin-II, the basic structure of the thick filament, is a dimer of myosin molecules with their long tails intertwined in a right-handed helix. Anti-parallel pairs of myosin-II dimers assemble to form a myosin thick filament. A basic property of a single helical structure is that it will unwind when force is applied to it. Bundles of right-handed helical structures will also exhibit torsional motion when they are subject to stress, either generated internally or caused by stretch, a fact illustrated in Fig. 3. The bundled black-white intertwined threads, all with a right-handed helix, are loaded by a heavy weight. Whereas each individual right-handed helix unwinds, the overall structure exhibits the formation of a left-handed helix. This illustrates that events on micro-level will have its impact on macro-level. We propose that torsional behavior of myocytes will be transmitted to the overall myocardial structure. To be important, such a tiny effect requires amplification. This might be found in the adaptation of myocyte size and orientation to mechanical load. Any tiny difference in myofiber orientation becomes amplified until one final form dominates. Because myocytes in SIT and situs solitus must have the same structure, this might explain that normal subjects as well as those with SIT have the same myofiber architecture of the LV apex. The fact that molecular signals are not the only forces that pattern and shape the developing embryo has also been shown by Scott and Stainier (2003) who demonstrated that mechanical stresses sensed by cells also seem to be involved in creating the body plan. Differences in the pattern of torsion between normal subjects and those with SIT cannot be explained by differences in heart rate variability or right and/or left ventricular loading conditions between the two groups. None of our subjects had conduction disturbances, myocardial infarct, or signs of (pulmonary) hypertension. In the normal heart, through slice motion will not affect observed torsion because the longitudinal gradient in rotation angle is about uniform. In SIT, some differences in torsion differences may be attributed to throughslice motion. The variation in measured patterns of torsion in SIT suggests that every single SIT patient has a unique fiber orientation pattern, especially in the more basal parts of the left ventricle. Deformation in these more basal parts of the left ventricle influences of course the torsion pattern in the apical part. Future more detailed studies on the deformation of both apical and basal slices might shed more light on the differences found for the apical torsion pattern within the SIT group. The fact that LV apical deformation in normal subjects and in those with SIT is not completely congruent might also be sought in the different, i.e. mirror-imaged, gross-anatomy at the basal part of the LV. In this region not only mechanical forces but also hemodynamical forces imposed by the gross anatomy will play a role. The direction of blood expelled from the LV is more or less concomitant with the long axis of the LV and, hence, will not induce any rotational movement of the LV. However, the RV outflow tract is directed oblique to the LV long axis. If blood is expelled from the RV, it will impose a rotational force on the LV in the direction opposite to the blood flow direction. This force will be maximal just below the equator of the heart and result in the elongation of subepicardial fibers during ejection, if these fibers are oriented in directions
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Fig. 3. The bundled black–white intertwined threads, all with a right-handed helix, are loaded by a heavy weight. Whereas each individual helix unwinds, the overall structure exhibits the formation of a left-handed helix.
other than a left-handed helix in the case of situs solitus and a right-handed one in SIT. Every single SIT patient has found a unique solution to deal with the mechanical and hemodynamical forces, and, hence, has a unique LV torsion pattern. The SIT-heart therefore provides a unique possibility to study regulatory mechanisms for cardiac fiber orientation, which is very important in understanding and predicting effects of cardiac remodeling in asynchronously activated hearts, after myocardial infarction, and after surgical interventions. Because myocardial structure is one of the determining factors for electrical conduction, the SIT heart might also be used to study cardiac mechano-electric interactions in more detail. In conclusion: (1) gross anatomy is mirror-imaged in SIT; (2) the LV apical deformation pattern as well as its helical myofiber pattern are not mirror-imaged in SIT; and (3) the LV apex might develop independently from the left-right controlling genetic pathway. In this developmental process mechanical forces imposed by the basic non-inverted right-handed helical structures of the contractile elements of the myocytes might play a major role. 5. Grants Supported by Grant 2000T036 from The Netherlands Heart Foundation and Grant PF155 from the University Hospital Maastricht. T.D. is a Clinical Fellow of The Netherlands Heart Foundation (Dr. E. Dekker Fund). Editor’s note See also related communications in this issue by Taggart and Lab (2008) and Cherubini et al. (2008). References Arts, T., Meerbaum, S., Reneman, R.S., Corday, E., 1984. Torsion of the left ventricle during the ejection phase in the intact dog. Cardiovasc. Res. 18, 183–193.
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