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(Circulation Research. 2006;98:1110.)
© 2006 American Heart Association, Inc.

Editorials

When Is a Fly in the Ointment a Solution and not a Problem?

Neal D. Epstein, Julien S. Davis

From the Molecular Physiology Section, Molecular Cardiology Laboratory, National Heart, Lung, and Blood Institute, Bethesda, Md.

Correspondence to Neal D. Epstein, MD, Molecular Physiology Section, Molecular Cardiology Laboratory, NHLBI, NIH, 10 Center Drive, MSC 1760, Building 10, Room 7B-15, Bethesda, MD 20892. E-mail epsteinn{at}mail.nih.gov

See related article, pages 1212–1218

Key Words: stretch-activation • myosin kinetics • cardiac • regulatory light chain phosphorylation

You never know from where and when the next clue to any particular scientific problem will arise. In the case of cardiac function, it may have been in 1948 from Professor J.W.S. Pringle, who was trying to figure out how flies manage to fly upside down.¹ Having mounted a truncated fly wing apparatus on a gyroscopic base, he serendipitously noted that when inertial and damping conditions were just right, the truncated wings oscillated at more than 100 s^–1 independent of neuronal innervation (Figure, A). This, he surmised, was attributable to matching an intrinsic resonant property of insect flight muscle to the elastic and inertial properties of the wing and exoskeletal structure, producing a resonant system generating oscillatory power. He called this intrinsic property of insect flight muscle "stretch–activation" because it is a recurrent stretching in the face of persistent Ca²⁺ levels, not Ca²⁺ pulses, that activate the actomyosin interaction in these insect flight muscles. This resonant system resembles a parent pushing a child on a swing. The parent must push at the correct time; that is, the addition of energy to the system must be matched to the intrinsic resonance of the system.

View larger version (11K): [in this window] [in a new window]   A, Graph of what Pringle called "free oscillation" of the truncated wing apparatus. The work loop is counter-clockwise, so that as the muscle oscillates, tension is greater during shortening than at the same length during the stretch part of the cycle. The muscle is performing work on the apparatus during shortening. Adapted from Pringle1 with permission of the publisher. B, Abstract tracing of an isometrically contracting rabbit slow muscle fiber after an imposed stretch showing the immediate spike in tension, decay, and second rise in tension.

The resonant system in flight muscle can also be seen through inspection of the mechanical transient of an isometrically contracting fiber after a stretch that is imposed and held at the new length. As shown in panel B of the Figure, there is an immediate increase in tension followed by an abrupt drop and then a second rise in tension. It is the time delay to this second rise in tension that defines the frequency of the resonant system. This can be visualized as a contraction of the biceps which initiates a stretch of the triceps and then the reversal, as the lower arm alternates between angles of 90° and 180°. The time delay is the length of time after the triceps stretch before the latter contracts and initiates a biceps stretch and so on. More recently, it has been shown that this property in insect flight muscle is regulated by the phosphorylation of the myosin regulatory light chain (RLC) in 2 distinct adjacent sites. Transgenic flies without these phosphorylation sites in the myosin RLC are rendered flightless because of diminished oscillatory power.²

In those insects in which wings beat at high rates (the wingbeat frequency of flies is 150 s^–1), nature has engineered a tradeoff between the sarcoplasmic reticulum (SR) and mitochondria because of limited space between the predominant thin and thick filaments in muscle fibers. The pumping of Ca²⁺ in and out of the sarcoplasm works well in fast skeletal muscle where muscles contract and hold for variable periods of time. If, however, the rate of muscle contraction is fast and periodic, as in flight muscle, the amount of SR apparatus necessary to pump Ca²⁺ back and forth in excess of 100 s^–1 would occupy the space needed for the additional mitochondria required to power flight. Thus, in flight muscle, a small SR apparatus keeps Ca²⁺ levels either "on" or "off" by neuronal innervation that is asynchronous with the rate of muscle contraction. Oscillatory power is produced through alternating stretch and shortening in opposing muscle groups as long as Ca²⁺ levels are on. Because the stretch–activation response is only active in the face of elevated Ca²⁺ levels, wingbeating can be stopped simply by the uptake of Ca²⁺ into the SR. The fact that we use the word "beating" to describe both wings and heart is merely a colloquial recognition of periodic motion and oscillatory power in both systems.

Pringle and his student, G.J. Steiger, went on to characterize stretch–activation in a variety of muscle types in many animals.^1,3 They found this property to be a general property of muscle, diminished in fast skeletal muscle but exaggerated in cardiac muscle. They demonstrated that at 20°C the time delay to the second rise in tension is on the order of 50 milliseconds in insect flight muscle and 1.2 seconds in mounted rabbit papillary muscle. Ca²⁺ and P_i levels were determined to affect resonant frequency. Furthermore, the stretch–activation apparent rate constants, when corrected for temperature, are linearly related to a plot of the range of physiologic heart rates in a variety of animals ranging from turtles to hummingbirds. Although the oscillatory power of heart is clearly driven by synchronous electrically paced Ca²⁺ transients, Pringle and Steiger proposed that the matching of the intrinsic stretch–activation rates, across species, to their respective ranges of physiologic heart rate, would certainly help increase cardiac efficiency. At the very least, a mismatch across species would be selected against as the heart evolved.

In a recent article from R. Moss and colleagues,⁴ the authors investigate the stretch–activation response in a mouse model in which cardiac myosin binding protein-C (cMyBP-C) has been deleted. It has long been appreciated that cMyBP-C has a functional role in modulating the contractile mechanism of cardiac muscle. More recently, interest in this protein has increased because of the identification of many nonsense mutations in cMyBP-C that cause hypertrophic cardiomyopathy (HCM) (for a review see reference ⁵). The authors find striking changes in the response to stretch in ventricular muscle strip preparations from these transgenic mice compared with wild-type. After a stretch of an isometrically contracting muscle strip, the decay in tension that occurs after the initial spike in tension is deeper and the subsequent rise in tension is faster in the KO preparation than in the wild-type. The decreased time delay (higher rate constant) in the second rise in tension is reminiscent of papillary muscle in transgenic mice harboring a mutant allele (Met149Val) of the human myosin essential light chain (ELC). However, unlike the mutant ELC preparation, the cMyBP-C preparation shows a much greater decay in tension.

The ELC mutation has likewise been shown to cause a variant of HCM, but in the latter case, many of the patients and virtually all of the transgenics with the Met149Val ELC mutation produced in our laboratory are associated with a dramatic hypertrophy of the papillary muscle and adjacent ventricular tissue.^6,7 Moreover, 2 families with a Glu22Lys mutation in the myosin regulatory light chain (RLC) show the same rare hypertrophic pattern.⁶ This mutation is adjacent to the human homologue (Ser-15) of the insect flight muscle RLC phosphorylation sites that regulate stretch–activation. The homologous mutation in mouse RLC has also been shown to inhibit phosphorylation of Ser-15, producing the same midcavity disease in transgenic mice.⁸ Nonlinear least square analysis of the mechanical transients of slow skeletal muscle fibers (which express the cardiac myosin isoform) has yielded classical Huxley Simmons (H-S) exponentials (rates and amplitudes) that provide a kinetic signature of muscle type.⁹ This analysis has been applied to single skinned rabbit slow skeletal muscle fibers to obtain the single exponential H-S phase 3, recognized to be the source of the stretch–activation response.^3,9,10 There is a damping of the normalized stretch activation amplitude of the fibers with fully phosphorylated RLC. Overall tension is significantly increased while the resonant frequency in this analysis remains constant. Can this type of analysis add to the findings of Stelzer et al? Will the extraction of all the H-S phases from the cMyBP-C ventricular strip mechanical transients yield additional insights? Can it explain why specific mutations in the ELC and RLC modifying the stretch–activation response result in a phenotype of midcavity ventricular hypertrophy whereas the nonsense cMyBP-C mutations result in a more generalized cardiac hypertrophy? It may even be possible that the comparison of extracted exponentials from hearts of different HCM mouse models can be used to address controversy in the field of myosin kinetics.

Our own use of nonlinear least square analysis of fast and slow muscle myosin fiber transients has depended on the use of a skeletal myosin light chain kinase (MLCK) we cloned from normal human, mouse, and rabbit heart that is identical to the respective skeletal MLCKs.^11,12 We subsequently identified a small family with a mutation in this gene producing a kinase with twice the V_max of the normal skeletal MLCK.¹³ The young man with this mutation coinherited a mutation in the cardiac myosin heavy chain adjacent to the ELC from his other parent. Both parents have incidental mild asymmetric HCM while their son, the proband, presented at age 13 with severe partial midcavity hypertrophy. Recently, a report of a skeletal MLCK KO mouse has called into question the presence of skeletal MLCK in the heart, citing a lack of obvious effect in mouse hearts of unspecified age.¹⁴ Our own previous work identified a spatial gradient of RLC phosphorylation (RLCP) with high levels at the epicardium and apex and lower levels at the endocardium and base in normal mouse heart. This gradient is matched by a gradient of MLCK detected by an antibody we raised to the whole expressed active enzyme.¹³ A spatial gradient of the Rho kinase–dependent phosphatase subunit (small myosin binding unit) that removes the phosphate from RLC has been reported in rat heart.¹⁵ This gradient is observed as the inverse of RLCP from apex to base. As such, the distribution of phosphatase is consistent with the pattern of MLCK and RLCP we observe. Our own skeletal MLCK KO has demonstrated no gradient but rather a uniform pattern of cardiac RLCP across the ventricle (S. Winitsky, unpublished observations, 2006). Taken together, this requires the existence of at least one additional kinase to phosphorylate the cardiac myosin RLC. However, it also suggests that phosphorylation-dephosphorylation rates are modulated by the presence of skeletal MLCK at the apex and epicardium. Observation of these KO mice as they age and/or crossing them with mild models of HCM transgenics will help disclose whether there is a role for skeletal muscle MLCK in the heart.

How might this gradient of RLCP interface with the stretch–activation response? Can this gradient unite cardiac myosin kinetics and cardiac anatomy? The counter-helical fiber orientations of the vertebrate heart have long been recognized and elegantly demonstrated by serial microscopic fiber orientation studies.¹⁶ The epicardial fibers have a left-handed orientation whereas the endocardial fibers are right-handed. Multiple MRI studies, however, produce a paradox with respect to this architecture. Tagged MRI studies show torsion of the ventricle in a counter-clockwise direction viewed from the apex.¹³ Because the endocardium is the first to be innervated, it initially rotates in the opposite direction; however, it is quickly captured by the hyperphosphorylated stronger epicardium and the full thickness of the ventricle rotates in the direction of epicardial fiber shortening. Thus, the epicardial fibers dominate and the endocardial region moves against its orientation. Paradoxically, the endocardium rotates at a higher angular velocity than the epicardium itself. To visualize an outer left-handed helix dominating an inner right-handed helix, see the supplemental animation (available online at http://circres.ahajournals.org). The gradient of RLCP is relevant to this paradox in two ways. First, the RLCP in the epicardium produces a vastly increased amount of tension which provides dominant epicardial force which overwhelms endocardium. Although MRI has not visualized stretch in systole outside of papillary muscles, the architecture of left-handed epicardium and right-handed endocardial fibers suggests endocardial winding or stretch in some portion of systole as the epicardium dominates endocardium. A drop in tension in the endocardium after as little as 0.1% stretch (well below the resolution of MRI) could reinforce epicardial dominance and help explain the faster angular velocity by the endocardium as it moves with decreased resistance.

Beyond the papillary muscles, which being linear, are easily seen to be stretched in systole, microsonometry experiments suggest that endocardium and portions deep to it also stretch slightly in systole.^17,18 This, too, presents a paradox, for although eccentric contraction (stretch of a contracting fiber) is exceedingly efficient, a fiber that is stretched presents an additional load to the fibers that are shortening. This is the last thing one would desire in an organ like a heart in which efficiency is so critical. How then does the heart take advantage of a torsion/wringing motion, yet escape the pitfall of what is essentially a brake, applied to shortening fibers? In this case, the application of nonlinear least square kinetic analysis to transients of fast- and slow-skinned muscle fibers (±RLC phosphorylation) suggests an answer. This analysis, in contrast to fast muscle myosin, shows that the force producing step of cardiac/slow skeletal myosin is readily reversible. Thus, myosin heads detached by strain, are likely to be in a higher energy state in these muscles.¹⁹ Because of the partial reversibility of the cardiac myosin ATPase cycle, this will occur once for each stretch without the hydrolysis of an additional ATP. Local instabilities in muscle length will make even greater use of this pathway. Additional energy is conserved through the recruitment of the stretch-activated second rise in tension to the overall work in systole. We have shown that the timing of this delayed tension fits the physiologic range of heart rate in mouse and rabbit.^7,13

A hybrid car is designed to convert an electric motor into a generator on application of the brakes, capturing inertial energy that would otherwise be converted to heat. In this respect, the reversibility of cardiac myosin resembles a hybrid motor, which is not surprising given the need for efficiency in the heart. Thus, in the big picture, perhaps the fly has climbed out of the ointment and into a Prius.

	Acknowledgments

 
We thank Steve O. Winitsky and Robert S. Adelstein for a careful reading and for comments on the manuscript. The work discussed from our laboratory is funded through the National Heart, Lung, and Blood Institute intramural institute.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top References

 

1. Pringle JWS. The Cronnian Lecture. Stretch activation of muscle: function and mechanism. Proc R Soc Lond B. 1977; 201: 107–130.
2. Tohtong R, Yamashita H, Graham M, Haeberle J, Simcox A, Maughan D. Impairment of muscle function caused by mutations of phosphorylation sites in myosin regulatory light chain. Nature. 1995; 374: 650–653.[CrossRef][Medline] [Order article via Infotrieve]
3. Steiger G. Insect Flight Muscle. Amsterdam: North Holland; 1977.
4. Stelzer JE, Dunning SB, Moss RL. Ablation of cardiac myosin-binding protein-C accelerates stretch activation in murine skinned myocardium. Circ Res. 2006; 98: 1212–1218.[Abstract/Free Full Text]
5. Flashman E, Redwood C, Moolman-Smook J, Watkins H. Cardiac myosin binding protein C: its role in physiology and disease. Circ Res. 2004; 94: 1279–1289.[Abstract/Free Full Text]
6. Poetter K, Jiang H, Hassanzadeh S, Master SR, Chang A, Dalakas MC, Rayment I, Sellers JR, Fananapazir L, Epstein ND. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet. 1996; 13: 63–69.[CrossRef][Medline] [Order article via Infotrieve]
7. Vemuri R, Lankford EB, Poetter K, Hassanzadeh S, Takeda K, Yu ZX, Ferrans VJ, Epstein ND. The stretch-activation response may be critical to the proper functioning of the mammalian heart. Proc Natl Acad Sci U S A. 1999; 96: 1048–1053.[Abstract/Free Full Text]
8. Szczesna-Cordary D, Guzman G, Zhao J, Hernandez O, Wei J, Diaz-Perez Z. The E22K mutation of myosin RLC that causes familial hypertrophic cardiomyopathy increases calcium sensitivity of force and ATPase in transgenic mice. J Cell Sci. 2005; 118: 3675–3683.[Abstract/Free Full Text]
9. Davis JS. Kinetic analysis of dynamics of muscle function. Methods Enzymol. 2000; 321: 23–37.[Medline] [Order article via Infotrieve]
10. Kawai M, Brandt PW. Sinusoidal analysis: a high resolution method for correlating biochemical reactions with physiological processes in activated skeletal muscles of rabbit, frog, and crayfish. J Muscle Res Cell Motil. 1980; 1: 279–303.[CrossRef][Medline] [Order article via Infotrieve]
11. Davis JS, Hassanzadeh S, Winitsky S, Wen H, Aletras A, Epstein ND. A gradient of myosin regulatory light-chain phosphorylation across the ventricular wall supports cardiac torsion. Cold Spring Harb Symp Quant Biol. 2002; 67: 345–352.[CrossRef][Medline] [Order article via Infotrieve]
12. Davis JS, Satorius CL, Epstein ND. Kinetic effects of myosin regulatory light chain phosphorylation on skeletal muscle contraction. Biophys J. 2002; 83: 359–370.[Abstract/Free Full Text]
13. Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, Epstein ND. The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation. Cell. 2001; 107: 631–641.[CrossRef][Medline] [Order article via Infotrieve]
14. Zhi G, Ryder JW, Huang J, Ding P, Chen Y, Zhao Y, Kamm KE, Stull JT. Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction. Proc Natl Acad Sci U S A. 2005; 102: 17519–17524.[Abstract/Free Full Text]
15. Rajashree R, Blunt BC, Hofmann PA. Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart. Am J Physiol Heart Circ Physiol. 2005; 289: H1736–H1743.[Abstract/Free Full Text]
16. Streeter DD Jr, Spotnitz HM, Patel DP, Ross J Jr, Sonnenblick EH. Fiber orientation in the canine left ventricle during diastole and systole. Circ Res. 1969; 24: 339–347.[Abstract/Free Full Text]
17. Ashikaga H, Omens JH, Ingels NB Jr, Covell JW. Transmural mechanics at left ventricular epicardial pacing site. Am J Physiol Heart Circ Physiol. 2004; 286: H2401–H2407.[Abstract/Free Full Text]
18. Buckberg GD, Mahajan A, Jung B, Markl M, Hennig J, Ballester-Rodes M MRI myocardial motion and fiber tracking: a confirmation of knowledge from different imaging modalities. Eur J Cardiothorac Surg. 2006; 29 (suppl 1): s165–s177.[Medline] [Order article via Infotrieve]
19. Davis JS, Epstein ND. Kinetic effects of fiber type on the two subcomponents of the Huxley-Simmons phase 2 in muscle. Biophys J. 2003; 85: 390–401.[Abstract/Free Full Text]

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