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Titin Regulation of Cardiac Stiffness: Molecular Mechanisms of Diastolic Dysfunction

By: , Posted on: March 20, 2014

Short Summary:  The sarcomere generates contractile force in cardiac muscle through the interaction between actin thin filaments and myosin thick filaments during systole. During diastole, in contrast, relaxation depends largely on the elastic properties of the sarcomere. It has hypothesized for some time that sarcomere elasticity is dependent on the properties of giant protein titin, a large filamentous protein which spans the sarcomere.  We discuss a number of recent studies which identified that the elastic properties of titin are modulated through isoform switiching and kinase-mediated phosphorylation, resulting in altered sarcomeric extensibility during diastole.  Recent studies of human heart failure suggest that these findings may underlie mechanisms found in patients with end-stage heart failure due to dilated (non-ischemic) cardiomyopathy.

The core mechanical function of the heart occurs at the level of the sarcomere involving myosin, actin, and titin.  The cyclic contractions of the heart actually occur at the level of the cross-bridges of myosin thick filaments and the actin thin filaments.  These forces generate the molecular basis of the heart’s systolic function, which has been extensively characterized.  Following the resulting cardiac contraction (phases 1 and 2 of the cardiac cycle, see Figure 1), diastolic relaxation occurs (see isovolemic relaxation, Figure 1).  However, our understanding of the mechanisms of sarcomere function during diastole is still evolving.

Figure 1: The Cardiac Cycle
The cardiac cycle. The left ventricular developed pressure over time during the human cardiac cycle. Isovolumetric filling is followed by ejection, isovolumetric relaxation, which then passively fills. Adapted from Stehle, et al., 2010 (1)

With the opening of the arterioventricular valve, isovolumetric relaxation ends, and filling starts.  Ventricular diastole initiates the closure of the aortic valve; the removal of calcium from the cytosol and from troponin C (which triggered systolic contraction) as recently reviewed 1.  After isovolumetric relaxation occurs, filling of the heart starts.  In healthy young adults, most of this filling occurs during the early diastolic filling phase (Phase 4, Figure 1).  During the early diastolic filling phase, ventricular pressure continues to decrease even with the myocytes completely stretched.  Once all the cross bridges in the sarcomere have detached, the early diastolic filling gives way to diastasis.  It is here that the heart mechanics of the fully relaxed sarcomere are determined completely by the elastic properties of titin 2, 3.  The sarcomere length affects the next isovolumetric contraction.  In this way, titin plays a significant role in determining the preload of the next cycle based on the dynamics of systolic contraction via Frank-Starling mechanisms4.

The presence of an elastic sarcomeric filament was first suggested when it was observed that the N2 line, a structure within the I band first described in 18785, moves the same proportional distance away from both the M and Z lines during sarcomere lengthening6, 7.  This elastic filament came to be known as titin, the largest protein discovered to date.  Located on chromosome 2, the titin gene is 294 kilobases comprised of 363 exons which code for a protein with a predicted total molecular mass of 4.2MDa8.  Most of titin is made up of immunoglobin (Ig) or fibrinectin-type-III-like globular domains, but unique sequences are distributed throughout the protein.  Titin spans half the sarcomere, with its N-terminus attached at the Z-disc and its C-terminus attached to the myosin rod at the M line (Figure 2).   The main functions of titin are to translate mechanical information to signaling pathways and moderate sarcomeric extensibility.

Figure 2: Titin in the Context of the Sarcomere
Titin in the context of the sarcomere. Titin extends from Z disk to M-line. Titin in the I band is largely responsible for the rigidity and relaxation of the heart during diastole. Adapted from Willis et al., 2009 (18).

The functional spring elements of the sarcomere are found between the Z disc and M line, made up of titin (Figure 2). The elastic force generated by these spring elements allows titin to restore the sarcomere to its equilibrium length during early diastolic filling and control the passive tension of the relaxed sarcomere.  Different titin isoforms, generated through alternative splicing, vary in the I band spring elements.  Both skeletal and cardiac titin isoforms contain proximal and distal Ig regions and the PEVK domain, named for its high content of proline (P), glutamic acid (E), valine (V), and lysine (K).  Cardiac titin isoforms contain an additional unique sequence, the N2B region.  Finally, the larger cardiac isoform, N2BA, contains two other regions, the middle-Ig and N2A regions 8, 9.  These structural differences are compared in Figure 3A.  The rigidity of the individual Ig, PEVK, and N2B spring regions in the I band domain of titin, determined by single-molecule force spectroscopy, suggests that the straightening of the I band domain occurs in two phases: 1) the linkers between individual Ig domains in the Ig regions extend at low stretch-forces followed by 2) the extension of the unique PEVK and N2B regions at high stretch-forces 8.  Applying forces to the Ig region which cause titin to extend to lengths greater than physiological stretch-length (1.8-2.4µm) leads to the unfolding of individual Ig domains 8.

Figure 3: The Relationship Between Titin Isoforms, Titin Subunit Phosphorylation, and Cardiac Stiffness
The relationship between titin isoforms, titin subunit phosphorylation, and cardiac stiffness. A. Cartoon representation of the N2B and N2BA cardiac titin isoforms. Spring elements are depicted as follows: red balls, Ig region; purple circles, N2B region; green zigzags, PEVK region; blue squares, N2A region. B. Recently identified mechanisms that may underlie titin-dependent short and long term alteration of sarcomeric passive tension. [Abbreviations: Ig, immunoglobin-like; PEVK, proline (P), glutamic acid (E), valine (V), and lysine (K)]
Ratio changes in titin isoforms and post-translational modifications of titin can both alter sarcomeric passive tension.  Isoform switching provides a long term modulation of sarcomere passive tension because it involves the degradation of one titin isoform and the expression of another (Figure 3B, left).  There are two main titin isoforms in the heart: the short N2B isoform (~3000 kDa) and the long N2BA isoform (3200-3700 kDa).  In the N2B isoform, a section of the I band region between Ig I27 and the PEVK region is omitted and in the N2BA isoform, additional middle-Ig and N2A regions are present.  The difference in length between the N2B and N2BA isoforms elicits a corresponding difference in titin-based passive tension: N2B increases sarcomeric passive tension because its expanded length is shorter than that of N2BA8.

The overall ratio of N2B to N2BA determines the stiffness of the heart.  Rodents require heightened restoring forces because they have rapid heart rates; therefore, they have a high N2B:N2BA ratio.  In contrast, larger animals have a low N2B:N2BA ratio because they have slow heart rates and do not require enhanced restoring forces 9. The N2B:N2BA ratio can change based on the needs of the animal.  For instance, the N2B:N2BA ratio is increased in hibernating grizzly bears to ensure that the increased diastolic filling which occurs during periods of decreased heart rate does not result in left ventricle dilatation 10.  Titin isoform switching also takes place during development–the N2B:N2BA is low during the early embryonic period and becomes progressively larger as development progresses.  This gradual switch to the shorter isoform serves to increase passive tension as the heart becomes larger and diastolic volume increases 11.  Titin isoform switching may be used as a compensatory mechanism in disease states: Warren et al. 12 shows that N2BA isoform expression is decreased in spontaneously hypertensive rats, suggesting that the heart attempts to maintain Frank-Starling equilibrium under high pressure conditions by increasing sarcomeric passive tension. Titin isoforms composition may also be involved in the progression to heart failure; the N2B:N2BA ratio is decreased in transplant hearts from patients with coronary artery disease compared to the N2B:N2BA ratio in transplant hearts from patients with nonischemic heart disease 13.  While much is known about cardiac titin composition under various conditions, the mechanism which triggers titin isoform switching remains to be understood.

In contrast to ratio changes in titin isoforms due to long term adaptations, post-translational modifications to titin transiently affect sacromeric passive tension (Figure 3B, right). Phosphorylation is the primary post-translational modification of titin which alters sarcomeric passive tension.  Protein kinase A (PKA) can phosphorylate the N2B region while protein kinase G (PKG) can phosphorylate the PEVK region.  Phosphorylation of N2B and PEVK affect titin’s overall passive tension in opposite ways.  Single-molecule force spectroscopy experiments show that PKA/PKG phosphorylation of the N2B element decreases titin’s passive tension, while PKC phosphorylation of the PEVK element increases titin’s passive tension (Figure 3B)14.  Anderson et al.14 postulate that the negatively-charged phosphoryl group attracts positively-charged amino acids within the PEVK element, adding an additional force which needs to be overcome to extend it.  In contrast, the phosphoryl group may repel negatively-charged amino acids within the N2B element, decreasing the force needed to extend it.  Like titin isoform composition, titin phosphorylation is also altered in heart disease, as suggested by the improved left-ventricular function of patients with diastolic heart failure after stimulating myocardial PKA activity through beta-adrenoreceptor activation 15.

This regulation of titin in the human may play a role in the development of heart failure.  In patients with end-stage heart failure due to dilated cardiomyopathy (non-ischemic), the N2B (more rigid) to N2BA (less rigid) expression ration was significantly increase compared to controls, without affecting total titin levels 16.  The N2B:N2BA ration was highest in patients with the greatest amount of impaired LV relaxation, determined by Doppler echocardiography and invasive hemodynamics.  Our understanding of how titin isoforms switch may help in the development of treating diastolic cardiac disease.  Furthermore, understanding titin’s role in metabolism, the compartmentalization of metabolic enzymes, and binding of chaperones can help us understand the broader role of titin in cardiac diseases 17.

About Monte S. Willis

Monte WillisMonte S. Willis, MD, PhD, is associate professor at the Department of Pathology and Laboratory Medicine, Director of Campus Health Services Laboratory, and Director of the McLendon Clinical Laboratories at the University of North Carolina in Chapel Hill, where he leads a research team studying the role of the ubiquitin proteasome system in metabolism and the pathophysiology of cardiac disease, teaches in the School of Medicine and Graduate School, and is currently completing his MBA at Kenan-Flagler Business School. His book, Cellular and Molecular Pathobiology of Cardiovascualar Diseasethat he authored with Dr. Jonathan Homeister and Dr. James Stone, published earlier this year.

References

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2.         Granzier HL, Labeit S. The giant protein titin: a major player in myocardial mechanics, signaling, and disease. Circ Res. 2004;94(3):284-295.

3.         Labeit S, Kolmerer B, Linke WA. The giant protein titin. Emerging roles in physiology and pathophysiology. Circ Res. 1997;80(2):290-294.

4.         Gaasch WH, Cole JS, Quinones MA, Alexander JK. Dynamic determinants of letf ventricular diastolic pressure-volume relations in man. Circulation. 1975;51(2):317-323.

5.         Engelmann TW. Pflugers Arch 1878;18:1-25.

6.         Page S. Fine Structure of Tortoise Skeletal Muscle. J Physiol. 1968;197:709-715.

7.         Locker R, Leet N. Histology of Highly Stretched Beef Muscle: IV. Evidence for Movement of Gap Filaments through the Z-line, using the N2-line and the M-line as markers. J Ultrastruct. Res. 1976;56:31-38.

8.         Linke WA. Sense and stretchability: the role of titin and titin-associated proteins in myocardial stress-sensing and mechanical dysfunction. Cardiovasc Res. 2008;77(4):637-648.

9.         LeWinter MM, Wu Y, Labeit S, Granzier H. Cardiac titin: structure, functions and role in disease. Clin Chim Acta. 2007;375(1-2):1-9.

10.       Nelson OL, Robbins CT, Wu Y, Granzier H. Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears. Am J Physiol Heart Circ Physiol. 2008;295(1):H366-371.

11.       Greaser ML, Krzesinski PR, Warren CM, Kirkpatrick B, Campbell KS, Moss RL. Developmental changes in rat cardiac titin/connectin: transitions in normal animals and in mutants with a delayed pattern of isoform transition. J Muscle Res Cell Motil. 2005;26(6-8):325-332.

12.       Warren CM, Jordan MC, Roos KP, Krzesinski PR, Greaser ML. Titin isoform expression in normal and hypertensive myocardium. Cardiovasc Res. 2003;59(1):86-94.

13.       Neagoe C, Kulke M, del Monte F, Gwathmey JK, de Tombe PP, Hajjar RJ, Linke WA. Titin isoform switch in ischemic human heart disease. Circulation. 2002;106(11):1333-1341.

14.       Anderson BR, Bogomolovas J, Labeit S, Granzier H. The effects of PKCalpha phosphorylation on the extensibility of titin’s PEVK element. J Struct Biol. 2010.

15.       Borbely A, van der Velden J, Papp Z, Bronzwaer JG, Edes I, Stienen GJ, Paulus WJ. Cardiomyocyte stiffness in diastolic heart failure. Circulation. 2005;111(6):774-781.

16.       Nagueh SF, Shah G, Wu Y, Torre-Amione G, King NM, Lahmers S, Witt CC, Becker K, Labeit S, Granzier HL. Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy. Circulation. 2004;110(2):155-162.

17.       Granzier H, Wu Y, Siegfried L, LeWinter M. Titin: physiological function and role in cardiomyopathy and failure. Heart Fail Rev. 2005;10(3):211-223.

18.       Willis MS, Schisler JC, Portbury AL, Patterson C. Build it up-Tear it down: protein quality control in the cardiac sarcomere. Cardiovasc Res. 2009;81(3):439-448.

 (C) Monte S. Willis 2014

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