In healthy hearts, myofilaments become more sensitive to Ca2+ as the myocardium is stretched. This effect is known as length-dependent activation and is an important cellular-level component of the Frank–Starling mechanism. Few studies have measured length-dependent activation in the myocardium from failing human hearts. We investigated whether ischemic and non-ischemic heart failure results in different length-dependent activation responses at physiological temperature (37°C). Myocardial strips from the left ventricular free wall were chemically permeabilized and Ca2+-activated at sarcomere lengths (SLs) of 1.9 and 2.3 µm. Data were acquired from 12 hearts that were explanted from patients receiving cardiac transplants; 6 had ischemic heart failure and 6 had non-ischemic heart failure. Another 6 hearts were obtained from organ donors. Maximal Ca2+-activated force increased at longer SL for all groups. Ca2+ sensitivity increased with SL in samples from donors (P < 0.001) and patients with ischemic heart failure (P = 0.003) but did not change with SL in samples from patients with non-ischemic heart failure. Compared with donors, troponin I phosphorylation decreased in ischemic samples and even more so in non-ischemic samples; cardiac myosin binding protein-C (cMyBP-C) phosphorylation also decreased with heart failure. These findings support the idea that troponin I and cMyBP-C phosphorylation promote length-dependent activation and show that length-dependent activation of contraction is blunted, yet extant, in the myocardium from patients with ischemic heart failure and further reduced in the myocardium from patients with non-ischemic heart failure. Patients who have a non-ischemic disease may exhibit a diminished contractile response to increased ventricular filling.
Sarcomere length–dependent effects on Ca 2+ -troponin regulation in myocardium expressing compliant titin
Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca 2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca 2+ -induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin–actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca 2+ -induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca 2+ -troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length–dependent enhancement of troponin regulation with both Ca 2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length–dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length–dependent Ca 2+ -troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca 2+ -troponin regulation of the myocardium.
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- Award ID(s):
- 1656450
- NSF-PAR ID:
- 10091389
- Date Published:
- Journal Name:
- The Journal of General Physiology
- Volume:
- 151
- Issue:
- 1
- ISSN:
- 0022-1295
- Page Range / eLocation ID:
- 30 to 41
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Force production by actin–myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca 2+ -activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca 2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.more » « less
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Background and Purpose Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca2+as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank–Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere‐length dependence of Ca2+‐sensitive isometric contraction to determine how mavacamten might modulate the Frank–Starling mechanism.
Experimental Approach Multicellular preparations from the left ventricular‐free wall of hearts from organ donors were chemically permeabilized and Ca2+activated in the presence or absence of 0.5‐μM mavacamten at 1.9 or 2.3‐μm sarcomere length (37°C). Isometric force and frequency‐dependent viscoelastic myocardial stiffness measurements were made.
Key Results At both sarcomere lengths, mavacamten reduced maximal force and Ca2+sensitivity of contraction. In the presence and absence of mavacamten, Ca2+sensitivity of force increased as sarcomere length increased. This suggests that the length‐dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca2+sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross‐bridge recruitment and speeding cross‐bridge detachment under maximally activated conditions (pCa 4.5).
Conclusion and Implications Mavacamten did not eliminate sarcomere length‐dependent increases in the Ca2+sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies.
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null (Ed.)Morbidity and mortality associated with heart disease is a growing threat to the global population, and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca 2+ sensitivity of contraction for both genotypes, but this reduction in pCa 50 was nearly twice as large for WT-RLC versus N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed up myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca 2+ sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM. NEW & NOTEWORTHY Mavacamten is a pharmaceutical that binds to myosin, and it is under investigation as a therapy for some forms of heart disease. We show that mavacamten reduces isometric tension and Ca 2+ sensitivity of contraction in skinned myocardial strips from a mouse model of hypertrophic cardiomyopathy that expresses the N47K mutation in cardiac myosin regulatory light chain. Mavacamten reduces contractility by decreasing strong cross-bridge binding, partially due to faster cross-bridge nucleotide handling rates that speed up myosin detachment.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1085/jgp.201812218
