Examining the in Vivo Role of the Amino Terminus of the Essential Myosin Light Chain

Abstract

The functional significance of the actin binding region at the amino terminus of the cardiac essential myosin light chain (ELC) remains obscure. Previous experiments carried out in vitro indicated that modulation of residues 5-14 could induce an inotropic effect, increasing maximal ATPase activity at submaximal Ca 2؉ concentrations (Rarick, H. M., Opgenorth, T. J., von Geldern, T. W., Wu-Wong, J. R., and Solaro, R. J. (1996) J. Biol. Chem. 271, 27039 -27043). Using transgenesis, we effected a cardiac-specific replacement of ELC with a protein containing a 10-amino acid deletion at positions 5-14. Both the ventricular (ELC1v⌬5-14) and atrial (ELC1a⌬5-14) isoforms lacking this peptide were stably incorporated into the sarcomere at high efficiencies. Surprisingly when the kinetics of skinned fibers isolated from the ELC1v⌬5-14 or ELC1a⌬5-14 mice were examined, no alterations in either unloaded shortening or maximum shortening velocities were apparent. Myofibrillar Mg 2؉ -ATPase activity was also unchanged in these preparations. No significant changes in the fiber kinetics in the cognate compartments were observed when either deletion-containing protein replaced endogenous ELC1v or ELC1a. The data indicate that the previously postulated importance of this region in mediating critical protein interactions between the cardiac ELCs and the carboxyl-terminal residues of actin in vivo should be reassessed.Myosin is the major motor protein in the contractile apparatus of all muscle types and cyclically interacts with the thin filament to generate force (1). The different "conventional" myosins, or myosin IIs, are thought to underlie the contractile properties of the different muscle types. Myosin II is a hexameric protein made up of two heavy chains (MyHCs) 1 (M r ϳ229,000) and four light chains (MLCs) (M r ϳ18,000 -27,000). The MyHCs consist of two separate domains: a globular head region and a rod region that assumes an ␣-helical coiled coil. The ATPase activity underlying muscle contraction is localized at the amino-terminal end that corresponds to the globular head and neck of the molecule. Also associated with this heavy chain domain are the MLCs (2).Myosin-actin cross-bridge interactions play an important role in determining cardiac systolic and diastolic function. Cardiac MyHC is encoded by two genes, ␣-and ␤-MyHC, and each heavy chain is associated with two MLC types, the regulatory MLC and essential MLCs (ELCs). X-ray crystallographic analyses have demonstrated that both the ELC and regulatory MLC are associated with the neck region of the MyHC (2, 3). Striated muscle can contain two ELC isoforms, ELC1f and ELC3f. The sequences of the two are quite similar except that the ELC1f isoform contains an amino-terminal extension of 40 amino acids. The cross-bridge kinetics of ELC3f fibers are significantly faster than ELC1f fibers (4), and the functional significance of the amino-terminal extension has been the subject of multiple investigations (5-9). We have shown previously that transgenic (TG) substitution of the at...

Analysis of Myosin Heavy Chain Functionality in the Heart

“…1C). These levels were relatively low compared with those that are sometimes observed when other contractile proteins were overexpressed using transgenesis (27,31,32). However, we have noted in other related studies that high copy numbers and high levels of MHC expression (Ͼ12-fold overexpression) can be lethal, presumably because of the relatively insoluble nature of the intact protein.…”

“…The two cardiac myosins were separated on denaturing gels in the presence of glycerol, essentially as described by Reiser and co-workers (25,26). Fiber isolation and their analyses have been described in detail (27). The in vitro motility assays (28) and determination of cardiac hemodynamics in the isolated working heart model and the closed chest intact mouse model were carried out as described (29,30).…”

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

“…The N-terminal extension of the ventricular ELC in the mouse is similar to that in the human (14,18), suggesting that their physiological roles are similar. In skinned atrial and ventricular strips from transgenic mice in which ELC residues 5-14 (ELC1v⌬5-14, referred to as 1v⌬5-14) were deleted, some of which interact with actin (10,(15)(16)(17), there was a surprising lack of morphological and functional differences between transgenic (1v⌬5-14) and non-transgenic (NTG) controls.…”

“…In skinned atrial and ventricular strips from transgenic mice in which ELC residues 5-14 (ELC1v⌬5-14, referred to as 1v⌬5-14) were deleted, some of which interact with actin (10,(15)(16)(17), there was a surprising lack of morphological and functional differences between transgenic (1v⌬5-14) and non-transgenic (NTG) controls. The calcium sensitivity of isometric tension of 1v⌬5-14 was similar to that of controls (absolute tensions were not reported), and there were no observed differences in MgATPase activity and shortening velocity at maximum calcium activation (pCa 5) (18). The lack of morphological and functional differences was unexpected because other experiments involving the addition of 10-residue peptides consisting of the deleted region or portions thereof (ELC residues 1-10 (19) and 5-14 (20, 21)) showed effects on cross-bridge kinetics.…”

“…A previous study examined the role of the actin-binding region at the N terminus of the ventricular ELC in a transgenic mouse model (18). The N-terminal extension of the ventricular ELC in the mouse is similar to that in the human (14,18), suggesting that their physiological roles are similar.…”