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Abstract
The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1vDelta5-14, referred to as 1vDelta5-14), a region that interacts with actin. 1vDelta5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vDelta5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1vDelta5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vDelta5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.
Highlights
Muscle myosin is a hexamer that contains two heavy chains (MHCs),3 with each heavy chain having two types of light chains: the essential light chain (ELC) and the regulatory light chain
Our results strongly suggest that an interaction between residues 5–14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance
Adding ELC residues 5–14 to rat ventricle myofibrils resulted in an ϳ2-fold increase in MgATPase at submaximum calcium concentrations [21]
Summary
Materials—All reagents were purchased from Sigma except where noted. Mouse Model—We used the same 1v⌬5–14 transgenic and control (NTG) lines as in previous studies [5, 18]. A mouse in which 70% of the ␣-MHC was replaced with -MHC through genetic manipulation [27] was used as a marker Both transcript and MHC analyses were performed on mice at 15 weeks, and an additional line (ELC1v-wt), defined in previous studies [5, 18], was used as a second control. For the x-ray diffraction experiments, 0 –10% (w/v) dextran T-500 (an osmotic compression agent) was added to relaxing solution to establish the concentration required to compress the skinned strip lattice to its intact spacing. X-ray Diffraction—Papillary muscle strips from four 1v⌬5–14 and four NTG mice at 13 weeks of age were prepared as described above, secured between adjustable hooks in a perfusion chamber that contained relaxing solution, and stretched to 2.2-m sarcomere length. Significant difference a Significantly different from young NTG mice (p Ͻ 0.05). b Significantly different from young 1v⌬5–14 mice (p Ͻ 0.05)
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