Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity.
OBJECTIVE
In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity.


RESULTS
We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac β-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rβ-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or β-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rβ-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged.


CONCLUSIONS
Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Daria
D
Petzhold
Janine
J
Lossie
Sandro
S
Keller
Sascha
S
Werner
Hannelore
H
Haase
Ingo
I
Morano
D019943:Amino Acid Substitution; D000818:Animals; D000831:Animals, Newborn; D024743:Atrial Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020013:Calcium Signaling / Q000502:physiology; D024722:Cardiac Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014162:Transfection; D024723:Ventricular Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018994:Myosin Light Chains; D020033:Protein Isoforms; D011994:Recombinant Proteins; D024743:Atrial Myosins; D024722:Cardiac Myosins; D024723:Ventricular Myosins