Analysis of myosin heavy chain functionality in the heart.
Comparison of mammalian cardiac alpha- and beta-myosin heavy chain isoforms reveals 93% identity. To date, genetic methodologies have effected only minor switches in the mammalian cardiac myosin isoforms. Using cardiac-specific transgenesis, we have now obtained major myosin isoform shifts and/or replacements. Clusters of non-identical amino acids are found in functionally important regions, i.e. the surface loops 1 and 2, suggesting that these structures may regulate isoform-specific characteristics. Loop 1 alters filament sliding velocity, whereas Loop 2 modulates actin-activated ATPase rate in Dictyostelium myosin, but this remains untested in mammalian cardiac myosins. Alpha --> beta isoform switches were engineered into mouse hearts via transgenesis. To assess the structural basis of isoform diversity, chimeric myosins in which the sequences of either Loop 1+Loop 2 or Loop 2 of alpha-myosin were exchanged for those of beta-myosin were expressed in vivo. 2-fold differences in filament sliding velocity and ATPase activity were found between the two isoforms. Filament sliding velocity of the Loop 1+Loop 2 chimera and the ATPase activities of both loop chimeras were not significantly different compared with alpha-myosin. In mouse cardiac isoforms, myosin functionality does not depend on Loop 1 or Loop 2 sequences and must lie partially in other non-homologous residues.
Krenz
Maike
M
Cincinnati Children's Hospital Medical Center, The Children's Hospital Research Foundation, MLC 7020, Cincinnati, Ohio 45229-3039, USA.
Sanbe
Atsushi
A
Bouyer-Dalloz
Florence
F
Gulick
James
J
Klevitsky
Raisa
R
Hewett
Timothy E
TE
Osinska
Hanna E
HE
Lorenz
John N
JN
Brosseau
Christine
C
Federico
Andrea
A
Alpert
Norman R
NR
Warshaw
David M
DM
Perryman
M Benjamin
MB
Helmke
Steve M
SM
Robbins
Jeffrey
J
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D006321:Heart / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000502:physiology*; D020033:Protein Isoforms / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D018995:Myosin Heavy Chains; D000252:Calcium-Transporting ATPases; D002118:Calcium