title: Dynamic changes in free Ca-calmodulin levels in adult cardiac myocytes.
abstract: Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca-CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca-CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca-CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca-CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca-CaM sensing probe for 24-48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca-CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca-CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon beta-adrenergic stimulation with isoproterenol, there was an increase in the amplitude of the signals during each beat (parallel to the shortening, which is an indirect measure of [Ca]i) and also a rise in the diastolic [Ca-CaM]. Total [CaM] measured by both competitive ELISA and semi-quantitative Western blots was 5-6 microM in isolated adult ventricular myocytes. Our results indicate that there are dynamic changes in free Ca-CaM levels (a phasic component tracking [Ca]i) as well as system memory that integrates the [Ca]i signals (a tonic component).
authors.affiliation: Department of Physiology, Stritch School of Medicine Loyola University, Chicago, 2160 South First Avenue, Maywood, IL 60153, USA.
authors.forename: Lars S
authors.initials: LS
authors.lastname: Maier
authors.forename: Mark T
authors.initials: MT
authors.lastname: Ziolo
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.forename: Anthony
authors.initials: A
authors.lastname: Persechini
authors.forename: Ruben
authors.initials: R
authors.lastname: Mestril
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000328:Adult; D000818:Animals; D015374:Biosensing Techniques; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008954:Models, Biological; D032342:Muscle Cells / Q000166:cytology*; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D002147:Calmodulin; D002118:Calcium