title: Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects.
abstract: Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H703-H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 microM) abolished the stimulation-dependent acceleration of twitch [Ca]i decline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]i decline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (tau) of [Ca]i decline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 +/- 6 vs. 188 +/- 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]i during rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 +/- 74 nM for WT, 1118 +/- 133 nM for PLB-KO, P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The tau for [Ca]i decline of the cafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 +/- 0.2 s in WT vs. 3.2 +/- 0.2 in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]i decline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.
authors.affiliation: Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: G
authors.initials: G
authors.lastname: Chu
authors.forename: E G
authors.initials: EG
authors.lastname: Kranias
authors.forename: D M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D015151:Immunoblotting; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics* / Q000378:metabolism*; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019831:Sodium-Calcium Exchanger; D000099211:Phospholamban