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Updated: Aug 19, 2026
Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling
Published on: August 1, 2016
Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects
L Li1, G Chu, E G Kranias
1Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
Insights
Heart rate increases accelerate relaxation via CaMKII, independent of phospholamban (PLB). Ablating PLB in myocytes still shows CaMKII-dependent acceleration, with faster Ca handling and altered ion exchange.
Area of Science:
- Cardiology
- Molecular Biology
- Physiology
Background:
- Increased heart rate accelerates myocyte relaxation, a process linked to sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase (CaMKII).
- Phosphorylation of phospholamban (PLB) by CaMKII is a potential mechanism for stimulating SR Ca transport.
Purpose of the Study:
- To investigate the role of PLB and its phosphorylation in CaMKII-dependent acceleration of relaxation and SR Ca handling.
- To determine if PLB is essential for the CaMKII-mediated acceleration of twitch Ca decline.
Main Methods:
- Ventricular myocytes from wild-type (WT) and PLB gene-ablated (PLB-KO) mice were used.
- Experiments involved steady-state (SS) stimulation, application of CaMKII inhibitor KN-93, and caffeine-induced contractures (CafC).
- Quantitative analysis of Ca transport, SR Ca load, and ion exchanger activity was performed.
Main Results:
- Both WT and PLB-KO myocytes exhibited faster relaxation and [Ca]i decline during SS stimulation compared to resting conditions.
- CaMKII inhibition abolished stimulation-dependent acceleration of [Ca]i decline in PLB-KO myocytes, indicating PLB independence.
- PLB-KO myocytes showed faster [Ca]i decline (112 ms vs. 188 ms), increased SR Ca load (1118 nM vs. 565 nM), and reduced Na/Ca exchange activity.
Conclusions:
- CaMKII-dependent acceleration of twitch [Ca]i decline and relaxation occurs independently of PLB and its phosphorylation.
- PLB-KO myocytes exhibit enhanced SR Ca-ATPase activity, increased SR Ca load, and diminished Na/Ca exchange function.
- These findings elucidate novel mechanisms regulating cardiac myocyte relaxation and Ca handling.
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.
