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Published on: March 22, 2019
Differential integration of Ca2+-calmodulin signal in intact ventricular myocytes at low and high affinity
Qiujing Song1, Jeffrey J Saucerman, Julie Bossuyt
1Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153, USA.
Abstract:
Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca2+ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca2+-bound CaM (Ca2+-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca2+ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (Kd = approximately 45 nm) and BsCaM-2 (Kd = approximately 2 nm), to monitor the real time Ca2+-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca2+-CaM alterations more closely following the Ca2+ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca2+-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca2+-CaM biosensors in beating cardiac myocytes revealed that the different Ca2+-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca2+-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca2+ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca2+ signaling pathways.
Insights
Cardiac calmodulin (CaM) dynamics are crucial for heart function. Different CaM target affinities shape cellular responses to calcium signals, influencing heart cell signaling pathways.
Area of Science:
- Cardiovascular Biology
- Molecular Cell Biology
- Biophysics
Background:
- Intracellular calcium in cardiac myocytes fluctuates beat-to-beat.
- Calmodulin (CaM) is a key transducer of calcium signals, regulating vital cellular processes through its targets.
- The dynamic behavior of CaM targets in response to calcium signaling in the heart remains poorly understood.
Purpose of the Study:
- To investigate how varying affinities of CaM targets for Ca2+-bound CaM (Ca2+-CaM) influence their responses to repetitive calcium signals in cardiac myocytes.
- To elucidate the mechanisms by which CaM targets exhibit dynamic and tonic interactions based on their affinity for Ca2+-CaM.
Main Methods:
- Utilized two Förster resonance energy transfer (FRET)-based biosensors, BsCaM-45 (low affinity, Kd ≈ 45 nm) and BsCaM-2 (high affinity, Kd ≈ 2 nm).
- Monitored real-time Ca2+-CaM dynamics in paced adult ventricular myocytes under varying pacing frequencies.
- Employed biochemically detailed computational models to simulate Ca2+-CaM biosensor behavior in beating cardiac myocytes.
Main Results:
- BsCaM-45 more closely tracked beat-to-beat Ca2+-CaM alterations compared to BsCaM-2.
- Increasing pacing frequency led to a significant elevation in diastolic Ca2+-CaM binding for the high-affinity BsCaM-2, but not for BsCaM-45.
- Computational models confirmed that differing Ca2+-CaM binding affinities adequately predict the distinct kinetics and diastolic integration observed for the biosensors.
Conclusions:
- CaM targets with distinct Ca2+-CaM affinities (e.g., low vs. high) exhibit differential responses to the same calcium signal, manifesting as phasic or integrating behaviors.
- These affinity-dependent responses are crucial for appropriately tuning CaM targets to their specific intracellular calcium signaling pathways in cardiac myocytes.
- The study provides insights into the dynamic regulation of cardiac cellular processes by calmodulin signaling.
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