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Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling
Published on: August 1, 2016
Free and bound intracellular calmodulin measurements in cardiac myocytes
1Department of Physiology, Loyola University Chicago, 2160 S First Ave., Maywood, IL 60153, United States.
Abstract:
Calmodulin (CaM) is a ubiquitous Ca2+ binding protein and Ca2+-CaM activates many cellular targets and functions. While much of CaM is thought to be protein bound, quantitative data in cardiac myocytes is lacking regarding CaM location, [CaM]free and CaM redistribution during changes in [Ca2+]i. Here, we demonstrated that in adult rabbit cardiac myocytes, CaM is highly concentrated at Z-lines (confirmed by Di-8-ANEPPS staining of transverse tubules) using three different approaches: immunocytochemistry (endogenous CaM), Alexa Fluor 488 conjugate CaM (F-CaM) in both permeabilized cells (exogenous CaM) and in patch clamped intact cells (via pipette dialysis). Using 100 nM [CaM]free we washed F-CaM into permeabilized myocytes and saw a two-phase (fast and slow) CaM binding curve with a plateau after 40 min of F-CaM wash-in. We also measured myocyte [CaM]free using two modified null-point titration methods, finding [CaM]free to be 50-75 nM (which is only 1% of total [CaM]). Higher [Ca2+]i increased CaM binding especially in the nucleus and at Z-lines and significantly slowed F-CaM dissociation rate when F-CaM was washed out of permeabilized myocytes. Additionally, in both permeabilized and intact myocytes, CaM moved into the nucleus when [Ca2+]i was elevated, and this was reversible. We conclude that [CaM]free is very low in myocytes even at resting [Ca2+]i, indicating intense competition of CaM targets for free CaM. Bound CaM is relatively concentrated at Z-lines at rest but translocates significantly to the nucleus upon elevation of [Ca2+]i, which may influence activation of different targets and cellular functions.
Insights
Free calmodulin (CaM) is scarce in cardiac cells, with most bound CaM concentrated at Z-lines. Elevated calcium triggers CaM translocation to the nucleus, influencing cellular functions.
Area of Science:
- Cellular Biology
- Cardiovascular Physiology
- Molecular Mechanisms
Background:
- Calmodulin (CaM) is a crucial calcium-binding protein regulating numerous cellular processes.
- Quantitative data on CaM localization, free concentration ([CaM]free), and dynamics in cardiac myocytes is limited.
- Understanding CaM behavior is vital for comprehending calcium-mediated signaling in the heart.
Purpose of the Study:
- To quantify free CaM concentration ([CaM]free) in cardiac myocytes.
- To determine the localization and redistribution of CaM under varying intracellular calcium levels ([Ca2+]i).
- To investigate the binding kinetics and dynamics of CaM in cardiac myocytes.
Main Methods:
- Immunocytochemistry for endogenous CaM localization.
- Alexa Fluor 488 conjugate CaM (F-CaM) in permeabilized and intact myocytes (via patch-clamp dialysis).
- Modified null-point titration methods to measure myocyte [CaM]free.
- Analysis of F-CaM binding and dissociation rates.
Main Results:
- CaM is highly concentrated at Z-lines in adult rabbit cardiac myocytes.
- Myocyte [CaM]free is low (50-75 nM), representing only ~1% of total CaM.
- Elevated [Ca2+]i enhances CaM binding at the nucleus and Z-lines, slowing dissociation.
- CaM translocates to the nucleus upon increased [Ca2+]i, a reversible process.
Conclusions:
- Cardiac myocytes maintain a very low [CaM]free, suggesting intense competition for CaM targets.
- Bound CaM primarily localizes to Z-lines at rest but redistributes to the nucleus when [Ca2+]i rises.
- CaM redistribution likely modulates the activation of specific targets and downstream cellular functions.
