Zinc controls RyR2 activity during excitation-contraction coupling

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Cardiac excitation-contraction (EC) coupling is a process which governs contractility of the heart through the controlled release of Ca²⁺ from the sarcoplasmic reticulum (SR). The type-2 ryanodine receptor (RyR2) is the route through which Ca²⁺ is released from the SR providing the necessary driving force for cellular contraction. In heart failure, RyR2-channels become abnormally active, or ‘leaky’, and are unable to remain closed during diastole resulting in unwanted irregular contractile and electrical activity.¹ Defective Zn²⁺ handling has been shown to contribute to the cellular pathology of certain cardiomyopathies which give rise to impaired contractility including heart failure.² This is likely a consequence of altered EC coupling as a result of modified RyR2 function. How zinc impacts upon the contractile force and the release of calcium from intracellular stores in heart is not fully understood.

In the recent study by Woodier and co-workers³ it was shown that cytosolic Zn²⁺ can act as a high affinity activator of RyR2. In the aforementioned study, single RyR2 channels were incorporated into phospholipid bilayers under voltage-clamp conditions and the direct action of Zn²⁺ at the cytosolic face of the channel studied. This approach enabled the study of RyR2 function under tight control of the chemical environment. Concentrations of free Zn²⁺ ≤ 1 nM potentiated RyR2 activity but the presence of activating levels of cytosolic Ca²⁺ was a requirement for channel activation. At concentrations of free Zn²⁺ > 1 nM, the main activating ligand became Zn²⁺ and the requirement of Ca²⁺ for channel activation was removed. Under these conditions channel gating was altered and RyR2 gated in exceptionally long-lived open states. The ability of Zn²⁺ at a concentration of 1 nM to directly activate RyR2 reveals that RyR2 has a much higher affinity for Zn²⁺ than Ca²⁺ (by ∼3-orders of magnitude). These data suggest that RyR2-mediated Ca²⁺-homeostasis is intimately related to intracellular Zn²⁺ levels. Woodier et al. also showed that Zn²⁺ modulated both the frequency and amplitude of Ca²⁺-waves in cardiomyocytes in a concentration-dependent manner.³ Reduction of the concentration of intracellular Ca²⁺ to sub-activating concentrations did not abolish Ca²⁺-waves in the presence of 1 nM Zn²⁺. This suggests that RyR2 gating is altered under these conditions whereby RyR2 gates in a Ca²⁺-independent manner with Zn²⁺ the sole activating ligand. These data indicate that channel dysregulation, through aberrant Zn²⁺ homeostasis, may play a fundamental role in the generation of heart failure and other arrhythmic diseases.

Cardiomyocytes contain a small but measurable pool of free Zn²⁺ in the cytosol reported to be ∼100 pM.⁴ Since small changes in the Zn²⁺ level will have a marked effect on RyR2 activity, this becomes highly relevant when we consider that concentrations of Zn²⁺ have recently been reported to be transiently elevated to ∼50 nM during Zn²⁺-signaling events.⁵ Live-cell detection of intracellular Zn²⁺ and Ca²⁺ using selective fluorophores reveal that intracellular Zn²⁺ concentrations are altered during cardiac EC coupling and that spatio-temporal fluctuations in free Zn²⁺ levels are comparable to those of Ca²⁺.⁶ Extracellular Zn²⁺ can also enter cardiomyocytes through the L-type Ca²⁺ channel in a similar manner to Ca²⁺.⁷ These Zn²⁺ fluctuations may serve to modulate RyR2 activity highlighting a potential role for Zn²⁺ in fine-tuning graded Ca²⁺-release events to control the force and duration of cardiac contractions. Under certain pathophysiological conditions including heart failure, diabetes and ischemia, concentrations of intracellular Zn²⁺ are chronically elevated and are reported to be in the high nanomolar range (c.a. 30 nM).^2,8 Under such conditions RyR2 will decouple from the regulatory effects of cytosolic Ca²⁺ and be under control of Zn²⁺. This may contribute toward abnormally high RyR2 channel activity which is associated with heart failure and fatal arrhythmias.

The role of Zn²⁺ as a high affinity activator of RyR2 able to modulate channel function in the absence of Ca²⁺ represents a paradigm shift in our understanding of how RyR2 is activated during EC coupling. These new data provide a plausible mechanistic explanation linking Zn²⁺ dyshomeostasis to certain cardiomyopathies characterized by defective contractility and dysregulated Ca²⁺-responses (Fig. 1). However, in order to substantiate a model for integrated Zn²⁺-signaling in the heart, a more detailed understanding of the molecular mechanism by which Zn²⁺ modulates RyR2 function is required and the further impact this has on cardiac function needs to be examined. Determining the origin of Zn²⁺ fluxes during the cardiac contraction-relaxation cycle in both health and disease states will also be crucial in advancing our understanding of how cellular Zn²⁺ shapes EC coupling. Understanding Zn²⁺ signaling in the heart and unveiling new mechanisms involved in regulating intracellular Ca²⁺ dynamics in cardiac tissue may highlight potential new drug targets in the fight against heart failure and fatal arrhythmias.

Figure 1. A model to show how zinc may regulate RyR2-mediated sarcoplasmic reticulum calcium release. Physiological conditions: During the resting phase of the cardiac cycle RyR2 is closed as the cytosolic [Ca²⁺] is sub-activating (100 nM). The normal trigger for RyR2 activation during systole is a transient rise in [Ca²⁺] (≥ 1 µM). Zn²⁺-dyshomeostasis: If the intracellular [Zn²⁺] rises >1 nM, the dependency on Ca²⁺ for RyR2 openings is removed and Zn²⁺ becomes the activating ligand. This leads to abnormally active channels.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

SJP is supported by a Royal Society of Edinburgh Biomedical Research Fellowship. This work was supported by the British Heart Foundation (grant no. FS/14/69/31001 to SJP).