FKBP12 binds to the cardiac ryanodine receptor with negative cooperativity: implications for heart muscle physiology in health and disease.

Cardiac ryanodine receptors (RyR2) release the Ca2+ from intracellular stores that is essential for cardiac myocyte contraction. The ion channel opening is tightly regulated by intracellular factors, including the FK506 binding proteins, FKBP12 and FKBP12.6. The impact of these proteins on RyR2 activity and cardiac contraction is debated, with often apparently contradictory experimental results, particularly for FKBP12. The isoform that regulates RyR2 has generally been considered to be FKBP12.6, despite the fact that FKBP12 is the major isoform associated with RyR2 in some species and is bound in similar proportions to FKBP12.6 in others, including sheep and humans. Here, we show time- and concentration-dependent effects of adding FKBP12 to RyR2 channels that were partly depleted of FKBP12/12.6 during isolation. The added FKBP12 displaced most remaining endogenous FKBP12/12.6. The results suggest that FKBP12 activates RyR2 with high affinity and inhibits RyR2 with lower affinity, consistent with a model of negative cooperativity in FKBP12 binding to each of the four subunits in the RyR tetramer. The easy dissociation of some FKBP12/12.6 could dynamically alter RyR2 activity in response to changes in in vivo regulatory factors, indicating a significant role for FKBP12/12.6 in Ca2+ signalling and cardiac function in healthy and diseased hearts. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Activation of RyR2 by class I kinase inhibitors.

BACKGROUND AND PURPOSE: Kinase inhibitors are a common treatment for cancer. Class I kinase inhibitors that target the ATP-binding pocket are particularly prevalent. Many of these compounds are cardiotoxic and can cause arrhythmias. Spontaneous release of Ca2+ via cardiac ryanodine receptors (RyR2), through a process termed store overload-induced Ca2+ release (SOICR), is a common mechanism underlying arrhythmia. We explored whether class I kinase inhibitors could modify the activity of RyR2 and trigger SOICR to determine if this contributes to the cardiotoxic nature of these compounds. EXPERIMENTAL APPROACH: The impact of class I and II kinase inhibitors on SOICR was studied in HEK293 cells and ventricular myocytes using single-cell Ca2+ imaging. A specific effect on RyR2 was confirmed using single channel recordings. Ventricular myocytes were also used to determine if drug-induced changes in SOICR could be reversed using anti-SOICR agents. KEY RESULTS: Class I kinase inhibitors increased the propensity of SOICR. Single channel recording showed that this was due to a specific effect on RyR2. Class II kinase inhibitors decreased the activity of RyR2 at the single channel level but had little effect on SOICR. The promotion of SOICR mediated by class I kinase inhibitors could be reversed using the anti-SOICR agent VK-II-86. CONCLUSIONS AND IMPLICATIONS: Part of the cardiotoxicity of class I kinase inhibitors can be assigned to their effect on RyR2 and increase in SOICR. Compounds with anti-SOICR activity may represent an improved treatment option for patients.