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.

Spontaneous Ca2+ transients in rat pulmonary vein cardiomyocytes are increased in frequency and become more synchronous following electrical stimulation.

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca2+) signalling are purported to play a role. Therefore, the intracellular Ca2+ concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca2+ in neighbouring cardiomyocytes; asynchronous spontaneous Ca2+ transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca2+ transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P < 0.001). Increasing the extracellular Ca2+ concentration enhanced this effect, with the frequency of spontaneous Ca2+ transients increasing from 0.45 ± 0.05 Hz to between 0.75 ± 0.06 and 0.94 ± 0.09 Hz after electrical stimulation at 3 to 9 Hz (P < 0.001), and this was accompanied by a significant increase in the velocity of Ca2+ transients that manifested as waves. Moreover, in the presence of high extracellular Ca2+, the spontaneous Ca2+ transients occurred more synchronously in the initial few seconds following electrical stimulation. The ryanodine receptors, which are the source of spontaneous Ca2+ transients in pulmonary vein cardiomyocytes, were found to be arranged in a striated pattern in the cell interior, as well as along the periphery of cell. Furthermore, labelling the sarcolemma with di-4-ANEPPS showed that over 90% of pulmonary vein cardiomyocytes possessed T-tubules. These findings demonstrate that the frequency of spontaneous Ca2+ transients in the rat pulmonary vein are increased following higher rates of electrical stimulation and increasing the extracellular Ca2+ concentration.

Differential sensitivity of Ca²+ wave and Ca²+ spark events to ruthenium red in isolated permeabilised rabbit cardiomyocytes.

Spontaneous Ca²(+) waves in cardiac muscle cells are thought to arise from the sequential firing of local Ca²(+) sparks via a fire-diffuse-fire mechanism. This study compares the ability of the ryanodine receptor (RyR) blocker ruthenium red (RuR) to inhibit these two types of Ca²(+) release in permeabilised rabbit ventricular cardiomyocytes. Perfusing with 600 nm Ca²(+) (50 µm EGTA) caused regular spontaneous Ca²(+) waves that were imaged with the fluorescence from Fluo-5F using a laser-scanning confocal microscope. Addition of 4 µm RuR caused complete inhibition of Ca²(+) waves in 50% of cardiomyocytes by 2 min and in 100% by 4 min. Separate experiments used 350 µm EGTA (600 nm Ca²(+)) to limit Ca²(+) diffusion but allow the underlying Ca(2+) sparks to be imaged. The time course of RuR-induced inhibition did not match that of waves. After 2 min of RuR, none of the characteristics of the Ca²(+) sparks were altered, and after 4 min Ca²(+) spark frequency was reduced ∼40%; no sparks could be detected after 10 min. Measurements of Ca(2+) within the SR lumen using Fluo-5N showed an increase in intra-SR Ca²(+) during the initial 2-4 min of perfusion with RuR in both wave and spark conditions. Computational modelling suggests that the sensitivity of Ca²(+) waves to RuR block depends on the number of RyRs per cluster. Therefore inhibition of Ca²(+) waves without affecting Ca²(+) sparks may be explained by block of small, non-spark producing clusters of RyRs that are important to the process of Ca²(+) wave propagation.

Assessment of sarcoplasmic reticulum Ca2+ depletion during spontaneous Ca2+ waves in isolated permeabilized rabbit ventricular cardiomyocytes.

In this study, Ca2+ release due to spontaneous Ca2+ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca2+ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca2+ signals during spontaneous Ca2+ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca2+] increase during a Ca2+ wave was 98.1% +/- 0.3% of that observed during caffeine application. Conversion to total Ca2+ release suggested that Ca2+ release from a Ca2+ wave was not significantly different from that released during caffeine application (104% +/- 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca2+ wave was 82.5% +/- 2.6% of that observed during caffeine application. Assuming a maximum free [Ca2+] of 1.1 mM, this translates to a 96.2% +/- 0.8% change in intra-SR free [Ca2+] and a 91.7% +/- 1.6% depletion of the total Ca2+. This equates to a minimum intra-SR free Ca2+ of 46 +/- 7 microM during a Ca2+ wave. Reduction of RyR2 Ca2+ sensitivity by tetracaine (50 microM) reduced the spontaneous Ca2+ release frequency while increasing the Ca2+ wave amplitude. This did not significantly change the total depletion of the SR (94.5% +/- 1.1%). The calculated minimum [Ca2+] during these Ca2+ waves (87 +/- 19 microM) was significantly higher than control (p < 0.05). A computational model incorporating this level of Ca2+ depletion during a Ca2+ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca2+ release. In conclusion, spontaneous Ca2+ release results in substantial but not complete local Ca2+ depletion of the SR. Furthermore, measurements suggest that Ca2+ release terminates when luminal [Ca2+] reaches approximately 50 microM.

Measurement and modeling of Ca2+ waves in isolated rabbit ventricular cardiomyocytes.

The time course and magnitude of the Ca(2+) fluxes underlying spontaneous Ca(2+) waves in single permeabilized ventricular cardiomyocytes were derived from confocal Fluo-5F fluorescence signals. Peak flux rates via the sarcoplasmic reticulum (SR) release channel (RyR2) and the SR Ca(2+) ATPase (SERCA) were not constant across a range of cellular [Ca(2+)] values. The Ca(2+) affinity (K(mf)) and maximum turnover rate (V(max)) of SERCA and the peak permeability of the RyR2-mediated Ca(2+) release pathway increased at higher cellular [Ca(2+)] loads. This information was used to create a computational model of the Ca(2+) wave, which predicted the time course and frequency dependence of Ca(2+) waves over a range of cellular Ca(2+) loads. Incubation of cardiomyocytes with the Ca(2+) calmodulin (CaM) kinase inhibitor autocamtide-2-related inhibitory peptide (300 nM, 30 mins) significantly reduced the frequency of the Ca(2+) waves at high Ca(2+) loads. Analysis of the Ca(2+) fluxes suggests that inhibition of CaM kinase prevented the increases in SERCA V(max) and peak RyR2 release flux observed at high cellular [Ca(2+)]. These data support the view that modification of activity of SERCA and RyR2 via a CaM kinase sensitive process occurs at higher cellular Ca(2+) loads to increase the maximum frequency of spontaneous Ca(2+) waves.