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Luminal Ca(2+) content regulates intracellular Ca(2+) release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers.

ABSTRACT: Ca(+)-induced Ca(2+) release tightly controls the function of ventricular cardiac myocytes under normal and pathological conditions. Two major factors contributing to the regulation of Ca(2+) release are the cytosolic free Ca(2+) concentration and sarcoplasmic reticulum (SR) Ca(2+) content. We hypothesized that the amount of Ca(2+) released from the SR during each heart beat strongly defines the refractoriness of Ca(2+) release. To test this hypothesis, EGTA AM, a high-affinity, slow-association rate Ca(2+) chelator, was used as a tool to modify luminal SR Ca(2+) content. An analysis of the cytosolic and luminal SR Ca(2+) dynamics recorded from the epicardial layer of intact mouse hearts indicated that the presence of EGTA reduced the diastolic SR free Ca(2+) concentration and fraction of SR Ca(2+) depletion during each beat. In addition, this maneuver shortened the refractory period and accelerated the restitution of Ca(2+) release. As a consequence of the accelerated restitution, the frequency dependence of Ca(2+) alternans was significantly shifted toward higher heart rates, suggesting a role of luminal SR Ca(2+) in the genesis of this highly arrhythmogenic phenomenon. Thus, intra-SR Ca(2+) dynamics set the refractoriness and frequency dependence of Ca(2+) transients in subepicardial ventricular myocytes.

SUBMITTER: Kornyeyev D

PROVIDER: S-EPMC2886618 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Luminal Ca(2+) content regulates intracellular Ca(2+) release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers.

Kornyeyev Dmytro D Reyes Mariano M Escobar Ariel L AL

American journal of physiology. Heart and circulatory physiology 20100409 6

Ca(+)-induced Ca(2+) release tightly controls the function of ventricular cardiac myocytes under normal and pathological conditions. Two major factors contributing to the regulation of Ca(2+) release are the cytosolic free Ca(2+) concentration and sarcoplasmic reticulum (SR) Ca(2+) content. We hypothesized that the amount of Ca(2+) released from the SR during each heart beat strongly defines the refractoriness of Ca(2+) release. To test this hypothesis, EGTA AM, a high-affinity, slow-association ...[more]

PMID: 20382849

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Project description:Background The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Objectives Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Methods Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. Results IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Conclusions Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

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Project description:Intracellular calcium (Ca(2+)) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca(2+) binding proteins (Ca(2+) buffers) play an important role in regulating Ca(2+) concentration ([Ca(2+)]). Buffers typically slow [Ca(2+)] temporal dynamics and increase the effective volume of Ca(2+) domains. Because fluctuations in [Ca(2+)] decrease in proportion to the square-root of a domain's physical volume, one might conjecture that buffers decrease [Ca(2+)] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca(2+) signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca(2+) influx with passive exchange of both Ca(2+) and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca(2+) and buffer and use these stochastic differential equations to determine the magnitude of [Ca(2+)] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca(2+) signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca(2+)] fluctuations during periods of Ca(2+) influx, whereas stationary (immobile) Ca(2+) buffers do not. Also contrary to expectations, we find that in the absence of Ca(2+) influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca(2+)] fluctuations. We derive an analytical formula describing the influence of rapid Ca(2+) buffers on [Ca(2+)] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca(2+) buffers alter the dynamics of [Ca(2+)] fluctuations in a nonintuitive manner. The finding that Ca(2+) buffers do not suppress intrinsic domain [Ca(2+)] fluctuations raises the intriguing question of whether or not [Ca(2+)] fluctuations are a physiologically significant aspect of local Ca(2+) signaling.

Dataset Information

Luminal Ca(2+) content regulates intracellular Ca(2+) release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers.

Publications

Luminal Ca(2+) content regulates intracellular Ca(2+) release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers.

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