β-Adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca ²⁺ channel

Abstract

As the most prototypical G protein-coupled receptor, ␤-adrenergic receptor (␤AR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca 2؉ influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca 2؉ release flux via ryanodine receptors (RyRs). However, whether and how ␤AR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca 2؉ imaging technology, we measured the response of a single RyR Ca 2؉ release unit, in the form of a Ca 2؉ spark, to its native trigger, the Ca 2؉ sparklet from a single LCC. We found that acute application of the selective ␤AR agonist isoproterenol (1 M, <20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca 2؉ release flux underlying a Ca 2؉ spark to SR Ca 2؉ content indicated that ␤AR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that ␤AR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca 2؉ content. The PKA antagonists Rp-8-CPT-cAMP (100 M) and H89 (10 M) both eliminated these effects, indicating that ␤AR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during ␤AR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered ␤AR effects in cardiovascular diseases.␤-adrenergic receptor ͉ calcium signaling ͉ excitation-contraction coupling ͉ isoproterenol ͉ protein kinase A T he ability of the heart to beat faster and stronger is central to the vertebrate survival instinct (1, 2). In the basal state, only a fraction of heart pumping power is used. During stress, ␤-adrenergic receptor (␤AR)-mediated sympathetic modulation of heart function releases the functional reserve of the excitation-contraction (E-C) coupling to meet the increased demands of blood pumping power (3, 4). The cardiac E-C coupling operates as a coordinated system, and the degree of coordination/synchronization among its component organelles and molecules is a major determinant of the system output (2). The Ca 2ϩ transient that governs the cardiac cell contraction is generated via the Ca 2ϩ -induced Ca 2ϩ release (CiCR) mechanism (1, 5, 6), in which the Ca 2ϩ influx through L-type Ca 2ϩ channels (LCCs) during excitation activates the ryanodine receptor (RyR) Ca 2ϩ release from the sarcoplasmic reticulum (SR) (7,8). At the molecular level, individual LCC opening and local RyR Ca 2ϩ release, in the form of Ca 2ϩ spark firing, are both stochastic processes (8, 9). This stochastic behavior allows a wide dynamic range of the CiCR system, i.e., from firing random Ca 2ϩ sparks to generating global Ca 2ϩ transients.During ␤AR stimulation, local Ca 2ϩ releases are ac...

Cardiac BIN1 (cBIN1) is a regulator of cardiac contractile function and an emerging biomarker of heart muscle health

“…As a result of cBIN1 redistribution, phosphorylated RyRs is recruited to cBIN1-microdomains to couple with LTCCs at the t-tubule membrane. Thus, cBIN1 controls β-AR-dependent LTCC-RyR coupling at dyads during acute stress (Fu et al, 2016), improving CICR gain and calcium transient as observed previously (Zhou et al, 2009). When BIN1 is reduced, RyR recruitment into dyads will be impaired, accumulation of uncoupled receptors outside of dyads not only limits efficient CICR but also forms leaky receptor clusters, impairing cardiac contractility and increasing arrhythmia risks (Hong et al, 2014).…”

Mitochondrial disruption occurs downstream from β-adrenergic overactivation by isoproterenol in differentiated, but not undifferentiated H9c2 cardiomyoblasts: Differential activation of stress and survival pathways

“…The use of H9c2 cells, which express β-adrenergic receptors ( A key mediator of the differences between undifferentiated and differentiated H9c2 cardiomyoblasts regarding ISO toxicity difference may be calcium-mediated downstream activation of signaling pathways, which follows through cAMP/PKA activation (Zhou et al, 2009, Mann et al, 1992. Increased basal calcium has been previously described in several models of cell differentiation, including cardiac cells (Fu et al, 2006), mouse embryonic stem cells (ES) (Metzger et al, 1994), human induced pluripotent stem cells (hiPSCs) (Lee et al, 2011) and rat neonatal cardiomyocytes (Gomez et al, 1994).…”

Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice

“…Phosphorylation of RyR2 at Ser2808 enhances the Po of the channel by enhancing its sensitivity to cytosolic [Ca 2+ ] in planar lipid bilayers (9,10). While it is well established that increasing I Ca,L and SR Ca 2+ load enhance SR Ca 2+ release (11), it is controversial as to whether increasing RyR2 Po has similar effects (3,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In this study, we have used mice engineered with RyR2 that cannot be PKA phosphorylated (21) to directly address the role of PKA phosphorylation of RyR2 in regulating cardiac contractility.…”