A Single Protein Kinase A or Calmodulin Kinase II Site Does Not Control the Cardiac Pacemaker Ca ²⁺ Clock

Abstract

Background Fight or flight heart rate (HR) increases depend on protein kinase A (PKA) and calmodulin kinase II (CaMKII) mediated enhancement of Ca2+ uptake and release from sarcoplasmic reticulum (SR) in sinoatrial nodal cells (SANC). However, the impact of specific PKA and CaMKII phosphorylation sites on HR is unknown. Methods and Results We systematically evaluated validated PKA and CaMKII target sites on phospholamban (PLN) and the ryanodine receptor (RyR2) using genetically modified mice. We found that knockin alanine replacement of RyR2 PKA (S2808) or CaMKII (S2814) target sites failed to affect HR responses to isoproterenol or spontaneous activity in vivo or in SANC. Similarly, selective mutation of PLN amino acids critical for enhancing SR Ca2+ uptake by PKA (S16) or CaMKII (T17) to alanines did not affect HR in vivo or in SANC. In contrast, CaMKII inhibition by expression of AC3-I has been shown to slow SANC rate responses to isoproterenol and decrease SR Ca2+ content. PLN deficiency rescued SR Ca2+ content and SANC rate responses to isoproterenol in mice with AC3-I expression, suggesting CaMKII affects HR by modulation of SR Ca2+ content. Consistent with this, mice expressing a superinhibitory PLN mutant had low SR Ca2+ content and slow HR in vivo and in SANC. Conclusions SR Ca2+ depletion reduces HR and SR Ca2+ repletion restores physiological SANC rate responses despite CaMKII inhibition. PKA and CaMKII do not affect HR by a unique target site governing SR Ca2+ uptake or release. HR acceleration may require an SR Ca2+ content threshold.

Functional Microdomains in Heart’s Pacemaker: A Step Beyond Classical Electrophysiology and Remodeling

“…cAMP-mediated, PKA- ( Vinogradova et al, 2006 ) and CaMKII-dependent phosphorylation ( Vinogradova et al, 2000 ; Li et al, 2016 ) of RyRs have been reported to affect the size and rhythm of LCRs in rabbit SANCs. At the same time, knock-in alanine replacement of RyR phosphorylation sites PKA (S2808) or CaMKII (S2814) did not affect heart rate responses to isoproterenol in vivo or in isolated SANCs in mice ( Wu et al, 2016 ). Furthermore, Wu et al (2016) demonstrated that selective mutations of PLN phosphorylation sites PKA (S16) or CaMKII (T17) in mice also did not affect heart rate.…”

The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms

“…Because the current model includes descriptions of both clock mechanisms, post-translation modification signaling cascades, and autonomic-nervous receptors, it should be useful to predict pathological alterations in the function of any of these components. Of note, a recent paper using mice as an experimental model found that PKA signaling played a minor role under basal conditions (Wu et al, 2016 ). The difference between the current and Wu et al results might be related to the mammal each group is working with.…”

Physiological and unappreciated roles of CaMKII in the heart

“…Interestingly, CaMKII inhibition only affects HR after β-adrenergic stimulation, and not at baseline. It must be noted that this effect did not depend on a single CaMKII in PLB or RyR2, but rather that the concerted action on multiple phosphorylation targets decreases SR Ca 2+ content below a certain threshold which seems to be required for the fight or flight response [ 110 ].…”

“…Ultimately, this can repress Ca 2+ transients and contractility and serve as a basis for arrhythmogenic effects. It must be noted that CaMKII and protein kinase A (PKA) share a number of phosphorylation targets, among which are RyR2 and PLN [ 23 , 110 ]. RyR2, for example, can be phosphorylated by CaMKII at Ser2815 and by PKA at Ser2809, and both phosphorylation events increase the open probability of the RyR2 channel and are therefore pro-arrhythmic.…”