Rationale: Ca 2+ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca 2+ alternans remains undefined. Increasing evidence suggests that Ca 2+ alternans results from alternations in the inactivation of cardiac ryanodine receptor (RyR2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown. Objective: To determine the role of calmodulin (CaM) on Ca 2+ alternans in intact working mouse hearts. Methods and Results: We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type (CaM-WT), a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 WT or mutant mouse hearts. We monitored Ca 2+ transients in ventricular myocytes near the adenovirus injection sites in Langendorff-perfused intact working hearts using confocal Ca 2+ imaging. We found that CaM-WT and CaM-M37Q promoted Ca 2+ alternans and prolonged Ca 2+ transient recovery in intact RyR2 WT and mutant hearts, whereas, CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca 2+ current, but had no significant impact on sarcoplasmic reticulum Ca 2+ content. Further, we developed a novel numerical myocyte model of Ca 2+ alternans that incorporates Ca 2+ -CaM-dependent regulation of RyR2 and the L-type Ca 2+ channel. Remarkably, the new model recapitulates the impact on Ca 2+ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca 2+ elevation as a result of rapid pacing triggers Ca 2+ -CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes SR Ca 2+ release, which in turn reduces diastolic cytosolic Ca 2+ , leading to alternations in diastolic cytosolic Ca 2+ , RyR2 inactivation, and SR Ca 2+ release (i.e. Ca 2+ alternans). Conclusions: Our results demonstrate that inactivation of RyR2 by Ca 2+ -CaM is a major determinant of Ca 2+ alternans, making Ca 2+ -CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/− mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/− mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Increasing evidence suggests that Alzheimer's disease (AD) progression is driven by a vicious cycle of soluble β‐amyloid (Aβ)‐induced neuronal hyperactivity. Thus, breaking this vicious cycle by suppressing neuronal hyperactivity may represent a logical approach to stopping AD progression. In support of this, we have recently shown that genetically and pharmacologically limiting ryanodine receptor 2 (RyR2) open time prevented neuronal hyperactivity, memory impairment, dendritic spine loss, and neuronal cell death in a rapid, early onset AD mouse model (5xFAD). Here, we assessed the impact of limiting RyR2 open time on AD‐related deficits in a relatively late occurring, slow developing AD mouse model (3xTG‐AD) that bears more resemblance (compared to 5xFAD) to that of human AD. Using behavioral tests, long‐term potentiation recordings, and Golgi and Nissl staining, we found that the RyR2‐E4872Q mutation, which markedly shortens the open duration of the RyR2 channel, prevented learning and memory impairment, defective long‐term potentiation, dendritic spine loss, and neuronal cell death in the 3xTG‐AD mice. Furthermore, pharmacologically shortening the RyR2 open time with R‐carvedilol rescued these AD‐related deficits in 3xTG mice. Therefore, limiting RyR2 open time may offer a promising, neuronal hyperactivity‐targeted anti‐AD strategy.