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The type 2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report 4 cryo-electron microscopy (cryo-EM) structures of porcine RyR2 bound to distinct modulators that, together with our published structures, provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the central domain that facilitates the dilation of the S6 bundle but is insufficient to open the pore. The small-molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state even in the presence of Ca2+ and PCB95. Although the channel is open when PCB95 is replaced by caffeine and adenosine 5′-triphosphate (ATP), neither of the modulators alone can sufficiently counter the antagonistic effect to open the channel. Our study marks an important step toward mechanistic understanding of the sophisticated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP + / − /PS1 + / − ). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca 2+ ) leak. RyR-mediated ER Ca 2+ leak activates Ca 2+ -dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca 2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aβ load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

Coordinated release of calcium (Ca 2+ ) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptor (RYR2) channels is essential for cardiomyocyte function. In catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disease characterized by stress‐induced ventricular arrhythmias in young patients with structurally normal hearts, autosomal dominant mutations in RYR2 or recessive mutations in calsequestrin lead to aberrant diastolic Ca 2+ release from the SR causing arrhythmogenic delayed after depolarizations (DADs). Here, we report the generation of induced pluripotent stem cells (iPSCs) from a CPVT patient carrying a novel RYR2 S406L mutation. In patient iPSC‐derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca 2+ concentrations, a reduced SR Ca 2+ content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes. This was due to increased frequency and duration of elementary Ca 2+ release events (Ca 2+ sparks). Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca 2+ spark properties and rescued the arrhythmogenic phenotype. This suggests defective inter‐domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation. Our work provides a new in vitro model to study the pathogenesis of human cardiac arrhythmias and develop novel therapies for CPVT.

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.

Duchenne muscular dystrophy (DMD) results from mutations in the gene encoding dystrophin which lead to impaired function of skeletal and cardiac muscle, but little is known about the effects of the disease on vascular smooth muscle cells (SMCs). Here we used the mdx mouse model to study the effects of mutant dystrophin on the regulation of cerebral artery and arteriole SMC contractility, focusing on an important Ca2+-signaling pathway composed of type 2 ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) and large-conductance Ca2+-activated K⁺ (BK) channels on the plasma membrane. Nanoscale superresolution image analysis revealed that RyR2 and BKα were organized into discrete clusters, and that the mean size of RyR2 clusters that colocalized with BKα was larger in SMCs from mdx mice (∼62 RyR2 monomers) than in controls (∼40 RyR2 monomers). We further found that the frequency and signal mass of spontaneous, transient Ca2+-release events through SR RyR2s (“Ca2+ sparks”) were greater in SMCs from mdx mice. Patch-clamp electrophysiological recordings indicated a corresponding increase in Ca2+-dependent BK channel activity. Using pressure myography, we found that cerebral pial arteries and parenchymal arterioles from mdx mice failed to develop appreciable spontaneous myogenic tone. Inhibition of RyRs with tetracaine and blocking of BK channels with paxilline restored myogenic tone to control levels, demonstrating that enhanced RyR and BK channel activity is responsible for the diminished pressure-induced constriction of arteries and arterioles from mdx mice. We conclude that increased size of RyR2 protein clusters in SMCs from mdx mice increases Ca2+ spark and BK channel activity, resulting in cerebral microvascular dysfunction.

Ion channels play a crucial role in cardiac functions, and their activities exhibit dynamic changes during heart development. However, the precise function of ion channels in human heart development remains elusive. In this study, we utilized human embryonic stem cells (hESCs) as a model to mimic the process of human embryonic heart development. During hESCs differentiation into cardiomyocytes, we observed differential expression of ion channel genes, including upregulation of ryanodine receptor 2 (RYR2), which encodes a calcium release channel. Subsequently, we discovered that Suramin, an activator of RyR2, efficiently promoted cardiac differentiation even in the absence of conventional WNT inhibitors. Furthermore, various modulators targeting sodium channels, potassium channels or chloride channels were examined under chemically defined conditions during cardiac differentiation. We found that DIDS, a chloride transport inhibitor, also enhanced hESCs differentiation into cardiomyocytes. Both Suramin and DIDS partially inhibited WNT signaling pathway, and RYR2 knockdown attenuated cardiac differentiation induced by WNT inhibitor treatment, or Suramin or DIDS administration. The resulting cardiomyocytes induced by these ion modulators exhibited specific expression patterns of cardiac genes and displayed typical electrophysiological signals. Notably, compared to WNT inhibitor treatment group, both Suramin and DIDS led to increased generation of atrial-like cardiomyocytes suggesting their potential as alternative inducers for specific cardiomyocyte lineage commitment during human cardiomyocyte induction processes. This study demonstrates that regulation of ion channels plays a crucial role in determining the fate of cardiac cells, providing an effective approach for inducing cardiomyocytes from hPSCs and highlighting their critical involvement in human heart development.

The incidence of sudden cardiac death (SCD) in people living with HIV infection (PLWH), especially those with inadequate viral suppression, is high and the reasons for this remain incompletely characterized. The timely opening and closing of type 2 ryanodine receptor (RyR2) is critical for ensuring rhythmic cardiac contraction–relaxation cycles, and the disruption of these processes can elicit Ca2+ waves, ventricular arrhythmias, and SCD. Herein, we show that the HIV protein Tat (HIV-Tat: 0–52 ng/mL) and therapeutic levels of the antiretroviral drugs atazanavir (ATV: 0–25,344 ng/mL), efavirenz (EFV: 0–11,376 ng/mL), and ritonavir (RTV: 0–25,956 ng/mL) bind to and modulate the opening and closing of RyR2. Abacavir (0–14,315 ng/mL), bictegravir (0–22,469 ng/mL), Rilpivirine (0–14,360 ng/mL), and tenofovir disoproxil fumarate (0–18,321 ng/mL) did not alter [3H]ryanodine binding to RyR2. Pretreating RyR2 with low HIV-Tat (14 ng/mL) potentiated the abilities of ATV and RTV to bind to open RyR2 and enhanced their ability to bind to EFV to close RyR2. In silico molecular docking using a Schrodinger Prime protein–protein docking algorithm identified three thermodynamically favored interacting sites for HIV-Tat on RyR2. The most favored site resides between amino acids (AA) 1702–1963; the second favored site resides between AA 467–1465, and the third site resides between AA 201–1816. Collectively, these new data show that HIV-Tat, ATV, EFV, and RTV can bind to and modulate the activity of RyR2 and that HIV-Tat can exacerbate the actions of ATV, EFV, and RTV on RyR2. Whether the modulation of RyR2 by these agents increases the risk of arrhythmias and SCD remains to be explored.

The Cardiomyopathy–associated gene 5 ( Cmya5 ) encodes myospryn, a large tripartite motif (TRIM)-related protein found predominantly in cardiac and skeletal muscle. Cmya5 is an expression biomarker for a number of diseases affecting striated muscle and may also be a schizophrenia risk gene. To further understand the function of myospryn in striated muscle, we searched for additional myospryn paralogs. Here we identify a novel muscle-expressed TRIM-related protein minispryn, encoded by Fsd2 , that has extensive sequence similarity with the C-terminus of myospryn. Cmya5 and Fsd2 appear to have originated by a chromosomal duplication and are found within evolutionarily-conserved gene clusters on different chromosomes. Using immunoaffinity purification and mass spectrometry we show that minispryn co-purifies with myospryn and the major cardiac ryanodine receptor (RyR2) from heart. Accordingly, myospryn, minispryn and RyR2 co-localise at the junctional sarcoplasmic reticulum of isolated cardiomyocytes. Myospryn redistributes RyR2 into clusters when co-expressed in heterologous cells whereas minispryn lacks this activity. Together these data suggest a novel role for the myospryn complex in the assembly of ryanodine receptor clusters in striated muscle.

Calsenilin is a calcium ion (Ca 2+ )-binding protein involved in regulating the intracellular concentration of Ca 2+ , a second messenger that controls multiple cellular signaling pathways. The ryanodine receptor (RyR) amplifies Ca 2+ signals entering the cytoplasm by releasing Ca 2+ from endoplasmic reticulum (ER) stores, a process termed calcium-induced calcium release (CICR). Here, we describe a novel mechanism, in which calsenilin controls the activity of neuronal RyRs. We show calsenilin co-localized with RyR2 and 3 in the ER of mouse hippocampal and cortical neurons using immunocytochemistry. The underlying protein-protein interaction between calsenilin and the RyR was determined in mouse central nervous system (CNS) neurons using immunoprecipitation studies. The functional relevance of this interaction was assayed with single-channel electrophysiology. At low physiological Ca 2+ concentrations, calsenilin binding to the cytoplasmic face of neuronal RyRs decreased the RyR’s open probability, while calsenilin increased the open probability at high physiological Ca 2+ concentrations. This novel molecular mechanism was studied further at the cellular level, where faster release kinetics of caffeine-induced Ca 2+ release were measured in SH-SY5Y neuroblastoma cells overexpressing calsenilin. The interaction between calsenilin and neuronal RyRs reveals a new regulatory mechanism and possibly a novel pharmacological target for the control of Ca 2+ release from intracellular stores.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, and oral squamous cell carcinoma (OSCC) is one of the most common types. There is strong evidence that ryanodine receptor 2 (RYR2) plays an important role in different types of cancer according to previous studies. Its expression is associated with survival in patients with HNSCC, but it is unknown whether altered RYR2 expression contributes to tumorigenesis. Therefore, we examined how RYR2 polymorphisms affect OSCC susceptibility and clinicopathological characteristics. Five single nucleotide polymorphisms (SNPs) of RYR2, rs12594, rs16835904, rs2779359, rs3765097, and rs3820216, were analyzed in 562 cases of OSCC and 332 healthy controls using real-time PCR. We demonstrated that RYR2 SNP rs12594 was significantly different between the case and control groups, but this difference was not significant after adjusting for personal habits. In contrast, we found that different genotypes of SNP rs2779359 were significantly associated with the characteristics of clinical stage and tumor size in OSCC patients, according to the odds ratios and the adjusted odds ratios; specifically, patients with the T genotype had 1.477-fold (95% CI, 1.043 to 2.091; p = 0.028) and 1.533-fold (95% CI, 1.087–2.162; p = 0.015) increases in clinical stage and tumor size, respectively, compared with patients with the C allele. The results of our study, in which RYR2 SNPs associated with OSCC progression and development were examined for the first time, suggest that clinicopathological characteristics may alter OSCC susceptibility. Finally, RYR2 SNP rs2779359 not only plays a role in both the prognosis and diagnosis of oral cancer but is also likely an important predictive factor for recurrence, response to treatment, and medication toxicity.