Ryanodine Receptor Signaling Research Articles

A New Cell Type in Pulmonary Veins

Pulmonary veins (PVs) carry oxygenated blood from lungs back to the heart and may play an important role in preventing blood backflow from the left atrium. Previous studies show that large PVs exhibit spontaneously pulsatile contractions in cardiomyocyte-like manner. However, the contractions of small PVs have not been investigated. We hypothesized that Ca2+ signaling mechanisms in the myocytes mediate small PV contractions. Small, 4th-order PVs from C57BL6 mice were obtained and imaged. Pressurized PVs showed ryanodine receptor-dependent Ca2+ signals and spontaneous contractions. Light sheet fluorescence microscopy and immunostaining showed the expression of troponin C, a cardiomyocyte marker, in the myocyte layer of small PVs, which was not observed in the pulmonary arteries, vena cava, or mesenteric veins. Based on these results, we termed these cells cardiomyocyte-like (CML) cells. In the study of mice with Myh11 Cre-driven tdTomato expression, Myh11 expression was observed in CML cells in small PVs, which was not seen in the smooth muscle cells (SMC) of small PAs. Endothelial protrusions crossing the connective tissue layer into the CML layer were also observed in PVs. Previous studies showed PV remodeling in pulmonary hypertension (PH), and impaired Ca2+ signaling in PAs contribute to higher pulmonary arterial pressure in PH. We further hypothesized that ryanodine receptor (RyR) Ca2+ signals in CML cells are impaired in PH. Results show that RyR Ca2+ signaling was reduced in CML cells of PH mice (injected with Su5416, subjected to chronic hypoxic conditions). Overall, our findings identify a new cell type in small PVs. Moreover, our data show that RyR Ca2+ signals in CML cells underlie spontaneous contractions in small PV, and are impaired in PH. We propose that RyR signaling mechanisms in CML cells may be possible targets for therapy in pulmonary vascular disorders. This work was supported by funding from the National Institute of Health to Swapnil Sonkusare (R01:HL146914, R01:HL157407). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calcium-Permeable AMPA Receptors Mediate Timing-Dependent LTP Elicited by Low Repeat Coincident Pre- and Postsynaptic Activity at Schaffer Collateral-CA1 Synapses.

High-frequency stimulation induced long-term potentiation (LTP) and low-frequency stimulation induced LTD are considered as cellular models of memory formation. Interestingly, spike timing-dependent plasticity (STDP) can induce equally robust timing-dependent LTP (t-LTP) and t-LTD in response to low frequency repeats of coincident action potential (AP) firing in presynaptic and postsynaptic cells. Commonly, STDP paradigms relying on 25-100 repeats of coincident AP firing are used to elicit t-LTP or t-LTD, but the minimum number of repeats required for successful STDP is barely explored. However, systematic investigation of physiologically relevant low repeat STDP paradigms is of utmost importance to explain learning mechanisms in vivo. Here, we examined low repeat STDP at Schaffer collateral-CA1 synapses by pairing one presynaptic AP with either one postsynaptic AP (1:1t-LTP), or a burst of 4 APs (1:4t-LTP) and found 3-6 repeats to be sufficient to elicit t-LTP. 6× 1:1t-LTP required postsynaptic Ca2+ influx via NMDARs and L-type VGCCs and was mediated by increased presynaptic glutamate release. In contrast, 1:4t-LTP depended on postsynaptic metabotropic GluRs and ryanodine receptor signaling and was mediated by postsynaptic insertion of AMPA receptors. Unexpectedly, both 6× t-LTP variants were strictly dependent on activation of postsynaptic Ca2+-permeable AMPARs but were differentially regulated by dopamine receptor signaling. Our data show that synaptic changes induced by only 3-6 repeats of mild STDP stimulation occurring in ≤10s can take place on time scales observed also during single trial learning.

Transcriptomic profiling of mTOR and ryanodine receptor signaling molecules in developing zebrafish in the absence and presence of PCB 95.

The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways regulate fundamental processes of neurodevelopment, and genetic mutations within these pathways have been linked to neurodevelopmental disorders. While previous studies have established that these signaling molecules are expressed in developing zebrafish, a detailed characterization of the ontogenetic profile of these signaling molecules is lacking. Thus, we evaluated the spatiotemporal expression of key transcripts in mTOR and RyR signaling pathways in wildtype zebrafish at 24, 72 and 120 hours post fertilization (hpf). We further determined whether transcriptional profiles of a subset of genes in both pathways were altered by exposure to PCB 95 (2,2′,3,5′,6-pentachlorobiphenyl), a pervasive environmental contaminant known to cause developmental neurotoxicity in mammalian systems via RyR-dependent mechanisms. Quantitative PCR revealed that transcription generally increased across development. Genes in the signaling pathway upstream of the mTORC1 complex, and the RyR-paralogs, ryr2a and ryr3, were robustly upregulated, and in situ hybridization of ryr3 coincided with a transcriptional shift from muscle to neuronal tissue after 24 hpf. Static waterborne exposure to PCB 95 beginning at 6 hpf significantly altered transcription of genes in both pathways. These changes were concentration- and time-dependent, and included downregulation of rptor, a member of the mTORC1 complex, at both 72 and 120 hpf, and increased transcript levels of the RyR paralog ryr2b and downstream target of RyR signaling, Wingless-type 2ba (wnt2ba) at 72 hpf. The detailed transcriptomic profiling of key genes within these two signaling pathways provides a baseline for identifying other environmental factors that modify normal spatiotemporal expression patterns of mTOR and RyR signaling pathways in the developing zebrafish, as illustrated here for PCB 95.

Endothelium- and smooth muscle-dependent vasodilator effects of Citrus aurantium L. var. amara: Focus on Ca(2+) modulation.

Neroli, the essential oil of Citrus aurantium L. var. amara, is a well-characterized alleviative agent used to treat cardiovascular symptoms. However, because it has been found to have multiple effects, its mechanism of action requires further exploration. We sought to clarify the mechanism underlying the actions of neroli in mouse aorta. In aortic rings from mice precontracted with prostaglandin F2 alpha, neroli induced vasodilation. However, relaxation effect of neroli was decreased in endothelium-denuded ring or pre-incubation with the nitric oxide synthase inhibitor NG-Nitro-l-arginine-methyl ester (L-NAME). And also, neroli-induced relaxation was also partially reversed by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), a soluble guanylyl cyclase (sGC) inhibitor. In addition, neroli inhibited extracellular Ca(2+)-dependent, depolarization-induced contraction, an effect that was concentration dependent. Pretreatment with the non-selective cation channel blocker, Ni(2+), attenuated neroli-induced relaxation, whereas the K(+) channel blocker, tetraethylammonium chloride, had no effect. In the presence of verapamil, added to prevent Ca(2+) influx via smooth muscle voltage-gated Ca(2+) channels, neroli-induced relaxation was reduced by the ryanodine receptor (RyR) inhibitor ruthenium red. Our findings further indicate that the endothelial component of neroli-induced vasodilation is partly mediated by the NO-sGC pathway, whereas the smooth muscle component involves modulation of intracellular Ca(2+) concentration through inhibition of cation channel-mediated extracellular Ca(2+) influx and store-operated Ca(2+) release mediated by the RyR signaling pathway.

In Vivo Suppression of MicroRNA-24 Prevents the Transition Toward Decompensated Hypertrophy in Aortic-Constricted Mice

During the transition from compensated hypertrophy to heart failure, the signaling between L-type Ca(2+) channels in the cell membrane/T-tubules and ryanodine receptors in the sarcoplasmic reticulum becomes defective, partially because of the decreased expression of a T-tubule-sarcoplasmic reticulum anchoring protein, junctophilin-2. MicroRNA (miR)-24, a junctophilin-2 suppressing miR, is upregulated in hypertrophied and failing cardiomyocytes. To test whether miR-24 suppression can protect the structural and functional integrity of L-type Ca(2+) channel-ryanodine receptor signaling in hypertrophied cardiomyocytes. In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively prevented the degradation of heart contraction, but not ventricular hypertrophy. Electrophysiology and confocal imaging studies showed that antagomir treatment prevented the decreases in L-type Ca(2+) channel-ryanodine receptor signaling fidelity/efficiency and whole-cell Ca(2+) transients. Further studies showed that antagomir treatment stabilized junctophilin-2 expression and protected the ultrastructure of T-tubule-sarcoplasmic reticulum junctions from disruption. MiR-24 suppression prevented the transition from compensated hypertrophy to decompensated hypertrophy, providing a potential strategy for early treatment against heart failure.

Prostaglandin FP receptor inhibitor reduces ischemic brain damage and neurotoxicity

Bioactive lipids such as the prostaglandins have been reported to have various cytoprotective or toxic properties in acute and chronic neurological conditions. The roles of PGF2α and its receptor (FP) are not clear in the pathogenesis of ischemic brain injury. Considering that this G-protein coupled receptor has been linked to intracellular calcium regulation, we hypothesized that its blockade would be protective. We used FP antagonist (AL-8810) and FP receptor knockout (FP−/−) mice in in vivo and in vitro stroke models. Mice that were treated with AL-8810 had 35.7±6.3% less neurologic dysfunction and 36.4±6.0% smaller infarct volumes than did vehicle-treated mice after 48h of permanent middle cerebral artery occlusion (pMCAO); FP−/− mice also had improved outcomes after pMCAO. Blockade of the FP receptor also protected against oxygen-glucose deprivation (OGD)-induced cell death and reactive oxygen species formation in slice cultures. Finally, we found that an FP receptor agonist dose dependently increased intracellular Ca2+ levels in cultured neurons and established that FP-related Ca2+ signaling is related to ryanodine receptor signaling. These results indicate that the FP receptor is involved in cerebral ischemia-induced damage and could promote development of drugs for treatment of stroke and acute neurodegenerative disorders.

Excitatory and inhibitory synaptic transmission is differentially influenced by two ortho-substituted polychlorinated biphenyls in the hippocampal slice preparation

Exposure to polychlorinated biphenyls impairs cognition and behavior in children. Two environmental PCBs 2,2',3,3',4,4',5-heptachlorobiphenyl (PCB170) and 2,2',3,5',6-pentachlorobiphenyl (PCB95) were examined in vitro for influences on synaptic transmission in rat hippocampal slices. Field excitatory postsynaptic potentials ( fEPSPs) were recorded in the CA1 region using a multi-electrode array. Perfusion with PCB170 (10 nM) had no effect on fEPSP slope relative to baseline period, whereas (100 nM) initially enhanced then depressed fEPSP slope. Perfusion of PCB95 (10 or 100 nM) persistently enhanced fEPSP slope > 200%, an effect that could be inhibited by dantrolene, a drug that attenuates ryanodine receptor signaling. Perfusion with picrotoxin (PTX) to block GABA neurotransmission resulted in a modest increase in fEPSP slope, whereas PTX + PCB170 (1–100 nM) persistently enhanced fEPSP slope in a dose dependent manner. fEPSP slope reached > 250% of baseline period in the presence of PTX + 100 nM PCB170, conditions that evoked marked epileptiform after-potential discharges. PCB95 and PCB170 were found to differentially influence the Ca 2+-dependence of [ 3H]ryanodine-binding to hippocampal ryanodine receptors. Non-coplanar PCB congeners can differentially alter neurotransmission in a manner suggesting they can elicit imbalances between inhibitory and excitatory circuits within the hippocampus. Differential sensitization of ryanodine receptors by Ca 2+ appears to mediate, at least in part, hippocampal excitotoxicity by non-coplanar PCBs.

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.

Intermolecular Failure of L-type Ca2+ Channel and Ryanodine Receptor Signaling in Hypertrophy

Pressure overload–induced hypertrophy is a key step leading to heart failure. The Ca2+-induced Ca2+ release (CICR) process that governs cardiac contractility is defective in hypertrophy/heart failure, but the molecular mechanisms remain elusive. To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca2+ channel (LCC) and ryanodine receptors (RyRs) in aortic stenosis rat models of compensated (CHT) and decompensated (DHT) hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of “intermolecular failure.” Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca2+ release, visualized as “Ca2+ spikes,” became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca2+ transients in CHT. These data suggested that, within a certain limit, termed the “stability margin,” mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of “hidden” intermolecular failure in CHT has important clinical implications.

Ryanodine receptor signaling is required for anti-CD3-induced T cell proliferation, interleukin-2 synthesis, and interleukin-2 receptor signaling.

Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression.