The biophysical properties of TRIC-A and TRIC-B and their interactions with RyR2.

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are thought to provide counter-ion currents to enable charge equilibration across the sarco/endoplasmic reticulum (SR) and nuclear membranes. However, there is also evidence that TRIC-A may interact directly with ryanodine receptor type 1 (RyR1) and 2 (RyR2) to alter RyR channel gating. It is therefore possible that the reverse is also true, where the presence of RyR channels is necessary for fully functional TRIC channels. We therefore coexpressed mouse TRIC-A or TRIC-B with mouse RyR2 in HEK293 cells to examine if after incorporating membrane vesicles from these cells into bilayers, the presence of TRIC affects RyR2 function, and to characterize the permeability and gating properties of the TRIC channels. Importantly, we used no purification techniques or detergents to minimize damage to TRIC and RyR2 proteins. We found that both TRIC-A and TRIC-B altered the gating behavior of RyR2 and its response to cytosolic Ca2+ but that TRIC-A exhibited a greater ability to stimulate the opening of RyR2. Fusing membrane vesicles containing TRIC-A or TRIC-B into bilayers caused the appearance of rapidly gating current fluctuations of multiple amplitudes. The reversal potentials of bilayers fused with high numbers of vesicles containing TRIC-A or TRIC-B revealed both Cl- and K+ fluxes, suggesting that TRIC channels are relatively non-selective ion channels. Our results indicate that the physiological roles of TRIC-A and TRIC-B may include direct, complementary regulation of RyR2 gating in addition to the provision of counter-ion currents of both cations and anions.

The ryanodine receptor mutational characteristics and its indication for cancer prognosis.

Ca2+ signaling is altered substantially in many cancers. The ryanodine receptors (RYRs) are among the key ion channels in Ca2+ signaling. This study aimed to establish the mutational profile of RYR in cancers and investigate the correlation between RYR alterations and cancer phenotypes. The somatic mutation and clinical data of 11,000 cancer patients across 33 cancer types was downloaded from The Cancer Genome Atlas (TCGA) database. Subsequent data processing was performed with corresponding packages of the R software. Mutational profile was analyzed and its correlation with tumor mutational burden (TMB), patient prognosis, age and smoking status was analyzed and compared. All three RYR isoforms exhibited random mutational distribution without hotspot mutations when all cancers were analyzed together. The number of mutations in RYR2 (2388 mutations) far overweight that of RYR1 (1439 mutations) and RYR3 (1573 mutations). Linear correlation was observed between cumulative TMB and cumulative number of mutations for all RYR isoforms. Patients with RYR mutations exhibited significantly higher TMB than those without RYR mutations for most cancer types. Strong correlation was also revealed in the average number of mutations per person between pairs of RYR isoforms. No stratification of patient overall survival (OS) by mutational status was found for all three RYR isoforms when all cancers were analyzed together, however, significant stratification of OS by RYR mutations was revealed in several individual cancers, most strikingly in LUAD (P = 0.0067, RYR1), BLCA (P = 0.00071, RYR2), LUSC (P = 0.036, RYR2) and KIRC (P = 0.0042, RYR3). Furthermore, RYR mutations were correlated with higher age, higher smoking history grading and higher number of pack years. Characteristic mutation profile of RYRs in cancers has been revealed for the first time. RYR mutations were correlated with TMB, age, smoking status and capable of stratifying the prognosis of patients in several cancer types.

Spatial and Temporal Distribution of Information Processing in the Human Dorsal Anterior Cingulate Cortex.

The dorsal anterior cingulate cortex (dACC) is a key node in the human salience network. It has been ascribed motor, pain-processing and affective functions. However, the dynamics of information flow in this complex region and how it responds to inputs remain unclear and are difficult to study using non-invasive electrophysiology. The area is targeted by neurosurgery to treat neuropathic pain. During deep brain stimulation surgery, we recorded local field potentials from this region in humans during a decision-making task requiring motor output. We investigated the spatial and temporal distribution of information flow within the dACC. We demonstrate the existence of a distributed network within the anterior cingulate cortex where discrete nodes demonstrate directed communication following inputs. We show that this network anticipates and responds to the valence of feedback to actions. We further show that these network dynamics adapt following learning. Our results provide evidence for the integration of learning and the response to feedback in a key cognitive region.

The V2475F CPVT1 mutation yields distinct RyR2 channel populations that differ in their responses to cytosolic Ca2+ and Mg2.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a lethal genetic disease causing arrhythmias and sudden cardiac death in children and young adults and is linked to mutations in the cardiac ryanodine receptor (RyR2). The effects of CPVT1 mutations on RyR2 ion-channel function are often investigated using purified recombinant RyR2 channels homozygous for the mutation. However, CPVT1 patients are heterozygous for the disease, so this approach does not reveal the true changes to RyR2 function across the entire RyR2 population of channels in the heart. We therefore investigated the native cardiac RyR2 single-channel abnormalities in mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2, and applied molecular modelling techniques to investigate the possible structural changes that could initiate any altered function. We observed that increased sensitivity of cardiac V2475F(+/-)-RyR2 channels to both activating and inactivating levels of cytosolic Ca2+ , plus attenuation of Mg2+ inhibition, were the most marked changes. Severity of abnormality was not uniform across all channels, giving rise to multiple sub-populations with differing functional characteristics. For example, 46% of V2475F(+/-)-RyR2 channels exhibited reduced Mg2+ inhibition and 23% were actually activated by Mg2+ . Using homology modelling, we discovered that V2475 is situated at a hinge between two regions of the RyR2 helical domain 1 (HD1). Our model proposes that detrimental functional changes to RyR2 arise because mutation at this critical site reduces the angle between these regions. Our results demonstrate the necessity of characterising the total heterozygous population of CPVT1-mutated channels in order to understand CPVT1 phenotypes in patients. KEY POINTS: RyR2 mutations can cause type-1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a lethal, autosomal-dominant arrhythmic disease. However, the changes in RyR2 ion-channel function that result from the many different patient mutations are rarely investigated in detail and often only recombinant RyR2, homozygous for the mutation, is studied. As CPVT1 is a heterozygous disease and the tetrameric RyR2 channels expressed in the heart will contain varying numbers of mutated monomers, we have investigated the range of RyR2 single-channel abnormalities found in the hearts of mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2. Specific alterations to ligand regulation of V2475F(+/-)-RyR2 were observed. Multiple sub-populations of channels exhibited varying degrees of abnormality. In particular, an increased sensitivity to activating and inactivating cytosolic [Ca2+ ], and reduced sensitivity to Mg2+ inhibition were evident. Our results provide mechanistic insight into the changes to RyR2 gating that destabilise sarcoplasmic reticulum Ca2+ -release causing life-threatening arrhythmias in V2475F(+/-)-CPVT1 patients.

Combining tissue engineering and optical imaging approaches to explore interactions along the neuro-cardiac axis.

Interactions along the neuro-cardiac axis are being explored with regard to their involvement in cardiac diseases, including catecholaminergic polymorphic ventricular tachycardia, hypertension, atrial fibrillation, long QT syndrome and sudden death in epilepsy. Interrogation of the pathophysiology and pathogenesis of neuro-cardiac diseases in animal models present challenges resulting from species differences, phenotypic variation, developmental effects and limited availability of data relevant at both the tissue and cellular level. By contrast, tissue-engineered models containing cardiomyocytes and peripheral sympathetic and parasympathetic neurons afford characterization of cellular- and tissue-level behaviours while maintaining precise control over developmental conditions, cellular genotype and phenotype. Such approaches are uniquely suited to long-term, high-throughput characterization using optical recording techniques with the potential for increased translational benefit compared to more established techniques. Furthermore, tissue-engineered constructs provide an intermediary between whole animal/tissue experiments and in silico models. This paper reviews the advantages of tissue engineering methods of multiple cell types and optical imaging techniques for the characterization of neuro-cardiac diseases.