Modeling CICR in rat ventricular myocytes: voltage clamp studies

<p>Abstract</p> <p>Background</p> <p>The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect <it>Ca</it>²⁺loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR <it>Ca</it>²⁺release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic <it>Ca</it>²⁺concentration ([<it>Ca</it>²⁺]<it>_myo</it>).</p> <p>Methods</p> <p>The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed <it>Ca</it>²⁺channels (trigger-channel and release-channel). It releases <it>Ca</it>²⁺flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[<it>Ca</it>²⁺]<it>_myo</it>.</p> <p>Results</p> <p>Our model reproduces measured VC data published by several laboratories, and generates graded <it>Ca</it>²⁺release at high <it>Ca</it>²⁺gain in a homeostatically-controlled environment where [<it>Ca</it>²⁺]<it>_myo</it>is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR <it>Ca</it>²⁺release, its activation by trigger <it>Ca</it>²⁺, and its refractory characteristics mediated by the luminal SR <it>Ca</it>²⁺sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence <it>Ca</it>²⁺homeostasis.</p> <p>Conclusions</p> <p>We examine the role of the above <it>Ca</it>²⁺regulating mechanisms in handling various types of induced disturbances in <it>Ca</it>²⁺levels by quantifying cellular <it>Ca</it>²⁺balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.</p

Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells: A novel mechanism of hypoxic pulmonary hypertension

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca2+ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca2+ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca2+ stores, caused dramatic increase in cation entry measured by Mn2+ quenching of fura-2 and by Ca2+ transients. OAG-induced responses were ≈700-fold more resistant to La3+ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca2+ or inhibition of store-operated Ca2+ entry with La3+ and SK&F-96365 reversed the elevated basal [Ca 2+]i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.link_to_subscribed_fulltex

Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca 2+ stores in pulmonary arterial smooth muscle cells

Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca 2+ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, ∼15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca 2+ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca 2+ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 ± 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca 2+ sparks (FDHM = 44.6 ± 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca 2+ stores; the molecular-complexity and spatial heterogeneity of RyRs may facilitate specific Ca 2+ regulation of cellular functions in PASMCs. Copyright © 2005 the American Physiological Society.link_to_subscribed_fulltex

Uncovering Resistant Genes in EGFR-mutated Lung Adenocarcinomas Prior to Targeted Therapy

Parallel Session 2 – Advanced Medical Research - no. S6Introduction & Project Objectives Lung cancer is the most lethal malignancy around the world. In Hong Kong, lung adenocarcinomas are driven by activating mutations of Epidermal Growth Factor Receptor (EGFR) in 20% male and 60% female patients. The metastatic mutant cancers are treated by targeted therapy using tyrosine kinase inhibitors (TKI) but drug responses are impaired by the presence of resistant mutations. Resistant mutations may involve EGFR downstream molecules or signaling cascades that bypass EGFR and support tumour cell proliferation and survival. These mutations are suggested to arise during TKI-selection implicating analysis of post treatment cancers is necessary for their identification but such tumours often have limited accessibility and small biopsy yield. On the other hand, it has been suggested resistant mutations are already present in cancers excised at the time of diagnosis preceding tumour metastasis or targeted therapy. This implicates analysis of excision samples which provide sufficient tumour quantity without imposing extra patient discomfort could be useful for uncovering second line treatment targets when TKI resistance occurs. This study aims to test the applicability of this approach. Methods The whole exome mutation profiles of 39 EGFR mutant lung adenocarcinomas was compared with the TKI response pattern of 16 non-responders and 23 responders. Four post-TKI tumors with acquired resistance were also analyzed. Results The tumours harboured 26 recurrent, non-synonymous single nucleotide variations (SNV) or insertion/deletion (INDEL) mutations of actionable targets and known cancer genes. Excluding EGFR, TP53 was the most common mutant. EGFR T790M was detected in all the post-TKI tumours but not pre-treatment samples from responders or non-responders. Various mutations of EGFR bypass cascades were involved in the non-responder group only, including recurrent PTEN, PIK3CA, NF1, and single case AKT1, ALK, RAF1 and KDR mutations. Notably, mutations in the β-catenin pathway were detected in the non-responders and post-TKI tumours, including APC, CTNNB1 and c-MYC. Putative novel resistant candidates were also observed. Conclusion and Discussion Our study demonstrated known and candidate TKI-resistant mutations could be uncovered by next generation sequencing of pre-treatment excision specimens of EGFR-mutant lung adenocarcinomas. The specific mutations identified in individual tumours could be useful for personalized medicine, offering customized targets for long term therapy and specific biomarkers for tumour surveillance

Dose-response relationship of nasopharyngeal carcinoma above conventional tumoricidal level: A study by the Hong Kong nasopharyngeal carcinoma study group (HKNPCSG)

Background and purpose: To define the dose-response relationship of nasopharyngeal carcinoma (NPC) above the conventional tumoricidal dose level of 66 Gy when the basic radiotherapy (RT) course was given by the 2D Ho's technique. Patients and methods: Data from all five regional cancer centers in Hong Kong were pooled for this retrospective study. All patients (n=2426) were treated with curative-intent RT with or without chemotherapy between 1996 and 2000 with the basic RT course using the Ho's technique. The primary endpoint was local control. The prognostic significance of dose-escalation ('boost') after 66 Gy, T-stage, N-stage, use of chemotherapy, sex and age (≤40 years vs >40 years) was studied. Both univariate and multivariate analyses were performed. Results: On multivariate analysis, T-stage (P< 0.01; hazard ratio [HR], 1.58) and optimal boost (P=0.01; HR, 0.34) were the only significant factors affecting local failure for the whole study population, and for the population of patients treated by radiotherapy alone, but not for patients who also received chemotherapy. The following were independent determinants of local failure for patient groups with different T-stages treated by radiotherapy alone: use of a boost in T1/T2a disease (P=0.01; HR, 0.33); use of a boost (P<0.01; HR, 0.60) and age (P=0.01; HR, 1.02) in T3/T4 tumors. Among patients with T2b tumors treated by radiotherapy alone and given a boost, the use of a 20 Gy-boost gave a lower local failure rate than a 10 Gy-boost. There was no apparent excess mortality attributed to RT complications. Conclusions: Within the context of a multi-center retrospective study, dose-escalation above 66 Gy significantly improved local control for T1/T2a and T3/4 tumors when the primary RT course was based on the 2D Ho's technique without additional chemotherapy. 'Boosting' in NPC warrants further investigation. Caution should be taken when boosting is considered because of possible increase in radiation toxicity. © 2006 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex