Rlip Depletion Alters Oncogene Transcription at Multiple Distinct Regulatory Levels.

Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status.

Anticancer Activity of Ω-6 Fatty Acids through Increased 4-HNE in Breast Cancer Cells.

Her2-amplified breast cancers resistant to available Her2-targeted therapeutics continue to be a challenge in breast cancer therapy. Dox is the mainstay of chemotherapy of all types of breast cancer, but its usefulness is limited by cumulative cardiotoxicity. Because oxidative stress caused by dox generates the pro-apoptotic Ω-6 PUFA metabolite 4-hydroxynonenal (4-HNE), we surmised that Ω-6 PUFAs would increase the effectiveness of dox chemotherapy. Since the mercapturic acid pathway enzyme RALBP1 (also known as RLIP76 or Rlip) that limits cellular accumulation of 4-HNE also mediates dox resistance, the combination of Ω-6 PUFAs and Rlip depletion could synergistically improve the efficacy of dox. Thus, we studied the effects of the Ω-6 PUFA arachidonic acid (AA) and Rlip knockdown on the antineoplastic activity of dox towards Her2-amplified breast cancer cell lines SK-BR-3, which is sensitive to Her2 inhibitors, and AU565, which is resistant. AA increased lipid peroxidation, 4-HNE generation, apoptosis, cellular dox concentration and dox cytotoxicity in both cell lines while sparing cultured immortalized cardiomyocyte cells. The known functions of Rlip including clathrin-dependent endocytosis and dox efflux were inhibited by AA. Our results support a model in which 4-HNE generated by AA overwhelms the capacity of Rlip to defend against apoptosis caused by dox or 4-HNE. We propose that Ω-6 PUFA supplementation could improve the efficacy of dox or Rlip inhibitors for treating Her2-amplified breast cancer.

RALBP1 in Oxidative Stress and Mitochondrial Dysfunction in Alzheimer's Disease.

The purpose of our study is to understand the role of the RALBP1 gene in oxidative stress (OS), mitochondrial dysfunction and cognition in Alzheimer's disease (AD) pathogenesis. The RALPB1 gene encodes the 76 kDa protein RLIP76 (Rlip). Rlip functions as a stress-responsive/protective transporter of glutathione conjugates (GS-E) and xenobiotic toxins. We hypothesized that Rlip may play an important role in maintaining cognitive function. The aim of this study is to determine whether Rlip deficiency in mice is associated with AD-like cognitive and mitochondrial dysfunction. Brain tissue obtained from cohorts of wildtype (WT) and Rlip+/- mice were analyzed for OS markers, expression of genes that regulate mitochondrial fission/fusion, and synaptic integrity. We also examined mitochondrial ultrastructure in brains obtained from these mice and further analyzed the impact of Rlip deficiency on gene networks of AD, aging, stress response, mitochondrial function, and CREB signaling. Our studies revealed a significant increase in the levels of OS markers and alterations in the expression of genes and proteins involved in mitochondrial biogenesis, dynamics and synapses in brain tissues from these mice. Furthermore, we compared the cognitive function of WT and Rlip+/- mice. Behavioral, basic motor and sensory function tests in Rlip+/- mice revealed cognitive decline, similar to AD. Gene network analysis indicated dysregulation of stress-activated gene expression, mitochondrial function and CREB signaling genes in the Rlip+/- mouse brain. Our results suggest that Rlip deficiency-associated increases in OS and mitochondrial dysfunction could contribute to the development or progression of OS-related AD processes.

Haploinsufficiency Interactions between RALBP1 and p53 in ERBB2 and PyVT Models of Mouse Mammary Carcinogenesis.

We recently reported that loss of one or both alleles of Ralbp1, which encodes the stress-protective protein RLIP76 (Rlip), exerts a strong dominant negative effect on both the inherent cancer susceptibility and the chemically inducible cancer susceptibility of mice lacking one or both alleles of the tumor suppressor p53. In this paper, we examined whether congenital Rlip deficiency could prevent genetically-driven breast cancer in two transgenic mouse models: the MMTV-PyVT model, which expresses the polyomavirus middle T antigen (PyVT) under control of the mouse mammary tumor virus promoter (MMTV) and the MMTV-Erbb2 model which expresses MMTV-driven erythroblastic leukemia viral oncogene homolog 2 (Erbb2, HER2/Neu) and frequently acquires p53 mutations. We found that loss of either one or two Rlip alleles had a suppressive effect on carcinogenesis in Erbb2 over-expressing mice. Interestingly, Rlip deficiency did not affect tumor growth but significantly reduced the lung metastatic burden of breast cancer in the viral PyVT model, which does not depend on either Ras or loss of p53. Furthermore, spontaneous tumors of MMTV-PyVT/Rlip+/+ mice showed no regression following Rlip knockdown. Finally, mice lacking one or both Rlip alleles differentially expressed markers for apoptotic signaling, proliferation, angiogenesis, and cell cycling in PyVT and Erbb2 breast tumors. Our results support the efficacy of Rlip depletion in suppressing p53 inactivated cancers, and our findings may yield novel methods for prevention or treatment of cancer in patients with HER2 mutations or tumor HER2 expression.

Dietary supplementation with sulforaphane ameliorates skin aging through activation of the Keap1-Nrf2 pathway.

Visible impairments in skin appearance, as well as a subtle decline in its functionality at the molecular level, are hallmarks of skin aging. Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-pathway, which is important in controlling inflammation and oxidative stress that occur during aging, can be triggered by sulforaphane (SFN), an isothiocyanate found in plants from the Brassicaceae family. This study aimed to assess the effects of SFN intake on age-related skin alterations. Male C57BL6 young (2 months) and old (21 months) mice were treated for 3 months with SFN diet (442.5 mg per kg) or control diet. The antioxidant capacities of the skin were increased in old SFN-treated animals as measured by mRNA levels of Nrf2 (P<.001) and its target genes NQO1 (P<.001) and HO1 (P<.01). Protein expression for Nrf2 was also increased in old SFN fed animals (P<.01), but not the protein expression of NQO1 or HO1. Additionally, ROS and MMP9 protein levels were significantly decreased (P<.05) in old SFN fed animals. Histopathological analysis confirmed that there was no difference in epidermal thickness in old, when compared to young, SFN treated animals, while the dermal layer thickness was lower in old vs. young, treated animals (P<.05). Moreover, collagen deposition was improved with SFN treatment in young (P<.05) and structurally significantly improved in the old mice (P<.001). SFN dietary supplementation therefore ameliorates skin aging through activation of the Nrf2-pathway.

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01-3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01-100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. SIGNIFICANCE STATEMENT: ATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

Multi-Omic Analysis Reveals Different Effects of Sulforaphane on the Microbiome and Metabolome in Old Compared to Young Mice.

Dietary factors modulate interactions between the microbiome, metabolome, and immune system. Sulforaphane (SFN) exerts effects on aging, cancer prevention and reducing insulin resistance. This study investigated effects of SFN on the gut microbiome and metabolome in old mouse model compared with young mice. Young (6-8 weeks) and old (21-22 months) male C57BL/6J mice were provided regular rodent chow ± SFN for 2 months. We collected fecal samples before and after SFN administration and profiled the microbiome and metabolome. Multi-omics datasets were analyzed individually and integrated to investigate the relationship between SFN diet, the gut microbiome, and metabolome. The SFN diet restored the gut microbiome in old mice to mimic that in young mice, enriching bacteria known to be associated with an improved intestinal barrier function and the production of anti-inflammatory compounds. The tricarboxylic acid cycle decreased and amino acid metabolism-related pathways increased. Integration of multi-omic datasets revealed SFN diet-induced metabolite biomarkers in old mice associated principally with the genera, Oscillospira, Ruminococcus, and Allobaculum. Collectively, our results support a hypothesis that SFN diet exerts anti-aging effects in part by influencing the gut microbiome and metabolome. Modulating the gut microbiome by SFN may have the potential to promote healthier aging.

Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling.

Age-associated mitochondrial dysfunction and oxidative damage are primary causes for multiple health problems including sarcopenia and cardiovascular disease (CVD). Though the role of Nrf2, a transcription factor that regulates cytoprotective gene expression, in myopathy remains poorly defined, it has shown beneficial properties in both sarcopenia and CVD. Sulforaphane (SFN), a natural compound Nrf2-related activator of cytoprotective genes, provides protection in several disease states including CVD and is in various stages of clinical trials, from cancer prevention to reducing insulin resistance. This study aimed to determine whether SFN may prevent age-related loss of function in the heart and skeletal muscle. Cohorts of 2-month-old and 21- to 22-month-old mice were administered regular rodent diet or diet supplemented with SFN for 12 weeks. At the completion of the study, skeletal muscle and heart function, mitochondrial function, and Nrf2 activity were measured. Our studies revealed a significant drop in Nrf2 activity and mitochondrial functions, together with a loss of skeletal muscle and cardiac function in the old control mice compared to the younger age group. In the old mice, SFN restored Nrf2 activity, mitochondrial function, cardiac function, exercise capacity, glucose tolerance, and activation/differentiation of skeletal muscle satellite cells. Our results suggest that the age-associated decline in Nrf2 signaling activity and the associated mitochondrial dysfunction might be implicated in the development of age-related disease processes. Therefore, the restoration of Nrf2 activity and endogenous cytoprotective mechanisms by SFN may be a safe and effective strategy to protect against muscle and heart dysfunction due to aging.

Rlip Depletion Suppresses Growth of Breast Cancer.

RLIP76 (RAL-binding protein-1, Rlip) is a stress-protective mercapturic-acid-pathway transporter protein that also plays a key role in regulating clathrin-dependent endocytosis as a Ral effector. Targeted inhibition or depletion of Rlip causes regression of xenografts of many cancers and is capable of abrogating tumor formation in p53-null mice. This is associated with the reversion of the abnormal methylomic profile of p53-null mice to wild-type. In a query of The Cancer Genome Atlas (TCGA) databases, we found that Rlip expression was associated with poor survival and with significant differences in the frequencies of PIK3CA mutation, MYC amplification, and CDKN2A/B deletion, which were the most commonly mutated, amplified, and deleted genes, respectively, among TCGA breast cancer patients. We conducted the present study to further examine the effects of Rlip inhibition and to evaluate the in vitro and in vivo efficacy in breast cancer. Using immunogold electron microscopy, we found that plasma-membrane Rlip was accessible to cell-surface antibodies in the MCF7 (ER+) breast cancer cell line. Rlip depletion resulted in decreased survival of MCF7 and MDA-MB-231 cells and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity and DNA laddering, indicating apoptotic cell death. Additionally, in vitro knockdown of Rlip inhibited EGF endocytosis and WNT/MAPK signaling. Xenograft studies in nude mice showed regression of breast cancer via antisense-mediated depletion of Rlip mRNA as well as by anti-Rlip antibody. Finally, knockdown of Rlip by antisense locked nucleic acid oligonucleotides increased markers for apoptotic signaling and decreased markers for proliferation, angiogenesis, and cell cycling in MCF7 and MDA-MB-231luc xenografts. Our findings validate Rlip as an attractive target in breast cancer.

Topical 2'-Hydroxyflavanone for Cutaneous Melanoma.

2'-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towardsmultiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermalimplants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growthof the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro.Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SKMEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signalingproteins TNFα, and phospho-PDGFR-ß were depleted in all three cell lines; MEKK-15 was depletedby 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-ß inhibitor) and AZD2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive,antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously beenshown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evidentfrom inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topicallyapplied 2HF-Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumorsimplanted in mice and caused no overt toxicity despite significant systemic absorption. 2HFtreatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas itincreased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled byendocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF-PLO gel maybe useful for topical therapy of cutaneous metastases of melanoma and could enhance theantineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF inmelanoma overlaps with RLI76 inhibitors.

High fat diet causes renal fibrosis in LDLr-null mice through MAPK-NF-κB pathway mediated by Ox-LDL.

BACKGROUND: Dyslipidemia, particularly increased LDL-cholesterol level in serum, is associated with atherosclerosis and fibrosis in different organs. This study was designed to investigate the effects of increase in LDL-cholesterol on renal fibrosis. METHODS: Wild-type (WT) and LDLr knockout (KO) mice were fed standard or high fat diet (HFD), and their kidneys were collected after 26 weeks of dietary intervention for identification of fibrosis and study of potential mechanisms. Additional studies were performed in cultured renal fibroblasts. RESULTS: We observed extensive and diffuse fibrosis in the kidneys of mice given HFD (P < 0.05 vs. standard chow). Fibrosis was associated with enhanced expression of fibronectin, nicotinamide adenine dinucleotide phosphate oxidases and activated p38 and p44/42 mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). There was evidence for accumulation of 4-hydroxynonenal, a lipid peroxidation product, in the kidneys and of ox-LDL in the arteries of LDLr KO mice given HFD. The expression of ox-LDL receptor LOX-1 and of transforming growth factor beta 1 (TGFß1) was increased in these kidneys. All these changes were more pronounced in LDLr KO mice than in the WT mice. In in vitro studies, treatment of fibroblasts from kidneys of LDLr KO mice with ox-LDL showed intense proliferation and collagen formation (all P < 0.05, fibroblasts from WT mice kidneys). Blockade of p38 MAPK, p44/42 MAPK, or NF-κB significantly attenuated expression of profibrotic signals, collagen formation, and proliferation of fibroblasts. CONCLUSIONS: HFD induces renal fibrosis in LDLr-null mice primarily through activation of the nicotinamide adenine dinucleotide phosphate oxidase MAPK-NF-κB pathway by ox-LDL.

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: a coupled electromechanical modeling study.

BACKGROUND: Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). METHODS: Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa,L); and the enhancement in SERCA pump activity via phosphorylation of PLB. RESULTS: Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated ß-adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa,L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. CONCLUSIONS: Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa,L as the key mechanisms underlying the aforementioned positive FFR.

Multiphysics model of a rat ventricular myocyte: a voltage-clamp study.

BACKGROUND: The objective of this study is to develop a comprehensive model of the electromechanical behavior of the rat ventricular myocyte to investigate the various factors influencing its contractile response. METHODS: Here, we couple a model of Ca2 + dynamics described in our previous work, with a well-known model of contractile mechanics developed by Rice, Wang, Bers and de Tombe to develop a composite multiphysics model of excitation-contraction coupling. This comprehensive cell model is studied under voltage clamp (VC) conditions, since it allows to focus our study on the elaborate Ca2 + signaling system that controls the contractile mechanism. RESULTS: We examine the role of various factors influencing cellular contractile response. In particular, direct factors such as the amount of activator Ca2 + available to trigger contraction and the type of mechanical load applied (resulting in isosarcometric, isometric or unloaded contraction) are investigated. We also study the impact of temperature (22 to 38°C) on myofilament contractile response. The critical role of myofilament Ca2 + sensitivity in modulating developed force is likewise studied, as is the indirect coupling of intracellular contractile mechanism with the plasma membrane via the Na + /Ca2 + exchanger (NCX). Finally, we demonstrate a key linear relationship between the rate of contraction and relaxation, which is shown here to be intrinsically coupled over the full range of physiological perturbations. CONCLUSIONS: Extensive testing of the composite model elucidates the importance of various direct and indirect modulatory influences on cellular twitch response with wide agreement with measured data on all accounts. Thus, the model provides mechanistic insights into whole-cell responses to a wide variety of testing approaches used in studies of cardiac myofilament contractility that have appeared in the literature over the past several decades.

Angiotensin II upregulates Ca(V)1.2 protein expression in cultured arteries via endothelial H(2)O(2) production.

BACKGROUND: We previously reported that angiotensin II caused an endothelial-dependent increase in L-type voltage-dependent Ca(2+) channel (Ca(V)1.2) in cultured arteries, but the signaling pathways are not clear. METHODS: Endothelial damage was generated by brief intra-arterial perfusion with 0.3% CHAPS. Ca(V)1.2 expression, function and H(2)O(2) were measured by Western blot, tension recording and Amplex Red H(2)O(2) assay kit, respectively. RESULTS: Angiotensin II dose-dependently upregulated Ca(V)1.2 expression in endothelium-intact arteries. The angiotensin II upregulation of Ca(V)1.2 expression in endothelium-intact arteries was blocked by NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI), apocynin, a more specific NAD(P)H oxidase inhibitor gp91ds-tat and also by catalase. H(2)O(2) similarly upregulated Ca(V)1.2 expression in endothelium-intact and endothelium-damaged arteries, and the latter effect was also blocked by DPI and apocynin. Angiotensin II increased H(2)O(2) production by endothelium-intact but not by endothelium-damaged arteries, and this effect was blocked by apocynin, catalase and gp91ds-tat. The upregulation of Ca(V)1.2 by angiotensin II and H(2)O(2) is accompanied by an increased tension response to KCl and the Ca(2+) channel activator FPL 64176, and this effect was also attenuated by gp91ds-tat. CONCLUSION: These results suggest that angiotensin II stimulates endothelial NAD(P)H oxidase-produced H(2)O(2,) which may additionally act through vascular smooth muscle NAD(P)H oxidase, to upregulate vascular Ca(V)1.2 protein.

Modeling CICR in rat ventricular myocytes: voltage clamp studies.

BACKGROUND: 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 Ca²(+) loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca²(+) 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 Ca²(+) concentration ([Ca²(+)](myo)). METHODS: 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 Ca²(+) channels (trigger-channel and release-channel). It releases Ca²(+) flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca²(+)](myo). RESULTS: Our model reproduces measured VC data published by several laboratories, and generates graded Ca²(+) release at high Ca²(+) gain in a homeostatically-controlled environment where [Ca²(+)](myo) is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca²(+) release, its activation by trigger Ca²(+), and its refractory characteristics mediated by the luminal SR Ca²(+) sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence Ca²(+) homeostasis. CONCLUSIONS: We examine the role of the above Ca²(+) regulating mechanisms in handling various types of induced disturbances in Ca²(+) levels by quantifying cellular Ca²(+) balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.

Cardiac hypertrophy during hypercholesterolemia and its amelioration with rosuvastatin and amlodipine.

Hypercholesterolemia is a common accompaniment of atherosclerosis and may be associated with cardiac hypertrophy. To define the mechanistic basis of cardiac hypertrophy in hypercholesterolemia, we fed low-density lipoprotein receptor knockout (LDLR KO) mice regular diet or high cholesterol (HC) diet for 26 weeks. There was clear evidence of cardiomyocyte hypertrophy and collagen deposition in the hearts of LDLR KO mice fed with HC diet, confirmed by histopathology (hematoxylin and eosin and Picrosirius staining) and upregulation of genes for brain natriuretic peptide, alpha-tubulin, transforming growth factor beta1, and connective tissue growth factor (CTGF). These changes were independent of change in blood pressure. The hypercholesterolemic mice hearts showed an upregulation of LOX-1, an oxidized low-density lipoprotein receptor, and angiotensin II type 1 receptor (AT1R) at messenger RNA level. In addition, there was a marked upregulation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappaB (NF-kappaB) messenger RNA, indicating overexpression of markers of oxidant stress. A separate group of LDLR KO mice were fed HC diet along with a potent 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor rosuvastatin or a dihydropyridine calcium channel blocker amlodipine. Administration of rosuvastatin or amlodipine reduced the overexpression of genes for LOX-1 and AT1R and associated NADPH oxidase and NF-kappaB. These phenomena were associated with a marked decrease in cardiomyocyte hypertrophy and collagen deposits in and around the cardiomyocytes. In conclusion, this study provides evidence of cardiac hypertrophy and fibrosis in hypercholesterolemia independent of blood pressure change LOX-1 and AT1R act as possible signals for oxidant stress leading to alterations in cardiac structure during hypercholesterolemia. Most importantly, rosuvastatin and amlodipine ameliorate cardiomyocyte hypertrophy and fibrosis.

Angiotensin II causes endothelial-dependent increase in expression of Ca(V)1.2 protein in cultured arteries.

Examination was made of the direct vascular effects of the hypertension-inducing pressor hormone angiotensin II on expression and activity of the voltage-gated calcium channel Ca(V)1.2. Freshly dissected rat superior mesenteric artery beds were maintained in organ culture unpressurized for 24 h in the presence or absence of angiotensin II. Relative to controls, angiotensin II increased Ca(V)1.2 protein expression and tension-inducing activity but not Ca(V)1.2 message. The increase in Ca(V)1.2 protein expression by angiotensin II was abrogated by damaging the endothelium. Thus, the endothelium is involved in regulating Ca(V)1.2 expression in the vascular wall.

High-frequency fatigue of skeletal muscle: role of extracellular Ca(2+).

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.