Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1.

BACKGROUND: The human p.G2434R variant of the RYR1 gene is most frequently associated with malignant hyperthermia (MH) in the UK. We report the phenotype of a knock-in mouse that expresses the RYR1 variant p.G2435R, which is isogenetic with the human variant. METHODS: We observed the general phenotype; determined the sensitivity of myotubes to caffeine-, KCl, and halothane-induced Ca2+ release; determined the in vivo response to halothane or increased ambient temperature; and determined the in vivo myoplasmic intracellular Ca2+ concentration in skeletal muscle before and during exposure to volatile anaesthetics. RESULTS: RYR1 pG2435R/MH normal (MHS-Heterozygous[Het]) or RYR1 pG2435R/pG2435R (MHS-Homozygous[Hom]) mice were fully viable under typical rearing conditions, although some male MHS-Hom mice died spontaneously. The normalised half-maximal effective concentration (95% confidence interval) for intracellular Ca2+ release in myotubes in response to KCl [MH normal, MHN, 21.4 (19.8-23.1) mM; MHS-Het 16.2 (15.2-17.2) mM; MHS-Hom 11.2 (10.2-12.2) mM] and caffeine (MHN, 5.7 (5-6.3) mM; MHS-Het 4.5 (3.9-5.0) mM; MHS-Hom 1.77 (1.5-2.1) mM] exhibited a gene dose-dependent decrease, and there was a gene dose-dependent increase in halothane sensitivity. Intact animals show a gene dose-dependent susceptibility to MH with volatile anaesthetics or to heat stroke. RYR1 p.G2435R mice had elevated skeletal muscle intracellular resting [Ca2+]i, (values are expressed as mean (SD)) (MHN 123 (3) nM; MHS-Het 156 (16) nM; MHS-Hom 265 (32) nM; P<0.001) and [Na+]i (MHN 8 (0.1) mM; MHS-Het 10 (1) mM; MHS-Hom 14 (0.7) mM; P<0.001) that was further increased by exposure to volatile anaesthetics. CONCLUSIONS: RYR1 pG2435R mice demonstrated gene dose-dependent in vitro and in vivo responses to pharmacological and environmental stressors that parallel those seen in patients with the human RYR1 variant p.G2434R.

Both isoforms of ketohexokinase are dispensable for normal growth and development.

Dietary fructose intake has dramatically increased over recent decades and is implicated in the high rates of obesity, hypertension, and type 2 diabetes (metabolic syndrome) in Western societies. The molecular determinants of this epidemiologic correlation are incompletely defined, but high-flux fructose catabolism initiated by ketohexokinase (Khk, fructokinase) is believed to be important. The Khk gene encodes two enzyme isoforms with distinctive substrate preferences, the independent physiological roles of which are unclear. To investigate this question, and for testing the importance of Khk in metabolic syndrome, isoform-selective genetic lesions would be valuable. Two deficiency alleles of the mouse Khk gene were designed. The first, Khk(3a), uses targeted "knock-in" of a premature termination codon to induce a selective deficiency of the minor Khk-A isoform, preserving the major Khk-C isoform. The second, the Khk(Δ) allele, ablates both isoforms. Mice carrying each of these Khk-deficiency alleles were generated and validated at the DNA, RNA, and protein levels. Comparison between normal and knockout animals confirmed the specificity of the genetic lesions and allowed accurate analysis of the cellular distribution of Khk within tissues such as gut and liver. Both Khk(3a/3a) and Khk(Δ/Δ) homozygous mice were healthy and fertile and displayed minimal biochemical abnormalities under basal dietary conditions. These studies are the first demonstration that neither Khk isoform is required for normal growth and development. The new mouse models will allow direct testing of various hypotheses concerning the role of this enzyme in metabolic syndrome in humans and the value of Khk as a pharmacological target.

Inhibition of double-strand break non-homologous end-joining by cisplatin adducts in human cell extracts.

The effect of cis-diaminedichloroplatinum(II) (cisplatin) DNA damage on the repair of double-strand breaks by non-homologous end-joining (NHEJ) was determined using cell-free extracts. NHEJ was dramatically decreased when plasmid DNA was damaged to contain multiple types of DNA adducts, along the molecule and at the termini, by incubation of DNA with cisplatin; this was a cisplatin concentration-dependent effect. We investigated the effect a single GTG cisplatination site starting 10 bp from the DNA termini would have when surrounded by the regions of AT-rich DNA which were devoid of the major adduct target sequences. Cisplatination of a substrate containing short terminal 13-15 bp AT-rich sequences reduced NHEJ to a greater extent than that of a substrate with longer (31-33 bp) AT-rich sequences. However, cisplatination at the single GTG site within the AT sequence had no significant effect on NHEJ, owing to the influence of additional minor monoadduct and dinucleotide adduct sites within the AT-rich region and owing to the influence of cisplatination at sites upstream of the AT-rich regions. We then studied the effect on NHEJ of one cis-[Pt(NH3)2{d(GpTpG)-N7(1),-N7(3)} [abbreviated as 1,3-d(GpTpG)] cisplatin adduct in the entire DNA molecule, which is more reflective of the situation in vivo during concurrent chemoradiation. The presence of a single 1,3-d(GpTpG) cisplatin adduct 10 bases from each of the two DNA ends to be joined resulted in a small (30%) but significant decrease in NHEJ efficiency. This process, which was DNA-dependent protein kinase and Ku dependent, may in part explain the radiosensitizing effect of cisplatin administered during concurrent chemoradiation.

Role of p53 in the responses of human urothelial cells to genotoxic damage.

Loss of p53 function is a feature of many types of malignancy, including transitional-cell carcinoma (TCC), where it is associated with high-grade lesions and the development of muscle-invasive disease. Genotoxic agents used as part of the treatment strategy may contribute to tumour progression by inducing further non-lethal DNA damage in surviving cells. To determine the role of p53 in cellular responses to genotoxic agents, we used cultured normal human urothelial (NHU) cells and NHU cells with disabled p53 function. Mitomycin C and gamma-radiation caused normal cells to undergo an extended period of cell-cycle arrest, followed by complete recovery of proliferative potential. In contrast, cells with disabled p53 function, whether karyotypically normal (HU-E6 cells) or post-crisis with karyotypic abnormalities (HU-E6P cells), underwent extensive apoptosis. Overall survival was dose-dependent, and surviving HU-E6 cells from low-dose treatments showed clonal karyotypic abnormalities. These findings demonstrate that p53 status is a crucial factor in determining the ability of urothelial cells to survive DNA damage and suggest caution in the use of genotoxic treatments for low-grade tumours as our data imply that malignancies that have not yet lost p53 function will show the same "repair-and-recovery" response as normal cells.

N;-3 and n;-6 polyunsaturated fatty acids induce cytostasis in human urothelial cells independent of p53 gene function.

The role of long-chain polyunsaturated fatty acids (PUFA) in the etiopathology and treatment of cancer is poorly understood. We have studied the effects of n;-3 and n;-6 PUFA on the proliferation and survival of normal human uroepithelial (NHU) cells, cells with disabled p53 function after stable transfection with the human papillomavirus 16 (HPV16) E6 gene (HU-E6), and p53-disabled cells that had passed through crisis and acquired karyotypic abnormalities (HU-E6P). The n;-3 and n;-6 PUFA had distinct reversible antiproliferative and irreversible cytostatic effects according to concentration and exposure time. The reversible antiproliferative effect was partly due to the production of lipoxygenase metabolites. NHU and HU-E6 cells were equally sensitive to n;-3 and n;-6 PUFA, but HU-E6P cells were more resistant to both the antiproliferative and cytostatic effects. Cytostatic concentrations of n;-3 and n;-6 PUFA did not induce apoptosis, but caused permanent growth arrest ("interphase" or "reproductive" cell death) and mRNA levels for genes involved in cell cycle control (p21, p16, p27, cdk1, cdk2, and cdk4) were not altered. Neither n;-3 nor n;-6 PUFA promoted acquisition of karyotypic abnormalities in HU-E6 cells, suggesting that n;-3 and n;-6 PUFA do not cause genotoxic damage. In conclusion, our studies show that the antiproliferative and cytostatic effects of n;-3 and n;-6 PUFA are not dependent on p53 function and, further, that transformation results in a loss of sensitivity to n;-3 and n;-6 PUFA-mediated growth inhibition.

Effects of long-chain fatty acids on human urothelial cells in organ culture.

It has been suggested that tumour-derived cells are differentially sensitive to the anti-proliferative and cytotoxic effects of long chain n-3 and n-6 polyunsaturated fatty acids (PuFAs). We have previously shown that PuFAs are also growth suppressive to highly proliferative normal human urinary bladder uro-epithelial (NHU) cells grown in monolayer culture. To determine if the effects on NHU cells are directly related to the proliferative index, we have studied the effects of long chain fatty acids in a bladder organ culture system, where proliferation and differentiation of the urothelium is under homeostatic control. A 50 microM concentration of fatty acids was chosen as this concentration of PuFA was profoundly growth inhibitory to NHU cells in monolayer culture. In organ culture, 50 microM PuFAs had no detectable effect on the proliferation or on the preservation of urothelial differentiated histioarchitecture, as assessed using a panel of phenotypic markers. These results suggest that the effects of PuFA may be modulated by the tissue microenvironment.