Jacob Hofman-Bang

Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation.

BACKGROUND Atrial fibrillation (AF) is the most common clinical arrhythmia and a major cause of cardiovascular morbidity and mortality. Among the gene defects previously associated with AF is a gain of function of the slowly activating delayed rectifier potassium current IKs, secondary to mutations in KCNQ1. Coexpression of KCNE5, the gene encoding the MiRP4 beta-subunit, has been shown to reduce IKs. OBJECTIVE The purpose of this study was to test the hypothesis that mutations in KCNE5 are associated with AF in a large cohort of patients with AF. METHODS One-hundred fifty-eight patients with AF were screened for mutations in the coding region of KCNE5. RESULTS A missense mutation involving substitution of a phenylalanine for leucine at position 65 (L65F) was identified in one patient. This patient did not have a history of familial AF, and neither KCNQ1 nor KCNE2 mutations were found. Transient transfection of Chinese hamster ovary (CHO) cells expressing IKs(KCNQ1+KCNE1) with KCNE5 suppressed the developing and tail currents of IKs in a concentration-dependent manner. Transient transfection with KCNE5-L65F failed to suppress IKs, yielding a current indistinguishable from that recorded in the absence of KCNE5. Developing currents recorded during a test pulse to +60 mV and tail currents recorded upon repolarization to -40 mV both showed a significant concentration-dependent gain of function in IKs with expression of KCNE5-L65F vs KCNE5-WT. CONCLUSION The results of this study suggest that a missense mutation in KCNE5 may be associated with nonfamilial or acquired forms of AF. The arrhythmogenic mechanism most likely is a gain of function of IKs.

Increased parathyroid expression of klotho in uremic rats.

Klotho is a protein of significant importance for mineral homeostasis. It helps to increase parathyroid hormone (PTH) secretion and in the trafficking of Na+/K+-ATPase to the cell membrane; however, it is also a cofactor for fibroblast growth factor (FGF)-23 to interact with its receptor, FGFR1 IIIC, resulting in decreased PTH secretion. Studies on the regulation of parathyroid klotho expression in uremia have provided varying results. To help resolve this, we measured klotho expression in the parathyroid and its response to severe uremia, hyperphosphatemia, and calcitriol treatment in the 5/6 nephrectomy rat model of secondary hyperparathyroidism. Parathyroid klotho gene expression and protein were significantly increased in severely uremic hyperphosphatemic rats, but not affected by moderate uremia and normal serum phosphorus. Calcitriol suppressed klotho gene and protein expression in severe secondary hyperparathyroidism, despite a further increase in plasma phosphate. Both FGFR1 IIIC and Na+/K+-ATPase gene expression were significantly elevated in severe secondary hyperparathyroidism. Parathyroid gland klotho expression and the plasma calcium ion concentration were inversely correlated. Thus, our study suggests that klotho may act as a positive regulator of PTH expression and secretion in secondary hyperparathyroidism.

High-efficiency multiplex capillary electrophoresis single strand conformation polymorphism (multi-CE-SSCP) mutation screening of SCN5A: a rapid genetic approach to cardiac arrhythmia

Mutations in the SCN5A gene coding for the α‐subunit of the cardiac Na+ ion channel cause long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, sick sinus node syndrome, progressive conduction disease, dilated cardiomyopathy and atrial standstill. These diseases exhibit variable expressivity, and identification of gene carriers is clinically important, particularly in sudden infant and adult death syndromes. The SCN5A gene comprises 28 exons distributed over 100 kbp of genomic sequence at chromosome 3p21. Disease‐causing mutations are private and scattered over the DNA sequence, making it difficult to screen for specific mutations. We developed a multiplex capillary‐electrophoresis single‐strand conformation polymorphism (Multi‐CE‐SSCP) mutation screening protocol on the ABI 3100 platform and applied it to 10 previously slab‐gel SSCP identified mutations and SNPs and used it to identify one novel deletion. The method is highly efficient, with a turnover of 23 patients per 24 h and a false positive rate of 0.5% of the analyzed amplicons. Each variant has a particular elution pattern, and all 20 carriers of the H558R polymorphism out of 57 persons were correctly identified. We suggest that the method could become part of routine work‐up of patients with suspicious syncope and of members of families with sudden unexplained death.

Key role of the kidney in the regulation of fibroblast growth factor 23

High circulating levels of fibroblast growth factor 23 (FGF23) have been demonstrated in kidney failure, but mechanisms of this are not well understood. Here we examined the impact of the kidney on the early regulation of intact FGF23 in acute uremia as induced by bilateral or unilateral nephrectomy (BNX and UNX, respectively) in the rat. BNX induced a significant increase in plasma intact FGF23 levels from 112 to 267 pg/ml within 15 min, which remained stable thereafter. UNX generated intact FGF23 levels between that seen in BNX and sham-operated rats. The intact to C-terminal FGF23 ratio was significantly increased in BNX rats. The rapid rise in FGF23 after BNX was independent of parathyroid hormone or FGF receptor signaling. No evidence of early stimulation of FGF23 gene expression in the bone was found. Furthermore, acute severe hyperphosphatemia or hypercalcemia had no impact on intact FGF23 levels in normal and BNX rats. The half-life of exogenous recombinant human FGF23 was significantly prolonged from 4.4 to 11.8 min in BNX rats. Measurements of plasma FGF23 in the renal artery and renal vein demonstrated a significant renal extraction. Thus the kidney is important in FGF23 homeostasis by regulation of its plasma level and metabolism.

The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity.

The involvement of sodium/potassium-ATPase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the α-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested to be linked to increased hormone secretion. In this study, we used an in vivo rat model to determine the importance of sodium/potassium-ATPase in PTH secretion. Glands were exposed and treated in situ with vehicle or ouabain, a specific inhibitor of sodium/potassium-ATPase. PTH secretion was significantly increased in response to ethylene glycol tetraacetic acid-induced acute hypocalcemia and to the same extent in both vehicle and ouabain groups. The glands were removed, and inhibition of the ATPase was measured by (86)rubidium uptake, which was found to be significantly decreased in ouabain-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also examined but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium-ATPase activity in vivo had no effect on the secretory response to acute hypocalcemia. Hence, the suggested importance of this ATPase in the regulation of PTH secretion could not be confirmed in this in vivo model.

Effect of chronic uremia on the transcriptional profile of the calcified aorta analyzed by RNA sequencing

The development of vascular calcification (VC) in chronic uremia (CU) is a tightly regulated process controlled by factors promoting and inhibiting mineralization. Next-generation high-throughput RNA sequencing (RNA-seq) is a powerful and sensitive tool for quantitative gene expression profiling and the detection of differentially expressed genes. In the present study, we, for the first time, used RNA-seq to examine rat aorta transcriptomes from CU rats compared with control rats. Severe VC was induced in CU rats, which lead to extensive changes in the transcriptional profile. Among the 10,153 genes with an expression level of >1 reads/kilobase transcript/million mapped reads, 2,663 genes were differentially expressed with 47% upregulated genes and 53% downregulated genes in uremic rats. Significantly deregulated genes were enriched for ontologies related to the extracellular matrix, response to wounding, organic substance, and ossification. The individually affected genes were of relevance to osteogenic transformation, tissue calcification, and Wnt modulation. Downregulation of the Klotho gene in uremia is believed to be involved in the development of VC, but it is debated whether the effect is caused by circulating Klotho only or if Klotho is produced locally in the vasculature. We found that Klotho was neither expressed in the normal aorta nor calcified aorta by RNA-seq. In conclusion, we demonstrated extensive changes in the transcriptional profile of the uremic calcified aorta, which were consistent with a shift in phenotype from vascular tissue toward an osteochondrocytic transcriptome profile. Moreover, neither the normal vasculature nor calcified vasculature in CU expresses Klotho.

Circulating FGF23 Levels in Response to Acute Changes in Plasma Ca 2

The regulation of fibroblast growth factor 23 (FGF23) synthesis and secretion is still incompletely understood. FGF23 is an important regulator of renal phosphate excretion and has regulatory effects on the calciotropic hormones calcitriol and parathyroid hormone (PTH). Calcium (Ca) and phosphate homeostasis are closely interrelated, and it is therefore likely that Ca is involved in FGF23 regulation. It has recently been reported that dietary Ca influenced FGF23 levels, with high Ca increasing FGF23. The mechanism remains to be clarified. It remains unknown whether acute changes in plasma Ca influence FGF23 levels and whether a close relationship, similar that known for Ca and PTH, exists between Ca and FGF23. Thus, the aim of the present study was to examine whether acute hypercalcemia and hypocalcemia regulate FGF23 levels in the rat. Acute hypercalcemia was induced by an intravenous Ca infusion and hypocalcemia by infusion of ethylene glycol tetraacetic acid (EGTA) in normal and acutely parathyroidectomized rats. Intact plasma FGF23 and intact plasma PTH and plasma Ca2+ and phosphate were measured. Acute hypercalcemia and hypocalcemia resulted as expected in adequate PTH secretory responses. Plasma FGF23 levels remained stable at all plasma Ca2+ levels; acute parathyroidectomy did not affect FGF23 secretion. In conclusion, Ca is not a regulator of acute changes in FGF23 secretion.

Ergocalciferol treatment and aspects of mineral homeostasis in patients with chronic kidney disease stage 4-5.

Abstract Background. Focus on non-classical effects and possible less side effects of treatment with nutritional vitamin D, raises the expectation of possible benefits from treating chronic kidney disease (CKD) patients with ergocalciferol (vitamin D2). Treatment with 1,25(OH)2vitamin D (calcitriol) induces elevated fibroblast growth factor 23 (FGF23), while epidemiological studies have found positive effects of nutritional and 25(OH)vitamin D on mortality in CKD. Disturbed mineral homeostasis in CKD is correlated to adverse outcome and cardiovascular mortality. The objective was to examine the possible effects of treatment with high doses of ergocalciferol on parameters of mineral homeostasis in predialysis CKD patients. Methods. A total of 43 adult patients with CKD stage 4–5, not receiving vitamin D supplementation, were studied, and allocated by simple randomization to either an intervention (n = 26) or a control group (n = 17). The intervention group received ergocalciferol, 50.000 IU/week for 6 weeks. Plasma FGF23, creatinine, parathyroid hormone (PTH), phosphate and ionized calcium were obtained at baseline and after the 6 weeks. Results. The intervention group had a significant increase in 25(OH)D2 concentration from < 10 to 90 ± 4 nmol/L, while 1,25(OH)2D (62 ± 6 at baseline and 67 ± 6 pmol/L at 6 weeks) remained stable. No changes were seen in the circulating vitamin D concentrations in the control group. After the 6 weeks of treatment no significant changes were seen in concentration of creatinine, phosphate, ionized calcium, PTH and FGF23 remained stable. Conclusion. No harmful effects of short-term treatment with high-dose ergocalciferol were observed on markers of mineral homeostasis and FGF23 in CKD patients stage 4–5.

Ventricular tachycardia in a Brugada syndrome patient caused by a novel deletion in SCN5A

The aim of the present study was to identify the molecular mechanism behind ventricular tachycardia in a patient with Brugada syndrome. Arrhythmias in patients with Brugada syndrome often occur during sleep. However, a 28-year-old man with no previously documented arrhythmia or syncope who experienced shortness of breath and chest pain during agitation is described. An electrocardiogram revealed monomorphic ventricular tachycardia; after he was converted to nodal rhythm, he spontaneously went into sinus rhythm, and showed classic Brugada changes with coved ST elevation in leads V(1) to V(2). Mutation analysis of SCN5A revealed a novel mutation, 3480 deletion T frame shift mutation, resulting in premature truncation of the protein. Heterologous expression of this truncated protein in human embryonic kidney 293 cells showed a markedly reduced protein expression level. By performing whole-cell patch clamp experiments using human embryonic kidney 293 cells transfected with the mutated SCN5A, no current could be recorded. Hence, the results suggest that the patient suffered from haploinsufficiency of Na(v)1.5, and that this mutation was the cause of his Brugada syndrome.

The flame retardant DE-71 (a mixture of polybrominated diphenyl ethers) inhibits human differentiated thyroid cell function in vitro

Background Normal thyroid function is essential for general growth and metabolism, but can be affected by endocrine disrupting chemicals (EDCs). Polybrominated diphenyl ethers (PBDEs) have been used worldwide to reduce flammability in different materials and are suspected to be EDCs. The production of the commercial Penta- and OctaBDE mixtures is banned, but DecaBDEs and existing products may leak PBDEs into the environment. Our aim was to investigate the effect of the PentaBDE mixture DE-71 on human thyroid cells in vitro. Materials and methods Primary human thyroid cells were obtained as paraadenomatous tissue and cultured in monolayers. The influence of DE-71 on cyclic adenosine monophosphate (cAMP) and thyroglobulin (Tg) production was examined in the culture medium by competitive radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Real-time quantitative PCR analysis of thyroid-specific genes was performed on the exposed cell cultures. PBDE concentrations were determined in cellular and supernatant fractions of the cultures. Results DE-71 inhibited Tg-release from TSH-stimulated thyrocytes. At 50 mg/L DE-71, mean Tg production was reduced by 71.9% (range: 8.5–98.7%), and cAMP by 95.1% (range: 91.5–98.8%) compared to controls). Expression of mRNA encoding Tg, TPO and TSHr were significantly inhibited (p<0.0001, p = 0.0079, and p = 0.0002, respectively). The majority of DE-71 added was found in the cell fraction. No cytotoxicity was found. Conclusions DE-71 inhibited differentiated thyroid cell functions in a two phase response manner and a concentration-dependent inhibition of Tg and cAMP production, respectively, as well as expression of mRNA encoding Tg, TPO and TSHr. Our findings suggest an inhibiting effect of PBDEs on thyroid cells.