Thomas P. Flagg

Cardiac sarcolemmal KATP channels: Latest twists in a questing tale!

Reconstitution of K(ATP) channel activity from coexpression of members of the pore-forming inward rectifier gene family (Kir6.1, KCNJ8, and Kir6.2 KCNJ11) with sulfonylurea receptors (SUR1, ABCC8, and SUR2, ABCC9) of the ABCC protein sub-family, has led to the elucidation of many details of channel gating and pore properties, as well as the essential roles of Kir6.2 and SUR2 subunits in generating cardiac ventricular K(ATP). However, despite this extensive body of knowledge, there remain significant holes in our understanding of the physiological role of the cardiac K(ATP) channel, and surprising new findings keep emerging. Recent findings from genetically modified animals include the apparent insensitivity of cardiac sarcolemmal channels to nucleotide levels, and unenvisioned complexities of the subunit make-up of the cardiac channels. This topical review focuses on these new findings and considers their implications.

Electrocardiographic Studies of Romidepsin Demonstrate Its Safety and Identify a Potential Role for KATP Channel

Purpose: Romidepsin is a histone deacetylase inhibitor (HDI) approved for the treatment of both cutaneous and peripheral T-cell lymphoma (CTCL and PTCL). During development, a thorough assessment of cardiac toxicity was conducted. Experimental Design: A phase II single-agent nonrandomized study of romidepsin was conducted in patients with CTCL or PTCL who had progressed after at least 1 prior systemic therapy. Results: Results for the first 42 patients enrolled on the NCI 1312 phase II study of romidepsin in CTCL or PTCL showed no cardiac toxicity based on serial electrocardiograms (ECG), troponins, and MUGA scans/echocardiograms. The cardiac assessments reported herein confirm the safety of romidepsin among 131 enrolled patients, while supporting a role for electrolyte replacement. Heart rate increased an average 11 bpm following romidepsin infusion; there was no evidence of increased arrhythmia. Criteria for potassium/magnesium replacement were met before 55% of 1365 romidepsin doses; an association with hypoalbuminemia was confirmed. We propose a mechanism for ST segment flattening and depression, the most common ECG abnormalities observed: HDI-induced alteration of the activity or expression of KATP channels. In addition, examination of the variants of the active transporter of romidepsin, ABCB1, showed a trend toward smaller heart rate changes in the peri-infusion period among wild-type than variant diplotypes. Conclusions: We conclude that in the context of appropriate attention to electrolyte levels, the data support the cardiac safety of romidepsin. Clin Cancer Res; 19(11); 3095–104. ©2013 AACR.

Promoter DNA Methylation Regulates Murine SUR1 (Abcc8) and SUR2 (Abcc9) Expression in HL-1 Cardiomyocytes

Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 µM 5-aza-2′-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.

Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis.

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic β-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.

Brief isoflurane administration as a post-exposure treatment for organophosphate poisoning

HighlightsOrganophosphate poisoning (OP) leads to uncontrolled seizures and CNS damage.A 4 min isoflurane treatment given 20 to 30 min post OP blocked seizure activity.The brief isoflurane treatment reduced mortality from 37% to zero.Isoflurane administered 30 min after paraoxon also prevented neurodegeneration.Brief isoflurane administration is an effective post‐exposure treatment for OP. &NA; Organophosphate chemical threat agents (OP‐CTA) exert toxic effects through cholinergic over‐activation. However, after the initial cholinergic phase, the pathophysiology shifts to a non‐cholinergic phase which leads to prolonged status epilepticus (SE), irreversible neuronal degeneration and long‐term damage to the central nervous system. The efficacy of delayed treatments against OP‐CTA is generally low due to the fact that most drugs fail to inhibit the later phase of non‐cholinergic activation. Recently, we reported that intranasal brain delivery of obidoxime (OBD) provides complete neuroprotection against a lethal dose of paraoxon when administered 5 min after intoxication. In follow‐up studies, we examined the window of effectiveness and found that OBD lost effectiveness around 15 min post‐exposure, which corresponds to the onset of the non‐cholinergic phase of intoxication. However, we observed that a brief isoflurane administration, the inhalation anesthetic used to facilitate intranasal drug administration, was effective against paraoxon‐induced neurotoxicity. Thus, the present study aimed to investigate the time‐course and dose‐response efficacy of a brief 4 min isoflurane administration as a treatment for neurotoxicity induced by OP‐CTA. We found that isoflurane is a potent anti‐seizure agent and neuroprotectant when administered between 20 and 30 min after paraoxon exposure, stopping SE within 10 min of administration and preventing acute neurodegeneration seen 24 h later. We also found that the seizure blocking and neuroprotectant properties of isoflurane, when administered 30 min after paraoxon, are dose‐dependent. The effectiveness and current clinical use of isoflurane support its use as an innovative approach for post exposure treatment of organophosphate poisoning.

Reply: Labeling and Drug Safety of Loperamide: Time for a Proactive Approach?

We thank Dr. Woronow and his colleagues at the U.S. Food and Drug Administration (FDA) for their interest in our study [(1)][1], and we commend them for their recently published series of torsade de pointes and sudden death cases from the FDA Adverse Event Reporting System [(2)][2]. This system has