Wang, Ruxing

Background: Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) channel contribute to heart failure (HF) and focal segmental glomerulosclerosis in humans. Our GWAS results indicated that the TRPC6 A404V variant, having a minor allele frequency of 12% in the population, is associated with doxorubicin-induced cardiotoxicity. However, the underlying ionic mechanisms of this variant have not been fully examined. Methods: Combining patch clamp recording, site-directed mutagenesis, and in-silico analysis, we evaluated the TRPC6 A404V variant function. Results: Whole-cell TRPC6 currents were elicited in HEK293 cells using a voltage ramp protocol from -100 mV to +100 mV with a holding potential of -60 mV, 48 hours after transfection with TRPC6 WT or A404V cDNAs. The inward-current densities at -100 mV and the outward-current densities at +100 mV for TRPC6 WT and A404V variant were similar at baseline. However, after exposure to 50 μM 1-oleoyl acetyl-sn-glycerol (OAG, a TRPC6 activator), these current densities were significantly increased by 2.6-fold and 4.5-fold respectively in the WT (n=20 cells), and by 8.4-fold and 8.9-fold respectively in the A404V variant (n=12 cells, p<0.05 vs. WT). The OAG effects were abolished by superfusion with 1 μM BI-749327 (a specific TRPC6 inhibitor). Moreover, a 24-hour treatment with 0.5 μM doxorubicin (DOX) further amplified the effects of OAG, increasing the total inward- and outward-current densities by 7.2-fold and 9.5-fold respectively in TRPC6 WT (n=11 cells), and by 13.9-fold and 14.7-fold respectively in the A404V variant (n=13 cells, p<0.05 vs. WT). In comparison, the OAG effects on TRPC6 WT and A404V channels were not potentiated by treatment with 0.5 μM doxorubicinol (DOXol, a metabolite of DOX) for 24 h (n=10-12 cells, p=N.S.). Molecular docking studies showed that OAG had a lower binding energy (-5.60 kcal/mol) and a smaller apparent equilibrium constant (Kd) of 0.079 mM with the A404V variant, compared to those of -4.49 kcal/mol and 0.511 mM respectively with TTRPC6 WT. Conclusion: TRPC6 A404V is a gain-of-function variant with a higher binding affinity to OAG. Treatment with DOX but not DOXol greatly enhances TRPC6 WT and A404V channel activation by OAG. Thus, cancer patients carrying the TRPC6 A404V variant may be more vulnerable to develop HF upon treatment with anthracycline.

BackgroundAlthough sudden death (SD) is a rare complication after atrioventricular junction (AVJ) ablation and permanent pacemaker implantation, the risk factors leading to this SD remain unknown. The purpose of this study was to investigate SD and its risk factors after ablate‐and‐pace strategy for rate control in atrial fibrillation (AF) patients during long‐term follow‐up.HypothesisMethodsFrom January 2005 to December 2009, we enrolled into this study 517 AF patients with AVJ ablation and right ventricular pacemaker implantation. Patients were divided into 2 groups, SD and non‐SD. Cox proportional hazards models were used to assess potential risk factors for overall mortality and SD.ResultsDuring a mean follow‐up of 25.8 ± 18.6 months (range, 3 days to 63.8 months), 53 patients died (15 with SD). Cox proportional hazards models showed that the presence of congestive heart failure, New York Heart Association functional class, chronic renal failure, and nonsustained ventricular tachycardia were risk factors that predicted overall mortality. For SD, the presence of dilated cardiomyopathy and mitral stenosis were associated risk factors. SD was exclusively seen in patients who had narrow QRS complex or right bundle branch block prior to AVJ ablation and pacemaker implantation; SD was not seen in any patient with preexisting complete left bundle branch block.ConclusionsDilated cardiomyopathy, mitral stenosis, and baseline QRS morphology should be examined as potential risk factors for SD after AVJ ablation and pacemaker implantation.

Rationale : The large conductance Ca 2+ -activated K + (BK) channel, a key determinant of vascular tone, is regulated by angiotensin II (Ang II) type 1 receptor signaling. Upregulation of Ang II functions and downregulation of BK channel activities have been reported in diabetic vessels. However, the molecular mechanisms underlying Ang II-mediated BK channel modulation, especially in diabetes mellitus, have not been thoroughly examined. Objectives : The aim in this study was to determine whether caveolae-targeting facilitates BK channel dysfunction in diabetic vessels. Methods and Results : Using patch clamp techniques and molecular biological approaches, we found that BK channels, Ang II type 1 receptor, G αq/11 (G protein q/11 α subunit), nonphagocytic NAD(P)H oxidases (NOX-1), and c-Src kinases (c-Src) were colocalized in the caveolae of rat arterial smooth muscle cells and the integrity of caveolae in smooth muscle cells was critical for Ang II-mediated BK channel regulation. Most importantly, membrane microdomain targeting of these proteins was upregulated in the caveolae of streptozotocin-induced rat diabetic vessels, leading to enhanced Ang II-induced redox-mediated BK channel modification and causing BK channel and coronary dysfunction. The absence of caveolae abolished the effects of Ang II on vascular BK channel activity and preserved BK channel function in diabetes. Conclusions : These results identified a molecular scheme of receptor/enzyme/channel/caveolae microdomain complex that facilitates the development of vascular BK channel dysfunction in diabetes.