Lü, Tong

Epoxyeicosatrienoic acids (EETs) potently and stereospecifically activate the cardiac ATP‐sensitive K + (Kir6.2) channels by reducing channel sensitivity to ATP. In this study, we determined the effects of EETs on the binding of ATP to Kir6.2 by using 2,4,6‐trinitrophenyl (TNP)‐ATP, which increases fluorescence emission >8‐fold with binding to protein, and by using GST fusion protein containing Kir6.2. TNP‐ATP binds to the GST fusion protein containing the Kir6.2C‐terminus but not to that with the Kir6.2N‐terminus. Binding of TNP‐ATP to GST‐Kir6.2C (K D 5.0 μ M) was unchanged by 5 μ M 11,12‐EET (K D 4.4 μ M) but B max was reduced by half, suggesting non‐competitive inhibition of ATP binding. The effect of 11,12‐EET was dose‐dependent and 8,9‐ and 14,15‐EETs were equipotent inhibitors of TNP‐ATP binding to GST‐Kir6.2C. Arachidonic acid and 11,12‐DHET were not effective inhibitors, whereas the methyl ester of 11,12‐EET was as effective as 11,12‐EET in inhibiting TNP‐ATP binding to GST‐Kir6.2C. R195A mutation in GST‐Kir6.2C abolished the EET effects on TNP‐ATP binding, while the K185A mutation, which reduced ATP binding to Kir6.2, had no effect. These findings are consistent with previous patch clamp results that showed the epoxide group in EET and R195 in Kir6.2 are critical for K ATP channel activation by EET. We conclude that EETs bind to the C‐terminus of the K ATP channel and allosterically alter the ATP binding site, reducing the channel sensitivity to ATP.

We examined the role of H 2 S on shear stress‐mediated dilation of isolated mouse coronary arteries. Na 2 S produced concentration‐dependent dilation in these vessels, which was significantly inhibited by iberiotoxin. BK currents in mouse coronary smooth muscle cells were directly activated by Na 2 S, suggesting H 2 S produced vasodilation through BK channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm 2 ), producing graded dilatory responses, but such effects were diminished in the presence of 100 μM Na 2 S. Pre‐incubation with the cystathionine γ‐lyase inhibitor, D,L‐propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress‐mediated vasodilation. However, in the presence of L‐NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress‐mediated vasodilation, suggesting an interaction between NO and H 2 S signaling. Using cultured bovine aortic endothelial cells, eNOS activity was inhibited by Na 2 S. These results suggest that both NO and H 2 S are important shear stress‐mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H 2 S through inhibition of NO generation. (Supported by grants from NIH)

Background Sorbs2b (sorbin and SH3 domain‐containing 2b) was recently identified as a cardiomyopathy gene from a zebrafish mutagenesis screen. However, cardiac functions of its mammalian ortholog remain elusive. Methods and Results We conducted a detailed expression and subcellular localization analysis of Sorbs2 ortholog in mice and a phenotypic characterization in Sorbs2 knockout mice. Sorbs2 is highly expressed in the mouse heart and encodes an adhesion junction/desmosome protein that is mainly localized to the intercalated disc. A mutation with near complete depletion of the Sorbs2 protein in mice results in phenotypes characteristic of human arrhythmogenic cardiomyopathy (ACM), including right ventricular dilation, right ventricular dysfunction, spontaneous ventricular tachycardia, and premature death. Sorbs2 is required to maintain the structural integrity of intercalated disc. Its absence resulted in profound cardiac electrical remodeling with impaired impulse conduction and action potential derangements. Targeted sequencing of human patients with ACM identified 2 rare splicing variants classified as likely pathogenic were in 2 unrelated individuals with ACM from a cohort of 59 patients with ACM. Conclusions The Sorbs2 knockout mouse manifests several key features reminiscent of human ACM. Although the candidacy of SORBS2 as a new ACM‐susceptibility gene is supported by preliminary human genetics study, future validation in larger cohorts with ACM is needed.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is a major cause of thromboembolic stroke in the United States. The risk of stroke is a 5-fold increase in patients with AF. The Sorbin and SH3 domain-containing protein 2 (Sorbs2) is a scaffolding protein known to interact with actin and several other cytoskeletal proteins in the intercalated discs and Z band of cardiomyocytes. However, the role of Sorbs2 gene in cardiovascular diseases is largely unknown. Using the loss-of-function of Sorbs2 ( Sorbs2 e8/e8 ) mice, we found that Sorbs2 e8/e8 mice had an abnormal lifespan and die at 4 to 7 months of age. Echocardiogram examine revealed that there was a significant decrease in right ventricle (RV) and left ventricular (LV) ejection fraction (EF), increase in RV end-diastolic volume (EDV) and end-systolic volume (ESV), and progressive chamber enlargement in left atria (LA) and RV, particularly in LA with severe fibrosis and intramural thrombus formation, in Sorbs2 e8/e8 mice after 3 months of age. Surface electrocardiograph also showed a significant conduction delay and spontaneous arrhythmias including those of bifid P wave configuration, right bundle branch block (RBBB), spontaneous AF and non-sustained ventricular tachycardia (VT) in Sorbs2 e8/e8 mice. Intracellular recordings in the freshly isolated LA demonstrated that Sorbs2 e8/e8 myocardium had a delayed action potential activation in response to electrical impulse excitation, reduced amplitude and upstroke velocity (dV/dt) at phase 0, slowed repolarization at phase 1 and phase 2, prolonged atrial effective refractory period (aERP), action potential durations at 50% (APD 50 ) and 90% (APD 90 ) repolarization, and decreased resting potentials. Moreover, the mRNA and protein expressions of voltage-gated Na + channels, L-type Ca 2+ channels, transient outward K + channels and inward rectifier K + channels were significantly downregulated in the LA of Sorbs2 e8/e8 mice, compared wo those of wild type mice. Our results indicate that Sorbs2 gene plays an important role in atrial myocardium architecture and electrophysiology. Sorbs2 dysfunction leads to atrial cardiomyopathy, spontaneous AF and cardiac embolism in mice. Hence, Sorbs2 is a new genetic association with cardioembolic stroke.

Abstract Aims Type 2 diabetes (T2D) is strongly associated with cardiovascular morbidity and mortality in patients. Vascular large conductance Ca2+-activated potassium (BK) channels, composed of four pore-forming α subunits (BK-α), and four regulatory β1 subunits (BK-β1), are densely expressed in coronary arterial smooth muscle cells (SMCs) and play an important role in regulating vascular tone and myocardial perfusion. However, the role of BK channels in coronary microvascular dysfunction of human subjects with diabetes is unclear. In this study, we examined BK channel function and protein expression, and BK channel-mediated vasodilation in freshly isolated coronary arterioles from T2D patients. Methods and results Atrial tissues were obtained from 16 patients with T2D and 25 matched non-diabetic subjects during cardiopulmonary bypass procedure. Microvessel videomicroscopy and immunoblot analysis were performed in freshly dissected coronary arterioles and inside-out single BK channel currents was recorded in enzymatically isolated coronary arteriolar SMCs. We found that BK channel sensitivity to physiological Ca2+ concentration and voltage was downregulated in the coronary arteriolar SMCs of diabetic patients, compared with non-diabetic controls. BK channel kinetics analysis revealed that there was significant shortening of the mean open time and prolongation of the mean closed time in diabetic patients, resulting in a remarkable reduction of the channel open probability. Functional studies showed that BK channel activation by dehydrosoyasaponin-1 was diminished and that BK channel-mediated vasodilation in response to shear stress was impaired in diabetic coronary arterioles. Immunoblot experiments confirmed that the protein expressions of BK-α and BK-β1 subunits were significantly downregulated, but the ratio of BK-α/BK-β1 was unchanged in the coronary arterioles of T2D patients. Conclusions Our results demonstrated for the first time that BK channel function and BK channel-mediated vasodilation were abnormal in the coronary microvasculature of diabetic patients, due to decreased protein expression and altered intrinsic properties of BK channels.

The mechanisms underlying dysfunction of cerebral microvasculature induced by type 1 diabetes (T1D) are not fully understood. We hypothesized that in cerebral microvascular endothelium, α-processing of amyloid precursor protein (APP) is impaired by T1D. In cerebral microvessels derived from streptozotocin (STZ)-induced T1D mice protein levels of APP and its α-processing enzyme, a disintegrin and metalloprotease 10 (ADAM10) were significantly decreased, along with down-regulation of adenylate cyclase 3 (AC3) and enhanced production of thromboxane A 2 (TXA 2 ). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA 2 receptor decreased APP expression. Furthermore, levels of soluble APPα (sAPPα, a product of α-processing of APP) were significantly reduced in hippocampus of T1D mice. In contrast, amyloidogenic processing of APP was not affected by T1D in both cerebral microvessels and hippocampus. Most notably, studies in endothelial specific APP knockout mice established that genetic inactivation of APP in endothelium was sufficient to significantly reduce sAPPα levels in the hippocampus. In aggregate, our findings suggest that T1D impairs non-amyloidogenic processing of APP in cerebral microvessels. This may exert detrimental effect on local concentration of neuroprotective molecule, sAPPα, in the hippocampus.

The large conductance Ca2+-activated K+ (BK) channel β1-subunit (BK-β1) is a key modulator of BK channel electrophysiology and the downregulation of BK-β1 protein expression in vascular smooth muscle cells (SMCs) underlies diabetic vascular dysfunction. In this study, we hypothesized that the nuclear factor erythroid-2–related factor 2 (Nrf2) signaling pathway plays a significant role in the regulation of coronary BK channel function and vasodilation in high-fat diet (HFD)–induced obese/diabetic mice. We found that the protein expressions of BK-β1 and Nrf2 were markedly downregulated, whereas those of the nuclear factor-κB (NF-κB) and the muscle ring finger protein 1 (MuRF1 [a ubiquitin E3 ligase for BK-β1]) were significantly upregulated in HFD mouse arteries. Adenoviral expression of Nrf2 suppressed the protein expressions of NF-κB and MuRF1 but enhanced BK-β1 mRNA and protein expressions in cultured coronary SMCs. Knockdown of Nrf2 resulted in reciprocal changes of these proteins. Patch-clamp studies showed that coronary BK-β1–mediated channel activation was diminished in HFD mice. Importantly, the activation of Nrf2 by dimethyl fumarate significantly reduced the body weight and blood glucose levels of HFD mice, enhanced BK-β1 transcription, and attenuated MuRF1-dependent BK-β1 protein degradation, which in turn restored coronary BK channel function and BK channel–mediated coronary vasodilation in HFD mice. Hence, Nrf2 is a novel regulator of BK channel function with therapeutic implications in diabetic vasculopathy.

BK channels are major ionic determinants of vasodilation. BK channel function is impaired in diabetic vessels due to accelerated proteolysis of its beta-1 (BK-β1) subunits in response to increased oxidative stress. The nuclear factor E2-related factor-2 (Nrf2) signalling pathway has emerged as a master regulator of cellular redox status, and we hypothesized that it plays a central role in regulating BK channel function in diabetic vessels. We found that Nrf2 expression was markedly reduced in db/db diabetic mouse aortas, and this was associated with significant downregulation of BK-β1. In addition, the muscle ring finger protein 1 (MuRF1), a known E-3 ligase targeting BK-β1 ubiquitination and proteasomal degradation, was significantly augmented. These findings were reproduced by knockdown of Nrf2 by siRNA in cultured human coronary artery smooth muscle cells. In contrast, adenoviral transfer of Nrf2 gene in these cells downregulated MuRF1 and upregulated BK-β1 expression. Activation of Nrf2 by dimethyl fumarate preserved BK-β1 expression and protected BK channel and vascular function in db/db coronary arteries. These results indicate that expression of BK-β1 is closely regulated by Nrf2 and vascular BK channel function can be restored by Nrf2 activation. Nrf2 should be considered a novel therapeutic target in the treatment of diabetic vasculopathy.

Background— Left atrial (LA) compliance and contractility influence left ventricular stroke volume. We hypothesized that diminished LA compliance and contractile function occur early during the development of heart failure with preserved ejection fraction (HFpEF) and impair overall cardiac performance. Methods and Results— Cardiac magnetic resonance imaging, echocardiography, left ventricular and LA pressure-volume studies, and tissue analyses were performed in a model of early HFpEF (elderly dogs, renal wrap-induced hypertension, exogenous aldosterone; n=9) and young control dogs (sham surgery; n=13). Early HFpEF was associated with LA enlargement, cardiomyocyte hypertrophy, and enhanced LA contractile function (median active emptying fraction 16% [95% confidence interval, 13–24]% versus 12 [10–14]%, P =0.008; end-systolic pressure-volume relationship slope 2.4 [1.9–3.2]mm Hg/mL HFpEF versus 1.5 [1.2–2.2]mm Hg/mL controls, P =0.01). However, atrioventricular coupling was impaired and the curvilinear LA end-reservoir pressure-volume relationship was shifted upward/leftward in HFpEF (LA stiffness constant [β LA ] 0.16 [0.11–0.18]mm Hg/mL versus 0.06 [0.04–0.10]mm Hg/mL controls; P =0.002), indicating reduced LA compliance. Impaired atrioventricular coupling and lower LA compliance correlated with lower left ventricular stroke volume. Total fibrosis and titin isoform composition were similar between groups; however, titin was hyperphosphorylated in HFpEF and correlated with β LA . LA microvascular reactivity was diminished in HFpEF versus controls. LA microvascular density tended to be lower in HFpEF and inversely correlated with β LA . Conclusions— In early-stage hypertensive HFpEF, LA cardiomyocyte hypertrophy, titin hyperphosphorylation, and microvascular dysfunction occur in association with increased systolic and diastolic LA chamber stiffness, impaired atrioventricular coupling, and decreased left ventricular stroke volume. These data indicate that maladaptive LA remodeling occurs early during HFpEF development, supporting a concept of global myocardial remodeling.

The large conductance Ca2+-activated K+ (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel β1-subunits (BK-β1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-β1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.