Chai, Qiang

Background: Reduced left atrial (LA) compliance (LAC) contributes to pulmonary venous hypertension in heart failure with preserved ejection fraction (HFpEF); however the underlying mechanisms are poorly defined. We hypothesized that microvascular inflammation with rarefaction and impaired nitric oxide (NO) signaling, LA titin hypophosphorylation and fibrosis may each contribute to reduced LAC in HFpEF. Methods: Invasive LA pressure-volume (PV) analyses (open-chest; pericardiotomy; admittance catheter) were performed in 9 aged dogs with experimental hypertension (bilateral renal wrap + DOCA; HFpEF) compared to 13 sham-operated young adult dogs (control). LAC was measured at end-LA reservoir during acute preload reduction (cava occlusion). LA tissue and LA microvascular function were analyzed. Results: Compared to controls, the curvilinear end-LA reservoir PV relationship ( Fig A ) was shifted upward/leftward in HFpEF (LA diastolic stiffness constant 0.08 vs 0.16 mmHg/mL in controls, p=0.005). LA microvascular density (MVD; CD31 stain) tended to be lower in HFpEF than controls (2154±578 vs 2650±523 vessels/mm 2 , p=0.07) and LA MVD negatively correlated with the LA diastolic stiffness constant (r=-0.66, p=0.006). LA microvessels from HFpEF dogs showed impaired endothelium-dependent shear stress-mediated dilation compared to controls, consistent with impaired NO signaling ( Fig B ). Titin isoform expression was similar between groups, but isoform N2B (stiffer) was (p=0.03) and N2BA (compliant) tended to be (p=0.09) hypophosphorylated in HFpEF LA appendage (LAA) tissue. Fibrosis (Picrosirius Red) was similar between groups in the LA free wall and LAA, but increased in areas adjacent to the pulmonary veins in HFpEF (27±12% vs controls 15±8%, p=0.01). Conclusion: In experimental HFpEF, LAC was reduced in association with evidence of LA microvascular rarefaction and impaired NO signaling, titin hypophosphorylation and regional increases in LA fibrosis.

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)

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 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.

PurposeAutoantibodies are implicated in the pathogenesis of cardiovascular diseases and cardiac arrhythmias. In this pilot study, we tested the hypothesis that autoantibodies are present in patients with postural orthostatic tachycardia syndrome (POTS).Experimental designSeven control subjects (6 F:1 M, average age 36.1 years) and ten patients with the diagnosis of POTS (7 F: 3 M, average age 35.1 years) provided informed consent and 30 mL of venous blood. Human heart membrane proteins were resolved by 2DE and immunoblotted against purified IgGs from controls and patients.ResultsEighteen protein spots immunoreactive specifically against patient IgGs were detected and they were excised from gels, trypsin‐digested, and analyzed by nanoLC‐electrospray MS/MS. Forty unique proteins were identified and these include proteins that are associated with cardiac hypertrophy (mimecan, myozenin), cardiac remodeling (periostin), cardiomyopathy (desmin, desmoplakin), cell survival (laminin), structural integrity (filamin), chaperone proteins (crystalline, HSP70), mitochondrial enzymes, and channel proteins. Ingenuity Pathway Analysis showed multiple pathways were involved including those that regulate energy metabolism, redox, fibrosis, cardiac hypertrophy, and degeneration.Conclusions and clinical relevanceAutoantibodies are present in patients with POTS. These autoantibodies cross‐react with a wide range of cardiac proteins and may induce alterations in cardiac function. Autoimmune pathogenetic mechanisms should be further explored in these patients.

Activity of the vascular large conductance Ca2+-activated K+ (BK) channel is tightly regulated by its accessory β1 subunit (BK-β1). Downregulation of BK-β1 expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)–dependent F-box–only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β1 degradation and leads to diabetic BK channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β1 expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator–activated receptor δ activator) reduced atrogin-1 expression and restored BK channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β1 degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β1 degradation and protected coronary function in diabetes.

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.