Citations

Background: We have found that the vascular large conductance Ca 2+ -activated K + channels (BK) are targeted to caveolae. The goal of this study is to determine the role of caveolae in BK regulation by angiotensin II (AII) in normal and diabetic rats. Results: In coronary arterial smooth muscle cells (CASMC) from control rats, AII (2 μ M) inhibited BK by 40.5% from 227±26 pA/pF to 135±14 pA/pF (HP −60 mV, TP 150 mV, n=7, p<0.05). However, AII had no effects on BK in CASMC transfected with caveolin-1 siRNA (vs 50% inhibition in cells transfected with control siRNA) or in cells from Cav1−/− mice, suggesting the AII effects are caveolae-dependent. Density gradient analysis of rat vessel homogenates showed that caveolin-1, BK, Gq, cSrc, and NOX-1 all reside in the caveolae-rich fraction, where AT 1 R was absent unless the animal was pre-treated with AII, suggesting receptor translocation upon agonist activation. However, CASMC from streptozotocin-induced diabetic rats showed significant reduction in BK (61.5±10.6 pA/pF) with no response to AII (61.2±15.7 pA/pF, n=8). Vascular BK, caveolin-1, AT 1 R, cSrc, and NOX-1 expression in caveolae were all upregulated by 1.6- to 3.1-fold in diabetes. Density gradient analysis of diabetic vessels showed the presence of AT 1 R in the caveolae-rich fraction without AII pretreatment, suggesting that the AT 1 R signaling pathway might be constitutively active in diabetes. This was accompanied by a 2-fold increase in BK tyrosine phosphorylation and BK tyrosine nitration. The AII effects on BK function in diabetic CASMC were abolished by pretreatment (1 hr) with PKC inhibitor or by LavA (cSrc inhibitor) and can be mimicked by incubation (2 hr) of control CASMC with peroxynitrite. Conclusion: AT 1 R-BK-enzyme complex in the caveolae microdomain is critical in vascular BK regulation. Enhanced AII-signaling facilitates BK post-translational modification, resulting in BK dysfunction in diabetes.

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.

Angiotensin II (Ang II) type I receptor (ATR 1 ) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca 2+ actviated K + (BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR 1 , and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR 1 inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β 1 subunit, ATR 1 , cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR 1 activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR 1 , Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR 1 and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.

GTP Cyclohydrolase‐I (GTPCHI) is the rate‐limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), which is an essential cofactor for the enzymatic activity of endothelial nitric oxide synthase (eNOS). To better understand the role of GTPCHI in maintaining eNOS function, we analyzed GTPCHI subcellular localization and activity in human umbilical cord vein endothelial cells (HUVEC). Western blot analysis showed similar levels of GTPCHI protein expression in both Triton X‐100 ‐soluble and ‐insoluble fractions, suggesting membrane targeting of GTPCHI. Further analysis of membrane‐associated proteins using sucrose gradient ultracentrifugation revealed that GTPCHI protein expression was localized to caveolar microdomains along with caveolin‐1(Cav‐1) and eNOS. HPLC analysis for GTPCHI activity confirmed the localization of GTPCHI to caveolar microdomains as well as golgi‐associated membrane fractions. Using an in vitro GST pull‐down assay with recombinantGST‐caveolin‐1 fusion protein incubated with the lysate of HUVEC, we demonstrated specific binding of GTPCHI to caveolin‐1. To determine if caveolin‐1 may regulate GTPCHI activity in vivo, we analyzed aortas from Cav‐1 −/ − mice and were able to detect a two‐fold increase in BH4 production and GTPCHI activity in the aortas of Cav‐1 −/ − mice when compared to eNOS −/ − mice or wild type mice. These studies reveal that BH4 synthesis can occur in specialized membrane compartments in close proximity to eNOS and may be subjected to posttranslational regulation by caveolin‐1 which has an inhibitory effect on GTPCHI enzyme activity. Funded by NIH grant HL‐53524.

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.

Rationale: Acute kidney injury (AKI) has a high prevalence and mortality in critically ill patients. It is also a powerful risk factor for heart failure incidence driven by hemodynamic changes and neurohormonal activation. However, no drugs have been approved by the Food and Drug Administration. Endogenous pGC-A (particulate guanylyl cyclase A receptor) activators were reported to preserve renal function and improve mortality in AKI patients, although hypotension accompanied by pGC-A activators have limited their therapeutic potential. Objective: We investigated the therapeutic potential of a nonhypotensive pGC-A activator/designer natriuretic peptide, CRRL269, in a short-term, large animal model of ischemia-induced AKI and also investigated the potential of uCNP (urinary C-type natriuretic peptide) as a biomarker for AKI. Methods and Results: We first showed that CRRL269 stimulated cGMP generation, suppressed plasma angiotensin II, and reduced cardiac filling pressures without lowering blood pressure in the AKI canine model. We also demonstrated that CRRL269 preserved glomerular filtration rate, increased renal blood flow, and promoted diuresis and natriuresis. Further, CRRL269 reduced kidney injury and apoptosis as evidenced by ex vivo histology and tissue apoptosis analysis. We also showed, compared with native pGC-A activators, that CRRL269 is a more potent inhibitor of apoptosis in renal cells and induced less decreases in intracellular Ca 2+ concentration in vascular smooth muscle cells. The renal antiapoptotic effects were at least mediated by cGMP/PKG pathway. Further, CRRL269 inhibited proapoptotic genes expression using a polymerase chain reaction gene array. Additionally, we demonstrated that AKI increased uCNP levels. Conclusions: Our study supports developing CRRL269 as a novel renocardiac protective agent for AKI treatment.

Key PointsSVECs support the development of hematopoiesis in MF. LCs and SVECs each have distinct properties and functions that differ between normal and MF spleens.

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.