Background Clinical trials of therapeutic angiogenesis with vascular endothelial growth factor (VEGF) have been disappointing, owing likely to endothelial dysfunction. We used a swine model of ...chronic ischemia and endothelial dysfunction to determine whether resveratrol coadministration would improve the angiogenic response to VEGF therapy. Methods Yorkshire swine fed a high-cholesterol diet underwent left circumflex ameroid constrictor placement, and were given either no drug (high cholesterol control HCC, n = 8), perivascular VEGF (2 μg sustained release high cholesterol VEGF-treated; HCV, n = 8), or VEGF plus oral resveratrol (10 mg/kg, high cholesterol VEGF- and resveratrol-treated; HCVR, n = 8). After 7 weeks, myocardial contractility, perfusion, and microvessel reactivity in the ischemic territory were assessed. Tissue was analyzed for vessel density, oxidative stress, and protein expression. Results Myocardial perfusion was significantly improved in the HCV group compared with the HCC group; resveratrol coadministration abrogated this improvement. There were no differences in regional myocardial contractility between groups. Endothelium-dependent microvessel relaxation was improved in the HCVR group, and endothelium-independent relaxation response was similar between groups. Arteriolar density was greatest in the HCV group, whereas capillary density was similar between groups. Expression of Akt and phospho-endothelial nitric oxide synthase were increased in the HCVR group. Total protein oxidative stress and myeloperoxidase expression were reduced in the HCVR group, but so was the oxidative-stress dependent phosphorylation of vascular endothelial cadherin (VE-cadherin) and β-catenin. Conclusion Although resveratrol coadministration decreases oxidative stress and improves endothelial function, it abolishes improvements in myocardial perfusion and arteriolar density afforded by VEGF treatment alone. This effect is due likely to inhibition of the oxidative stress-dependent phosphorylation of VE-cadherin, an essential step in the initiation of arteriogenesis.
Mitochondrial Ca(2+)-activated large-conductance K(+) (BKCa) channels are thought to provide protection during ischemic insults in the heart. Rottlerin (mallotoxin) has been implicated as a potent ...BKCa activator. The purpose of this study was twofold: 1) to investigate the efficacy of BKCa channel activation as a cardioprotective strategy during ischemic cardioplegic arrest and reperfusion (CP/R) and 2) to assess the specificity of rottlerin for BKCa channels. Wild-type (WT) and BKCa knockout (KO) mice were subjected to an isolated heart model of ischemic CP/R. A mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential and reactive oxygen species (ROS) production. CP/R decreased left ventricular developed pressure, positive and negative first derivatives of left ventricular pressure, and coronary flow (CF) in WT mice. Rottlerin dose dependently increased the recovery of left ventricular function and CF to near baseline levels. BKCa KO hearts treated with or without 500 nM rottlerin were similar to WT CP hearts. H9c2 cells subjected to in vitro CP/R displayed reduced mitochondrial membrane potential and increased ROS generation, both of which were significantly normalized by rottlerin. We conclude that activation of BKCa channels rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.
Abstract Background We tested the short-term effects of completely nonpulsatile versus pulsatile circulation after ventricular excision and replacement with total implantable pumps in an animal model ...on peripheral vascular permeability. Methods Ten calves underwent cardiac replacement with two HeartMate III continuous-flow rotary pumps. In five calves, the pump speed was rapidly modulated to impart a low-frequency pulse pressure in the physiologic range (10–25 mm Hg) at a rate of 40 pulses per minute (PP). The remaining five calves were supported with a pulseless systemic circulation and no modulation of pump speed (NP). Skeletal muscle biopsies were obtained before cardiac replacement (baseline) and on postoperative days (PODs) 1, 7, and 14. Skeletal muscle-tissue water content was measured, and morphologic alterations of skeletal muscle were assessed. VE-cadherin, phospho–VE-cadherin, and CD31 were analyzed by immunohistochemistry. Results There were no significant changes in tissue water content and skeletal muscle morphology within group or between groups at baseline, PODs 1, 7, and 14, respectively. There were no significant alterations in the expression and/or distribution of VE-cadherin, phospho–VE-cadherin, and CD31 in skeletal muscle vasculature at baseline, PODs 1, 7, and 14 within each group or between the two groups, respectively. Although continuous-flow total artificial heart (CFTAH) with or without a pulse pressure caused slight increase in tissue water content and histologic damage scores at PODs 7 and 14, it failed to reach statistical significance. Conclusions There was no significant adherens-junction protein degradation and phosphorylation in calf skeletal muscle microvasculature after CFTAH implantation, suggesting that short term of CFTAH with or without pulse pressure did not cause peripheral endothelial injury and did not increase the peripheral microvascular permeability.
We examined the impact of acute myocardial ischemia followed by reperfusion (AMI-R) on local and circulating renin–angiotensin system (RAS) in a swine model. The mid left anterior descending artery (
...n
= 6) was occluded for 1 h, followed by reperfusion for 2 h. Monastryl blue/triphenyl tetrazolium chloride staining identified the area-at-risk (AAR) and infarction. A second group of control animals underwent sham operations (C:
n
= 4). Myocardial expression of angiotensinogen (AGT), renin, chymase, angiotensin converting enzyme (ACE), angiotensin II (Ang II), Ang II type1 receptor (AT1R) and Ang II type 2 receptor (AT2R) in the AAR and the non-ischemic left ventricle (NLV) was assessed. Serum level of these proteins at baseline and at the end of reperfusion was also examined. Chymase (
P
< 0.05), ACE (
P
< 0.05), Ang II (
P
< 0.05), AT1R (
P
< 0.05) and AT2R (
P
< 0.05) expressions were found to be significantly higher in the AAR compared to the NLV and C whereas no significant differences were found for AGT (
P
= 0.58) and renin (
P
= 0.38). Serum concentration of ACE was significantly higher at the end of reperfusion than at baseline (
P
< 0.01), whereas no significant difference was found for chymase (
P
= 0.71), AGT (
P
= 0.57) and Ang II (
P
= 0.19). Immunohistochemistry of myocardial sections demonstrated significantly higher expression of ACE (
P
= 0.02), AT1R (
P
= 0.01), AT2R (
P
= 0.02) and Ang II (
P
< 0.01) in the AAR as compared to the NLV, whereas no significant difference was found for renin (
P
= 0.39). In conclusion, AMI-R resulted in significantly higher expression of specific cardiac RAS components in AAR compared to the NLV in the acute period.
We investigated the impact of hydrogen sulfide (H(2)S) on myocardium in the setting of cold crystalloid cardioplegia and cardiopulmonary bypass (CP/CPB). Eighteen male Yorkshire pigs underwent 1 h ...CP/CPB followed by 2 h of reperfusion. Pigs received either: placebo (control, n=9), or H(2)S (as NaHS) as a bolus/infusion (bolus/infusion, n=6), or as an infusion (infusion, n=6). The expression pattern of various myocardial effector pathways was investigated. Coronary microvascular relaxation to endothelium-dependent and -independent agonists was assessed. No differences in cardiac function were observed among groups. Endothelium-dependent microvascular relaxation to adenosine diphosphate was improved in the H(2)S bolus/infusion group only (P<0.05). The expression of hemeoxygenase-1, phospho-heat shock proteins27 and phospho-p44/42 MAPK extracellular signal-regulated kinase were higher in H(2)S-treated groups (P<0.05). Phospho-endothelial nitric oxide synthase (P=0.08), phospho-B-cell lymphoma 2 (P=0.09), and phospho-Bad (P=0.06) all displayed a trend to be higher with H(2)S treatment. The expressions of apoptosis inducing factor and Bcl 2/adenovirus E1B 19 kDa-interacting protein were lower in H(2)S treated groups (P<0.05). The microtubule-associated protein 1 light chain 3 ratio was lower in the infusion group vs. control animals (P<0.05). There was a trend for lower phospho-mammalian target of rapamycin expression in the infusion group (P=0.07), whereas phosphorylation of p70S6K1 was higher with H(2)S-treatment (P=0.09). This study demonstrates that H(2)S-treatment may offer biochemical myocardial protection via attenuation of caspase-independent apoptosis and autophagy in the setting of CP/CPB.
Background We investigated the contractile response of human coronary microvasculature to thromboxane A-2 (TXA-2), with and without the blockade of TXA-2 receptors or the inhibition of ...phospholipase-C (PLC) or of protein kinase C-α (PKC-α) in the human coronary microvasculature before and after cardioplegia, followed by reperfusion (CP/Rep). Protein/gene expression and localization of TXA-2 receptors, TXA-2 synthase, PLC, and other TXA-2–related proteins was also examined. Methods Right atrial tissue was harvested before and after cold blood cardioplegia, followed by about 10 minutes of reperfusion, from 28 patients undergoing cardiac operations. Coronary arterioles (90 to 170 μm in diameter) were dissected from the harvested tissue. Results The post-CP/Rep contractile response of coronary arterioles to TXA-2 analog U-46619 was significantly impaired vs pre-CP/Rep ( p < 0.05). The TXA-2 receptor antagonist SQ-29548 (10–6 M) prevented the contractile response to U-46619 ( p < 0.05). Pretreatment with the PLC inhibitor U73122 (10–6 M) significantly inhibited the U-46619–induced contractile response ( p < 0.05). Administration of the PKC-α inhibitor safingol failed to affect U-46619–induced contraction. Total protein levels and gene expression of TXA-2 receptors, TXA-2 synthase, PLC-β3, phospho–PLC-β3, PLC-γ1, and phospho–PLC-γ1 were not altered after CP/Rep. Confocal microscopy showed no significant differences in the expression of TXA-2 receptors or PLC-β3 in the microcirculation. TXA-2 receptors and PLC-β3 were both present in smooth muscle and endothelium. Conclusions Cardioplegia/Rep decreases the contractile response of human coronary arterioles to TXA-2 soon after cardiac operations. The contractile response to the TXA-2 analog U-46619 is through activation of TXA-2 receptors and PLC.
Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca2+ handling and impaired mitochondrial function accompanied with reduced ...mitochondrial calcium concentration (mito-Ca2+). Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca2+ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca2+ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-Ca2+ restores systolic Ca2+ release without adverse effects in diastole, we overexpressed mitochondrial Ca2+ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-Ca2+ using genetic probe mtRCamp1h revealed that mito-Ca2+ in TAB VMs paced at 2 Hz under β-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-Ca2+. However, it failed to improve cytosolic Ca2+ handling and reduce proarrhythmic spontaneous Ca2+ waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-Ca2+, reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca2+ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca2+ mishandling. These data establish nonlinear mito-Ca2+/mito-ROS relationship, whereby partial restoration of mito-Ca2+ in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca2+ uptake exacerbates damaging mito-ROS emission. NEW & NOTEWORTHY Defective intracellular Ca2+ homeostasis and aberrant mitochondrial function are common features in cardiac disease. Here, we directly compared potential benefits of mito-ROS scavenging and restoration of mito-Ca2+ uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca2+ biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca2+ homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.
Objective Growth factor and cell-based angiogenesis are attractive therapeutic options for diabetic patients with end-stage coronary disease. Reduced collateral vessel formation observed in diabetes ...is associated with increased expression of anti-angiogenic proteins, angiostatin and endostatin. The aim of this study was to determine the effects of insulin treatment on the diabetic angiogenic response to chronic myocardial ischemia. Methods Yucatan miniswine were treated with alloxan (pancreatic β-cell specific toxin, 150 mg/kg) and divided into two groups. In the diabetic group (DM, n = 8), blood glucose levels were kept greater than 250 mg/dL, and in the insulin-treated group (IDM, n = 6), intramuscular insulin was administered daily to keep blood glucose less than 150 mg/dL. A third group of age-matched swine served as nondiabetic controls (ND; n = 8). Eight weeks later, all animals underwent circumflex artery ameroid constrictor placement to induce chronic ischemia. Myocardial perfusion was assessed at 3 and 7 weeks after ameroid placement using microspheres. Microvascular function, capillary density, and myocardial expression of anti-angiogenic mediators were evaluated. Results Diabetic animals exhibited significant impairments in endothelium-dependent microvessel relaxation to adenosine diphosphate and substance P, which were reversed in insulin-treated animals. Collateral-dependent perfusion in the ischemic circumflex territory, which was profoundly reduced in diabetic animals (−0.18 ± 0.02 vs +0.23 ± 0.07 mL · min−1 · g−1 ; P < .001), improved significantly with insulin treatment (0.12 ± 0.05 mL · min−1 · g−1 ; P < .01). Myocardial expression of anti-angiogenic proteins, angiostatin and endostatin, showing a 4.3- and 3.6-fold increase in diabetic animals respectively (both P < .01 vs ND), was markedly reduced in insulin-treated animals (2.3- and 1.8-fold vs ND; both P < .01). Conclusions Insulin treatment successfully reversed diabetic coronary endothelial dysfunction and significantly improved the endogenous angiogenic response. These pro-angiogenic effects may be mediated through downregulation of anti-angiogenic mediators. Insulin therapy appears to be a promising modality to enhance the angiogenic response in diabetic patients.
Key points
Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.
SK channels are voltage independent and ...their gating is controlled by intracellular Ca2+ in a biphasic manner. Submicromolar Ca2+ activates the channel via constitutively‐bound calmodulin, whereas higher Ca2+ exerts inhibitory effect during depolarization.
Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function.
PKA phosphorylation attenuates ISK rectification by reducing the Ca2+/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar Ca2+i.
This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive.
Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta‐adrenergic stimulation (isoproterenol, 100 nmol L–1) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild‐type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine‐465 as the site that elicits PKA‐dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+/voltage‐dependent inhibition of the channels at high Ca2+ without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA‐dependent phosphorylation at serine‐465.
Key points
Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.
SK channels are voltage independent and their gating is controlled by intracellular Ca2+ in a biphasic manner. Submicromolar Ca2+ activates the channel via constitutively‐bound calmodulin, whereas higher Ca2+ exerts inhibitory effect during depolarization.
Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function.
PKA phosphorylation attenuates ISK rectification by reducing the Ca2+/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar Ca2+i.
This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive.
PDF
