Chronic kidney disease and left ventricular diastolic dysfunction (CKD-LVDD) alter cardiac expression of mitochondria-related genes in swine.

Cardiovascular disease and heart failure doubles in patients with chronic kidney disease (CKD), but the underlying mechanisms remain obscure. Mitochondria are central to maintaining cellular respiration and modulating cardiomyocyte function. We took advantage of our novel swine model of CKD and left ventricular diastolic dysfunction (CKD-LVDD) to investigate the expression of mitochondria-related genes and potential mechanisms regulating their expression. CKD-LVDD and normal control pigs (n=6/group, 3 males/3 females) were studied for 14 weeks. Renal and cardiac hemodynamics were quantified by multidetector-CT, echocardiography, and pressure-volume loop studies, respectively. Mitochondrial morphology (electron microscopy) and function (Oroboros) were assessed ex vivo. In randomly selected pigs (n=3/group), cardiac mRNA-, MeDIP-, and miRNA-sequencing (seq) were performed to identify mitochondria-related genes and study their pre- and post -transcriptional regulation. CKD-LVDD exhibited cardiac mitochondrial structural abnormalities and elevated mitochondrial H2O2 emission but preserved mitochondrial function. Cardiac mRNA-seq identified 862 mitochondria-related genes, of which 69 were upregulated and 33 downregulated (fold-change ≥2, false discovery rate≤0.05). Functional analysis showed that upregulated genes were primarily implicated in processes associated with oxidative stress, whereas those downregulated mainly participated in respiration and ATP synthesis. Integrated mRNA/miRNA/MeDIP-seq analysis showed that upregulated genes were modulated predominantly by miRNAs, whereas those downregulated were by miRNA and epigenetic mechanisms. CKD-LVDD alters cardiac expression of mitochondria-related genes, associated with mitochondrial structural damage but preserved respiratory function, possibly reflecting intrinsic compensatory mechanisms. Our findings may guide the development of early interventions at stages of cardiac dysfunction in which mitochondrial injury could be prevented, and the development of LVDD ameliorated.

Insulin-like growth factor 1 reduces coronary atherosclerosis in pigs with familial hypercholesterolemia.

Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque's fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis, consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress, and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis showed that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1's effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.

Western diet-induced obesity results in brain mitochondrial dysfunction in female Ossabaw swine.

Diet-induced obesity is implicated in the development of a variety of neurodegenerative disorders. Concurrently, the loss of mitochondrial Complex I protein or function is emerging as a key phenotype across an array of neurodegenerative disorders. Therefore, the objective of this study was to determine if Western diet (WD) feeding in swine [carbohydrate = 40.8% kCal (17.8% of total calories from high fructose corn syrup), protein = 16.2% kcal, fat = 42.9% kCal, and 2% cholesterol] would result in Complex I syndrome pathology. To characterize the effects of WD-induced obesity on brain mitochondria in swine, high resolution respirometry measurements from isolated brain mitochondria, oxidative phosphorylation Complex expression, and indices of oxidative stress and mitochondrial biogenesis were assessed in female Ossabaw swine fed a WD for 6-months. In line with Complex I syndrome, WD feeding severely reduced State 3 Complex I, State 3 Complex I and II, and uncoupled mitochondrial respiration in the hippocampus and prefrontal cortex (PFC). State 3 Complex I mitochondrial respiration in the PFC inversely correlated with serum total cholesterol. WD feeding also significantly reduced protein expression of oxidative phosphorylation Complexes I-V in the PFC. WD feeding significantly increased markers of antioxidant defense and mitochondrial biogenesis in the hippocampi and PFC. These data suggest WD-induced obesity may contribute to Complex I syndrome pathology by increasing oxidative stress, decreasing oxidative phosphorylation Complex protein expression, and reducing brain mitochondrial respiration. Furthermore, these findings provide mechanistic insight into the clinical link between obesity and mitochondrial Complex I related neurodegenerative disorders.

Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

Mechanism-Driven Modeling to Aid Non-invasive Monitoring of Cardiac Function via Ballistocardiography.

Left ventricular (LV) catheterization provides LV pressure-volume (P-V) loops and it represents the gold standard for cardiac function monitoring. This technique, however, is invasive and this limits its applicability in clinical and in-home settings. Ballistocardiography (BCG) is a good candidate for non-invasive cardiac monitoring, as it is based on capturing non-invasively the body motion that results from the blood flowing through the cardiovascular system. This work aims at building a mechanistic connection between changes in the BCG signal, changes in the P-V loops and changes in cardiac function. A mechanism-driven model based on cardiovascular physiology has been used as a virtual laboratory to predict how changes in cardiac function will manifest in the BCG waveform. Specifically, model simulations indicate that a decline in LV contractility results in an increase of the relative timing between the ECG and BCG signal and a decrease in BCG amplitude. The predicted changes have subsequently been observed in measurements on three swine serving as pre-clinical models for pre- and post-myocardial infarction conditions. The reproducibility of BCG measurements has been assessed on repeated, consecutive sessions of data acquisitions on three additional swine. Overall, this study provides experimental evidence supporting the utilization of mechanism-driven mathematical modeling as a guide to interpret changes in the BCG signal on the basis of cardiovascular physiology, thereby advancing the BCG technique as an effective method for non-invasive monitoring of cardiac function.

KCa3.1 Inhibition Decreases Size and Alters Composition of Atherosclerotic Lesions Induced by Low, Oscillatory Flow.

Low, oscillatory flow/shear patterns are associated with atherosclerotic lesion development. Increased expression of KCa3.1 has been found in Vascular Smooth Muscle (VSM), macrophages and T-cells in lesions from humans and mice. Increased expression of KCa3.1, is also required for VSM cell proliferation and migration. Previously, we showed that the specific KCa3.1 inhibitor, TRAM-34, could inhibit coronary neointimal development following balloon injury in swine. Atherosclerosis develops in regions with a low, oscillatory (i.e. atheroprone) flow pattern. Therefore, we used the Partial Carotid Ligation (PCL) model in high-fat fed, Apoe-/- mice to determine the role of KCa3.1 in atherosclerotic lesion composition and development. PCL was performed on 8-10 week old male Apoe-/- mice and subsequently placed on a Western diet (TD.88137, Teklad) for 4 weeks. Mice received daily s.c. injections of TRAM-34 (120 mg/kg) or equal volumes of vehicle (peanut oil, PO). 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) treatment reduced lesion size ~50% (p < 0.05). In addition, lesions from TRAM-34 treated mice contained less collagen (6% ± 1% vs. 15% ± 2%; p < 0.05), fibronectin (14% ± 3% vs. 32% ± 3%; p < 0.05) and smooth muscle content (19% ± 2% vs. 29% ± 3%; p < 0.05). Conversely, TRAM-34 had no effect on total cholesterol (1455 vs. 1334 mg/dl, PO and TRAM, resp.) or body weight (29.1 vs. 28.8 g, PO and TRAM, resp.). Medial smooth muscle of atherosclerotic carotids showed diminished RE1-Silencing Transcription Factor (REST)/Neural Restrictive Silencing Factor (NRSF) expression, while REST overexpression in vitro inhibited smooth muscle migration. Together, these data support a downregulation of REST/NRSF and upregulation of KCa3.1 in determining smooth muscle and matrix content of atherosclerotic lesions.

Mineralocorticoid receptor blockade normalizes coronary resistance in obese swine independent of functional alterations in Kv channels.

Impaired coronary microvascular function (e.g., reduced dilation and coronary flow reserve) predicts cardiac mortality in obesity, yet underlying mechanisms and potential therapeutic strategies remain poorly understood. Mineralocorticoid receptor (MR) antagonism improves coronary microvascular function in obese humans and animals. Whether MR blockade improves in vivo regulation of coronary flow, a process involving voltage-dependent K+ (Kv) channel activation, or reduces coronary structural remodeling in obesity is unclear. Thus, the goals of this investigation were to determine the effects of obesity on coronary responsiveness to reductions in arterial PO2 and potential involvement of Kv channels and whether the benefit of MR blockade involves improved coronary Kv function or altered passive structural properties of the coronary microcirculation. Hypoxemia increased coronary blood flow similarly in lean and obese swine; however, baseline coronary vascular resistance was significantly higher in obese swine. Inhibition of Kv channels reduced coronary blood flow and augmented coronary resistance under baseline conditions in lean but not obese swine and had no impact on hypoxemic coronary vasodilation. Chronic MR inhibition in obese swine normalized baseline coronary resistance, did not influence hypoxemic coronary vasodilation, and did not restore coronary Kv function (assessed in vivo, ex vivo, and via patch clamping). Lastly, MR blockade prevented obesity-associated coronary arteriolar stiffening independent of cardiac capillary density and changes in cardiac function. These data indicate that chronic MR inhibition prevents increased coronary resistance in obesity independent of Kv channel function and is associated with mitigation of obesity-mediated coronary arteriolar stiffening.

Oleanolic acid causes reversible contraception in male mice by increasing the permeability of the germinal epithelium.

The effects of oleanolic acid (OA) on the fertility of male mice were investigated using both invivo and invitro experimental models. The experimental group (n=12) was treated with a daily dose of 30mgOAkg-1 bodyweight (i.p.), while the control group (n=6) received a daily dose of 10% ethanol solution (1mLkg-1 bodyweight). The effect of OA on the permeability status of TM4 Sertoli monolayers was investigated by measuring the transepithelial electrical resistance (TER), intracellular electrical resistance and semiquantitative RT-PCR. After 45 days, OA-treated males produced no pregnancies but in the control group, all 12 females were impregnated (69 offspring). Male mice, which demonstrated sterility when exposed to OA, recovered their fertility after 30 days (78 offspring). Testicular histological observations of OA-treated mice showed detachment of adjacent Sertoli-Sertoli cells. A control monolayer developed TER of 300-400 Ω.cm2, but OA (50, 100, 200µgL-1) treated monolayers developed TER of approximately 100Ω.cm2. Intracellular electrophysiological and RT-PCR data supported the premise that OA compromised tight junctional permeability. The study demonstrated reversible contraception in male mice by increasing the permeability of the germinal epithelium and further postulates that contraceptive reversibility is brought about by the reconstitution of the paracellular junctions between adjacent Sertoli cells.

Differential impact of severe familial hypercholesterolemia on regional skeletal muscle and organ blood flows during exercise: Effects of PDE5 inhibition.

OBJECTIVE: Swine with familial hypercholesterolemia (FH) exhibit attenuated exercise-induced systemic vasodilation that is restored by phosphodiesterase 5 (PDE5) inhibition. Whether the impacts of FH and PDE5 inhibition to impair and restore exercise-induced vasodilation, respectively, results from tissue-specific or generalized effects remains unclear. Thus, we hypothesized that FH induces generalized impairment of skeletal muscle vasodilation that would be alleviated by PDE5 inhibition. METHODS: Systemic vascular responses to exercise were assessed in chronically instrumented normal and FH swine before and after PDE5 inhibition with EMD360527. Skeletal muscle and organ blood flows and conductances were determined via the microsphere technique. RESULTS: As previously reported, vs normal swine, FH swine have pronounced elevation of total cholesterol and impaired exercise-induced vasodilation that is restored by PDE5 inhibition. Blood flows to several, not all, skeletal muscle vascular beds were severely impaired by FH associated with reduced blood flow to many visceral organs. PDE5 inhibition differentially impacted skeletal muscle and organ blood flows in normal and FH swine. CONCLUSIONS: These data indicate that FH induces regional, not generalized, vasomotor dysfunction and that FH and normal swine exhibit unique tissue blood flow responses to PDE5 inhibition thereby adding to accumulating evidence of vascular bed-specific dysfunction in co-morbid conditions.

Endurance exercise training does not limit coronary atherosclerosis in familial hypercholesterolemic swine.

Human studies demonstrate that physical activity reduces both morbidity and mortality of coronary heart disease (CHD) including decreased progression and/or regression of CHD with life-style modification which includes exercise. However, evidence supporting an intrinsic, direct effect of exercise in attenuating the development of CHD is equivocal. One limitation has been the lack of a large animal model with clinically evident CHD disease. Thus, we examined the role of endurance exercise in CHD development in a swine model of familial hypercholesterolemia (FH) that exhibits robust, complex atherosclerosis. FH swine were randomly assigned to either sedentary (Sed) or exercise trained (Ex) groups. At 10 months of age, Ex pigs began a 10 months, moderate-intensity treadmill-training intervention. At 14 months, all pigs were switched to a high-fat, high-cholesterol diet. CHD was assessed by intravascular ultrasound (IVUS) both prior to and after completion of 6 months on the HFC diet. Prior to HFC diet, Ex resulted in a greater coronary artery size in the proximal and mid sections of the LCX compared to SED, with no effect in the LAD. After 6 months on HFC diet, there was a 5-6 fold increase in absolute plaque volume in all segments of the LCX and LAD in both groups. At 20 months, there was no difference in vessel volume, lumen volume, absolute or relative plaque volume in either the LCX or LAD between Sed and Ex animals. These findings fail to support an independent, direct effect of exercise in limiting CHD progression in familial hypercholesterolemia.

Vascular cell transcriptomic changes to exercise training differ directionally along and between skeletal muscle arteriolar trees.

EXT-induced arteriolar adaptations in skeletal muscle are heterogeneous because of spatial variations in muscle fiber type composition and fiber recruitment patterns during exercise. The purpose of this report is to summarize a series of experiments conducted to test the hypothesis that changes in vascular gene expression are signaled by alterations in shear stress resulting from increases in blood flow, muscle fiber type composition, and fiber recruitment patterns. We also report results from a follow-up study of Ankrd23, one gene whose expression was changed by EXT. We expected to see differences in magnitude of changes in gene expression along arteriolar trees and between/among arteriolar trees but similar directional changes. However, transcriptional profiles of arterioles/arteries from OLETF rats exposed to END or SIT reveal that EXT does not lead to similar directional changes in the transcriptome among arteriolar trees of different skeletal muscles or along arteriolar trees within a particular muscle. END caused the most changes in gene expression in 2A arterioles of soleus and white gastrocnemius with little to no changes in the FAs. Ingenuity Pathway Analysis across vessels revealed significant changes in gene expression in 18 pathways. EXT increased expression of some genes (Shc1, desert hedgehog protein (Dhh), adenylate cyclase 4 (Adcy4), G protein-binding protein, alpha (Gnat1), and Bcl2l1) in all arterioles examined, but decreased expression of ubiquitin D (Ubd) and cAMP response element modulator (Crem). Many contractile and/or structural protein genes were increased by SIT in the gastrocnemius FA, but the same genes exhibited decreased expression in red gastrocnemius arterioles. Ankrd23 mRNA levels increased with increasing branch order in the gastrocnemius arteriolar tree and were increased 19-fold in gastrocnemius muscle FA by SIT. Follow-up experiments indicate that Ankrd23 mRNA level was increased 14-fold in cannulated gastrocnemius FA when intraluminal pressure was increased from 90 and 180 cm H2O for 4 hours. Also, Ankrd23-/- mice exhibit limited ability to form collateral arteries following femoral artery occlusion compared to WT mice (angioscore WT=0.18±0.03; Ankrd23-/- =0.04±0.01). Further research will be required to determine whether Ankrd23 plays an important role in mechanically induced vascular remodeling of the arterial tree in skeletal muscle.

Severe familial hypercholesterolemia impairs the regulation of coronary blood flow and oxygen supply during exercise.

Accelerated development of coronary atherosclerosis is a defining characteristic of familial hypercholesterolemia (FH). However, the recent data highlight a significant cardiovascular risk prior to the development of critical coronary stenosis. We, therefore, examined the hypothesis that FH produces coronary microvascular dysfunction and impairs coronary vascular control at rest and during exercise in a swine model of FH. Coronary vascular responses to drug infusions and exercise were examined in chronically instrumented control and FH swine. FH swine exhibited ~tenfold elevation of plasma cholesterol and diffuse coronary atherosclerosis (20-60 % plaque burden). Similar to our recent findings in the systemic vasculature in FH swine, coronary smooth muscle nitric oxide sensitivity was increased in vivo and in vitro with maintained endothelium-dependent vasodilation in vivo in FH. At rest and during exercise, FH swine exhibited increased myocardial O2 extraction resulting in reduced coronary venous SO2 and PO2 versus control. During exercise in FH swine, the transmural distribution of coronary blood flow was unchanged; however, a shift toward anaerobic cardiac metabolism was revealed by increased coronary arteriovenous H(+) concentration gradient. This shift was associated with a worsening of cardiac efficiency (relationship between cardiac work and O2 consumption) in FH during exercise owing, in part, to a generalized reduction in stroke volume which was associated with increased left atrial pressure in FH. Our data highlight a critical role for coronary microvascular dysfunction as a contributor to impaired myocardial O2 balance, cardiac ischemia, and impaired cardiac function prior to the development of critical coronary stenosis in FH.

Upregulation of intermediate-conductance Ca2+-activated K+ channels (KCNN4) in porcine coronary smooth muscle requires NADPH oxidase 5 (NOX5).

AIMS: NADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca(2+)-activated K(+) channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors. METHODS AND RESULTS: Dihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration. CONCLUSIONS: Our findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.

Ovariectomy increases L-type Ca(2+) channel activity in porcine coronary smooth muscle.

OBJECTIVE: Sex hormone status has been demonstrated to play a role in the regulation of ion channel activity. We previously demonstrated increased L-type Ca channel current (ICa) in the coronary smooth muscle cells (SMCs) of male swine compared with female swine. In male swine, endogenous testosterone increases ICa in SMCs by enhanced expression of the pore-forming α1 subunit Cav1.2. Conversely, the role of sex hormones in female swine has not previously been investigated. Therefore, the purpose of the current study was to determine the effect of ovariectomy (OVX) on L-type Ca channel activity and expression in female Yucatan miniature swine. METHODS: Sexually mature female swine were obtained from a breeder and either left intact (intact female [IF]; n = 5) or ovariectomized (n = 6). RESULTS: Sensitivity to depolarization-induced contractions was increased by OVX. Accordingly, mean (SEM) ICa was enhanced in the OVX group (-9.5 [0.6] pA/pF) compared with the IF group (-4.5 [0.3] pA/pF), although L-type Ca channel α1 subunit (Cav1.2; α1c) messenger RNA (mRNA) and protein expressions were unchanged.Among the L-type Ca channel ß subunits, ß1 (188 [31]) and ß2a (561 [79]) had higher mRNA expression levels (target/18S) than ß3 (9 [1]) and ß4 (2 [0.1]). Although ß2a, ß3, and ß4 mRNA and protein expressions were not different between groups, protein expression of the ß1 subunit (Cavß1) was decreased in the OVX group compared with the IF group. CONCLUSIONS: Endogenous female hormones inhibit L-type Ca channel activity in coronary SMCs potentially via the up-regulation of Cavß1 subunit expression.

Reduced contribution of endothelin to the regulation of systemic and pulmonary vascular tone in severe familial hypercholesterolaemia.

Vascular dysfunction has been associated with familial hypercholesterolaemia (FH), a severe form of hyperlipidaemia. We recently demonstrated that swine with FH exhibit reduced exercise-induced systemic, but not pulmonary, vasodilatation involving reduced nitric oxide (NO) bioavailability. Since NO normally limits endothelin (ET) action, we examined the hypothesis that reduced systemic vasodilatation during exercise in FH swine results from increased ET-mediated vasoconstriction. Systemic and pulmonary vascular responses to exercise were examined in chronically instrumented normal and FH swine in the absence and presence of the ETA/B receptor antagonist tezosentan. Intrinsic reactivity to ET was further assessed in skeletal muscle arterioles. FH swine exhibited ∼9-fold elevation in total plasma cholesterol versus normal swine. Similar to our recent findings, systemic, not pulmonary, vasodilatation during exercise was reduced in FH swine. Blockade of ET receptors caused marked systemic vasodilatation at rest and during exercise in normal swine that was significantly reduced in FH swine. The reduced role of ET in FH swine in vivo was not the result of decreased arteriolar ET responsiveness, as responsiveness was increased in isolated arterioles. Smooth muscle ET receptor protein content was unaltered by FH. However, circulating plasma ET levels were reduced in FH swine. ET receptor antagonism caused pulmonary vasodilatation at rest and during exercise in normal, but not FH, swine. Therefore, contrary to our hypothesis, FH swine exhibit a generalised reduction in the role of ET in regulating vascular tone in vivo probably resulting from reduced ET production. This may represent a unique vascular consequence of severe familial hypercholesterolaemia.

Familial hypercholesterolemia impairs exercise-induced systemic vasodilation due to reduced NO bioavailability.

Hypercholesterolemia impairs endothelial function [e.g., the nitric oxide (NO)-cyclic GMP-phosphodiesterase 5 (PDE5) pathway], limits shear stress-induced vasodilation, and is therefore expected to reduce exercise-induced vasodilation. To assess the actual effects of hypercholesterolemia on endothelial function and exercise-induced vasodilation, we compared the effects of endothelial NO synthase (eNOS) and PDE5 inhibition in chronically instrumented Yucatan (Control) and Rapacz familial hypercholesterolemic (FH) swine, at rest and during treadmill exercise. The increases in systemic vascular conductance produced by ATP (relative to nitroprusside) and exercise were blunted in FH compared with Control swine. The vasoconstrictor response to eNOS inhibition, with nitro-l-arginine (NLA), was attenuated in FH compared with Control swine, both at rest and during exercise. Furthermore, whereas the vasodilator response to nitroprusside was enhanced slightly, the vasodilator response to PDE5 inhibition, with EMD360527, was reduced in FH compared with Control swine. Finally, in the pulmonary circulation, FH resulted in attenuated vasodilator responses to ATP, while maintaining the responses to both NLA and EMD360527. In conclusion, hypercholesterolemia reduces exercise-induced vasodilation in the systemic but not the pulmonary circulation. This reduction appears to be the principal result of a decrease in NO bioavailability, which is mitigated by a lower PDE5 activity.

Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca²âº-sensitive K⁺ current in miniature swine with LV hypertrophy.

Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P < 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ET(A)) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K(+) currents (I(K(+))) in coronary smooth muscle cells. Raising internal Ca(2+) from 200 to 500 nM increased Ca(2+)-sensitive K(+) current in HF-TR and control, but not HF animals. In conclusion, an ET(A)-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca(2+)-sensitive I(K(+)) was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca(2+)-sensitive I(K(+)), illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy.

The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin.

Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise alpha and beta subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype.

Endogenous testosterone attenuates neointima formation after moderate coronary balloon injury in male swine.

AIMS: Previous studies from our laboratory have demonstrated that testosterone increases coronary smooth muscle protein kinase C delta (PKC delta) both in vivo and in vitro and inhibits coronary smooth muscle proliferation by inducing G(0)/G(1) cell cycle arrest in a PKC delta-dependent manner. The purpose of the present study was to determine whether endogenous testosterone limits coronary neointima (NI) formation in a porcine model of post-angioplasty restenosis. METHODS AND RESULTS: Sexually mature, male Yucatan miniature swine were either left intact (IM), castrated (CM), or castrated with testosterone replacement (CMT; Androgel, 10 mg/day). Angioplasty was performed in both the left anterior descending and left circumflex coronary arteries with balloon catheter overinflation to induce either moderate (1.25-1.3 x diameter; 3 x 30 s) or severe (1.4x diameter; 3 x 30 s) injury, and animals were allowed to recover for either 10 or 28 days. Injured coronary sections were dissected, fixed, stained (Verheoff-Van Gieson, Ki67, PKC delta, p27), and analysed. Vessels without internal elastic laminal rupture were excluded. Following moderate injury, intimal area, intima-to-media ratio (I/M), and I/M normalized to rupture index (RI) were increased in CM compared with IM and CMT. RI, medial area, and intimal/medial thickness (IMT) were not different between groups. NI formation was inversely related to serum testosterone concentration. Conversely, following severe injury, there were no significant differences between the groups. Testosterone inhibited proliferation and stimulated PKC delta and p27(kip1) expression during NI formation (10 days post-injury). CONCLUSION: These findings demonstrate that endogenous testosterone limits coronary NI formation in male swine and provides support for a protective role for testosterone in coronary vasculoproliferative diseases, such as restenosis and atherosclerosis.

PKCdelta mediates testosterone-induced increases in coronary smooth muscle Cav1.2.

Sex hormones have emerged as important modulators of cardiovascular physiology and pathophysiology. Our previous studies demonstrated that testosterone increases expression and activity of L-type, voltage-gated calcium channels (Cav1.2) in coronary arteries of males. The purpose of the present study was to determine whether testosterone (T) alters coronary protein kinase C delta (PKCdelta) expression and whether PKCdelta plays a role in coronary Cav1.2 expression. For in vitro studies, porcine right coronary arteries (RCA) and post-confluent (passages 3-6) 5-day, serum-restricted coronary smooth muscle cell cultures (CSMC) were incubated in the presence and absence of T or dihydrotestosterone (10 and 100 nm) for 18 h at 37 degrees C in a humidified chamber. For sex and endogenous testosterone-dependent effects, RCA were obtained from intact males, castrated males, castrated males with T replacement, and intact females. In vitro T and dihydrotestosterone caused an approximately 2-3-fold increase in PKCdelta protein levels, approximately 1.5-2-fold increase in PKCdelta kinase activity, and localization of PKCdelta toward the plasma membrane and nuclear envelope. PKCdelta protein levels were higher in coronary arteries of intact males compared with intact females. Elimination of endogenous testosterone by castration reduced RCA PKCdelta protein levels, an effect partially (approximately 45%) reversed by exogenous T (castrated males with T replacement). In CSMC, PKC inhibition with either the general PKC inhibitor, cheylerythrine, or the putative PKCdelta inhibitor, rottlerin, completely inhibited the T-mediated increase in coronary Cav1.2 protein levels. Conversely, Go6976, a conventional PKC isoform inhibitor, failed to inhibit T-induced increases in coronary Cav1.2 protein levels. PKCdelta short interference RNA completely blocked T-induced increases in Cav1.2 protein levels in CSMC. These results demonstrate for the first time that 1) endogenous T is a primary modulator of coronary PKCdelta protein and activity in males and 2) T increases Cav1.2 protein expression in a PKCdelta-dependent manner.