Yucatan Miniature Swine Research Articles

Female Sex Hormones and Cardiac Pressure Overload Independently Contribute to the Cardiogenic Dementia Profile in Yucatan Miniature Swine.

Post-menopausal women with heart failure (HF) frequently exhibit cardiogenic dementia. Using a pre-clinical swine model of post-menopausal HF, we recently demonstrated that experimental menopause (ovariectomy; OVX) and HF (6-month cardiac pressure overload/aortic banding; AB) independently altered cerebral vasomotor control and together impaired cognitive function. The purpose of this study was to examine the prefrontal cortex and hippocampus tissues from these animals to assess whether OVX and HF are associated with neurologic alterations that may contribute to cardiogenic dementia. We hypothesized that OVX and HF would independently alter neuronal cell signaling in swine with post-menopausal cardiogenic dementia. Immunoblot analyses revealed OVX was associated with reduced estrogen receptor-α in both brain regions and HF tended to exacerbate OVX-induced deficits in the hippocampus. Further, OVX was associated with a reduction in the ratio of phosphorylated:total Akt and ERK in the hippocampus as well as decreased total Akt and synaptophysin in the prefrontal cortex. In contrast, HF was associated with a trend toward reduced phosphorylated:total ERK in the prefrontal cortex. In addition, HF was associated with decreased β-amyloid (1–38) in the prefrontal cortex and increased β-amyloid (1–38) in the hippocampus. Regional brain lipid analysis revealed OVX tended to increase total, saturated, and monounsaturated fatty acid content in the prefrontal cortex, with the greatest magnitude of change occurring in the AB-OVX group. The data from this study suggest that OVX and HF are independently associated with regional-specific neurologic changes in the brain that contribute to the cardiogenic dementia profile in this model. This pre-clinical swine model may be a useful tool for better understanding post-menopausal cardiogenic dementia pathology and developing novel therapies.

Electrophysiological Assessment of Cardiac Complication in Chronic Diabetic Minipigs

Abundant evidence shows that patients with type 1 diabetes are at high risk for several cardiovascular disorders. Our objective was to assess cardiac electro-pathophysiology changes linked to chronic insulin-dependent diabetes in the Yucatan miniature swine (total N = 22). Diabetic animals were divided into different groups based on duration of diabetes (Group 1: normal; Groups 2, 3, 4 and 5: diabetes duration of 3-4, 4-5, 5-6 and >6 years). Cardiac electrophysiological parameters were acquired in conscious diabetic and normal control animals using a standard lead II configuration. Routine measurements of electrocardiograms, including HR (bpm), RR, PR, QRS, QT and QTc, were done (all in msec). A heart rate correction for the QT interval (QTc) was calculated using the Fridericia method [QTc=QT/(cubed root of RR)]. Mean heart rate was decreased for the diabetic Groups 2, 4, and 5 compared to the mean heart rate for normal animals (52, 54 and 54 vs. 75 bpm, respectively). The mean PR interval was increased in all diabetic animals compared to normal animals and the effect increase with the duration of diabetes (132 vs. 138, 140, 152 and 172 msec for Groups 1, 2, 3, 4 and 5, respectively). The mean QRS interval was increased for all of the diabetic animals compared to normal animals (47 vs. 56, 66, 52 and 64 msec for Groups 1, 2, 3, 4 and 5, respectively). There were no pronounced QTc abnormalities in this study when comparing diabetic to the normal animals although one animal in Group 3 did have a QTc prolongation of 43 msec. In addition, one animal in Group 5 had a prolonged PR segment (224 msec) associated with frequent ventricular escape complexes. In conclusion, chronic diabetes in Yucatan miniature swine manifests with progressive effects on heart rate, PR interval, and QRS duration. This indicates that the diabetic minipig could provide a good model to test preventative approaches for progressive cardiac therapies in diabetes, using electrocardiography segments as markers of early heart damage. Disclosure A. Stricker-Krongrad: None. J. Liu: None. G. Bouchard: None.

Loss of sex hormones alters indices of prefrontal cortex and hippocampal insulin signaling and increases lipid content in a region‐specific manner independent of cardiac pressure overload in female aortic‐banded Yucatan miniature swine

Estimates indicate ~50% of patients with heart failure with preserved ejection fraction (HFpEF) experience dementia. Interestingly, postmenopausal women represent the largest cohort of patients with HFpEF. Impaired insulin signaling in the brain has been implicated in dementia, and may be related to regional changes in the neuronal lipid composition. How such processes are effected by the loss of female sex hormones remains to be elucidated. Using a translationally relevant large animal model of pressure overload‐induced heart failure, we tested the hypotheses that the loss of female sex hormones alters proteins involved in brain insulin signaling, and that such changes are associated with an altered lipid profile. Twenty‐eight female Yucatan miniature swine were divided into four groups (n=7): control (CON), aortic banding (AB; pressure overload model of heart failure), ovariectomy (OVX), and AB with OVX (AB‐OVX). Pigs underwent OVX and AB at 7 and 8 months of age, respectively, and prefrontal cortex and hippocampus tissues were collected at 14 months. Estrogen receptor‐α (p<0.05) content was reduced with OVX in both cortical and hippocampal brain regions. In the cortex, OVX reduced total Akt (p<0.05) and synaptophysin (p<0.10), and in the hippocampus OVX reduced phospho‐Akt Ser473 (p<0.05) and phospho‐ERK (p<0.05). Neuronal lipid composition in the prefrontal cortex and hippocampus revealed increases in total lipids (p<0.10), saturated (p<0.10), and monounsaturated fatty acids (p<0.05) with OVX. The hippocampus had increased total (p<0.10), monounsaturated (p<0.05), and polyunsaturated fats (p<0.10). No changes were observed in OVX for polyunsaturated fats. In conclusion, the loss of female sex hormones alters Akt and MAPK/ERK protein content/pathway activation predominantly in the hippocampus, independent of cardiac pressure overload. Impaired indices of hippocampal insulin signaling were associated with a local accumulation of saturated and monounsaturated fats. Overall, the loss of female sex hormones may contribute to the development of dementia in a translational swine model of pressure overload HF with potential relevance to human HFpEF through metabolic pathways influencing cognition.Support or Funding InformationNIH R01 HL112998, PI: CAEAmerican Heart Association #16POST2776005, PI: TDOAlzheimer Society of Brant, Haldimand, Norfolk, Hamilton, and Halton, PI: REKMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of a Fully Synthetic Electrospun Matrix to a Bi-Layered Xenograft in Healing Full Thickness Cutaneous Wounds in a Porcine Model.

A fully synthetic electrospun matrix was compared to a bi-layered xenograft in the healing of full thickness cutaneous wounds in Yucatan miniature swine. Full thickness wounds were created along the dorsum, to which these matrices were applied. The wound area was measured over the course of healing and wound tissue was scored for evidence of inflammation and healing. Animals were sacrificed at Day 15 and Day 30 and tissue samples from the wound site were harvested for histopathological analysis to evaluate inflammation and tissue healing as evidenced by granulation tissue, collagen maturation, vascularization, and epithelialization. Average wound area was significantly smaller for treatment group wounds compared to control group wounds at 15 and 30 days ([7.7 cm2 ± 0.9]/[3.8 cm2 ± 0.8]) and ([2.9 cm2 ± 1.1]/[0.2 cm2 ± 0.0]) (control/treatment) (p = 0.002/p = 0.01). Histopathological analysis of wound sections revealed superior quality of healing with treatment group wounds, as measured by inflammatory response, granulation tissue, and re-epithelialization. A fully synthetic electrospun matrix was associated with faster rates of wound closure characterized by granulation tissue, deposition of mature collagen and vascularization at earlier time points than in wounds treated with a bi-layered xenograft. Treatment with this fully synthetic material may represent a new standard of care by facilitating full-thickness wound closure while eliminating the risks of inflammatory response and disease transmission associated with biologic modalities.

Intrauterine growth-restricted Yucatan miniature pigs experience early catch-up growth, leading to greater adiposity and impaired lipid metabolism as young adults.

Early nutrition has critical influences on cardiovascular disease risk in adulthood. The study objectives were to evaluate the impact of low birth weight on fasting and postprandial lipid metabolism and endothelium function in Yucatan miniature pigs. Intrauterine growth-restricted (IUGR) piglets (n = 6; 3 days old, 0.73 ± 0.04 kg) were paired with normal-weight (NW) same-sex littermates (n = 6; 1.11 ± 0.05 kg) and fed milk replacer ad libitum for 4 weeks. Thereafter, all pigs were fed a standard diet ad libitum for 5 h/day with growth, intakes, and blood samples collected for 8 months. At 9 months old, pigs were surgically fitted with venous catheters and an oral fat tolerance test was performed. At 10 months old, pigs were killed and endothelium-dependent and -independent vasodilations of isolated coronary arteries were measured using wire-myographs. IUGR pigs demonstrated catch-up growth (P < 0.05) in body weight and abdominal circumference prior to sexual maturity (<7 months old) and had more (P < 0.05) subcutaneous fat at 10 months old compared with NW pigs. IUGR pigs had consistently higher fasting plasma triglyceride concentrations from 5 to 10 months old and higher liver triglyceride and total cholesterol concentrations at 10 months old (P < 0.05). The fat tolerance test revealed delayed postprandial triglyceride clearance in IUGR pigs, but no differences in plaque formation or vascular reactivity. To conclude, IUGR and early postnatal catch-up growth are associated with increased overall body fat deposition and altered triglyceride metabolism in adult Yucatan miniature swine.

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity. Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications. We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability. The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

A model of acute renal allograft rejection in outbred Yorkshire piglets

Pigs represent a desirable animal model for the study of rejection in kidney transplantation with inbred Yucatan miniature swine (YMS) the most commonly studied strain due to well defined swine leukocyte antigen (SLA) genotypes. However, limitations to YMS may include cost and availability. Outbred Yorkshire pigs are widely available and significantly cheaper than YMS. Recent advances in SLA genotyping have allowed its application to outbred strains. On this basis, we theorized that Yorkshire pigs would be a viable alternative to YMS for the study of rejection in kidney transplantation. To address this question, we performed auto (Auto) and allotransplants (Allo) in 24 Yorkshire pigs, and assessed SLA genotypes and acute rejection after 72h. At sacrifice, and when compared to autotransplants, allotransplants had significant elevations in serum creatinine (8.4±1.3 vs 2.8±2.0mg/dL for Allo vs autotransplants, respectively) and BUN (61±9 vs 19.2±15mg/dL for Allo vs autotransplants, respectively). Warm ischemia times between the two groups did not differ (24±2.3 vs 26.4±1.4min for Auto vs Allo, respectively). There were 16 distinct SLA haplotypes identified from pigs undergoing allotransplantion, no matched donor-recipient pairs, and all allografts demonstrated rejection. Type IIA cellular rejection (Banff) was the most common. One allograft demonstrated hyperacute rejection due a blood group incompatibility. Histologically, the expression of regulatory Tcells and dendritic cells was increased in allografts. These data suggest that Yorkshire pigs may be a useful model for the study of acute rejection in experimental kidney transplantation.

Recipient-derived angiogenesis with short term immunosuppression increases bone remodeling in bone vascularized composite allotransplantation: A pilot study in a swine tibial defect model.

Current vascularized composite allotransplantation (VCA) transplantation protocols rely upon life-long immune modulation to maintain tissue perfusion. Alternatively, bone-only VCA viability may be maintained in small animal models using surgical angiogenesis from implanted autogenous vessels to develop a neoangiogenic bone circulation that will not be rejected. This study tests the method's efficacy in a large animal model as a bridge to clinical practice, quantifying the remodeling and mechanical properties of porcine tibial VCAs. A segmental tibial defect was reconstructed in Yucatan miniature swine by transplantation of a matched tibia segment from an immunologically mismatched donor. Microsurgical repair of nutrient vessels was performed in all pigs, with simultaneous intramedullary placement of an autogenous arteriovenous (AV) bundle in Group 2. Group 1 served as a no-angiogenesis control. All received 2 weeks of immunosuppression. After 16 weeks, micro-CT and histomorphometric analyses were used to evaluate healing and remodeling. Axial compression and nanoindentation studies evaluated bone mechanical properties. Micro-CT analysis demonstrated significantly more new bone formation and bone remodeling at the distal allotransplant/recipient junction and on the endosteal surfaces of Group 2 tibias (p = 0.03). Elastic modulus and hardness were not adversely affected by angiogenesis. The combination of 2 weeks of immunosuppression and autogenous AV-bundle implantation within a microsurgically transplanted tibial allotransplant permitted long-term allotransplant survival over the study period of 16 weeks in this large animal model. Angiogenesis increased bone formation and remodeling without adverse mechanical effects. The method may allow future composite-tissue allotransplantation of bone without the risks associated with long-term immunosuppression. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1242-1249, 2017.

Autologous Vascularized Tissue-Engineered Liver Replacement

METHODS: A segment of jejunum from a 6-week-old, 6.4-kg Yucatan miniature swine was mechanically and enzymatically digested to form organoid units (OU) consisting of epithelium and mesenchyme. These were loaded onto a biodegradable scaffold and implanted into either the omentum or subcutaneous tissue of the autologous animal. After 4 weeks, TESI was harvested and analyzed by histology and immunofluorescence for epithelium (villin, e-cadherin, Muc2, chromogranin A), lamina propria (PDGFRa), smooth muscle (a-SMA), nerves (Tuj), and progenitor cells (Sox9).

ENGLISH

The present study was designed to determine whether adult swine with peripheral artery insufficiency (PAI) would exhibit vascular dysfunction in vessels distinct from the affected distal limbs, the coronary conduit arteries. Moreover, we sought to evaluate the effect of exercise training on coronary vasomotor function in PAI. Eighteen female healthy young Yucatan miniature swine were randomly assigned to either occluded exercise trained (Occl-Ex, n=7), or occluded-sedentary (Occl-Sed, n=5), or non-occluded, non-exercised control (Non-Occl-Con, n=6) groups. Occl-Ex pigs were progressively trained by running on a treadmill (5days/week, 12 weeks). The left descending artery (LAD) and left circumflex (LCX) coronary arteries were harvested. Vasorelaxation to adenosine diphosphate (ADP), bradykinin (BK), and sodium nitro-prusside (SNP) were assessed in LAD's; while constrictor responses to phenylephrine (PE), angiotensin II (Ang II), and endothelin-1 (ET-1) were assessed in LCX's. Vasorelaxation to ADP was reduced in LADs from Occl-Sed and Occl-Ex pigs (P<0.001) as compared to Non-Occl-Con pigs; however, Occl-Ex pigs exhibited partial recovery (P<0.001) intermediate to the other two groups. BK induced relaxation was reduced in LADs from Occl-Ex and Occl-Sed pigs (P<0.001), compared to Non-Occl-Con, and exercise modestly increased responses to BK (P<0.05). In addition, SNP, PE, Ang II, and ET-1 responses were not significantly different among the groups. Our results indicate that 'simple' occlusion of the femoral arteries induces vascular dysfunction in conduit vessels distinct from the affected hindlimbs, as evident in blunted coronary vasorelaxation responses to ADP and BK. These findings imply that PAI, even in the absence of frank atherogenic vascular disease, contributes to vascular dysfunction in the coronary arteries that could exacerbate disease outcome in patients with peripheral artery disease. Further, regular daily physical activity partially recovered the deficit observed in the coronary arteries.

Viability assessment after conventional coronary angiography using a novel cardiovascular interventional therapeutic CT system: Comparison with gross morphology in a subacute infarct swine model

BackgroundGiven the lack of promptness and inevitable use of additional contrast agents, the myocardial viability imaging procedures have not been used widely for determining the need to performing revascularization. ObjectiveThis study is aimed to evaluate the feasibility of myocardial viability assessment, consecutively with diagnostic invasive coronary angiography (ICA) without use of additional contrast agent, using a novel hybrid system comprising ICA and multislice CT (MSCT). MethodsIn all, 14 Yucatan miniature swine models (female; age, 3 months; weight, 28–30 kg) were subjected to ICA followed by balloon occlusion (90 minutes) and reperfusion of the left anterior descending coronary artery. Two weeks after induction of myocardial infarction, delayed hyperenhancement (DHE) images were obtained, using a novel combined machine comprising ICA and 320-channel MSCT scanner (Aquilion ONE, Toshiba), after 2, 5, 7, 10, 15, and 20 minutes after conventional ICA. The heart was sliced in 10-mm consecutive sections in the short-axis plane and was embedded in a solution of 1% triphenyltetrazolium chloride (TTC). Infarct size was determined as TTC-negative areas as a percentage of total left ventricular area. On MSCT images, infarct size per slice was calculated by dividing the DHE area by the total slice area (%) and compared with histochemical analyses. ResultsSerial MSCT scans revealed a peak CT attenuation of the infarct area (222.5 ± 36.5 Hounsfield units) with a maximum mean difference in CT attenuation between the infarct areas and normal myocardium of at 2 minutes after contrast injection (106.4; P for difference = 0.002). Furthermore, the percentage difference of infarct size by MSCT vs histopathologic specimen was significantly lower at 2 (8.5% ± 1.8%) and 5 minutes (9.5% ± 1.9%) than those after 7 minutes. Direct comparisons of slice-matched DHE area by MSCT demonstrated excellent correlation with TTC-derived infarct size (r = 0.952; P < .001). Bland-Altman plots of the differences between DHE by MSCT and TTC-derived infarct measurements plotted against their means showed good agreement between the 2 methods. ConclusionThe feasibility of myocardial viability assessment by DHE using MSCT after conventional ICA was proven in experimental models, and the optimal viability images were obtained after 2 to 5 minutes after the final intracoronary injection of contrast agent for conventional ICA.

Clinical Biopotency of Insulin Analogues: Comparative Assessment in Different Species

ObjectiveThe clinical potency of human insulin has classically been evaluated following the U.S. Pharmacopeia (USP) guideline for comparison to an international standard (IS) of activity (IU). However, since insulin analogues cannot be standardized against IS, there is a need for a bioassay to assess clinical specific activity (U). To this extent, we compared the biopotency (U) of different insulin products in different species and used human (recombinant) and pork (natural) insulin as reference standards.Methods: New Zealand White rabbits were fasted and injected subcutaneously (s.c.) at a dose level of 0.5 U/kg. Diabetic Yucatan miniature swine (Sus scrofa) were injected s.c. at dose level of 0.1 U/kg. Normal insulin suppression tests (nIST) were conducted in primates (Macaca fascicularis) receiving a bolus intravenous (i.v.) infusion of glucose. Insulin products were given at dose level of 0.05 U/kg. Glucose levels were recorded using handheld glucometer devices. The blood glucose kinetic (BGPK), the blood glucose area under the curve (BGAUC) and the slope of the blood glucose clearance rate (kG) were used to assess clinical biopotency.Results: The BGPK for short‐acting human and pork insulin were similar in the rabbit assay but their respective BGAUCs were different (ratio of 1.2). Accordingly pork insulin was more potent during nIST in the non‐human primate (slope of ‐0.012 vs. ‐0.010; pork and human, respectively).ConclusionOur data indicate that the clinical biopotency of insulin products can be assessed using BGAUC and kG, but that only the type 1 diabetic miniature swine can discriminate between differences in clinical biopotency for all aspects of BGPK.

Adaptations of the endothelin system after exercise training in a porcine model of ischemic heart disease.

To the test the hypothesis that exercise training would increase endothelin-mediated vasoconstriction in collateral-dependent arteries via enhanced contribution of ET(A). An ameroid constrictor was surgically placed around the proximal LCX artery to induce gradual occlusion in Yucatan miniature swine. Eight weeks postoperatively, pigs were randomized into sedentary or exercise-training (treadmill; 5 days/week; 14 weeks) groups. Subsequently, arteries (~150 μm diameter) were isolated from collateral-dependent and nonoccluded myocardial regions and studied. Following exercise training, ET-1-mediated contraction was significantly enhanced in collateral-dependent arteries. Exercise training induced a disproportionate increase in the ET(A) contribution to the ET-1 contractile response in collateral-dependent arteries, with negligible contributions by ET(B). In collateral-dependent arteries of sedentary pigs, inhibition of ET(A) or ET(B) did not significantly alter ET-1 contractile responses in collateral-dependent arteries, suggesting compensation by the functionally active receptor. These adaptations occurred without significant changes in ET(A), ET(B), or ECE mRNA levels but with significant exercise-training-induced elevations in endothelin levels in both nonoccluded and collateral-dependent myocardial regions. Taken together, these data reveal differential adaptive responses in collateral-dependent arteries based upon physical activity level. ET(A) and ET(B) appear to compensate for one another to maintain contraction in sedentary pigs, whereas exercise-training favors enhanced contribution of ET(A).

The effect of purinergic P2 receptor blockade on skeletal muscle exercise hyperemia in miniature swine.

ATP could play an important role in skeletal muscle blood flow regulation by inducing vasodilation via purinergic P2 receptors. This study investigated the role of P2 receptors in exercise hyperemia in miniature swine. We measured regional blood flow with radiolabeled-microsphere technique and systemic hemodynamics before and after arterial infusion of the P2 receptor antagonist reactive blue 2 during treadmill exercise (5.2 km/h, ~60 % VO2max) and arterial ATP infusion in female Yucatan miniature swine (~29 kg). Mean blood flow during exercise from the 16 sampled skeletal muscle tissues was 138 ± 18 mL/min/100 g (mean ± SEM), and it was reduced in 11 (~25 %) of the 16 sampled skeletal muscles after RB2 was infused. RB2 also lowered diaphragm blood flow and kidney blood flow, whereas lung tissue blood flow was increased (all P < 0.05). Infusion of RB2 increased arterial lactate concentration during exercise from 1.6 ± 0.5 to 3.4 ± 0.6 mmol/L and heart rate from 216 ± 12 to 230 ± 9 beats/min, whereas blood pressure was unaltered. Arterial ATP infusion caused a ~twofold increase in blood flow in 15 of the 16 sampled muscle tissues and this effect was abolished after RB2 infusion. These results indicate that P2 receptors play a role in regulating skeletal muscle blood flow during exercise in miniature swine.

Myosin phosphatase isoforms and related transcripts in the pig coronary circulation and effects of exercise and chronic occlusion

Myosin phosphatase (MP) is a key target of signaling pathways that regulate smooth muscle tone and blood flow. Alternative splicing of MP targeting subunit (MYPT1) exon 24 (E24) generates isoforms with variable presence of a C-terminal leucine zipper (LZ) required for activation of MP by NO/cGMP. Here we examined the expression of MP and associated genes in a disease model in the coronary circulation. Female Yucatan miniature swine remained sedentary or were exercise-trained beginning eight weeks after placement of an ameroid constrictor around the left circumflex (LCX) artery. Fourteen weeks later epicardial arteries (~1mm) and resistance arterioles (~125μm) were harvested and assayed for gene expression. MYPT1 isoforms were distinct in the epicardial arteries (E24−/LZ+) and resistance arterioles (E24+/LZ−) and unchanged by exercise training or coronary occlusion. MYPT1, CPI-17 and PDE5 mRNA levels were not different between arteries and arterioles while Kir2.1 and eNOS were 6.6-fold and 3.9-fold higher in the arterioles. There were no significant changes in transcript abundance in epicardial arteries of the collateralized (LCX) vs. non-occluded left anterior descending (LAD) territories, or in exercise-trained vs. sedentary pigs. There was a significant 1.2 fold increase in CPI-17 in collateral-dependent arterioles, independent of exercise, and a significant 1.7 fold increase in PDE5 in arterioles from exercise-trained pigs, independent of occlusion. We conclude that differences in MYPT1 E24 (LZ) isoforms, eNOS, and Kir2.1 distinguish epicardial arteries and resistance coronary arterioles. Up-regulation of coronary arteriolar PDE5 by exercise and CPI-17 by chronic occlusion could contribute to altered vasomotor responses and requires further study.

Exercise training enhances multiple mechanisms of relaxation in coronary arteries from ischemic hearts

Exercise training of coronary artery disease patients is of considerable interest, since it has been shown to improve vascular function and, thereby, enhance blood flow into compromised myocardial regions. However, the mechanisms underlying exercise-induced improvements in vascular function have not been fully elucidated. We tested the hypothesis that exercise training increases the contribution of multiple mediators to endothelium-dependent relaxation of coronary arteries in the underlying setting of chronic coronary artery occlusion. To induce gradual occlusion, an ameroid constrictor was placed around the proximal left circumflex coronary artery in Yucatan miniature swine. At 8 wk postoperatively, pigs were randomly assigned to sedentary or exercise (treadmill, 5 days/wk) regimens for 14 wk. Exercise training significantly enhanced the contribution of nitric oxide, prostanoids, and large-conductance Ca(2+)-dependent K(+) (BKCa) channels to endothelium-dependent, bradykinin-mediated relaxation in nonoccluded and collateral-dependent arteries. Combined nitric oxide synthase, prostanoid, and BKCa channel inhibition ablated the enhanced relaxation associated with exercise training. Exercise training significantly increased nitric oxide levels in response to bradykinin in endothelial cells isolated from nonoccluded and collateral-dependent arteries. Bradykinin treatment significantly increased PGI2 levels in all artery treatment groups and tended to be further enhanced after nitric oxide synthase inhibition in exercise-trained pigs. No differences were found in whole cell BKCa channel currents, BKCa channel protein levels, or arterial cyclic nucleotide levels. Although redundant, upregulation of parallel vasodilator pathways appears to contribute to enhanced endothelium-dependent relaxation, potentially providing a more refined control of blood flow after exercise training.

Exercise does not increase skeletal muscle FNDC5 protein or mRNA in pigs

Endurance exercise training (EX) is reported to increase FNDC5 expression in skeletal muscle (SkM) of mice (Nature 481:463, 2012). We tested the hypothesis that EX increases FNDC5 expression in pig SkM. Castrated male Rapacz hypercholesterolemic (FH) and normal Yucatan miniature swine (Y) were divided into EX and unexercised (SED) groups. After 16–20 weeks pigs were anesthetized and tissue samples collected and FNDC5 gene expression measured by RT‐PCR. Citrate synthase activity (CS) (index of EX‐induced adaptation) was increased in SkM samples of both FH and Y EX. FNDC5 mRNA appeared 64% less deltoid and 25% less in long head of triceps brachii (LHT) SkM of FH EX pigs. EX did not appear to affect FNDC5 mRNA expression in fat or heart muscle. FNDC5 mRNA expression in subcutaneous fat was comparable to that in the SkM. EX enhanced PGC1α mRNA levels which were much higher in the SkM samples than in fat samples. The changes in PGC1α mRNA levels were not correlated with FNDC5 mRNA. Despite the increase in CS activity in SkM, there was no difference in FNDC5 protein content (Western blot) between SED and EX SkM of Y pigs. We conclude that EX does not increase FNDC5 mRNA or protein in pig SkM.

Exercise Training‐Enhanced, Endothelium‐Dependent Dilation Mediated by Altered Regulation of BKCa Channels in Collateral‐Dependent Porcine Coronary Arterioles

Test the hypothesis that exercise training increases the contribution of BK(Ca) channels to endothelium-mediated dilation in coronary arterioles from collateral-dependent myocardial regions of chronically occluded pig hearts and may function downstream of H2O2. An ameroid constrictor was placed around the proximal left circumflex coronary artery to induce gradual occlusion in Yucatan miniature swine. Eight weeks postoperatively, pigs were randomly assigned to sedentary or exercise training (treadmill; 14 week) regimens. Exercise training significantly enhanced bradykinin-mediated dilation in collateral-dependent arterioles (~125 μm diameter) compared with sedentary pigs. The BK(Ca) -channel blocker, iberiotoxin alone or in combination with the H2O2 scavenger, polyethylene glycol catalase, reversed exercise training-enhanced dilation in collateral-dependent arterioles. Iberiotoxin-sensitive whole-cell K+ currents (i.e., BK(Ca)-channel currents) were not different between smooth muscle cells of nonoccluded and collateral-dependent arterioles of sedentary and exercise trained groups. These data provide evidence that BK(Ca)-channel activity contributes to exercise training-enhanced endothelium-dependent dilation in collateral-dependent coronary arterioles despite no change in smooth muscle BK(Ca)-channel current. Taken together, our findings suggest that a component of the bradykinin signaling pathway, which stimulates BK(Ca) channels, is enhanced by exercise training in collateral-dependent arterioles and suggest a potential role for H2O2 as the mediator.

Porcine model to evaluate local tissue tolerability associated with subcutaneous delivery of protein

IntroductionThe conversion from intravenous (IV) to subcutaneous (SC) delivery of biotherapeutics has increased in recent years. Some of the reasons for this shift in route of delivery are due to patient convenience, reduced adverse systemic effects, lack of a need for vascular access, and reduced cost of patient care, which ultimately lead to improved patient quality of life. One caveat to SC delivery is the limited volumes that can be administered at a single site and the associated local tolerability. To characterize factors that affect subcutaneous delivery of large volumes of therapeutic proteins, a porcine model was developed. Model endpoints included measurement of interstitial pressure, assessment of local skin visco-elasticity, and the qualitative assessment of local infusion sites. MethodsImmunoglobulin G (IgG) was subcutaneously infused into the abdominal region of Yucatan miniature swine. Changes in interstitial pressure were measured, using an in-line pressure transducer, during and after infusions. Additionally, pre- and post-infusion changes in local skin visco-elasticity were measured using a Cutometer®. Lastly, infusion sites were assessed for post-infusion local skin reactions such as erythema and swelling. Similar assessments were made following SC IgG delivery with the permeation enhancer recombinant human hyaluronidase PH20 (rHuPH20). ResultsSubcutaneous infusions of IgG, in the presence of rHuPH20, significantly reduced average interstitial pressures by 55% during the infusion period and by 67% during the post-infusion period, compared to the control. Infusions in the presence of rHuPH20 also maintained better local skin elasticity as seen by a 42% increase in local skin pliability compared to the control. Finally, infusions with rHuPH20 resulted in an 80% reduction in swelling area compared to the control. DiscussionA large animal model was developed that incorporates both quantitative and qualitative assessment methods to aid in understanding SC delivery of proteins.

Heart failure with preserved ejection fraction: chronic low-intensity interval exercise training preserves myocardial O2 balance and diastolic function

We have previously reported chronic low-intensity interval exercise training attenuates fibrosis, impaired cardiac mitochondrial function, and coronary vascular dysfunction in miniature swine with left ventricular (LV) hypertrophy (Emter CA, Baines CP. Am J Physiol Heart Circ Physiol 299: H1348-H1356, 2010; Emter CA, et al. Am J Physiol Heart Circ Physiol 301: H1687-H1694, 2011). The purpose of this study was to test two hypotheses: 1) chronic low-intensity interval training preserves normal myocardial oxygen supply/demand balance; and 2) training-dependent attenuation of LV fibrotic remodeling improves diastolic function in aortic-banded sedentary, exercise-trained (HF-TR), and control sedentary male Yucatan miniature swine displaying symptoms of heart failure with preserved ejection fraction. Pressure-volume loops, coronary blood flow, and two-dimensional speckle tracking ultrasound were utilized in vivo under conditions of increasing peripheral mean arterial pressure and β-adrenergic stimulation 6 mo postsurgery to evaluate cardiac function. Normal diastolic function in HF-TR animals was characterized by prevention of increased time constant of isovolumic relaxation, normal LV untwisting rate, and enhanced apical circumferential and radial strain rate. Reduced fibrosis, normal matrix metalloproteinase-2 and tissue inhibitors of metalloproteinase-4 mRNA expression, and increased collagen III isoform mRNA levels (P < 0.05) accompanied improved diastolic function following chronic training. Exercise-dependent improvements in coronary blood flow for a given myocardial oxygen consumption (P < 0.05) and cardiac efficiency (stroke work to myocardial oxygen consumption, P < 0.05) were associated with preserved contractile reserve. LV hypertrophy in HF-TR animals was associated with increased activation of Akt and preservation of activated JNK/SAPK. In conclusion, chronic low-intensity interval exercise training attenuates diastolic impairment by promoting compliant extracellular matrix fibrotic components and preserving extracellular matrix regulatory mechanisms, preserves myocardial oxygen balance, and promotes a physiological molecular hypertrophic signaling phenotype in a large animal model resembling heart failure with preserved ejection fraction.