Ossabaw Swine Research Articles

Mitochondrial and Microvascular Impairments in a Novel Swine Model of Peripheral Artery Disease

Introduction: Impaired skeletal muscle mitochondria and microvascular function have been reported in human peripheral artery disease (PAD). However, no large animal models of PAD have mimicked these impairments. Purpose: The purpose of this study was to examine the skeletal muscle microvasculature and mitochondria in a novel swine model of PAD. Hypothesis: We hypothesized that hindlimb ischemia leads to dysfunction of skeletal muscle microvessels and mitochondria. Methods: Ossabaw swine (n=5) with metabolic syndrome underwent endovascular coil occlusion of the right external iliac artery to generate hindlimb ischemia (ISC). The left leg served as a non-ischemic control (CON). After 4-weeks, biopsies were collected from both legs (lateral gastrocnemius). Skeletal muscle arterioles were assessed using video microscopy. Microvascular vasodilatory function was assessed in response to flow (Shear stress), acetylcholine (ACh), and sodium nitroprusside (SNP). Skeletal muscle mitochondrial function was assessed using high-resolution respirometry. Substrates were administered as follows: malate (M) & glutamate (G), adenosine diphosphate (ADP), succinate (S), oligomycin, and ascorbate (Asc) & N,N,N,N-tetramethyl-phenylenediamine (TMPD). Complex I state 2 (CI-S2) was assessed as M+G, complex I state 3 (CI-S3) was assessed as (M+G+ADP)-(M+G), complex II state 3 (CII-S3) was assessed as (M+G+ADP+S)-(M+G+ADP), complex I&II state 3 (CI&II S3) was assessed as (M+G+ADP+S)-(M+G), state 4 (S4) was assessed after oligomycin, and complex IV (CIV) respiration was assessed after Asc+TMPD. The respiratory control ratio (RCR) was calculated as CI&II-S3/S4. Results: Vasodilation in response to flow was reduced in ISC vs. CON (Δ-13.1±13.7%, p=0.01). but not in response to ACh (Δ-6.2±17.7%, p=0.23) or SNP (Δ1.7±11.0%, p=0.53). Additionally, CI-S2 (Δ-0.3±3.7 pmolO2·s−1·mg−1, p=0.65), S4 (Δ-1.7±4.4 pmolO2·s−1·mg−1, p=0.14), and CIV (Δ-2.8±20.9 pmolO2·s−1·mg−1, p=0.65) were not different between ISC and CON. However, CI-S3 (Δ-4.7±5.0 pmolO2·s−1·mg−1, p<0.01), CII-S3 (Δ-3.5±5.0 pmolO2·s−1·mg−1, p=0.02), CI&II-S3 (Δ-8.5±8.7 pmolO2·s−1·mg−1, p<0.01), and RCR (Δ-0.8±1.3 pmolO2·s−1·mg−1, p=0.04) were all reduced in ISC vs. CON. Conclusions: We found that skeletal muscle microvessels and mitochondria were impaired in our swine model. Based on the present findings, we conclude that our novel porcine model closely replicates the pathophysiology PAD, making it a promising large animal model for studying human PAD. This research was supported by the National Institutes of Health (R01HD106911, R01AG062198, R01AG0778031) and the Great Plains IdeA-CTR (U54 GM115458). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1154: Length And Proximal Extent Of Occlusion Dictates Severity Of Disease In A Mini-swine Model Of PAD

Background: There is ongoing need for large animal models of peripheral artery disease (PAD) that replicate human disease. We present mild (MI), moderate (MD) and Severe (S) phenotypes of PAD in an Ossabaw mini-swine platform. Methods: Metabolic syndrome was induced in swine (N=16; age= 4 mo) with a high fat/fructose/sodium diet for 8 weeks. Ischemia induction (T0) consisted of: (1) open ligation and resection of R Superficial Femoral Artery (SFA) for MI(N=4); (2) endovascular coil occlusion of R External Iliac Artery (REIA), RSFA and popliteal artery for MD (N=9); and (3) coil occlusion of REIA, RSFA, and right internal iliac artery (RIIA) for S (N=4). At 4 weeks (T4) and 8 (T8), all swine underwent angiography via carotid access, and bilateral gastrocnemius biopsy. All swine had measurements of ankle brachial indices (ABIs), and calf muscle tissue oximetry (STO 2 ) measurements at T0, T4 and T8, along with weekly measurement of walking performance on a treadmill (WP). Results: At T4 and T8 R ABI were increasingly decreased from MI to MD to S (table 1). Mean STO 2 difference between R and L calf at T4 and T8 had no changes in MI, while the differences were larger in S compared to MD (table 1). WP was increasingly decreased from MI to MD to S (table 1). The artery that grew the most on angiography at T4 and T8 were R profunda artery (RPFA) in MI; RIIA in MD; and LIIA in S (table 1). In MI, R PFA had faster flow at T4 and T8 compared to L PFA. At T4, MD and S had similar delays of RPFA reconstitution, this improved at T8 in MD but did not in S (table 1). On histology, MI had no obvious myopathy, MD and S had moderate and severe myopathy respectively. All S subjects developed poorly-healing ischemic ulcers. Conclusion: Length and proximal extent of occlusion dictated the characteristics and severity of phenotypes in our Ossabaw swine model of PAD. This mirrors PAD in humans and can be used to develop therapies for different disease severities.

A pathway to precision medicine: coronary and cerebral arterial transcriptomic analysis reveals distinct pathophysiological adaptations in two clinically relevant porcine models of heart failure

Traditional treatments for heart failure (HF) with reduced ejection fraction (HFrEF) are ineffective at treating HF with preserved ejection fraction (HFpEF), establishing a need for more targeted therapeutic approaches. To date, the comparative vascular adaptions induced by these two distinct forms of HF have been largely unexplored. Therefore, the objective of this study was to compare HFrEF and HFpEF pathophysiological adaptions in both coronary and cerebral microvasculature in two clinically relevant swine models of HF. We hypothesized that HFrEF and HFpEF will induce differing pathophysiological adaptations within the microvasculature of the heart as well as peripheral tissues, such as the brain. Intact female Ossabaw swine (2 months old) were fed a Western Diet for 4 months to develop metabolic syndrome (HOMA-IR: HFrEF 2.94 ± 0.34; HFpEF 2.95 ± 0.59). At 6 months of age, animals were either subjected to 90 minutes ischemia followed by reperfusion (I/R) to induce a myocardial infarction (MI) recapitulating cardiometabolic HFrEF (38.0 ± 3.9% EF) or were aortic banded to induce pressure-overload with relevance to cardiometabolic HFpEF (61.5 ± 1.7% EF). Following humane euthanasia, coronary arterioles (139.5 ± 4.9 μm inner diameter) and middle cerebral artery second order pial arterioles (281.1 ± 16.7 μm) from each model were dissected and either cannulated and pressurized for myography experiments or flash frozen for RNA isolation, sequencing, and predictive pathway analysis. Smooth muscle dependent U46619 (thromboxane A2 agonist)-induced constriction of cerebral arterioles was not different between groups (HFrEF 46.9 ± 10.3%; HFpEF 50.7 ± 4.8%), however, smooth muscle dependent dilation to sodium nitroprusside (SNP, nitric oxide mimetic) was significantly reduced in HFrEF (37.1 ± 14.0%) compared to HFpEF (83.6 ± 4.6%), suggesting the dilatory capacity of peripheral vessels is impaired when EF% is reduced. Furthermore, RNA-sequencing comparison between HFrEF and HFpEF uncovered 35 differentially expressed genes in the coronary (n=3) and 23 differentially expressed genes in cerebral (n=3) arterioles (q ≤ 0.1). Ingenuity pathway analysis predicted upstream regulators that code for angiotensinogen, STAT3, and interferon-Ƴ were inhibited (z-score > -2.0) in the HFrEF model, which was associated with decreased proliferation of smooth muscle cells and vasculogenesis with increased organismal death and inflammation in the coronary arterioles. Furthermore, analysis of the cerebral arterioles predicted inhibition of angiotensinogen (z-score > -2.0) in the HFrEF model which was associated with increased fibroblast cell death, dedifferentiation of smooth muscle cells, and recruitment of macrophages. Together, these data reveal distinct pathophysiological functional and transcriptomic adaptions between HFrEF and HFpEF in the coronary and cerebral arterioles. Additionally, these data provide three candidate genes that regulate these pathophysiological processes and could be targeted for potential therapeutic intervention. Funding: REGENCOR and Department of Defense (DOD) W81XWH-18-1-0179. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Abstract 539: Mini-swine Model Of Hindlimb Ischemia On A Background Of Metabolic Syndrome Mimics Peripheral Artery Disease And Claudication

Background: There is an ongoing need for an animal model of PAD that is similar to the human patients in its size, anatomy, physiology, comorbidities and suitability for invasive/non-invasive testing and treatment. Methods: Three months old Ossabaw swine (N=9) were fed a high fat/fructose/sodium diet for 8 weeks. Right hindlimb ischemia was then induced (N=6, 2 female) using Right Superficial Femoral Artery (RSFA cutdown, right external iliac artery endovascular (R EIA) coil embolization/occlusion and RSFA resection (T0) vs sham (N=3, 1 female). At 4 weeks (T4), all swine underwent R carotid cutdown, 2-D angiography, and bilateral(b/l) gastrocnemius open biopsy. At 8 weeks (T8) swine underwent L carotid cutdown, 2-D angiography, b/l sampling of the gastrocnemius. During all procedures, b/l ankle brachial indices (ABIs), and tissue oximetry on the calf and thigh were measured. All swine underwent weekly measurement of their treadmill walking distance. Gastrocnemius muscle mitochondrial respiration was measured with an Oroboros Oxygraph-2k. Results: Blood draws showed hyperlipidemia and hyperglycemia in all swine at T0, T4, and T8 after a 12-hour fasting period. Hindlimb ischemia lowered right-sided ABIs at T4 and T8 by 45 and 39% (p<0.001) , and tissue oximetry on R calf (28 and 18% p<0.01) and thigh (22 and 18% P<.01 ) with no differences in the sham. Ischemia reduced treadmill times from T0 to T8 from 24 to 9.4 minutes (p<0.01) vs sham increase from 23.3 to 32.2 (p<0.09) . R internal Iliac artery had the largest growth of cross-sectional area at T4 and T8 (111.7 and 115.2%) compared to L EIA (40.6 and 46.1% - p< 0.02 and <0.5 ) and aorta (14.5 and 29% p <0.02 and <0.02 ). R profunda artery angiographic appearance was delayed at T4, which improved at T8 after normalization of the timing ratio from right to left (3.25 vs 2.3 p<0.008 ). Mitochondrial oxygen consumption in the ischemic right gastrocnemius was consistently lower ex-vivo (21.96±6.41 vs 15.11±11.1 p<0.03 ) with no differences in the sham. We had no fatalities. Discussion: We present a porcine model of hindlimb ischemia (iliofemoral artery occlusion) which recapitulates key aspects of the pathophysiology of human PAD/ claudication, and can support the development of new therapies for PAD.

A novel RSK3/mAKAPß signalosome-focused gene therapy inhibits developing heart failure in aortic-banded, Western diet-fed female Ossabaw swine

RSK3 (p90 ribosomal S6 kinase 3) in the cardiomyocyte is required for the induction of pathological cardiac remodeling through its action at perinuclear signalosomes organized by the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ). At mAKAPβ, RSK3 phosphorylates the transcription factor serum response factor in response to adrenergic stimulation, promoting concentric cardiac hypertrophy and interstitial myocardial fibrosis. We have developed a novel preclinical Ossabaw swine model of cardiometabolic heart failure featuring metabolic syndrome (obesity, insulin resistance, dyslipidemia) and cardiovascular dysfunction including concentric hypertrophy, diastolic dysfunction, preserved ejection fraction, and pulmonary edema. The purpose of this study was to examine a novel gene therapy that displaces RSK3 on mAKAPβ by adeno-associated virus (AAV)-based expression of a RSK3|mAKAPβ anchoring disruptor peptide (RBD) in cardiomyocytes (AAV9sc.RBD). We hypothesized that disruption of RSK3|mAKAPβ by AAV9sc.RBD would improve cardiac function and decrease pulmonary congestion. Female Ossabaw pigs were assigned to HF control (HF; n=4-5) and HF AAV9sc.RBD-treated (HF+RBD; n=4-7) groups. Animals were fed a Western diet (3 mo. old) and aortic banded (6 mo. old) prior to terminal experiments (12 mo. old). Peripheral intravenous saline containing 3 x 1014 viral genomes (vg) of AAV9sc.RBD biologic was infused immediately after aortic banding. Pressure-volume hemodynamics were used to measure cardiac function, and group comparisons were made using student’s t-test or Mann-Whitney test with significance reported at the P≤0.05 level. 0.9-2.5 x104 vg|μg genomic DNA was delivered in the heart, equivalent to ~0.4-1.2 virion per adult swine cardiomyocyte. Wet lung weight was decreased in HF+RBD animals compared to HF (283±19 vs. 423±64 g) and associated with improved diastolic function evident by a decrease in the withend diastolic pressure-volume relationship (EDPVR: 0.039±0.004 vs. 0.022±0.003 mmHg|mL). Stroke volume was increased in the HF+RBD group (40±2 vs. 50±2 mL) and associated improved hemodynamic coupling between the left ventricle and peripheral vasculature demonstrated by decreased arterial elastance (Ea; 2.3±0.2 vs. 1.7±0.2 mmHg|mL) and the ventricular-arterial coupling ratio (Ea|ESPVR; 0.14±0.03 vs. 0.06±0.0.01, P=0.06). These data suggest AAV9sc.RBD-mediated disruption of RSK3|mAKAPβ signalosomes in the cardiomyocyte improves cardiac function and ventricular-vascular interaction, highlighting the potential of gene therapy for treating cardiometabolic heart failure Department of Defense (DOD) W81XWH-18-1-0179 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High fat diet feeding induces brain mitochondrial dysfunction in female Ossabaw swine

Study objective: Emerging evidence now suggest reduced mitochondrial function as a key initiator in Alzheimer’s disease (AD)-like pathology increasing the need for models of AD that display mitochondrial dysfunction. Dyslipidemia is a common aliment from overconsumption of a high fat diet (HFD) that increases the risk of AD and may contribute to mitochondrial dysfunction. Rodent studies utilize HFDs to induce brain pathology resembling that of AD. Unsurprisingly, rodent studies are rarely recapitulated in humans, which leads to a very low clinical success rate and highlights the need for more translatable models to study AD-like pathology. Swine seem to share many characteristics of humans and might offer a more translatable model to study HFD induced pathology. Thus, the objective of this study was to determine if HFD feeding in swine could offer a more translatable model to study mitochondrial dysfunction during neurodegeneration. Hypothesis: 6-months of HFD feeding will decrease indices of mitochondrial function and biogenesis in female Ossabaw swine. Methods: To characterize the effects of a HFD on brain mitochondrial health in a highly translatable swine model, high resolution respirometry measurements were taken from isolated brain mitochondria from in female Ossabaw swine fed a HFD for 6-months. Additionally, indices of mitochondrial biogenesis in whole tissue was measured (transcriptional markers of biogenesis and citrate synthase activity). Results: HFD feeding significantly increased body weight (p<0.05) with trending increases in blood glucose (p<0.10). Maximal uncoupled mitochondrial respiration was significantly decreased (p<0.05) with state 3-complex I and state 3 complex I & II trending down in the dorsal hippocampus (DH) after HFD diet feeding (p<0.10). Additionally, state 2 respiration was significantly decreased in the prefrontal cortex (PFC) after HFD feeding (p<0.05). HFD feeding increased transcriptional markers of mitochondrial biogenesis in the DH and PFC (p<0.05), without altering whole homogenate citrate synthase activity in either brain region. Conclusions: These data indicate HFD feeding in Ossabaw pigs could offer a potential model to study pathologies relating to mitochondrial dysfunction during neurodegenerative process. Additionally, the DH was found to be more vulnerable to HFD-induced perturbations, evident by the exacerbated deficits in mitochondrial respiration in this brain region. Finally, these data suggest HFD-induced increase in transcriptional markers of mitochondrial biogenesis may be a compensatory response to HFD-induced mitochondrial respiration deficits. However, citrate synthase activity was not different between groups indicating this upregulation of transcriptional markers was unable to translate to an increase in mitochondrial content in the DH and PFC. The project was supported by the MU Office of Postdoctoral Education Research Grant Program. Additional funding was also generously provided Scott Rector. Images in figure 2 adapted and modified from “Krebs cycle template”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates . This work was also supported with resources and the use of facilities at the University of Missouri and Harry S. Truman Memorial Veterans Hospital in Columbia, Missouri. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Decreased arterial elastance following a novel RSK3/mAKAPß gene therapy is associated with improved bradykinin-dependent pial artery dilation in aortic-banded, Western diet-fed female Ossabaw swine

Cardiogenic dementia is associated with increased risk of mortality in heart failure. Cognitive decline is more prevalent in females, of which cardiac function is a strong predictor. We have demonstrated cerebrovascular insufficiency in female Ossabaw swine with cardiometabolic heart failure (CM-HF). The purpose of this study was to examine the effect of a novel gene therapy that displaces p90 ribosomal S6 kinase 3 (RSK3) from perinuclear muscle A-kinase anchoring protein β (mAKAPβ) signalosomes using a systemically delivered adeno-associated virus (AAV) to express a RSK3/mAKAPβ anchoring disruptor peptide (RBD) controlled by a cardiomyocyte-specific promoter (AAV9.RBD). We hypothesized that disruption of RSK3/mAKAPβ by AAV9.RBD would improve cardiac and cerebrovascular function in parallel, diminishing cardiogenic pathology. Female Ossabaw pigs were assigned to HF control (HF; n=4) and HF AAV9.RBD-treated (HF+RBD; n=5) groups. Animals were fed a Western diet (3 mo. old) and aortic banded (6 mo. old) prior to euthanasia (12 mo. old). Pressure-volume hemodynamics were used to measure arterial elastance (Ea), the ratio of end systolic pressure:stroke volume (SV). Pressure myography was used to assess in vitro cerebral vascular function (2a pial arterioles; inner diameter 267 ± 19 μm HF & 248 ± 9 μm HF+RBD). Group comparisons were made using student’s t-test and repeated measures ANOVA with significance reported at the P≤0.05 level. Ea was decreased in HF+RBD animals compared to HF (mmHg/mL; 2.3 ± 0.2 HF & 1.7 ± 0.2 HF+RBD) and driven by increased SV (mL; 40 ± 2 HF & 50 ± 2 HF+RBD). Decreased arterial load was associated with increased endothelial-dependent dilation to bradykinin (BK) in the HF+RBD group compared to HF (Group x Dose interaction) in isolated pial arteries. BK-induced dilation was decreased in the presence of L-NAME (nitric oxide synthase inhibitor) in HF, but not HF+RBD animals (Group x Dose interaction). In contrast, BK-induced dilation was decreased in the presence of indomethacin (prostaglandin synthesis inhibitor) only in the HF+RBD group (Group x Dose interaction). In conclusion, decreased cardiac arterial load (Ea) was coupled with improved endothelial-dependent cerebral vascular function that was mechanistically characterized by altered nitric oxide/prostaglandin balance in female Ossabaw swine with CM-HF. These findings suggest AAV9.RBD-mediated disruption of RSK3/mAKAPβ signalosomes in cardiomyocytes has a positive cardiogenic effect on cerebrovascular function. Department of Defense (DOD) W81XWH-18-1-0179 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mesenteric fat cryolipolysis attenuates insulin resistance in the Ossabaw swine model of the metabolic syndrome.

The rising prevalence of insulin resistance (IR), metabolic syndrome, and type 2 diabetes are associated with increases in abdominal mesenteric fat. Adipocytes are sensitive to low temperatures, making cryolipolysis of mesenteric fat an attractive treatment modality to potentially reduce IR. We aimed to determine whether (1) cryolipolysis is safe in reducing the volume of the mesenteric fat and (2) reduction in mesenteric fat volume reduces indices of IR and glycemic dysfunction. Indiana University School of Medicine. A novel cooling device and method delivered cryolipolysis in a controlled manner to avoid tissue ablative temperatures. Ossabaw pigs (n = 8) were fed a high-fat diet for 9 months to develop visceral obesity, IR, and metabolic syndrome. Following laparotomy, mesenteric fat cryolipolysis (MFC) was performed in 5 pigs, while 3 served as sham surgery controls. The volume of the mesenteric fat was measured by computed tomography and compared with indices of glucose intolerance before and at 3 and 6 months postprocedure. MFC safely reduced mesenteric fat volume by ∼30% at 3 months, which was maintained at 6 months. Body weight did not change in either the MFC or sham surgery control groups. Measure of glycemic control, insulin sensitivity, and blood pressure significantly improved after MFC compared with sham controls. MFC reduces the volume of mesenteric fat and improves glycemic control in obese, IR Ossabaw pigs, without adverse effects.

Chronic high-rate pacing induces heart failure with preserved ejection fraction-like phenotype in Ossabaw swine.

The lack of pre-clinical large animal models of heart failure with preserved ejection fraction (HFpEF) remains a growing, yet unmet obstacle to improving understanding of this complex condition. We examined whether chronic cardiometabolic stress in Ossabaw swine, which possess a genetic propensity for obesity and cardiovascular complications, produces an HFpEF-like phenotype. Swine were fed standard chow (lean; n = 13) or an excess calorie, high-fat, high-fructose diet (obese; n = 16) for ~ 18weeks with lean (n = 5) and obese (n = 8) swine subjected to right ventricular pacing (180 beats/min for ~ 4weeks) to induce heart failure (HF). Baseline blood pressure, heart rate, LV end-diastolic volume, and ejection fraction were similar between groups. High-rate pacing increased LV end-diastolic pressure from ~ 11 ± 1mmHg in lean and obese swine to ~ 26 ± 2mmHg in lean HF and obese HF swine. Regression analyses revealed an upward shift in LV diastolic pressure vs. diastolic volume in paced swine that was associated with an ~ twofold increase in myocardial fibrosis and an ~ 50% reduction in myocardial capillary density. Hemodynamic responses to graded hemorrhage revealed an ~ 40% decrease in the chronotropic response to reductions in blood pressure in lean HF and obese HF swine without appreciable changes in myocardial oxygen delivery or transmural perfusion. These findings support that high-rate ventricular pacing of lean and obese Ossabaw swine initiates underlying cardiac remodeling accompanied by elevated LV filling pressures with normal ejection fraction. This distinct pre-clinical tool provides a unique platform for further mechanistic and therapeutic studies of this highly complex syndrome.

Hypoglycosylated Follistatin‐Like 1 Attenuates the Loss of Coronary and Cerebral Vascular Functional Capacity in Ossabaw Swine with Cardiometabolic Heart Failure with Reduced Ejection Fraction

Ischemic heart disease is a leading cause of death, often resulting from heart failure caused by myocardial infarction. Diabetic cardiomyopathy is increasingly problematic clinically, with the combination of metabolic syndrome and chronic inflammation greatly increasing the risk for both coronary and cerebral vascular disease. Follistatin‐like 1 (FSTL1) is a protein that, when non‐glycosylated, displays regenerative properties including pro‐angiogenic effects and prevention of abnormal vascular remodeling. Thus, the objective of the current study was to assess the therapeutic benefit of human recombinant non‐glycosylated FSTL1 protein on both coronary and cerebral vascular function in a pre‐clinical Ossabaw swine model of myocardial infarction (MI). We hypothesized FSTL1 would attenuate the development of coronary and cerebral vascular dysfunction in an experimental setting of cardiometabolic heart failure with reduced ejection fraction (HFrEF). Intact female Ossabaw swine (2 months old) were fed a Western Diet for 4 months to develop metabolic syndrome. At 6 months of age, animals were subjected to 90 minutes ischemia followed by reperfusion (I/R) to induce MI. One month post‐MI, ALZET osmotic pumps were implanted and either vehicle (MI group) or FSTL1 (MI+FSTL1 group) was delivered over two weeks, with terminal vascular experiments performed 2 months post‐MI. In vitroassessment of isolated coronary (n=5‐6 for infarct, border, and remote regions of the left ventricle; 139.5 ± 4.9 μm diameter) and cerebral (n=2‐6 for middle cerebral artery second order pial; 385.3 ± 18.7 μm diameter) arteriole function was examined using pressure myography. Dose response curves for: 1) U46619 (thromboxane A2 agonist); and 2) NS‐1619 (large‐conductance calcium‐activated potassium channel activator; BKCa) were conducted. A 2x2 ANOVA (Group X Dose) was used to determine significance at the p < 0.05 level. Cardiometabolic HFrEF was indicated by a combined ejection fraction of 38 ± 2% and a fasting HOMA‐IR of 3.8 ± 0.4 (vs. 0.6 ± 0.1 historical control). In coronary arterioles, the NS‐1619 induced vasodilatory capacity was increased ~18‐fold in MI+FSTL1 animals relative to MI (100 ± 13% vs. 1832 ± 3% in MI and MI+FSTL1, respectively) in the remote region of the heart (interaction effect), but not the infarct nor border regions. In cerebral arterioles, the vasoconstrictor capacity in response to U46619 was increased ~2‐fold in MI+FSTL1 relative to MI animals (100 ± 19% vs. 213 ± 25% in MI and MI+FSTL1, respectively; interaction effect). In conclusion, FSTL1 partially attenuates the loss of functional capacity in both coronary and cerebral arterioles, demonstrating its potential to improve vascular function in an experimental setting of cardiometabolic HFrEF.

ST segment elevation: the lost measure of early ischemic preconditioning in the landmark study of Murry et al. 1986

BackgroundIn their landmark study Murry et al. (Circulation 74:1124, 1986) showed dramatic ST segment elevation of the ECG during the first occlusion of a dog coronary artery. They made the insightful observation that “… in subsequent occlusions, these changes were slower to develop and frequently did not reach the magnitude seen during the first occlusion.” This electrophysiological preconditioning has been almost completely dismissed as irrelevant to ischemic preconditioning in animal models. We found in the Ossabaw miniature swine model of metabolic syndrome that brief coronary occlusion often led to ST elevation that quickly progressed to ventricular tachycardia and fibrillation.HypothesisAn AMPKγ3 mutation in Ossabaw swine will explain the extreme ST elevation and provide evidence for the relevance of ST segment ischemic preconditioning.Methods and ResultsLean mutants had impaired glucose tolerance compared to wild type Ossabaw swine. Myocardial ischemia induced by balloon occlusion of the circumflex artery elicited profound electrocardiographic ST elevation in lean mutant compared to wild type swine. Infusion of an AMPK agonist, AICAR (0.5 mM), prior to occlusion significantly reduced ST elevation in lean mutants, while completely preventing ST segment elevation in wild type. Ischemic preconditioning of the ST segment changes occurred in wild type pigs during the course of 5 occlusions, but only occurred in mutant pigs with AICAR infusion. Infusion of compound C (30 μM), an AMPK antagonist, prior to occlusion blocked the AICAR‐induced attenuation of ST elevation in lean mutants. The KATP channel agonist pinacidil increased ST elevation and the antagonist glibenclamide attenuated ST elevation during occlusion. While the degree of diet‐induced MetS (obesity, further impaired glucose tolerance, insulin resistance, and hypertension) was not different between mutant and wild type swine, except for greater plasma lipids in mutants, there was no difference in ST elevation during myocardial occlusion.ConclusionsLean Ossabaw swine with the V199l AMPK mutation have impaired glucose tolerance and exacerbated KATP channel‐mediated ST elevation during myocardial ischemia. Excess calorie diet induces MetS and abolishes cardioprotection in wild type AMPK swine. ST segment changes are an excellent measure of ischemic preconditioning in this model, thus showing the relevance of Murry’s landmark discovery.

Chronic High‐Rate Pacing Induces Heart Failure with Preserved Ejection Fraction‐Like Phenotype in Obese Ossabaw Swine

The lack of pre‐clinical large animal models of heart failure with preserved ejection fraction (HFpEF) remains a growing, yet unmet obstacle to improving understanding of this complex condition. This study tested the hypothesis that chronic, high‐rate pacing induces a HFpEF‐like phenotype in obese Ossabaw swine. Swine were fed standard chow or an excess calorie, high‐fat, high‐fructose diet for ~18 weeks. Obese swine were implanted with a pacemaker to increase heart rate to 180 beats/min for 4 weeks (obese HF). Compared to lean swine, chronic pacing did not affect baseline (un‐paced) blood pressure or heart rate, but increased heart weight (P= 0.03), fibrosis (P< 0.001), and tended to reduce capillary density (P= 0.06). Cardiac output was increased in obese HF (P= 0.02) and associated with ~45% increase in ventricular stroke volume (P= 0.01), elevated end‐diastolic pressure (25 ± 2 mmHg; P< 0.001), and a normal ejection fraction of 56 ± 7% (P= 0.57). Baseline coronary blood flow was increased in obese HF (P= 0.03); however, MVO2 (P= 0.48) and coronary venous PO2 (P= 0.14) remained unchanged. Hemorrhage studies revealed impairment of the chronotropic response (59 ± 10 to 158 ± 19 bpm in lean control vs. 70 ± 5 vs. 84 ± 5 bpm in obese HF; P< 0.001) and augmented reductions in coronary blood flow (0.39 ± 0.03 to 0.19 ± 0.04 ml/min/g in lean control vs. 0.60 ± 0.05 to 0.21 ± 0.04 ml/min/g in obese HF) as blood pressure decreased from 109 ± 3 to 42 ± 1 mmHg in lean control vs. 102 ± 4 to 42 ± 1 mmHg in obese HF swine. These findings support that chronic high‐rate pacing of obese Ossabaw swine induces key phenotypic features of the human HFpEF condition and provides a distinct preclinical tool for future mechanistic and therapeutic study.

SIRT1 Inhibition Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background: SIRT1 is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 increases sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity that is essential to buffer the increase in Ca2+ induced by release from the sarcoplasmic reticulum (SR). Our lab has shown that metabolic syndrome (MetS) impairs SERCA activity in coronary smooth muscle cells and causes coronary artery disease in Ossabaw miniature swine. We hypothesized that SIRT1 inhibition and MetS would impair Ca2+ buffering. 
 Methods: CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT) and mimic the naturally occurring mutation in humans and decrease SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. The cells were exposed to 5 mM caffeine to maximally release stores of Ca2+ from the SR. Ca2+ buffering capacity of each cell was analyzed after the caffeine-induced peak increase to assess Ca2+ efflux and SERCA activity. 
 Results: MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. The rapid phase of Ca2+ buffering due to Ca2+ efflux was not affected by SIRT1 mutation or MetS. The slower phase of Ca2+ buffering due to SERCA activity was impaired only by SIRT1 mutation (p<0.0005), not by MetS. 
 Conclusion: SIRT1 mutation alone inhibited SERCA buffering of Ca2+ in coronary smooth muscle. (Support: NIH T35HL110854, DK120240, DK09751.)

Metabolic Syndrome and SIRT1 Mutation Impair Ca2+ Channels in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background:
 Changes in Ca2+ regulation have been implicated in various pathologies such as coronary artery disease and metabolic syndrome (MetS), thereby potentiating these diseases. Our lab has shown that MetS decreases voltage-gated Ca2+ channel (VGCC) activity and sarcoplasmic reticulum (SR) Ca2+ release in coronary smooth muscle cells and increases coronary artery disease in Ossabaw miniature swine. Furthermore, decreased SIRT1 enzyme function can impair Ca2+ signaling and increase coronary disease and MetS. We hypothesized that impaired SIRT1 and MetS would decrease VGCC function and SR calcium store.
 Methods:
 CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT), mimicking the naturally occurring mutation in humans which decreases SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. Depolarization of cells with perfusion of 80 mM K+ was used to elicit Ca2+ influx through VGCC. Caffeine (5 mM) exposure activated the Ca2+ release channel (ryanodine receptor) on the SR.
 Results:
 MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. A two-way analysis of variance revealed statistically significant overall effects of genotype (p=0.02), diet (p<0.0001), and an interaction (p<0.0001) between these variables to decrease VGCC function. In contrast, no effect was observed on SR Ca2+ release.
 Conclusion and Potential Impact:
 SIRT1 inhibition and MetS decreased VGCC function independently, but not additively or synergistically. (Support: NIH T35HL110854, DK120240, DK09751.)

Intracellular Ca2+ Dysregulation in Coronary Smooth Muscle Is Similar in Coronary Disease of Humans and Ossabaw Miniature Swine.

Intracellular free Ca2+ ([Ca2+]i) dysregulation occurs in coronary smooth muscle (CSM) in atherosclerotic coronary artery disease (CAD) of metabolic syndrome (MetS) swine. Our goal was to determine how CAD severity, arterial structure, and MetS risk factors associate with [Ca2+]i dysregulation in human CAD compared to changes in Ossabaw miniature swine. CSM cells were dispersed from coronary arteries of explanted hearts from transplant recipients and from lean and MetS swine with CAD. CSM [Ca2+]i elicited by Ca2+ influx and sarcoplasmic reticulum (SR) Ca2+ release and sequestration was measured with fura-2. Increased [Ca2+]i signaling was associated with advanced age and a greater media area in human CAD. Decreased [Ca2+]i signaling was associated with a greater number of risk factors and a higher plaque burden in human and swine CAD. Similar [Ca2+]i dysregulation exhibited in human and Ossabaw swine CSM provides strong evidence for the translational relevance of this large animal model.

Cerebrovascular insufficiency and amyloidogenic signaling in Ossabaw swine with cardiometabolic heart failure.

Individuals with heart failure (HF) frequently present with comorbidities, including obesity, insulin resistance, hypertension, and dyslipidemia. Many patients with HF experience cardiogenic dementia, yet the pathophysiology of this disease remains poorly understood. Using a swine model of cardiometabolic HF (Western diet+aortic banding; WD-AB), we tested the hypothesis that WD-AB would promote a multidementia phenotype involving cerebrovascular dysfunction alongside evidence of Alzheimer’s disease (AD) pathology. The results provide evidence of cerebrovascular insufficiency coupled with neuroinflammation and amyloidosis in swine with experimental cardiometabolic HF. Although cardiac ejection fraction was normal, indices of arterial compliance and cerebral blood flow were reduced, and cerebrovascular regulation was impaired in the WD-AB group. Cerebrovascular dysfunction occurred concomitantly with increased MAPK signaling and amyloidogenic processing (i.e., increased APP, BACE1, CTF, and Aβ40 in the prefrontal cortex and hippocampus) in the WD-AB group. Transcriptomic profiles of the stellate ganglia revealed the WD-AB group displayed an enrichment of gene networks associated with MAPK/ERK signaling, AD, frontotemporal dementia, and a number of behavioral phenotypes implicated in cognitive impairment. These provide potentially novel evidence from a swine model that cerebrovascular and neuronal pathologies likely both contribute to the dementia profile in a setting of cardiometabolic HF.

Tissue‐Specific Differences in Myocardial and Coronary Vascular ACE2 and TMPRSS2 mRNA Levels are Dependent Upon Sex, Comorbidities, Pressure‐Overload, and Pig Species

SARS‐COV‐2, or COVID‐19, is a respiratory virus infecting over 86 million people worldwide. In addition to respiratory infections, SARS‐COV‐2 has been shown to include cardiovascular (CV) complications, including myocarditis and acute coronary syndrome. Risk of severe complications from SARS‐COV‐2 in individuals with existing CV and metabolic disease has been shown to be increased. Evidence indicates SARS‐COV‐2 enters tissues via the angiotensin‐converting enzyme 2 (ACE2) receptor and that the virus is primed and activated by transmembrane protease, serine 2 (TMPRSS2). The goal of this study was to determine ACE2 and TMPRSS2 mRNA levels in pre‐clinical swine models of heart failure (HF). We hypothesized sex, pressure‐overload, and comorbidities would increase ACE2 and TMPRSS2 mRNA levels. A retrospective analysis was conducted in previously completed studies in our lab including: 1) Female, intact Ossabaw swine that were either lean control or western diet‐fed aortic‐banded (N=4‐5/group); 2) Female Yucatan mini‐swine subject to ovariectomy and/or aortic banding (N=5‐8/group); and 3) Sedentary and exercise trained male, intact Yucatan mini‐swine that were aortic banded. ACE2 and TMPRSS2 mRNA levels were evaluated in the left ventricle (LV), right ventricle (RV), and coronary vasculature using qRT‐PCR. Linear regression analysis was used to determine differences between the following variables: pig species, sex hormones, aortic banding, comorbidities, exercise training, and tissue. Data was log‐transformed to meet linear regression assumptions. ACE2 and TMPRSS2 mRNA levels were significantly influenced by sex, comorbidity, and tissue type. TMPRSS2 mRNA levels were also influenced by species and disease status. Specifically, ACE2 mRNA levels decreased 57.1% in the LV and increased 169.9% in the RV of males compared to coronary vessels in intact females. TMPRSS2 mRNA levels increased in the LV and RV of males (1,218.6% and 5,479.8%, respectively) compared to coronary vessels in intact females. ACE2 and TMPRSS2 mRNA levels increased 344% and 453.4%, respectively, in the LV of Ossabaw swine fed a Western Diet compared to coronary vessels from Yucatan and Ossabaw swine without comorbidities. Species differences indicated TMPRSS2 mRNA levels increased 449.2% in the RV and 498.6% in the LV in Yucatan mini‐swine compared to coronary vessels in Ossabaw swine. A 107.3% increase in TMPRSS2 mRNA level was observed in male swine without HF compared to female intact swine with HF highlighting the importance of sex and disease state. Exercise training did not impact ACE2 or TMPRSS2 mRNA levels irrespective of tissue. In conclusion, these results suggest differences in RV, LV and coronary mRNA levels of ACE2 and TMPRSS2 are dependent upon sex and comorbidities. TMPRSS2 levels are additionally influenced by pig species and pressure‐overload. These results provide insight into how ACE2 and TMPRSS2 mRNA levels may influence the cardiovascular involvement of SARS‐COV‐2 infection in an experimental setting of pre‐clinical HF incorporating different swine species, sex, and comorbidities.

Cluster Analysis Identifies Phosphatidylcholine Species in the Liver and Heart as Prediction Hubs in Ossabaw Swine with Cardio‐Metabolic Heart Failure

We recently developed an Ossabaw swine model (Western Diet and aortic banding; WD-AB) that exhibits features consistent with cardio-metabolic heart failure (HF). This pre-clinical model has a pattern of findings possibly useful for enhancing our understanding of the interactions between metabolic syndrome and HF. The aim of this study is to use advanced quantitative methods to reveal novel relationships and candidate predictors we hypothesize may be important to cardio-metabolic HF. A large-scale data repository was created containing 142 hemodynamic, structural, and functional cardiovascular measures, 413 lipid species identified using shotgun lipidomics, and 28,386 mRNA-transcripts revealed by RNA-seq. Repository data was generated from heart, vascular, hepatic, and blood samples collected from 2 groups of intact female Ossabaw swine: 1) lean control (CON; N=5); and 2) WD-AB (N=4). Logistic regression models with AUC scores identified a subset of measurements predictive of HF status that included 1,237 measurements (4.3%). Log2 fold changes were then calculated and statistically significant changes (p<0.05) with absolute value log2 fold changes >1 were considered. Pearson correlations for these 153 measurements were analyzed to identify redundant information using k-medoids clustering analysis with an average silhouette approach. Twenty-eight left ventricle (LV) genes and 125 lipids in 2 clusters (Fig. 1 Circos plot) with center points were identified. Cluster 1 (7 mRNA-transcripts, 70 lipids) was composed of strong correlations with center point phosphatidylcholine (PC) alkyl ether (a) species 32 from the liver (LIVER_PC­_32.0a_754) that increased 10-fold in the WD-AB group (P<0.05; 25±4 CON vs. 277±56 WD-AB pmols/mg). Cluster 2 (21 mRNA-transcripts, 55 lipids) had center point PC species 38.4a from the LV (HEART_PC_38.4a_830) that increased ~3-fold in the WD-AB group (P<0.05; 37±3 CON vs. 102±3 WD-AB pmols/mg). Identified PCa species containing arachidonic acid are storage depots for bioactive oxylipids (eicosanoids) and a precursor for platelet activating factor production. Increased alkyl ether phospholipids may also indicate increased peroxisome proliferation (the site of alkyl ether biosynthesis) and the potential to increase unknown peroxisome proliferator-activated receptor (PPAR) endogenous ligands. In conclusion, PCa species identified from the heart and liver of WD-AB animals may be underappreciated and have predictive significance for cardio-metabolic HF. Ongoing work includes enrichment analysis to determine biological processes underlying clusters.

Urodynamic Characterization of Aged Ossabaw Miniature Pigs Mimics Human Detrusor Underactivity

Detrusor underactivity (DU) and its clinical correlate, underactive bladder (UAB), are common urologic conditions experienced by up to 20 million Americans, however, its pathophysiology is poorly understood and treatment is limited. This highlights the need for a high fidelity animal model that has similar bladder characteristics that will allow direct testing of new treatment modalities. Building on our previous study in metabolic syndrome pigs (Comp. Med. PMID 32972487), we hope to further establish the Ossabaw swine as a translational model for human bladder dysfunction, highlighting its comparable bladder capacity and function as major strengths. We hypothesized that the aged Ossabaw model will demonstrate detrusor underactivity on urodynamic measures due to similar age-related loss of bladder contractility and degeneration of neural and non-neural function. Ossabaw pigs were catheterized via transurethral access when possible or suprapubic cystostomy under general anesthesia. Urodynamic studies were conducted on old (N=3; 10.7 ± 2.6 y) and young (N=10; 3.2 ± 1.0 y) pigs to confirm detrusor underactivity. Fisher's exact test and two sample t-test were performed for comparisons between demographic and urodynamic parameters. Intravesical pressure at capacity with saline was significantly reduced in old vs. young pigs (6.4 ± 3.5 vs. 27.2 ± 17.5 cm H2O; p < 0.001), while compliance was significantly increased (201.4 ± 120.9 vs. 74.6 ± 73.4 mL/cm H2O; p = 0.03). Carbachol infusion revealed significantly reduced maximal pressure during active contraction in aged pigs (29.1 ± 4.0 vs. 44.5 ± 16.7; p = 0.03), but no significant changes in compliance. This characteristic hallmark of detrusor underactivity in aged Ossabaw miniature swine demonstrates its powerful utility as a translational model for age-related bladder dysfunction due to its human-sized bladder more closely approximating human urinary physiology.