Search

Inflammation and disruption of cardiac metabolism are prevalent in the setting of myocardial ischemia. Canagliflozin, a sodium-glucose costransporter-2 inhibitor, has beneficial effects on the heart, though the precise mechanisms are unknown. This study investigated the effects of canagliflozin therapy on metabolic pathways and inflammation in ischemic myocardial tissue using a swine model of chronic myocardial ischemia. Sixteen Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic ischemia. Two weeks later, pigs received either no drug (n = 8) or 300 mg canagliflozin (n = 8) daily. Five weeks later, pigs underwent terminal harvest and tissue collection. Canagliflozin treatment was associated with a trend toward decreased expression of fatty acid oxidation inhibitor acetyl-CoA carboxylase and decreased phosphorylated/inactivated acetyl-CoA carboxylase, a promotor of fatty acid oxidation, compared with control ischemic myocardium (P =.08, P =.03). There was also a significant modulation in insulin resistance markers p -IRS1, p -PKCα, and phosphoinositide 3-kinase in ischemic myocardium of the canagliflozin group compared with the control group (all P <.05). Canagliflozin treatment was associated with a significant increase in inflammatory markers interleukin 6, interleukin 17, interferon-gamma, and inducible nitric oxide synthase (all P <.05). There was a trend toward decreased expression of the anti-inflammatory cytokines interleukin 10 (P =.16) and interleukin 4 (P =.31) with canagliflozin treatment. The beneficial effects of canagliflozin therapy appear to be associated with inhibition of fatty acid oxidation and enhancement of insulin signaling in ischemic myocardium. Interestingly, canagliflozin appears to increase the levels of several inflammatory markers, but further studies are required to better understand how canagliflozin modulates inflammatory signaling pathways. [ABSTRACT FROM AUTHOR]

Sodium-glucose cotransporter-2 inhibitor, canagliflozin, improves myocardial perfusion to ischemic territory without accompanying changes in vascular density. We aimed to (1) characterize effects on angiogenic pathways, (2) use multiomics to identify gene expression and metabolite profiles relevant to regulation of myocardial blood flow, and (3) investigate drug effect on coronary microvascular reactivity. A nondiabetic swine model of chronic myocardial ischemia and nondiabetic rat model were used to study functional and molecular effects of canagliflozin on myocardium and in vitro microvascular reactivity. Canagliflozin resulted in increased coronary microvascular vasodilation and decreased vasoconstriction (P <.05) without changes in microvascular density (P >.3). Expression of the angiogenic modulator, endostatin, increased (P =.008), along with its precursor, collagen 18 (P <.001), and factors that increase its production, including cathepsin L (P =.004). Endostatin and collagen 18 levels trended toward an inverse correlation with blood flow to ischemic territory at rest. Proangiogenic fibroblast growth factor receptor was increased (P =.03) and matrix metalloproteinase-9 was decreased (P <.001) with canagliflozin treatment. Proangiogenic vascular endothelial growth factor A (P =.13), Tie-2 (P =.10), and Ras (P =.18) were not significantly altered. Gene expression related to the cardiac renin-angiotensin system was significantly decreased. In chronic myocardial ischemia, canagliflozin increased absolute blood flow to the myocardium without robustly increasing vascular density or proangiogenic signaling. Canagliflozin resulted in altered coronary microvascular reactivity to favor vasodilation, likely through direct effect on vascular smooth muscle. Downregulation of cardiac renin-angiotensin system demonstrated local regulation of perfusion. [Display omitted] [Display omitted] [ABSTRACT FROM AUTHOR]

Our recent studies using a porcine model of metabolic syndrome (MS) and chronic myocardial ischemia show that extracellular vesicle (EV) therapy improves blood flow and arteriogenesis in ischemic myocardium, although mechanisms of these changes are unclear. We hypothesized that in the setting of MS, EV therapy would decrease antiangiogenic signaling to mediate increased blood flow to chronically ischemic myocardium. Yorkshire swine were fed a high-fat diet for 4 weeks to induce MS, then underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs underwent intramyocardial injection of vehicle (control, n = 6) or human bone marrow-derived EVs (n = 8). Five weeks later, left ventricular myocardium in ischemic territory was harvested. Protein expression was measured using immunoblot analysis, and data were analyzed using Wilcoxon rank sum test. Myocardial perfusion was measured with isotope-labeled microspheres, and correlation data were analyzed using Spearman rank correlation coefficient. EV treatment was associated with decreased expression of antiangiogenic proteins, angiostatin (P <.001) and endostatin (P =.043) in ischemic myocardium compared with control. In EV-treated pigs, there was a negative correlation between blood flow to ischemic myocardium and angiostatin (r s = −0.76; P =.037), but not endostatin expression (r s =.02; P =.98). EV treatment was also associated with decreased cathepsin D, which cleaves precursors to produce angiostatin and endostatin, in ischemic myocardium (P =.020). In the setting of MS and chronic myocardial ischemia, EV therapy is associated with decreased expression of antiangiogenic proteins, which might contribute to increased blood flow to chronically ischemic myocardium. [Display omitted] [Display omitted] [ABSTRACT FROM AUTHOR]

Extracellular vesicle (EV) therapy has been shown to mitigate inflammation in animal models of acute myocardial ischemia/reperfusion. This study evaluates the effect of EV therapy on inflammatory signaling in a porcine model of chronic myocardial ischemia and metabolic syndrome. Yorkshire swine were fed a high-cholesterol diet for 4 weeks to induce metabolic syndrome, then underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs received intramyocardial injection of either saline (control) (n = 6) or EVs (n = 8). Five weeks later, pigs were put to death and left ventricular myocardial tissue in ischemic and nonischemic territories were harvested. Protein expression was measured with immunoblotting, and macrophage count was determined by immunofluorescent staining of cluster of differentiation 68. Data were statistically analyzed via Wilcoxon rank-sum test. EV treatment was associated with decreased expression of proinflammatory markers nuclear factor kappa B (P =.002), pro-interleukin (IL) 1ß (P =.020), and cluster of differentiation 11c (P =.001) in ischemic myocardium, and decreased expression of nuclear factor kappa B in nonischemic myocardium (P =.03) compared with control. EV treatment was associated with increased expression of anti-inflammatory markers IL-10 (P =.020) and cluster of differentiation 163 (P =.043) in ischemic myocardium compared with control. There were no significant differences in expression of IL-6, tumor necrosis factor alpha, arginase, HLA class II histocompatibility antigen DR alpha chain, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, or phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in ischemic myocardium or pro-IL1ß, IL-6, tumor necrosis factor alpha, IL-10, or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in nonischemic myocardium of EV-treated pigs compared with control. There were no differences in macrophage count in ischemic myocardium between EV-treated pigs and control. In the setting of metabolic syndrome and chronic myocardial ischemia, intramyocardial EV therapy attenuates proinflammatory signaling. [Display omitted] [Display omitted] [ABSTRACT FROM AUTHOR]

The burden of mortality and morbidity of cardiovascular disease is in part due to substantial fibrosis accelerated by coexisting risk factors. This study aims to evaluate the effect of extracellular vesicle therapy on diastolic function and myocardial fibrosis in the setting of chronic myocardial ischemia with and without a high-fat diet. Forty male Yorkshire swine were administered a normal or high-fat diet. At 11 weeks of age, they underwent placement of an ameroid constrictor on their left circumflex coronary artery. Both dietary groups then received either intramyocardial injection of vehicle saline as controls or extracellular vesicles as treatment into the ischemic territory (normal diet control, n = 8; high-fat diet controls, n = 11) or extracellular vesicles (normal diet extracellular vesicles, n = 9; high-fat diet extracellular vesicles, n = 12). Five weeks later, hemodynamic parameters, histology, and selected protein expression were evaluated. Extracellular vesicles reduced end-diastolic pressure volume relationship (P =.002), perivascular collagen density (P =.031), calcium mineralization (P =.026), and cardiomyocyte diameter (P <.0001), and upregulated osteopontin (P =.0046) and mechanistic target of rapamycin (P =.021). An interaction between extracellular vesicles and diet was observed in the vimentin area (P =.044) and fraction of myofibroblast markers to total vimentin (P =.049). Significant changes across diet were found with reductions in muscle fiber area (P =.026), tumor necrosis factor α (P =.0002), NADPH oxidase 2 and 4 (P =.0036, P =.008), superoxide dismutase 1 (P =.034), and phosphorylated glycogen synthase kinase 3β (P =.020). Extracellular vesicle therapy improved the myocardium's ability to relax and is likely due to structural improvements at the extracellular matrix and cellular levels. [Display omitted] [ABSTRACT FROM AUTHOR]

Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium. Yorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and CI were continued for 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content. In the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters. Calpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities. [ABSTRACT FROM AUTHOR]

Background Calpain inhibition has an enhancing effect on myocardial perfusion and improves myocardial density by inhibiting glycogen synthase kinase 3β (GSK-3β) and up-regulating downstream signaling pathways, including the insulin/PI3K and WNT/β-catenin pathways, in a pig model of chronic myocardial ischemia in the setting of metabolic syndrome. Methods Pigs were fed a high-fat diet for 4 weeks, then underwent placement of an ameroid constrictor to the left circumflex artery. Three weeks later, the animals received no drug (high-cholesterol controls [HCC]), a high-dose calpain inhibitor (HCI), a low-dose calpain inhibitor (LCI), or a GSK-3β inhibitor (GSK-3βI). The diets and drug regimens were continued for 5 weeks and the myocardial tissue was harvested. Results Calpain and GSK-3β inhibition caused an increase in myocardial perfusion ratios at rest and during pacing compared with controls. Pigs in the LCI and HCI groups had increased vessel density in the ischemic myocardium, and pigs in the GSK-3βI group had increased vessel density in the ischemic and nonischemic myocardium compared with the HCC group. Calpain inhibition modulates proteins involved in the insulin/PI3K and WNT/β-catenin pathways. Quantitative proteomics revealed that calpain and GSK-3β inhibition significantly modulated the expression of proteins enriched in cytoskeletal regulation, metabolism, respiration, and calcium-binding pathways. Conclusions In the setting of metabolic syndrome, calpain or GSK-3β inhibition increases vessel density in both ischemic and nonischemic myocardial tissue. Calpain inhibition may exert these effects through the inhibition of GSK-3β and up-regulation of downstream signaling pathways, including the insulin/PI3K and WNT/β-catenin pathways. [ABSTRACT FROM AUTHOR]

Purpose Calpain overexpression is implicated in aberrant angiogenesis. We hypothesized that calpain inhibition (MDL28170) would improve collateral perfusion in a swine model with hypercholesterolemia and chronic myocardial ischemia. Methods Yorkshire swine fed a high cholesterol diet for 4 weeks underwent surgical placement of an ameroid constrictor to their left circumflex coronary artery. Three weeks later, animals received no drug, high cholesterol control group (n = 8); low-dose calpain inhibition (0.12 mg/kg; n = 9); or high-dose calpain inhibition (0.25 mg/kg; n = 8). The heart was harvested after 5 weeks. Results Myocardial perfusion in ischemic myocardium significantly improved with high-dose calpain inhibition at rest and with demand pacing ( P = .016 and .011). Endothelium-dependent microvessel relaxation was significantly improved with low-dose calpain inhibition ( P = .001). There was a significant increase in capillary density, with low-dose calpain inhibition and high-dose calpain inhibition ( P = .01 and .01), and arteriolar density with low-dose calpain inhibition ( P = .001). Calpain inhibition significantly increased several proangiogenic proteins, including vascular endothelial growth factor ( P = .02), vascular endothelial growth factor receptor 1 ( P = .003), vascular endothelial growth factor receptor 2 ( P = .003), and talin, a microvascular structural protein ( P = .0002). There was a slight increase in proteins implicated in endothelial-dependent (nitric oxide mediated) relaxation, including extracellular signal-regulated kinase, phosphorylated extracellular signal-regulated kinase, and inducible nitric oxide synthase with calpain inhibition. Conclusions In the setting of hypercholesterolemia, calpain inhibition improved perfusion, with a trend toward increased collateralization on angiography and increased capillary and arteriolar densities in ischemic myocardium. Calpain inhibition also improved endothelium-dependent microvessel relaxation and increased expression of proteins implicated in angiogenesis and vasodilatation. [ABSTRACT FROM AUTHOR]

Introduction Calpain is a family of cysteine proteases that has an important role in the initiation, regulation, and execution of cell death. Our recent studies using a hypercholesterolemic swine model demonstrated that in the setting of the metabolic syndrome, calpain inhibition (CI) improved collateral-dependent perfusion and increased expression of proteins implicated in angiogenesis and vasodilation. In this study, we hypothesized that CI (by MLD28170) would decrease myocardial apoptosis in the same model. Methods Yorkshire swine, all fed a high-cholesterol diet for 4 weeks underwent placement of an ameroid constrictor on the left circumflex coronary artery. Three weeks later, animals received either no drug, termed the high-cholesterol control group (HCC; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and the CI were continued for 5 weeks, after which the pig was humanely killed and the left ventricular myocardium was harvested and analyzed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, oxyblot analysis, and Western blots. Data were analyzed using the Kruskal–Wallis test. Results The percentage of apoptotic cells to total cells in ischemic myocardial territory was decreased in the LCI and HCI groups compared with the HCC group as shown by TUNEL staining ( P = .018). There was a decrease in proapoptotic proteins, including cleaved caspase 3, caspase 9, cleaved caspase 9, Bax, BAD, p-BAD, and Erk 1/2 ( P ≤ .049 each), but no decrease in caspase 3 ( P = .737). There was also an increase in antiapoptotic proteins, including BCL-2 and p-BCL2 ( P ≤ .025 each). In the ischemic myocardium, several proangiogenic proteins were increased in the LCI and HCI groups compared with the HCC group, including p-AKT, p-eNOS, and eNOS ( P ≤ .006 each) but there was no increase in AKT ( P = .311). CI decreased tissue oxidative stress in both the LCI and HCI groups compared to the HCC group as shown by oxyblot analysis ( P = .021). Conclusion In the setting of hypercholesterolemia, CI decreases apoptosis and the expression of proteins in proapoptotic signaling pathways. CI also increased expression of proteins implicated in anti apoptotic pathways and improves oxidative stress in ischemic myocardial tissue. [ABSTRACT FROM AUTHOR]

Calpain overexpression is implicated in mitochondrial damage leading to tissue oxidative stress and myocardial ischemic injury. The aim of this study was to determine the effects of calpain inhibition (CI) on mitochondrial impairment and oxidative stress in a swine model of chronic myocardial ischemia and metabolic syndrome. Yorkshire swine were fed a high-fat diet for 4 weeks to induce metabolic syndrome then underwent placement of an ameroid constrictor to the left circumflex artery. Three weeks later, animals received: no drug (control, "CON"; n= 7); a low-dose calpain inhibitor (0.12 mg/kg; "LCI", n= 7); or high-dose calpain inhibitor (0.25 mg/kg; "HCI", n=7). Treatment continued for 5 weeks, followed by tissue harvest. Cardiac tissue was assayed for protein carbonyl content, as well as antioxidant and mitochondrial protein expression. Reactive oxygen species (ROS) and mitochondrial respiration was measured in H9c2 cells following exposure to normoxia or hypoxia (1%) for 24 h with or without CI. In ischemic myocardial tissue, CI was associated with decreased total oxidative stress compared to control. CI was also associated with increased expression of mitochondrial proteins superoxide dismutase 1, SDHA, and pyruvate dehydrogenase compared to control. 100 nM of calpain inhibitor decreased ROS levels and respiration in both normoxic and hypoxic H9c2 cardiomyoblasts. In the setting of metabolic syndrome, CI improves oxidative stress in chronically ischemic myocardial tissue. Decreased oxidative stress may be via modulation of mitochondrial proteins involved in free radical scavenging and production. [Display omitted] • Calpain inhibition reduces oxidative stress in ischemic myocardium and hypoxic cells. • Calpain inhibition modulates mitochondrial protein markers and respiration in ischemic myocardium. • Calpain inhibition may protect against myocardial injury and dysfunction by preserving ATP synthase. • Calpain inhibition is a potential therapeutic strategy for chronic myocardial ischemia. [ABSTRACT FROM AUTHOR]