Abstract
Background:
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.
Methods:
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 (CON, n=8) or 300mg canagliflozin (CAN, n=8) daily. Five weeks later, pigs underwent terminal harvest and tissue collection.
Results:
CAN treatment was associated with a trend towards decreased expression of fatty acid oxidation inhibitor ACC and decreased phosphorylated/inactivated ACC, a promotor of fatty acid oxidation, compared to CON ischemic myocardium (p=0.08, p=0.03). There was also a significant modulation in insulin resistance markers p-IRS1, p-PKCα, and PI3K in ischemic myocardium of the CAN group compared to CON (all p<0.05). CAN treatment was associated with a significant increase in inflammatory markers IL-6, IL-17, IFN-γ, and iNOS (all p<0.05). There was a trend towards decreased expression of the anti-inflammatory cytokines IL-10 (p=0.16) and IL-4 (p=0.31) with CAN treatment.
Conclusions:
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 signing pathways.
Introduction:
Coronary artery disease (CAD) continues to be one of the leading causes of death worldwide contributing to over nine million deaths annually (1). However, limited medical therapies exist for end-stage CAD that is not amenable to surgical or endovascular intervention. Without adequate treatment, CAD can progress to acute infarction or congestive heart failure (CHF) (2,3).
Sodium-glucose co-transporter 2 (SGLT2) inhibitors are a relatively new class of diabetes medications that have shown benefit in CAD and CHF (4). SGLT2 inhibitors function by blocking the SGLT2 receptor in the kidney, decreasing glucose reabsorption in the proximal tubule and increasing urinary exertion of glucose (5). Clinical data has shown a decrease cardiovascular mortality, non-fatal myocardial infarction, and stroke in diabetic patients treated with SGLT2 inhibitors. SGLT2 inhibitors have also been shown to decrease readmission, symptom burden, and cardiovascular mortality in patents with CHF with and without diabetes (6-15). Growing clinical evidence supporting the use of SGLT2 inhibitors in ischemic heart disease has resulted in the United States Food and Drug Administration expanding indications for the use of SGLT2 inhibitors in ischemic heart disease and CHF (16).
The clinical evidence to support the use of SGLT2 inhibitors continues to grow; however, the biochemical mechanism driving the increase in cardiac function is poorly understood. Small animal studies using mice and zebrafish have further demonstrated increased cardiac output in animals treated with SGLT2 inhibitors in models of acute ischemia and reperfusion injury (17-19). While these small animal models have shown that SGLT2 inhibitors decrease myocardial remodeling and reduce oxidative stress, the definitive mechanisms for the increase in myocardial function have yet to be identified (20-21).
Given the gap in knowledge, our group seeks to further understand the role of SGLT2 inhibitors in chronic myocardial ischemia using SGLT2 inhibitor canagliflozin (CAN) and a swine model of chronic myocardial ischemia. We have recently demonstrated decreased myocardial fibrosis, increased myocardial function, and increased coronary prefusion in swine treated with CAN (22). The objective of this study is to further investigate the biochemical effects of CAN therapy in ischemic myocardium by analyzing the effects of canagliflozin on myocardial metabolism and inflammatory signaling in a swine model of chronic myocardial ischemia.
Methods:
Model:
This study is a further analysis of myocardial tissue samples obtained from the 16 swine presented in our previously published manuscript studying the effects of CAN on cardiac function and fibrosis (22). To model chronic myocardial ischemia, sixteen Yorkshire swine (Cummings School of Veterinary Medicine of Tufts University Farm, North Grafton, MA) underwent thoracotomy for placement of an ameroid constrictor (Research Instruments SW, Escondido, CA) around the proximal left circumflex artery at age eleven weeks. Swine recovered for two weeks and were assigned to either control (CON) or treatment (CAN) groups (22). This two-week period allowed for ameroid constrictor closure to model the use of CAN as a treatment for existing CAD. The CON group received vehicle with no drug (n=8, 5 male, 3 female) daily. The treatment group received 300mg oral CAN daily (n=8, 4 male, 4 female). After five weeks, animals where euthanized for tissue harvest.
Humane Animal Care/Institutional Animal Care and Use Committee:
The local Institutional Animal Care and Use Committee approved all experiments in this study. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.
Ameroid Placement:
The pre-op care and anesthesia was administered as previously reported (22). The swine were placed in a modified right lateral decubitus position. The area was prepped with betadine and draped in a sterile fashion. A left thoracotomy was performed in the second intercostal space. The pericardium was opened and secured with silk sutures to expose the left ventricle and atrium. The left atrium was retracted with silk suture to expose the left circumflex artery (LCx) and left anterior descending artery (LAD). The LCx and LAD were traced back to the left main coronary artery. The LCx was exposed using a combination of blunt and sharp dissection at the take-off of the LCx from the left main coronary artery. After exposure the animal was systemically heparinized (80 IU/kg), and a vessel loop was placed around the LCx. The area of ischemia was mapped by occluding the LCx for two minutes by lifting the vessel loop while injecting 5mL of gold microspheres (BioPal, Worcester, MA) into the left atrium. LCx occlusion was confirmed by monitoring for electrocardiogram changes on the anesthesia monitor. The vessel loop was relaxed, and nitroglycerin was applied topically over the vessel to reverse vasospasm. An ameroid constrictor (Research Instruments SW, Escondido, CA) was placed around the LCx.
The pericardium, ribs, and muscle were closed in layers with vicryl sutures. The skin was closed with a running subcuticular and dressed with a betadine-soaked dressing. Post-op pain control, antibiotics, and thrombosis prophylaxis were the same as previously reported (22).
Tissue Harvest:
The swine underwent the same pre-op preparation and induction of anesthesia as previously described and were placed in a supine position. The area was prepped with betadine and draped in a sterile fashion. The chest was open with a median sternotomy, and care was taken to open the pericardium and expose the left ventricle, left atrium, and right atrium. After exposure of the left atrium, the swine were systemically heparinized (80 IU/kg). A pressure-volume catheter (Transonic, Ithica, NY) was placed via the apex of the left ventricle using Seldinger technique.
The right femoral artery was exposed with a groin cut down. A 6-French sheath was placed using Seldinger technique. Blood was drawn from the catheter and a pressure monitor (Transonic, Ithica, NY) was placed. Blood flow was calculated as previously described by injecting 5ml of isotope-labeled microspheres (BioPal, Worcester, MA) into the left atrium and withdrawing 10mL of blood from the femoral artery (22). This was performed at rest and while pacing the heart at 150 bpm.
After completion of hemodynamic measurements, the anesthesia was deepened. The pig was euthanized, and the heart was excised. The heart was divided into sixteen sections based on position relative to the LCx and LAD. Tissue was flash frozen in liquid nitrogen for immunoblot and immunohistochemistry (22).
Immunoblotting:
Ischemic and non-ischemic myocardial tissue was converted to a lysate using RIPA Lysis and Extraction Buffer, Halt Protease Inhibitor Cocktail (ThermoFisher Scientific, Waltham, MA) and an ultrasonic homogenizer (22). Protein concentration was quantified using BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA). The lysate was loaded (40μg) and ran on a 4-12% Bis-Tris gel (ThermoFisher Scientific, Waltham, MA), and transferred to a nitrocellulose or polyvinylidene difluoride membrane (ThermoFisher Scientific, Waltham, MA). The membranes were blocked with 5% non-fat dry milk in tris-buffered saline (TBST) (Boston BioProducts, Milford, MA) and incubated with 1:1,000 dilutions of primary antibodies in 3% albumin in TBST. Horseradish peroxidase-linked mouse or rabbit secondary antibodies (Cell Signaling, Danvers, MA) were prepared as 2.5:10,000 dilutions in 3% albumin in TBST. The membrane was developed with ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA). Imaging was performed on a ChemiDoc Imaging System (Bio-Rad, Hercules, CA). Restore PLUS Western Blot Stripping Buffer (ThermoFisher Scientific, Waltham, MA) was used to strip membranes and allow for repeat probing. Immunoblot band intensity was measured using NIH Image J software.
Primary antibodies:
Primary antibodies to fatty acid synthase (FAS), phospho-acetyl-CoA carboxylase (pACC), Acetyl-CoA carboxylase (ACC), phospho-protein kinase C alpha (pPKCα), protein kinase C alpha (PKCα), phospho-glycogen synthase kinase 3 beta (pGSK3b), glycogen synthase kinase 3 beta (pGSK3b), phospho-mammalian target of rapamycin (pmTOR), Glucose Transporter Type 4 (GLUT 4), carnitine palmitoyltransferase 1A (CPT1α), pyruvate dehydrogenase (PDH), phosphoinositide 3-kinase (PI3K), phospho-forkhead box O1 (pFOX01), forkhead box O1 (FOX01), phospho-insulin receptor substrate-1 (pIRS-1), insulin receptor substrate-1 (IRS-1), phospho-Fructose-2,6-Biphosphatase 2 (pPFKFB2), Fructose-2,6-Biphosphatase 2 (PFKFB2), Retinol binding protein 4 (RBP4), interleukin 1 (IL-1), phospho-protein kinase C alpha (pPKCα), protein kinase C alpha (PKCα) nuclear factor kappa B (NFκB), tumor necrosis factor alpha (TNFα), CD163, CD11c, toll like receptor 2 (TLR2), inducible nitric oxide synthase (iNOS), cluster of differentiation 40 (CD40), HLA class II histocompatibility antigen DR alpha chain (HLA-DRA) from Cell Signaling (Cell Signaling, Danvers, MA). Primary antibodies to interleukin- 6 (IL-6), and phospho-nuclear factor kappa B (p-NFκB) were obtained from Abcam (Abcam, Cambridge, UK). Primary antibodies to interleukin-10 (IL-10) and toll like receptor 4 (TLR4) where obtained from Novus Biologicals (Novus Biologicals, Centennial, CO). Primary antibodies to interleukin-4 (IL-4) interleukin-8 (IL-8), interleukin-12b (IL-12b) interleukin-17 (IL-17), interferon gamma (INF-γ), and NLR family pyrin domain containing 3 (NLRP-3) were obtained from Proteintech (Proteintech, Rosemont, IL).
Immunohistochemistry:
Immunohistochemistry was performed as previously described using frozen section slides (23). Slides where fixed with 10% paraformaldehyde, blocked, and incubated with an antibody to CD68 (Cell Signaling, Danvers, MA) for macrophage staining. Images were analyzed with an Olympus VS200 Slide Scanner (Olympus, Tokyo, Japan) at 20X magnification. Macrophages were counted for each specimen using QuPath software (24).
Statistical Analysis:
All data are presented as mean value with standard deviation. Immunoblot data is reported as mean fold change compared to the average control with standard deviation. Data were analyzed for normality using Shapiro-Wilk test. Normal data was analyzed with Student's t-test. Non-parametric data was analyzed with Wilcoxon rank-sum. Multigroup comparison was performed using Kruskal-Wallis test. Post-hoc analysis was preformed using Holm correction test. Analysis was performed using Prism 9 (GraphPad Software, San Diego, CA) and R software (Vienna, Austria). Outliers greater than two standard deviations from the mean were excluded.
Results:
Myocardial Metabolism:
Insulin signaling:
CAN treatment was associated with a decrease and normalization of the expression of pPKCα in the ischemic segment of CAN (CAN-I) compared to the ischemic segment of CON (CON-I) (p=0.04)(Table 1). CAN treatment was associated with a decrease in PI3K in CAN-I group compared to CON-I group (p=0.03). CAN treatment was associated with an increase in PDH expression in the non-ischemic segment of CAN (CAN-N) compared to non-ischemic segment of CON (CON-N) (p=0.01), and this increase was maintained between the CON-I and CAN-I groups (p=0.03). CON-I was associated with a trend towards increased expression of pIRS compared to CON-N (p=0.08). CAN treatment was associated with a decrease in pIRS1 and IRS1 expression in CAN-I compared to CON-I (p=0.04, p=0.009). There was a significant decrease in pPFKFB2 in CAN-N compared to CAN-I (p=0.04) and a trend toward decreased pPFKFB2 in CAN-I compared to CON-I (Table 1).
Table 1: Multigroup Comparison for Metabolism Markers.
Data values are presented as mean ± standard deviation. Immunoblotting data is fold change normalized to average control. Multigroup comparison was performed using Kruskal-Wallis test. Post-hoc analysis was preformed using Holm correction test. Data points greater than two standard deviations from the mean are excluded from analysis. Phospho-Acetyl-CoA carboxylase (pACC), Acetyl-CoA carboxylase (ACC), canagliflozin treatment ischemic (CAN-I), canagliflozin treatment nonischemic (CAN-N), control treatment ischemic (CON-I), control treatment non-ischemic (CON-N), carnitine palmitoyltransferase 1A (CPT1α), Fatty acid synthase (FAS), phospho-forkhead box 01 (pFOX01), forkhead box 01 (FOX01), phospho-glycogen synthase kinase 3 beta (pGSK3b), glycogen synthase kinase 3 beta (GSK3b), Glucose Transporter Type 4 (GLUT 4), phospho-insulin receptor substrate-1 (pIRS - 1), insulin receptor substrate-1 (IRS - 1), phospho-mammalian target of rapamycin (pmTOR), pyruvate dehydrogenase (PDH), phospho-Protein kinase C alpha (pPKCα), Protein kinase C alpha (PKCα), phospho-Fructose-2,6-Biphosphatase 2 (pPFKFB2), Fructose-2,6-Biphosphatase 2 (PFKFB2), Phosphoinositide 3-kinase (PI3K), Retinol binding protein 4 (RBP4).
| Marker | CON-N | CAN-N | CON-I | CAN-I | CON-N vs CAN-N p Value |
CON-N vs CON-I p Value |
CAN-N vs CAN-I p Value |
CON-I vs CAN-I p Value |
|---|---|---|---|---|---|---|---|---|
| pACC | 1.00 ± 0.43 | 0.92 ± 0.33 | 1.67 ± 0.95 | 0.62 ± 0.56 | 0.82 | 0.20 | 0.64 | 0.03 |
| ACC | 1.00 ± 0.34 | 1.00 ± 0.20 | 1.23 ± 0.27 | 0.89 ± 0.13 | 0.85 | 0.29 | 0.85 | 0.08 |
| pACC / ACC | 1.00 ± 043 | 1.19 ± 0.47 | 1.84 ± 1.16 | 0.84 ± 0.66 | 0.69 | 0.22 | 0.69 | 0.13 |
| CPT1α | 1.00 ± 0.43 | 0.90 ± 0.14 | 1.00 ± 0.17 | 0.56 ± 0.15 | 1.00 | 1.00 | 0.03 | 0.009 |
| FAS | 1.00 ± 0.79 | 0.59 ± 0.44 | 1.88 ± 1.70 | 1.27 ± 0.47 | 0.58 | 0.50 | 0.58 | 0.58 |
| pFOXO1 | 1.00 ± 0.22 | 0.82 ± 0.15 | 1.40 ± 0.25 | 0.93 ± 0.25 | 0.26 | 0.08 | 0.26 | 0.06 |
| FOXO1 | 1.00 ± 0.32 | 1.07 ± 0.29 | 1.60 ± 0.32 | 1.18 ± 0.33 | 1.00 | 0.06 | 1.00 | 0.20 |
| pFOXO1 / FOXO1 | 1.00 ± 3.12 | 0.76 ± 0.21 | 0.87 ± 0.26 | 0.78 ± 0.24 | 0.53 | 1.00 | 1.00 | 1.00 |
| pGSK3β | 1.00 ± 0.33 | 0.77 ± 0.18 | 1.18 ± 0.42 | 0.91 ± 0.50 | 0.96 | 1.00 | 1.00 | 1.00 |
| GSK3β | 1.00 ± 0.18 | 0.99 ± 0.04 | 0.90 ± 0.09 | 0.78 ± 0.11 | 0.48 | 0.40 | 0.009 | 0.48 |
| pGSK3β / GSK3β | 1.00 ± 0.25 | 0.78 ± 0.18 | 1.30 ± 0.35 | 1.16 ± 0.57 | 0.40 | 0.37 | 0.97 | 0.97 |
| GLUT 4 | 1.00 ± 0.34 | 1.52 ± 0.17 | 0.78 ± 0.28 | 0.88 ± 0.25 | 0.08 | 0.79 | 0.009 | 0.79 |
| pIRS-1 | 1.00 ± 0.78 | 1.17 ± 0.52 | 2.17 ± 0.72 | 0.77 ± 0.59 | 0.70 | 0.08 | 0.48 | 0.04 |
| IRS-1 | 1.00 ± 0.63 | 0.82 ± 0.26 | 1.45 ± 0.31 | 0.58 ± 0.34 | 0.93 | 0.93 | 0.93 | 0.009 |
| pIRS-1 / IRS-1 | 1.00 ± 0.29 | 1.58 ± 0.39 | 1.68 ± 0.28 | 1.37 ± 0.52 | 0.12 | 0.009 | 0.31 | 0.26 |
| pmTOR | 1.00 ± 0.60 | 1.15 ± 0.79 | 2.05 ± 1.23 | 1.04 ± 0.76 | 1.00 | 0.72 | 1.00 | 0.72 |
| PDH | 1.00 ± 0.48 | 1.59 ± 0.16 | 2.44 ± 0.32 | 1.64 ± 0.52 | 0.01 | 0.009 | 0.94 | 0.03 |
| pPKCα | 1.00 ± 0.47 | 0.87 ± 0.20 | 1.03 ± 0.13 | 0.77 ± 0.14 | 0.94 | 0.93 | 0.93 | 0.04 |
| PKCα | 1.00 ± 0.31 | 1.47 ± 0.23 | 1.43 ± 0.38 | 1.25 ± 0.11 | 0.06 | 0.12 | 0.26 | 0.59 |
| pPKCα / PKCα | 1.00 ± 0.66 | 0.52 ± 0.10 | 0.66 ± 0.19 | 0.53 ± 0.07 | 0.53 | 1.00 | 1.00 | 1.00 |
| PPFKFB2 | 1.00 ± 0.28 | 0.98 ± 0.22 | 1.16 ± 0.34 | 0.67 ± .103 | 1.00 | 0.62 | 0.04 | 0.12 |
| PFKFB2 | 1.00 ± 0.34 | 1.23 ± 0.28 | 1.59 ± 0.57 | 0.98 ± 0.16 | 0.48 | 0.28 | 0.16 | 0.28 |
| pPFKFB2 / PFKFB2 | 1.00 ± 0.07 | 0.78 ± 0.09 | 0.74 ± 0.09 | 0.68 ± 0.07 | 0.01 | 0.009 | 0.13 | 0.48 |
| PI3K | 1.00 ± 0.11 | 1.15 ± 0.10 | 1.36 ± 0.10 | 1.16 ± 0.14 | 0.08 | 0.009 | 0.94 | 0.03 |
| RBP4 | 1.00 ± 0.14 | 0.74 ± 0.16 | 0.67 ± 0.16 | 0.71 ± 0.15 | 0.12 | 0.04 | 1.00 | 1.00 |
There was a trend toward increased expression of GLUT 4 in CAN-N compared to CON-N (p = 0.08), but no difference between the ischemic groups (p=0.79). There was no significant change in GLUT 4 in CON-N compared to CON-I (p=0.79), but there was a significant decrease in GLUT-4 in the CAN-I compared to CAN-N (p=0.009). There was a trend toward decreased expression of pFOXO1 and FOXO1 between CON-I and CAN-I (p=0.06, p=0.20). There was a trend towards increased expression of pFOXO1 and FOXO1 in CON-N vs CON-I group (p=0.08, p=0.06), and no significant difference in expression of pFOXO1 and FOXO1 in CAN-N vs CAN-I group (p=0.26, p=1.00) (Table 1). There was no change in expression of pGSK3b, GSK3b, pMTOR, PFKFB2, and PKCα between the CON-N and CAN-N or CON-I and CAN-I groups (all p>0.05)(Table 1).
Fatty acid oxidation
CAN treatment was associated with a decrease in pACC and trend towards decreased ACC in CAN-I compared to CON-I (p=0.03, p=0.08). There was a significant decrease in carnitine palmitoyltransferase 1A (CPT1α) in CAN-I compared to CON-I and CAN-N (p=0.009, p=0.03)(Table 1).
Myocardial Inflammation:
In the ischemic swine myocardium, CAN treatment was associated with a significant increase in inflammatory markers IL-6, IL-17, IFN-γ and iNOS (p<0.05)(Table 2). There was a trend towards increased expression of the inflammatory markers IL-8 (p=0.14) and TNF–α (p=0.24). CAN treatment was associated with a trend towards decreased expression of the anti-inflammatory cytokines IL-10 (p=0.16) and IL-4 (p=0.12). There was a trend towards decreased expression of the pro-inflammatory signaling NFκB (p=0.06) in the CAN group. There was no significant difference in expression of the proinflammatory markers IL–1, NLRP–3, CD11c, TLR4, HLA–DRA, pNFκB and eNOS (Table 2).
Table 2: Inflammatory Changes in Ischemic Myocardium.
Data values are presented as mean ± standard deviation. Immunoblotting data is fold change normalized to average control. P values are calculated using Student's t-test or Wilcoxon rank-sum based on the results of Shapiro-Wilk test. Endothelial nitric oxide synthase (eNOS), HLA class II histocompatibility antigen DR alpha chain (HLA-DRA), Interleukin - 1 (IL - 1), interleukin - 4 (IL - 4), interleukin - 6 (IL - 6), interleukin - 6 (IL - 8) interleukin - 10 (IL - 10), interleukin – 17 (IL – 17), INF- γ (Interferon gamma), inducible nitric oxide synthase (iNOS), phospho-nuclear factor kappa B (p-NFκB), nuclear factor kappa B (NFκB), NLR family pyrin domain containing 3 (NLRP-3), toll like receptor 4 (TLR4), tumor necrosis factor alpha (TNFα).
| Marker | Mean Change in Expression | p - Value |
|---|---|---|
| CD - 11c | 1.18 ± 0.38 | 0.32 |
| eNOS | 0.97 ± 0.27 | 0.85 |
| HLA - DRA | 1.10 ± 0.29 | 0.52 |
| IL - 1 | 1.03 ± 0.20 | 0.81 |
| IL - 4 | 0.83 ± 0.25 | 0.12 |
| IL - 6 | 1.62 ± 0.53 | 0.02 |
| IL - 8 | 1.07 ± 0.27 | 0.14 |
| IL - 10 | 0.72 ± 0.42 | 0.16 |
| IL - 17 | 1.48 ± 0.26 | 0.001 |
| INF - γ | 1.15 ± 0.15 | 0.009 |
| iNOS | 1.44 ± 0.35 | 0.01 |
| p-NF - κB | 0.87 ± 0.30 | 0.45 |
| NF - κB | 0.82 ± 0.11 | 0.06 |
| p-NF - κB/NF - κB | 1.07 ± 0.40 | 0.89 |
| NLRP-3 | 0.99 ± 0.16 | 0.94 |
| TLR - 4 | 1.00 ± 0.13 | 0.99 |
| TNF - α | 1.18 ± 0.17 | 0.24 |
In the non-ischemic swine myocardium, CAN treatment was associated with a significant increase in pro-inflammatory markers IL-6 and IL-17 (p<0.001, p=0.05) and decrease in pro-inflammatory marker NLRP-3 (p=0.002). There was a trend toward increased anti-inflammatory IL-10 (p=0.14), and a trend toward a decrease in pro-inflammatory iNOS and eNOS (p=0.16, p=0.08). There was no significant difference in expression of IL–1, NLRP–3, CD11c, TLR4, HLA–DRA, pNFκB, NFκB, IL-8, TNF–α and IFN-γ (Table 3).
Table 3: Inflammatory Changes in Non-ischemic Myocardium.
Data values are mean ± standard deviation. Immunoblotting data is fold change normalized to average control. P values are calculated using Student's t-test or Wilcoxon rank-sum based on the results of Shapiro-Wilk test. Endothelial nitric oxide synthase (eNOS), HLA class II histocompatibility antigen DR alpha chain (HLA-DRA), Interleukin - 1 (IL - 1), interleukin - 4 (IL - 4), interleukin - 6 (IL - 6), interleukin - 6 (IL - 8) interleukin - 10 (IL - 10), interleukin – 17 (IL – 17), INF- γ (Interferon gamma), inducible nitric oxide synthase (iNOS), phospho-nuclear factor kappa B (p-NFκB), nuclear factor kappa B (NFκB), NLR family pyrin domain containing 3 (NLRP-3), toll like receptor 4 (TLR4), tumor necrosis factor alpha (TNFα).
| Marker | Mean Change in Expression | p - Value |
|---|---|---|
| CD - 11c | 0.62 ± 0.78 | 0.80 |
| eNOS | 0.71 ± 0.24 | 0.08 |
| HLA - DRA | 0.85 ± 0.26 | 0.18 |
| IL - 1 | 0.78 ± 0.27 | 0.46 |
| IL - 4 | 1.10 ± 0.17 | 0.51 |
| IL - 6 | 1.70 ± 0.24 | <0.001 |
| IL - 8 | 0.56 ± 0.11 | 0.24 |
| IL - 10 | 1.16 ± 0.17 | 0.14 |
| IL - 17 | 1.35 ± 0.34 | 0.05 |
| INF - γ | 1.58 ± 0.63 | 0.13 |
| iNOS | 0.61 ± 0.19 | 0.16 |
| p-NF - κB | 1.17± 0.42 | 0.57 |
| NF - κB | 1.02 ± 0.14 | 0.76 |
| p-NF - κB/NF - κB | 1.07 ± 0.31 | 0.68 |
| NLRP-3 | 0.68 ± 0.13 | 0.002 |
| TLR - 4 | 0.55 ± 0.25 | 0.40 |
| TNF - α | 0.93 ± 0.16 | 0.61 |
CD 68 staining for macrophage invasion showed no significant difference in macrophage invasion between the CAN-I and CON-I groups (Figure 1).
Figure 1: Macrophage Invasion.
CD 68 staining for macrophages showed no significant deference between ischemic myocardium treated with canagliflozin and ischemic control myocardium. Red, CD 68 stain; Blue, DAPI (4′,6-diamidino-2-phenylindole) nuclear staining.
Discussion:
SGLT-2 inhibitor treatment, including CAN, has emerged as a potential therapy for advanced CAD and CHF. Clinical data has shown decreased cardiovascular mortality, heart failure symptom burden, and heart failure readmission in patients treated with SGLT-2 inhibitors (6-15). We have recently demonstrated decreased myocardial fibrosis, increased cardiac output, and increased coronary perfusion in the CAN treated swine in this cohort. However, the exact mechanisms of these benefits are unclear (22). This study revealed several metabolic and inflammatory changes that may help us better understand how CAN is functioning in the myocardium.
Myocardial ischemia can result in a dysregulation of myocardial metabolism. This dysregulation can be increased fatty acid oxidation, as is seen in CHF, or decreased fatty acid oxidation and increased glycolysis, as is seen in myocardial ischemia (25). CAN treatment was associated with decreases in CPT1α and pACC enzymes that are essential for fatty acid oxidation as well as a trend towards decreased inhibitory ACC in the ischemic myocardium. The seemingly contradictory changes in CPT1α and pACC compared to ACC likely represent an attempt to return the myocardium to a normal balance of fatty acid oxidation. PDH was increased in CON-N compared to CON-I indicating an increase in glycolysis with chronic ischemia while PDH was increased in both CAN-N and CAN-I. This represents a decrease in glycolysis with CAN treatment that is unchanged with ischemia.
Myocardial insulin resistance is a major consequence of myocardial ischemia that contributes to the development of CHF (26). In our study, CAN treatment was associated with a significant decrease in key markers for myocardial insulin resistance including pPKCα, PI3K, pIRS-1, and IRS-1 in the ischemic myocardium. The activation of FOX01 has also been implicated in myocardial insulin resistance and cardiac dysfunction (27). In our study, we found a trend toward decreased expression of pFOX01 and FOX01. This decrease in markers for insulin resistance including pPKCα, PI3K, pIRS-1, IRS-1, pFOX01 and FOX01, suggest that CAN may improve cardiac function by reducing insulin resistance in ischemic myocardium.
Therefore, the overall metabolic changes seen with CAN treatment in ischemic myocardium shows a trend towards normalization of the ischemia-related changes. This normalization of ischemic metabolic changes is likely related to both increased coronary perfusion and direct drug effects as represented by metabolic changes in nonischemic CAN myocardium. The signaling pathway contributing changes in myocardial metabolism is likely complex as swine hearts have no native SGLT-2 receptors, but our prior work has shown changes in intermediate signaling molecules including AMPK and ERK that could play a role in regulating cellular metabolism (22).
The effects of CAN on inflammation in chronic myocardial ischemia have not been studied previously. In ischemic myocardium, CAN treatment resulted in increased expression of pro-inflammatory markers IL-6, IL-17, IFN-γ, and iNOS, with a trend towards increased IL-8 and TNF–α. CAN treatment was also associated with a trend towards decreased anti-inflammatory cytokines IL-10 and IL-4 in the ischemic myocardium. Importantly, these changes in inflammatory markers appear to be independent of macrophage invasion. CAN treatment resulted in an increase in pro-inflammatory cytokines IL-6 and IL-17, and a decrease in NLRP-3 in the non-ischemic myocardium. Therefore, CAN treatment resulted in increased expression of inflammatory markers in both ischemic and non-ischemic myocardium, with a greater effect in the ischemic territory. Together, these findings suggest that the increased inflammatory signaling is due to a drug effect that can be modulated by chronic myocardial ischemia.
Inflammation is typically regarded as a maladaptive process that results in several derangements including apoptosis and fibrosis; however, inflammation in myocardial infarction has been shown to increase recruitment of inflammatory cells and promote infarct healing, angiogenesis, and ventricular remodeling (28). Given the increased cardiac function seen with CAN treatment and decreased fibrosis, it is possible that the inflammatory changes in chronically ischemic myocardium treated with CAN may play a role in structured remodeling rather than traditional maladaptive remodeling. IL-6, IL-17 and IFN-γ have been implicated in potentially reducing fibrosis in specific settings. The most well described is IFN-γ. IFN-γ is believed to have an antifibrotic effect by decreasing the signal transducers and activators of transcription (STAT) pathway and extracellular matrix deposition (28-30).
SGLT2 inhibitors have been shown to have several benefits in ischemic heart disease and CHF and are quickly being adopted into clinical practice, particularly in the setting of heart failure (6-16). The results of our study further validate the use of SGLT-2 inhibitors in humans with CAD. SGLT-2 inhibitors have been shown in humans to increase cardiac function and decrease overall cardiac mortality. Our prior work has validated this observation using a large animal model. This continuation of our prior work further characterizes the metabolic and inflammatory changes that could play a role in the improvement of cardiac function. Particularly with respect to metabolism, the disruption of myocardial metabolism is an essential component of the pathology and CAN appears to play a role in normalizing the metabolic derangement of chronic myocardial ischemia (25-27). The inflammatory results of our study are paradoxical at first, but increases in markers such as IFN-γ could play a role in remodeling that requires further investigation. The results of our study continue to support the use of SGLT-2 inhibitors in patients with myocardial ischemia.
This study has several limitations worth discussing. Given the nature of large animal work, this study has a relatively small sample size with a total of 16 subjects. This can result in analyses being underpowered. The study uses a mix of male and female swine but given the small sample size, the study is not powered to identify potential sex specific changes in metabolism or inflammation. We assume that the ameroid constrictor will be close to the point of hemodynamically significant stenosis within two weeks. However, due to the lack of definitive testing such as coronary angiography and the variable timeline for ameroid constrictor closure, treatment could be initiated before critical ischemia in some animals. The ameroid constrictor fully closes in all animals during the study, making this a model of progressive CAD, not stable CAD. We used systemic heparinization at the time of harvest. Heparin will elevate plasma free fatty acids but should have minimal effect on the enzymes in the myocardium. The pathways studied are limited by the number of markers that can be studied using traditional methods, thus it is possible that important markers or changes were missed.
Ultimately further large animal and clinical studies are needed to answer key questions regarding the cardiac benefits of SGLT2 inhibitors. This study, along with our previous work, has shown significant changes in the myocardium using a normal diet model. However, further research is needed to investigate the response to SGLT2 inhibitor treatment in diabetes and metabolic syndrome. Additionally, there is a lack of literature on clinical studies evaluating SGLT-2 inhibitors in the context of revascularization procedures such as percutaneous coronary intervention or coronary artery bypass grafting.
Conclusion:
SGLT2 inhibitor treatment has emerged as a potential therapy for advanced CAD and CHF; however, the myocardial effects of SGLT2 inhibitor therapy are not fully understood. In our study, the SGLT-2 inhibitor CAN was associated with significant changes in myocardial metabolism and inflammatory pathways in chronically ischemic swine myocardium. Ischemic myocardium in the CAN treated group showed a trend towards normalization of ischemia-induced metabolic changes in insulin signaling and fatty acid metabolism. CAN treatment was also associated with an increase in myocardial inflammatory markers in both ischemic and nonischemic myocardium independent of macrophage invasion. Therefore, the increase in inflammation and normalization of ischemic metabolic changes seen with CAN treatment may contribute in part to the increase in cardiac output and coronary perfusion seen with CAN treatment.
Supplementary Material
Canagliflozin therapy is associated with inhibition of fatty acid oxidation, enhancement of insulin signaling, and inflammation in ischemic myocardium. The importance of this finding is the expansion of our knowledge of the underling mechanism for the cardiac benefits seen with canagliflozin.
Funding/Support:
This research was funded by NIH T32HL160517 (D.D.H., M.B.); the National Heart, Lung, and Blood Institute (NHLBI) 1F32HL160063-01 (S.A.S.); T32 GM065085-10 (C.M.X.); R01HL133624 and R56HL133624-05 (M.R.A.); R01HL46716 and R01HL128831 (F.W.S.).
Footnotes
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Conflict of Interest/Disclosure:
The authors have no conflicts of interest to disclose.
Presentation: This paper contains data that was presented in two presentations at 19th Annual Academic Surgical Congress, February 6-8, 2023. Houston, TX.
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