Richard T. Clements

The Effects of Therapeutic Sulfide on Myocardial Apoptosis in Response to Ischemia – Reperfusion Injury

OBJECTIVE Ischemia-reperfusion (I/R) injury, often encountered clinically, results in myocardial apoptosis and necrosis. Hydrogen sulfide (H(2)S) is produced endogenously in response to ischemia and thought to be cardioprotective, although its mechanism of action is not fully known. This study investigates cardioprotection provided by exogenous H2S, generated as sodium sulfide on apoptosis following myocardial I/R injury. METHODS The mid-LAD coronary artery in Yorkshire swine (n=12) was occluded for 60 min, followed by reperfusion for 120 min. Controls (n=6) received placebo, and treatment animals (n=6) received sulfide 10 min prior to and throughout reperfusion. Hemodynamic, global, and regional functional measurements were obtained. Evans blue/TTC staining identified the area-at-risk (AAR) and infarction. Serum CK-MB, troponin I, and FABP were assayed. Tissue expression of bcl-2, bad, apoptosis-inducing-factor (AIF), total and cleaved caspase-3, and total and cleaved PARP were assessed. PAR and TUNEL staining were performed to assess apoptotic cell counts and poly-ADP ribosylation, respectively. RESULTS Pre-I/R hemodynamics were similar between groups. Post-I/R, mean arterial pressure (mmHg) was reduced by 30.2+/-4.3 in controls vs 8.2+/-6.9 in treatment animals (p=0.01). +LV dP/dt (mmHg/s) was reduced by 1308+/-435 in controls vs 403+/-283 in treatment animals (p=0.001). Infarct size (% of AAR) in controls was 47.4+/-6.2% vs 20.1+/-3.3% in the treated group (p=0.003). In treated animals, CK-MB and FABP were lower by 47.0% (p=0.10) and 45.1% (p=0.01), respectively. AIF, caspase-3, and PARP expression was similar between groups, whereas cleaved caspase-3 and cleaved PARP was lower in treated animals (p=0.04). PAR staining was significantly reduced in sulfide treated groups (p=0.04). TUNEL staining demonstrated significantly fewer apoptotic cells in sulfide treated animals (p=0.02). CONCLUSIONS Sodium sulfide is efficacious in reducing apoptosis in response to I/R injury. Along with its known effects on reducing necrosis, sulfides effects on apoptosis may partially contribute to providing myocardial protection. Exogenous sulfide may have therapeutic utility in clinical settings in which I/R injury is encountered.

Hydrogen Sulfide Therapy Attenuates the Inflammatory Response in a Porcine Model of Myocardial Ischemia – Reperfusion Injury

INTRODUCTION Hydrogen sulfide is produced endogenously in response to myocardial ischemia and thought to be cardioprotective. The mechanism underlying this protection has yet to be fully elucidated, but it may be related to sulfides ability to limit inflammation. This study investigates the cardioprotection provided by exogenous hydrogen sulfide and its potential anti-inflammatory mechanism of action. METHODS The mid left anterior descending coronary artery in 14 Yorkshire swine was acutely occluded for 60 minutes, followed by reperfusion for 120 minutes. Controls (n = 7) received placebo, and treatment animals (n = 7) received sulfide 10 minutes before and throughout reperfusion. Hemodynamic and functional measurements were obtained. Evans blue and triphenyl tetrazolium chloride staining identified the area at risk and infarction. Coronary microvascular reactivity was assessed. Tissue was assayed for myeloperoxidase activity and proinflammatory cytokines. RESULTS Pre-ischemia/reperfusion hemodynamics were similar between groups, whereas post-ischemia/reperfusion mean arterial pressure was reduced by 28.7 +/- 5.0 mm Hg in controls versus 6.7 +/- 6.2 mm Hg in treatment animals (P = .03). Positive first derivative of left ventricular pressure over time was reduced by 1325 +/- 455 mm Hg/s in controls versys 416 +/- 207 mm Hg/s in treatment animals (P = .002). Segmental shortening in the area at risk was better in treatment animals. Infarct size (percent of area at risk) in controls was 41.0% +/- 7.8% versus 21.2% +/- 2.5% in the treated group (P = .036). Tissue levels of interleukin 6, interleukin 8, tumor necrosis factor-alpha, and myeloperoxidase activity decreased in the treatment group. Treated animals demonstrated improved microvascular reactivity. CONCLUSIONS Therapeutic sulfide provides protection in response to ischemia/reperfusion injury, improving myocardial function, reducing infarct size, and improving coronary microvascular reactivity, potentially through its anti-inflammatory properties. Exogenous sulfide may have therapeutic utility in clinical settings in which ischemia/reperfusion injury is encountered.

Effect of hydrogen sulfide in a porcine model of myocardial ischemia-reperfusion: Comparison of different administration regimens and characterization of the cellular mechanisms of protection

Objective: We investigate the impact of different regimens of parenteral hydrogen sulfide (H2S) administration on myocardium during ischemia-reperfusion (IR) and the molecular pathways involved in its cytoprotective effects. Methods: Eighteen male Yorkshire pigs underwent 60 minutes of mid-left anterior descending coronary artery occlusion followed by 120 minutes of reperfusion. Pigs received either placebo (control, n = 6) or H2S as a bolus (bolus group, n = 6, 0.2 mg/kg over 10 seconds at the start of reperfusion) or as an infusion (infusion group, n = 6, 2 mg·kg−1·h−1 initiated at the onset of ischemia and continued into the reperfusion period). Myocardial function was monitored throughout the experiment. The area at risk and myocardial necrosis was determined by Monastral blue/triphenyl tetrazolium chloride staining. Apoptosis and the expression pattern of various intracellular effector pathways were investigated in the ischemic territory. Coronary microvascular reactivity to endothelium-dependent and endothelium-independent factors was measured. Results: H2S infusion but not bolus administration markedly reduce myocardial infarct size (P < 0.05) and improve regional left ventricular function, as well as endothelium-dependent and endothelium-independent microvascular reactivity (P < 0.05). The expression of B-cell lymphoma 2 (P = 0.059), heat shock protein 27 and αB-crystallin (P < 0.05) were lower in H2S-treated groups. Infusion of H2S caused higher expression of phospho-glycogen synthase kinase-3 β isoform(P < 0.05) and lower expression of mammalian target of rapamycin and apoptosis-inducing factor (P < 0.05). Bolus of H2S caused higher expression of phospho-p44/42 MAPK extracellular signal-regulated kinase and lower expression of Beclin-1 (P < 0.05). The expression of caspase 3 and cleaved caspase 3 were lower (P < 0.05), whereas the expression of phospho-Bad(Ser136) was higher in the bolus group versus control and infusion groups (P < 0.05). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cell count was lower in both H2S-treated groups compared with the control (P < 0.05). Conclusions: This study demonstrates that infusion of H2S is superior to a bolus alone in reducing myocardial necrosis after IR injury, even though some markers of apoptosis and autophagy were affected in both H2S-treated groups. Thus, the current results indicate that infusion of H2S throughout IR may offer better myocardial protection from IR injury.

Resveratrol Improves Myocardial Perfusion in a Swine Model of Hypercholesterolemia and Chronic Myocardial Ischemia

Background— Resveratrol may provide protection against coronary artery disease. We hypothesized that supplemental resveratrol will improve cardiac perfusion in the ischemic territory of swine with hypercholesterolemia and chronic myocardial ischemia. Methods and Results— Yorkshire swine were fed either a normal diet (control, n=7), a hypercholesterolemic diet (HCC, n=7), or a hypercholesterolemic diet with supplemental resveratrol (100 mg/kg/d orally, HCRV, n=7). Four weeks later, an ameroid constrictor was placed on the left circumflex artery. Animals underwent cardiac MRI and coronary angiography 7 weeks later before euthanasia and tissue harvest. Total cholesterol was lowered about 30% in HCRV animals (P<0.001). Regional wall motion analysis demonstrated a significant decrease in inferolateral function from baseline to 7 weeks in HCC swine (P=0.04). There was no significant change in regional function in HCRV swine from baseline to 7 weeks (P=0.32). Tissue blood flow during stress was 2.8-fold greater in HCRV swine when compared with HCC swine (P=0.04). Endothelium-dependent microvascular relaxation response to Substance P was diminished in HCC swine, which was rescued by resveratrol treatment (P=0.004). Capillary density (PECAM-1 staining) demonstrated fewer capillaries in both HCC and HCRV swine versus control swine (P=0.02). Immunoblot analysis demonstrated significantly greater expression in HCRV versus HCC swine of the following markers of angiogenesis: VEGF (P=0.002), peNOS (ser1177) (P=0.04), NFkB (P=0.004), and pAkt (thr308) (P=0.001). Conclusions— Supplemental resveratrol attenuates regional wall motion abnormalities, improves myocardial perfusion in the collateral dependent region, preserves endothelium-dependent coronary vessel function, and upregulates markers of angiogenesis associated with the VEGF signaling pathway.

Functional, Cellular, and Molecular Characterization of the Angiogenic Response to Chronic Myocardial Ischemia in Diabetes

Background— Ischemic heart disease is the most common cause of mortality in diabetic patients. Although therapeutic angiogenesis is an attractive option for these patients, they appear to have reduced collateral formation in response to myocardial ischemia. The aims of this study were to establish a large animal model of diabetes and chronic myocardial ischemia, evaluate the effects of diabetes on the angiogenic response, and elucidate the molecular pathways involved. Methods and Results— Diabetes was induced in male Yucatan miniswine using a pancreatic β-cell specific toxin, alloxan (150 mg/kg; n=8). Age-matched swine served as controls (n=8). Eight weeks after induction, chronic ischemia was induced by ameroid constrictor placement around the circumflex coronary artery. Myocardial perfusion and function were assessed at 3 and 7 weeks after ameroid placement using isotope-labeled microspheres. Endothelial cell density and myocardial expression of angiogenic mediators was evaluated. Diabetic animals exhibited significant endothelial dysfunction. Collateral dependent perfusion and LV function were significantly impaired in diabetic animals. Diabetic animals also demonstrated reduced endothelial cell density (173±14 versus 234±23 cells/hpf, P=0.03). Expression of VEGF, Ang-1, and Tie-2 was reduced, whereas antiangiogenic proteins, angiostatin (4.4±0.9-fold increase, P<0.001), and endostatin (2.9±0.4-fold increase, P=0.03) were significantly elevated in the diabetic myocardium. Conclusions— Diabetes results in a profound impairment in the myocardial angiogenic response to chronic ischemia. Pro- and antiangiogenic mediators identified in this study offer novel targets for the modulation of the angiogenic response in diabetes.

Endostatin and angiostatin are increased in diabetic patients with coronary artery disease and associated with impaired coronary collateral formation

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Because of the diffuse nature of their disease, diabetic patients may be at risk for incomplete revascularization, highlighting a potential role for proangiogenic therapy in this group. This study investigates molecular mechanisms of angiogenesis in diabetic patients. Myocardial tissue was harvested from patients undergoing coronary artery bypass grafting [nondiabetic (ND) 11, type 2 diabetic (DM) 10]. Expression of angiostatin, endostatin, their precursors (plasminogen and collagen XVIII, respectively), enzymes leading to their production [matrix metalloprotease (MMP)-2 and -9, cathepsin L], and an inhibitor of MMPs (tissue inhibitor of metalloproteinase) was assessed with Western blotting. MMP activity was assessed. Coronary collateralization was graded by Rentrop scoring of angiograms. Plasminogen and collagen XVIII expression were similar between groups. Angiostatin expression trended to increase 1.24-fold (P = 0.07), and endostatin expression increased 2.02-fold in DM patients relative to ND (P = 0.02). MMP-9 expression was no different between groups, whereas MMP-2 expression decreased 1.8-fold in diabetics (P = 0.003). MMP-2 and -9 activity decreased 1.33-fold (P = 0.03) and 1.57-fold (P = 0.04), respectively, in diabetic patients. Cathepsin L expression was 1.38-fold higher in diabetic patients (P = 0.02). Coronary collateralization scores were ND 2.1 +/- 0.37 vs. DM 1.0 +/- 0.4 (P = 0.05). Myocardial endostatin expression correlated strongly with the percentage of hemoglobin A(1c) (r = 0.742, P = 0.0001). Myocardial expression of angiostatin and endostatin demonstrated significant negative linear correlations with coronary collateralization (angiostatin r = -0.531, P = 0.035, endostatin r = -0.794, P = 0.0002). Diabetic patients with CAD exhibit increased levels of the antiangiogenic proteins angiostatin and endostatin and differential regulation of the enzymes governing their production relative to ND patients. Myocardial levels of these proteins show significant correlation to coronary collateralization. These findings offer potential new therapeutic targets for enhancing proangiogenic therapy and insight into the angiogenic impairments seen in diabetes.

Calcium-Activated Potassium Channels Contribute to Human Coronary Microvascular Dysfunction After Cardioplegic Arrest

Background— Cardioplegic arrest (CP) followed by reperfusion after cardiopulmonary bypass induces coronary microvascular dysfunction. We investigated the role of calcium-activated potassium (KCa) channels in this dysfunction in the human coronary microvasculature. Methods and Results— Human atrial tissue was harvested before CP from a nonischemic segment and after CP from an atrial segment exposed to hyperkalemic cold blood CP (mean CP time, 58 minutes) followed by 10-minute reperfusion. In vitro relaxation responses of precontracted arterioles (80 to 180 μm in diameter) in a pressurized no-flow state were examined in the presence of KCa channel activators/blockers and several other vasodilators. We also examined expression and localization of KCa channel gene products in the coronary microvasculature using reverse transcriptase–polymerase chain reaction, immunoblot, and immunofluorescence photomicroscopy. Post-CP reperfusion relaxation responses to the activator of intermediate and small conductance KCa channels (IKCa/SKCa), NS309 (10−5 M), and to the endothelium-dependent vasodilators, substance P (10−8 M) and adenosine 5′diphosphate (10−5 M), were significantly reduced compared with pre-CP responses (P<0.05, n=8/group). In contrast, relaxation responses to the activator of large conductance KCa channels (BKCa), NS1619 (10−5 M), and to the endothelium-independent vasodilator, sodium nitroprusside (10−4 M), were unchanged pre- and post-CP reperfusion (n=8/group). Endothelial denudation significantly diminished NS309-induced vasodilatation and abolished substance P- or adenosine 5′ diphosphate-induced relaxation (P<0.05), but had no effect on relaxation induced by either NS1619 or sodium nitroprusside. The total polypeptide levels of BKCa, IKCa, and SKCa and the expression of IKCa mRNA were not altered post-CP reperfusion. Conclusion— Cardioplegic arrest followed by reperfusion after cardiopulmonary bypass causes microvascular dysfunction associated with and likely in part due to impaired function of SKCa and IKCa channels in the coronary microcirculation. These results suggest novel mechanisms of endothelial and smooth muscle microvascular dysfunction after cardiac surgery.

Effect of Hypercholesterolemia on Myocardial Necrosis and Apoptosis in the Setting of Ischemia-Reperfusion

Background— Hypercholesterolemia is prevalent in patients who experience myocardial ischemia-reperfusion injury (IR). We investigate the impact of dietary-induced hypercholesterolemia on the myocardium in the setting of acute IR. Methods and Results— In normocholesterolemic (NC, n=7) and hypercholesterolemic (HC, n=7) Yucatan male pigs, the left anterior descending coronary artery was occluded for 60 minutes, followed by reperfusion for 120 minutes. Hemodynamic values were recorded, and TTC staining was used to assess necrosis. Oxidative stress was measured. Specific cell death and survival signaling pathways were assessed by Western blot and TUNEL staining. Infarct size was 45% greater in HC versus NC (42% versus 61%, P<0.05), whereas the area at risk (AAR) was similar in both groups (P=0.61). Whereas global LV function (+dP/dt, P<0.05) was higher during entire period of IR in HC versus NC, regional function deteriorated more following reperfusion in HC (P<0.05). Ischemia increased indices of myocardial oxidative stress such as protein oxidation (P<0.05), lipid peroxidation (P<0.05), and nitrotyrosylation in HC versus NC, as well as the expression of phospho-eNOS (P<0.05). The expression of myeloperoxidase, p38 MAPK, and phospho-p38 MAPK was higher in HC versus NC (all P<05). Ischemia caused higher expression of the proapoptotic protein PARP (P<0.05), and lower expression of the prosurvival proteins Bcl2 (P<0.05), phospho-Akt, (P<0.05), and phospho-PKCϵ (P<0.05) in the HC versus NC. TUNEL-positive cell count was 3.8-fold (P<0.05) higher in the AAR of HC versus NC. Conclusions— This study demonstrates that experimental hypercholesterolemia is associated with increased myocardial oxidative stress and inflammation, attenuation of cell survival pathways, and induction of apoptosis in the ischemic territory, which together may account for the expansion of myocardial necrosis in the setting of acute IR.

Hypercholesterolemia is associated with hyperactive cardiac mTORC1 and mTORC2 signaling

Nutritional excess and hyperlipidemia increase the heart’s susceptibility to ischemic injury. Mammalian target of rapamycin (mTOR) controls the cellular response to nutritional status and may play a role in ischemic injury. To explore the effect of hypercholesterolemia on cardiac mTOR signaling, we assessed mTOR signaling in hypercholesterolemic swine (HC) that are also susceptible to increased cardiac ischemia-reperfusion injury. Yucatan pigs were fed a high-fat/high-cholesterol diet for 4 weeks to induce hypercholesterolemia, and mTOR signaling was measured by immunoblotting and immunofluorescence in the non-ischemic left ventricular area. Total myocardial mTOR and raptor levels were markedly increased in the HC group compared to the normocholesterolemic group, and directly correlated with serum cholesterol levels. mTOR exhibited intense perinuclear staining in myocytes only in the HC group. Hypercholesterolemia was associated with hyperactive signaling upstream and downstream of both mTOR complexes, including myocardial Akt, S6K1, 4EBP1, S6, and PKC-alpha, increased levels of cardiac hypertrophy markers, and a trend toward lower levels of myocardial autophagy. Hypercholesterolemia can now be added to the growing list of conditions associated with aberrant mTOR signaling. Hypercholesterolemia produces a unique profile of alterations in cardiac mTOR signaling, which is a potential target in cardiac diseases associated with hypercholesterolemia and nutritional excess.

Indices of Apoptosis Activation After Blood Cardioplegia and Cardiopulmonary Bypass

Background— Cardioplegic arrest (CA) using cold blood cardioplegia (CBC) has been reported to reduce ischemia-reperfusion (IR)-induced myocardial injury via apoptosis. We studied key apoptotic mediators via the caspase-dependent and intrinsic pathways as well as poly(ADP)-ribosylating protein (PARP) activity in myocardial and peripheral tissues after CA and cardiopulmonary bypass (CBP). Methods and Results— Right atrial (RA) and skeletal muscle(SM) was harvested from cardiac surgical patients with similar baseline characteristics (N =6) before and after CPB and CBC. Total and modified caspase-3, Bcl-2, Bad, apoptosis-inducing factor (AIF), and PARP were quantified by immunoblotting. Terminal caspase-3 activity was assessed and immunohistochemistry was performed for PARP and AIF. TUNEL staining was used for identification of apoptotic cells. Microarray gene expression analysis was performed using Affymetrix U95 GeneChip. In RA tissue, CA with CBC significantly increased phosphorylation of Bcl-2 (Ser70), Bad (Ser112) (2.63±0.4 and 1.77±0.3-fold respectively; P<0.05), and cleavage of the downstream caspase 3 (1.45±0.1-fold; P<0.05). There was no significant change in total protein levels. Also, there was an increase in mature AIF (57 kDa) levels (1.22±0.01-fold; P<0.05) and a trend toward nuclear translocation on histological staining. Caspase 3 activity was increased 1.5±0.14-fold (P<0.05). The number of apoptotic cells in atrial tissue increased after compared with before CPB/CA using TUNEL staining (1.55±0.66 versus 0.325±0.05%, respectively; P=0.03). In contrast, SM samples did not show any of the changes observed in RA tissue after CPB. Conclusion— Despite optimal current surgical myocardial protection, we found that CA with CBC induced both programmed cell death and survival signaling in myocardial tissue.