Costas Schulze

Intracellular Action of Matrix Metalloproteinase-2 Accounts for Acute Myocardial Ischemia and Reperfusion Injury

Background—Matrix metalloproteinases are best recognized for their ability to degrade the extracellular matrix in both physiological and pathological conditions. However, recent findings indicate that some of them are also involved in mediating acute processes such as platelet aggregation and vascular tone. The acute contractile defect of the heart after ischemia-reperfusion may involve the proteolytic degradation of the thin filament protein troponin I; however, the protease responsible for this remains obscure. Methods and Results—Here we report that matrix metalloproteinase-2 is colocalized with troponin I within the thin myofilaments of cardiomyocytes in ischemic-reperfused hearts and that troponin I is a novel intracellular target for proteolytic cleavage by matrix metalloproteinase-2. Inhibition of matrix metalloproteinase-2 activity prevented ischemia-reperfusion-induced troponin I degradation and improved the recovery of mechanical function of the heart. Conclusions—These data reveal for the first time a novel molecular mechanism by which matrix metalloproteinase-2 causes acute myocardial dysfunction after ischemia-reperfusion-injury and that matrix metalloproteinase-2 has a biological action within the cell.

Degradation of Myosin Light Chain in Isolated Rat Hearts Subjected to Ischemia-Reperfusion Injury A New Intracellular Target for Matrix Metalloproteinase-2

Background—Matrix metalloproteinase-2 (MMP-2) contributes to cardiac dysfunction resulting from ischemia-reperfusion (I/R) injury. MMP-2 not only remodels the extracellular matrix but also acts intracellularly in I/R by degrading troponin I. Whether other intracellular targets exist for MMP-2 during I/R is unknown. Methods and Results—Isolated rat hearts were subjected to 20 minutes of ischemia and 30 minutes of reperfusion. The impaired recovery of mechanical function of the heart was attenuated by the MMP inhibitors o-phenanthroline or doxycycline. Quantitative 2D electrophoresis of homogenates of aerobically perfused hearts (control) or those subjected to I/R injury (in the presence or absence of MMP inhibitors) showed 3 low-molecular-weight proteins with levels that were significantly increased upon I/R injury and normalized to control levels by MMP inhibitors. Mass spectrometry analysis identified all 3 proteins as fragments of myosin light chain 1, which possesses theoretical cleavage recognition sequences for MMP-2 and is rapidly degraded by it in vitro. The association of MMP-2 with the thick myofilament in fractions prepared from I/R hearts was observed with immunogold electron microscopy, gelatin zymography for MMP-2 activity, and immunoprecipitation. MMP-2 was found to cleave myosin light chain 1 between tyrosine 189 and glutamine 190 at the C terminus. Conclusions—Our results demonstrate that myosin light chain 1 is another novel substrate for MMP-2 in the cardiomyocyte and that its degradation may contribute to contractile dysfunction resulting from I/R injury to the heart.

Matrix metalloproteinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro

Matrix metalloproteinases (MMPs) are traditionally known for their role in extracellular matrix remodeling. Increasing evidence reveals several alternative substrates and novel biological roles for these proteases. Recent evidence showed the intracellular localization of MMP‐2 within cardiac myocytes, colocalized with troponin I within myofilaments. Here we investigated the presence of MMP‐2 in the nucleus of cardiac myocytes using both immunogold electron microscopy and biochemical assays with nuclear extracts. The gelatinase activity found in both human heart and rat liver nuclear extracts was blocked with MMP inhibitors. In addition, the ability of MMP‐2 to cleave poly (ADP‐ribose) polymerase (PARP) as a substrate was examined as a possible role for MMP‐2 in the nucleus. PARP is a nuclear matrix enzyme involved in the repair of DNA strand breaks, which is known to be inactivated by proteolytic cleavage. PARP was susceptible to cleavage by MMP‐2 in vitro in a concentration‐dependent manner, yielding novel degradation products of ~66 and <45 kDa. The cleavage of PARP by MMP‐2 was also blocked by MMP inhibitors. This is the first characterization of MMP‐2 within the nucleus and we hereby suggest its possible role in PARP degradation.

Imbalance Between Tissue Inhibitor of Metalloproteinase-4 and Matrix Metalloproteinases During Acute Myoctardial Ischemia-Reperfusion Injury

Background—We have previously reported that matrix metalloproteinase-2 (MMP-2) contributes to myocardial ischemia-reperfusion injury by degradation of troponin I, a regulatory element of the contractile proteins. MMP activities are also tightly regulated by tissue inhibitors of metalloproteinase (TIMPs). The change in TIMPs during acute myocardial ischemia-reperfusion injury is not clear. Methods and Results—Isolated rat hearts were perfused either aerobically for 75 minutes or subjected to 15, 20, or 25 minutes of global, no-flow ischemia followed by 30 minutes of aerobic reperfusion. During reperfusion after ischemia, there was a rapid, enhanced release of TIMP-4, the most abundant TIMP in the heart, into the coronary effluent, as shown both by reverse zymography and Western blot. There was a negative correlation between the recovery of cardiac mechanical function and the release of TIMP-4 during reperfusion in hearts subjected to different durations of ischemia. Immunogold electron microscopy revealed a close association of TIMP-4 with the sarcomeres in aerobically perfused hearts. Moreover, TIMP-4 was present only in thin myofilaments prepared from aerobically perfused hearts but not in ischemic-reperfused hearts. An enhanced MMP activity was shown in ischemic-reperfused hearts by in situ zymography. Conclusions—Loss of TIMP-4 from the cardiac myocyte leads to an increase in net myocardial MMP activity that contributes to acute myocardial stunning injury.

Difference between pre-operative and cardiopulmonary bypass mean arterial pressure is independently associated with early cardiac surgery-associated acute kidney injury

BackgroundCardiac surgery-associated acute kidney injury (CSA-AKI) contributes to increased morbidity and mortality. However, its pathophysiology remains incompletely understood. We hypothesized that intra-operative mean arterial pressure (MAP) relative to pre-operative MAP would be an important predisposing factor for CSA-AKI.MethodsWe performed a prospective observational study of 157 consecutive high-risk patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). The primary exposure was delta MAP, defined as the pre-operative MAP minus average MAP during CPB. Secondary exposure was CPB flow. The primary outcome was early CSA-AKI, defined by a minimum RIFLE class - RISK. Univariate and multivariate logistic regression were performed to explore for association between delta MAP and CSA-AKI.ResultsMean (± SD) age was 65.9 ± 14.7 years, 70.1% were male, 47.8% had isolated coronary bypass graft (CABG) surgery, 24.2% had isolated valve surgery and 16.6% had combined procedures. Mean (± SD) pre-operative, intra-operative and delta MAP were 86.6 ± 13.2, 57.4 ± 5.0 and 29.4 ± 13.5 mmHg, respectively. Sixty-five patients (41%) developed CSA-AKI within in the first 24 hours post surgery. By multivariate logistic regression, a delta MAP≥26 mmHg (odds ratio [OR], 2.8; 95%CI, 1.3-6.1, p = 0.009) and CPB flow rate ≥54 mL/kg/min (OR, 0.2, 0.1-0.5, p < 0.001) were independently associated with CSA-AKI. Additional variables associated with CSA-AKI included use of a side-biting aortic clamp (OR, 3.0; 1.3-7.1, p = 0.012), and body mass index ≥25 (OR, 4.2; 1.6-11.2, p = 0.004).ConclusionA large delta MAP and lower CPB flow during cardiac surgery are independently associated with early post-operative CSA-AKI in high-risk patients. Delta MAP represents a potentially modifiable intra-operative factor for development of CSA-AKI that necessitates further inquiry.

Reduction of systemic and cardiac adhesion molecule expression after off-pump versus conventional coronary artery bypass grafting.

ABSTRACT Cardiopulmonary bypass (CPB) and operative trauma are associated with increased expression of pro‐ inflammatory mediators. We determined the relative contribution of CPB on activation of cytokines and adhesion molecules in patients undergoing coronary revascularization by comparing them with patients receiving off‐pump coronary artery bypass grafting (OPCAB). Twenty‐six patients were assigned to either the OPCAB procedure using a suction device and regular sternotomy (n = 13), or were treated conventionally using extracorporeal circulation, blood cardioplegia, and hypothermic arrest (29°C‐31°C; n = 13). Systemic levels of TNF‐&agr; and the soluble adhesion molecules P‐selectin and intracellular adhesion molecule 1 (ICAM‐1) were assayed. Immunohistochemistry was used to account for cardiac‐specific expression of adhesion molecules in interventricular endomyocardial sections. Both systemic and endomyocardial expres‐ sion of adhesion molecules were lower in the OPCAB group. Coronary revascularization with CPB resulted in a significant higher expression of TNF‐&agr;, which was associated with P‐selectin and ICAM‐1 expression. This was accompanied with higher catecholamine requirement in the CPB group in the early postoperative period. Despite comparable surgical trauma, the OPCAB procedure without the use of CPB and cardioplegic arrest significantly reduces systemic and cardiac adhesion molecule expression and catecholamine requirement. Since the clinical course in the early postoperative period was comparable, larger trials are required to select the appropria te patient who benefits most from one or the other treatment regime.

Minimally invasive coronary artery bypass grafting: port-access approach versus off-pump techniques

BACKGROUNDnWithin the past 5 years several surgical techniques have been developed for less invasive surgical treatment of coronary artery disease. The aim of this study was to define specific indications for the various minimally invasive coronary artery surgical procedures.nnnMETHODSnMinimally invasive direct coronary artery bypass grafting through a minithoracotomy was performed in 67 patients. The left internal mammary artery was anastomosed on the beating heart with the use of a pressure or suction stabilizer without the use of extracorporeal circulation. In 58 other patients with multivessel disease, the off-pump coronary artery bypass grafting technique through a sternotomy was applied with a left internal mammary artery to left anterior descending artery and additional vein grafts without extracorporeal circulation. In a third group, Port-Access (Heartport Inc, Redwood City, CA) coronary artery bypass grafting was performed through a left minithoracotomy with the use of an endovascular extracorporeal circulation system and cardioplegic arrest. Angiographic follow-up was complete in 64% of the patients.nnnRESULTSnThere was minimal perioperative or postoperative mortality (0.5%). The medium surgical procedure time for all minimally invasive and off-pump procedures was 2.5 hours; it was 4.5 hours for Port-Access procedures. The median postoperative intensive care unit stay was 1.0 days, and the median hospitalization was 5.0 days. Overall graft patency was 97.3%; in 8 patients (4.1%) a stenosis either at or distal to the graft anastomosis was dilated with coronary angioplasty.nnnCONCLUSIONSnFor single-vessel disease of the left anterior descending artery, the minimally invasive coronary artery bypass grafting procedure can be performed safely without the use of extracorporeal circulation. In case of hemodynamic instability or anatomic variation, the Port-Access procedure can be applied without additional necessity for sternotomy. For multivessel disease, the off-pump bypass grafting procedure with sternotomy can be recommended depending on the coronary arteries involved. In case of necessary grafts to the lateral marginal or circumflex branches, Port-Access grafting can be recommended and may play an important role in the future for the development of fully endoscopic robot-assisted coronary artery bypass grafting.

Coronary vasomotor dysfunction in the cardiac allograft: impact of different immunosuppressive regimens.

Immunosuppression may have an important impact on early graft coronary endothelial injury. We investigated functional and morphologic coronary alterations, myocardial expression, and cardiac release of possible mediators of allograft vasculopathy within 6 months after cardiac transplantation with respect to different immunosuppressive regimens. Epicardial and microvascular endothelium-dependent and endothelium-independent vasomotor function and epicardial intimal thickening were measured in 8 transplant recipients treated with cyclosporin A (CyA), azathioprine, and prednisone (group 1), 9 transplant recipients treated with tacrolimus (TKL), azathioprine, and prednisone (group 2), and 14 patients treated with TKL, mycophenolate mofetil (MMF), and prednisone (group 3). The gene expressions of inducible and endothelial nitric oxide synthase (iNOS and eNOS), endothelin-1, prostacyclinsynthase, and thromboxansynthase were analyzed in endomyocardial biopsy specimens using semiquantitative reverse transcription polymerase chain reaction. Transcardiac cytokine release, endothelin-1, and nitrate-release were determined from plasma samples. Epicardial endothelial dysfunction (vasoconstriction to acetylcholine > 10%) and microvascular smooth muscle cell dysfunction (flow velocity increase to adenosine and nifedipine < 2.0) were enhanced in heart transplant recipients immunosuppressed with TKL, azathioprine, and prednisone. The prevalence of epicardial dysfunction was 78% in group 2 versus 44% and 46% in group 1 and 3 (p < 0.05), respectively. The prevalence of microvascular dysfunction was 56% in group 2 versus 13% and 7% in group 1 and 3 (p < 0.02), respectively. Coronary vasomotor dysfunction was associated with increased myocardial iNOS expression (p < 0.05), decreased eNOS expression (p < 0.05), and enhanced cardiac immunoreactive interleukin-6 (p < 0.01). Coronary intimal thickening was not different between the groups. The combination of TKL and MMF appears to be superior to TKL and azathioprine (and comparable to CyA and azathioprine) concerning preservation of early coronary vasomotor function, eNOS expression, iNOS suppression as well as cardiac interleukin-6 release. This may have an important impact on subsequent development of transplant coronary atherosclerosis.

Doxycycline reduces cardiac matrix metalloproteinase-2 activity but does not ameliorate myocardial dysfunction during reperfusion in coronary artery bypass patients undergoing cardiopulmonary bypass.

Objectives:Matrix metalloproteinase-2 proteolyzes intracellular proteins in the heart and induces acute myocardial contractile dysfunction in ischemia-reperfusion injury. Doxycycline, a matrix metalloproteinase inhibitor, prevented matrix metalloproteinase-2-induced troponin I cleavage in rat hearts and improved contractile function following ischemia-reperfusion. In patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass, increased atrial matrix metalloproteinase-2 activity was inversely correlated with cardiac mechanical function at 3 hours reperfusion. We performed a study in patients with coronary artery disease undergoing primary elective coronary artery bypass graft surgery with cardiopulmonary bypass to determine whether doxycycline reduces cardiac mechanical dysfunction, matrix metalloproteinase activity, and troponin I degradation after reperfusion. Design:Randomized, double-blinded, placebo-controlled study. Setting:University of Alberta Hospital. Patients:Forty-two patients with coronary artery disease undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. Interventions:Patients were randomized to receive either oral administration of 20 mg of doxycycline or matching placebo pill twice a day at least 2 days prior to surgery, on the day of surgery, and for the first 3 postoperative days. Measurements and Main Results:Left ventricular stroke work index was examined prior to cardiopulmonary bypass and at 24 hours reperfusion. Right atrial biopsies were collected before cardiopulmonary bypass and 10 minutes after aortic cross-clamp release to determine matrix metalloproteinase-2 activity and troponin I level. Blood was collected to determine matrix metalloproteinase activity and interleukin-6, C-reactive protein, and troponin I levels. Cardiac 72-kDa matrix metalloproteinase-2 activity was lower upon reperfusion in biopsies from the doxycycline group (p = 0.01), and the increase of matrix metalloproteinase-2 activity in the placebo group due to reperfusion did not appear in the doxycycline group (p = 0.05). Doxycycline, however, did not ameliorate cardiac mechanical dysfunction following reperfusion or the cardiopulmonary bypass-coronary artery bypass graft-induced increased plasma matrix metalloproteinase-9, interleukin-6, and C-reactive protein levels. Cardiopulmonary bypass-coronary artery bypass graft or doxycycline did not change tissue or plasma troponin I levels at 10 minutes reperfusion. Conclusions:Although doxycycline did not improve myocardial stunning following coronary artery bypass graft surgery with cardiopulmonary bypass, it reduced cardiac matrix metalloproteinase-2 activity in these patients. A larger trial and/or higher dose of doxycycline may yet be warranted.

Phosphorylcholine-coated circuits improve preservation of platelet count and reduce expression of proinflammatory cytokines in CABG: a prospective randomized trial.

Abstractu2002 Background: The interaction of blood with foreign artificial surfaces during cardiopulmonary bypass (CPB) has been recognized as a major stimulus in evoking a systemic inflammatory and metabolic response. Phosphorylcholine (PC) is a new‐generation coating material designed to ameliorate biocompatibility and thereby to reduce the detrimental interactions of CPB. We studied the effects of PC‐coated perfusion circuits on platelet function and the humoral and cellular response to CPB. Methods: Thirty patients undergoing coronary artery bypass grafting were randomized to PC‐coated (PC group, n = 15) and noncoated (control group, n = 15) circuit groups. Clinical data, total blood loss, and pre‐ and postoperative platelet counts were recorded and IL‐6 and TNF‐α, CD41a, CD42b, and CD62p were measured at induction of anesthesia, after the initiation of CPB and at termination of CPB. Results: There was a significantly improved preservation of platelet count following CPB in the PC group (p = 0.028), which was sustained over a period of 72 hours. The use of PC‐coated circuits further resulted in a significant attenuation of TNF‐α and IL‐6 expression (p < 0.05 and p < 0.01); however, we were unable to detect any differences in clinical outcomes. Conclusions: Despite similar clinical outcome, the obvious reduction of cytokine expression and improved preservation of platelet count suggest superior biocompatibility of PC‐coated circuits.