Michael Marber

Na(+)/H(+) exchange inhibitors for cardioprotective therapy: progress, problems and prospects.

Extensive pre-clinical work indicates that inhibition of the sarcolemmal Na(+)/H(+) exchanger (NHE) affords significant protection to myocardium subjected to ischemia and reperfusion, predominantly through reduced intracellular accumulation of Na(+) and consequently Ca(2+). In contrast, recent clinical studies with the NHE inhibitors cariporide and eniporide in patients with evolving myocardial infarction (MI) and those at risk of MI have provided mixed and somewhat contradictory data. The experimental evidence suggests that the key mechanism through which NHE inhibitors afford protection consists in slowing the progression of myocardial injury during ischemia and thereby enhancing myocardial salvage by reperfusion. It follows from this that, to obtain maximum cardioprotective benefit, 1) the NHE inhibitor must be present in jeopardized myocardium, at a concentration sufficient to inhibit NHE activity, before (or as soon as possible after) the onset of ischemia, and 2) ischemia must be terminated by timely reperfusion. Thus, in the GUARDIAN trial, the cardioprotective efficacy of cariporide was limited to the subset of high-risk patients who underwent coronary artery bypass graft surgery, in whom both prerequisites could be readily fulfilled. In contrast, no cardioprotective benefit was observed in the ESCAMI trial, in which eniporide was administered late as an adjunct to reperfusion therapy in patients with evolving MI. Ongoing clinical studies will determine whether NHE inhibition will find therapeutic application in the setting of cardiac surgery, while pre-clinical investigations continue to test the potential of NHE inhibitors in the treatment of other cardiovascular diseases such as heart failure.

Involvement of Nox2 NADPH Oxidase in Adverse Cardiac Remodeling After Myocardial Infarction

Oxidative stress plays an important role in the development of cardiac remodeling after myocardial infarction (MI), but the sources of oxidative stress remain unclear. We investigated the role of Nox2-containing reduced nicotinamide-adenine dinucleotide phosphate oxidase in the development of cardiac remodeling after MI. Adult Nox2−/− and matched wild-type (WT) mice were subjected to coronary artery ligation and studied 4 weeks later. Infarct size after MI was similar in Nox2−/− and WT mice. Nox2−/− mice exhibited significantly less left ventricular (LV) cavity dilatation and dysfunction after MI than WT mice (eg, echocardiographic LV end-diastolic volume: 75.7±5.8 versus 112.4±12.3 μL; ejection fraction: 41.6±3.7 versus 32.9±3.2%; both P<0.05). Similarly, in vivo LV systolic and diastolic functions were better preserved in Nox2−/− than WT mice (eg, LV dP/dtmax: 7969±385 versus 5746±234 mm Hg/s; LV end-diastolic pressure: 12.2±1.3 versus 18.0±1.8 mm Hg; both P<0.05). Nox2−/− mice exhibited less cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis; reduced increases in expression of connective tissue growth factor and procollagen 1 mRNA; and smaller increases in myocardial matrix metalloproteinase–2 activity than WT mice. These data suggest that the Nox2-containing reduced nicotinamide-adenine dinucleotide phosphate oxidase contributes significantly to the processes underlying adverse cardiac remodeling and contractile dysfunction post-MI.

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes

Activation of protein kinase C (PKC) and more recently mitogen‐activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC‐δ results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC‐δ, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal‐simulated ischemia activated only p38 (2.8±0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC‐δ or by preconditioning (0.48 ±0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0± 3.1%, LDH release 77.3±4.0%, and MTT bioreduction 127.1 ±4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild‐type p38a or p38β. Transfected p38a and β show differential activation (P<0.001) during sustained simulated ischemia, with p38a remaining activated (1.48±0.36 vs. basal) but p38β deactivated (0.36± 0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38a (0.65±0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38α, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9±3.9% and MTT bioreduction 130.2±6.5% of control, n=8, P<0.05). Thus, inhibition of p38α activation during ischemia reduces injury and may contribute to preconditioning‐induced cardioprotection in this model.—Saurin, A. T., Martin, J. L., Heads, R. J., Foley, C., Mockridge, J. W., Wright, M. J., Wang, Y., Marber, M. S. The role of differential activation of p38‐mitogen‐activated protein kinase in preconditioned ventricular myocytes. FASEB J. 14, 2237–2246 (2000)

Circulating Humoral Factors and Endothelial Progenitor Cells in Patients With Differing Coronary Collateral Support

Background—The mechanisms underlying the variation in collateral formation between patients, even with similar patterns of coronary artery disease, remain unclear. This study investigates whether circulating humoral or cellular factors can provide an insight into this variation. Methods and Results—Thirty patients with isolated left anterior descending coronary artery disease underwent percutaneous coronary intervention with collateral flow index (CFI) determined using a pressure wire. Patients with inadequate (CFI <0.25) compared with those with adequate (CFI ≥0.25) collateral support had, or tended to have, lower concentrations of coronary sinus growth factors and plasma exerting a weaker effect on endothelial cell migration and angiogenesis in vitro. However, there was an inverse correlation between serum mitogenicity and CFI (r =−0.61, P <0.01). No significant differences were detected between the 2 groups in plasma levels of total vascular endothelial growth factor, vascular endothelial growth factor165, or placental growth factor. There was a strong positive correlation between numbers of CD34/CD133-positive circulating hemopoietic precursor cells and CFI (r =0.75, P <0.001). In patients with inadequate, compared with those with adequate, CFI, the numbers of differentiated endothelial progenitor cells (EPCs) appearing in the circulation and in culture were significantly reduced by 75% (P <0.05) and 70% (P <0.05), respectively. Conclusions—In this study, inadequate coronary collateral development is associated with reduced numbers of circulating EPCs and impaired chemotactic and proangiogenic but not mitogenic activity. These findings are consistent with current efforts to enhance collateral formation by augmentation of circulating EPCs.

Systemic inflammation in unstable angina is the result of myocardial necrosis

OBJECTIVES We investigated whether the source of the acute phase response in unstable angina (UA) lay within the culprit coronary plaque or distal myocardium. BACKGROUND An inflammatory response is an important component of the acute coronary syndromes. However, its origin and mechanism remain unclear. METHODS In 94 stable patients undergoing coronary angiography, the relationship between systemic levels of tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6) and C-reactive protein (CRP) and extent of atherosclerosis was studied. The temporal relationship between these markers and troponin T (TnT) was determined in 91 patients with UA. Cytokine levels were measured in the aortic root and coronary sinus of 36 unstable patients. RESULTS There was no relationship found between stable coronary atherosclerosis and inflammatory marker levels. Compared with this group, admission levels of IL-6 (3.6 +/- 0.3 ng/ml vs. 10.7 +/- 1.7 ng/ml, p < 0.05) and CRP (2.3 +/- 0.1 mg/l vs. 4.6 +/- 0.6 mg/l, p < 0.05) were elevated in patients with UA. In this group, IL-6 and CRP remained elevated in those who subsequently experienced major adverse cardiac events. This inflammatory response occurred in parallel to the appearance of TnT. Both TNF-alpha (19.2 +/- 3.4 ng/ml vs. 17.1 +/- 3.3 ng/ml, p < 0.001) and IL-6 (10.3 +/- 1.4 ng/ml vs. 7.7 +/- 1.1 ng/ml, p < 0.01) were elevated in the coronary sinus compared with aortic root in patients with UA. This was principally observed in those who were TnT positive. There was no cytokine gradient across the culprit plaque. CONCLUSIONS There is an intracardiac inflammatory response in UA that appears to be the result of low-grade myocardial necrosis. The ruptured plaque does not appear to contribute to the acute phase response.

High-Resolution Magnetic Resonance Myocardial Perfusion Imaging at 3.0-Tesla to Detect Hemodynamically Significant Coronary Stenoses as Determined by Fractional Flow Reserve

OBJECTIVES The objective of this study was to compare visual and quantitative analysis of high spatial resolution cardiac magnetic resonance (CMR) perfusion at 3.0-T against invasively determined fractional flow reserve (FFR). BACKGROUND High spatial resolution CMR myocardial perfusion imaging for the detection of coronary artery disease (CAD) has recently been proposed but requires further clinical validation. METHODS Forty-two patients (33 men, age 57.4 ± 9.6 years) with known or suspected CAD underwent rest and adenosine-stress k-space and time sensitivity encoding accelerated perfusion CMR at 3.0-T achieving in-plane spatial resolution of 1.2 × 1.2 mm(2). The FFR was measured in all vessels with >50% severity stenosis. Fractional flow reserve <0.75 was considered hemodynamically significant. Two blinded observers visually interpreted the CMR data. Separately, myocardial perfusion reserve (MPR) was estimated using Fermi-constrained deconvolution. RESULTS Of 126 coronary vessels, 52 underwent pressure wire assessment. Of these, 27 lesions had an FFR <0.75. Sensitivity and specificity of visual CMR analysis to detect stenoses at a threshold of FFR <0.75 were 0.82 and 0.94 (p < 0.0001), respectively, with an area under the receiver-operator characteristic curve of 0.92 (p < 0.0001). From quantitative analysis, the optimum MPR to detect such lesions was 1.58, with a sensitivity of 0.80, specificity of 0.89 (p < 0.0001), and area under the curve of 0.89 (p < 0.0001). CONCLUSIONS High-resolution CMR MPR at 3.0-T can be used to detect flow-limiting CAD as defined by FFR, using both visual and quantitative analyses.

Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts

OBJECTIVE Activation of protein kinase C (PKC) isoforms is associated with the cardioprotective effect of early ischaemic preconditioning (IP). PKC consists of at least 10 different isoforms, encoded by separate genes, which mediate distinct physiological functions. Although the PKC-epsilon isoform has been implicated in preconditioning, uncertainty remains. We investigated whether preconditioning still occurs in a mouse line lacking cardiac PKC-epsilon protein due to a targeted disruption within the pkc-epsilon allele. METHODS The isolated buffer-perfused hearts from knockout mice lacking PKC-epsilon (-/-) and sibling heterozygous mice (+/-), with a normal PKC-epsilon complement, were preconditioned by 4 x 4 min ischaemia/6 min reperfusion. Hearts then underwent 45 min of global ischaemia followed by 1.5 h of reperfusion. RESULTS In PKC-epsilon (+/-) hearts ischaemic preconditioning reduced infarction volume as a percentage of myocardial volume (24.3+/-4.5 vs. 41.3+/-4.7%, P<0.001). In contrast, in PKC-epsilon (-/-) hearts preconditioning failed to diminish infarction (36.4+/-2.9 vs. 38.8+/-4.5%). Surprisingly however, although preconditioning did not reduce infarct size, it did enhance contractile recovery in PKC-epsilon (-/-) mice (43.1+/-3.9 vs. 24.9+/-5.1%, P<0.05), similar to the level seen in PKC-epsilon (+/-) hearts (35.2+/-3.9 vs. 20.9+/-5.0%, P<0.05). CONCLUSIONS These data suggest that PKC-epsilon is essential for the reduction in infarction that follows early ischaemic preconditioning, but is not associated with the improvement in functional recovery.

Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2–deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti–MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

Peripheral Augmentation Index Defines the Relationship Between Central and Peripheral Pulse Pressure

Peripheral systolic blood pressure is amplified above central aortic systolic pressure, but the late systolic shoulder of the peripheral pulse may approximate central systolic pressure. Because late systolic pressure also determines the peripheral augmentation index, a measure of pressure wave reflection within the systemic circulation, this implies a direct relationship between amplification and augmentation. We compared the late systolic shoulder of the peripheral pressure waveform with estimates of central systolic pressure obtained using a transfer function in 391 subjects undergoing diagnostic coronary angiography and/or elective angioplasty (30% with insignificant coronary artery disease). In a subset (n=12) we compared the late systolic shoulder of the peripheral pulse with central pressure obtained with a catheter placed in the aortic root. Measurements were made at baseline, during atrial pacing, and during administration of nitroglycerin. Late systolic shoulder pressure closely approximated transfer function estimates of central pressure (R=0.96; P<0.0001; mean difference±SD: 0.5±5.2 mm Hg). Despite changes in waveform morphology induced by pacing and nitroglycerin (reducing mean values±SE of the augmentation index from 76±3.8% to 66±4.6% and 60±3.3%, respectively), there was close agreement between the late systolic shoulder of the peripheral pulse and measured values of central pressure (R=0.96; P<0.001; mean difference: 1.7±4.8 mm Hg). In conclusion, the late systolic shoulder of the peripheral pulse closely approximates central systolic pressure and peripheral augmentation index, the ratio of central:peripheral pulse pressure. Interventions to lower augmentation index and peripheral vascular resistance will have multiplicative effects in lowering central blood pressure.

Diverse Mechanisms of Myocardial p38 Mitogen-Activated Protein Kinase Activation Evidence for MKK-Independent Activation by a TAB1-Associated Mechanism Contributing to Injury During Myocardial Ischemia

Abstract— The ischemic activation of p38&agr; mitogen-activated protein kinase (p38&agr;-MAPK) is thought to contribute to myocardial injury. Under other circumstances, activation is through dual phosphorylation by MAPK kinase 3 (MKK3). Therefore, the mkk3−/− murine heart should be protected during ischemia. In retrogradely perfused mkk3−/− and mkk3+/+ mouse hearts subjected to 30 minutes of global ischemia and 120 minutes of reperfusion, infarction/risk volume was similar (50±5 versus 51±4, P =0.93, respectively), as was intraischemic p38-MAPK phosphorylation (10 minutes ischemia as percent basal, 608±224 versus 384±104, P =0.43, respectively). This occurred despite undetectable activation of MKK3/6 in mkk3−/− hearts. However, tumor necrosis factor (TNF)-induced p38-MAPK phosphorylation was markedly diminished in mkk3−/− vs mkk3+/+ hearts (percent basal, 127±23 versus 540±267, respectively, P =0.04), suggesting an MKK-independent activation mechanism by ischemia. Hence, we examined p38-MAPK activation by TAB1-associated autophosphorylation. In wild-type mice and mkk3−/− mice, the p38-MAPK catalytic site inhibitor SB203580 (1 &mgr;mol/L) diminished phosphorylation during ischemia versus control (10 minutes ischemia as percent basal, 143±2 versus 436±96, P =0.003, and 122±25 versus 623±176, P =0.05, respectively) and reduced infarction volume (infarction/risk volume, 57±5 versus 36±3, P <0.001, and 50±5 versus 29±3, P =0.003, respectively) but did not alter TNF-induced activation, although in homogenates of ischemic hearts but not TNF-exposed hearts, p38-MAPK was associated with TAB1. Furthermore, adenovirally expressed wild-type and drug-resistant p38&agr;-MAPK, lacking the SB203580 binding site, was phosphorylated when H9c2 myoblasts were subjected to simulated ischemia. However, SB203580 (1 &mgr;mol/L) did not prevent the phosphorylation of resistant p38&agr;-MAPK. These findings suggest the ischemic activation of p38-MAPK contributing to myocardial injury is by TAB1-associated autophosphorylation.