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Background: The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has protective functions in the cardiovascular system. TERT is not only present in the nucleus but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection, and the appropriate tools are missing to dissect this., Methods: We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart, and cellular functions of cardiomyocytes, fibroblasts, and endothelial cells, as well, were determined., Results: All mice were phenotypically normal. Although respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wild-type mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after 1, 2, and 4 weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial nitric oxide synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits, explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The telomerase activator TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation., Conclusions: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves the migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection, and its increase could serve as a therapeutic strategy.

Coronary microembolization from the erosion or rupture of a vulnerable atherosclerotic plaque occurs spontaneously in acute coronary syndromes and iatrogenically during percutaneous coronary interventions. Typical consequences of coronary microembolization are microinfarcts with an inflammatory response, contractile dysfunction, and reduced coronary reserve. Apart from transient elevations of creatine kinase and troponin, microemboli can be visualized by intracoronary Doppler and the resulting microinfarcts by late-enhancement nuclear magnetic resonance. Statins, antiplatelet agents, and coronary vasodilators protect against microembolization and microinfarction when started before percutaneous coronary interventions. Distal protection devices can retrieve atherothrombotic debris and prevent its embolization into the microcirculation, but their effect on clinical outcome has been disappointing so far, except for saphenous vein bypass grafts. Devices for aspiration of thrombi and thrombus-derived vasoconstrictor, thrombogenic, and inflammatory substances, however, reduce thrombus burden, improve perfusion, and provide protection in patients with acute myocardial infarction.

Background: Survivin inhibits apoptosis and regulates cell division in many organs, but its function in the heart is unknown., Methods and Results: We show that cardiac-specific deletion of survivin resulted in premature cardiac death. The underlying cause was a dramatic reduction in total cardiomyocyte numbers as determined by a stereological method for quantification of cells per organ. The resulting increased hemodynamic load per cell led to progressive heart failure as assessed by echocardiography, magnetic resonance imaging, positron emission tomography, and invasive catheterization. The reduction in total cardiomyocyte number in alpha-myosin heavy chain (MHC)-survivin(-/-) mice was due to an approximately 50% lower mitotic rate without increased apoptosis. This occurred at the expense of DNA accumulation because survivin-deficient cardiomyocytes displayed marked DNA polyploidy indicative of consecutive rounds of DNA replication without cell division. Survivin small interfering RNA knockdown in neonatal rat cardiomyocytes also led to polyploidization and cell cycle arrest without apoptosis. Adenoviral overexpression of survivin in cardiomyocytes inhibited doxorubicin-induced apoptosis, induced DNA synthesis, and promoted cell cycle progression. The phenotype of the alphaMHC-survivin(-/-) mice also allowed us to determine the minimum cardiomyocyte number sufficient for normal cardiac function. In human cardiomyopathy, survivin was potently induced in the failing heart and downregulated again after hemodynamic support by a left ventricular assist device. Its expression positively correlated with the mean cardiomyocyte DNA content., Conclusions: We suggest that the ontogenetically determined cardiomyocyte number may be an independent factor in the susceptibility to cardiac diseases. Through its profound impact on both cardiomyocyte replication and apoptosis, survivin may emerge as a promising new target for myocardial regeneration.

Background: The frequency and importance of microembolization in patients with acute coronary syndromes and during coronary interventions have recently been appreciated. Experimental microembolization induces immediate ischemic dysfunction, which recovers within minutes. Subsequently, progressive contractile dysfunction develops over several hours and is not associated with reduced regional myocardial blood flow (perfusion-contraction mismatch) but rather with a local inflammatory reaction. We have now studied the effect of antiinflammatory glucocorticoid treatment on this progressive contractile dysfunction., Methods and Results: Microembolization was induced by injecting microspheres (42-microm diameter) into the left circumflex coronary artery. Anesthetized dogs were followed up for 8 hours and received placebo (n=7) or methylprednisolone 30 mg/kg IV either 30 minutes before (n=7) or 30 minutes after (n=5) microembolization. In addition, chronically instrumented dogs received either placebo (n=4) or methylprednisolone (n=4) 30 minutes after microembolization and were followed up for 1 week. In acute placebo dogs, posterior systolic wall thickening was decreased from 20.0+/-2.1% (mean+/-SEM) at baseline to 5.8+/-0.6% at 8 hours after microembolization. Methylprednisolone prevented the progressive myocardial dysfunction. Increased leukocyte infiltration in the embolized myocardium was prevented only when methylprednisolone was given before microembolization. In chronic placebo dogs, progressive dysfunction recovered from 5.0+/-0.7% at 4 to 6 hours after microembolization back to baseline (19.1+/-1.6%) within 5 days. Again, methylprednisolone prevented the progressive myocardial dysfunction., Conclusions: Methylprednisolone, even when given after microembolization, prevents progressive contractile dysfunction.

Background: Coronary flow reserve (CFR) is not normalized shortly after coronary stenting. We hypothesized that alpha-adrenergic coronary vasoconstriction acts to limit CFR., Methods and Results: We assessed flow velocity by Doppler wires and cross-sectional area by angiography in 46 patients undergoing coronary culprit lesion stenting (81+/-4% stenosis). Hyperemia was induced by adenosine (24 micro g IC or 140 micro g/kg per minute IV) before and after stenting. Finally, either the alpha(1)-antagonist urapidil (10 mg IC) or the alpha(2)-antagonist yohimbine (3 mg IC) was randomly combined with adenosine. In 8 subjects with angiographically normal coronary arteries, CFR was increased from 3.21+/-0.30 to 3.74+/-0.43 by yohimbine and to 4.58+/-0.65 by urapidil, respectively (P=0.0001). Patients were divided according to the cutoff of CFR > or =3.0 (n=18) or <2.5 (n=28). Revascularization per se did not change CFR. However, 15 minutes after stenting, CFR decreased to 2.05+/-0.55 from CFR 3.64+/-0.58, whereas in patients with CFR 2.39+/-0.51, it remained unchanged. Yohimbine improved CFR to 3.26+/-0.42 and to 3.41+/-0.58 in patients with >3.0 and <2.05+/-0.55 baseline CFR, respectively. Urapidil improved CFR to 3.52+/-0.30 and 3.98+/-1.07, respectively., Conclusions: Urapidil and yohimbine attenuated the CFR impairment occurring after revascularization by increasing both the epicardial vasodilator effect of adenosine and the blood flow velocity, thus suggesting that the adrenergic system plays an important role in limiting the capacity of the coronary circulation to dilate.

Background: Stenting-related myocardial injury has been recognized as a frequent and prognostically important event, the extent of which depends on microcirculatory impairment in association with platelet aggregation, inflammation, and increased oxidative stress. Recent studies underscored the non-lipid-lowering effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) with antithrombotic, antiinflammatory, and antioxidative aspects. Thus, we tested the hypothesis that preprocedural statin therapy is associated with a reduction in the extent of stenting-related myocardial injury., Methods and Results: We stratified 296 consecutive patients who were undergoing stenting of a de novo stenosis according to the preprocedural status of statin therapy (229 statin-treated and 67 control patients). Incidence of periprocedural myocardial injury was assessed by analysis of creatine kinase (CK; upper limit of normal [ULN] 70 IU/L for women, 80 IU/L for men) and cardiac troponin T (cTnT; bedside test; threshold 0.1 ng/mL) before and 6, 12, and 24 hours after the intervention. Relative to control patients, the incidence of CK elevation >3x ULN was more than 90% lower in statin-treated patients (0.4% versus 6.0%, P=0.01). Statin therapy was the only factor independently associated with a lower risk of CK elevation >3x ULN (OR: 0.08, 95% CI: 0.01 to 0.75; P=0.03). The overall incidences of CK and cardiac troponin T elevation were slightly lower in statin-treated than in control patients (14.4% versus 20.9%, P=0.3, and 17.9% versus 22.4%, P=0.5, respectively)., Conclusions: Preprocedural statin therapy is associated with a reduction in the incidence of larger-sized, stenting-related myocardial infarctions. Prospective, randomized trials are warranted to further assess this cardioprotective effect of statins in coronary intervention.

Background: Although adrenergic activation plays a major role in the initiation of experimental myocardial ischemia, the significance of alpha-adrenergic coronary constriction in humans has been questioned. The present study assessed the impact of selective alpha-adrenergic receptor activation in patients with normal or atherosclerotic coronary arteries., Methods and Results: In 39 patients, coronary blood flow (CBF, mL/min) was determined from combined angiography and Doppler measurements. In 8 patients with normal coronary arteries (group 1) and 9 with single coronary artery stenosis (group 2), doses of 1, 2.5, 5, and 10 mg IC of the alpha1-agonist methoxamine (M) were injected. Identical doses of the alpha2-agonist BHT933 (B) were injected in 8 patients with normal coronary arteries (group 3) and 8 with single stenosis (group 4). In 6 additional patients with single stenosis (group 5), aortocoronary sinus lactate differences were measured in response to M and B. CBF remained unchanged in group 1. In contrast, CBF was decreased dose-dependently in group 2, with a maximum at 10 mg M (39.0+/-9.4 versus 15.2+/-7.0). In groups 3 and 4, CBF was also decreased dose-dependently, with a maximum at 10 mg B (63.3+/-24.8 versus 49. 1+/-27.9 and 41.5+/-19.0 versus 12.7+/-8.0, respectively). In group 5, there was more net lactate production with B than with M (-0. 34+/-0.11 versus -0.04+/-0.09 mmol/L)., Conclusions: In normal coronary arteries, alpha1-adrenergic activation does not reduce CBF, whereas alpha2-adrenergic activation reduces CBF by microvascular constriction. Both alpha1- and alpha2-adrenergic epicardial and microvascular constriction are augmented by atherosclerosis and can induce myocardial ischemia.

Background: AMI reperfusion by thrombolysis does not improve TIMI flow and LV function. The role of infarct-related artery (IRA) stenosis and superimposed changes in coronary vasomotor tone in maintaining LV dysfunction must be elucidated., Methods and Results: Forty patients underwent diagnostic angiography 24 hours after thrombolysis. Seventy-two hours after thrombolysis, the culprit lesion was dilated with coronary stenting. During angioplasty, LV function was monitored by transesophageal echocardiography. Percent regional systolic thickening was quantitatively assessed before PTCA, soon after stenting, 15 minutes after stenting, and after phentolamine 12 microg/kg IC (n=10), the alpha1-blocker urapidil 600 microg/kg IV (n=10), or saline (n=10). Ten patients pretreated with beta-blockers received urapidil 10 mg IC. Coronary stenting significantly improved thickening in IRA-dependent and in non-IRA-dependent myocardium (from 27+/-15% to 38+/-16% and from 40+/-15% to 45+/-15%, respectively). Simultaneously, TIMI frame count decreased from 39+/-11 and 40+/-11 in the IRA and non-IRA, respectively, to 23+/-10 and 25+/-7 (P<0.05). Fifteen minutes after stenting, thickening worsened in both IRA- and non-IRA-dependent myocardium (to 19+/-14% and 28+/-14%, P<0.05), and TIMI frame count returned, in both the IRA and non-IRA, to the values obtained before stenting. Phentolamine and urapidil increased thickening to 36+/-17% and 41+/-14% in IRA and to 48+/-11% and 49+/-17% in non-IRA myocardium respectively, and TIMI frame count decreased to 16+/-6 and to 17+/-5, respectively. Changes were attenuated with beta-blocker pretreatment., Conclusions: Our finding that alpha-adrenergic blockade attenuates vasoconstriction and postischemic LV dysfunction supports the hypothesis of an important role of neural mechanisms in this phenomenon.

Background: Mibefradil is a calcium antagonist with few negative inotropic effects at therapeutic concentrations., Methods and Results: The effect of mibefradil on infarct size (IS) was compared with those of placebo, amlodipine, and verapamil in 64 anesthetized pigs. In placebo pigs, after 90 minutes of ischemia and 120 minutes of reperfusion, IS (by triphenyl tetrazolium chloride staining) was 15.3+/-10.8% (SD) of the area at risk. Mibefradil (0.60 mg/kg IV) reduced heart rate and left ventricular (LV) pressure, and IS was 1. 9+/-3.9% (P<0.05 versus placebo). Verapamil (0.15 mg/kg IV) also decreased heart rate, LV pressure, and IS (6.1+/-4.2%, P<0.05 versus placebo). Amlodipine (0.20 mg/kg IV) did not alter heart rate, LV pressure, or IS (9.9+/-5.4%, P=NS versus placebo). When heart rate was maintained constant by left atrial pacing and LV pressure was adjusted to that of the placebo group by an intra-aortic balloon, mibefradil still decreased IS (3.8+/-3.0%, P<0.05 versus placebo), but verapamil did not (11.6+/-8.3%, P=NS versus placebo). With glibenclamide infusion, mibefradil no longer reduced IS (13.1+/-4.3% versus 17.8+/-5.6% with glibenclamide alone, P=NS)., Conclusions: The IS-limiting effect of mibefradil, in contrast to that of verapamil, was not dependent on favorable hemodynamics but was abolished by glibenclamide, suggesting a direct cardioprotective action of mibefradil.

Background: A threshold concept for ischemic preconditioning (IPc) has been proposed. It is unclear, however, whether IPc, above a certain threshold, is an all-or-nothing or a graded phenomenon., Methods and Results: In 71 enflurane-anesthetized swine, severe left anterior descending coronary artery hypoperfusion for 90 minutes followed by 2 hours of reperfusion resulted in an infarct size (IS, by triphenyltetrazolium chloride) of 16.7+/-3.4% (SEM) of the area at risk. IPc by 2 minutes of low-flow ischemia and 15 minutes of reperfusion before the 90-minute target ischemia did not reduce IS (21.9+/-7.0%). IS was decreased to 9.0+/-2.6% (P<0.05) by 3 minutes of IPc and reduced further to 1.9+/-0.9% (P<0.05) by 10 minutes of IPc. The interstitial adenosine concentration (microdialysis, high-performance liquid chromatography) was unchanged with 2 and 3 minutes of IPc but increased with 10 minutes of IPc (by 573+/-144%). The interstitial bradykinin concentration (microdialysis, radioimmunoassay) remained unchanged with 2 minutes of IPc but increased to a similar extent with 3 minutes (by 198+/-32%) and 10 minutes (by 224+/-30%) of IPc. The IS reduction by 3 minutes of IPc was abolished by blockade of the bradykinin B2 receptor with intracoronary HOE 140 (16.6+/-4.3%) but not with intracoronary infusion of adenosine deaminase (8.4+/-2.5%, P<0.05). HOE 140, however, did not affect the IS reduction (3.5+/- 1.1%, P<0.05) by 10 minutes of IPc. Combined infusion of HOE 140 and adenosine deaminase abolished the IS reduction by 10 minutes of IPc (15.4+/-6.7%)., Conclusions: IS reduction by IPc is a graded phenomenon. Whereas bradykinin is essential during preconditioning ischemia of shorter duration, adenosine is more important during preconditioning ischemia of longer duration.

Background: Myocardial fractional flow reserve (FFR) is based on pressure measurements. We have now sought to establish a Doppler-based concept of relative flow velocity reserve (RFVR) for the functional assessment of stenosis severity in epicardial coronary arteries. A clear threshold value to discriminate the functional severity of a coronary stenosis does not exist for coronary flow velocity reserve (CVR) based on intracoronary Doppler measurements. In contrast, the concept of FFR, which is based on intracoronary pressure measurements, has been extensively validated. An FFR value below 0.75 reliably indicates a significant stenosis., Methods and Results: RFVR is calculated as the ratio between distal CVR in the stenosed target vessel and distal CVR in a nonstenotic reference vessel. In 21 patients, RFVR was determined in 24 target vessels by use of intracoronary adenosine and correlated to the FFR, determined as the ratio of mean poststenotic to aortic pressures, in the target vessel. Stenosis severity was classified according to quantitative coronary angiography analysis. Reference diameter was 3.0+/-0.4 mm (mean+/-SD), and area stenosis was 74+/-15% (range, 40% to 95%). CVRs in the target and reference vessels were 2.1+/-0.5 and 2.6+/-0.7, respectively. FFR ranged from 0.49 to 0.99 (mean, 0.81+/-0.15) and RFVR from 0.53 to 1.0 (mean, 0.82+/-0.13). Poststenotic CVR did not correlate with either percent area stenosis (r=0.27, P=NS) or FFR (r=0.33, P=NS). In contrast, FFR as well as RFVR showed a curvilinear relation to percent area stenosis (r=0.89, P<0.0001 and r=0.79, P<0.0001, respectively). There was a close linear correlation between FFR and RFVR (r=0.91, P<0.0001)., Conclusions: RFVR correlates closely to FFR and to percent area stenosis, whereas the correlation of CVR with FFR and percent area stenosis is rather poor. RFVR is a promising new concept for assessment of coronary stenosis severity and clinical decision making based on Doppler measurements.

Background: Modeling of the time-[1-(11)C]acetate activity curve assumes a constant concentration of labeled tricarboxylic acid cycle intermediates and associated metabolites, such as glutamate and aspartate, which may, however, decrease in short-term hibernating myocardium., Methods and Results: In 12 anesthetized pigs, [1-(11)C]acetate was injected as a bolus into the cannulated left anterior descending coronary artery during normoperfusion, inotropic stimulation, and early (5 to 45 minutes) and prolonged ischemia (60 to 90 minutes). Regional myocardial oxygen consumption (MVO2, microliters per minute per gram) was measured, and the absence of necrosis was verified by triphenyl tetrazolium chloride staining. Inotropic stimulation increased MVO2 from 52.5+/-7.4 to 195.4+/-36.2 (mean+/-SD) and the rate constant (kmono, minutes[-1]) of [1-(11)C]acetate clearance from 0.094+/-0.018 to 0.322+/-0.076. During early ischemia, MVO2 and kmono were decreased to 24.3+/-8.5 and 0.061+/-0.011, respectively. Kmono closely correlated to MVO2 during normoperfusion, inotropic stimulation, and early ischemia. In short-term hibernating myocardium, however, at an unchanged MVO2, kmono increased toward control values (0.080+/-0.014). Myocardial glutamate and aspartate concentrations (biopsies) decreased to 47+/-26% and 77+/-18%; the peak count rate decreased to 66+/-22% of its respective control value. After correction for the decreases in glutamate and aspartate or in peak count rate, kmono was again decreased (0.050+/-0.016 or 0.052+/-0.014, respectively), and a close relationship to MVO2 was restored., Conclusions: Kmono correlates to MVO2 in short-term hibernating myocardium when the decreases in aspartate and glutamate or in peak count rate are considered.

Background: We tested the hypothesis that decreased calcium responsiveness is responsible for the reduction in contractile function in regional hibernating and stunned myocardium in situ., Methods and Results: In 19 anesthetized swine, the left anterior descending coronary artery flow was reduced to decrease anterior myocardial work index (sonomicrometry) by approximately 60%. During 90 minutes of hypoperfusion, creatine phosphate recovered (as determined by biopsy specimens and bioluminescence) and no necrosis developed (as determined by staining with triphenyl tetrazolium chloride). In 10 swine, changes in the intracellular calcium concentration were induced by systematic variation of the postextrasystolic time interval at a constant prematurity. In 9 additional swine, a graded IC calcium infusion was performed. Under control conditions, anterior myocardial work increased with a fully compensated postextrasystolic time interval from 380+/-93 (mean+/-SD) to 523+/-98 mm Hg . mm. IC calcium infusion increased anterior myocardial work under control conditions from 356+/-85 to a maximum of 428+/-93 mm Hg . mm. Although the maximal responses were decreased during postextrasystolic potentiation (222+/-68 versus 523+/-98 mm Hg . mm) and calcium infusion (176+/-32 versus 428+/-93 mm Hg . mm) after 90 minutes of ischemia, the relationships between increases in anterior myocardial work and, respectively, postextrasystolic time interval and IC calcium were not different. The same was true after 30 minutes of reperfusion., Conclusions: Both regional hibernating myocardium and stunned myocardium in situ are characterized by a decrease in overall myocardial calcium responsiveness; however, there appears to be no significant myocardial desensitization to calcium.

Background: The purpose of the study was to characterize the functional and metabolic adjustments of a myocardial region subjected to low-flow ischemia. In addition, studies tested whether such myocardium retains an inotropic reserve., Methods and Results: Anesthetized swine were studied in which the left anterior descending coronary artery was cannulated and perfused at a constant low level causing regional contractile dysfunction (sonomicrometry for wall thickness) and the appearance of metabolic indicators of ischemia (decrease in creatine phosphate and lactate production) with only slight loss of ATP and glycogen (transmural biopsies). After 85 minutes of low-flow ischemia, dobutamine was infused into the hypoperfused artery as an inotropic challenge. Coronary hypoperfusion for 5 minutes resulted in a 54% reduction of regional systolic wall thickening, reversal of lactate consumption to lactate production, and a significant decrease in creatine phosphate. Subendocardial blood flow was reduced from 0.62 +/- 0.11 (+/- SD) to 0.16 +/- 0.07 mL.min-1.g-1. Prolonged hypoperfusion for 85 minutes resulted in no further change in regional blood flow but a partial recovery of metabolic parameters. Dobutamine infusion after 85 minutes of hypoperfusion increased regional myocardial work. However, again lactate production was significantly increase and creatine phosphate was decreased. Regional coronary hypoperfusion produces a downregulation of regional contractile function in proportion to the blood flow decrease. With prolonged hypoperfusion, after the initial adjustment phase, there is little further change in function, and metabolic markers of ischemia improve. Although the ischemic downregulated myocardium retains a significant inotropic reserve, primarily anaerobic energy production is utilized., Conclusions: These data are consistent with downregulation being a protective mechanism for the ischemic myocardium to restore an energy supply-demand balance in the face of reduced blood flow. Inotropic stimulation of the downregulated myocardium enhances regional function but at the cost of worsening its metabolic status. Thus, inotropic stimulation of the hypoperfused and downregulated myocardium is probably detrimental to long-term viability.