Laura Comini

Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury.

Background—Apoptosis contributes to cell loss after ischemia/reperfusion injury in the heart. This study describes the time course and level of apoptosis in different cell types in the intact heart during ischemia/reperfusion injury. Methods and Results—Isolated Langendorff-perfused rat hearts were subjected to perfusion alone (control) or to 35 minutes of regional ischemia, either alone or followed by 5, 60, or 120 minutes of reperfusion. Sections were stained by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) and propidium iodide and with anti-von Willebrand factor, anti-desmin, or anti-active caspase 3 antibodies; they were then visualized by confocal microscopy. Sections were also examined by electron microscopy. No TUNEL-positive cells were seen in control hearts or hearts exposed to ischemia alone. Early in reperfusion, TUNEL staining was colocalized with endothelial cells from small coronary vessels. Endothelial apoptosis peaked at 1 hour of reperfusion and, at this time, there was clear perivascular localization of apoptotic cardiac myocytes, whose number was inversely proportional to their distance from a positive vessel. After 2 hours of reperfusion, apoptotic cardiac myocytes assumed a more homogeneous distribution. Active caspase 3 labeling was seen independent of DNA fragmentation during ischemia alone, but it colocalized with TUNEL staining over the 3 time points of reperfusion. Immunocytochemical findings were confirmed by electron microscopy and Western blotting. Conclusions—In the very early stages of reperfusion, apoptosis is first seen in the endothelial cells from small coronary vessels. The radial spread of apoptosis to surrounding cardiac myocytes suggests that reperfusion induces the release of soluble pro-apoptotic mediators from endothelial cells that promote myocyte apoptosis.

Serum From Patients With Severe Heart Failure Downregulates eNOS and Is Proapoptotic Role of Tumor Necrosis Factor-α

BACKGROUND Cytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis. METHODS AND RESULTS We studied 21 patients with severe CHF. Levels of tumor necrosis factor-alpha (TNF-alpha) and several neuroendocrine parameters were assessed. eNOS was measured by Western Blot analysis and apoptosis by optical microscopy and flow cytometry. We observed (1) eNOS downregulation (difference versus healthy subjects at 24 hours [P<0.05] and 48 hours [P<0.001]), (2) nuclear morphological changes typical of apoptosis; and (3) a high apoptotic rate with propidium iodide (increasing from 2.1+/-0.4% to 11.3+/-1.2% at 48 hours; P<0.001 versus healthy subjects) and annexin V. An anti-human TNF-alpha antibody did not completely counteract these effects. A strong correlation existed between eNOS downregulation and apoptosis (r = -0.89; P<0.001). CONCLUSIONS Serum from patients with severe CHF downregulates eNOS expression and increases apoptosis. High levels of TNF-alpha likely play a role, but they cannot be the only factor responsible.

Different Signaling Pathways Induce Apoptosis in Endothelial Cells and Cardiac Myocytes During Ischemia/Reperfusion Injury

Apoptosis contributes, with necrosis, to the cardiac cell loss after ischemia/reperfusion injury. The apoptotic cascade is initiated either by mitochondrial damage and activation of caspase-9 or by death receptor ligation and activation of caspase-8. In the present study, performed in the isolated rat heart exposed either to ischemia alone or ischemia followed by reperfusion, cleavage of caspase-9 was observed primarily in endothelial cells. Conversely, caspase-8 cleavage was only found in cardiomyocytes, where it progressively increased throughout reperfusion. Addition of a specific caspase-9 inhibitor to the perfusate before ischemia prevented endothelial apoptosis, whereas preischemic infusion of a specific caspase-8 inhibitor affected only myocyte apoptosis. Additionally, caspase-8–mediated BID processing was observed only during reperfusion. Production of tBID then sustains mitochondrial injury and perpetuates caspase-9 activation.

K-ATP channel gene expression is induced by urocortin and mediates its cardioprotective effect

Background—Urocortin is a novel cardioprotective agent that can protect cardiac myocytes from the damaging effects of ischemia/reperfusion both in culture and in the intact heart and is effective when given at reperfusion. Methods and Results—We have analyzed global changes in gene expression in cardiac myocytes after urocortin treatment using gene chip technology. We report that urocortin specifically induces enhanced expression of the Kir 6.1 cardiac potassium channel subunit. On the basis of this finding, we showed that the cardioprotective effect of urocortin both in isolated cardiac cells and in the intact heart is specifically blocked by both generalized and mitochondrial-specific KATP channel blockers, whereas the cardioprotective effect of cardiotrophin-1 is unaffected. Conversely, inhibiting the Kir 6.1 channel subunit greatly enhances cardiac cell death after ischemia. Conclusions—This is, to our knowledge, the first report of the altered expression of a KATP channel subunit induced by a cardioprotective agent and demonstrates that KATP channel opening is essential for the effect of this novel cardioprotective agent.

Serum From Patients With Acute Coronary Syndromes Displays a Proapoptotic Effect on Human Endothelial Cells: A Possible Link to Pan-Coronary Syndromes

Background—Endothelial apoptosis of atherosclerotic lesions is a possible determinant for the stable-to-vulnerable plaque transition. Recent data support the notion that plaque activation may be a pan-coronary process, advocating the existence of circulating triggers. Methods and Results—Serum from 40 healthy subjects (group 1) and 73 patients with stable angina (n=32; group 2) or acute coronary syndromes (n=41; group 3) was incubated with human umbilical vein endothelial cells. The percentage of apoptosis by flow cytometry and Fas, Bax, and Bcl-2 protein expression by immunoblotting were evaluated at entry in patients and control subjects and repeated after 12 months in group 3. At baseline, apoptotic nuclei were higher in group 3 (14±6%) than in group 2 (3.3±1.8%) and group 1 (1.35±0.8%) (P <0.001). Fas and Bcl-2 were increased in group 3 with respect to groups 1 and 2 (P <0.01). Coincubation of group 3 serum with anti–tumor necrosis factor-&agr; and anti–interleukin-6 monoclonal antibodies did not affect the human umbilical vein endothelial cell apoptotic process, whereas addition of Trolox decreased apoptosis to <50%. The percentage of apoptosis in group 3 significantly correlated to the numbers of coronary complex lesions at angiography (r =0.58, P <0.0005). In group 3, apoptosis and the Bax/Bcl-2 ratio decreased at 1 year (P <0.0001, P <0.05 respectively). Conclusions—Serum from patients with acute coronary syndromes displays a proapoptotic effect on human endothelial cells, supporting the theory of the existence of circulating triggers potentially able to activate atherosclerotic lesions.

Heart rate reduction with ivabradine prevents the global phenotype of left ventricular remodeling

The aim of this study was to investigate the effect of chronic heart rate (HR) reduction with the hyperpolarization-activated current inhibitor ivabradine on the global phenotype of left ventricular (LV) remodeling in a ligated rat model. Seven days after coronary artery ligation, Wistar rats received ivabradine (10 mg · kg(-1) · day(-1) administered in drinking water) [myocardial infarction + ivabradine (MI+IVA), n = 22] or vehicle only (drinking water) (MI, n = 20) for 90 days. A sham group (n = 20) was included for model validation. MI+IVA rats had 12% lower HR (P < 0.01), improved LV volumes, 15% higher LV ejection fraction (LVEF, P < 0.01) than MI rats, and 33% reductions in both plasma atrial natriuretic peptide (ANP, P = 0.052) and cardiac hydroxyproline. Using patch-clamp, action potential duration was reduced and transient outward current density increased (P < 0.05). Cardiac energy metabolism was also improved (+33% creatine phosphate, P < 0.001; +15% ATP; and +9% energy charge, P < 0.05). Significant correlations were found between HR and parameters of cardiac metabolism, ANP, and LVEF (all P < 0.05). The HR-reducing properties of ivabradine prevent changes in the global phenotype of LV remodeling in the rat, optimize energy consumption, and avoid electrophysiological and structural remodeling.

Differences in the Effect of Angiotensin-converting Enzyme Inhibitors on the Rate of Endothelial Cell Apoptosis: In Vitro and In Vivo Studies

AimAn imbalance between endothelial apoptosis and regeneration is one of the initiating events in atherosclerosis. Angiotensin-converting enzyme (ACE) inhibition corrects the endothelial dysfunction observed in coronary artery disease, and this could be the consequence of a reduction in the rate of endothelial apoptosis. The aim of this study was to investigate the effect of different ACE inhibitors on endothelial apoptosis.MethodsWe examined the effect of five ACE inhibitors (enalapril, perindopril, quinapril, ramipril, and trandolapril) on the rate of endothelial apoptosis, either in vitro in human umbilical vein endothelial cells (HUVECs), using a serum deprivation method to induce apoptosis, or in vivo in rats, inducing apoptosis via endotoxic shock with Escherichia coli lipopolysaccharides (LPS).ResultsWe were unable to detect any significant effect of ACE inhibition on the rate of in vitro endothelial apoptosis at concentrations ranging from 5 × 10−8 to 10−6 M. In contrast, chronic in vivo administration of ACE inhibitors to rats at dosages that had similar hypotensive effects reduced the rate of LPS-induced apoptosis significantly for perindopril (P < 0.001) and nonsignificantly for the other ACE inhibitors. The order of potency of the ACE inhibitors tested was perindopril > ramipril ≫ quinapril = trandolapril = enalapril, with significant differences between perindopril and quinapril (P < 0.01), trandolapril (P < 0.001), and enalapril (P < 0.001). The difference between perindopril and ramipril did not reach significance.ConclusionOur experiments suggest differences between ACE inhibitors in terms of inhibition of endothelial apoptosis in vivo.

Hypertension, Aging, and Myocardial Synthesis of Heat-Shock Protein 72

We determined the temperature-induced synthesis of the 72-kD heat-shock protein (hsp72) in hearts of normotensive and spontaneously hypertensive rats (SHR) subjected to whole-body hyperthermia (42.0 +/- 0.5 degrees C for 15 minutes). The animals were studied at three different ages: young (2 months), adult (6 months), and old (18 months). The hsp72 was determined by Western blot analysis using a monoclonal antibody. The results were calculated densitometrically as a percentage of a commercial standard. Young SHR responded to hyperthermic stress with increased synthesis of hsp72 compared with age-matched normotensive rats (298.8 +/- 70.0% versus 88.3 +/- 25.5%). This trend was maintained in adult rats (118.1 +/- 31.0% versus 54.8 +/- 21.3%) but not in old rats (65.3 +/- 29.4% versus 43.6 +/- 15.1%). Aging caused a reduction of hsp72 expression in response to hyperthermic stress in both SHR (4.6-fold) and normotensive rats (twofold). These data show that hearts of young and adult SHR respond to heat shock with enhanced synthesis of hsp72. This abnormal response, attenuated by aging, is independent of the presence and degree of hypertension or hypertrophy and is potentially linked to the genetic determination of the disease.

Long‐term treatment with ivabradine in post‐myocardial infarcted rats counteracts f‐channel overexpression

BACKGROUND AND PURPOSE Recent clinical data suggest beneficial effects of ivabradine, a specific heart rate (HR)‐lowering drug, in heart failure patients. However, the mechanisms responsible for these effects have not been completely clarified. Thus, we investigated functional/molecular changes in If, the specific target of ivabradine, in the failing atrial and ventricular myocytes where this current is up‐regulated as a consequence of maladaptive remodelling.

Home-Based Versus In-Hospital Cardiac Rehabilitation After Cardiac Surgery: A Nonrandomized Controlled Study

Background Exercise rehabilitation after cardiac surgery has beneficial effects, especially on a long-term basis. Rehabilitative programs with telemedicine plus appropriate technology might satisfy the needs of performing rehabilitation at home. Objective The purpose of this study was to compare exercise capacity after home-based cardiac rehabilitation (HBCR) or in-hospital rehabilitation in patients at low to medium risk for early mortality (EuroSCORE 0–5) following cardiac surgery. Design A quasi-experimental study was conducted. Methods At hospital discharge, patients were given the option to decide whether to enroll in the HBCR program. Clinical examinations (electrocardiography, cardiac echo color Doppler, chest radiography, blood samples) of patients in the HBCR group were collected during 4 weeks of rehabilitation, and exercise capacity (assessed using the Six-Minute Walk Test [6MWT]) was assessed before and after rehabilitation. A group of patients admitted to the in-hospital rehabilitation program was used as a comparison group. Patients in the HBCR group were supervised at home by a medical doctor and telemonitored daily by a nurse and physical therapist by video conference. Periodic home visits by health staff also were performed. Results One hundred patients were recruited into the HBCR group. An equal number of patients was selected for the comparison group. At the end of the 4-week study, the 2 groups showed improvement from their respective baseline values only in the 6MWT. No difference was found in time × group interaction. Limitations Because patients self-selected to enroll in the HBCR program and because they were enrolled from a single clinical center, the results of the study cannot be generalized. Conclusions In patients who self-selected HBCR, the program was found to be effective and comparable to the standard in-hospital rehabilitative approach, indicating that rehabilitation following cardiac surgery can be implemented effectively at home when coadministered with an integrated telemedicine service.