Evasio Pasini

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.

The protective role of heat stress in the ischaemic and reperfused rabbit myocardium

Cells subjected to increases in temperature induce the expression of several proteins known as heat shock or stress proteins. This process enhances the cells ability to overcome the effects of further stress. In this respect, the effects of heat stress have been reported to protect the hearts of rats following ischaemia and reperfusion. We have confirmed and extended this observation, not only using different indices of myocardial injury but also in another species, namely the rabbit. Animals were anaesthetized and the body temperature raised to 42 degrees C for a 15-min period. Controls were treated in the same way but without heating. Twenty-four hours later the rabbits were re-anaesthetized and the hearts removed for either heat stress protein analysis or perfusion with Krebs buffer using an isolated perfused heart apparatus. Hearts were subjected to 60 min of low flow (1 ml/min) ischaemia followed by 30 min of reperfusion. All hearts subjected to heat stress showed an enhanced recovery of function upon reperfusion as measured by improvements in developed pressure (27.3 +/- 3.6 vs 16.3 +/- 3.0 mmHg) and diastolic pressure (37.3 +/- 7.4 vs 54.7 +/- 3.1 mmHg). In addition, creatine kinase release, associated with reperfusion, was significantly reduced in the heat-stressed hearts (532 +/- 102 vs 1138 +/- 73 mU/min/g wet wt). Myocardial accumulation and release of oxidized glutathione, an index of oxidative stress, was significantly reduced in the heat-stressed group (0.003 +/- 0.003 vs 0.376 +/- 0.113 nmol/min/g wet wt). The improved metabolic status of the reperfused heat-stressed hearts was further demonstrated by a significant conservation in the levels of ATP (6.1 +/- 0.9 vs 2.8 +/- 0.8 mumol/g dry wt) and CP (36.9 +/- 6.4 vs 16.4 +/- 5.1 mumol/g dry wt). Finally, isolated mitochondrial function in terms of respiratory control index (RCI) was maintained in the heat-stressed hearts (9.2 +/- 0.9 vs 5.7 +/- 0.2) and overloading with calcium was reduced. These data extend the hypothesis that heat stress protects the heart following ischaemia and reperfusion in this in vitro model, in a way as yet undetermined.

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.

Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis

We have previously demonstrated that STAT‐1 plays a critical role in promoting apoptotic cell death in cardiac myocytes following ischemia/reperfusion (I/R) injury. Epigallocatechin‐3‐gallate (EGCG), the major constituent of green tea, has recently been reported to inhibit STAT‐1 activity in noncardiac cells. In the present study, we have assessed the protective effects of EGCG and green tea extract (GTE) infusion on both cultures of cardiac myocytes and the isolated rat heart. EGCG reduced STAT‐1 phosphorylation and protected cardiac myocytes against I/R‐induced apoptotic cell death. Moreover, EGCG reduced the expression of a known STAT‐1 pro‐apoptotic target gene, Fas receptor. More interestingly, oral administration of GTE as well as EGCG infusion limited the extent of infarct size and attenuated the magnitude of myocyte apoptosis in the isolated rat heart exposed to I/R injury. This reduction cell death was associated with improved hemodynamic recovery and ventricular function in the ischemic/reperfused rat heart. This is the first report to show that consumption of green tea is able to mediate cardioprotection and enhance cardiac function during I/R injury. Because GTE‐mediated cardioprotection is achieved, at least in part, through inhibition of STAT‐1 activity, we may postulate that a similar action can be implemented in the clinical setting to minimize STAT‐1 activation levels in patients with acute coronary artery disease (CAD).

Is nutritional intake adequate in chronic heart failure patients

OBJECTIVES The goal of this study was to investigate the nutrition adequacy and energy availability for physical activity in free-living, clinically stable patients with chronic heart failure (CHF). BACKGROUND Little information exists regarding the nutrition adequacy and alimentary habits of patients with clinically stable CHF. We hypothesized that CHF patients have an inadequate intake of calories and protein, leading to a negative calorie and nitrogen balance, an expression of increased tissue breakdown. METHODS In 57 non-obese patients with CHF (52 males and 5 females; 52 +/- 3 years; body mass index <25 kg/m(2)) and in 49 healthy subjects (39 males and 10 females) matched for age, body mass index, and sedentary life style we evaluated total energy expenditure (TEE), calorie intake (kcal(I)), and nitrogen intake (N(I)) from a seven-day food diary, total nitrogen excretion (TNE), and energy availability (EA = kcal(I) - resting energy expenditure). A zero calorie balance (CB) occurred when kcal(I) = TEE; a nitrogen balance (NB) in equilibrium was set at NB (= N(I) - TNE) 0 +/- 1 g/day. RESULTS In patients and controls kcal(I) and N(I) were similar. However, in CHF patients the kcal(I) was <TEE with a consequent negative CB (-186 +/- 305 kcal/day vs. + 104.2 +/- 273 kcal/day of controls; p < 0.01). Nitrogen balance resulted negative in CHF (-1.7 +/- 3.2 g/24 h vs. + 2.2 +/- 3.6 g/24 h in controls; p < 0.01). Energy availability in CHF patients was 41% lower than in controls (p < 0.05). CONCLUSIONS Non-obese, free-living patients with clinically stable CHF have an inadequate intake of calories and protein and reduced energy availability for physical activity.

Micronutrient deficiencies: an unmet need in heart failure.

Heart failure (HF) is a common, disabling, and costly disease. Despite major advances in medical therapy, morbidity and mortality remain high, in part because current pharmacological regimens may not fully address some unique requirements of the heart for energy. The heart requires a continuous supply of energy-providing substrates and amino acids in order to maintain its function. In HF, defects in substrate metabolism and cardiac energy and substrate utilization may contribute to contractile dysfunction. HF is often accompanied by a deficiency in key micronutrients required for unimpeded energy transfer. Correcting these deficits has been proposed as a method to limit or even reverse the progressive myocyte dysfunction and/or necrosis in HF. This review summarizes the existing HF literature with respect to supplementation trials of key micronutrients involved in cardiac metabolism: coenzyme Q10, l-carnitine, thiamine, and amino acids, including taurine. Studies using a broader approach to supplementation are also considered. Although some of the results are promising, none are conclusive. There is a need for a prospective trial to examine the effects of micronutrient supplementation on morbidity and mortality in patients with HF.

Acute and Chronic Effects of Propionyl-L-Carnitine on the Hemodynamics, Exercise Capacity, and Hormones in Patients with Congestive Heart Failure

Carnitine is an important cofactor in the intermediary metabolism of the heart, and carnitine deficiency is associated with congestive heart failure. We therefore studied the effects of acute (IV bolus, 30 mg/kg body weight) and chronic administration (1.5 mg/d for 1 month) of propionyl-L-carnitine on hemodynamics, hormone levels, ventricular function, exercise capacity, and peak oxygen consumption in 30 patients with chronic congestive heart failure (NYHA II–III, mean EF 29.5 ± 7%) in a phase II, parallel, single-blind, randomized, and placebo-controlled study. Acute administration of propionyl-L-carnitine caused a significant reduction in pulmonary artery and pulmonary wedge pressures at both day 1 (P < 0.001) and day 30 (P < 0.05) of the study but no other hemodynamics changes. Hormone levels did not change following acute administration of the drug. Chronic administration of propionyl-L-carnitine increased peak oxygen consumption by 45% (from 16.0 ± 3 to 23.5 ± 2 mL/kg/min, P ± 0.001), exercise time by 21% (from 8.1 ± 0.5 to 9.8 ± 0.4 minutes, P < 0.01), and peak exercise heart rate by 12% (P < 0.01). These changes were concomitant with a reduction of pulmonary artery pressure. In the treated group, there was a slight, but significant (P < 0.01), reduction in left ventricular dimensions. Hemodynamics and hormones measured after 1 month of oral therapy remained unchanged, except for a fall in pulmonary artery pressures, with a nonsignificant trend towards a fall in filling pressures and plasma norepinephrine. The chronic changes in the propionyl-L-carnitine group were seen at 15 days of treatment, and no further changes in these parameters were seen at 1 month. We conclude that propionyl-L-carnitine increases exercise capacity and reduces ventricular size in patients with congestive heart failure. The drug has no significant effects on hemodynamics or neurohormone levels. The use of a single-blind design reduces the impact of the positive finding on exercise capacity.

Metabolic Adaptation During a Sequence of No-Flow and Low-Flow Ischemia A Possible Trigger for Hibernation

BACKGROUND Myocardial hibernation is an adaptive phenomenon occurring in patients with a history of acute ischemia followed by prolonged hypoperfusion. METHODS AND RESULTS We investigated, in isolated rabbit heart, whether a brief episode of global ischemia followed by hypoperfusion maintains viability. Four groups were studied; group 1,300 minutes of aerobia; group 2,240 minutes of total ischemia and 60 minutes of reperfusion; group 3, 10 minutes of total ischemia, 230 minutes of hypoperfusion (90% coronary flow reduction), and 60 minutes of reperfusion; and group 4, 240 minutes of hypoperfusion followed by reperfusion. In group 3, viability was maintained. Ten minutes of ischemia caused quiescence, a fall in interstitial pH (from 7.2 +/- 0.01 to 6.1 +/- 0.8), creatine phosphate (CP), and ATP (from 54.5 +/- 5.0 and 25.0 +/- 1.9 to 5.0 +/- 1.1 and 15.3 +/- 2.5 mumol/g dry wt, P < .01). Subsequent hypoperfusion failed to restore contraction and pH but improved CP (from 5.0 +/- 1.1 to 20.1 +/- 3.4, P < .01). Reperfusion restored pH, developed pressure (to 92.3%), and NAD/NADH and caused a washout of lactate and creatine phosphokinase with no alterations of mitochondrial function or oxidative stress. In group 4, hypoperfusion resulted in progressive damage. pH fell to 6.2 +/- 0.7, diastolic pressure increased to 34 +/- 5.6 mm Hg, CP and ATP became depressed, and oxidative stress occurred. Reperfusion partially restored cardiac metabolism and function (47%). CONCLUSIONS A brief episode of total ischemia without intermittent reperfusion maintains viability despite prolonged hypoperfusion. This could be mediated by metabolic adaptation, preconditioning, or both.

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.

Adequate energy-protein intake is not enough to improve nutritional and metabolic status in muscle-depleted patients with chronic heart failure

An adequate energy‐protein intake (EPI) when combined with amino acid supplementation may have a positive impact nutritional and metabolic status in patients with chronic heart failure (CHF).