Ricardo J. Gelpi

Ischemic postconditioning reduces infarct size by activation of A1 receptors and K+ATP channels in both normal and hypercholesterolemic rabbits

The effect of ischemic postconditioning (Postcon) in hypercholesterolemic animals is unknown. The objectives were to determine if ischemic preconditioning (IPC) and Postcon reduce infarct size in hypercholesterolemic animals and to assess if A1 receptors and K+ATP channels are involved in Postcon mechanisms. Isolated rabbit hearts were perfused according to the Langendorff technique and subjected to 30 minutes of ischemia and 30 minutes of reperfusion (G1). In Group 2, IPC was performed (1 cycle of 5 minutes ischemia/reperfusion) before 30 minutes of ischemia. In Group 3 (G3), Postcon was performed (2 cycles of 30-second reperfusion/ischemia) after 30 minutes of ischemia. The G3 protocol was repeated in G4 and G5, but during Postcon, an A1 receptor blocker (DPCPX, 200 nM) and glybenclamide (K+ATP, blocker, 0.3 μM) were administered, respectively. The G1 to G5 protocols were repeated in animals fed with an enriched cholesterol diet (1%) for 4 weeks (G6 to G10). The infarct size was measured by triphenyltetrazolium. The infarct size was 16.6 ± 4.6% in G1 and 25.8 ± 7.3% in G6, and IPC and Postcon reduced the infarct area in both normal and hypercholesterolemic animals (G2: 5.1 ± 1.7% [P < 0.05] and G3: 5.4 ± 0.9% [P < 0.05] in normal animals; G7: 4.1 ± 1.6% [P < 0.05] and G8 4.8 ± 0.9% [P < 0.05], in hypercholesterolemic animals). Both DPCPX and glybenclamide abolished the effect reached by Postcon. Thus, Postcon reduces infarct size in normal and hypercholesterolemic animals through the activation of A1 and K+ATP channels.

Effect of early versus late AT1 receptor blockade with losartan on postmyocardial infarction ventricular remodeling in rabbits

To characterize the temporal activation of the renin-angiotensin system after myocardial infarction (MI) in rabbits, we examined cardiac ANG II type 1 receptor (AT(1)R) expression and ANG II levels from 3 h to 35 days. The effects of losartan (12.5 mg.kg(-1).day(-1)) on functional and histomorphometric parameters when treatment was initiated early (3 h) and late (day 15) post-MI and maintained for different periods of time [short term (4 days), midterm (20 days), and long term (35 days)] were also studied. AT(1)R expression increased in the MI zone at 15 and 35 days (P < 0.05). ANG II levels increased (P < 0.05) in the non-MI zone at 24 h and in the MI zone as well as in plasma at 4 days and then progressively decreased until 35 days. The survival rate was significantly lower in untreated MI and early long-term-treated animals. Diastolic pressure-volume curves in MI at 35 and 56 days shifted to the right (P < 0.05). This shift was even more pronounced in long-term-treated groups (P < 0.05). Contractility decreased (P < 0.05 vs. sham) in the untreated and long-term-treated groups and was attenuated in the midterm-treated group. The early administration of losartan reduced RAM 11-positive macrophages from 4.15 +/- 0.05 to 3.05 +/- 0.02 cells/high-power field (HPF; P < 0.05) and CD45 RO-positive lymphocytes from 2.23 +/- 0.05 to 1.48 +/- 0.01 cells/HPF (P < 0.05) in the MI zone at 4 days. Long-term treatment reduced the scar collagen (MI: 70.50 +/- 2.35% and MI + losartan: 57.50 +/- 2.48, P < 0.05), determined the persistency of RAM 11-positive macrophages (3.02 +/- 0.13 cells/HPF) and CD45 RO-positive lymphocytes (2.77 +/- 0.58 cells/HPF, P < 0.05 vs. MI), and reduced the scar thinning ratio at 35 days (P < 0.05). Consequently, the temporal expressions of cardiac AT(1)R and ANG II post-MI in rabbits are different from those described in other species. Long-term treatment unfavorably modified post-MI remodeling, whereas midterm treatment attenuated this harmful effect. The delay in wound healing (early reduction and late persistency of inflammatory infiltrate) and adverse remodeling observed in long-term-treated animals might explain the unfavorable effect observed in rabbits.

Rosuvastatin Given During Reperfusion Decreases Infarct Size and Inhibits Matrix Metalloproteinase-2 Activity in Normocholesterolemic and Hypercholesterolemic Rabbits

There is evidence that statin treatment before ischemia protects myocardium from ischemia/reperfusion injury. The objective is to determine whether rosuvastatin administered during reperfusion modifies infarct size and the recovery of postischemic ventricular dysfunction in normocholesterolemic and hypercholesterolemic rabbits. In addition, we also evaluated the role of matrix metalloproteinase type 2 (MMP)-2 activation. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion. In group 2, we added rosuvastatin after 30 minutes of ischemia and from the beginning of reperfusion. In group 3, an MMP inhibitor (doxycycline) was administered during the first 2 minutes of reperfusion. Finally, we repeated these groups but in hypercholesterolemic rabbits (groups 4, 5, and 6). The infarct size was 16.6% ± 3.9% in group 1 and 25.6% ± 2.7% in group 4. Rosuvastatin reduced infarct size to 4.5% ± 1.1% and 6.1% ± 1.5% in groups 2 and 5, respectively (P < 0.05). Rosuvastatin significantly decreased MMP-2 activity during reperfusion, and doxycycline induced an inhibition of MMP-2 activity and a reduction of infarct size in normocholesterolemic (4.9% ± 0.9%) and hypercholesterolemic animals (8.3% ± 1.6%) (P < 0.05). Rosuvastatin reduces infarct size and attenuates MMP-2 activity. These data and the correlation between MMP-2 and infarct size suggest that MMP-2 plays an important role in the mechanisms of cardioprotection afforded by rosuvastatin.

Dissociation between myocardial relaxation and diastolic stiffness in the stunned heart: its prevention by ischemic preconditioning

The effects of myocardial stunning and ischemic preconditioning on left-ventricular developed pressure and end-diastolic pressure (diastolic stiffness) as well as on coronary-perfusion pressure were examined in isolated isovolumic rabbit hearts. The isovolumic relaxation was evaluated, and the time constant of pressure decay during the isovolumic period was calculated. Our experimental protocol comprised: 1) myocardial stunning-global ischemia (15 min) followed by reperfusion (30 min); 2) myocardial stunning-global ischemia (20 min) followed by reperfusion (30 min); and 3) ischemic preconditioning — a single cycle of brief global ischemia and reperfusion (5 min each), before a second ischemic period, of 20-min duration. There was no effect upon systolic and diastolic parameters when 15 and 20 minutes of ischemia were evaluated. In both stunned groups the left ventricular developed pressure first recovered to near control values, but then stabilized at only 60% of the control values. Whereas the isovolumic relaxation time constant was increased after 5 min of reperfusion, and return to control values at late reperfusion, the end diastolic pressure remained elevated during the entire period. Values of dP/dV calculated at common pressure levels, were used as a second index of diastolic stiffness. They were increased after stunning, as also was the coronary perfusion pressure. When the heart was preconditioned with a single episode of ischemia, the systolic and diastolic alterations were completely abolished. We thus concluded that diastolic abnormalities incurred by myocardial stunning consist in both an increase in diastolic stiffness and an early impairment of isovolumic relaxation. The increase in stiffness cannot result from incomplete relaxation since these two parameters become temporally dissociated during the reperfusion period.

Is stunning prevented by ischemic preconditioning

In a model of global ischemia in the isolated perfused rat heart, a 20 min ischemic period followed by 30 min of reperfusion induces a decrease in isovolumic developed pressure (LVDP) and +dP/dtmax to 61 ± 6% and 61 ± 7% of baseline, respectively. Left ventricular end-diastolic pressure (LVEDP) increases to 36 ± 4 mmHg at the end of the reperfusion period. No significant necrotic area as assessed by triphenyltetrazolium chloride (TTC) was detected at the end of the reperfusion period. By an immunohistochemical method using antiactin monoclonal antibodies 10.8 ± 1.9% of unstained cells were detected in the stunned hearts and 10.3 ± 1.2% in control hearts. Preceding the ischemic episode with a cycle of 5 min of ischemia followed by 10 min of reperfusion (ischemic preconditioning) protected contractile function. LVDP and +dP/dtmax now stabilized at 89 ± 5% and 94 ± 5% of baseline respectively. LVEDP was 20 ± 2 mmHg at the end of the reperfusion period. The protection of contractile dysfunction after 20 min of ischemia was achieved also by early reperfusion of low Ca2+-low pH perfusate. With this intervention LVDP stabilized at 87 ± 5% of baseline. LVEDP was 12 ± 2 mmHg at the end of the reperfusion period. A positive inotropic intervention induced by a modified postextrasystolic potentiation protocol at the end of the reperfusion period increases LVDP to levels higher than baseline in the stunned hearts. However, these values were less than those obtained in control hearts. Ischemic preconditioning significantly increased the maximal inotropic response. Therefore, ischemic preconditioning diminishes the contractile dysfunction of early stunning. (Mol Cell Biochem 186: 123-129, 1998)

Atrial natriuretic peptide behaviour and myocyte hypertrophic profile in combined pressure and volume-induced cardiac hypertrophy.

Objective To investigate cardiomyocyte hypertrophy and hormonal profile in cardiac hypertrophy resulting from sequentially applied overloads. Methods We studied Sprague–Dawley rats with renovascular hypertension (RV), where pressure overload predominates, or deoxycorticosterone acetate (DOCA)-salt (DS), where volume overload predominates, at 2 and 4 weeks of treatment, and the combination of both models in inverse sequence: RV 2 weeks/DS 2 weeks (RV2/DS2) and DS 2 weeks/RV 2 weeks (DS2/RV2), and their sham controls (Sh). Results Blood pressure and cardiomyocyte diameter increased to a similar extent in RV and DS at 2 and 4 weeks and in combined models. Cardiomyocyte length increased remarkably in the DS4 group. Circulating atrial natriuretic peptide (ANP) was elevated in all hypertensive groups after 2 and 4 weeks. The RV2/DS2 group showed similar plasma ANP levels to RV4, but DS2/RV2 exhibited a three-fold increase in ANP levels (P < 0.001 versus Sh4, DS2 and DS4). Atrial ANP mRNA remained unchanged in all groups. DS treatment alone or in combination with RV increased left ventricular ANP mRNA, meanwhile only RV treatment increased left ventricular B-type natriuretic peptide (BNP) mRNA. Ventricular ANP expression levels, but not circulating ANP, correlated with both cardiomyocyte diameter (r = 0.859, P < 0.01) and length (r = 0.848, P < 0.01). Renal expression of natriuretic peptide receptor C (NPR-C) was unchanged in RV4 but decreased to a similar extent in the DS4 group and both combined treatments. Conclusion Morphometric patterns seem to be more related to the paracrine function of the heart than to the secretion of ANP and the endocrine function. Plasma ANP in the DS2/RV2 group could indicate a different evolution of the remodelling process. ANP expression seems to be a more sensitive marker for volume than for pressure overload.

Ventricular function and natriuretic peptides in sequentially combined models of hypertension

Hemodynamic parameters and natriuretic peptide levels were evaluated in cardiac hypertrophy produced by sequentially applied renovascular (RV) and deoxycorticosterone acetate-salt (DS) models of hypertension. We studied hypertensive rats by RV or DS treatment at 2 and 4 wk, as well as by the combination of 2 wk of each treatment in an inverse sequence: RV 2 wk/DS 2 wk (RV2/DS2) and DS 2 wk/RV 2 wk (DS2/RV2). The in vivo cardiac function, interstitial fibrosis, and synthesis and secretion of types A (ANP) and B (BNP) natriuretic peptides were monitored in hypertensive models compared with their corresponding sham (Sh2, Sh4). There were no differences in relaxation parameters among RV or DS groups and combined treatments. Left ventricular +dP/dt(max) increased only in RV4 (P < 0.01 vs. Sh4), and this increase was abolished in RV2/DS2. Interstitial collagen concentration increased after 4 wk in both RV4 and RV2/DS2 groups. Although there were no changes in collagen concentration in either DS2 or DS4 groups, clipping after 2 wk of DS (DS2/RV2) remarkably stimulated interstitial fibrosis (P < 0.01 vs. DS2). Plasma BNP increased in RV treatment at 4 wk (P < 0.001 vs. Sh4), but not in DS. Interestingly, RV applied after the 2 wk of DS treatment induced a marked increase in BNP levels (P < 0.001 vs. Sh4). In this regard, plasma BNP appears to be a reliable indicator of pressure overload. Our results suggest that the second stimulus of mechanical overload in combined models of hypertension determines the evolution of hypertrophy and synthesis and secretion of ANP and BNP.

High cholesterol diet effects on ischemia–reperfusion injury of the heart

Ischemic heart disease is the leading cause of morbi-mortality in developed countries. Both ischemia-reperfusion injury and mechanisms of cardioprotection have been studied for more than 50 years. It is known that the physiopathological mechanism of myocardial ischemia involves several factors that are closely related to its development, of which hypercholesterolemia is one of the main ones. Therefore, the objective of this review was to elucidate the effects of a high-cholesterol diet on normal ventricular function and ischemia-reperfusion injury associated phenomenon such as post-ischemic ventricular dysfunction (stunned myocardium). Although there exist many studies considering several aspects of this physiopathological entity, the majority were carried out on normal animals. Thus, experiments carried out on hypercholesterolemic models are controversial, in particular those evaluating different mechanisms of cardioprotection such as ischemic preconditioning and postconditioning, and cardioprotection granted by drugs such as statins, which apart from exerting a lipid-lowering effect, exert pleiotropic effects providing cardioprotection against ischemia-reperfusion injury. These controversial results concerning the mechanisms of cardioprotection vary according to quality, composition, and time of administration of the high-cholesterol diet, as well as the species used in each experiment. Thus, to compare the results it is necessary to take all of these variables into account, since they can change the obtained results.

Apoptosis in severe, compensated pressure overload predominates in nonmyocytes and is related to the hypertrophy but not function

It is widely held that myocyte apoptosis in left ventricular hypertrophy (LVH) contributes to left ventricle (LV) dysfunction and heart failure. The main goal of this investigation was to determine if there is a statistical relationship among LV hypertrophy, apoptosis and LV function, and importantly whether the apoptosis occurs in myocytes or nonmyocytes in the heart. We used both rat and canine models of severe LVH induced by chronic thoracic aortic banding with resultant LV-aortic pressure gradients 145-155 mmHg and increases in LV/body weight of 58 and 70%. These models also provided the ability to examine transmural apoptosis in LVH. In both models, the overwhelming majority (88%) of apoptotic cells were nonmyocytes. The regressions for apoptosis vs. LVH were stronger for nonmyocytes than myocytes and also stronger in the subendocardium than the subepicardium. Importantly, LV systolic and diastolic wall stresses were normal, indicating that the apoptosis could not be attributed to LV stretch or heart failure. In addition, there was no relationship between the extent of apoptosis and LV ejection fraction, which actually increased (P < 0.05), in the face of elevated LV systolic pressure, indicating that greater apoptosis did not result in a decrease in LV function. Thus, in response to chronic, severe pressure overload, LVH in the absence of LV dilation, and elevated LV wall stress, apoptosis occurred predominantly in nonmyocytes in the myocardial interstitium, more in the subendocardium than the subepicardium. The extent of apoptosis was linearly related to the amount of LV hypertrophy, but not to LV function.

Altered Load: An Important Component of Impaired Diastolic Function in Hypertension and Heart Failure

The effects of altered load and contractile state on commonly employed indices of left ventricular (LV) function have been studied extensively in the normal myocardium. However, when these very same indices are applied to the study of diastolic abnormalities in cardiovascular disease states, the influences of load and impaired contractility are often overlooked in favor of the assumption that intrinsic myocardial abnormalitis, not altered load, are the dominant determinants of diastolic dysfunction. Such conclusions are based on the fact that the preponderance of data regarding diastolic dysfunction are derived, on the one hand, from in vitro studies of isolated myocytes, trabeculae, or isolated heart preparations in which loading conditions can be controlled and, on the other hand, from studies in patients where loading conditions were not controlled.