Germán E. González

Histopathologic time course of myocardial infarct in rabbit hearts

INTRODUCTION The histopathologic evolution of myocardial infarct and of remote zones in rabbit hearts was studied. METHODS The left coronary artery of 55 rabbits was ligated and rabbits were sacrificed at 2, 4, 6, 8, 12, 14, 16, 18, 26, 35 and 56 days post-ligature (n=5 per group). Two rabbits were used as control and four were sham-operated. The hearts were excised, cut in slices and stained with hematoxylin-eosin, Massons trichrome and picrosirius red. The histological evaluation was semiquantitative (scale: 0 to ++). RESULTS At day 2, the presence of neutrophils was ++, decreasing suddenly at day 4 and disappearing completely at day 6. The proliferation of cells with features of fibroblasts increased from days 4 to 14 post-occlusion. Coagulation necrosis in mid-myocardium during the first week was ++. Subendocardial myocytolysis was evident from day 2 up to day 56 post-infarction. During the second week, proliferation of lymphocytes and macrophages (++), granulation tissue formation (++) and incipient traces of fibrosis that peaked at day 35 were observed. Scarring was complete at day 56 (++). In remote zones (right ventricle and septum), the proliferation of cells+ on Vimentin was observed at day 2, and perivascular, interstitial and endocardial fibrosis started to increase at day 6 and peaked at day 16. CONCLUSION Although myocardial infarction in rabbits maintains the essence of the infarct chronology, some differences as the early presence of cells+ on Vimentin and subendocardial fibrosis in infarcted areas, and also the rapid increase and early disappearance of neutrophils appear when other species are considered. An interesting finding was the early proliferation of cells with features of fibroblasts in remote zones.

Deletion of interleukin-6 prevents cardiac inflammation, fibrosis and dysfunction without affecting blood pressure in angiotensin II-high salt-induced hypertension.

Objective: Inflammation has been proposed as a key component in the development of hypertension and cardiac remodeling associated with different cardiovascular diseases. However, the role of the proinflammatory cytokine interleukin-6 in the chronic stage of hypertension is not well defined. Here, we tested the hypothesis that deletion of interleukin-6 protects against the development of hypertension, cardiac inflammation, fibrosis, remodeling and dysfunction induced by high salt diet and angiotensin II (Ang II). Methods: Male C57BL/6J and interleukin-6-knock out (KO) mice were implanted with telemetry devices for blood pressure (BP) measurements, fed a 4% NaCl diet, and infused with either vehicle or Ang II (90 ng/min per mouse subcutaneously) for 8 weeks. We studied BP and cardiac function by echocardiography at baseline, 4 and 8 weeks. Results: Myocyte cross-sectional area (MCSA), macrophage infiltration, and myocardial fibrosis were also assessed. BP increased similarly in both strains when treated with Ang II and high salt (Ang II-high salt); however, C57BL/6J mice developed a more severe decrease in left ventricle ejection fraction, fibrosis, and macrophage infiltration compared with interleukin-6-KO mice. No differences between strains were observed in MCSA, capillary density and MCSA to capillary density ratio. Conclusion: In conclusion, absence of interleukin -6 did not alter the development of Ang II-high salt-induced hypertension and cardiac hypertrophy, but it prevented the development of cardiac dysfunction, myocardial inflammation, and fibrosis. This indicates that interleukin-6 plays an important role in hypertensive heart damage but not in the development of hypertension.

Galectin-1 Controls Cardiac Inflammation and Ventricular Remodeling during Acute Myocardial Infarction

Galectin-1 (Gal-1), an evolutionarily conserved β-galactoside-binding lectin, plays essential roles in the control of inflammation and neovascularization. Although identified as a major component of the contractile apparatus of cardiomyocytes, the potential role of Gal-1 in modulating heart pathophysiology is uncertain. Here, we aimed to characterize Gal-1 expression and function in the infarcted heart. Expression of Gal-1 was substantially increased in the mouse heart 7 days after acute myocardial infarction (AMI) and in hearts from patients with end-stage chronic heart failure. This lectin was localized mainly in cardiomyocytes and inflammatory infiltrates in peri-infarct areas, but not in remote areas. Both simulated hypoxia and proinflammatory cytokines selectively up-regulated Gal-1 expression in mouse cardiomyocytes, whereas anti-inflammatory cytokines inhibited expression of this lectin or had no considerable effect. Compared with their wild-type counterpart, Gal-1-deficient (Lgals1(-/-)) mice showed enhanced cardiac inflammation, characterized by increased numbers of macrophages, natural killer cells, and total T cells, but reduced frequency of regulatory T cells, leading to impaired cardiac function at baseline and impaired ventricular remodeling 7 days after nonreperfused AMI. Treatment of mice with recombinant Gal-1 attenuated cardiac damage in reperfused AMI. Taken together, our results indicate a protective role for Gal-1 in normal cardiac homeostasis and postinfarction remodeling by preventing cardiac inflammation. Thus, Gal-1 treatment represents a potential novel strategy to attenuate heart failure in AMI.

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.

Angiotensin II‐induced dilated cardiomyopathy in Balb/c but not C57BL/6J mice

Balb/c mice, which are T‐helper lymphocyte 2 (Th2) responders, are highly susceptible to infectious and non‐infectious heart diseases, whereas C57BL/6 mice (Th1 responders) are not. Angiotensin II (Ang II) is not only a vasopressor but also a pro‐inflammatory factor that leads to cardiac hypertrophy, fibrosis and dysfunction. We hypothesized that Ang II exacerbates cardiac damage in Balb/c but not in C57BL/6 mice even though both strains have a similar level of hypertension. Twelve‐week‐old male C57BL/6J and Balb/c mice received either vehicle or Ang II (1.4 mg kg−1 day−1, s.c. via osmotic minipump) for 8 weeks. At baseline, Balb/c mice exhibited the following: (1) a lower heart rate; (2) an enlarged left ventricular chamber; (3) a lower ejection fraction and shortening fraction; and (4) twice the left ventricular collagen deposition of age‐matched C57BL/6J mice. Angiotensin II raised systolic blood pressure (to ∼150 mmHg) and induced cardiomyocyte hypertrophy in a similar manner in both strains. While C57BL/6J mice developed compensatory concentric hypertrophy and fibrosis in response to Ang II, Balb/c mice demonstrated severe left ventricular chamber dilatation, wall thinning and fibrosis, leading to congestive heart failure as evidenced by dramatically decreased ejection fraction and lung congestion (significant increase in lung weight), which are both characteristic of dilated cardiomyopathy. Our study suggests that the Th phenotype plays an active role in cardiac remodelling and function both in basal conditions and in hypertension. Angiotensin II‐induced dilated cardiomyopathy in Balb/c mice is an ideal animal model for studying the impact of the adaptive immune system on cardiac remodelling and function and for testing strategies to prevent or treat hypertension‐associated heart failure.

Galectin-3 is essential for early wound healing and ventricular remodeling after myocardial infarction in mice☆☆☆

Fil: Gonzalez, German Esteban. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiopatologia Cardiovascular; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Bioquimica y Medicina Molecular; Argentina

Treatment with N-acetyl-seryl-aspartyl-lysyl-proline prevents experimental autoimmune myocarditis in rats

Myocarditis is commonly associated with cardiotropic infections and has been linked to development of autoimmunity. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases; however, its effect on autoimmune-mediated cardiac diseases remains unknown. We studied the effects of Ac-SDKP in experimental autoimmune myocarditis (EAM), a model of T cell-mediated autoimmune disease. This study was conducted to test the hypothesis that Ac-SDKP prevents autoimmune myocardial injury by modulating the immune responses. Lewis rats were immunized with porcine cardiac myosin and treated with Ac-SDKP or vehicle. In EAM, Ac-SDKP prevented both systolic and diastolic cardiac dysfunction, remodeling as shown by hypertrophy and fibrosis, and cell-mediated immune responses without affecting myosin-specific autoantibodies or antigen-specific T cell responses. In addition, Ac-SDKP reduced cardiac infiltration by macrophages, dendritic cells, and T cells, pro-inflammatory cytokines [interleukin (IL)-1α, tumor necrosis factor-α, IL-2, IL-17] and chemokines (cytokine-induced neutrophil chemoattractant-1, interferon-γ-induced protein 10), cell adhesion molecules (intercellular adhesion molecule-1, L-selectin), and matrix metalloproteinases (MMP). Ac-SDKP prevents autoimmune cardiac dysfunction and remodeling without reducing the production of autoantibodies or T cell responses to cardiac myosin. The protective effects of Ac-SDKP in autoimmune myocardial injury are most likely mediated by inhibition of 1) innate and adaptive immune cell infiltration and 2) expression of proinflammatory mediators such as cytokines, chemokines, adhesion molecules, and MMPs.

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.

Ludwig: The Bioengineer [Retrospectroscope]

On the basis of the strict exclusion of the vis vitalis, the demand was raised by Carl Ludwig, Helmholtz, Du Bois-Reymond, and Brucke for a physiology which was causal-analytical and physically and chemically experimental. If, out of these four investigators, we pick Ludwig as the actual founder of modern physiology, the grounds for this must be justified specifically. That modern physiology is not to be contemplated without the works of the three great students of Johannes Muller is explicitly emphasized. However, Carl Ludwig occupies a special position for physiology. [1]