Susana Ravassa

New Targets to Treat the Structural Remodeling of the Myocardium

Classical therapy of heart failure is based on treatment of its pre-disposing/triggering factors and of the neurohumoral activation secondary to the deterioration of cardiac function. A new view is emerging that proposes the direct intervention on the pathological structural remodeling of the myocardium as part of heart failure therapy. In fact, in conditions of chronic injury, the cardiomyocytic and the noncardiomyocytic components of the myocardium undergo a series of structural lesions (i.e., cardiomyocyte growth and death, inflammation, alterations of collagen matrix, and microvascular rarefaction) that are governed by a complex interplay of mechanisms. Our increasing knowledge of the role of these mechanisms in remodeling enables us not only to better understand how our more successful therapies work but also to explore novel therapies for the future. In this paper, we will examine recent insights from experimental and pilot clinical studies that have provided new targets for interventions to prevent or reverse inflammation, alterations of collagen matrix, and cardiomyocyte death.

Cardiomyocyte apoptosis in hypertensive cardiomyopathy

It is widely accepted that there are two principal forms of cell death; namely, necrosis and apoptosis. According to the classical view, necrosis is the major mechanism of cardiomyocyte death in cardiac diseases. However, in the past few years observations have been made showing that cardiomyocyte apoptosis occurs in diverse conditions and that apoptosis may be a contributing cause of the loss and functional abnormalities of cardiomyocytes with important pathophysiological consequences. In this regard, although a number of formal proofs are pending, it is conceivable that cardiomyocyte apoptosis may be an important variable in the clinical evolution of hypertensive cardiomyopathy. This review summarizes recent evidence demonstrating that cardiomyocyte apoptosis is abnormally stimulated in the heart of animals and humans with arterial hypertension. In addition, the potential mechanisms of cardiomyocyte apoptosis in hypertension and its detrimental impact on cardiac function will be addressed. Finally, the perspectives of strategies aimed to detect and modulate apoptosis of cardiomyocytes in hypertensive cardiomyopathy will be considered.

Apoptosis in hypertensive heart disease.

Numerous hypotheses have been considered to explain the fundamental mechanism(s) for the development of systolic dysfunction and heart failure in animals and humans with arterial hypertension. Besides contractile disturbances of cardiomyocytes and interstitial and perivascular fibrosis, cardiomyocyte loss is now being considered as one of the determinants of the maladaptive processes implicated in the transition from compensated to decompensated left ventricular hypertrophy. A number of experimental evidence suggest that exaggerated apoptosis may account for the loss of cardiomyocytes in the hypertensive left ventricle. Furthermore, some factors intrinsic and extrinsic to the cardiomyocyte emerge as potential candidates to trigger apoptosis. The elucidation of the possible interactions between these factors may be of major interest to prevent the progression to heart failure in patients with hypertensive heart disease.

GLP-1 and cardioprotection. From bench to bedside

During myocardial infarction (MI), a variety of mechanisms contribute to the activation of cell death processes in cardiomyocytes, determining the final MI size, subsequent mortality, and post-MI remodelling. The deleterious mechanisms accompanying the ischaemic and reperfusion phases in MI include deprivation of oxygen, nutrients, and survival factors, accumulation of waste products, generation of oxygen free radicals, calcium overload, neutrophil infiltration of the ischaemic area, depletion of energy stores, and opening of the mitochondrial permeability transition pore, all of which contribute to the activation of apoptosis and necrosis in cardiomyocytes. During the last few years, glucagon-like peptide-1 (GLP-1) (7-36)-based therapeutic strategies have been incorporated into the treatment of patients with type 2 diabetes mellitus. Cytoprotection is among the pleiotropic actions described for GLP-1 in different cell types, including cardiomyocytes. This paper reviews the most relevant experimental and clinical studies that have contributed to a better understanding of the molecular mechanisms and intracellular pathways involved in the cardioprotection induced by GLP-1, analysing in depth its potential role as a therapeutic target in the ischaemic and reperfused myocardium as well as in other pathologies that are associated with myocardial remodelling and heart failure.

Biochemical markers of myocardial remodelling in hypertensive heart disease

The intricate mechanisms responsible for the structural remodelling of the myocardium that facilitates the evolution to heart failure in hypertensive patients, namely in those with left ventricular hypertrophy, requires from clinicians the utilization of a multibiomarker approach for short-term and long-term stratification as well as prognostication of patients. Biochemical markers may also help to identify patients with no clinical evidence of hypertensive heart disease, and provide information about the need for more aggressive therapy during different stages of the disease, and potentially provide valuable biochemical data for the specialist. Although there is a continuous and complex interplay between biochemical and imaging markers, perhaps their use will also have the potential to modify the medical management of patients with hypertensive heart disease and therapeutic decision-making by tailoring a targeted therapy according to the predominant mechanism of myocardial remodelling. This article will review in brief the most relevant information on a panel of circulating molecules that may accomplish the criteria required to be considered as biochemical markers of the cardiomyocyte and non-cardiomyocyte structural changes that occur in the hypertensive myocardium.

Antiapoptotic effects of GLP-1 in murine HL-1 cardiomyocytes

Activation of apoptosis contributes to cardiomyocyte dysfunction and death in diabetic cardiomyopathy. The peptide glucagon-like peptide-1 (GLP-1), a hormone that is the basis of emerging therapy for type 2 diabetic patients, has cytoprotective actions in different cellular models. We investigated whether GLP-1 inhibits apoptosis in HL-1 cardiomyocytes stimulated with staurosporine, palmitate, and ceramide. Studies were performed in HL-1 cardiomyocytes. Apoptosis was induced by incubating HL-1 cells with staurosporine (175 nM), palmitate (135 μM), or ceramide (15 μM) for 24 h. In staurosporine-stimulated HL-1 cardiomyocytes, phosphatidylserine exposure, Bax-to-Bcl-2 ratio, Bad phosphorylation (Ser(136)), BNIP3 expression, mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, DNA fragmentation, and mammalian target of rapamycin (mTOR)/p70S6K phosphorylation (Ser(2448) and Thr(389), respectively) were assessed. Apoptotic hallmarks were also measured in the absence or presence of low (5 mM) and high (10 mM) concentrations of glucose. In addition, phosphatidylserine exposure and DNA fragmentation were analyzed in palmitate- and ceramide-stimulated cells. Staurosporine increased apoptosis in HL-1 cardiomyocytes. GLP-1 (100 nM) partially inhibited staurosporine-induced mitochondrial membrane depolarization and completely blocked the rest of the staurosporine-induced apoptotic changes. This cytoprotective effect was mainly mediated by phosphatidylinositol 3-kinase (PI3K) and partially dependent on ERK1/2. Increasing concentrations of glucose did not influence GLP-1-induced protection against staurosporine. Furthermore, GLP-1 inhibited palmitate- and ceramide-induced phosphatidylserine exposure and DNA fragmentation. Incretin GLP-1 protects HL-1 cardiomyocytes against activation of apoptosis. This cytoprotective ability is mediated mainly by the PI3K pathway and partially by the ERK1/2 pathway and seems to be glucose independent. It is proposed that therapies based on GLP-1 may contribute to prevent cardiomyocyte apoptosis.

Mechanisms of increased susceptibility to angiotensin II-induced apoptosis in ventricular cardiomyocytes of spontaneously hypertensive rats.

Previous findings have shown that hypotensive doses of losartan prevent the excess of apoptosis present in the hypertrophied left ventricle of adult spontaneously hypertensive rats (SHR). This study was designed to determine whether angiotensin II facilitates apoptosis in cardiomyocytes of adult SHR. Primary cultures of ventricular cardiomyocytes from 30-week-old normotensive Wistar-Kyoto rats (WKY) and SHR with left ventricular hypertrophy were exposed to 10−9 mol/L angiotensin II for 24 hours. Apoptotic cells were assessed by terminal deoxynucleotidyl transferase assay and confirmed by Annexin V detection. The expression of Bax-&agr;, Bcl-2, p53 , and caspase-3 proteins was assessed by Western blot assays. The expression of BAX gene was assessed by Northern blot. Angiotensin II increased (P <0.01) cardiomyocyte apoptosis, and this effect was higher (P <0.001) in SHR cells than in WKY cells. Whereas losartan (10−7 mol/L) blocked the apoptotic effect of the octapeptide in cells from the two strains of rats, PD123319 (10−7 mol/L) inhibited angiotensin II–mediated apoptosis only in SHR cells. Angiotensin II stimulated (P <0.01) Bax-&agr; protein, and this effect was higher (P <0.01) in SHR cells than in WKY cells. Angiotensin II did not modify Bcl-2, p53, and BAX mRNA in cells from the two strains of rats. Angiotensin II induced a similar increase (P <0.05) in the ratio caspase-3/procaspase-3 (an index of caspase-3 activation) in cardiomyocytes from the two strains of rats. The present in vitro results indicate that SHR cardiomyocytes exhibit enhanced susceptibility to angiotensin II–induced apoptosis. Ligand binding to angiotensin II type 1 and type 2 receptors leading to changes in posttranscriptional processing of Bax-&agr; and accumulation of this proapoptotic protein may be involved in the abnormal response of SHR cardiomyocytes. These data support a role for angiotensin II in apoptosis observed in the left ventricle of these rats.

Osteopontin-mediated myocardial fibrosis in heart failure: a role for lysyl oxidase?

AIMS We investigated whether the pro-fibrotic matricellular protein osteopontin (OPN) is associated with the enzymes involved in the extracellular synthesis of fibril-forming collagen type I (i.e. procollagen C-proteinase, PCP) and its cross-linking to form insoluble fibrils (i.e. lysyl oxidase, LOX) in heart failure (HF) of hypertensive origin. METHODS AND RESULTS OPN, PCP, and LOX were assessed by histochemical and molecular methods in the myocardium of 21 patients with hypertensive heart disease (HHD) and HF. Whereas the myocardial expression of OPN was very scarce in control hearts (n = 10), it was highly expressed in HF patients (P < 0.0001). OPN was directly correlated with LOX (r = 0.460, P = 0.041), insoluble collagen (r = 0.534, P = 0.015), pulmonary capillary wedge pressure (r = 0.558; P = 0.009), and left-ventricular (LV) chamber stiffness (r = 0.458, P = 0.037), and inversely correlated with LV ejection fraction (r = -0.513, P = 0.017) in all patients. OPN did not correlate with PCP and other parameters assessing collagen synthesis by fibroblasts or degradation by matrix metalloproteinases. In vitro studies showed that OPN significantly (P < 0.05) increases the expression and activity of LOX in human cardiac and dermal fibroblasts. CONCLUSION An excess of OPN is associated with increased LOX and insoluble collagen, as well as with LV stiffness and systolic dysfunction in patients with HHD and HF. In addition, OPN up-regulates LOX in human fibroblasts. It is suggested that the OPN-LOX axis might facilitate the formation of insoluble collagen (i.e. stiff and resistant to degradation) and the subsequent alteration in LV mechanical properties and function in patients with HHD and HF.

Association of depressed cardiac gp130-mediated antiapoptotic pathways with stimulated cardiomyocyte apoptosis in hypertensive patients with heart failure

Objective To investigate whether the glycoprotein (gp130)-mediated survival pathway, which protects cardiomyocytes from apoptosis, is depressed in left ventricular hypertrophy hypertensive patients with chronic heart failure. Methods Transvenous endomyocardial biopsies were obtained in 52 hypertensive patients with left ventricular hypertrophy: 28 without heart failure and 24 with heart failure. gp130 and gp130-dependent antiapoptotic pathways p42/44 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) as well as gp130 agonist cardiotrophin-1 were analyzed by reverse transcriptase-polymerase chain reaction and western blot. Apoptosis was assessed by DNA end-labeling (TUNEL), caspase-3 immunostaining and caspase substrate poly(ADP-ribose) polymerase cleavage. Results gp130 protein expression (P < 0.05) and p42/44 MAPK and PI3K/Akt activation (P < 0.01) were decreased in heart-failure hypertensive patients compared with nonheart-failure hypertensive individuals. No changes in gp130 mRNA expression were found between the two groups. Cardiotrophin-1 was increased (P < 0.05) at both the mRNA and protein levels in heart-failure hypertensive individuals compared with nonheart-failure hypertensive individuals. Cardiomyocyte apoptosis was increased (P < 0.01) in heart-failure hypertensive individuals compared with nonheart-failure hypertensive individuals. Inverse correlations (P < 0.01) occurred between cardiomyocyte apoptosis and p42/44 MAPK and PI3K/Akt activation in all hypertensive patients. Cardiotrophin-1 correlated inversely (r = −0.554, P < 0.05) with gp130 in all hypertensive individuals. In cultured HL-1 cardiomyocytes, cardiotrophin-1 decreased (P < 0.05) the gp130:phosphorylated gp130 (at Ser782) ratio and increased (P < 0.05) gp130ubiquitination. Conclusions An association exists between depression of the gp130 cytoprotective pathway and stimulation of cardiomyocyte apoptosis in hypertensive patients that develop heart failure. Whether the excess of cardiotrophin-1 induces ligand-induced receptor down-regulation in these patients requires further study.

Torasemide Inhibits Angiotensin II–Induced Vasoconstriction and Intracellular Calcium Increase in the Aorta of Spontaneously Hypertensive Rats

Torasemide is a loop diuretic that is effective at low once-daily doses in the treatment of arterial hypertension. Because its antihypertensive mechanism of action may not be based entirely on the elimination of salt and water from the body, a vasodilator effect of this drug can be considered. In the present study, the ability of different concentrations of torasemide to modify angiotensin II (Ang II)-induced vascular responses was examined, with the use of an organ bath system, in endothelium-denuded aortic rings from spontaneously hypertensive rats. Ang II-induced increases of intracellular free calcium concentration ([Ca(2+)](i)) were also examined by image analysis in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. A dose-response curve to Ang II was plotted for cumulative concentrations (from 10(-9) to 10(-6) mol/L) in endothelium-denuded aortic rings (pD(2)=7.5+/-0.3). Isometric contraction induced by a submaximal concentration of Ang II (10(-7) mol/L) was reduced in a dose-dependent way by torasemide (IC(50)=0.5+/-0.04 micromol/L). Incubation of VSMCs with different concentrations of Ang II (from 10(-10) to 10(-6) mol/L) resulted in a dose-dependent rise of [Ca(2+)](i) (pD(2)=7.5+/-0.3). The stimulatory effect of [Ca(2+)](i) induced by a submaximal concentration of Ang II (10(-7) mol/L) was blocked by torasemide (IC(50)=0.5+/-0.3 nmol/L). Our findings suggest that torasemide blocks the vasoconstrictor action of Ang II in vitro. This action can be related to the ability of torasemide to block the increase of [Ca(2+)](i) induced by Ang II in VSMCs. It is proposed that these actions might be involved in the antihypertensive effect of torasemide observed in vivo.