Antonio Lax

Metformin protects against doxorubicin-induced cardiotoxicity: involvement of the adiponectin cardiac system.

Doxorubicin has cardiotoxic effects that limit its clinical benefit in cancer patients. Metformin exerts cardioprotective actions via AMP-activated protein kinase (AMPK) and increases the expression of adiponectin and its receptors (adipoR1 and adipoR2) in skeletal muscle and adipose tissue, but its effect on cardiac tissue is still unknown. This work aimed to study whether metformin exerts any protective action against the cardiotoxicity of doxorubicin and whether the cardiac system of adiponectin is involved in any such action. The addition of doxorubicin (5μM) to adult mouse cardiomyocytes (HL-1 cell line) induced apoptosis, which was characterized by a loss of cell viability, activation of caspases, and fragmentation of the genetic material. Doxorubicin treatment also caused a decrease in the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase. Pretreatment with metformin (4mM, 24h) provided protection against doxorubicin-induced damage. This pretreatment significantly increased cell viability, attenuated the activation of caspases and the fragmentation of genetic material, and restored the antioxidant activity. In addition, metformin up-regulated the expression of adiponectin and its receptors, adipoR1 and adipoR2, in cardiomyocytes. In contrast, silencing either adipoR1 or adipoR2 with siRNA inhibited the AMPK activation and the protective effects of metformin. Taken together, these results demonstrate that metformin protects cardiomyocytes from doxorubicin-induced damage and that the cardiac adiponectin system plays an important role in this protective action.

Mineralocorticoid Receptor Antagonists Modulate Galectin-3 and Interleukin-33/ST2 Signaling in Left Ventricular Systolic Dysfunction After Acute Myocardial Infarction

OBJECTIVES This study aimed to evaluate the specific role of the 2 available mineralocorticoid receptor antagonists (MRAs), eplerenone and spironolactone, on the modulation of galectin-3 (Gal-3) and interleukin (IL)-33/ST2 signaling in an experimental model of left ventricular systolic dysfunction after acute myocardial infarction (MI). BACKGROUND The molecular mechanisms of benefits of MRAs in patients with left ventricular systolic dysfunction after MI not well understood. METHODS MI and left ventricular systolic dysfunction were induced by permanent ligation of the anterior coronary artery in 45 male Wistar rats, randomly assigned to no therapy (MI group, n = 15) or to receive MRAs (100 mg/kg/day) for 4 weeks; either eplerenone (n = 15) or spironolactone (n = 15) was used. A sham group was used as a control (n = 8). Elements of the pathway for Gal-3 including transforming growth factor (TGF)-β and SMAD3, as well as that for IL-33/ST2 (including IL-33 and soluble ST2 [sST2]) were analyzed in the infarcted and noninfarcted myocardium by quantitative real-time reverse transcription polymerase chain reaction. Expression of markers of fibrosis (collagen types I and III, tissue inhibitor of metalloproteinase-1) and inflammation (IL-6, tumor necrosis factor-α, monocyte chemotactic protein-1) was also examined. RESULTS In the infarcted myocardium, compared with sham animals, the MI group had higher concentrations of Gal-3, TGF-β, SMAD3, IL-33, and sST2, as well as higher concentrations of markers of fibrosis and inflammation. Treatment with MRAs down-regulated Gal-3, TGF-β, and SMAD3 and enhanced IL-33/ST2 signaling with lower expression of sST2; protective IL-33 up-regulation was unaffected by MRAs. Modulation of Gal-3 and IL-33/ST2 signaling induced by MRAs correlated with lower expression levels of fibrosis and inflammatory markers. No differences were found between eplerenone and spironolactone. In the noninfarcted myocardium, compared with sham animals, the MI group exhibited a higher expression of Gal-3 and IL-33, but no signs of inflammation or fibrosis were observed; in the presence of MRAs, IL-33 expression was significantly up-regulated, but Gal-3 was unaffected. CONCLUSIONS MRAs play a pivotal role in the Gal-3 and IL-33/ST2 modulation in post-MI cardiac remodeling.

Red blood cell distribution width predicts new-onset anemia in heart failure patients☆

BACKGROUND Hematologic abnormalities such as elevated red blood cell distribution width (RDW) as well as anemia are prognostically meaningful among heart failure (HF) patients. The inter-relationship between these hematologic abnormalities in HF is unclear, however. We therefore aimed to assess whether RDW is predicting changes in hemoglobin concentrations as well as onset of anemia. METHODS 268 consecutive non-anemic patients with acutely decompensated HF (ADHF) were enrolled at hospital discharge and RDW was measured. At 6 month follow-up, change in hemoglobin as well as new-onset anemia was studied as a function of RDW at discharge. RESULTS RDW at discharge correlated negatively with hemoglobin values at 6 months (r=-0.220; p<0.001); a greater decrease in hemoglobin concentration occurred in those with higher values of RDW at discharge (p=0.004), independently of baseline hemoglobin concentration and other risk factors. At 6 months, 54 patients (20%) developed new-onset anemia. RDW values at discharge were significantly higher among patients who developed new-onset anemia (15.1 ± 2.2 vs. 14.2 ± 1.4, p=0.005). In integrated discrimination improvement analyses, the addition of RDW measurement improved the ability to predict new-onset anemia (IDI 0.0531, p<0.001), beyond known risk factors as hemoglobin, renal function, age, diabetes mellitus, sex and HF symptom severity. In adjusted analyses, patients with RDW>15% (derived from receiver operating characteristic analysis) had a tripling of the risk of new-onset anemia (OR=3.1, 95% CI 1.5-5.1, p=0.002). CONCLUSION Among non-anemic patients with ADHF, RDW measurement at the time of hospital discharge independently predicts lower hemoglobin concentrations and new-onset anemia over a 6-month follow up period.

Involvement of ferritin heavy chain in the preventive effect of metformin against doxorubicin-induced cardiotoxicity.

Doxorubicin is a wide-spectrum chemotherapeutic agent, although a cumulative dose may cause cardiac damage and lead to heart failure. Doxorubicin cardiotoxicity is dependent on the intracellular iron pool and manifests itself by increasing oxidative stress. Our group has recently shown the ability of metformin, an oral antidiabetic with cardiovascular benefits, to protect cardiomyocytes from doxorubicin-induced damage. This work aimed to study whether metformin is able to modulate the expression of ferritin, the major intracellular iron storage protein, in cardiomyocytes and whether it is involved in their protection. The addition of metformin to adult mouse cardiomyocytes (HL-1 cell line) induced both gene and protein expression of the ferritin heavy chain (FHC) in a time-dependent manner. The silencing of FHC expression with siRNAs inhibited the ability of metformin to protect cardiomyocytes from doxorubicin-induced damage, in terms of the percentage of cell viability, the levels of reactive oxygen species, and the activity of antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase). In addition, metformin induced the activation of NF-κB in HL-1 cells, whereas preincubation with SN50, an inhibitor of NF-κB, blocked the upregulation of the FHC and the protective effect mediated by metformin. Taken together, these results provide new knowledge on the protective actions of metformin against doxorubicin-induced cardiotoxicity by identifying FHC and NF-κB as the major mediators of this beneficial effect.

Soluble ST2 Is a Marker for Acute Cardiac Allograft Rejection

BACKGROUND Soluble ST2 (sST2), an interleukin (IL)-1 receptor family member, has a role in immunologic tolerance and has also emerged as a biomarker of cardiac stretch and remodeling. The sST2 role in heart transplantation is still unknown. METHODS From the heart transplantation population at our institution (n = 74), we selected a subset of 26 patients who had an acute rejection episode in the first year after transplantation (35%; 52 ± 14 years; 76% men). Endomyocardial biopsy (EMB) results obtained at the time of the first rejection episode represented the rejection cohort (n = 26). Each patient served as a control to himself or herself, with EMB without rejection obtained before and after the rejection episode (n = 52). All laboratory measurements and blood samples were obtained at the time of EMB. RESULTS sST2 concentrations rose significantly in the context of acute rejection (130 [60 to 238] versus 51 ng/mL [28 to 80]; p = 0.002). Tertile analyses of sST2 concentrations revealed a graded association with rejection (p = 0.002) and repeated measurement analyses showed that sST2 concentrations were significantly modulated by the presence of rejection (p = 0.001). In receiver operator characteristic (ROC) analysis, sST2 had an area under the curve (AUC) of 0.72; the optimal cutoff point was 68 ng/mL (positive predictive value of 53%, negative predictive value of 83%), which predicted acute cellular rejection (odds ratio [OR] 4.9; 95% confidence interval [CI], 1.7 to 14.5; p = 0.004). The addition of sST2 values to those for the N-terminal pro B-type natriuretic peptide (NT-proBNP) resulted in a significant improvement on the integrated discrimination index (IDI) for rejection (relative improvement of 24%; p = 0.021). CONCLUSIONS sST2 concentrations are modulated by the presence of acute rejection and provide complementary predictive ability to NT-proBNP for the biochemical identification of rejection.

Modulation of IL‐33/ST2 system in postinfarction heart failure: correlation with cardiac remodelling markers

Interleukin (IL)‐33 and sST2 are molecules with an opposite pathophysiologic implications in the myocardial response after acute myocardial infarction (AMI). Both may be a target for therapeutic interventions. The kinetics of IL‐33 and sST2 expression in infarcted myocardium and their correlation with the ongoing processes of fibrosis, inflammation and apoptosis remains poorly defined.

Differential Actions of Eplerenone and Spironolactone on the Protective Effect of Testosterone Against Cardiomyocyte Apoptosis In Vitro

INTRODUCTION AND OBJECTIVES Testosterone deficiency is associated with a poor prognosis in patients with heart failure. It is not clear whether testosterone reduces cardiomyocyte apoptosis or whether the effect of spironolactone, an aldosterone receptor blocker with progestogenic and anti-androgen activity, differs from that of the selective aldosterone blocker eplerenone. METHODS Apoptosis induced by hyperosmotic stress in the embryonic rat heart cell line H9c2 was monitored by measuring cell viability, DNA fragmentation and caspase-3, -8 and -9 activation. The effect of testosterone was investigated in the presence or absence of spironolactone and eplerenone. RESULTS Exposure to sorbitol (0.6 M, 3 h) decreased cell viability and increased DNA fragmentation and caspase-3, -8 and -9 activation. These effects were all significantly reduced by testosterone, 100 nM (P< .01). Pretreatment with spironolactone, 10 .M, blocked the effects of testosterone, decreased cell viability (P< .01) and increased caspase activation (P< .01). In contrast, eplerenone, 10 .M, increased cell viability (P< .001) without altering the effect on caspase activation. These actions were not modified by the androgen receptor blocker flutamide. They were mediated by SAPK/JNK and ERK1/2 signaling pathways (P< .01). CONCLUSIONS Testosterone appears to have a protective effect against cardiomyocyte apoptosis which is antagonized by spironolactone but not by eplerenone. These effects await confirmation in in vivo models, but their presence could have clinical and therapeutic implications.

Clinical relevance of sST2 in cardiac diseases.

Abstract ST2 has two main isoforms, ST2L and soluble isoform of ST2 (sST2), by alternative splicing. The interaction between interleukin (IL)-33 and the transmembrane isoform ST2L is up-regulated in response to myocardial stress and exerts cardio-protective actions in the myocardium by reducing fibrosis, hypertrophy and enhancing survival. The circulating isoform sST2, by sequestering IL-33, abrogates these favorable actions and will be elevated as a maladaptive response to cardiac diseases. Indeed, circulating sST2 concentrations correlate with a worse phenotype of disease including adverse remodeling and fibrosis, cardiac dysfunction, impaired hemodynamics and higher risk of progression. In patients with acute and chronic heart failure, sST2 concentrations are strongly predictive of death, regardless of the cause and left ventricle (LV) ejection fraction, and contribute relevant information in addition to other prognosticators and biomarkers, as natriuretic peptides or troponins. sST2 also retains prognostic information in the setting of acute myocardial infarction (AMI) and predicts cardiovascular death and risk of heart failure (HF) development in these patients. sST2 could also be a promising tool to stratify the risk of sudden cardiac death (SCD) in patients with depressed LV ejection fraction. Therefore, sST2 represents a clinically relevant biomarker reflecting pathophysiological processes and contributing predictive information in the setting of several cardiovascular diseases, and especially in patients with HF.

Ferritin heavy chain as main mediator of preventive effect of metformin against mitochondrial damage induced by doxorubicin in cardiomyocytes

The efficacy of doxorubicin (DOX) as an antitumor agent is greatly limited by the induction of cardiomyopathy, which results from mitochondrial dysfunction and iron-catalyzed oxidative stress in the cardiomyocyte. Metformin (MET) has been seen to have a protective effect against the oxidative stress induced by DOX in cardiomyocytes through its modulation of ferritin heavy chain (FHC), the main iron-storage protein. This study aimed to assess the involvement of FHC as a pivotal molecule in the mitochondrial protection offered by MET against DOX cardiotoxicity. The addition of DOX to adult mouse cardiomyocytes (HL-1 cell line) increased the cytosolic and mitochondrial free iron pools in a time-dependent manner. Simultaneously, DOX inhibited complex I activity and ATP generation and induced the loss of mitochondrial membrane potential. The mitochondrial dysfunction induced by DOX was associated with the release of cytochrome c to the cytosol, the activation of caspase 3, and DNA fragmentation. The loss of iron homeostasis, mitochondrial dysfunction, and apoptosis induced by DOX were prevented by treatment with MET 24h before the addition of DOX. The involvement of FHC and NF-κB was determined through siRNA-mediated knockdown. Interestingly, the presilencing of FHC or NF-κB with specific siRNAs blocked the protective effect induced by MET against DOX cardiotoxicity. These findings were confirmed in isolated primary neonatal rat cardiomyocytes. In conclusion, these results deepen our knowledge of the protective action of MET against DOX-induced cardiotoxicity and suggest that therapeutic strategies based on FHC modulation could protect cardiomyocytes from the mitochondrial damage induced by DOX by restoring iron homeostasis.

Doxorubicin-induced oxidative stress: The protective effect of nicorandil on HL-1 cardiomyocytes

The primary cardiotoxic action of doxorubicin when used as antitumor drug is attributed to the generation of reactive oxygen species (ROS) therefore effective cardioprotection therapies are needed. In this sense, the antianginal drug nicorandil has been shown to be effective in cardioprotection from ischemic conditions but the underlying molecular mechanism to cope with doxorubicin-induced ROS is unclear. Our in vitro study using the HL-1 cardiomyocyte cell line derived from mouse atria reveals that the endogenous nitric oxide (NO) production was stimulated by nicorandil and arrested by NO synthase inhibition. Moreover, while the NO synthase activity was inhibited by doxorubicin-induced ROS, the NO synthase inhibition did not affect doxorubicin-induced ROS. The inhibition of NO synthase activity by doxorubicin was totally prevented by preincubation with nicorandil. Nicorandil also concentration-dependently (10 to 100 μM) decreased doxorubicin-induced ROS and the effect was antagonized by 5-hydroxydecanoate. The inhibition profile of doxorubicin-induced ROS by nicorandil was unaltered when an L-arginine derivative or a protein kinase G inhibitor was present. Preincubation with pinacidil mimicked the effect of nicorandil and the protection was eliminated by glibenclamide. Quantitative colocalization of fluorescence indicated that the mitochondrion was the target organelle of nicorandil and the observed response was a decrease in the mitochondrial inner membrane potential. Interference with H+ movement across the mitochondrial inner membrane, leading to depolarization, also protected from doxorubicin-induced ROS. The data indicate that activation of the mitochondrial ATP-sensitive K+ channel by nicorandil causing mitochondrial depolarization, without participation of the NO donor activity, was responsible for inhibition of the mitochondrial NADPH oxidase that is the main contributor to ROS production in cardiomyocytes. Impairment of the cytosolic Ca2+ signal induced by caffeine and the increase in lipid peroxidation, both of which are indicators of doxorubicin-induced oxidative stress, were also prevented by nicorandil.