This study aims to determine the predictive value of the soluble suppression of tumorigenicity 2 ... more This study aims to determine the predictive value of the soluble suppression of tumorigenicity 2 (sST2) biomarker in atrial fibrillation (AF) recurrence. This prospective, observational study included patients with AF referred for electrical cardioversion (ECV) or pulmonary vein isolation (PVI) procedures. Baseline characteristics were collected, and sST2 was determined at baseline and at 3 and 6 months of follow-up. sST2 was determined at baseline in a matched control group. Left atrial voltage mapping was performed in patients undergoing PVI. The sST2 maximal predictive capacity of AF recurrence was at the 3-month FU in the cohort of patients undergoing ECV with respect to 6-month AF recurrence with an AUC of 0.669, a cut-off point of 15,511 pg/mL, a sensitivity of 60.97%, and a specificity of 69.81%. The ROC curve of the sST2 biomarker at baseline and 3 months in the cohort of patients undergoing PVI showed AUCs of 0.539 and 0.490, respectively. The logistic regression model iden...
Anthracycline-induced cardiotoxicity is the most severe collateral effect of chemotherapy origina... more Anthracycline-induced cardiotoxicity is the most severe collateral effect of chemotherapy originated by an excess of oxidative stress in cardiomyocytes that leads to cardiac dysfunction. We assessed clinical data from patients with breast cancer receiving anthracyclines and searched for discriminating microRNAs between patients that developed cardiotoxicity (cases) and those that did not (controls), using RNA sequencing and regression analysis. Serum levels of 25 microRNAs were differentially expressed in cases versus controls within the first year after anthracycline treatment, as assessed by three different regression models (elastic net, Robinson and Smyth exact negative binomial test and random forest). MiR-4732-3p was the only microRNA identified in all regression models and was downregulated in patients that experienced cardiotoxicity. MiR-4732-3p was also present in neonatal rat cardiomyocytes and cardiac fibroblasts and was modulated by anthracycline treatment. A miR-4732-3p...
The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 D... more The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial evidenced the potential of sodium-glucose cotransporter 2 (SGLT2) inhibitors for the treatment of patients with diabetes and cardiovascular disease. Recent evidences have shown the benefits of the SGLT2 inhibitor empagliflozin on improving liver steatosis and fibrosis in patients with T2DM. Metabolomic studies have been shown to be very useful to improve the understanding of liver pathophysiology during the development and progression of metabolic hepatic diseases, and because the effects of empagliflozin and of other SGLT2 inhibitors on the complete metabolic profile of the liver has never been analysed before, we decided to study the impact on the liver of male Zucker diabetic fatty (ZDF) rats of a treatment for 6 weeks with empagliflozin using an untargeted metabolomics approach, with the purpose to help to clarify the benefits of the use of empagliflozi...
QT interval prolongation is an important marker for the development of cardiac arrhythmias (CAs).... more QT interval prolongation is an important marker for the development of cardiac arrhythmias (CAs). Optimal methods to estimate QT/QTc intervals in patients with ventricular pacing (VP) and its correlation with CA have not been widely investigated. We aimed to validate the currently available formulas for QT determination during VP and to compare their abilities in predicting the occurrence of CA (atrial fibrillation [AF] and malignant ventricular arrhythmias [VAs] in patients with advanced heart failure and cardiac resynchronization therapy). Consecutive patients with advanced heart failure who underwent cardiac resynchronization therapy implantation between August 2001 and April 2015 were included in a retrospective study. Four proposed formulas for QT correction in VP rhythms were evaluated. One hundred eighty patients were enrolled. During 44 months of follow-up, 43 patients (37.7%) developed AF and 16 patients (8.9%) developed VA. There was no correlation between corrected QT inc...
The pig-to-primate model is increasingly being utilized as the final preclinical means of assessi... more The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. To obtain information about the nature of cytotoxic response in pig-to-baboon xenotransplants, we sought to determine if serum cytotoxicity in this model was assay dependent. Sera from nine kidney or heart xenotransplanted baboons were obtained before transplantation and at the time of acute humoral xenograft rejection (AHXR). Cytotoxicity was measured by an anti-pig haemolytic assay (APHA) and by a flow cytometry complement-dependent assay (FCCA), using pig blood lymphocytes (PBLs). Serum samples showing inter-assay differences were absorbed with pig erythrocytes and assayed by APHA and FCCA, as well as by measuring anti-alphaGal and total anti-pig xenoantibodies. The results showed that in four AHXR samples, FCCA cytotoxicity was higher than APHA cytotoxicity. Absorption with pig erythrocytes diminished FCCA and removed APHA cytotoxicity. Residual FCCA activity was due to total anti-pig and IgM anti-alphaGal and non-Gal antibodies. Our results indicate that some cytotoxic antibodies present in the sera of xenotransplanted baboons at time of AHXR are IgM antibodies directed against pig PBL antigens not detected by APHA.
Atrial fibrillation is the most prevalent tachyarrhythmia in clinical practice, with very high ca... more Atrial fibrillation is the most prevalent tachyarrhythmia in clinical practice, with very high cardiovascular morbidity and mortality with a high-cost impact in health systems. Currently, it is one of the main causes of stroke and subsequent heart failure and sudden death. miRNAs mediate in several processes involved in cardiovascular disease, including fibrosis and electrical and structural remodeling. Several studies suggest a key role of miRNAs in the course and maintenance of atrial fibrillation. In our study, we aimed to identify the differential expression of circulating miRNAs and their predictive value as biomarkers of recurrence in atrial fibrillation patients undergoing catheter pulmonary vein ablation. To this effect, 42 atrial fibrillation patients were recruited for catheter ablation. We measured the expression of 84 miRNAs in non-recurrent and recurrent groups (45.2%), both in plasma from peripheral and left atrium blood. Expression analysis showed that miRNA-451a is d...
In Brugada syndrome, even within the same family where all affected individuals share the same mu... more In Brugada syndrome, even within the same family where all affected individuals share the same mutation, phenotypic variation is prominent, with variable penetrance and expressivity, presenting different degrees of involvement. It is difficult to establish a direct correlation between genotype and phenotype to predict prognosis in complications and risk of sudden death. The factors that modulate this inter- and intra-familial phenotypic variability remain to be determined. With the intention of testing whether other genetic factors, in addition to the causal mutation in SCN5A, may have a modulating effect on the Brugada phenotype and the risk of sudden death, we have studied 8 families with a causal variant in SCN5A with at least two affected individuals, one of whom has suffered cardiac arrest or sudden death. Whole exome sequencing was performed looking for additional variants that modify the phenotype and allow us to predict a better or worse prognosis for the evolution of the di...
Immunocytochemistry analysis of IGF-1 and HGF expression in paMSC-IGF- 1-GFP (A) and paMSC-HGF-Ch... more Immunocytochemistry analysis of IGF-1 and HGF expression in paMSC-IGF- 1-GFP (A) and paMSC-HGF-Cherry (B). (+) indicates cells transfected with pRRL-sin-IGF-1-IRES-GFP (A) or pRRL-sin-HGF-IRES-Cherry vectors (B); (–) negative controls. GFP- (A-V) or Cherry-positive cells (B-V) were tested for IGF-1 (A-Vi) or HGF (B-Vi) expression; nuclei were DAPI stained (iii and Vii), showing that all paMSC-IGF-1 and HGF were positive for GFP/Cherry and IGF-1/HGF, respectively, and that paMSC-IGF-1 and HGF are negative for HGF and IGF-1, respectively. HEK293T-derived populations were used as controls (A and B; upper panels); controls confirm that HEK-293 do not express GFP/Cherry or IGF-1/HGF, and that HEK293-IGF-1 and HEK293-HGF express GFP/Cherry and IGF-1/HGF, respectively. Alexa 488- (green) and Alexa 568- (red) conjugated secondary antibodies were used. Scale bars = 20 μm. (TIF 414 kb)
Immunocytochemistry analysis of IGF-1 and HGF expression post-sorting in negative populations. Po... more Immunocytochemistry analysis of IGF-1 and HGF expression post-sorting in negative populations. Post-sorting immunocytochemistry for IGF-1 in GFP-negative population (A) and Cherry-negative population (B). Images confirm that IGF-1-negative cells do not express GFP/Cherry or IGF-1, and that HGF-negative cells do not express GFP/Cherry but demonstrated basal HGF levels. (C) HGF expression was monitored in paMSC-IGF-1-GFP populations (positive and negative). Positive populations were negative for HGF expression but the negative fraction showed basal HGF levels. (D) IGF-1 expression was monitored in paMSC-HGF-Cherry (positive and negative); both were positive for IGF-1 expression showing basal levels for this growth factor. (+) corresponds to primary and secondary antibodies ICC; (–) corresponds to ICC only with secondary antibodies. Secondary antibodies were Alexa-conjugated as in Additional file 4 (Figure S2). Scale bars = 20 μm. (TIF 242 kb)
(A) Immunohistocytochemistry analysis in control animals (transplanted but not infarcted). Animal... more (A) Immunohistocytochemistry analysis in control animals (transplanted but not infarcted). Animal transplanted with 30 × 106 paMSC + GFP + SPIO cells. Left panels show negative controls, incubation with Alexa 568-secondary antibody (top) and DAPI staining (bottom). Center and right images show isolated GFP-positive cells (anti-GFP-A568) in the Heart 2 section. (B) Immunohistocytochemistry analysis of control animals (no treatment). Left and center images show negative controls (DAPI staining and incubation with Alexa 568-secondary antibody). Right image shows negative results for GFP immunofluorescence. (TIF 1493 kb)
Cardiac resynchronization therapy represents a therapeutic option for heart failure drug-refracto... more Cardiac resynchronization therapy represents a therapeutic option for heart failure drug-refractory patients. However, due to the lack of success in 30% of the cases, there is a demand for an in-depth analysis of individual heterogeneity. In this study, we aimed to evaluate the prognostic value of circulating miRNA differences. Responder patients were defined by a composite endpoint of the presence of left ventricular reverse remodelling (a reduction ≥15% in telesystolic volume and an increment ≥10% in left ventricular ejection fraction). Circulating miRNAs signature was analysed at the time of the procedure and at a 6-month follow-up. An expression analysis showed, both at baseline and at follow-up, differences between responders and non-responders. Responders presented lower baseline expressions of miR-499, and at follow-up, downregulation of miR-125b-5p, both associated with a significant improvement in left ventricular ejection fraction. The miRNA profile differences showed a ma...
Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in ca... more Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin), OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-cou...
This study aims to determine the predictive value of the soluble suppression of tumorigenicity 2 ... more This study aims to determine the predictive value of the soluble suppression of tumorigenicity 2 (sST2) biomarker in atrial fibrillation (AF) recurrence. This prospective, observational study included patients with AF referred for electrical cardioversion (ECV) or pulmonary vein isolation (PVI) procedures. Baseline characteristics were collected, and sST2 was determined at baseline and at 3 and 6 months of follow-up. sST2 was determined at baseline in a matched control group. Left atrial voltage mapping was performed in patients undergoing PVI. The sST2 maximal predictive capacity of AF recurrence was at the 3-month FU in the cohort of patients undergoing ECV with respect to 6-month AF recurrence with an AUC of 0.669, a cut-off point of 15,511 pg/mL, a sensitivity of 60.97%, and a specificity of 69.81%. The ROC curve of the sST2 biomarker at baseline and 3 months in the cohort of patients undergoing PVI showed AUCs of 0.539 and 0.490, respectively. The logistic regression model iden...
Anthracycline-induced cardiotoxicity is the most severe collateral effect of chemotherapy origina... more Anthracycline-induced cardiotoxicity is the most severe collateral effect of chemotherapy originated by an excess of oxidative stress in cardiomyocytes that leads to cardiac dysfunction. We assessed clinical data from patients with breast cancer receiving anthracyclines and searched for discriminating microRNAs between patients that developed cardiotoxicity (cases) and those that did not (controls), using RNA sequencing and regression analysis. Serum levels of 25 microRNAs were differentially expressed in cases versus controls within the first year after anthracycline treatment, as assessed by three different regression models (elastic net, Robinson and Smyth exact negative binomial test and random forest). MiR-4732-3p was the only microRNA identified in all regression models and was downregulated in patients that experienced cardiotoxicity. MiR-4732-3p was also present in neonatal rat cardiomyocytes and cardiac fibroblasts and was modulated by anthracycline treatment. A miR-4732-3p...
The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 D... more The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial evidenced the potential of sodium-glucose cotransporter 2 (SGLT2) inhibitors for the treatment of patients with diabetes and cardiovascular disease. Recent evidences have shown the benefits of the SGLT2 inhibitor empagliflozin on improving liver steatosis and fibrosis in patients with T2DM. Metabolomic studies have been shown to be very useful to improve the understanding of liver pathophysiology during the development and progression of metabolic hepatic diseases, and because the effects of empagliflozin and of other SGLT2 inhibitors on the complete metabolic profile of the liver has never been analysed before, we decided to study the impact on the liver of male Zucker diabetic fatty (ZDF) rats of a treatment for 6 weeks with empagliflozin using an untargeted metabolomics approach, with the purpose to help to clarify the benefits of the use of empagliflozi...
QT interval prolongation is an important marker for the development of cardiac arrhythmias (CAs).... more QT interval prolongation is an important marker for the development of cardiac arrhythmias (CAs). Optimal methods to estimate QT/QTc intervals in patients with ventricular pacing (VP) and its correlation with CA have not been widely investigated. We aimed to validate the currently available formulas for QT determination during VP and to compare their abilities in predicting the occurrence of CA (atrial fibrillation [AF] and malignant ventricular arrhythmias [VAs] in patients with advanced heart failure and cardiac resynchronization therapy). Consecutive patients with advanced heart failure who underwent cardiac resynchronization therapy implantation between August 2001 and April 2015 were included in a retrospective study. Four proposed formulas for QT correction in VP rhythms were evaluated. One hundred eighty patients were enrolled. During 44 months of follow-up, 43 patients (37.7%) developed AF and 16 patients (8.9%) developed VA. There was no correlation between corrected QT inc...
The pig-to-primate model is increasingly being utilized as the final preclinical means of assessi... more The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. To obtain information about the nature of cytotoxic response in pig-to-baboon xenotransplants, we sought to determine if serum cytotoxicity in this model was assay dependent. Sera from nine kidney or heart xenotransplanted baboons were obtained before transplantation and at the time of acute humoral xenograft rejection (AHXR). Cytotoxicity was measured by an anti-pig haemolytic assay (APHA) and by a flow cytometry complement-dependent assay (FCCA), using pig blood lymphocytes (PBLs). Serum samples showing inter-assay differences were absorbed with pig erythrocytes and assayed by APHA and FCCA, as well as by measuring anti-alphaGal and total anti-pig xenoantibodies. The results showed that in four AHXR samples, FCCA cytotoxicity was higher than APHA cytotoxicity. Absorption with pig erythrocytes diminished FCCA and removed APHA cytotoxicity. Residual FCCA activity was due to total anti-pig and IgM anti-alphaGal and non-Gal antibodies. Our results indicate that some cytotoxic antibodies present in the sera of xenotransplanted baboons at time of AHXR are IgM antibodies directed against pig PBL antigens not detected by APHA.
Atrial fibrillation is the most prevalent tachyarrhythmia in clinical practice, with very high ca... more Atrial fibrillation is the most prevalent tachyarrhythmia in clinical practice, with very high cardiovascular morbidity and mortality with a high-cost impact in health systems. Currently, it is one of the main causes of stroke and subsequent heart failure and sudden death. miRNAs mediate in several processes involved in cardiovascular disease, including fibrosis and electrical and structural remodeling. Several studies suggest a key role of miRNAs in the course and maintenance of atrial fibrillation. In our study, we aimed to identify the differential expression of circulating miRNAs and their predictive value as biomarkers of recurrence in atrial fibrillation patients undergoing catheter pulmonary vein ablation. To this effect, 42 atrial fibrillation patients were recruited for catheter ablation. We measured the expression of 84 miRNAs in non-recurrent and recurrent groups (45.2%), both in plasma from peripheral and left atrium blood. Expression analysis showed that miRNA-451a is d...
In Brugada syndrome, even within the same family where all affected individuals share the same mu... more In Brugada syndrome, even within the same family where all affected individuals share the same mutation, phenotypic variation is prominent, with variable penetrance and expressivity, presenting different degrees of involvement. It is difficult to establish a direct correlation between genotype and phenotype to predict prognosis in complications and risk of sudden death. The factors that modulate this inter- and intra-familial phenotypic variability remain to be determined. With the intention of testing whether other genetic factors, in addition to the causal mutation in SCN5A, may have a modulating effect on the Brugada phenotype and the risk of sudden death, we have studied 8 families with a causal variant in SCN5A with at least two affected individuals, one of whom has suffered cardiac arrest or sudden death. Whole exome sequencing was performed looking for additional variants that modify the phenotype and allow us to predict a better or worse prognosis for the evolution of the di...
Immunocytochemistry analysis of IGF-1 and HGF expression in paMSC-IGF- 1-GFP (A) and paMSC-HGF-Ch... more Immunocytochemistry analysis of IGF-1 and HGF expression in paMSC-IGF- 1-GFP (A) and paMSC-HGF-Cherry (B). (+) indicates cells transfected with pRRL-sin-IGF-1-IRES-GFP (A) or pRRL-sin-HGF-IRES-Cherry vectors (B); (–) negative controls. GFP- (A-V) or Cherry-positive cells (B-V) were tested for IGF-1 (A-Vi) or HGF (B-Vi) expression; nuclei were DAPI stained (iii and Vii), showing that all paMSC-IGF-1 and HGF were positive for GFP/Cherry and IGF-1/HGF, respectively, and that paMSC-IGF-1 and HGF are negative for HGF and IGF-1, respectively. HEK293T-derived populations were used as controls (A and B; upper panels); controls confirm that HEK-293 do not express GFP/Cherry or IGF-1/HGF, and that HEK293-IGF-1 and HEK293-HGF express GFP/Cherry and IGF-1/HGF, respectively. Alexa 488- (green) and Alexa 568- (red) conjugated secondary antibodies were used. Scale bars = 20 μm. (TIF 414 kb)
Immunocytochemistry analysis of IGF-1 and HGF expression post-sorting in negative populations. Po... more Immunocytochemistry analysis of IGF-1 and HGF expression post-sorting in negative populations. Post-sorting immunocytochemistry for IGF-1 in GFP-negative population (A) and Cherry-negative population (B). Images confirm that IGF-1-negative cells do not express GFP/Cherry or IGF-1, and that HGF-negative cells do not express GFP/Cherry but demonstrated basal HGF levels. (C) HGF expression was monitored in paMSC-IGF-1-GFP populations (positive and negative). Positive populations were negative for HGF expression but the negative fraction showed basal HGF levels. (D) IGF-1 expression was monitored in paMSC-HGF-Cherry (positive and negative); both were positive for IGF-1 expression showing basal levels for this growth factor. (+) corresponds to primary and secondary antibodies ICC; (–) corresponds to ICC only with secondary antibodies. Secondary antibodies were Alexa-conjugated as in Additional file 4 (Figure S2). Scale bars = 20 μm. (TIF 242 kb)
(A) Immunohistocytochemistry analysis in control animals (transplanted but not infarcted). Animal... more (A) Immunohistocytochemistry analysis in control animals (transplanted but not infarcted). Animal transplanted with 30 × 106 paMSC + GFP + SPIO cells. Left panels show negative controls, incubation with Alexa 568-secondary antibody (top) and DAPI staining (bottom). Center and right images show isolated GFP-positive cells (anti-GFP-A568) in the Heart 2 section. (B) Immunohistocytochemistry analysis of control animals (no treatment). Left and center images show negative controls (DAPI staining and incubation with Alexa 568-secondary antibody). Right image shows negative results for GFP immunofluorescence. (TIF 1493 kb)
Cardiac resynchronization therapy represents a therapeutic option for heart failure drug-refracto... more Cardiac resynchronization therapy represents a therapeutic option for heart failure drug-refractory patients. However, due to the lack of success in 30% of the cases, there is a demand for an in-depth analysis of individual heterogeneity. In this study, we aimed to evaluate the prognostic value of circulating miRNA differences. Responder patients were defined by a composite endpoint of the presence of left ventricular reverse remodelling (a reduction ≥15% in telesystolic volume and an increment ≥10% in left ventricular ejection fraction). Circulating miRNAs signature was analysed at the time of the procedure and at a 6-month follow-up. An expression analysis showed, both at baseline and at follow-up, differences between responders and non-responders. Responders presented lower baseline expressions of miR-499, and at follow-up, downregulation of miR-125b-5p, both associated with a significant improvement in left ventricular ejection fraction. The miRNA profile differences showed a ma...
Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in ca... more Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin), OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-cou...
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