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Pisano F.,Coronary Care Unit | Pisano F.,Laboratory of Experimental Cardiology for Cell and Molecular Therapy | Altomare C.,Laboratory of Experimental Cardiology for Cell and Molecular Therapy | Cervio E.,Coronary Care Unit | And 20 more authors.
Stem Cells | Year: 2015

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499-‰+-‰133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499-‰+-‰133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. © 2015 Alpha Med Press.


Ciuffreda M.C.,Fondazione IRCCS IRCCS Institute for Treatment and Research Policlinico San Matteo | Ciuffreda M.C.,Laboratory of Experimental Cardiology for Cell and Molecular Therapy | Tolva V.,Istituto Auxologico Italiano | Casana R.,Istituto Auxologico Italiano | And 8 more authors.
PLoS ONE | Year: 2014

Rationale: During the past 30 years, myocardial ischemia/reperfusion injury in rodents became one of the most commonly used model in cardiovascular research. Appropriate pain-prevention appears critical since it may influence the outcome and the results obtained with this model. However, there are no proper guidelines for pain management in rats undergoing thoracic surgery. Accordingly, we evaluated three analgesic regimens in cardiac ischemia/reperfusion injury. This study was strongly focused on 3R's ethic principles, in particular the principle of Reduction. Methods: Rats undergoing surgery were treated with pre-surgical tramadol (45 mg/kg intra-peritoneal), or carprofen (5 mg/ kg sub-cutaneous), or with pre-surgical administration of carprofen followed by 2 post-surgery tramadol injections (multimodal group). We assessed behavioral signs of pain and made a subjective evaluation of stress and suffering one and two hours after surgery. Results: Multi-modal treatment significantly reduced the number of signs of pain compared to carprofen alone at both the first hour (61±42 vs 123±47; p<0.05) and the second hour (43±21 vs 74±24; p<0.05) post-surgery. Tramadol alone appeared as effective as multi-modal treatment during the first hour, but signs of pain significantly increased one hour later (from 66±72 to 151±86, p<0.05). Carprofen alone was more effective at the second hour post-surgery when signs of pain reduced to 74±24 from 113±40 in the first hour (p<0.05). Stress behaviors during the second hour were observed in only 20% of rats in the multimodal group compared to 75% and 86% in the carprofen and tramadol groups, respectively (p<0.05). Conclusions: Multi-modal treatment with carprofen and tramadol was more effective in preventing pain during the second hour after surgery compared with both tramadol or carprofen. Our results suggest that the combination of carprofen and tramadol represent the best therapy to prevent animal pain after myocardial ischemia/reperfusion. We obtained our results accordingly with the ethical principle of Reduction. © 2014 Ciuffreda et al.


Danieli P.,Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology | Danieli P.,Laboratory of Experimental Cardiology for Cell and Molecular Therapy | Malpasso G.,Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology | Malpasso G.,Laboratory of Experimental Cardiology for Cell and Molecular Therapy | And 22 more authors.
Stem Cells Translational Medicine | Year: 2015

The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardio-protection and angiogenesis. Moreover, we aimed to identify the putative active paracrine medi-ators.hAMCswereisolated,expanded,andcharacterized.Invitro,conditionedmediumfromhAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, andstrongly promoted capillary formation at theinfarct border zone. Genearray analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factorswere detected in hAMC-CMbycytokine array.Ourresultsstronglysupport theconcept thattheadministrationof hAMC-CM favors the repair process after acute myocardial infarction. © Alpha Med Press 2015.


Lavatelli F.,Amyloidosis Research and Treatment Center | Lavatelli F.,University of Pavia | Lavatelli F.,Clinical Chemistry Laboratory | Imperlini E.,Centro Of Ricerca Of Ingegneria Genetica Ceinge Biotecnologie Avanzate | And 22 more authors.
FASEB Journal | Year: 2015

In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into lifethreatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiplemyeloma without amyloidosis (the nonamyloidogenic/ noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy. © FASEB.

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