The aging process is a general event involving all organisms and organs, characterized by physiological and metabolic dysfunctions. In cardiac muscle aging represents the major risk factor for cardiac disease onset. A number of hallmarks characterize the ageing process, nevertheless among those the main is represented by an excessive mitochondrial ROS (radical oxygen species) production. For this reason, it has been hypothesized that ROS lead to mitochondrial DNA (mtDNA) damages, inducing cellular dysfunction and organ failure. Furthermore, mitochondria being responsible for the fine tuning between mitochondrial fusion, fission and autophagy, which are essential processes for cellular functioning, their role in aging become predominant. To understand molecular bases of cardiac muscle aging in a physiological model, the proteomic profiles of Sprague Dawley rat hearts of 6, 22 and 30 months old were analyzed by 2D-DIGE technology (two dimensional Differential In Gel Electrophoresis). To contribute to clarify the role of mitochondria in aging, fusion, fission and autophagy were investigated by antigen antibody reactions on total muscle extracts. In addition, mitochondria were isolated by classical methodologies including differential centrifugation and density gradient for the study of proteomic profile by 2D-DIGE of subsarcolemmal (SSM) and intermiofibrillar (IFM) mitochondria. Results on total cardiac muscle, indicate a number of changed proteins, particularly of specific protein isoforms, even though changes in abundance are modest. These results suggest that this model could be representative of a successful aging, like it occurs in human centenarians. In particular, variations involve myosin binding proteins and the troponin I, mitochondrial aldehyde dehydrogenase (Aldh2) and a group of serpins, suggesting that these proteins could be possible putative biomarkers of the aging process. Concerning mitochondrial dynamic in aging, it is impaired with a decreased fission, an increased fusion and a decreased autophagy. The latter through the increment of LC3, could indicate a protective activation of the non-canonical autophagic process. Furthermore, it has been observed that in course of aging there is an increment of Sirtuin 3, which protects mtDNA from mitochondrial ROS attacks, and of CypD, which promotes the opening of mitochondrial permeability transition pore (mtPTP), with a harmful effect for the cell. The changes, that resulted be protective from senescence are in line with the hypothesis of a successful aging, even though the changes in mitochondrial dynamics are remarkable. However, this study reveals an incoherence between mitochondrial and cardiac tissue proteomic results, particularly on respiratory chain proteins. To avoid unreliable conclusions after proteomics we tested the integrity of isolated mitochondria. We observed that matrix proteins were not enriched in mitochondrial extracts, while the mitochondrial membrane proteins were enriched up to 600%. Further validations were obtained with the analysis of the supernatant (after SS mitochondria enrichment) and by isolation of mitochondria with commercial kits. Our observations are supported by recent papers (Picard et al.), which highlighted the drawbacks of mitochondria isolation by classical methodology demonstrating that the tridimensional mitochondrial network and their interactions with other cellular compartments were destroyed. Also our study underlines technical problems associated with mitochondrial isolation and we suggest, to guarantee results reliability, a careful evaluation of mitochondria integrity (considering both mitochondrial spaces and membranes proteins), before any quantitative differential analysis. The cardiac muscle proteomic changes in this physiological model of aging indicate that some variations are associated to an intrinsic cardiac aging. Further progresses to validate the reliability of proposed biomarkers of aging will be possible by testing them in animal models affected by cardiovascular disorders.
ANALISI PROTEOMICA DEL MUSCOLO CARDIACO DI RATTO IN CORSO D'INVECCHIAMENTO / R. Leone ; tutore: C. Gelfi ; coordinatore: M. S. Clerici. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE BIOMEDICHE PER LA SALUTE, 2014 Feb 04. ((26. ciclo, Anno Accademico 2013.
|Titolo:||ANALISI PROTEOMICA DEL MUSCOLO CARDIACO DI RATTO IN CORSO D'INVECCHIAMENTO|
|Supervisori e coordinatori interni:||CLERICI, MARIO SALVATORE|
|Data di pubblicazione:||4-feb-2014|
|Parole Chiave:||proteomics; cardiac muscle ; aging ; mitochondria|
|Settore Scientifico Disciplinare:||Settore BIO/12 - Biochimica Clinica e Biologia Molecolare Clinica|
|Citazione:||ANALISI PROTEOMICA DEL MUSCOLO CARDIACO DI RATTO IN CORSO D'INVECCHIAMENTO / R. Leone ; tutore: C. Gelfi ; coordinatore: M. S. Clerici. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE BIOMEDICHE PER LA SALUTE, 2014 Feb 04. ((26. ciclo, Anno Accademico 2013.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/leone-roberta_phd2014-02-04|
|Appare nelle tipologie:||Tesi di dottorato|