Abstract-Chronological myocardial aging is viewed as the inevitable effect of time on the functional reserve of the heart.Cardiac failure in elderly patients is commonly interpreted as an idiopathic or secondary myopathy superimposed on the old heart independently from the aging process. Thus, aged diseased hearts were studied to determine whether cell regeneration was disproportionate to the accumulation of old dying cells, leading to cardiac decompensation. Endomyocardial biopsies from 19 old patients with a dilated myopathy were compared with specimens from 7 individuals of similar age and normal ventricular function. Ten patients with idiopathic dilated cardiomyopathy were also analyzed to detect differences with aged diseased hearts. Senescent cells were identified by the expression of the cell cycle inhibitor p16 INK4a and cell death by hairpin 1 and 2. Replication of primitive cells and myocytes was assessed by MCM5 labeling, myocyte mitotic index, and telomerase function. Aged diseased hearts had moderate hypertrophy and dilation, accumulation of p16INK4a positive primitive cells and myocytes, and no structural damage. Cell death markedly increased and occurred only in cells expressing p16INK4a that had significant telomeric shortening. Cell multiplication, mitotic index and telomerase increased but did not compensate for cell death or prevented telomeric shortening. Idiopathic dilated cardiomyopathy had severe hypertrophy and dilation, tissue injury, and minimal level of p16INK4a labeling. In conclusion, telomere erosion, cellular senescence, and death characterize aged diseased hearts and the development of cardiac failure in humans. Key Words: aging Ⅲ telomeric shortening and telomerase activity Ⅲ p16INK4a marker of cellular senescence Ⅲ cardiac primitive cells Ⅲ heart failure M yocardial aging in humans has been studied extensively and two major conclusions have been reached. Aging effects decrease the functional reserve of the heart 1,2 and loss of myocytes 3 contributes to the attenuation of the response of the old heart to sudden changes in ventricular loading. However, the selection of patients or hearts to be analyzed has always been based on the chronological age of otherwise healthy individuals. [1][2][3] This understandable approach has neglected several variables of the aging process that cannot be easily quantified but may have a significant impact on organ and/or organism aging. It is a wellestablished fact that chronological age and physical age do not necessarily coincide. An 80-year-old man or woman may appear as young as a 60-year-old person. Similarly, organism and organ age do not proceed at the same pace. 4 -6 In general, there is little appreciation of these unpredictable factors and aged patients with cardiac decompensation are diagnosed according to classified diseases, excluding that aging per se can be the etiology of the pathological condition.To establish whether aging alone results in a dilated myopathy with characteristics different from idiopathic dilated cardiomyopathy (...

The aim of this work was to identify micro-RNAs (miRNAs) involved in the pathological pathways activated in skeletal muscle damage and regeneration by both dystrophin absence and acute ischemia. Eleven miRNAs were deregulated both in MDX mice and in Duchenne muscular dystrophy patients (DMD signature). Therapeutic interventions ameliorating the mdx-phenotype rescued DMD-signature alterations. The significance of DMD-signature changes was characterized using a damage/regeneration mouse model of hind-limb ischemia and newborn mice. According to their expression, DMD-signature miRNAs were divided into 3 classes. 1) Regeneration miRNAs, miR-31, miR-34c, miR-206, miR-335, miR-449, and miR-494, which were induced in MDX mice and in DMD patients, but also in newborn mice and in newly formed myofibers during postischemic regeneration. Notably, miR-206, miR-34c, and miR-335 were up-regulated following myoblast differentiation in vitro. 2) Degenerative-miRNAs, miR-1, miR-29c, and miR-135a, that were down-modulated in MDX mice, in DMD patients, in the degenerative phase of the ischemia response, and in newborn mice. Their down-modulation was linked to myofiber loss and fibrosis. 3) Inflammatory miRNAs, miR-222 and miR-223, which were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. These findings show an important role of miRNAs in physiopathological pathways regulating muscle response to damage and regeneration.

Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles 1,2 . We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and a-sarcoglycan (a-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin 3 in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies.Enlarging fiber size in dystrophic muscles produces beneficial effects in dystrophin-deficient MDX mice, a model of Duchenne muscular dystrophy (DMD) 2,4-6 . Previous studies have shown that three structurally unrelated deacetylase inhibitors-trichostatin A (TSA), valproic acid (VPA) and phenylbutyrate (PhB)-share the ability to promote myoblast fusion into hypernucleated myotubes with an increased size relative to myotubes formed in the absence of drugs 7,8 . To select a compound for long-term treatment of dystrophic mice, we compared the results of pilot experiments in which MDX mice were exposed to TSA (0.6 mg per kg body weight per day), VPA (160 mg/kg per day) or PhB (90 mg/kg per day) by daily intraperitoneal injections. We chose to begin the treatment when the first manifestations of the disease were already evident, as we sought to evaluate the efficacy of deacetylase inhibitors in a situation simulating the clinical stage at which human patients typically receive the diagnosis of muscular dystrophy 9 . Increased histone acetylation, which reflects the bioactivity of deacetylase inhibitors, was detected in muscles and other peripheral organs (for example, brain) a few hours after injection, indicating rapid uptake of the compounds (Supplementary Figure 1 online). We next evaluated the ability of satellite cells from MDX mice exposed to the deacetylase inhibitors for 10 d to differentiate into multinucleated myotubes. After 24 h in differentiation medium, myotubes were present only in cultures of satellite cells isolated from mice exposed to deacetylase inhibitors (Supplementary Figure 2 online). Notably, satellite cells derived from TSA-treated mice formed myotubes with the highest efficiency and showed an increased expression, relative to that in untreated controls, of myosin heavy chain (MyHC)-a marker of terminal differentiation-and of regeneration markers, such as follistatin and embryonic and perinatal MyHC (Supplementary Figure 2). Only satellite cells from TSA-treated mice showed reduced levels of myostatin mRNA relative to those from untreated controls. Treatment of 12-week-old mice with deacetylase inhibitors for an additional three months prevented an increase in serum concentrations of creatine kinase, a biomarker for the severity of the disease (Supplementary Figure 2). The decline of creatine kinase concentrations was more pronounced i...

Cells induced to apoptosis extrude glutathione in the reduced form concomitantly with (U937 cells) or before (HepG2 cells) the development of apoptosis, much earlier than plasma membrane leakage. Two specific inhibitors of carrier-mediated GSH extrusion, methionine or cystathionine, are able to decrease apoptotic GSH efflux across the intact plasma membrane, demonstrating that in these cell systems GSH extrusion occurs via a specific mechanism. While decreasing GSH efflux, cystathionine or methionine also decrease the extent of apoptosis. They fail to exert anti-apoptotic activity in cells previously deprived of GSH, indicating that the target of the protection is indeed GSH efflux. The cells rescued by methionine or cystathionine remained viable after removal of the apoptogenic inducers and were even able to replicate. This shows that a real rescue to perfect viability and not just a delay of apoptosis is achieved by forcing GSH to stay within the cells during apoptogenic treatment. All this evidence indicates that extrusion of reduced glutathione precedes and is responsible for the irreversible morphofunctional changes of apoptosis, probably by altering the intracellular redox state without intervention of reactive oxygen species, thus giving a rationale for the development of redox-dependent apoptosis under anaerobic conditions.