HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractSince aging increases oxidative stress, we analyzed the contribution of reactive oxygen species (ROS) to the contractile dysfunction of aged ventricular myocytes and investigated whether short-term interference with ROS formation could normalize contractile performance.Isolated ventricular myocytes from young (2-4 months) and aged (24-26 months) male mice (C57BL/6) were used. We analyzed sarcomere shortening and calcium transients We found that aged myocytes showed decelerated shortening/ relengthening without changes in fractional shortening. Calcium transient decay was similarly decelerated, but the amplitude of calcium transients was increased with aging. Calcium sensitivity of myofilaments of aged myocytes was reduced. These age-dependent changes occurred without altered calcium handling protein expression but were reversed by the superoxide scavenger tiron. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPTAged myocytes showed increased NADPH oxidase expression and activity.Pharmacological inhibition of NADPH oxidase (diphenylene iodonium; apocynin) normalized age-dependent deceleration of shortening/ relengthening.In summary, we show that increased superoxide formation by upregulated NADPH oxidase contributes significantly to age-dependent alterations in calcium handling and contractility of murine ventricular myocytes.

Cyclophilins interact directly with the Alzheimer's disease peptide Aβ (amyloid β-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aβ binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aβ(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aβ(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aβ(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aβ(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aβ fibril formation by cyclophilins.

Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of β-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of β-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes.

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