Leader: Tommaso Angelone (UNICAL); Other collaborator(s):
Within this task murine and human cell models of cardiac aging will be employed for testing novel cardioprotective therapies and individuate early biomarker of cardiovascular aging. This will allow to investigate the effects of combination therapy directed to multiple molecular targets aiming to booster the endogenous antioxidant capacity, target both cardiomyocyte and non-cardiomyocyte components (endothelial cells and fibroblasts), and act with a combined pharmacological action (novel antioxidants, such as selenoproteins, and anti-inflammatory agents). Finally, a proof-of-concept clinical study for testing canonical and novel biomarkers of aging predisposing to cardiovascular diseases will be set up.
Brief description of the activities and of the intermediate results
Selected contribution in Age-It General Meeting (20-21 May 2024, Venice):
Molecular analyses were conducted on human AC16 cardiomyocytes exposed to low doses of doxorubicin (DOX) to induce a senescent phenotype and treated with PSELT and/or NLRP3 inflammasome inhibitors. These analyses aimed to assess the expression levels of senescence hallmarks (i.e., p53 and p21) and markers of mitochondrial dysfunction caused by imbalances in fusion/fission processes (i.e., OPA1 and DRP1). Additionally, the expression levels of SELENOT and NLRP3 were evaluated to elucidate their pathophysiological role in the context of senescence. A paper entitled: "Elucidating emerging signaling pathways driving endothelial dysfunction in cardiovascular aging" was drafted, addressing the latest evidence on the emerging molecular events underlying cardiac and vascular dysfunction during ageing. The aim is to provide insights into the therapeutic potential of targeting novel molecular determinants to develop strategies capable of controlling and reducing cardiovascular risk in the elderly population.
Molecular analyses were conducted to assess the potential protective action of the selenoprotein T (SELENOT)-derived mimetic peptide, PSELT, in mitigating endoplasmic reticulum stress (ERS) and DNA damage in senescent human cardiomyocytes. These analyses focused on evaluating the expression levels of specific molecular markers of ERS (i.e BiP, IRE1α, ERO1α, and Calnexin) and indicators of DNA damage (e.g., Lamin B1 and p-γH2AX). In the context of doxorubicin (Dox)-induced senescence, the role of PSELT as a post-conditioning agent was further examined by analyzing molecular markers of the senescent phenotype. To gain deeper insights into the potential protective effect of PSELT in the context of cardiac senescence and aging, alternative cellular senescence models, beyond doxorubicin, were established. These included exposure to D-galactose for 48 hours and an assessment of PSELT’s impact on distinct molecular traits of cellular senescence. Additionally, in vivo experimental protocols were started on a physiologically ageing mouse model (C57BL/6J at 22-24 months of age) to explore the role of the endogenous protein SELENOT in the intracellular mechanisms underlying cardiovascular ageing.
Molecular analyses were conducted to evaluate the potential protective action of the selenoprotein T (SELENOT)-derived peptide, PSELT, in counteracting senescence-associated secretory phenotype (SASP) in senescent human cardiomyocytes. These analyses focused on assessing the expression levels of specific molecular markers including MMP-3, IL-6 and TNF-α. In the context of doxorubicin (Dox)-induced cardiomyocyte senescence, the potential beneficial effects of PSELT against oxidative stress were also investigated. These assessments included the evaluation of intracellular reactive oxygen species (ROS), mitochondrial superoxide production, and the activity of endogenous antioxidant enzymes such as superoxide dismutase (SOD) and catalase.
To gain deeper insights into the role of PSELT/SELENOT in the context of cardiomyocyte senescence, and in order to discriminate the contribution of the endogenous protein SELENOT in PSELT-induced cell protection, the effects of SELENOT knockdown were analyzed in terms of cardiomyocyte viability and DNA damage in senescent cardiomyocytes. These included the evaluation of protein expression of a selective markers of DNA damage as p-γH2AX in both control and SELENOT siRNA- transfected senescent cardiomyocytes.
In vivo experimental protocols using a physiologically ageing mouse model (C57BL/6J at 22-24 months of age) have been completed. Organs and tissues were collected for further histological, biochemical and molecular analyses, aimed at investigating the effects of PSELT on intracellular mechanisms underlying cardiovascular ageing.
Scientific papers:
Abstract to national/international conferences:
2024