The application of doxorubicin (DOX) is hampered by cardiotoxicity, with diastolic dysfunction as the earliest manifestation. Fibrosis leads to impaired relaxation, but the mechanisms that operate shortly after DOX exposure are not clear. We asked whether the activation of cardiac fibroblasts (CFs) anticipates myocardial dysfunction and evaluated the effects of DOX on CF metabolism. CFs were isolated from the hearts of rats after the first injection of DOX. In another experiment, CFs were exposed to DOX in vitro. Cell phenotype and metabolism were determined. Early effects of DOX consisted of diastolic dysfunction and unchanged ejection fraction. Markers of pro-fibrotic remodeling and evidence of CF transformation were present immediately after treatment completion. Oxygen consumption rate and extracellular acidification revealed an increased metabolic activity of CFs and a switch to glycolytic energy production. These effects were consistent in CFs isolated from the hearts of DOX-treated animals and in naïve CFs exposed to DOX in vitro. The metabolic switch was paralleled with the phenotype change of CFs that upregulated markers of myofibroblast differentiation and the activation of pro-fibrotic signaling. In conclusion, the metabolic switch and activation of CFs anticipate DOX-induced damage and represent a novel target in the early phase of anthracycline cardiomyopathy.

Sarcopenia is defined as the loss of muscle mass associated with reduced strength leading to poor quality of life in elderly people. The decline of skeletal muscle performance is characterized by bioenergetic impairment and severe oxidative stress, and does not always strictly correlate with muscle mass loss. We chose to investigate the ability of the metabolic modulator Ranolazine to counteract skeletal muscle dysfunctions that occur with aging. For this purpose, we treated aged C57BL/6 mice with Ranolazine/vehicle for 14 days and collected the tibialis anterior and gastrocnemius muscles for histological and gene expression analyses, respectively. We found that Ranolazine treatment significantly increased the muscle strength of aged mice. At the histological level, we found an increase in centrally nucleated fibers associated with an up-regulation of genes encoding MyoD, Periostin and Osteopontin, thus suggesting a remodeling of the muscle even in the absence of physical exercise. Notably, these beneficial effects of Ranolazine were also accompanied by an up-regulation of antioxidant and mitochondrial genes as well as of NADH-dehydrogenase activity, together with a more efficient protection from oxidative damage in the skeletal muscle. These data indicate that the protection of muscle from oxidative stress by Ranolazine might represent a valuable approach to increase skeletal muscle strength in elderly populations.

Leydig cells (LCs) represent a somatic testicular population responsible for testosterone synthesis, a hormone essential for spermatogenesis and male fertility. The obesity condition impairs LC steroidogenic activity, contributing to testicular oxidative stress and male reproductive dysfunctions. Using a high-fat-diet (HFD) murine model, we investigated the regulatory role of the nuclear factor of activated T cells 5 (NFAT5s) in the obesity-induced LC damage and the resulting alterations in intergenerationally inherited sperm circRNA cargo. Our findings reveal a significant upregulation of both circNFAT5 and NFAT5 protein levels in HFD testis. This molecular signature correlated with decreased antioxidant defense system, increased LC apoptosis, and impaired steroidogenesis. In vitro experiments, performed in TM3 cells, confirmed that NFAT5 nuclear shuttling drives proapoptotic gene activation, while NFAT5 silencing promotes LC survival. The analysis of HFD progeny (F1H) revealed a full recovery of testis oxidative status and LC apoptosis, linked with the recovery of NFAT5 expression. However, a steroidogenic deficiency persisted in F1H offspring. Notably, HFD and F1H epididymides exhibited NFAT5 overexpression concomitantly with impaired sperm morphology, motility, viability, and altered sperm circRNA profiles alongside a deregulated 4-hydroxy-2-nonenal (4HNE) profile, a marker of sperm oxidative stress. Lastly, an enhanced FUS-related amplification of circRNA perturbations was highlighted in F1H spermatozoa. Collectively, our findings reveal a dual functional role of NFAT5 as a testicular regulator of LC fate and an epididymal sentinel of metabolic stress, in turn linking paternal obesity to the persistent transmission of sperm epigenetic anomalies across the offspring.