Effects of sacubitril-valsartan on aging-related cardiac dysfunction.

Heart failure (HF) remains a huge medical burden worldwide, with aging representing a major risk factor. Here, we report the effects of sacubitril/valsartan, an approved drug for HF with reduced EF, in an experimental model of aging-related HF with preserved ejection fraction (HFpEF). Eighteen-month-old female Fisher 344 rats were treated for 12 weeks with sacubitril/valsartan (60 mg/kg/day) or with valsartan (30 mg/kg/day). Three-month-old rats were used as control. No differential action of sacubitril/valsartan versus valsartan alone, either positive or negative, was observed. The positive effects of both sacubitril/valsartan and valsartan on cardiac hypertrophy was evidenced by a significant reduction of wall thickness and myocyte cross-sectional area. Contrarily, myocardial fibrosis in aging heart was not reduced by any treatment. Doppler echocardiography and left ventricular catheterization evidenced diastolic dysfunction in untreated and treated old rats. In aging rats, both classical and non-classical renin-angiotensin-aldosterone system (RAAS) were modulated. In particular, with respect to untreated animals, both sacubitril/valsartan and valsartan showed a partial restoration of cardioprotective non-classical RAAS. In conclusion, this study evidenced the favorable effects, by both treatments, on age-related cardiac hypertrophy. The attenuation of cardiomyocyte size and hypertrophic response may be linked to a shift towards cardioprotective RAAS signaling. However, diastolic dysfunction and cardiac fibrosis persisted despite of treatment and were accompanied by myocardial inflammation, endothelial activation, and oxidative stress.

Glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter 2 inhibitors, anti-diabetic drugs in heart failure and cognitive impairment: potential mechanisms of the protective effects.

Heart failure and cognitive impairment emerge as public health problems that need to be addressed due to the aging global population. The conditions that often coexist are strongly related to advancing age and multimorbidity. Epidemiological evidence indicates that cardiovascular disease and neurodegenerative processes shares similar aspects, in term of prevalence, age distribution, and mortality. Type 2 diabetes increasingly represents a risk factor associated not only to cardiometabolic pathologies but also to neurological conditions. The pathophysiological features of type 2 diabetes and its metabolic complications (hyperglycemia, hyperinsulinemia, and insulin resistance) play a crucial role in the development and progression of both heart failure and cognitive dysfunction. This connection has opened to a potential new strategy, in which new classes of anti-diabetic medications, such as glucagon-like peptide-1 receptor (GLP-1R) agonists and sodium-glucose cotransporter 2 (SGLT2) inhibitors, are able to reduce the overall risk of cardiovascular events and neuronal damage, showing additional protective effects beyond glycemic control. The pleiotropic effects of GLP-1R agonists and SGLT2 inhibitors have been extensively investigated. They exert direct and indirect cardioprotective and neuroprotective actions, by reducing inflammation, oxidative stress, ions overload, and restoring insulin signaling. Nonetheless, the specificity of pathways and their contribution has not been fully elucidated, and this underlines the urgency for more comprehensive research.

Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in 'Land of Fires', Italy.

STUDY QUESTION: Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? SUMMARY ANSWER: Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. WHAT IS KNOWN ALREADY: Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood-testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the 'Land of Fires', has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. STUDY DESIGN, SIZE, DURATION: Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the 'Land of Fires' (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80-40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. MAIN RESULTS AND THE ROLE OF CHANCE: Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. LIMITATIONS, REASONS FOR CAUTION: Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. WIDER IMPLICATIONS OF THE FINDINGS: Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by 'Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania' (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Cytochalasin D restores nuclear size acting on F-actin and IZUMO1 localization in low-quality spermatozoa.

In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events.

Dapagliflozin protects the kidney in a non-diabetic model of cardiorenal syndrome.

Cardiorenal syndrome encompasses a spectrum of disorders involving heart and kidney dysfunction, and sharing common risk factors, such as hypertension and diabetes. Clinical studies have shown that patients with and without diabetes may benefit from using sodium-glucose cotransporter 2 inhibitors to reduce the risk of heart failure and ameliorate renal endpoints. Because the underlying mechanisms remain elusive, we investigated the effects of dapagliflozin on the progression of renal damage, using a model of non-diabetic cardiorenal disease. Dahl salt-sensitive rats were fed a high-salt diet for five weeks and then randomized to dapagliflozin or vehicle for the following six weeks. After treatment with dapagliflozin, renal function resulted ameliorated as shown by decrease of albuminuria and urine albumin-to-creatinine ratio. Functional benefit was accompanied by a decreased accumulation of extracellular matrix and a reduced number of sclerotic glomeruli. Dapagliflozin significantly reduced expression of inflammatory and endothelial activation markers such as NF-κB and e-selectin. Upregulation of pro-oxidant-releasing NADPH oxidases 2 and 4 as well as downregulation of antioxidant enzymes were also counteracted by drug treatment. Our findings also evidenced the modulation of both classic and non-classic renin-angiotensin-aldosterone system (RAAS), and effects of dapagliflozin on gene expression of ion channels/transporters involved in renal homeostasis. Thus, in a non-diabetic model of cardiorenal syndrome, dapagliflozin provides renal protection by modulating inflammatory response, endothelial activation, fibrosis, oxidative stress, local RAAS and ion channels.

Initial Phase of Anthracycline Cardiotoxicity Involves Cardiac Fibroblasts Activation and Metabolic Switch.

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.

Ranolazine Counteracts Strength Impairment and Oxidative Stress in Aged Sarcopenic Mice.

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.

Sodium-Glucose Cotransporter 2 Inhibitors and Heart Failure: A Bedside-to-Bench Journey.

Type 2 diabetes mellitus (T2DM) and heart failure (HF) are multifactorial diseases sharing common risk factors, such as obesity, hyperinsulinemia, and inflammation, with underlying mechanisms including endothelial dysfunction, inflammation, oxidative stress, and metabolic alterations. Cardiovascular benefits of sodium-glucose cotransporter 2 (SGLT2) inhibitors observed in diabetic and non-diabetic patients are also related to their cardiac-specific, SGLT-independent mechanisms, in addition to the metabolic and hemodynamic effects. In search of the possible underlying mechanisms, a research campaign has been launched proposing varied mechanisms of action that include intracellular ion homeostasis, autophagy, cell death, and inflammatory processes. Moreover, the research focus was widened toward cellular targets other than cardiomyocytes. At the moment, intracellular sodium level reduction is the most explored mechanism of direct cardiac effects of SGLT2 inhibitors that mediate the benefits in heart failure in addition to glucose excretion and diuresis. The restoration of cardiac Na+ levels with consequent positive effects on Ca2+ handling can directly translate into improved contractility and relaxation of cardiomyocytes and have antiarrhythmic effects. In this review, we summarize clinical trials, studies on human cells, and animal models, that provide a vast array of data in support of repurposing this class of antidiabetic drugs.

Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides.

Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies.

Paracrine recruitment and activation of fibroblasts by c-Myc expressing breast epithelial cells through the IGFs/IGF-1R axis.

Fibroblasts are among the most abundant stromal cells in the tumor microenvironment (TME), progressively differentiating into activated, motile, myofibroblast-like, protumorigenic cells referred to as Cancer-Associated Fibroblasts (CAFs). To investigate the mechanisms by which epithelial cells direct this transition, the early stages of tumorigenesis were exemplified by indirect cocultures of WI-38 or human primary breast cancer fibroblasts with human mammary epithelial cells expressing an inducible c-Myc oncogene (MCF10A-MycER). After c-Myc activation, the conditioned medium (CM) of MCF10A-MycER cells significantly enhanced fibroblast activation and mobilization. As this was accompanied by decreased insulin-like growth factor binding protein-6 (IGFBP-6) and increased insulin-like growth factor-1 and IGF-II (IGF-I, IGF-II) in the CM, IGFs were investigated as key chemotactic factors. Silencing IGFBP-6 or IGF-I or IGF-II expression in epithelial cells or blocking Insulin-like growth factor 1 receptor (IGF-1R) activity on fibroblasts significantly altered fibroblast mobilization. Exposure of WI-38 fibroblasts to CM from induced MCF10A-MycER cells or to IGF-II upregulated FAK phosphorylation on Tyr397 , as well as the expression of α-smooth muscle actin (α-SMA), features associated with CAF phenotype and increased cell migratory/invasive behavior. In three-dimensional (3D)-organotypic assays, WI-38 or human primary fibroblasts, preactivated with either CM from MCF10A-MycER cells or IGFs, resulted in a permissive TME that enabled nontransformed MCF10A matrix invasion. This effect was abolished by inhibiting IGF-1R activity. Thus, breast epithelial cell oncogenic activation and stromal fibroblast transition to CAFs are linked through the IGFs/IGF-1R axis, which directly promotes TME remodeling and increases tumor invasion.