Mediterranean Diet Adherence Is Associated with Mitochondrial Microproteins Humanin and SHMOOSE; Potential Role of the Humanin–Nox2 Interaction in Cardioprotection

Roberto Vicinanza^1,2*Vittoria Cammisotto²Junxiang Wan¹Kelvin Yen¹Daniele Pastori²Francesco Violi²Pasquale Pignatelli²Pinchas Cohen¹

• ¹University of Southern California Leonard Davis School of Gerontology, Los Angeles, United States
• ²Department of Clinical, Internal, Anaesthesiologic and Cardiovascular Sciences, Universita degli Studi di Roma La Sapienza, Rome, Italy

Background: The health benefits of the Mediterranean Diet (Med-Diet) have been demonstrated in observational studies and randomized controlled trials. Emerging evidence suggests that the biological effects of the Med-Diet may be mediated by the modulation of mitochondrial function. Human mitochondrial DNA (mtDNA) encodes microproteins, which have been shown to regulate aging, cardiometabolic functions, and neuroprotection. Objectives: To investigate Humanin and SHMOOSE (Small Human Mitochondrial ORF Over SErine tRNA), as potential mitochondrial biomarkers of Med-Diet adherence and their associations with oxidative stress biomarkers. Methods: Cross-sectional analysis of 49 patients (mean age 78.4 ± 8.7 years; 57% female) selected from an observational study of non-valvular atrial fibrillation (AF) conducted at the Atherothrombosis Center of Sapienza University of Rome. Patients were categorized into low-medium (0-6) and high (7-9) adherence to the Med-Diet based on the 9-item Med-Diet questionnaire. Oxidative stress was evaluated by measuring soluble Nox2-derived peptide (sNox2-dp) and plasma 8-iso-prostaglandin F2α (8-iso-PGF2α) using enzyme-linked immunosorbent assays (ELISA). Circulating Humanin and SHMOOSE levels were measured using an in-house sandwich ELISA. Results: High Med-Diet adherence was observed in 21 patients (43%), while 28 patients (57%) had low–medium adherence. Patients with high adherence exhibited higher plasma levels of SHMOOSE (p=0.046) and Humanin (p=0.045). The analysis of the dietary components of the Med-Diet revealed higher levels of SHMOOSE with olive oil consumption (p=0.020) and low intake of refined bread (p=0.029), while Humanin positively correlated with olive oil (p=0.0069), fish (p=0.038), and legumes (p=0.0282). Additionally, Humanin was inversely associated with sNox2-dp (p=0.019), which remained significant after adjusting for sex and BMI (B = -0.010; β = -0.302; p=0.040), and 8-iso-PGF2α (p=0.049). Conclusions: This study indicates i) a positive association between adherence to the Med-Diet and circulating levels of mitochondrial microproteins SHMOOSE and Humanin supporting their role as potential mediators of Med-Diet benefits; ii) a putative crosstalk between Humanin signaling and Nox2 activity, suggesting a novel cardioprotective mechanism of the Med-Diet. Collectively, these findings support mitochondrial microproteins as promising biomarkers for tailoring nutritional strategies for healthy aging. Further studies are warranted to elucidate the underlying mechanisms and determine the causal nature of these associations.