SARCOPENIA, LIVER AND CARDIOVASCULAR ALTERATIONS IN A MULTICENTRIC COHORT OF PATIENTS WITH MASLD: EVALUATION OF CLINICAL PRESENTATION AND GENETIC PREDISPOSITION

Background and Aims: Sarcopenia, characterized by the progressive loss of skeletal muscle mass (SMM), strength, and function, is not solely an age-related condition but is also associated with several diseases, including chronic liver disease (CLD) and cardiovascular (CV) disease. Metabolic-dysfunction associated steatotic liver disease (MASLD) is defined by the presence of liver steatosis combined with, at least, one cardiometabolic risk factor, with liver fibrosis serving as a critical determinant of liver-related and CV-related events and mortality. Sarcopenia is highly prevalent in advanced MASLD, although recent studies have highlighted myosteatosis, the accumulation of fat within muscle, as a key driver of liver disease progression. Studies conducted mainly in Asia have reported a high prevalence of sarcopenia even in early MASLD, though it is not routinely assessed. Both sarcopenia and MASLD contribute to atherosclerotic CV damage, especially in the presence of advanced liver fibrosis, and share common pathogenetic mechanisms such as insulin-resistance (IR), low-grade chronic systemic inflammation, mitochondrial dysfunction, and oxidative stress. Additionally, genetic predisposition plays a role in MASLD, with specific polymorphisms in PNPLA3, TM6SF2, MBOAT7, and GCKR promoting severe liver disease, while the HSD17B13 variant appears protective against MASLD. Despite this background, most studies evaluating the relationship between sarcopenia, MASLD, and CV disease are cross-sectional, primarily involving Asian populations, limiting the generalizability of findings and the ability to determine whether the relationship is bidirectional. Furthermore, the absence of standardized criteria for diagnosing sarcopenia in MASLD complicates comparisons across studies, leading to conflicting results. To date, no studies have yet explored the role of genetic variants predisposing to MASLD in the risk of developing sarcopenia and CV damage. This project aims to address these gaps by 1) evaluating the impact of sarcopenia on liver and CV damage in non-cirrhotic MASLD patients, 2) investigating the influence of MASLD-related genetic variants on muscle mass and CV damage, and 3) assessing changes in muscle, liver, and CV parameters over a 5-year follow-up in a subset of patients. Methods: 856 non-cirrhotic MASLD patients were recruited from liver outpatient clinics in Milan, Palermo, and London. At enrollment, anthropometric, demographic, and laboratory data were collected. Liver steatosis was assessed using abdominal ultrasound (US) and controlled attenuation parameter (CAP) measurements via Fibroscan, while liver fibrosis was evaluated using liver stiffness measurement (LSM) at Fibroscan. Severe steatosis was defined as CAP > 280 dB/m, and advanced fibrosis was identified as LSM ≥ 8 kPa. Liver biopsies were performed on 370 patients. Sarcopenia was assessed using bioelectrical impedance analysis (BIA), a non-invasive and radiation-free technique, with the skeletal muscle index (SMI) calculated as SMM/height^2. Patients were divided into gender-specific tertiles based on SMI (lowest, middle, and highest). CV damage was evaluated using carotid doppler US, with carotid intima-media thickness (cIMT) ≥ 0.9 mm or carotid plaques indicating CV damage. In 502 patients, epicardial fat thickness (EFT) was measured via echocardiography, and carotid-femoral pulse wave velocity (cfPWV) via tonometry. EFT ≥ 9.5/7.5 mm in men/women was considered a marker of visceral adiposity, while EFT ≥ 5.2 mm was associated with coronary artery disease. cfPWV > 10 m/sec was used as a marker of arterial stiffness. CV risk was calculated for all patients using SCORE2 and SCORE2-OP algorithms according to European Society of Cardiology (ESC) guidelines, with SCORE2-Diabetes applied to diabetic patients. Genetic polymorphisms in PNPLA3, TM6SF2, MBOAT7, GCKR, and HSD17B13 were analyzed in 437 patients. Finally, 104 patients underwent a 5-year follow-up to reassess muscle mass at BIA, CAP and LSM, cIMT and carotid plaques, and EFT. Results: Patients had a mean age of 51 years, with 63% males. Patients in the lowest SMI tertile were older (54 vs 48 years, p<0.001), had lower body mass index (BMI) (27 vs 33.4 kg/m2, p<0.001), lower waist circumference (WC) (99 vs 110 cm, p<0.001), higher prevalence of dyslipidemia (56% vs 46%, p=0.03) compared to those in the highest tertile. Regarding liver damage, patients in the lowest SMI tertile had lower CAP (293 vs 317 dB/m, p<0.001), LSM (4.9 vs 6.4 kPa, p<0.001), grade 3 fibrosis prevalence (20% vs 25%, p=0.009), and MASH rates (30% vs 53%, p=0.04) compared to those in the highest tertile. For CV damage, patients in the lowest SMI tertile had a higher percentage of patients with increased cIMT (p=0.01). No significant associations were observed between SMI and the genetic polymorphisms studied. In the lowest SMI tertile, PNPLA3 CG/GG variant was linked to increased LSM (OR 1.82, p=0.03), lower cIMT (OR 0.39, p=0.04), and increased cfPWV (OR 2.1, p=0.01). The TM6SF2 wild-type allele was associated with increased cIMT (OR 3.4, p=0.004), while the CT/TT variant correlated with reduced hypertension (OR 0.09, p=0.02) and lower WC (OR 0.25, p=0.02) in the lowest SMI tertile. In multivariate analysis, low SMI was significantly associated with lower BMI (OR 0.61, p<0.001), lower WC (OR 0.92, p=0.001), female sex (OR 2.21, p=0.008), increased cIMT (OR 2.10, p=0.02), and slightly older age (OR 1.04, p=0.002). Among hypertensive MASLD patients, low SMI was independently associated with increased cIMT (OR 2.06, p=0.03) and cfPWV (OR 2.73, p=0.03). In non-diabetic MASLD patients, low muscle mass was associated with increased cIMT (OR 1.66, p=0.03), carotid plaques (OR 1.56, p=0.02), less fibrosis on histology (OR 0.29, p=0.04), lower ALT levels (OR 0.54, p=0.001), higher LDL (OR 4.38, p=0.02), and lower HDL (OR 1.03, p=0.002). At 5-year follow-up, a significant reduction in mean BMI (29.1 vs 28.4, p=0.001), a decrease in patients in the lowest SMI tertile (42% vs 22%, p<0.001), and a regression of steatosis in 9% of patients, with significant reductions in CAP (300 vs 289 dB/m, p=0.02) and LSM≥8 kPa prevalence (17% vs 7%, p<0.001) were observed. Regarding CV damage, a significant increase in carotid plaques (34% vs 54%, p<0.001), mean EFT (7.5 vs 9.0 mm, p<0.001), and high CV risk categorization (56% vs 63%, p=0.004) were observed. Baseline low muscle mass was significantly associated with changes in delta LSM (HR 2.30, p=0.013), BMI (HR 0.61, p=<0.001), and dyslipidemia (HR 5.74, p=0.007) at follow-up. Discussion: This multicentric study revealed a significant independent relationship between sarcopenia and increased markers of atherosclerotic damage in non-cirrhotic MASLD patients, particularly among those with hypertension and without diabetes. Notably, patients with low muscle mass exhibited a lower prevalence of liver damage, possibly due to reduced visceral adiposity. However, longitudinal analysis demonstrated that low muscle mass was independently associated with an increase in mean LSM, suggesting that sarcopenia contributes to liver damage progression over time. Additionally, while persistent low muscle mass appeared to exacerbate CV damage, a definitive independent association was not observed, possibly due to the relatively short duration of follow-up. The study also identified a protective effect of the TM6SF2 polymorphism against CV damage, underscoring its potential role in mitigating CV risk. In conclusion, this research highlights the importance of integrating imaging techniques to evaluate both liver and CV damage alongside genetic profiling for a more holistic understanding of the interplay between sarcopenia and CV damage in non-cirrhotic MASLD patients. Preserving muscle mass emerges as a key strategy for preventing both liver and CV diseases, and future public health initiatives should prioritize interventions aimed at supporting and enhancing muscle mass health.