Introduction: Hypertension induces progressive cardiac remodeling, including myocardial fibrosis and impaired ventricular function [1]. Increasing evidence suggests that organ protection independent of blood pressure reduction is clinically relevant. Betaine, a naturally occurring osmolyte, is depleted in hypertensive states and may contribute to organ vulnerability [2, 3]. However, the long-term effects of betaine supplementation on cardiac structure and function remain unclear.

Aim: This study aimed to determine whether chronic betaine supplementation improves cardiac function and attenuates markers of organ damage in aged spontaneously hypertensive rats (SHR).

Methods: Male SHR (n=20) were treated from 8 to 78 weeks of age with either tap water (control) or betaine supplementation (500 mg/l in drinking water, a dose of approximately 78 ± 15 mg/kg per day.  Due to attrition unrelated to the current study, number of animals included in the final analysis were n=8 (control) and n=9 (betaine). Metabolic cage studies assessed 24-hour water and electrolyte balance. Echocardiography was performed. Arterial blood pressure (BP) and heart rate were recorded under urethane anaesthesia (1.5 g/kg, i.p.). Blood and urine samples were collected for biochemical analyses. Post-euthanasia, cardiac and renal tissues were harvested for histopathological and molecular evaluation. Tissue expression of renin-angiotensin system (RAS) components was analyzed via RT-qPCR, and plasma/urine choline metabolites were measured using UPLC. Data were analyzed using Student’s t-test or Mann-Whitney U test (p<0.05). All procedures complied with Directive 2010/63/EU and were approved by the local ethics committee.

Results: Betaine supplementation significantly increased 24-hour urine output (42.4±18.7 vs 33.3±6.9 mL, p=0.02) without affecting plasma or urinary electrolyte levels. Echocardiography revealed improved cardiac function, with higher ejection fraction in betaine-treated rats (69.0±13.01% vs 56.13±12.15%, p=0.048). Histopathological analysis demonstrated reduced signs of heart and kidney injury after betaine treatment (Figure 1). Above all, morphometric analysis revealed decreased myocardial fibrosis (p=0.014). Molecular analysis revealed minor alterations in RAS components, with increased cardiac AT1A receptor expression (p=0.047) and reduced renin expression in the renal medulla (p=0.05). Notably, betaine increased circulating choline-derived metabolites, including trimethylamine N-oxide (TMAO) and trimethylamine (TMA)(Table 1).

Conclusions: Chronic betaine supplementation improves cardiac function and reduces myocardial fibrosis, indicating a BP-independent cardioprotective effect. These effects may relate to increased diuresis and attenuation of pathological remodeling. These benefits may be linked to increased diuresis and the attenuation of pathological remodeling. However, the concomitant increase in potentially pro-atherogenic choline-derived metabolites suggests a complex metabolic profile that warrants further investigation.

Figure 1. Histopathological findings in Spontaneously Hypertensive Rats on water (SHR-Water) or betaine (SHR-Betaine). Values are means±SE, p values by t-test or U Mann Whitney, depending on the normality of the data distribution.

Table 1. Plasma and urinary concentrations of choline-derived metabolites, including trimethylamine N-oxide (TMAO), trimethylamine (TMA), betaine, glycerophosphocholine (GPC), and carnitine, in experimental groups.