Aspalathus linearis (Rooibos) regulates mitochondrial bioenergetics and dynamics in cardiomyoblasts under angiotensin II-induced stress

• Rooibos protects against Ang-II-induced mitochondrial stress. • Rooibos improves mitochondrial efficiency by reducing proton leak and enhancing complex I-linked oxidative phosphorylation and ATP production. • Rooibos reduces mitochondrial apoptosis by downregulating VDAC1 and caspase-3 activity and restoring cytochrome c response. • Rooibos modulates mitochondrial dynamics by increasing inhibitory phosphorylation of Drp1, reducing mitochondrial fragmentation. • Rooibos demonstrates similar protective mechanisms to Losartan. Cardiovascular diseases are closely associated with maladaptive cardiac remodelling processes, including mitochondrial dysfunction, redox imbalance, and cellular hypertrophy. Although conventional pharmacotherapies are widely used, they often come with undesirable side effects, prompting interest in safer and more accessible alternatives. Aspalathus linearis (Rooibos), a South African herbal tea recognized for its antioxidant capacity, has shown promise in cardiovascular research. However, its influence on mitochondrial quality control and bioenergetics under pathological stress remains unclear. This study explored the mitochondrial-protective properties of Rooibos in H9C2 cardiomyoblasts exposed to angiotensin II (Ang-II)-induced stress. Cardiomyoblasts were incubated for 48 hours under the following conditions: Ang-II (20 μM), Rooibos extract (100μg/ml), a combination of Ang-II and Rooibos, and Ang-II with Losartan (10 μM) as a positive control. Mitochondrial function was assessed using high-resolution respirometry to evaluate respiratory states, while ATP levels, citrate synthase activity, autophagy, and caspase-3 activity were quantified through biochemical assays. Mitochondrial dynamics and membrane integrity were examined by assessing proteins involved in fission and fusion using western blotting. Ang-II enhanced routine and leak respiration, complex I-linked OXPHOS, electron transport system (ETS) capacity, and the contribution of the S-pathway, F-pathway, and complex-II to the ETS capacity, while decreasing ATP levels and promoting mitochondrial apoptosis and autophagy. It also disrupted mitochondrial dynamics and membrane stability, evidenced by altered Drp1, reduced Mitofusin 1 and OPA1, and enhanced VDAC1. Rooibos co-treatment improved mitochondrial respiration, preserved mitochondrial membrane integrity, attenuated mitochondrial apoptotic responses, and rebalanced mitochondrial dynamics. These findings highlight that the protective effects of Rooibos are mediated by its ability to improve mitochondrial function despite the presence of Ang-II-induced stress. This is the first in-depth report of how RB can modulate mitochondrial pathways to induce cardioprotection in conditions of Ang-II-induced stress.