Abstract 19341: The Role of Mul1, a Mitochondrial Localized E3 Ligase, in the Heart

Background: Mitochondrial (Mito) quality is a key determinant of cardiac health and pathology. Mito quality control is maintained in part through fusion and fission of the Mito network and removal of damaged organelles through mitophagy. However, the molecular underpinnings of maintaining a healthy intracellular Mito pool are unclear. A Mito localized ubiquitin/SUMO E3 ligase, Mul1, has been suggested as a critical regulator of Mito quality control; however, the role of Mul1 in the heart, a Mito-dependent organ, has not yet been characterized. Methods: We used adenovirus-mediated delivery of Mul1 overexpression (Adv-Mul1) and short hairpin RNA Mul1 knockdown (Adv-ShMul1) to neonatal rat ventricular cardiomyocytes (CMs) and assessed Mito morphology, function and overall cell survival. Results: Adv-Mul1 localized to the outer Mito membrane, similar to endogenous Mul1. Adv-Mul1 dramatically altered Mito morphology from tubular to punctate, as well as reduced Mito membrane potential (ΔΨm). Electron microscopy demonstrated that Adv-Mul1 induced cristae remodeling, which may explain the loss of respiratory function analyzed by the Seahorse system. In addition, Adv- Mul1 increased mitochondrial calcium levels and cytochrome c release, resulting in increased cell death. Importantly, Mul1 knockdown using Adv-ShMul1 rescued CCCP-induced reduction in Mito ΔΨm, suggesting Mul1 critically regulates Mito quality control in CMs. Adv- Mul1 results in degradation of Mfn2 and Drp1, key regulators of Mito dynamics. In Adv-Mul1 CMs, adenovirus-mediated normalization of Drp1 levels restored Mito ΔΨm and cell death, but not Mito fragmentation, suggesting a requirement of both Mfn2 and Drp1 proteins. To determine if Mul1 functions in part through its post-translational modification of substrates via RING domain, we constructed a RING domain truncated Mul1, which was unable to alter the Mito respiration or induce cell death. This data suggests that Mul1 mediated Mito structural and functional alterations are dependent on its RING domain. Conclusion: Mul1 induces Mito dysfunction and cell death in CMs, which is mediated by its RING domain and downregulation of Drp1. Thus, our work helps characterize a novel regulator of Mito quality control in the heart.