Michael L. Kamradt

BACKGROUND: Calcium (Ca ²⁺ ) dysregulation is a hallmark of heart failure, impairing excitation-contraction coupling and contributing to pathological remodeling. The sarco/endoplasmic reticulum Ca ²⁺ ATPase isoform 2a (SERCA2a) mediates Ca ²⁺ reuptake into the sarcoplasmic reticulum (SR) during diastole, but its activity declines in failing hearts. DWORF (dwarf open reading frame), a newly identified cardiac microprotein, enhances SERCA2a activity and improves cardiomyocyte Ca ²⁺ cycling and contractility. SR Ca ²⁺ release also influences mitochondrial metabolism and ATP production. Here, we investigated whether DWORF overexpression improves SR Ca ²⁺ handling, augments mitochondrial Ca ²⁺ signaling, and protects against heart failure progression. METHODS: Transgenic and adeno-associated virus approaches were used to overexpress DWORF in the heart. Mice underwent transverse aortic constriction (TAC) to model pressure overload–induced heart failure. Cardiac function, mitochondrial metabolism, SR Ca ²⁺ uptake, and remodeling were assessed. RESULTS: Mitochondria from DWORF transgenic hearts displayed increased basal respiration, maximal respiration, and spare respiratory capacity, correlating with enhanced mitochondrial Ca ²⁺ uptake kinetics. Western blot analysis showed elevated levels of active PDH (pyruvate dehydrogenase) and mitochondrial Ca ²⁺ uniporter expression in DWORF transgenic hearts, supporting a role for DWORF in Ca ²⁺ -driven metabolic regulation. Similarly, MyoAAV (myo-adeno-associated virus)-mediated DWORF overexpression enhanced mitochondrial respiration and increased levels of active PDH in adult mice. Following TAC, MyoAAV-DWORF–treated mice maintained higher left ventricular function and were protected from further deterioration compared with controls. This benefit was observed when DWORF gene therapy was delivered preventively at the time of pressure overload or after heart failure was already established. DWORF gene therapy also attenuated remodeling, with lower heart weight and lung weight-to-tibia length ratios. Seahorse analysis confirmed sustained mitochondrial improvements in both treatment paradigms. CONCLUSIONS: DWORF overexpression enhances SR Ca ²⁺ dynamics, improves mitochondrial energetics, and attenuates pathological remodeling and heart failure progression in response to pressure overload. These findings support DWORF as a promising therapeutic target for heart failure.

Mitochondria rely upon the coordination of protein import, protein translation, and proper functioning of oxidative phosphorylation (OXPHOS) complexes I–V to sustain the activities of life for an organism. Each process is dependent upon the function of profoundly large protein complexes found in the mitochondria [translocase of the outer mitochondrial membrane (TOMM) complex, translocase of the inner mitochondrial membrane (TIMM) complex, OXPHOS complexes, mitoribosomes]. These massive protein complexes, in some instances more than one megadalton, are built up from numerous protein subunits of varying sizes, including many proteins that are ≤100–150 amino acids. However, these small proteins, termed microproteins, not only act as cogs in large molecular machines but also have important steps in inhibiting or promoting the intrinsic pathway of apoptosis, coordinate responses to cellular stress, and even act as hormones. This review focuses on microproteins that occupy the mitochondria and are critical for its function. Although the microprotein field is relatively new, researchers have long recognized the existence of these mitochondrial proteins as critical components of virtually all aspects of mitochondrial biology. Thus, recent studies estimating that hundreds of new microproteins of unknown function exist and are missing from current genome annotations suggests that the mitochondrial “microproteome” is a rich area for future biological investigation.

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and β (IKKα/β) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK^−/− cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.