Stephanie M. Adaniya

Mitochondrial fragmentation frequently occurs in chronic pathological conditions as seen in various human diseases. In fact, abnormal mitochondrial morphology and mitochondrial dysfunction are hallmarks of heart failure (HF) in both human patients and HF animal models. A link between mitochondrial fragmentation and cardiac pathologies has been widely proposed, but the physiological relevance of mitochondrial fission and fusion in the heart is still unclear. Recent studies have increasingly shown that posttranslational modifications (PTMs) of fission and fusion proteins are capable of directly modulating the stability, localization, and/or activity of these proteins. These PTMs include phosphorylation, acetylation, ubiquitination, conjugation of small ubiquitin-like modifier proteins, O-linked- N-acetyl-glucosamine glycosylation, and proteolysis. Thus, understanding the PTMs of fission and fusion proteins may allow us to understand the complexities that determine the balance of mitochondrial fission and fusion as well as mitochondrial function in various cell types and organs including cardiomyocytes and the heart. In this review, we summarize present knowledge regarding the function and regulation of mitochondrial fission and fusion in cardiomyocytes, specifically focusing on the PTMs of each mitochondrial fission/fusion protein. We also discuss the molecular mechanisms underlying abnormal mitochondrial morphology in HF and their contributions to the development of cardiac diseases, highlighting the crucial roles of PTMs of mitochondrial fission and fusion proteins. Finally, we discuss the future potential of manipulating PTMs of fission and fusion proteins as a therapeutic strategy for preventing and/or treating HF.

Key points Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent G q protein‐coupled receptor (G q PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of G q PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin‐like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD‐specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent G q PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking G q PCR signalling with the regulation of mitochondrial morphology and function. Abstract Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of G q protein‐coupled receptor (G q PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that G q PCR stimulation induced by α 1 ‐adrenergic stimulation mediates mitochondrial fragmentation in a fission‐ and PKD‐dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon G q PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin‐like protein 1 (DLP1), at S637. PKD‐dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac‐specific overexpression of constitutively active Gα q protein. In conclusion, G q PCR‐PKD signalling induces mitochondrial fragmentation and dysfunction via PKD‐dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD‐specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD‐dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and G q PCR signalling.

Abnormal mitochondrial morphology, especially fragmented mitochondria, and mitochondrial dysfunction are hallmarks of a variety of human diseases including heart failure (HF). Although emerging evidence suggests a link between mitochondrial fragmentation and cardiac dysfunction, it is still not well described which cardiac signaling pathway regulates mitochondrial morphology and function under pathophysiological conditions such as HF. Mitochondria change their shape and location via the activity of mitochondrial fission and fusion proteins. This mechanism is suggested as an important modulator for mitochondrial and cellular functions including bioenergetics, reactive oxygen species (ROS) generation, spatiotemporal dynamics of Ca2+ signaling, cell growth, and death in the mammalian cell- and tissue-specific manners. Recent reports show that a mitochondrial fission protein, dynamin-like/related protein 1 (DLP1/Drp1), is post-translationally modified via cell signaling pathways, which control its subcellular localization, stability, and activity in cardiomyocytes/heart. In this review, we summarize the possible molecular mechanisms for causing post-translational modifications (PTMs) of DLP1/Drp1 in cardiomyocytes, and further discuss how these PTMs of DLP1/Drp1 mediate abnormal mitochondrial morphology and mitochondrial dysfunction under adrenergic signaling activation that contributes to the development and progression of HF.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.