Transcriptomic Profiling Identifies Ferroptosis-Related Gene Signatures in Ischemic Muscle Satellite Cells Affected by Peripheral Artery Disease-Brief Report.

BACKGROUND: We hypothesized that transcriptomic profiling of muscle satellite cells in peripheral artery disease (PAD) would identify damage-related pathways contributing to skeletal muscle myopathy. We identified a potential role for ferroptosis-a form of programmed lytic cell death by iron-mediated lipid peroxidation-as one such pathway. Ferroptosis promotes myopathy in ischemic cardiac muscle but has an unknown role in PAD. METHODS: Muscle satellite cells from donors with PAD were obtained during surgery. cDNA libraries were processed for single-cell RNA sequencing using the 10X Genomics platform. Protein expression was confirmed based on pathways inferred by transcriptomic analysis. RESULTS: Unsupervised cluster analysis of over 25 000 cells aggregated from 8 donor samples yielded distinct cell populations grouped by a shared unique transcriptional fingerprint. Quiescent cells were diminished in ischemic muscle while myofibroblasts and apoptotic cells were prominent. Differential gene expression demonstrated a surprising increase in genes associated with iron transport and oxidative stress and a decrease in GPX4 (glutathione peroxidase 4) in ischemic PAD-derived cells. Release of the danger signal HMGB1 (high mobility group box-1) correlated with ferroptotic markers including surface transferrin receptor and were higher in ischemia. Furthermore, lipid peroxidation in muscle satellite cells was modulated by ferrostatin, a ferroptosis inhibitor. Histology confirmed iron deposition and lipofuscin, an inducer of ferroptosis in PAD-affected muscle. CONCLUSIONS: This report presents a novel finding that genes known to be involved in ferroptosis are differentially expressed in human skeletal muscle affected by PAD. Targeting ferroptosis may be a novel therapeutic strategy to reduce PAD myopathy.

Inflammatory Caspase Activity Mediates HMGB1 Release and Differentiation in Myoblasts Affected by Peripheral Arterial Disease.

Introduction: We previously showed that caspase-1 and -11, which are activated by inflammasomes, mediate recovery from muscle ischemia in mice. We hypothesized that similar to murine models, inflammatory caspases modulate myogenicity and inflammation in ischemic muscle disease. Methods: Caspase activity was measured in ischemic and perfused human myoblasts in response to the NLRP3 and AIM2 inflammasome agonists (nigericin and poly(dA:dT), respectively) with and without specific caspase-1 or pan-caspase inhibition. mRNA levels of myogenic markers and caspase-1 were assessed, and protein levels of caspases-1, -4, -5, and -3 were measured by Western blot. Results: When compared to perfused cells, ischemic myoblasts demonstrated attenuated MyoD and myogenin and elevated caspase-1 mRNA. Ischemic myoblasts also had significantly higher enzymatic caspase activity with poly(dA:dT) (p < 0.001), but not nigericin stimulation. Inhibition of caspase activity including caspase-4/-5, but not caspase-1, blocked activation effects of poly(dA:dT). Ischemic myoblasts had elevated cleaved caspase-5. Inhibition of caspase activity deterred differentiation in ischemic but not perfused myoblasts and reduced the release of HMGB1 from both groups. Conclusion: Inflammatory caspases can be activated in ischemic myoblasts by AIM2 and influence ischemic myoblast differentiation and release of pro-angiogenic HMGB1. AIM2 inflammasome involvement suggests a role as a DNA damage sensor, and our data suggest that caspase-5 rather than caspase-1 may mediate the downstream mediator of this pathway.

Lessons learned on a new procedure: Nonsternotomy minimally invasive pulmonary embolectomy.

The endpoint in emergent management of acute massive pulmonary embolism (PE) has traditionally been with embolectomy through a standard median sternotomy. This approach is limited in both exposure and concomitant functional morbidity associated with sternotomy. In a previous publication, we described a novel minimally invasive, thoracoscopically assisted approach to pulmonary embolectomy. This approach utilized a small 5-cm left upper parasternal thoracotomy and femoral cardiopulmonary bypass to conduct thoracoscopically assisted surgical pulmonary embolectomy. The first publication featured three patients that had a massive pulmonary embolus that was treated with minimally invasive pulmonary embolectomy, and the initial data was positive and suggested that this approach is safe and feasible. We now broaden our experience with another two patients who underwent this approach, and highlight a number of technical and management modifications that have been made to optimize the procedure. These lessons learned will ideally benefit future surgeons as this approach is more heavily implemented in practice.

Protein Degradome of Spinal Cord Injury: Biomarkers and Potential Therapeutic Targets.

Degradomics is a proteomics sub-discipline whose goal is to identify and characterize protease-substrate repertoires. With the aim of deciphering and characterizing key signature breakdown products, degradomics emerged to define encryptic biomarker neoproteins specific to certain disease processes. Remarkable improvements in structural and analytical experimental methodologies as evident in research investigating cellular behavior in neuroscience and cancer have allowed the identification of specific degradomes, increasing our knowledge about proteases and their regulators and substrates along with their implications in health and disease. A physiologic balance between protein synthesis and degradation is sought with the activation of proteolytic enzymes such as calpains, caspases, cathepsins, and matrix metalloproteinases. Proteolysis is essential for development, growth, and regeneration; however, inappropriate and uncontrolled activation of the proteolytic system renders the diseased tissue susceptible to further neurotoxic processes. In this article, we aim to review the protease-substrate repertoires as well as emerging therapeutic interventions in spinal cord injury at the degradomic level. Several protease substrates and their breakdown products, essential for the neuronal structural integrity and functional capacity, have been characterized in neurotrauma including cytoskeletal proteins, neuronal extracellular matrix glycoproteins, cell junction proteins, and ion channels. Therefore, targeting exaggerated protease activity provides a potentially effective therapeutic approach in the management of protease-mediated neurotoxicity in reducing the extent of damage secondary to spinal cord injury.

Degradomics in Neurotrauma: Profiling Traumatic Brain Injury.

Degradomics has recently emerged as a subdiscipline in the omics era with a focus on characterizing signature breakdown products implicated in various disease processes. Driven by promising experimental findings in cancer, neuroscience, and metabolomic disorders, degradomics has significantly promoted the notion of disease-specific "degradome." A degradome arises from the activation of several proteases that target specific substrates and generate signature protein fragments. Several proteases such as calpains, caspases, cathepsins, and matrix metalloproteinases (MMPs) are involved in the pathogenesis of numerous diseases that disturb the physiologic balance between protein synthesis and protein degradation. While regulated proteolytic activities are needed for development, growth, and regeneration, uncontrolled proteolysis initiated under pathological conditions ultimately culminates into apoptotic and necrotic processes. In this chapter, we aim to review the protease-substrate repertoires in neural injury concentrating on traumatic brain injury. A striking diversity of protease substrates, essential for neuronal and brain structural and functional integrity, namely, encryptic biomarker neoproteins, have been characterized in brain injury. These include cytoskeletal proteins, transcription factors, cell cycle regulatory proteins, synaptic proteins, and cell junction proteins. As these substrates are subject to proteolytic fragmentation, they are ceaselessly exposed to activated proteases. Characterization of these molecules allows for a surge of "possible" therapeutic approaches of intervention at various levels of the proteolytic cascade.