Replacement substance P reduces cardiac fibrosis in monkeys with type 2 diabetes.

BACKGROUND: Type 2 diabetes mellitus (T2DM)-associated cardiac fibrosis contributes to heart failure. We previously showed that diabetic mice with cardiomyopathy, including cardiac fibrosis, exhibit low levels of the neuropeptide substance P; exogenous replacement of substance P reversed cardiac fibrosis, independent of body weight, blood glucose and blood pressure. We sought to elucidate the effectiveness and safety of replacement substance P to ameliorate or reverse cardiac fibrosis in type 2 diabetic monkeys. METHODS: Four female T2DM African Green monkeys receive substance P (0.5 mg/Kg/day S.Q. injection) for 8 weeks. We obtained cardiac magnetic resonance imaging and blood samples to assess left ventricular function and fibrosis by T1 map-derived extracellular volume as well as circulating procollagen type I C-terminal propeptide. Hematological parameters for toxicities were also assessed in these monkeys and compared with three female T2DM monkeys receiving saline S.Q. as a safety comparison group. RESULTS: Diabetic monkeys receiving replacement substance P exhibited a ∼20% decrease in extracellular volume (p = 0.01), concomitant with ∼25% decrease procollagen type I C-terminal propeptide levels (p = 0.008). Left ventricular ejection fraction was unchanged with substance P (p = 0.42); however, circumferential strain was improved (p < 0.01). Complete blood counts, glycosylated hemoglobin A1c, lipids, liver and pancreatic enzymes, and inflammation markers were unchanged (p > 0.05). CONCLUSIONS: Replacement substance P reversed cardiac fibrosis in a large preclinical model of type 2 diabetes, independent of glycemic control. No hematological or organ-related toxicity was associated with replacement substance P. These results strongly support a potential application for replacement substance P as safe therapy for diabetic cardiac fibrosis.

Itaconate and Its Derivatives Repress Early Myogenesis In Vitro and In Vivo.

A Krebs cycle intermediate metabolite, itaconate, has gained attention as a potential antimicrobial and autoimmune disease treatment due to its anti-inflammatory effects. While itaconate and its derivatives pose an attractive therapeutic option for the treatment of inflammatory diseases, the effects outside the immune system still remain limited, particularly in the muscle. Therefore, we endeavored to determine if itaconate signaling impacts muscle differentiation. Utilizing the well-established C2C12 model of in vitro myogenesis, we evaluated the effects of itaconate and its derivatives on transcriptional and protein markers of muscle differentiation as well as mitochondrial function. We found itaconate and the derivatives dimethyl itaconate and 4-octyl itaconate disrupt differentiation media-induced myogenesis. A primary biological effect of itaconate is a succinate dehydrogenase (SDH) inhibitor. We find the SDH inhibitors dimethyl malonate and harzianopyridone phenocopie the anti-myogenic effects of itaconate. Furthermore, we find treatment with exogenous succinate results in blunted myogenesis. Together our data indicate itaconate and its derivatives interfere with in vitro myogenesis, potentially through inhibition of SDH and subsequent succinate accumulation. We also show 4-octyl itaconate suppresses injury-induced MYOG expression in vivo. More importantly, our findings suggest the therapeutic potential of itaconate, and its derivatives could be limited due to deleterious effects on myogenesis.

It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly.

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.

Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex.

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.