The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability.

Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.

Glutamine uptake and catabolism is required for myofibroblast formation and persistence.

BACKGROUND: Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. METHODS: Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1-/-) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. RESULTS: Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFß-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. CONCLUSIONS: These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.

Vitamin C and D Deficiency in Urban America: A Case Report.

Introduction: Scurvy is caused by vitamin C deficiency and manifests with a variety of symptoms including generalized fatigue, apathy, anemia, myalgias, easy bruising, and poor wound healing. It is generally thought of as a disease of the past, especially in developed countries. However, vitamin C deficiency still occurs, especially in patients with lack of access to fruits and vegetables. Other micronutrient deficiencies, including vitamin D deficiency, are also prevalent and can cause a multitude of signs and symptoms including osteomalacia, muscle weakness, and increased risk of many chronic illnesses. Case Report: Here we present a case of vitamin C and D deficiency in a previously healthy 26-year-old man during the coronavirus disease 2019 pandemic in urban America. Conclusion: Severe nutritional deficiencies still exist today. Emergency clinicians should be aware of the signs and symptoms to promptly diagnose and initiate treatment.

Impaired mitochondrial calcium efflux contributes to disease progression in models of Alzheimer's disease.

Impairments in neuronal intracellular calcium (iCa2+) handling may contribute to Alzheimer's disease (AD) development. Metabolic dysfunction and progressive neuronal loss are associated with AD progression, and mitochondrial calcium (mCa2+) signaling is a key regulator of both of these processes. Here, we report remodeling of the mCa2+ exchange machinery in the prefrontal cortex of individuals with AD. In the 3xTg-AD mouse model impaired mCa2+ efflux capacity precedes neuropathology. Neuronal deletion of the mitochondrial Na+/Ca2+ exchanger (NCLX, Slc8b1 gene) accelerated memory decline and increased amyloidosis and tau pathology. Further, genetic rescue of neuronal NCLX in 3xTg-AD mice is sufficient to impede AD-associated pathology and memory loss. We show that mCa2+ overload contributes to AD progression by promoting superoxide generation, metabolic dysfunction and neuronal cell death. These results provide a link between the calcium dysregulation and metabolic dysfunction hypotheses of AD and suggest mCa2+ exchange as potential therapeutic target in AD.

Mitochondrial calcium exchange links metabolism with the epigenome to control cellular differentiation.

Fibroblast to myofibroblast differentiation is crucial for the initial healing response but excessive myofibroblast activation leads to pathological fibrosis. Therefore, it is imperative to understand the mechanisms underlying myofibroblast formation. Here we report that mitochondrial calcium (mCa2+) signaling is a regulatory mechanism in myofibroblast differentiation and fibrosis. We demonstrate that fibrotic signaling alters gating of the mitochondrial calcium uniporter (mtCU) in a MICU1-dependent fashion to reduce mCa2+ uptake and induce coordinated changes in metabolism, i.e., increased glycolysis feeding anabolic pathways and glutaminolysis yielding increased α-ketoglutarate (αKG) bioavailability. mCa2+-dependent metabolic reprogramming leads to the activation of αKG-dependent histone demethylases, enhancing chromatin accessibility in loci specific to the myofibroblast gene program, resulting in differentiation. Our results uncover an important role for the mtCU beyond metabolic regulation and cell death and demonstrate that mCa2+ signaling regulates the epigenome to influence cellular differentiation.

Astrocytic metabolic switch is a novel etiology for Cocaine and HIV-1 Tat-mediated neurotoxicity.

Calcium (Ca2+) dynamics and oxidative signaling control mitochondrial bioenergetics in the central nervous system, where astrocytes are a major energy source for neurons. Cocaine use exacerbates HIV-associated neurocognitive disorders, but little is known about disruptions in astrocyte metabolism in this context. Our data show that the HIV protein Tat and cocaine induce a metabolic switch from glucose to fatty acid oxidation in astrocytes, thereby limiting lactate transport to neurons. Mechanistic analyses revealed increased Mitochondrial Ca2+ Uniporter (MCU)-mediated Ca2+ uptake in astrocytes exposed to Tat and cocaine due to oxidation of MCU. Since our data suggest that mitochondrial oxidation is dependent in part on MCU-mediated Ca2+ uptake, we targeted MCU to restore glycolysis in astrocytes to normalize extracellular lactate levels. Knocking down MCU in astrocytes prior to Tat and cocaine exposure prevented metabolic switching and protected neurons. These findings identify a novel molecular mechanism underlying neuropathogenesis in HIV and cocaine use.

AUF-1 and YB-1 independently regulate ß-globin mRNA in developing erythroid cells through interactions with poly(A)-binding protein.

The normal expression of ß-globin protein in mature erythrocytes is critically dependent on post-transcriptional events in erythroid progenitors that ensure the high stability of ß-globin mRNA. Previous work has revealed that these regulatory processes require AUF-1 and YB-1, two RNA-binding proteins that assemble an mRNP ß-complex on the ß-globin 3'UTR. Here, we demonstrate that the ß-complex organizes during the erythropoietic interval when both ß-globin mRNA and protein accumulate rapidly, implicating the importance of this regulatory mRNP to normal erythroid differentiation. Subsequent functional analyses link ß-complex assembly to the half-life of ß-globin mRNA in vivo, providing a mechanistic basis for this regulatory activity. AUF-1 and YB-1 appear to serve a redundant post-transcriptional function, as both ß-complex assembly and ß-globin mRNA levels are reduced by coordinate depletion of the two factors, and can be restored by independent rescue with either factor alone. Additional studies demonstrate that the ß-complex assembles more efficiently on polyadenylated transcripts, implicating a model in which the ß-complex enhances the binding of PABPC1 to the poly(A) tail, inhibiting mRNA deadenylation and consequently effecting the high half-life of ß-globin transcripts in erythroid progenitors. These data specify a post-transcriptional mechanism through which AUF1 and YB1 contribute to the normal development of erythropoietic cells, as well as to non-hematopoietic tissues in which AUF1- and YB1-based regulatory mRNPs have been observed to assemble on heterologous mRNAs.

A reverse time-course method for transcriptional chase analyses of mRNA half-lives in cultured cells.

Standard methods for assessing mRNA stabilities in intact cells are labor-intensive and can generate half-life (t(1/2)) measures that are both imprecise and inaccurate. We describe modifications to a conventional tetracycline-conditional transcriptional chase method for analyzing mRNA stability that significantly simplify its conduct, while generating highly reproducible and accurate t(1/2) values. The revised method-which is conducted as a reverse time course, and which accounts for interval expansion in the number of cultured cells-is validated for the analyses of mRNAs with both short and long half-lives. This approach facilitates accurate assessment of mRNA metabolism, providing a user-friendly tool for detailed investigations into their structures and functions, as well as the processes that contribute to their post-transcriptional regulation.