Integrated Functions of Cardiac Energetics, Mechanics, and Purine Nucleotide Metabolism.

Purine nucleotides play central roles in energy metabolism in the heart. Most fundamentally, the free energy of hydrolysis of the adenine nucleotide adenosine triphosphate (ATP) provides the thermodynamic driving force for numerous cellular processes including the actin-myosin crossbridge cycle. Perturbations to ATP supply and/or demand in the myocardium lead to changes in the homeostatic balance between purine nucleotide synthesis, degradation, and salvage, potentially affecting myocardial energetics and, consequently, myocardial mechanics. Indeed, both acute myocardial ischemia and decompensatory remodeling of the myocardium in heart failure are associated with depletion of myocardial adenine nucleotides and with impaired myocardial mechanical function. Yet there remain gaps in the understanding of mechanistic links between adenine nucleotide degradation and contractile dysfunction in heart disease. The scope of this article is to: (i) review current knowledge of the pathways of purine nucleotide depletion and salvage in acute ischemia and in chronic heart disease; (ii) review hypothesized mechanisms linking myocardial mechanics and energetics with myocardial adenine nucleotide regulation; and (iii) highlight potential targets for treating myocardial metabolic and mechanical dysfunction associated with these pathways. It is hypothesized that an imbalance in the degradation, salvage, and synthesis of adenine nucleotides leads to a net loss of adenine nucleotides in both acute ischemia and under chronic high-demand conditions associated with the development of heart failure. This reduction in adenine nucleotide levels results in reduced myocardial ATP and increased myocardial inorganic phosphate. Both of these changes have the potential to directly impact tension development and mechanical work at the cellular level. © 2024 American Physiological Society. Compr Physiol 14:5345-5369, 2024.

Higher mitochondrial oxidative capacity is the primary molecular differentiator in muscle of rats with high and low intrinsic cardiorespiratory fitness.

OBJECTIVE: Cardiorespiratory fitness (CRF) is tightly linked with health and longevity and is implicated in metabolic flexibility and substrate metabolism. The high capacity runner (HCR) and low capacity runner (LCR) rat lines are a genetically heterogeneous rat model selected and bred for CRF that reflect CRF in humans by exhibiting differences in nutrient handling. This study aims to differentiate the intrinsic substrate preference of the HCR compared to LCR rats to better understand the intersection of mitochondrial respiration and intrinsic CRF. METHODS: We performed bulk skeletal muscle RNA-Sequencing on male and female HCR and LCR rats and assessed the effect of rat line on mitochondrial gene expression pathways using the MitoCarta3.0 database. In a separate cohort of rats, mitochondria were isolated from skeletal and cardiac muscle and maximal oxidation rates were measured using an Oroboros O2k when provided either pyruvate or fatty acid substrates. RESULTS: The expression of mitochondrial genes are significantly upregulated in HCR skeletal muscle in both male and female rats. In respirometry experiments, fatty acid oxidative capacities were greater in HCR compared to LCR, and male compared to female rats, as a function of both mitochondrial quality and mitochondrial density. This effect was greater in the skeletal muscle than in the heart. Pyruvate oxidation did not differ significantly between lines. CONCLUSIONS: The capacity for increased fatty acid oxidation in the HCR rat is a result of selection for running capacity and is likely a key contributor to the healthy metabolic phenotype of individuals with high CRF.

Understanding Travel Considerations and Barriers for People with Disabilities to Using Current Modes of Transportation Through Journey Mapping.

This study aimed to apply a journey mapping methodology to identify travel considerations and barriers for people with disabilities (PWDs) at each travel stage, from considering a trip through to arriving at the destination for their current modes of transportation, with the objective of understanding and avoiding "pain points" during a transition to autonomous driving systems. Twenty PWDs, including those with physical, visual, aural, cognitive, and combined physical/visual impairments, participated in a semistructured one-on-one interview. Descriptive statistics were used for demographic information, and qualitative content analysis was used to analyze the transcribed interviews and extract themes. Themes were further organized by the modes of transportation used. The top four themes in considering and planning a trip were third-party assistance availability (private vehicle, public transportation, and paratransit), finding an accessible or suitable parking space (private vehicle), access to a service location (public transportation and paratransit), and transportation schedules (public transportation and paratransit). The top four travel barriers to locating, entering, riding, and exiting transportation and arriving at the destination were vehicle ingress/egress (private vehicle and public transportation), concerns about wheelchair securement (public transportation and paratransit), requiring third-party assistance (private vehicle and public transportation), and accessibility to service locations (public transportation). The study suggests that to mitigate travel considerations and barriers for PWDs, vehicle-specific barriers and infrastructure issues should be addressed simultaneously. We anticipate that the findings will provide insights into the design and development of autonomous vehicles, to better accommodate the needs of PWDs.

Donor-Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light.

Tryptophan (Trp) plays a variety of critical functional roles in protein biochemistry; however, owing to its low natural frequency and poor nucleophilicity, the design of effective methods for both single protein bioconjugation at Trp as well as for in situ chemoproteomic profiling remains a challenge. Here, we report a method for covalent Trp modification that is suitable for both scenarios by invoking photo-induced electron transfer (PET) as a means of driving efficient reactivity. We have engineered biaryl N-carbamoyl pyridinium salts that possess a donor-acceptor relationship that enables optical triggering with visible light whilst simultaneously attenuating the probe's photo-oxidation potential in order to prevent photodegradation. This probe was assayed against a small bank of eight peptides and proteins, where it was found that micromolar concentrations of the probe and short irradiation times (10-60 min) with violet light enabled efficient reactivity toward surface exposed Trp residues. The carbamate transferring group can be used to transfer useful functional groups to proteins including affinity tags and click handles. DFT calculations and other mechanistic analyses reveal correlations between excited state lifetimes, relative fluorescence quantum yields, and chemical reactivity. Biotinylated and azide-functionalized pyridinium salts were used for Trp profiling in HEK293T lysates and in situ in HEK293T cells using 440 nm LED irradiation. Peptide-level enrichment from live cell labeling experiments identified 290 Trp modifications, with 82% selectivity for Trp modification over other π-amino acids, demonstrating the ability of this method to identify and quantify reactive Trp residues from live cells.

Next generation effects of female adolescent morphine exposure: sex-specific alterations in response to acute morphine emerge before puberty.

Prescription opiate use by adolescent girls has increased significantly in the past decade. Preclinical studies using rats report alterations in morphine sensitivity in the adult offspring of adolescent morphine-exposed females (MOR-F1) when compared with the offspring of adolescent saline-exposed females (SAL-F1). To begin to elucidate the development of these next generation modifications, the present study examined the effects of acute morphine administration on sedation and corticosterone secretion in prepubescent SAL-F1 and MOR-F1 male and female rats. In addition, alterations in proopiomelanocortin (POMC) gene expression in the arcuate nucleus, as well as in tyrosine hydroxylase (TH) and µ-opioid receptor (OPRM1) gene expressions in the ventral tegmental area, were analyzed using quantitative PCR, to determine whether differential regulation of these genes was correlated with the observed behavioral and/or endocrine effects. Increased morphine-induced sedation, coupled with an attenuation of morphine-induced corticosterone secretion, was observed in MOR-F1 males. Significant alterations in both POMC and OPRM1 gene expressions were also observed in MOR-F1 males, with no change in TH mRNA expression. Overall, these data suggest that the transgenerational effects of adolescent morphine exposure can be discerned before pubertal development and are more pronounced in males, and suggest dysregulation of the hypothalamic-pituitary-adrenal axis in the offspring of adolescent morphine-exposed females.

Functional proteomics approach to investigate the biological activities of cDNAs implicated in breast cancer.

Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events which occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cell-based assays that monitor changes in cell proliferation, migration, and morphogenesis in MCF-10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.

G1/S cell cycle arrest provides anoikis resistance through Erk-mediated Bim suppression.

Proper attachment to the extracellular matrix is essential for cell survival. Detachment from the extracellular matrix results in an apoptotic process termed anoikis. Anoikis induction in MCF-10A mammary epithelial cells is due not only to loss of survival signals following integrin disengagement, but also to consequent downregulation of epidermal growth factor (EGFR) and loss of EGFR-induced survival signals. Here we demonstrate that G(1)/S arrest by overexpression of the cyclin-dependent kinase inhibitors p16(INK4a), p21(Cip1), or p27(Kip1) or by treatment with mimosine or aphidicolin confers anoikis resistance in MCF-10A cells. G(1)/S arrest-mediated anoikis resistance involves suppression of the BH3-only protein Bim. Furthermore, in G(1)/S-arrested cells, Erk phosphorylation is maintained in suspension and is necessary for Bim suppression. Following G(1)/S arrest, known proteins upstream of Erk, including Raf and Mek, are not activated. However, retained Erk activation under conditions in which Raf and Mek activation is lost is observed, suggesting that G(1)/S arrest acts at the level of Erk dephosphorylation. Thus, anoikis resistance by G(1)/S arrest is mediated by a mechanism involving Bim suppression through maintenance of Erk activation. These results provide a novel link between cell cycle arrest and survival, and this mechanism could contribute to the survival of nonreplicating, dormant tumor cells that avert apoptosis during early stages of metastasis.

The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini.

We have utilized in vitro three-dimensional epithelial cell cultures to analyze the role of apoptosis in the formation and maintenance of a hollow glandular architecture. Lumen formation is associated with the selective apoptosis of centrally located cells; this apoptosis follows apicobasal polarization and precedes proliferative suppression during acinar development. Notably, either inhibiting apoptosis (by exogenously expressing antiapoptotic Bcl family proteins) or enhancing proliferation (via Cyclin D1 or HPV E7 overexpression) does not result in luminal filling, suggesting glandular architecture is resistant to such isolated oncogenic insults. However, the lumen is filled when oncogenes that enhance proliferation are coexpressed with those that inhibit apoptosis, or when ErbB2, which induces both activities, is activated by homodimerization. Hence, apoptosis can counteract increased proliferation to maintain luminal space, suggesting that tumor cells must restrain apoptosis to populate the lumen.