Biology subjects

Kumar, R. A.

Publications and source records attributed to Kumar, R. A..

5 recordsLinked to original sources

bioRxiv2026

Genome-scale modelling and stable isotope tracing reveal dynamic metabolic reprogramming in a human cardiac hypertrophy model

Pathological cardiac hypertrophy is associated with metabolic remodelling; however the temporal dynamics of this remodelling remain poorly defined in human cardiomyocyte systems. Here, we combined transcriptomic profiling, genome-scale metabolic modelling and stable isotope-resolved metabolomics (SIRM) to map the metabolic consequences of endothelin-1 (ET-1) stimulation in human iPSC-derived cardiomyocytes (hiPSC-CMs). Integration of RNA sequencing data obtained after 8 and 48 hours of ET-1 treatment with the Human2.0 genome-scale metabolic model predicted time-dependent remodelling, characterised by progressive increases in glycolytic flux, enhanced glucose carbon entry into the TCA cycle, and a decrease in fatty acid {beta}-oxidation. Computational flux predictions were independently confirmed by extracellular flux analysis and 13C-substrate tracing, with concordance between modelled and measured fluxes. Extracellular flux analysis showed increased glycolytic capacity and mitochondrial respiration after 48 hours, with greater sensitivity to inhibition of mitochondrial pyruvate transport. Glucose tracing revealed increased 13C enrichment of glycolytic, pentose phosphate pathway, serine-glycine and TCA cycle metabolites, consistent with altered glucose-derived carbon allocation. Increased m+3 labelling of selected TCA-related metabolites was compatible with pyruvate anaplerosis, although pathway-specific fluxes were not resolved. In three-dimensional engineered heart tissue (EHTs) treated with ET-1 for 7 days, increased hypertrophic marker expression was accompanied by a blunted inotropic response, and metabolomics showed similar changes in metabolite abundance and glucose-derived labelling to 2D cultures. These findings support hiPSC-CMs and EHTs as complementary platforms for studying early metabolic remodelling in pathological cardiac hypertrophy and provide an integrated framework linking model predictions to functional and isotope-tracing measurements.

cell biology↗

bioRxiv2023

H2S preconditioning induces long-lived perturbations in O2 metabolism

Hydrogen sulfide exposure in moderate doses can induce profound but reversible hypometabolism in mammals. At a cellular level, H2S inhibits the electron transport chain (ETC), augments aerobic glycolysis, and glutamine-dependent carbon utilization via reductive carboxylation; however, the durability of these changes is unknown. We report that despite its volatility, H2S preconditioning increases P50(O2), the O2 pressure for half maximal cellular respiration, and has pleiotropic effects on oxidative metabolism that persist up to 24-48 h later. Notably, cyanide, another complex IV inhibitor, does not induce this type of metabolic memory. Sulfide-mediated prolonged fractional inhibition of complex IV by H2S is modulated by sulfide quinone oxidoreductase, which commits sulfide to oxidative catabolism. Since induced hypometabolism can be beneficial in disease settings that involve insufficient or interrupted blood flow, our study has important implications for attenuating reperfusion-induced ischemic injury, and/or prolonging shelf life of biologics like platelets.

biochemistry↗

bioRxiv2022

Structural analysis and ensemble docking revealed the binding modes of selected progesterone receptor modulators

Uterine fibroids (UF) are benign smooth muscle neoplasm of uterus that have a significant impact on a womans quality of life as they perturb hormonal homeostasis resulting in heavy menstrual bleeding, impaired fertility, pregnancy complications and loss. UF can be surgically removed through invasive procedures, but their recurrence rate is often high. Progesterone receptor (PR) has an imperative role in UF management. Mifepristone, ulipristal acetate (UPA) and asoprisnil (ASO) are some promising selective progesterone receptor modulators (SPRMs), acts on PR, but due to their side effects in long term use, they were withdrawn from the market. Hence, there is a dire need for novel, highly efficient with least side effects, therapeutics for the treatment of UF. To contribute towards the drug discovery for UF, we made an extensive structural comparison of reported PR crystal structures, also elucidated the binding modes of four existing SPRMs against human PR through ensemble docking approach. Our studies revealed the presence of 5 highly repeating water molecules that has an important role in ligand binding and structural stability. Our ensemble docking and MD simulation revealed that studied ligands have preferential selectivity towards the specific conformation of PR. It is anticipated that our study will be a useful resource to all the drug discovery scientists who are engaged in the identification of lead molecules against UF.

bioinformatics↗

bioRxiv2022

Skeletal muscle Nox4 knockout prevents and Nox2 knockout blunts loss of maximal diaphragm force in mice with heart failure with reduced ejection fraction

Patients with heart failure with reduced ejection fraction (HFrEF) experience diaphragm weakness that contributes to the primary disease symptoms of fatigue, dyspnea, and exercise intolerance. Weakness in the diaphragm is related to excessive production of reactive oxygen species (ROS), but the exact source of ROS remains unknown. NAD(P)H Oxidases (Nox), particularly the Nox2 and 4 isoforms, are important sources of ROS within skeletal muscle that contribute to optimal cell function. There are reports of increased Nox activity in the diaphragm of patients and animal models of HFrEF, implicating these complexes as possible sources of diaphragm dysfunction in HFrEF. To investigate the role of these proteins on diaphragm weakness in HFrEF, we generated inducible skeletal muscle specific knockouts of Nox2 or Nox4 using the Cre-Lox system and assessed diaphragm function in a mouse model of HFrEF induced by myocardial infarction. Diaphragm maximal specific force measured in vitro was depressed by ~20% with HFrEF. Knockout of Nox4 provided full protection against the loss of maximal force (p < 0.01), while the knockout of Nox2 provided partial protection (7% depression, p < 0.01). Mitochondrial respiration measured in permeabilized diaphragm muscle bundles increased with HFrEF or the knockout of Nox4 from skeletal muscle fibers (p < 0.05). Knockout of Nox2 from skeletal myofibers improved survival from 50 to 80% following myocardial infarction (p = 0.026). Our findings show an important role for skeletal muscle NAD(P)H Oxidases contributing to loss of diaphragm maximal force in HFrEF, along with systemic pathophysiological responses following myocardial infarction.

physiology↗

bioRxiv2022

Reversible thiol oxidation increases mitochondrial electron transport complex enzyme activity but not respiration in cardiomyocytes from patients with end-stage heart failure

Cardiomyocyte dysfunction in patients with end-stage heart failure with reduced ejection fraction (HFrEF) stems from mitochondrial dysfunction that contributes to an energetic crisis. Mitochondrial dysfunction reportedly relates to increased markers of oxidative stress, but the impact of reversible thiol oxidation on myocardial mitochondrial function in patients with HFrEF has not been investigated. In the present study, we assessed mitochondrial function in ventricular biopsies from patients with end-stage HFrEF in the presence and absence of the thiol reducing agent dithiothreitol (DTT). Isolated mitochondria exposed to DTT had increased enzyme activity of complexes I (p = 0.009) and III (p = 0.018) of the electron transport system, while complexes II (p = 0.630) or IV (p = 0.926) showed no changes. However, increased enzyme activity did not carry over to measurements of mitochondrial respiration in permeabilized bundles. Oxidative phosphorylation conductance (p = 0.439), maximal respiration (p = 0.312), and ADP sensitivity (p = 0.514) were unchanged by 5 mM DTT treatment. These results indicate that mitochondrial function can be modulated through reversible thiol oxidation, but other components of mitochondrial energy transfer are rate limiting in end-stage HFrEF. Optimal therapies to normalize cardiac mitochondrial respiration in patients with end-stage HFrEF may benefit from interventions to reverse thiol oxidation that limits complex I and III activities.

physiology↗