Biology subjects

Ashok, D.

Publications and source records attributed to Ashok, D..

4 recordsLinked to original sources

bioRxiv2026

Formate reduces ischemic injury in the male heart by increasing protein S-nitrosation

Ischemic heart disease is a leading cause of death for both men and women in the United States. We and others have demonstrated that nitric oxide (NO) signaling and associated protein S-nitrosation (SNO) play a key role in reducing ischemic injury in the heart. We also find that while females typically exhibit endogenous protection from ischemic injury, this protection is abrogated with the loss of the formate-generating enzyme alcohol dehydrogenase 5 (ADH5), but formate supplementation provided a rescue. Here, we investigate the cardioprotective efficacy of formate in male hearts. Hearts were Langendorff-perfused and subjected to ischemia/reperfusion (I/R) injury with and without formate. Formate-mediated protection was also examined using an in vitro model of coverslip-induced ischemic injury to identify molecular underpinnings. We found that formate yields protection from I/R injury in ex vivo and in vitro models by increasing post-ischemic protein SNO levels, while NO synthase inhibition blocked this formate-mediated increase in protein SNO in vitro, and attenuated protection from I/R injury ex vivo. Moreover, post-ischemic levels of tetrahydrobiopterin (BH4), a cofactor necessary for NOS function, were preserved in formate-treated hearts. Furthermore, inhibition of dihydrofolate reductase (DHFR), a one-carbon enzyme critical for BH4 recycling, blunted formate-mediated protection ex vivo. Collectively, our findings suggest that formate is a potent cardioprotective agent that confers protection by preserving post-ischemic BH4 levels, and enhancing protein SNO levels through a NOS-dependent mechanism. These findings have significant implications for the clinical prevention and treatment of ischemic heart disease in males.

physiology↗

bioRxiv2024

Innate Immune Activation and Mitochondrial ROS Invoke Persistent Cardiac Conduction System Dysfunction after COVID-19

BackgroundCardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome in humans, but the mechanisms behind COVID-19-linked arrhythmias are unknown. This study explores the acute and long term effects of SARS-CoV-2 on the cardiac conduction system (CCS) in a hamster model of COVID-19. MethodsRadiotelemetry in conscious animals was used to non-invasively record electrocardiograms and subpleural pressures after intranasal SARS-CoV-2 infection. Cardiac cytokines, interferon-stimulated gene expression, and macrophage infiltration of the CCS, were assessed at 4 days and 4 weeks post-infection. A double-stranded RNA mimetic, polyinosinic:polycytidylic acid (PIC), was used in vivo and in vitro to activate viral pattern recognition receptors in the absence of SARS-CoV-2 infection. ResultsCOVID-19 induced pronounced tachypnea and severe cardiac conduction system (CCS) dysfunction, spanning from bradycardia to persistent atrioventricular block, although no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped after the pulmonary infection was resolved, indicating persistent CCS injury. Increased cardiac cytokines, interferon-stimulated gene expression, and macrophage remodeling in the CCS accompanied the electrophysiological abnormalities. Interestingly, the arrhythmia phenotype was reproduced by cardiac injection of PIC in the absence of virus, indicating that innate immune activation was sufficient to drive the response. PIC also strongly induced cytokine secretion and robust interferon signaling in hearts, human iPSC-derived cardiomyocytes (hiPSC-CMs), and engineered heart tissues, accompanied by alterations in electrical and Ca2+ handling properties. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by in vivo inhibition of JAK/STAT signaling or by a mitochondrially-targeted antioxidant. ConclusionsThe findings indicate that long term dysfunction and immune cell remodeling of the CCS is induced by COVID-19, arising indirectly from oxidative stress and excessive activation of cardiac innate immune responses during infection, with implications for long COVID Syndrome.

pathology↗

bioRxiv2021

PDE9 Inhibition Activates PPARa to Stimulate Mitochondrial Fat Metabolism and Reduce Cardiometabolic Syndrome

Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show inhibiting cyclic-GMP selective phosphodiesterase-9A (PDE9-I) suppresses established diet-induced obesity and CMS in ovariectomized female and male mice. PDE9-I reduces abdominal, hepatic, and myocardial fat accumulation, stimulates mitochondrial activity in brown and white fat, and improves CMS, without altering activity or food intake. PDE9 localizes to mitochondria, and its inhibition stimulates lipolysis and mitochondrial respiration coupled to PPAR-dependent gene regulation. PPAR upregulation is required for PDE9-I metabolic efficacy and is absent in non-ovariectomized females that also display no metabolic benefits from PDE9-I. The latter is compatible with estrogen receptor- altering PPAR chromatin binding identified by ChIPSeq. In humans with heart failure and preserved ejection fraction, myocardial expression of PPARA and its regulated genes is reduced versus control. These findings support testing PDE9-I to treat obesity/CMS in men and postmenopausal women. SummaryOral inhibition of phosphodiesterase type 9 stimulates mitochondrial fat metabolism and lipolysis, reducing central obesity without changing appetite

physiology↗

bioRxiv2020

MitoWave: Spatiotemporal analysis of mitochondrial membrane potential fluctuations during ischemia-reperfusion

Mitochondria exhibit non-stationary unstable membrane potential ({Delta}{Psi}m) when subjected to stress, such as during Ischemia/Reperfusion (I/R). Understanding the mechanism of {Delta}{Psi}m instability involves characterizing and quantifying this phenomenon in response to I/R stress in an unbiased and reproducible manner. We designed a simple ImageJ-MATLAB-based workflow called MitoWave to unravel dynamic mitochondrial {Delta}{Psi}m changes that occur during ischemia and reperfusion. MitoWave employs MATLABs wavelet transform toolbox. In-vitro Ischemia was effected by placing a glass coverslip for 60 minutes on a monolayer of neonatal mouse ventricular myocytes (NMVMs). Removal of the coverslip allowed for reperfusion. {Delta}{Psi}m response to I/R was recorded on a confocal microscope using TMRM as the indicator. As proof-of-principle, we used MitoWave analysis on ten invitro I/R experiments. Visual observations corroborated quantitative MitoWave analysis results in classifying the ten I/R experiments into five outcomes that were observed based on the oscillatory state of {Delta}{Psi}m throughout the reperfusion time period. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion shows significant differences between five different outcomes (p< 0.001). Features such as time-points of {Delta}{Psi}m depolarization during I/R, area of mitochondrial clusters and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. We found that mitochondria from NMVMs subjected to I/R oscillate in the frequency range of 8.6-45mHz, with a mean of 8.73{+/-}4.35mHz. Oscillating clusters had smaller areas ranging from 49.78{+/-}40.64 m2 while non-oscillating clusters had larger areas 65.97{+/-}42.07m2. A negative correlation between frequency and mitochondrial cluster area was seen. We also observed that late {Delta}{Psi}m loss during ischemia correlated with early {Delta}{Psi}m stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a way to quantify complex time-resolved mitochondrial behavior. It provides an easy to follow workflow to automate microscopy analysis and allows for unbiased, reproducible quantitation of complex nonstationary cellular phenomena. Statement of SignificanceUnderstanding mitochondrial instability in Ischemia Reperfusion injury is key to determining efficacy of interventions. The MitoWave analysis is a powerful yet simple tool that enables even beginner MATALAB-Image J users to automate analysis of time-series from microscopy data. While we used it to detect {Delta}{Psi}m changes during I/R, it can be adapted to detect any such spatio-temporal changes. It standardizes the quantitative analysis of complex biological signals, opens the door to in-depth screening of the genes, proteins and mechanisms underlying metabolic recovery after ischemia-reperfusion.

cell biology↗