Biology subjects

Singh, H.

Publications and source records attributed to Singh, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Characterization of spatiotemporal dynamics of binary and graded tonic pain in humans using intracranial recordings

Pain is a complex experience involving sensory, emotional, and cognitive aspects, and multiple networks manage its processing in the brain. Examining how pain transforms into a behavioral response can shed light on the networks relationships and facilitate interventions to treat chronic pain. However, studies using high spatial and temporal resolution methods to investigate the neural encoding of pain and its psychophysical correlates have been limited. We recorded from intracranial stereo-EEG (sEEG) electrodes implanted in sixteen different brain regions of twenty patients who underwent psychophysical pain testing consisting of a tonic thermal stimulus to the hand. Broadband high-frequency local field potential amplitude (HFA; 70-150 Hz) was isolated to investigate the relationship between the ongoing neural activity and the resulting psychophysical pain evaluations. Two different generalized linear mixed-effects models (GLME) were employed to assess the neural representations underlying binary and graded pain psychophysics. The first model examined the relationship between HFA and whether the patient responded "yes" or "no" to whether the trial was painful. The second model investigated the relationship between HFA and how painful the stimulus was rated on a visual analog scale. GLMEs revealed that HFA in the inferior temporal gyrus (ITG), superior frontal gyrus (SFG), and superior temporal gyrus (STG) predicted painful responses at stimulus onset. An increase in HFA in the orbitofrontal cortex (OFC), SFG, and striatum predicted pain responses at stimulus offset. Numerous regions including the anterior cingulate cortex, hippocampus, IFG, MTG, OFC, and striatum, predicted the pain rating at stimulus onset. However, only the amygdala and fusiform gyrus predicted increased pain ratings at stimulus offset. We characterized the spatiotemporal representations of binary and graded painful responses during tonic pain stimuli. Our study provides evidence from intracranial recordings that the neural encoding of psychophysical pain changes over time during a tonic thermal stimulus, with different brain regions being predictive of pain at the beginning and end of the stimulus. Significance StatementWe investigated the neural encoding of pain psychophysics across 16 brain regions during a continuous thermal stimulus in humans. Mixed-effects models were used to analyze trends across 20 human subjects. Using intracranial electrodes, we show a parametric relationship between behavioral responses and HFA during ongoing pain. We found that HFA in cognitive and emotional pain processing regions was closely associated with pain evaluation at the stimulus onset, end, or both. The neural encoding of subjective pain intensity, measured by a visual analog scale, differed from that of binary pain intensity. Perception and psychophysical correlates to pain depend on how patients are asked to evaluate it. Our findings provide evidence that HFA can serve as a neural marker within specific brain regions of behavioral pain responses, as measured by sEEG.

neuroscience↗

bioRxiv2023

Intermittent Fasting induced ketogenesis inhibits mouse epithelial ovarian tumors by promoting anti-tumor T cell response.

Epithelial Ovarian Cancer (EOC) is the most lethal gynecologic cancer with limited genetic alterations identified that can be therapeutically targeted. In tumor bearing mice, short-term fasting, fasting mimicking diet and calorie restriction enhance the activity of antineoplastic treatment by modulating systemic metabolism and boosting anti-tumor immunity. We tested the outcome of sixteen-hour intermittent fasting (IF) on mouse EOC progression with focus on fasting driven antitumor immune responses. IF resulted in consistent decrease of tumor promoting metabolic growth factors and cytokines, recapitulating changes that creates a tumor antagonizing environment. Immune profiling revealed that IF profoundly reshapes anti-cancer immunity by inducing increase in CD4+ and CD8+ cells, paralleled by enhanced antitumor Th1 and cytotoxic responses, by enhancing their metabolic fitness. Metabolic studies revealed that IF generated bioactive metabolite BHB which can be a potential substitute for simulating the antitumor benefits of IF. However, in a direct comparison, IF surpassed exogenous BHB therapy in improving survival and activating anti-tumor immune response. Thus, our data provides strong evidence for IF and its metabolic mediator BHB for ameliorating EOC progression and as a viable approach in maintaining and sustaining an effective anti-tumor T cell response.

cancer biology↗

bioRxiv2023

Population-level comparisons of gene regulatory networks modeled on high-throughput single-cell transcriptomics data.

Single-cell technologies enable high-resolution studies of phenotype-defining molecular mechanisms. However, data sparsity and cellular heterogeneity make modeling biological variability across single-cell samples difficult. We present SCORPION, a tool that uses a message-passing algorithm to reconstruct comparable gene regulatory networks from single cell/nuclei RNA-seq data that are suitable for population-level comparisons by leveraging the same baseline priors. Using synthetic data, we found that SCORPION outperforms 12 other gene regulatory network reconstruction techniques. Using supervised experiments, we show that SCORPION can accurately identify differences in regulatory networks between wild-type and transcription factor-perturbed cells. We demonstrate SCORPIONs scalability to population-level analyses using a single-cell RNA-seq atlas containing 200,436 cells from colorectal cancer and adjacent healthy tissues. The differences detected by SCORPION between tumor regions are consistent across population cohorts, as well as with our understanding of disease progression and elucidate phenotypic regulators that may impact patient survival.

bioinformatics↗

bioRxiv2022

Heritability and pre-adult survivorship costs of ectoparasite resistance in the naturally occurring Drosophila-Gamasodes mite system

Our understanding of the evolutionary significance of ectoparasites in natural communities is limited by a paucity of information concerning the mechanisms and heritability of resistance to this ubiquitous and diverse assemblage of organisms. Here, we report the results of artificial selection for increasing ectoparasite resistance in replicate lines of Drosophila melanogaster derived from a field-fresh population. Resistance, as ability to avoid infestation by naturally occurring Gamasodes queenslandicus mites, increased significantly in response to selection, and realized heritability (s.e.) was estimated to be 0.11 (0.0090). Ability to deploy energetically expensive bursts of flight from the substrate was a main mechanism of resistance that responded to selection, aligning with previously documented metabolic costs of fly behavioral defenses. Host body size, which affects parasitism rate in some fly-mite systems, was not shifted by selection. In contrast, resistant lines expressed significant reductions in larva-to-adult survivorship with increasing toxic (ammonia) stress, identifying an environmentally modulated pre-adult cost of resistance. Flies resistant to G. queenslandicus were also more resistant to a different mite, Macrocheles subbadius, suggesting that we documented genetic variation and a pleiotropic cost of broad-spectrum behavioral immunity against ectoparasites. This study demonstrates significant evolutionary potential of an ecologically important trait.

evolutionary biology↗

bioRxiv2022

COVID-19db linkage maps of cell surface proteins and transcription factors in immune cells

The highly contagious SARS-CoV-2 and its associated disease (COVID-19) are a threat to global public health and economies. To develop effective treatments for COVID-19, we must understand the host cell types, cell states and regulators associated with infection and pathogenesis such as dysregulated transcription factors (TFs) and surface proteins, including signalling receptors. To link cell surface proteins with TFs, we recently developed SPaRTAN (Single-cell Proteomic and RNA-based Transcription factor Activity Network) by integrating parallel single-cell proteomic and transcriptomic data based on Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and gene cis-regulatory information. We apply SPaRTAN to CITE-seq datasets from patients with varying degrees of COVID-19 severity and healthy controls to identify the associations between surface proteins and TFs in host immune cells. Here, we present COVID-19db of Immune Cell States (https://covid19db.streamlit.app/), a web server containing cell surface protein expression, SPaRTAN-inferred TF activities, and their associations with major host immune cell types. The data include four high-quality COVID-19 CITE-seq datasets with a toolset for userfriendly data analysis and visualization. We provide interactive surface protein and TF visualizations across major immune cell types for each dataset, allowing comparison between various patient severity groups for the discovery of potential therapeutic targets and diagnostic biomarkers.

bioinformatics↗

bioRxiv2022

Mrj an Hsp40 family chaperone regulates oligomerization of Orb2 and long-term memory

Orb2 the Drosophila homolog of Cytoplasmic polyadenylation element binding protein (CPEB) forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation are dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2As prion-like oligomerization forms long-term memory but fails to maintain it over time. Since, this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Amongst these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, the knockdown of Mrj in the mushroom body neurons results in a deficit in long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating polysomes.

neuroscience↗

bioRxiv2022

Epigenetic CpG Duplex Marks Probed by an Evolved DNA Reader via a Well-Tempered Conformational Plasticity

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel "CpG duplex marks" could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.

genomics↗

bioRxiv2022

Chaperonin activity of Plasmodium prefoldin complex is essential to guard proteotoxic stress response and presents a new target for drug discovery

The intraerythrocytic growth of malaria parasite is challenged by the presence of proteotoxic stress and intrinsically unstructured proteins in the cytoplasm due to formation of toxic heme during haemoglobin digestion. To overcome the unavoidable stress and maintain the cellular protein homeostasis, parasite encodes for a number of chaperones and co-chaperones. Here, we functionally characterize the Plasmodium falciparum prefoldins (PfPFD1-6), a hexameric co-chaperone complex, for their role in protein homeostasis. We demonstrate that PfPFD1-6 localise to cytosol of the parasite and the subunits perform an orchestrated interaction (-PFD3-PFD2-PFD1-PFD5-PFD6-PFD4-) to form an active jelly-fish like complex. Biperiden, an N-propylpiperidine analogue identified by chemotype search from FDA, strongly binds and restricts the formation of prefoldin complex and inhibited its interaction with the substrates, PfMSP-1 and -tubulin-I. Biperiden treatment potently inhibited the in vitro (IC50: 1M) and in vivo growth of malaria parasite. Thus, this study provides novel virtues towards understanding the role of PfPFDs in regulating protein homeostasis and opens new avenues for drug discovery against malaria.

molecular biology↗

bioRxiv2022

Diagnostic tool to identify and treat DNA repair deficient gastroesophageal adenocarcinomas

Background and aimsDNA repair deficiency is a common feature of cancer. Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in solid tumors. HR deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of DNA repair pathway deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Furthermore, whether DNA repair deficient GEA have enhanced responsiveness to platinum chemotherapy and sensitivity to PARP inhibitors is not well characterized. MethodsUsing whole exome and genome sequencing data, we measured various HR deficiency-associated mutational signatures in patient specimen of gastric, esophageal and colorectal cancer specimens and gastric cancer cell lines. Gold-standard immunofluorescence assays were used to confirm HR and NER deficiency in cancer cell lines. The relationship between PARP inhibitor treatment and tumor response was evaluated in patients with gastric cancer. Drug sensitivity was determined using standard in vitro cell culture assays. Single-cell RNA-sequencing was performed to evaluate gastric cancer response to commonly used chemotherapeutics. ResultsWe found that a significant subset of GEA, but very few colorectal tumors, show evidence of HR deficiency by mutational signature analysis (HRD score). Gastric cancer cell lines with high HRD mutational signature scores demonstrated functional HR deficiency by RAD51 assay and increased sensitivity to platinum and PARP inhibitors. There was a positive association between HRD scores and tumor response in patients with gastric cancer treated with a PARP inhibitor on a clinical trial. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by DDB2 proteo-probe assay. Single-cell RNA-sequencing revealed that, in addition to inducing general apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, which may explain the outlier sensitivity. ConclusionA subset of upper gastrointestinal tumors have genomic features of HR and NER deficiency and therefore may be more likely to benefit from platinum chemotherapy and PARP inhibition.

cancer biology↗

bioRxiv2022

Human malaria parasite cold shock protein plays an essential role in asexual and sexual stage development and presents an excellent druggable target

Cold shock proteins are well characterized in bacteria, plants and humans, however there is no information on their existence and role in malaria parasite. Here, we have delineated the function of a novel cold shock protein of Plasmodium falciparum (Pf) which we have annotated PfCoSP. Our results show that recombinant PfCoSP has both DNA and RNA binding activity, and also interacts with alpha and beta tubulin of Pf. PfCoSP binds with RNA and alpha tubulin simultaneously to form a complex. Expression of PfCoSP was found during asexual blood stages and gametocyte stages of malaria parasite. PfCoSP expression up-regulates many folds upon cold treatment, suggesting its role during hypothermic cold shock. Interestingly, PfCoSP showed binding with human cold shock protein LIN28A inhibitor LI71 that also inhibits PfCSP -alpha/beta tubulin interactions. LI71 showed antimalarial activity against asexual blood stage and gametocyte stage suggesting its multi-stage, transmission-blocking potential. We propose that PfCoSP may form a workbench for translation during cold stress via its interactions with target mRNAs and the cytoskeleton protein tubulin.

molecular biology↗

bioRxiv2022

T-cell potential for CD19-expressing malignancies revealed by multi-dimensional single-cell profiling

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-cell malignancies serves as a model for identifying subsets with superior clinical activity. We profiled the infusion products (IP) of 16 patients with large B-cell lymphoma (LBCL) using an integrated suite of single-cell assays to reveal the therapeutic potential of CD19-specific CAR+ T cells. Timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed migration (persistent motion for at least one body length), and migration was associated with serial killing capacity. In addition, confocal microscopy revealed that migration is linearly correlated with both mitochondrial volume and lysosomal volume; and scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering. A marker-free sorting strategy enriched T cells with migratory capacity and validated serial killing, bioenergetics, and in vivo efficacy. In aggregate, we demonstrate that migration is a cell-intrinsic biomarker independent of CAR design or biomanufacturing, desired in the bioactivity of CAR+ T cells associated with clinical antitumor efficacy.

immunology↗

bioRxiv2022

miRNA408 and its encoded peptide, miPEP408, regulate arsenic stress response in Arabidopsis

MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in regulating various developmental and biological processes. The expression of miRNAs is differentially modulated in response to various stresses. Based on the recent findings, it has been shown that some of the pri-miRNAs encode small regulatory peptides, microRNA-encoded peptides (miPEP). miPEPs are reported to regulate the growth and development of plants by modulating corresponding miRNA expression; however, the role of these peptides in different stresses has not been explored yet. Here, we reported that pri-miR408 encodes a small peptide, miPEP408, that regulates the expression of miR408, its targets, and associated phenotype in Arabidopsis. Plants overexpressing miR408 showed severe sensitivity under low sulphur (LS), Arsenite As(III) and LS+As(III) stress, while miR408 mutant developed through the CRISPR/Cas9 approach showed tolerance. Transgenic lines showed phenotypic alteration and modulation in the expression of genes involved in the sulphur reduction pathway and affect sulphate and glutathione accumulation. Similar to miR408 overexpressing lines, the exogenous application of synthetic miPEP408 or miPEP408 overexpression led to sensitivity in plants under LS, As(III) and combined LS+As(III) stress compared to control. This study suggests the involvement of miR408 and miPEP408 in heavy metal and nutrient deficiency responses. One-sentence summarymiR408 and peptide encoded by miR408, miPEP408, regulate arsenic stress and low sulphur responses in Arabidopsis.

plant biology↗

bioRxiv2022

Immune and Clotting Dysfunction Detected in Saliva and Blood Plasma after COVID-19.

A growing number of studies indicate that coronavirus disease 2019 (COVID-19) is associated with inflammatory sequelae, but molecular signatures governing the normal vs. pathologic convalescence process have not been well-delineated. We characterized global immune and proteome responses in matched plasma and saliva samples obtained from COVID-19 patients collected between 4-6 weeks after initial clinical symptoms resolved. Convalescent subjects showed robust IgA and IgG responses and positive antibody correlations between matched saliva and plasma samples. However, global shotgun proteomics revealed persistent inflammatory patterns in convalescent samples including dysfunction of salivary innate immune cells and clotting factors in plasma (e.g., fibrinogen and antithrombin), with positive correlations to acute COVID-19 disease severity. Saliva samples were characterized by higher concentrations of IgA, and proteomics showed altered pathways that correlated positively with IgA levels. Our study positions saliva as a viable fluid to monitor immunity beyond plasma to document COVID-19 immune, inflammatory, and coagulation-related sequelae.

immunology↗

bioRxiv2022

Multidimensional single-cell analysis identifies a role of CD2-CD58 interactions for clinical antitumor T cell responses

The in vivo persistence of adoptively transferred T cells is predictive of anti-tumor response. Identifying functional properties of infused T cells that lead to in vivo persistence and tumor eradication has remained elusive. We profiled CD19-specific CAR T cells that comprise the infusion products used to treat large B cell lymphomas using high-throughput single-cell technologies based on Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates killing, cytokine secretion, and transcriptional profiling. Our results show that the directional migration of CD19-specific CAR T cells is correlated with polyfunctionality. We identified that CD2 on T cells is associated with directional migration and that the interaction between CD2 on T cells and CD58 on lymphoma cells accelerates killing and serial killing. Consistent with this, we observed elevated CD58 expression on pre-treatment tumor samples in patients with relapsed or refractory large B cell lymphomas treated with CD19-specific CAR T cell therapy was associated with complete clinical response and survival. These results highlight the importance of studying dynamic T-cell tumor cell interactions in identifying optimal antitumor responses. KEY POINTS- Profiling patient infusion products revealed that polyfunctional CAR T cells show directional migration, which is associated with higher CD2 expression - The ligand for CD2, CD58 is expressed at higher levels in the tumors of lymphoma patients who respond better to CAR T cell treatment

immunology↗

bioRxiv2022

Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.

immunology↗

bioRxiv2022

Tobacco HY5, NtHY5, positively regulates flavonoid biosynthesis and enhances salt stress tolerance

Tobacco remains one of the most commercially important crops due to the parasympathomimetic alkaloid nicotine used in cigarettes. Most genes involved in nicotine biosynthesis are expressed in root tissues; however, their light-dependent regulation has not been studied. We identified the ELONGATED HYPOCOTYL 5 homolog, NtHY5, from Nicotiana tabacum and demonstrated its role in nicotine biosynthesis. We report the development of CRISPR/Cas9-based mutant plants, NtHY5CR, and show down-regulation of the nicotine biosynthetic pathway, whereas NtHY5 overexpression (NtHY5OX) plants show the opposite effect. Grafting experiments using wild type, NtHY5CR, and NtHY5OX indicated that NtHY5 moves from shoot-to-root to regulate nicotine biosynthesis in the root tissue. We conclude that shoot HY5, directly or through enhancing expression of the HY5 in the root, promotes nicotine biosynthesis. The CRISPR/Cas9-based mutants developed, in this study; with low nicotine accumulation in leaves could help people to overcome their nicotine addiction and the risk of death from tobacco use.

plant biology↗

bioRxiv2021

Diverse digital and fuzzy composite transcriptional elements are prevalent features of mammalian cis-regulomes

Mammalian transcriptional regulatory sequences are comprised of complex combinations of simple transcription factor (TF) motifs. Stereospecific juxta-positioning of simple TF motifs generates composite elements (CEs), that increase combinatorial and regulatory specificity of TF-DNA interactions. Although a small number of CEs and their cooperative or anti-cooperative modes of TF binding have been thoroughly characterized, a systematic analysis of CE diversity, prevalence and properties in cis-regulomes has not been undertaken. We developed a computational pipeline termed CEseek to discover >20,000 CEs in open chromatin regions of diverse immune cells and validated many using CAP-SELEX, ChIP-Seq and STARR-seq datasets. Strikingly, the CEs manifested a bimodal distribution of configurations, termed digital and fuzzy, based on their stringent or relaxed stereospecific constraints, respectively. Digital CEs mediate cooperative as well as anti-cooperative binding of structurally diverse TFs that likely reflect AND/OR genomic logic gates. In contrast, fuzzy CEs encompass a less diverse set of TF motif pairs that are selectively enriched in p300 associated, multi-genic enhancers. The annotated CEs greatly expand the regulatory DNA motif lexicon and the universe of TF-TF interactions that underlie combinatorial logic of gene regulation.

genomics↗

bioRxiv2021

Inhibition of BKCa channels protects neonatal hearts against myocardial ischemia and reperfusion injury

BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channels activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.

physiology↗