About
Harry Ischiropoulos, Ph.D., is a Research Professor of Pediatrics specializing in Neonatology and Newborn Services at the University of Pennsylvania's Perelman School of Medicine. He is also the Director of Neonatology Research at the Children’s Hospital of Philadelphia Research Institute. His research focuses on the biological chemistry and molecular mechanisms of nitric oxide signaling, a free radical that mediates blood flow and various physiological responses across major organ systems. His work involves exploring nitric oxide-mediated post-translational modifications, such as cysteine S-nitrosation and tyrosine nitration, and their effects on protein function in metabolic processes and mitochondrial bioenergetics. Dr. Ischiropoulos employs mass spectrometry-based proteomics and structure-function analysis to investigate these biochemical processes, generating inventories of mouse brain proteomes, phosphoproteomes, S-nitrosoproteomes, and secretomes to understand neuron physiology and neurodegeneration.
Research topics
- Biology
- Computational biology
- Medicine
- Genetics
- Bioinformatics
- Cell biology
- Chemistry
- Neuroscience
- Biochemistry
- Mathematics
- Obstetrics
Selected publications
JACC Heart Failure · 2025-03-31 · 6 citations
articleOpen accessBACKGROUND: The etiology of exercise intolerance in heart failure with preserved ejection fraction (HFpEF) is multifactorial. Several contributing pathways may be improved by ketone ester (KE). OBJECTIVES: This study aims to determine whether KE improves exercise tolerance in HFpEF. METHODS: ) during incremental cardiopulmonary exercise testing and time to exhaustion during an additional constant-intensity exercise (75% peak workload) bout. RESULTS: -glucose infusions during constant-intensity exercise, plasma glucose appearance rate before and during exercise was lower with KE (-0.24 mg/kg/min; P < 0.001). During both exercise protocols, KE lowered: 1) respiratory exchange ratios, demonstrating decreased systemic carbohydrate use; 2) nonesterified fatty acids and glucose; and 3) estimated left ventricular filling pressures (E/e'). CONCLUSIONS: or constant-intensity exercise in HFpEF. (Ketogenic Exogenous Therapies in HFpEF [KETO-HFpEF]; NCT04633460).
Communications Biology · 2025-05-27 · 3 citations
articleOpen accessSenior authorDiminished bioavailability of nitric oxide (NO) contributes to the pathogenesis of cardiometabolic disorders. However, the alterations in signaling under NO deficiency remain mostly unknown. We combined genetics and proteomics to quantify changes in the heart proteome, phosphoproteome, and S-nitrosocysteine proteome in mice lacking nitric oxide synthases (NOS1, NOS2, NOS3), lacking all three enzymes (tNOS), or the alpha 1-regulatory subunit of the soluble guanylate cyclase (sGCα1). Modest changes of less than 1% in the proteome and 4% in the phosphoproteome in single NOS gene or sGCα1 null mouse hearts indicate sufficient biological compensation. In contrast, the number of S-nitrosylated proteins declined by 80%, 57%, and 35% in NOS3, NOS1, and NOS2 null mice, respectively. A 21% remodeling of the proteome and 9% of the phosphoproteome in the tNOS null mice included integral kinases that provide adaptive rewiring of signaling. The data revealed the emergence of enhanced mitogen-activated-kinases Mapk3/Mapk1 signaling, documented by increased phosphorylation of these kinases and their downstream targets. The data highlight that adaptive compensation of signaling prevents overt phenotypes during NO signaling deficits. In contrast, maladaptive signaling via Mapk3/Mapk1 may promote pathological cardiomyopathy that progressively develops in the tNOS null mice.
SSRN Electronic Journal · 2025-01-01
preprintOpen accessSenior authorJournal of the American College of Cardiology · 2025-03-29
articleOpen accessNitric Oxide · 2025-12-29
articleOpen accessSenior authorCorrespondingNitric oxide metabolites (NOm) in biological fluids have been widely used as surrogate markers to inform changes in nitric oxide-mediated signaling, which is critical for many biological functions. One method for quantifying NOm employs chemical reduction to convert the various metabolites into nitric oxide, followed by ozone-based chemiluminescence detection. Herein, we tested and quantified the bioanalytical parameters of the reduction-chemiluminescence assay and propose conditions for optimizing the assay. The slopes of the standard curves used for quantifying the metabolites showed a 3.8% intra-assay and 7.6% inter-assay relative standard deviation (%RSD). An 8% and 8.5% RSD was measured for two separate quality control serum samples, and a Levey-Jennings plot showed that all individual values fell within 1 standard deviation of the mean of all measurements. Repeat analysis of human serum samples (n=51) resulted in an average incurred sample reanalysis of 7.7%, with 49 samples having repeated results above the original value, indicating no loss of NOm over the four-week period. This was further confirmed by recovery experiments, which indicated average recovery rates of 100.4 ± 8.2% and 99.3 ± 4.3% for two operators, respectively. ANOVA of data from two operators indicated that sample-to-sample variability was the main contributor to the total variance. Overall, our results demonstrate that the assay provides appropriate and reproducible quantification of NOm, supporting its use for both exploratory and routine laboratory applications. Highlights : • Performance characteristics for the reductive-chemistry gas-phase chemiluminescence method for the detection of nitric oxide metabolites in biological samples. • Practical recommendations for maximizing analytical performance. • Optimized chemiluminescence assay is a reliable method to quantify NOm in serum.
Potassium Nitrate in Heart Failure With Preserved Ejection Fraction
JAMA Cardiology · 2024-12-18 · 12 citations
articleOpen accessImportance: Nitric oxide deficiency may contribute to exercise intolerance in patients with heart failure with preserved ejection fraction (HFpEF). Prior pilot studies have shown improvements in exercise tolerance with single-dose and short-term inorganic nitrate administration. Objective: To assess the impact of chronic inorganic nitrate administration on exercise tolerance in a larger trial of participants with HFpEF. Design, Setting, and Participants: This multicenter randomized double-blinded crossover trial was conducted at the University of Pennsylvania, the Philadelphia Veterans Affairs Medical Center, and Northwestern University between October 2016 and July 2022. Participants included patients with symptomatic (New York Heart Association class II/III) HFpEF who had objective signs of elevated left ventricular filling pressures. Image quantification, physiological data modeling and biochemical measurements, unblinding, and statistical analyses were completed in 2024. Intervention: Potassium nitrate (KNO3) (6 mmol 3 times daily) vs equimolar doses of potassium chloride (KCl) for 6 weeks, each with a 1-week washout in between. MAIN OUTCOMES AND MEASURES: The coprimary end points included peak oxygen uptake and total work performed during a maximal effort incremental cardiopulmonary exercise test. Secondary end points included the exercise systemic vasodilatory reserve (ie, reduction in systemic vascular resistance with exercise) and quality of life assessed using the Kansas City Cardiomyopathy Questionnaire. Results: Eighty-four participants were enrolled. Median age was 68 years and 58 participants were women (69.0%). Most participants had NYHA class II disease (69%) with a mean 6-minute walk distance of 335.5 (SD, 97.3) m. Seventy-seven participants received the KNO3 intervention and 74 received the KCl intervention. KNO3 increased trough levels of serum nitric oxide metabolites after 6 weeks (KNO3, 418.4 [SD, 26.9] uM vs KCl, 40.1 [SD, 28.3] uM; P < .001). KNO3 did not improve peak oxygen uptake (KNO3, 10.23 [SD, 0.43] mL/min/kg vs KCl, 10.17 [SD, 0.43] mL/min/kg; P = .73) or total work performed (KNO3, 25.9 [SD, 3.65] kilojoules vs KCl, 23.63 [SD, 3.63] kilojoules; P = .29). KNO3 nitrate did not improve the vasodilatory reserve or quality of life, though it was well-tolerated. Conclusions and Relevance: In this study, potassium nitrate did not improve aerobic capacity, total work, or quality of life in participants with HFpEF. Trial Registration: ClinicalTrials.gov Identifier: NCT02840799.
Redox Biochemistry and Chemistry · 2024-05-31 · 8 citations
articleOpen access1st authorCorrespondingProtein tyrosine nitration is a post-translational modification originating from the biological chemistry of nitric oxide. This article highlights key milestones, discusses apparent controversies and perspectives that have emerged in the last 35 years of research on protein tyrosine nitration. Since the execution of nitric oxide signaling is accomplished entirely by protein post translational modifications (PTMs), the prospect that protein tyrosine nitration augments nitric oxide signaling remains an intriguing but incomplete concept deserving further consideration.
Advanced Science · 2024-01-18 · 20 citations
articleOpen accessIn Alzheimer's disease (AD), dysfunctional mitochondrial metabolism is associated with synaptic loss, the major pathological correlate of cognitive decline. Mechanistic insight for this relationship, however, is still lacking. Here, comparing isogenic wild-type and AD mutant human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neurons (hiN), evidence is found for compromised mitochondrial energy in AD using the Seahorse platform to analyze glycolysis and oxidative phosphorylation (OXPHOS). Isotope-labeled metabolic flux experiments revealed a major block in activity in the tricarboxylic acid (TCA) cycle at the α-ketoglutarate dehydrogenase (αKGDH)/succinyl coenzyme-A synthetase step, metabolizing α-ketoglutarate to succinate. Associated with this block, aberrant protein S-nitrosylation of αKGDH subunits inhibited their enzyme function. This aberrant S-nitrosylation is documented not only in AD-hiN but also in postmortem human AD brains versus controls, as assessed by two separate unbiased mass spectrometry platforms using both SNOTRAP identification of S-nitrosothiols and chemoselective-enrichment of S-nitrosoproteins. Treatment with dimethyl succinate, a cell-permeable derivative of a TCA substrate downstream to the block, resulted in partial rescue of mitochondrial bioenergetic function as well as reversal of synapse loss in AD-hiN. These findings have therapeutic implications that rescue of mitochondrial energy metabolism can ameliorate synaptic loss in hiPSC-based models of AD.
Cell chemical biology · 2023-07-20 · 22 citations
articleOpen accessBlood · 2023-11-02
articleOpen accessObservational and randomized studies have associated platelet transfusions with increased morbidity and mortality in preterm neonates. Clinical practice changes have been hindered by a limited understanding of why adult donor-derived platelets might be inappropriate or harmful to infants. Neonatal and adult platelets differ in reactivity and function, but we lack an understanding of underlying molecular differences. As proteins and kinase-driven phosphorylation signaling dictate platelet biology, we aimed to characterize altered protein and phosphoprotein content between neonatal and adult platelets to help explain differences in platelet reactivity and function. We isolated resting platelets from full term cord blood (n=9) and adult peripheral blood (n=7) and used mass spectrometry-based platforms to ascertain protein and phosphoprotein content. We identified 4745 high-confidence proteins in adult and neonatal platelets, including 331 differentially abundant proteins (≥1.5-fold change, p&lt;0.05). Neonatal platelets had increased metabolic and ribosomal proteins, consistent with transcriptional data. Adult platelets were enriched for inflammatory proteins, including complement components, consistent with pro-inflammatory function. We also identified 2115 phospho-proteins (17852 unique phosphopeptides) across all samples. These impacted actin cytoskeletal biology, cell adhesion, and GTPase signaling that regulate platelet functions. Kinase enrichment analysis identified FYN, SRC, ABL1, and AKT1 as kinases most responsible for platelet phosphorylation activities. Granule trafficking and degranulation proteins were among 1183 differentially abundant phosphoproteins in neonatal vs adult samples (p&lt;0.05). For example, 31 different Reticulon 1 (RTN1) phosphopeptides were enriched in adult platelets (p&lt;0.05). RTN1 regulates membrane trafficking and participates in SNARE-mediated exocytosis. Increased RTN1-directed membrane trafficking may promote increased activation and degranulation in adult vs neonatal platelets. Indeed, SNARE complex protein deficiency was previously suggested to mediate hyporeactive degranulation in neonatal platelets. We detected 2 phosphopeptides in adult samples that were absent from any neonatal sample. We reasoned that these could represent developmental stage-specific differences that exist only in adult platelets. One of the adult-specific phosphopeptides was in Rap1GAP2 (phospho-S588). Rap1GAP2 is a highly phosphorylated GTPase-activating protein that inhibits Rap1, a small guanine-nucleotide-binding protein that facilitates integrin activation in platelets. Rap1GAP2 binds synaptotagmin-like protein 1 (Slp1) to regulate dense granule secretion. These findings may link altered developmentally regulated Rap1GAP2 activities with platelet degranulation. A Platelet Cell Adhesion Molecule (PECAM) phosphopeptide (phospho-S726) was also found exclusively in adult platelets. PECAM facilitates inflammation and leukocyte transendothelial migration. PECAM phosphorylation in adult platelets may reflect an enhanced ability to participate in these processes. These findings reveal key differences in protein content and signaling that underlie differential neonatal vs adult platelet reactivity and function, including pro-inflammatory activities and enhanced activation and/or degranulation propensity in adult platelets. RTN1-mediated SNARE complex function and Rap1 signaling activities represent novel and potentially modifiable targets to manipulate platelet activation and/or degranulation. Our results also help provide a biological rationale for the higher morbidity and mortality observed in preterm infants transfused with adult platelets, including the inadvertent introduction of serum complement and other pro-inflammatory molecules within transfusions of donated adult platelets.
Recent grants
NIH · $16.7M · 2007
NIH · $840k · 2002
Neurotoxicity Mechanisms of Reactive Intermediates
NIH · $4.9M · 1997–2016
Fibrin Structures and Lung Injury
NIH · $1.6M · 2011–2015
Reactive Species in Vascular Disease: Mechanisms of Injury
NIH · $8.1M · 1997–2025
Frequent coauthors
- 287 shared
Paschalis‐Thomas Doulias
University of Ioannina
- 101 shared
Jonathan S. Stamler
Case Western Reserve University
- 101 shared
Stanley L. Hazen
Cleveland Clinic Lerner College of Medicine
- 100 shared
Shuichi Hirai
Nihon University
- 100 shared
Kasra Moazzami
Emory University
- 100 shared
Kohei Ikeda
- 100 shared
Evangelia G. Kranias
University of Cincinnati
- 100 shared
Patrick Sips
Ghent University
Education
- 1984
B.S., Chemistry
Wagner College
- 1987
M.S., Pathology
New York Medical College
- 1990
Ph.D., Experimental Pathology
New York Medical College
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