Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing.

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Physiological Effects of N95 FFP and PPE in Healthcare Workers in COVID Intensive Care Unit: A Prospective Cohort Study.

BACKGROUND: Healthcare workers (HCWs) are at increased risk of exposure to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Personal protective equipment (PPE) is mandated for HCWs. However, the physiological effects on the HCWs while working in the protective gear remains unexplored. This study aimed to assess the physiological effects of the prolonged use of PPE on HCWs. MATERIALS AND METHODS: Seventy-five HCWs, aged 18-50 years were enrolled in this prospective, observational, cohort study. The physiological variables [heart rate, oxygen saturation, and perfusion index (PI)] were recorded at the start of duty, 4 hours after wearing N95 filtering facepiece respirator (FFR), pre-donning, and post-doffing. The rating of perceived exertion (RPE) score and modified Borg scale for dyspnea was evaluated. The physiological variables were represented as the mean ± standard deviation. Wilcoxon signed-rank test was used to show any difference in RPE and modified Borg scale for dyspnea. A p value of <0.05 was considered significant. RESULTS: There is a statistically significant difference in the physiological parameters post-doffing compared with baseline: Heart rate (p < 0.001); oxygen saturation (p < 0.001); PI (p < 0.001). RPE score showed increased discomfort with continuous use of N95 FFR. However, exertion increased only marginally. The major adverse effects noted with PPE use were fogging, headache, tiredness, difficulty in breathing, and mask soakage, with a resultant mean duration of donning to be 3.1 hours. CONCLUSION: The use of PPE can result in considerable changes in the physiological variables of healthy HCWs. The side effects may lead to excessive exhaustion and increased tiredness after prolonged shifts in the intensive care unit (ICU) while wearing PPE. HOW TO CITE THIS ARTICLE: Choudhury A, Singh M, Khurana DK, Mustafi SM, Ganapathy U, Kumar A, et al. Physiological Effects of N95 FFP and PPE in Healthcare Workers in COVID Intensive Care Unit: A Prospective Cohort Study. Indian J Crit Care Med 2020;24(12):1169-1173.

Anesthesia for subcutaneous implantable cardioverter-defibrillator implantation: Perspectives from the clinical experience of a U.S. panel of physicians.

BACKGROUND AND OBJECTIVE: Worldwide adoption of the subcutaneous implantable cardioverter-defibrillator (S-ICD) for preventing sudden cardiac death continues to increase, as longer-term evidence demonstrating the safety and efficacy of the S-ICD expands. As a relatively new technology, comprehensive anesthesia guidance for the management of patients undergoing S-ICD placement is lacking. This article presents advantages and disadvantages of different periprocedural sedation and anesthesia options for S-ICD implants including general anesthesia, monitored anesthesia care, regional anesthesia, and nonanesthesia personnel administered sedation and analgesia. METHODS: Guidance, for approaches to anesthesia care during S-ICD implantation, is presented based upon literature review and consensus of a panel of high-volume S-ICD implanters, a regional anesthesiologist, and a cardiothoracic anesthesiologist with significant S-ICD experience. The panel developed suggested actions for perioperative sedation, anesthesia, surgical practices, and a decision algorithm for S-ICD implantation. CONCLUSIONS: While S-ICD implantation currently requires higher sedation than transvenous ICD systems, the panel consensus is that general anesthesia is not required or is obligatory for the majority of patients for the experienced S-ICD implanter. The focus of the implanting physician and the anesthesia services should be to maximize patient comfort and take into consideration patient-specific comorbidities, with a low threshold to consult the anesthesiology team.

Tidal Volume Requirement in Mechanically Ventilated Infants with Meconium Aspiration Syndrome.

OBJECTIVE: The aim of the study is to test the hypothesis that increased physiologic dead space and functional residual capacity seen in meconium aspiration syndrome (MAS) results in higher tidal volume (VT) requirement to achieve adequate ventilation. STUDY DESIGN: Retrospective review of infants with MAS admitted to our hospital from 2000 to 2010 managed with conventional ventilation. Demographics, ventilator settings, VT, respiratory rate (RR), and blood gas values were recorded. Minute ventilation (MV) was calculated as RR × VT. Only VT values with corresponding partial pressure of carbon dioxide (Paco 2) between 35 and 60 mm Hg were included. Mean VT/kg and MV/kg were calculated for each patient. Forty infants ventilated for lung disease other than MAS or pulmonary hypoplasia served as controls. RESULTS: Birth weights of the 28 MAS patients and 40 control infants were similar (3,330 ± 500 g and 3,300 ± 640 g). Two patients in each group required extracorporeal membrane oxygenation. Infants with MAS required 26% higher VT and 42% higher MV compared with controls to maintain equal Paco 2. CONCLUSION: Infants with MAS require larger VT and higher total MV to achieve similar alveolar ventilation, consistent with pathophysiology of MAS. Our findings provide the first reference data to guide selection of VT in infants with MAS.

Tidal volume in infants with congenital diaphragmatic hernia supported with conventional mechanical ventilation.

OBJECTIVE: This study aims to test the hypothesis that the tidal volume (VT) required for maintaining eucapnia in infants with congenital diaphragmatic hernia (CDH) is not reduced to the same degree as their lung mass. STUDY DESIGN: Records of infants with CDH admitted to our hospital from 1997 to 2009 managed with conventional ventilation were reviewed. Demographics, ventilator settings, observed VT, respiratory rate (RR), and blood gas values pre- and postsurgery were recorded. Minute ventilation (MV) was calculated as a product of RR × VT. Only VT values with corresponding Paco 2 between 35 and 60 mm Hg were included. Mean VT/kg and MV/kg were calculated for each patient. Forty term/late preterm infants ventilated for lung disease other than CDH or pulmonary hypoplasia served as controls. RESULTS: Birth weights of the 19 patients with CDH and 40 control infants were similar (3,360 ± 480 g and 3,300 ± 640 g). Mean gestational age was 38.5 ± 2 and 37.4 ± 1.5 week, p = 0.02. Infants with CDH required similar VT and MV as controls to maintain equal Paco 2. CONCLUSIONS: Infants with CDH require similar VT to clear their CO2 production compared with infants of similar size without pulmonary hypoplasia. These are the first reference values to guide selection of VT in infants with CDH.

Targeting PfCLK3 with Covalent Inhibitors: A Novel Strategy for Malaria Treatment.

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase PfCLK3 with the reversible inhibitor TCMDC-135051 (1), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

The Diagnostic Accuracy of Serum and Urine Human Epididymis Protein 4 (HE4) in Ovarian Cancer in 15,394 Subjects: An Updated Meta-Analysis.

Background We aim to determine the diagnostic accuracy of both serum and urinary human epididymis protein 4 (HE4) in the diagnosis of ovarian cancer. Methods Electronic databases and search engines such as PubMed, Cochrane Library, and Google Scholar were searched systematically by two independent reviewers to retrieve articles published from inception to June 11, 2022. The diagnostic accuracy of serum and urinary HE4 was computed using the random-effects model in terms of pooled sensitivity, pooled specificity, and diagnostic odds ratio (DOR) with 95% confidence interval (CI). To explain any source of possible heterogeneity, meta-regression and subgroup analyses were performed. Risk of bias assessment was conducted using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tools recommended by the Cochrane Library. Result and conclusion This meta-analysis included a total of 38 studies of serum HE4 involving 14,745 subjects and five studies for urinary HE4 involving 649 subjects. We observed acceptable pooled sensitivity, specificity, summary receiver operating characteristics (SROC), and diagnostic odds ratio (DOR) at 0.79 (95% CI: 0.75-0.82), 0.92 (95% CI: 0.87-0.95), 0.88 (95% CI: 0.85-0.91), and 43 (95% CI: 25-72), respectively, for serum HE4 for discriminating ovarian cancer. For urine HE4, the pooled sensitivity, specificity, SROC, and DOR were 0.80 (95% CI: 0.64-0.90), 0.93 (95% CI: 0.83-0.98), 0.94 (95% CI: 0.91-0.95), and 55 (95% CI: 15-198), respectively. Therefore, HE4 is a promising biomarker with a high degree of specificity and acceptable sensitivity for the diagnosis of ovarian cancer. Registration number This meta-analysis was performed after the registration of the protocol in the PROSPERO database with registration number CRD42022324947.

Usefulness of Multisite Ventricular Pacing in Nonresponders to Cardiac Resynchronization Therapy.

Cardiac resynchronization therapy (CRT) is an established treatment for heart failure patients with myocardial dysfunction and delayed ventricular activation, but approximately 25% to 40% of patients do not respond to CRT. Left ventricular (LV) multisite pacing (MSP) has been proposed as a tool to improve CRT response. The goal of this study is to examine the safety and efficacy of LV MSP in CRT nonresponders. Between January 2018, and September 2019, the Strategic Management to Improve CRT Using Multi-Site Pacing trial prospectively enrolled 584 CRT-defibrillator recipients for established indications at 52 sites across the United States and evaluated their response at 6 months using the clinical composite score (CCS). Of the nonresponders, 102 patients had the LV MSP feature turned on and 78 patients completed the 12-month CCS evaluation. The LV MSP feature-related complication-free rate was 99.0% with a lower 95% confidence interval limit of 94.9%, which was higher than the performance goal of 90%. The proportion of nonresponders with an improved CCS from 6 to 12 months was 51.3% with a lower 95% confidence interval limit of 41.4%, which was higher than the performance goal of 5%. The estimated mean reduction in battery longevity with the LV MSP feature was about 3.6 months (estimated battery longevity of 8.87 ± 2.08 years at 6 months and 8.07 ± 2.23 years at 12 months). In conclusion, in CRT nonresponders, the use of the LV MSP feature is safe and associated with a ∼50% conversion rate with a small projected reduction in CRT-defibrillator battery longevity. LV MSP should be considered in the management of CRT nonresponders.

Obstetric and Maternal Outcomes After B-Lynch Compression Sutures: A Meta-Analysis.

This review article aimed to determine the obstetric and maternal outcomes after B-Lynch compression sutures to control atonic postpartum hemorrhage (PPH). This meta-analysis was performed after registering the protocol in the PROSPERO database with the registration number CRD42022355358. Two independent reviewers systematically searched electronic databases and search engines (PubMed, Cochrane Library, and Google Scholar) to retrieve published articles from inception to July 2022. The obstetric and maternal outcomes after the B-Lynch compression suture were computed using the random-effects model in pooled proportion with a 95% confidence interval (CI). Meta-regression analysis and subgroup analysis were performed to explain any source of possible heterogeneity. Quality assessment of the included studies was done using Joanna Briggs Institute (JBI) tools which are critical appraisal tools for systematic reviews and meta-analyses. This meta-analysis included a total of 30 studies involving 1,270 subjects. The pooled proportion of B-Lynch suture alone was 91% (95% CI = 82-97%). The combined proportion of B-Lynch suture plus another compression suture was 1% (95% CI = 0-3%), and the pooled proportion of B-Lynch suture plus vessel ligation was 3% (95% CI = 1-6%). The pooled proportions of PPH controlled and hysterectomies were 94% (95% CI = 91-97%, I2 = 65.3%) and 7% (95% CI = 4-10%, I2 = 72.13%), respectively. Therefore, B-Lynch suture (either alone or in combination with other techniques) is a simple and effective measure to control atonic PPH.

Role of adaptive radiation therapy for pediatric patients with diffuse pontine glioma.

We investigate the role of adaptive radiation therapy in pediatric patients with diffuse pontine glioma and the impact of steroid-related weight gain on treatment parameters utilizing cone-beam CT. Fifteen patients with diffuse pontine glioma were treated with three-dimensional conformal radiation therapy and enrolled on a daily localization protocol. The median age was 6 years (range: 2-13 years). Patient charts were examined to obtain the prescribed daily dose of dexamethasone and weight. The original treatment plan was recalculated based on the data obtained from the daily cone-beam CT. The changes in target and critical structure doses were calculated using gEUD. Correlations between prescribed dexamethasone, weight gain, source-to-skin distance (SSD) changes and dosimetric changes were investigated. Eleven of the 15 patients gained weight during radiation therapy, with an average gain of 2.2 kg (8.0%). The mean gEUD decreased was 0.57 Gy (range: 0.24-1.4 Gy) for the PTV, and the mean gEUD increase for critical structures was 1.14%. No strong correlations between prescribed dexamethasone doses, weight gain and dosimetric changes were found. Change in SSD vs. dose to PTV was correlated (R² = 0.51). Weight gain and changes to the external surface are apparent in these patients; however, the dosimetric changes to the target and critical structures were small and in most cases did not warrant an adaptive plan. The potential exists for a decrease in target dose in these patients; therefore, they should be monitored to assess for replanning when necessary.

CRISPR Cas9 based genome editing in inherited retinal dystrophies.

BACKGROUND: Precision genome engineering, with targeted therapy towards patient-specific mutations is predicted to be the future of personalized medicine. Ophthalmology is in the frontiers of development of targeted therapy since the eye is an accessible organ and has the ease of both delivery as well as monitoring effects of therapy. MATERIALS AND METHODS: We reviewed literature using keywords CRISPR, precision medicine, genomic editing, retinal dystrophies, retinitis pigmentosa, Usher syndrome, Stargardt's Disease. Further, we collated data on current clinical trials. RESULTS: There is growing evidence on the role of genomic editing in retinal dystrophies, the various methods used, and stage of development of different therapies have been summarized in this paper. CONCLUSIONS: The CRISPR-Cas9 system has revolutionized genome editing, and opened avenues in drug discovery. It is important to understand the role of this system along with its applicability in the field of ophthalmology. In this review article, we briefly describe its methodology, the strategies of employing it for making genetic perturbations, and explore its applications in inherited retinal dystrophies.

The Effect of Joint Surface Preparations and Chemical Surface Treatment on the Transverse Strength of Repaired Denture Base Resin.

AIM: The aim of the study is to investigate the effect of joint surface preparations and chemical surface treatment given to prepared surface on the transverse strength of repaired denture base resin. METHODOLOGY: Two hundred and twenty specimens (DPI India) were randomly allocated into eight groups (n = 10), according to the combination of joint surface preparation (Butt joint, 45° bevel, and rounded joint) and chemical surface treatment (methylene chloride, ethyl acetate, and methyl methacrylate) for 30 and 60 s each. Specimens, after surface treatment, were repaired with self-cured resin (DPI India) and then subjected to 3-point bending test to measure transverse strength. Fractured surfaces were viewed under stereomicroscope and subjected to AutoCAD analysis to determine type of failure (adhesive or cohesive) and measure their area. Measurement data were analyzed by one-way analysis of variance, paired t-test, Dunnett's D test, and multiple comparison Bonferroni t-test, with a 5% significance level. RESULTS: Most of the comparisons between the test specimens were statistically significant. Specimens (methylene chloride for 60 s and rounded joint) show 60%-70% gain of strength. CONCLUSIONS: For providing better bond strength to a repaired specimen, the segment of fractured specimens to be provided "Rounded joint" preparation with etching by methylene chloride for 60 s.

Performance and outcomes of transvenous rotational lead extraction: Results from a prospective, monitored, international clinical study.

BACKGROUND: Transvenous lead extraction (TLE) plays a critical role in managing patients with cardiovascular implantable electronic devices. Mechanical TLE tools, including rotational sheaths, are used to overcome fibrosis and calcification surrounding leads. Prospective clinical data are limited regarding the safety and effectiveness of use of mechanical TLE devices, especially rotational tools. OBJECTIVE: To prospectively investigate the safety and effectiveness of mechanical TLE in real-world usage. METHODS: Patients were enrolled at 10 sites in the United States and Europe to evaluate the use of mechanical TLE devices. Clinical success, complete procedural success, and complications were evaluated through follow-up (median, 29 days). Patient data were source verified and complications were adjudicated by an independent clinical events committee (CEC). RESULTS: Between October 2018 and January 2020, mechanical TLE tools, including rotational sheaths, were used to extract 460 leads with a median indwell time of 7.4 years from 230 patients (mean age 64.3 ± 14.4 years). Noninfectious indications for TLE were more common than infectious indications (61.5% vs 38.5%, respectively). The extracted leads included 305 pacemaker leads (66.3%) and 155 implantable cardioverter-defibrillator leads (33.7%), including 85 leads with passive fixation (18.5%). A bidirectional rotational sheath was needed for 368 leads (88.0%). Clinical success was obtained in 98.7% of procedures; complete procedural success was achieved for 96.3% of leads. CEC-adjudicated device-related major complications occurred in 6 of 230 (2.6%) procedures. No isolated superior vena cava injury or procedural death occurred. CONCLUSION: This prospective clinical study demonstrates that use of mechanical TLE tools, especially bidirectional rotational sheaths, are effective and safe.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

Glucose phosphorylation is required for insulin-dependent mTOR signalling in the heart.

OBJECTIVE: Insulin regulates both glucose uptake and postnatal cardiac growth. The anabolic effects of insulin are mediated by the mammalian target of rapamycin (mTOR), an evolutionarily conserved kinase which is also a convergence point between nutrient sensing and cell growth. We postulated that mTOR signalling in the heart requires the metabolism of glucose. METHODS: We interrogated the insulin-mediated mTOR signalling pathway in response to different metabolic interventions regulating substrate metabolism in the isolated working rat heart and in isolated cardiomyocytes. RESULTS: Although insulin enhanced Akt activity, phosphorylation of mTOR and its downstream targets (p70S6K and 4EBP1) required the addition of glucose. Glucose-dependent p70S6K phosphorylation was independent of the hexosamine biosynthetic pathway, the AMP kinase pathway, and the pentose phosphate pathway. However, inhibition of glycolysis downstream of hexokinase markedly enhanced p70S6K phosphorylation. Furthermore, 2-deoxyglucose activated p70S6K suggesting that phosphorylation of glucose is required for carbohydrate-mediated mTOR signalling in the heart. Lastly, we also found enhanced p70S6K phosphorylation in the hearts of diabetic rats. CONCLUSION: Phosphorylation of glucose is necessary for insulin-dependent mTOR activity in the heart, suggesting a link between intermediary metabolism and cardiac growth.

Safety and Efficacy of Transvenous Lead Extraction Utilizing the Evolution Mechanical Lead Extraction System: A Single-Center Experience.

OBJECTIVES: The goal of this study is to assess the safety and efficacy of mechanical lead extraction utilizing the Evolution system. BACKGROUND: Compared with other techniques commonly used for lead extraction, data regarding the safety and efficacy of mechanical lead extraction using the Evolution system is limited and needs further evaluation. METHODS: Between June 1, 2009 and September 30, 2016, we retrospectively analyzed 400 consecutive patients who exclusively underwent mechanical lead extraction utilizing the Evolution system. RESULTS: A total of 400 patients underwent mechanical lead extraction of 683 leads. Mean age of extracted leads was 6.77 ± 4.42 years (range 1 to 31 years). The extracted device system was an implantable cardioverter-defibrillator in 274 patients (68.5%) and a pacemaker system in 126 patients (31.5%). Complete lead removal rate was 97% with a clinical success rate of 99.75%. Incomplete lead removal with <4-cm remnant was associated with older leads (lead age >8 years). Failure to achieve clinical success was noted in 1 patient (0.25%). Cardiac papillary avulsion, system-related infection, and cardiac tamponade were the major complications noted in 6 patients (1.5%). Minor complications were encountered in 24 patients (6%), of which hematoma requiring evacuation was the most common minor complication. There were no patient deaths. CONCLUSIONS: In our single-center study, lead extractions utilizing the Evolution mechanical lead extraction system were safe and effective and resulted in high clinical and procedural success, with low complication rates and no fatalities.

Downregulation of peroxisome proliferator-activated receptor-alpha gene expression in a mouse model of ischemic cardiomyopathy is dependent on reactive oxygen species and prevents lipotoxicity.

BACKGROUND: The peroxisome proliferators-activated receptor-alpha (PPARalpha), a transcription factor that modulates fatty acid metabolism, regulates substrate preference in the heart. Although in acute ischemia there is a switch in substrate preference from fatty acids to glucose, metabolic gene expression in repetitive ischemia is not well described. In a mouse model of ischemic cardiomyopathy induced by repetitive ischemia/reperfusion (I/R), we postulated that downregulation of PPARalpha is regulated by reactive oxygen species and is necessary for maintaining contractile function in the heart. METHODS AND RESULTS: Repetitive closed-chest I/R (15 minutes) was performed daily in C57/BL6 mice, mice overexpressing extracellular superoxide dismutase, and mice treated with the PPARalpha agonist-WY-14,643. Echocardiography, histology, and candidate gene expression were measured at 3, 5, 7, and 28 days of repetitive I/R and 15 and 30 days after discontinuation of I/R. Repetitive I/R was associated with a downregulation of PPARalpha-regulated genes and both myosin heavy chain isoform transcript levels, which was reversible on discontinuation of I/R. Overexpression of EC-SOD prevented the downregulation of PPARalpha-regulated genes and myosin iso-genes by repetitive I/R. Furthermore, reactivation of PPARalpha in mice exposed to repetitive I/R worsened contractile function, induced microinfarctions, and increased intramyocardial triglyceride deposition, features suggestive of cardiac lipotoxicity. CONCLUSIONS: Metabolic and myosin isoform gene expression in repetitive I/R is mediated by reactive oxygen species. Furthermore, we suggest that downregulation of PPARalpha in repetitive I/R is an adaptive mechanism that is able to prevent lipotoxicity in the ischemic myocardium.

Induction of antioxidant gene expression in a mouse model of ischemic cardiomyopathy is dependent on reactive oxygen species.

Ischemia and reperfusion (I/R) are characterized by oxidative stress as well as changes in the antioxidant enzymes of the heart. However, little is known about the transcriptional regulation of myocardial antioxidant enzymes in repetitive I/R and hibernating myocardium. In a mouse model of ischemic cardiomyopathy induced by repetitive I/R, we postulated that induction of antioxidant gene expression was dependent on reactive oxygen species (ROS). Repetitive closed-chest I/R (15 min) was performed daily in C57/BL6 mice and in mice overexpressing extracellular superoxide dismutase (EC-SOD). Antioxidant enzyme expression was measured at 3, 5, 7, and 28 days of repetitive I/R as well as 15 and 30 days after discontinuation of I/R. In order to determine whether ROS directly modulates antioxidant gene expression, transcript levels were measured in cardiomyocytes exposed to hydrogen peroxide. Repetitive I/R caused an early and sustained increase in glutathione peroxidase (GPX) transcript levels, while heme oxygenase-1 (HO-1) expression increased only after 7 days of repetitive I/R. Overexpression of EC-SOD prevented the upregulation of GPX and HO-1 transcript levels by repetitive I/R, suggesting that both genes are regulated by ROS. However, while HO-1 transcript levels increased in cardiomyocytes exposed to hydrogen peroxide, oxidative stress failed to induce the expression of GPX implying that ROS regulates GPX transcript levels only indirectly in repetitive I/R. In conclusion, repetitive I/R was associated with an early upregulation of GPX expression as well as a delayed increase of HO-1 transcript levels in the heart. The induction of both antioxidant genes was dependent on ROS, suggesting that alterations in redox balance mediate not only tissue injury but also components of "programmed cell survival" in hibernating myocardium.