Xenotransplantation of Mitochondrial Electron Transfer Enzyme, Ndi1, in Myocardial Reperfusion Injury

A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Physical, cognitive, and mental health impacts of COVID-19 after hospitalisation (PHOSP-COVID): a UK multicentre, prospective cohort study

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research

TAT-Ndi1 overcomes effects of complex I dysfunction.

<p><b>A</b>. ATP levels in rat heart tissue +/− TAT-Ndi1 following 30 min ischemia and 15min reperfusion or without treatment (veh). TAT-Ndi1 prevents depletion of ATP stores in I/R hearts. (n = 4, *p<0.05, ***p<0.0005). <b>B</b>. Dihydroethidium stained 1mm rat heart sections +/−TAT-Ndi1 following 30 min no-flow ischemia and 15 min reperfusion. TAT-Ndi1 reduces superoxide production following I/R (representative image, n = 3). <b>C</b>. Total free MDA levels normalized to total protein in hearts perfused 20 min with or without TAT-Ndi1 and subjected to 30 min ischemia and 15 min reperfusion or perfused constantly with vehicle (n = 3, **p<0.005). <b>D</b>. NAD⁺/NADH ratios from rat hearts perfused 15 min +/− TAT-Ndi1 then subjected to 30 min ischemia and 15 min reperfusion or perfused continuously with vehicle (n = 4, *p<0.05, **p<0.005).</p

Mitochondrial integrity and function is preserved by TAT-Ndi1.

<p><b>A</b>. The absorbance of cardiac mitochondrial suspension from rat heart tissue was measured in the presence or absence of TAT-Ndi1. Hearts were perfused +/− TAT-Ndi1 for 20 min prior to isolating mitochondria. TAT-Ndi1 protects against calcium-induced mitochondrial swelling and this inhibition is abolished by Ndi1 inhibitor, flavone (representative trace, n = 4). <b>B</b>. Slope and V_max of mitochondrial swelling are reduced in mitochondria with TAT-Ndi1 (n = 4, p<0.05). <b>C</b>. Oxygen consumption of mitochondria isolated from rat hearts with or without TAT-Ndi1 and subjected to I/R (I/R+TAT-Ndi1:double line, I/R alone: dashed line) or constantly perfused (Con:thick line, +TAT-Ndi1:thin line). Oxygen levels were continuously monitored using a platinum Clark-type oxygen electrode. Changes of O<b>₂</b> concentration in chamber are shown with administration of treatments indicated (n = 4, representative trace). <b>D</b>. Rate of oxygen consumption following addition of complex I substrates palmitoyl-L-carnitine/malate and ADP (1mM final) prior to (black bars) and following (grey bars) addition of rotenone (*p<0.05). Mitochondria were isolated from hearts +/− TAT-Ndi1 subjected to I/R or constantly perfused (control and Ndi1 alone).</p

Generation of TAT-Ndi1 and expression <i>in vitro</i>.

<p><b>A</b>. Map of TAT-Ndi1 construct generated from inserting full length NDI1 gene (1,539bp) from pHook(NDI1) into the 6xHis-TAT-HA cloning vector. <b>B</b>. Lysates of adult rat ventricular myocytes were transduced with TAT-Ndi1 at 500nM in complete maintenance media for 20 min. Cell lysates were probed with anti-HA antibody to detect TAT-Ndi1. <b>C</b>. Adult cardiac myocytes (first and second rows) and HL-1 cells (third row) were transduced with TAT-Ndi1 at 500nM for 1 or 15 min as indicated, fixed and double-labeled with affinity-purified rabbit antibody to <i>S. cerevisiae</i> Ndi1 and mouse monoclonal cytochrome <i>c</i> antibody.</p

TAT-Ndi1 is cardioprotective in the Langendorff-perfused rat heart model of ischemia/reperfusion.

<p><b>A</b>. Rat hearts were perfused with or without TAT-Ndi1 for 20 min prior to 30 min ischemia and 2 hour reperfusion. Frozen sections were stained with TTC. TAT-Ndi1 reduced infarct size 61.5%±8.01. Mean and S.D. from at least 5 hearts per condition. (*p<0.05). <b>B</b>. Perfusate collected prior to ischemia (baseline) and 15 min following onset of reperfusion. Creatine kinase release was reduced 51.6%±9.8 following ischemia/reperfusion in hearts perfused with TAT-Ndi1. Mean and S.D. from at least 4 hearts per condition. (**p<0.01). <b>C</b>. Hearts were subjected to 30 min ischemia and perfused with or without TAT-Ndi1 at the onset of reperfusion. Hearts were reperfused for 2 hours. Sections were stained with TTC (representative image, n = 5). TAT-Ndi1 reduced infarct size 67.1%±17.1. Mean and S.D. from at least 5 hearts per condition (*p<0.05).</p

Ndi1-mediated cytoprotection following simulated ischemia reperfusion is specific.

<p><b>A</b>. pHook(Ndi1) and pDsRed2-mito were transiently transfected into HL-1 cells. Ndi1 (stained with anti-HA antibody) co-localized with mitochondria (DsRed-mito) (merged). <b>B</b>. Ndi1-transfected and empty pHook-transfected HL-1 cells and neonatal rat cardiomyocytes were subjected to 2 hours simulated ischemia and 24 hours reperfusion. Cell death was scored by permeability to Yo-Pro-1 stain and only transfected cells were scored. (≥250 cells scored per experiment, n = 3, *p<0.05). <b>C</b>. Neonatal rat cardiomyocytes transfected with empty pHook or pHook(Ndi1) were subjected to 2 hours simulated ischemia and 24 hours reperfusion. Pretreatment with Ndi1-inhibitor flavone abolished the cytoprotective effect of Ndi1 expression. Cell death was scored by permeability to Yo-Pro-1 stain and only transfected cells were scored. (≥250 cells scored per experiment, n = 3, **p<0.005).</p