Docosahexaenoic acid lowers cardiac mitochondrial enzyme activity by replacing linoleic acid in the phospholipidome

Cardiac mitochondrial phospholipid acyl chains regulate respiratory enzymatic activity. In several diseases, the rodent cardiac phospholipidome is extensively rearranged; however, whether specific acyl chains impair respiratory enzyme function is unknown. One unique remodeling event in the myocardium of obese and diabetic rodents is an increase in docosahexaenoic acid (DHA) levels. Here, we first confirmed that cardiac DHA levels are elevated in diabetic humans relative to controls. We then used dietary supplementation of a Western diet with DHA as a tool to promote cardiac acyl chain remodeling and to study its influence on respiratory enzyme function. DHA extensively remodeled the acyl chains of cardiolipin (CL), mono-lyso CL, phosphatidylcholine, and phosphatidylethanolamine. Moreover, DHA lowered enzyme activities of respiratory complexes I, IV, V, and I+III. Mechanistically, the reduction in enzymatic activities were not driven by a dramatic reduction in the abundance of supercomplexes. Instead, replacement of tetralinoleoyl-CL with tetradocosahexaenoyl-CL in biomimetic membranes prevented formation of phospholipid domains that regulate enzyme activity. Tetradocosahexaenoyl-CL inhibited domain organization due to favorable Gibbs free energy of phospholipid mixing. Furthermore, in vitro substitution of tetralinoleoyl-CL with tetradocosahexaenoyl-CL blocked complex-IV binding. Finally, reintroduction of linoleic acid, via fusion of phospholipid vesicles to mitochondria isolated from DHA-fed mice, rescued the major losses in the mitochondrial phospholipidome and complexes I, IV, and V activities. Altogether, our results show that replacing linoleic acid with DHA lowers select cardiac enzyme activities by potentially targeting domain organization and phospholipid-protein binding, which has implications for the ongoing debate about polyunsaturated fatty acids and cardiac health

Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation

Docosahexaenoic acid (DHA, 22:6) is an n-3 polyunsaturated fatty acid (n-3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state 2H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

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

DISCRIMINATING BETWEEN CARDIOLIPIN CONCENTRATION AND ACYL CHAIN COMPOSITION ON MEMBRANE BIOPHYSICAL ORGANIZATION

The pathogenesis of cardiovascular diseases (CVDs) is driven, in part, from impairment in myocardial energy metabolism. There is convincing evidence that myocardial metabolic abnormalities are fundamentally driven by mitochondrial dysfunction. One poorly studied mechanism of mitochondrial dysfunction involves potential defects in the biophysical organization of the inner mitochondrial membrane (IMM), which is a critical regulator of mitochondrial function and energy metabolism. Many studies show that the mitochondrial specific phospholipid cardiolipin (CL) plays a central role in maintaining the structure of the IMM and thereby protein clustering and activity. Though, in several CVDs, such as diabetic cardiomyopathy, ischemia-reperfusion injury and heart failure, CL’s unique structure is considerably altered which directly diminishes CL’s function within the IMM. Two key alterations of CL that directly contribute towards mitochondrial dysfunction are a loss of CL content and aberrant CL acyl chain remodeling. However, it is currently debated as to whether a loss of CL content or CL acyl chain remodeling has a greater impact on the structure-function of the IMM. Therefore in this study, we discriminate between decreased CL content versus CL acyl chain composition on key biophysical membrane properties of the IMM. The central hypothesis for this study is that a loss of CL content, rather than CL’s acyl chain composition, disrupts mitochondrial inner membrane lipid organization by directly diminishing protein clustering and activity. Using an innovative biophysical approach, which relied on the construction of biomimetic and native mitochondrial membranes, we demonstrate that the biophysical organization of the IMM is highly dependent upon specific lipid-protein interactions. More specifically we demonstrate that specific membrane associated mitochondrial proteins induce the formation of proteolipid microdomains that are sensitive to both CL concentration and extreme CL acyl chain remodeling. Collectively, our results have strong implications for the ongoing debate about surrounding CL alterations and their impact on mitochondrial inner membrane biophysical organization. By providing a connection between specific CL alterations and mitochondrial inner membrane organization, and thereby function, our results ultimately offer strategies for clinically addressing pathophysiological CL abnormalities through the design of specific CL-targeting therapeutics

A Calorimetric and Spectroscopic Study of the Interactions Between Model Lipid Membrane Bilayers and Simple Sugars

It is well known that various organisms, such as the tardigrade, naturally use carbohydrates, particularly disaccharides, to help preserve their cell structural and functional integrity during freezing or dehydration. There have been many different studies that have focused on investigating how certain sugars may help to preserve the structural and functional integrity of biological cells throughout such extreme conditions. These studies have used lipids as a simple and effective alternative to studying how small molecules, such as disaccharides, interact with model lipid membrane bilayers. Lipid bilayers, also known as liposomes, have classically been used to study and better understand the structure of membrane bilayers since their phase behavior is very well understood. Therefore, liposomes are often used to investigate how small molecules interact with membrane bilayers, and how these interactions translate into physical, structural, and potential biological changes. Differential scanning calorimetry (DSC) and infrared spectroscopy were used to examine the thermotropic phase behavior of model liposome membranes in the presence of either sucrose or sucralose. The data indicates that membranes are dehydrated and form an interdigitated layer in the presence of sucralose, but not in the presence of sucrose. We hypothesize that this behavior is due to the different hydrophobic properties of the sugars and that sucralose may penetrate the bilayer whereas sucrose will not. Additionally, at higher concentrations of sucralose the data suggests that sucralose may hydrogen bond to the polar head groups, particularly to the phosphate region of the lipid molecule, in an effort to stabilize the membrane bilayer and Van de Waals forces upon interdigitation. This study demonstrates that sucralose has a significant effect on the phase behavior of liposomes, and its usefulness as a possible cryoprotecting agent for the preservation of biological cells and tissues is of great interest and needs to be comprehensively investigated.  M.S

DISCRIMINATING BETWEEN CARDIOLIPIN CONCENTRATION AND ACYL CHAIN COMPOSITION ON MEMBRANE BIOPHYSICAL ORGANIZATION

The pathogenesis of cardiovascular diseases (CVDs) is driven, in part, from impairment in myocardial energy metabolism. There is convincing evidence that myocardial metabolic abnormalities are fundamentally driven by mitochondrial dysfunction. One poorly studied mechanism of mitochondrial dysfunction involves potential defects in the biophysical organization of the inner mitochondrial membrane (IMM), which is a critical regulator of mitochondrial function and energy metabolism. Many studies show that the mitochondrial specific phospholipid cardiolipin (CL) plays a central role in maintaining the structure of the IMM and thereby protein clustering and activity. Though, in several CVDs, such as diabetic cardiomyopathy, ischemia-reperfusion injury and heart failure, CL’s unique structure is considerably altered which directly diminishes CL’s function within the IMM. Two key alterations of CL that directly contribute towards mitochondrial dysfunction are a loss of CL content and aberrant CL acyl chain remodeling. However, it is currently debated as to whether a loss of CL content or CL acyl chain remodeling has a greater impact on the structure-function of the IMM. Therefore in this study, we discriminate between decreased CL content versus CL acyl chain composition on key biophysical membrane properties of the IMM. The central hypothesis for this study is that a loss of CL content, rather than CL’s acyl chain composition, disrupts mitochondrial inner membrane lipid organization by directly diminishing protein clustering and activity. Using an innovative biophysical approach, which relied on the construction of biomimetic and native mitochondrial membranes, we demonstrate that the biophysical organization of the IMM is highly dependent upon specific lipid-protein interactions. More specifically we demonstrate that specific membrane associated mitochondrial proteins induce the formation of proteolipid microdomains that are sensitive to both CL concentration and extreme CL acyl chain remodeling. Collectively, our results have strong implications for the ongoing debate about surrounding CL alterations and their impact on mitochondrial inner membrane biophysical organization. By providing a connection between specific CL alterations and mitochondrial inner membrane organization, and thereby function, our results ultimately offer strategies for clinically addressing pathophysiological CL abnormalities through the design of specific CL-targeting therapeutics

A Calorimetric and Spectroscopic Study of the Interactions Between Model Lipid Membrane Bilayers and Simple Sugars

It is well known that various organisms, such as the tardigrade, naturally use carbohydrates, particularly disaccharides, to help preserve their cell structural and functional integrity during freezing or dehydration. There have been many different studies that have focused on investigating how certain sugars may help to preserve the structural and functional integrity of biological cells throughout such extreme conditions. These studies have used lipids as a simple and effective alternative to studying how small molecules, such as disaccharides, interact with model lipid membrane bilayers. Lipid bilayers, also known as liposomes, have classically been used to study and better understand the structure of membrane bilayers since their phase behavior is very well understood. Therefore, liposomes are often used to investigate how small molecules interact with membrane bilayers, and how these interactions translate into physical, structural, and potential biological changes. Differential scanning calorimetry (DSC) and infrared spectroscopy were used to examine the thermotropic phase behavior of model liposome membranes in the presence of either sucrose or sucralose. The data indicates that membranes are dehydrated and form an interdigitated layer in the presence of sucralose, but not in the presence of sucrose. We hypothesize that this behavior is due to the different hydrophobic properties of the sugars and that sucralose may penetrate the bilayer whereas sucrose will not. Additionally, at higher concentrations of sucralose the data suggests that sucralose may hydrogen bond to the polar head groups, particularly to the phosphate region of the lipid molecule, in an effort to stabilize the membrane bilayer and Van de Waals forces upon interdigitation. This study demonstrates that sucralose has a significant effect on the phase behavior of liposomes, and its usefulness as a possible cryoprotecting agent for the preservation of biological cells and tissues is of great interest and needs to be comprehensively investigated

DISCRIMINATING BETWEEN CARDIOLIPIN CONCENTRATION AND ACYL CHAIN COMPOSITION ON MEMBRANE BIOPHYSICAL ORGANIZATION

The pathogenesis of cardiovascular diseases (CVDs) is driven , in part , from impairment in myocardial energy metabolism. There is convincing evidence that myocardial metabolic abnormalities are fundamentally driven by mitochondrial dysfunction. One poorly studied mechanism of mitochondrial dysfunction involves potential defects in the biophysical organization of the inner mitochondrial membrane (IMM) , which is a critical regulator of mitochondrial function and energy metabolism. Many studies show that the mitochondrial specific phospholipid cardiolipin (CL) plays a central role in maintaining the structure of the IMM and thereby protein clustering and activity. Though , in several CVDs , such as diabetic cardiomyopathy , ischemia-reperfusion injury and heart failure , CL's unique structure is considerably altered which directly diminishes CL's function within the IMM. Two key alterations of CL that directly contribute towards mitochondrial dysfunction are a loss of CL content and aberrant CL acyl chain remodeling. However , it is currently debated as to whether a loss of CL content or CL acyl chain remodeling has a greater impact on the structure-function of the IMM. Therefore in this study , we discriminate between decreased CL content versus CL acyl chain composition on key biophysical membrane properties of the IMM. The central hypothesis for this study is that a loss of CL content , rather than CL's acyl chain composition , disrupts mitochondrial inner membrane lipid organization by directly diminishing protein clustering and activity. Using an innovative biophysical approach , which relied on the construction of biomimetic and native mitochondrial membranes , we demonstrate that the biophysical organization of the IMM is highly dependent upon specific lipid-protein interactions. More specifically we demonstrate that specific membrane associated mitochondrial proteins induce the formation of proteolipid microdomains that are sensitive to both CL concentration and extreme CL acyl chain remodeling. Collectively , our results have strong implications for the ongoing debate about surrounding CL alterations and their impact on mitochondrial inner membrane biophysical organization. By providing a connection between specific CL alterations and mitochondrial inner membrane organization , and thereby function , our results ultimately offer strategies for clinically addressing pathophysiological CL abnormalities through the design of specific CL-targeting therapeutics