Implementation of a group-based lifestyle intervention programme (Healthy Heart) in general practices in The Netherlands: a mixed-methods study.

BACKGROUND: Lifestyle intervention programmes target behavioural risk factors that contribute to cardiovascular diseases (CVDs). Unfortunately, sustainable implementation of these programmes can be challenging. Gaining insights into the barriers and facilitators for successful implementation is important for maximising public health impact of these interventions. The Healthy Heart (HH) programme is an example of a combined lifestyle intervention programme. AIM: To analyse the reach, adoption, and implementation of the HH programme. DESIGN & SETTING: A mixed-methods study conducted in a general practice setting in The Netherlands. METHOD: Quantitative data were collected from the Healthy Heart study (HH study), a non-randomised cluster stepped-wedge trial to assess the effect of the HH programme on patients at high risk of developing CVDs at practice level. Qualitative data were obtained through focus groups. RESULTS: Out of 73 approached general practices, 55 implemented the HH programme. A total of 1082 patients agreed to participate in the HH study, of whom 64 patients were referred to the HH programme and 41 patients participated. Several barriers for participation were identified such as time investment, lack of risk perception, and being confident in changing lifestyle on their own. Important barriers for healthcare providers (HCPs) to refer a patient were time investment, lack of information to sufficiently inform patients, and preconceived notions regarding which patients the programme was suitable for. CONCLUSION: This study has offered insights from a patient and HCP perspective regarding barriers and facilitators for implementation of the group-based lifestyle intervention programme. The identified barriers and facilitators, and the suggested improvements, can be used by others who wish to implement a similar programme.

Molecular changes in hippocampal energy metabolism in mice selectively bred for extremes in stress reactivity: Relevance of mitochondrial dysfunction for affective disorders.

Affective disorders, such as major depression, are frequently associated with metabolic disturbances involving mitochondria. Although dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is known to alter energy metabolism, the precise mechanisms linking stress and metabolic disturbances are not sufficiently understood. We used a mouse model of affective disorders to investigate the impact of a genetic predisposition for extremes in stress reactivity on behavioural and metabolic phenotypes as well as energy metabolism. Adult males of three independent mouse lines selectively bred for high, intermediate or low HPA axis reactivity were tested for exploratory and locomotor activity as well as stress-coping behaviour. Additionally, basal and stress-induced plasma corticosterone levels, body weight, food intake and body composition were measured. At the molecular level, the hippocampal transcriptome was analysed using microarray, serial analysis of gene expression and qRT-PCR. Finally, mitochondrial DNA copy number, damages and mitochondrial respiration were assessed. We found clear effects of the differential stress reactivity on the behavioural, morphometric and metabolic measures. Remarkably, the hyperactive behavioural and neuroendocrine stress-coping style of high-reactivity mice was associated with significant changes in the expression of an extended list of genes involved in energy metabolism and several mitochondrial functions. Yet, only minor changes were found in mitochondrial DNA copy number, damages and respiration. Thus, our findings support a prominent role of glucocorticoids in shaping the major endophenotypes of the stress reactivity mouse model and contribute towards understanding the important role of HPA axis dysregulation and changes in energy metabolism in the pathophysiology of affective disorders.

PGC-1α Is a Master Regulator of Mitochondrial Lifecycle and ROS Stress Response.

Mitochondria play a major role in ROS production and defense during their life cycle. The transcriptional activator PGC-1α is a key player in the homeostasis of energy metabolism and is therefore closely linked to mitochondrial function. PGC-1α responds to environmental and intracellular conditions and is regulated by SIRT1/3, TFAM, and AMPK, which are also important regulators of mitochondrial biogenesis and function. In this review, we highlight the functions and regulatory mechanisms of PGC-1α within this framework, with a focus on its involvement in the mitochondrial lifecycle and ROS metabolism. As an example, we show the role of PGC-1α in ROS scavenging under inflammatory conditions. Interestingly, PGC-1α and the stress response factor NF-κB, which regulates the immune response, are reciprocally regulated. During inflammation, NF-κB reduces PGC-1α expression and activity. Low PGC-1α activity leads to the downregulation of antioxidant target genes resulting in oxidative stress. Additionally, low PGC-1α levels and concomitant oxidative stress promote NF-κB activity, which exacerbates the inflammatory response.

The Effect and Cost-Effectiveness of Offering a Combined Lifestyle Intervention for the Prevention of Cardiovascular Disease in Primary Care: Results of the Healthy Heart Stepped-Wedge Trial.

OBJECTIVE: To evaluate the effectiveness and cost-effectiveness of offering the combined lifestyle programme "Healthy Heart", addressing overweight, diet, physical activity, smoking and alcohol, to improve lifestyle behaviour and reduce cardiovascular risk. DESIGN: A practice-based non-randomised stepped-wedge cluster trial with two-year follow-up. Outcomes were obtained via questionnaires and routine care data. A cost-utility analysis was performed. During the intervention period, "Healthy Heart" was offered during regular cardiovascular risk management consultations in primary care in The Hague, The Netherlands. The period prior to the intervention period served as the control period. RESULTS: In total, 511 participants (control) and 276 (intervention) with a high cardiovascular risk were included (overall mean ± SD age 65.0 ± 9.6; women: 56%). During the intervention period, 40 persons (15%) participated in the Healthy Heart programme. Adjusted outcomes did not differ between the control and intervention period after 3-6 months and 12-24 months. Intervention versus control (95% CI) 3-6 months: weight: ß -0.5 (-1.08-0.05); SBP ß 0.15 (-2.70-2.99); LDL-cholesterol ß 0.07 (-0.22-0.35); HDL-cholesterol ß -0.03 (-0.10-0.05); physical activity ß 38 (-97-171); diet ß 0.95 (-0.93-2.83); alcohol OR 0.81 (0.44-1.49); quit smoking OR 2.54 (0.45-14.24). Results were similar for 12-24 months. Mean QALYs and mean costs of cardiovascular care were comparable over the full study period (mean difference (95% CI) QALYs: -0.10 (-0.20; 0.002); costs: EUR 106 (-80; 293)). CONCLUSIONS: For both the shorter (3-6 months) and longer term (12-24 months), offering the Healthy Heart programme to high-cardiovascular-risk patients did not improve their lifestyle behaviour nor cardiovascular risk and was not cost-effective on a population level.

Loss of respiratory complex I subunit NDUFB10 affects complex I assembly and supercomplex formation.

The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH2) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress. Complex I, the NADH-dehydrogenase, is part of the largest supercomplex (CI + CIII2 + CIVn). Here, we demonstrate the role of NDUFB10, a subunit of the membrane arm of complex I, in complex I and supercomplex assembly on the one hand and bioenergetics function on the other. NDUFB10 knockout was correlated with a decrease of SCAF1, a supercomplex assembly factor, and a reduction of respiration and mitochondrial membrane potential. This likely is due to loss of proton pumping since the CI P P -module is downregulated and the P D -module is completely abolished in NDUFB10 knock outs.

Pathophysiological Mechanisms of Cardiac Dysfunction in Transgenic Mice with Viral Myocarditis.

Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.

Inner mitochondrial membrane structure and fusion dynamics are altered in senescent human iPSC-derived and primary rat cardiomyocytes.

Dysfunction of the aging heart is a major cause of death in the human population. Amongst other tasks, mitochondria are pivotal to supply the working heart with ATP. The mitochondrial inner membrane (IMM) ultrastructure is tailored to meet these demands and to provide nano-compartments for specific tasks. Thus, function and morphology are closely coupled. Senescent cardiomyocytes from the mouse heart display alterations of the inner mitochondrial membrane. To study the relation between inner mitochondrial membrane architecture, dynamics and function is hardly possible in living organisms. Here, we present two cardiomyocyte senescence cell models that allow in cellular studies of mitochondrial performance. We show that doxorubicin treatment transforms human iPSC-derived cardiomyocytes and rat neonatal cardiomyocytes in an aged phenotype. The treated cardiomyocytes display double-strand breaks in the nDNA, have ß-galactosidase activity, possess enlarged nuclei, and show p21 upregulation. Most importantly, they also display a compromised inner mitochondrial structure. This prompted us to test whether the dynamics of the inner membrane was also altered. We found that the exchange of IMM components after organelle fusion was faster in doxorubicin-treated cells than in control cells, with no change in mitochondrial fusion dynamics at the meso-scale. Such altered IMM morphology and dynamics may have important implications for local OXPHOS protein organization, exchange of damaged components, and eventually the mitochondrial bioenergetics function of the aged cardiomyocyte.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system.

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.

Lima1 mediates the pluripotency control of membrane dynamics and cellular metabolism.

Lima1 is an extensively studied prognostic marker of malignancy and is also considered to be a tumour suppressor, but its role in a developmental context of non-transformed cells is poorly understood. Here, we characterise the expression pattern and examined the function of Lima1 in mouse embryos and pluripotent stem cell lines. We identify that Lima1 expression is controlled by the naïve pluripotency circuit and is required for the suppression of membrane blebbing, as well as for proper mitochondrial energetics in embryonic stem cells. Moreover, forcing Lima1 expression enables primed mouse and human pluripotent stem cells to be incorporated into murine pre-implantation embryos. Thus, Lima1 is a key effector molecule that mediates the pluripotency control of membrane dynamics and cellular metabolism.

Experiences with tailoring of primary diabetes care in well-organised general practices: a mixed-methods study.

BACKGROUND: Dutch standard diabetes care is generally protocol-driven. However, considering that general practices wish to tailor diabetes care to individual patients and encourage self-management, particularly in light of current COVID-19 related constraints, protocols and other barriers may hinder implementation. The impact of dispensing with protocol and implementation of self-management interventions on patient monitoring and experiences are not known. This study aims to evaluate tailoring of care by understanding experiences of well-organised practices 1) when dispensing with protocol; 2) determining the key conditions for successful implementation of self-management interventions; and furthermore exploring patients' experiences regarding dispensing with protocol and self-management interventions. METHODS: in this mixed-methods prospective study, practices (n = 49) were invited to participate if they met protocol-related quality targets, and their adult patients with well-controlled type 2 diabetes were invited if they had received protocol-based diabetes care for a minimum of 1 year. For practices, study participation consisted of the opportunity to deliver protocol-free diabetes care, with selection and implementation of self-management interventions. For patients, study participation provided exposure to protocol-free diabetes care and self-management interventions. Qualitative outcomes (practices: 5 focus groups, 2 individual interviews) included experiences of dispensing with protocol and the implementation process of self-management interventions, operationalised as implementation fidelity. Quantitative outcomes (patients: routine registry data, surveys) consisted of diabetes monitoring completeness, satisfaction, wellbeing and health status at baseline and follow-up (24 months). RESULTS: Qualitative: In participating practices (n = 4), dispensing with protocol encouraged reflection on tailored care and selection of various self-management interventions A focus on patient preferences, team collaboration and intervention feasibility was associated with high implementation fidelity Quantitative: In patients (n = 126), likelihood of complete monitoring decreased significantly after two years (OR 0.2 (95% CI 0.1-0.5), p < 0.001) Satisfaction decreased slightly (- 1.6 (95% CI -2.6;-0.6), p = 0.001) Non-significant declines were found in wellbeing (- 1.3 (95% CI -5.4; 2.9), p = 0.55) and health status (- 3.0 (95% CI -7.1; 1.2), p = 0.16). CONCLUSIONS: To tailor diabetes care to individual patients within well-organised practices, we recommend dispensing with protocol while maintaining one structural annual monitoring consultation, combined with the well-supported implementation of feasible self-management interventions. Interventions should be selected and delivered with the involvement of patients and should involve population preferences and solid team collaborations.

Mitochondrial F1 FO ATP synthase determines the local proton motive force at cristae rims.

The classical view of oxidative phosphorylation is that a proton motive force (PMF) generated by the respiratory chain complexes fuels ATP synthesis via ATP synthase. Yet, under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected these two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions. pH profiles of mitochondrial sub-compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH sensor directly at ATP synthase's F1 and FO subunits, complex IV and in the matrix. Our results clearly show that ATP synthase activity substantially controls the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible ΔpH. In addition, we show how this changes lateral, transmembrane, and radial pH gradients in glycolytic and respiratory cells.

Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development.

During implantation, the murine embryo transitions from a "quiet" into an active metabolic/proliferative state, which kick-starts the growth and morphogenesis of the post-implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine-tunes the expression of genes that encode ribosomal proteins and is required for proper tissue-scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage's high-energy demands for cell proliferation and morphogenesis.

The receptor subunit Tom20 is dynamically associated with the TOM complex in mitochondria of human cells.

The outer membrane translocase (TOM) is the import channel for nuclear-encoded mitochondrial proteins. The general import pore contains Tom40, Tom22, Tom5, Tom6, and Tom7. Precursor proteins are bound by the (peripheral) receptor proteins Tom20, Tom22, and Tom70 before being imported by the TOM complex. Here we investigated the association of the receptor Tom20 with the TOM complex. Tom20 was found in the TOM complex, but not in a smaller subcomplex. In addition, a subcomplex was found without Tom40 and Tom7 but with Tom20. Using single particle tracking of labeled Tom20 in overexpressing human cells, we show that Tom20 has, on average, higher lateral mobility in the membrane than Tom7/TOM. After ligation of Tom20 with the TOM complex by post-tranlational protein trans-splicing using the traceless, ultrafast cleaved Gp41-1 integrin system, a significant decrease in the mean diffusion coefficient of Tom20 was observed in the resulting Tom20-Tom7 fusion protein. Exposure of Tom20 to high substrate loading also resulted in reduced mobility. Taken together, our data show that the receptor subunit Tom20 interacts dynamically with the TOM core complex. We suggest that the TOM complex containing Tom20 is the active import pore and that Tom20 is associated when substrate is available.

Selective prevention of cardiovascular disease using integrated lifestyle intervention in primary care: protocol of the Healthy Heart stepped-wedge trial.

INTRODUCTION: Lifestyle interventions are shown to be effective in improving cardiovascular disease (CVD) risk factors. It has been suggested that general practitioners can play an essential role in CVD prevention. However, studies into lifestyle interventions for primary care patients at high cardiovascular risk are scarce and structural implementation of lifestyle interventions can be challenging. Therefore, this study aims to (1) evaluate (cost-)effectiveness of implementation of an integrated group-based lifestyle programme in primary care practices; (2) identify effective intervention elements and (3) identify implementation determinants of an integrated group-based lifestyle intervention for patients with high cardiovascular risk. METHODS AND ANALYSIS: The Healthy Heart study is a non-randomised cluster stepped-wedge trial. Primary care practices will first offer standard care during a control period of 2-6 months, after which practices will switch (step) to the intervention, offering participants a choice between a group-based lifestyle programme or standard care. Participants enrolled during the control period (standard care) will be compared with participants enrolled during the intervention period (combined standard care and group-based lifestyle intervention). We aim to include 1600 primary care patients with high cardiovascular risk from 55 primary care practices in the area of The Hague, the Netherlands. A mixed-methods process evaluation will be used to simultaneously assess effectiveness and implementation outcomes. The primary outcome measure will be achievement of individual lifestyle goals after 6 months. Secondary outcomes include lifestyle change of five lifestyle components (smoking, alcohol consumption, diet, weight and physical activity) and improvement of quality of life and self-efficacy. Outcomes are assessed using validated questionnaires at baseline and 3, 6, 12 and 24 months of follow-up. Routine care data will be used to compare blood pressure and cholesterol levels. Cost-effectiveness of the lifestyle intervention will be evaluated. Implementation outcomes will be assessed using the RE-AIM model, to assesses five dimensions of implementation at different levels of organisation: reach, efficacy, adoption, implementation and maintenance. Determinants of adoption and implementation will be assessed using focus groups consisting of professionals and patients. ETHICS AND DISSEMINATION: This study is approved by the Ethics Committee of the Leiden University Medical Center (P17.079). Results will be shared with the primary care group, healthcare providers and patients, and will be disseminated through journal publications and conference presentations. TRIAL REGISTRATION NUMBER: NL60795.058.17. Status: pre-results.

Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors.

Our group has previously established a strategy utilizing fluorescence lifetime probes to image membrane protein supercomplex (SC) formation in situ. We showed that a probe at the interface between individual mitochondrial respiratory complexes exhibits a decreased fluorescence lifetime when a supercomplex is formed. This is caused by electrostatic interactions with the adjacent proteins. Fluorescence lifetime imaging microscopy (FLIM) records the resulting decrease of the lifetime of the SC-probe. Here we present the details of our method for performing SC-FLIM, including the evaluation of fluorescence lifetimes from the FLIM images. To validate the feasibility of the technique for monitoring adaptive SC formation, we compare data obtained under different metabolic conditions. The results confirm that SC formation is dynamic.

The first versatile human iPSC-based model of ectopic virus induction allows new insights in RNA-virus disease.

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.

In Cellulo Protein Semi-Synthesis from Endogenous and Exogenous Fragments Using the Ultra-Fast Split Gp41-1 Intein.

Protein semi-synthesis inside live cells from exogenous and endogenous parts offers unique possibilities for studying proteins in their native context. Split-intein-mediated protein trans-splicing is predestined for such endeavors and has seen some successes, but a much larger variety of established split inteins and associated protocols is urgently needed. We characterized the association and splicing parameters of the Gp41-1 split intein, which favorably revealed a nanomolar affinity between the intein fragments combined with the exceptionally fast splicing rate. Following bead-loading of a chemically modified intein fragment precursor into live mammalian cells, we fluorescently labeled target proteins on their N- and C-termini with short peptide tags, thus ensuring minimal perturbation of their structure and function. In combination with a nuclear-entrapment strategy to minimize cytosolic fluorescence background, we applied our technique for super-resolution imaging and single-particle tracking of the outer mitochondrial protein Tom20 in HeLa cells.