The Brief Solastalgia Scale: A Psychometric Evaluation and Revision.

Witnessing degradation and loss to one's home environment can cause the negative emotional experience of solastalgia. We review the psychometric properties of the 9-item Solastalgia subscale from the Environmental Distress Scale (Higginbotham et al. (EcoHealth 3:245-254, 2006)). Using data collected from three large, independent, adult samples (N = 4229), who were surveyed soon after the 2019/20 Australian bushfires, factor analyses confirmed the scale's unidimensionality, while analyses derived from Item Response Theory highlighted the poor psychometric performance and redundant content of specific items. Consequently, we recommend a short-form scale consisting of five items. This Brief Solastalgia Scale (BSS) yielded excellent model fit and internal consistency in both the initial and cross-validation samples. The BSS and its parent version provide very similar patterns of associations with demographic, health, life satisfaction, climate emotion, and nature connectedness variables. Finally, multi-group confirmatory factor analysis demonstrated comparable construct architecture (i.e. configural, metric, and scalar invariance) across validation samples, gender categories, and age. As individuals and communities increasingly confront and cope with climate change and its consequences, understanding related emotional impacts is crucial. The BSS promises to aid researchers, decision makers, and practitioners to understand and support those affected by negative environmental change.

Informed consent-patients' understanding of risk.

AIMS: The central concept of informed consent is communication of the chance of a successful outcome. The risks and benefits are probabilistic concepts derived from populations; they do not map with any certainty to the individual. We tested patients' comprehension of basic probability concepts that are needed for informed consent. METHODS: Patients (n=478) completed five questions designed to test risk estimates that are relevant to informed consent. The questions posed non-medical scenarios to avoid patients associating them with their clinical care. The questionnaire was in English and was only offered to patients whose nurse felt that they had sufficient English literacy to understand the questions. RESULTS: Out of a possible total of five correct answers, Asian patients scored lowest, and significantly less than Pakeha/Europeans (average total score 2.6±1.7 vs 3.6±1.4, p<0.001, 95% confidence interval 0.5 to 1.38). The total score for Maori/Pasifika was intermediate (3.2±1.4), yet they had the lowest deprivation index. This discordant finding may be due to poorer English literacy among Asian participants. On multiple linear regression, Asian ethnicity and advancing age were the independent predictors of a low score. Socio-economic deprivation decile and sex were not. CONCLUSIONS: When answering questions constructed according to best practice, many (but not all) patients have reasonable risk comprehension. Further improvement could target older patients, those of Asian ethnicity and probably all patients where English is a second language. Liberal use of interpreters is suggested.

Predictors of individual mental health and psychological resilience after Australia's 2019-2020 bushfires.

AIMS: We assessed the mental health effects of Australia's 2019-2020 bushfires 12-18 months later, predicting psychological distress and positive psychological outcomes from bushfire exposure and a range of demographic variables, and seeking insights to enhance disaster preparedness and resilience planning for different profiles of people. METHODS: We surveyed 3083 bushfire-affected and non-affected Australian residents about their experiences of bushfire, COVID-19, psychological distress (depression, anxiety, stress, post-traumatic stress disorder) and positive psychological outcomes (resilient coping, wellbeing). RESULTS: We found high rates of distress across all participants, exacerbated by severity of bushfire exposure. For people who were bushfire-affected, being older, having less financial stress, and having no or fewer pre-existing mental disorders predicted both lower distress and higher positive outcomes. Being male or having less income loss also predicted positive outcomes. Severity of exposure, higher education and higher COVID-19-related stressors predicted both higher distress and higher positive outcomes. Pre-existing physical health diagnosis and previous bushfire experience did not significantly predict distress or positive outcomes. RECOMMENDATIONS: To promote disaster resilience, we recommend investment in mental health, particularly for younger adults and for those in rural and remote areas. We also recommend investment in mechanisms to protect against financial distress and the development of a broader definition of bushfire-related impacts than is currently used to capture brushfires' far-reaching effects.

Reduction in Junctophilin 2 Expression in Cardiac Nodal Tissue Results in Intracellular Calcium-Driven Increase in Nodal Cell Automaticity.

BACKGROUND: Spontaneously depolarizing nodal cells comprise the pacemaker of the heart. Intracellular calcium (Ca2+) plays a critical role in mediating nodal cell automaticity and understanding this so-called Ca2+ clock is critical to understanding nodal arrhythmias. We previously demonstrated a role for Jph2 (junctophilin 2) in regulating Ca2+-signaling through inhibition of RyR2 (ryanodine receptor 2) Ca2+ leak in cardiac myocytes; however, its role in pacemaker function and nodal arrhythmias remains unknown. We sought to determine whether nodal Jph2 expression silencing causes increased sinoatrial and atrioventricular nodal cell automaticity due to aberrant RyR2 Ca2+ leak. METHODS: A tamoxifen-inducible, nodal tissue-specific, knockdown mouse of Jph2 was achieved using a Cre-recombinase-triggered short RNA hairpin directed against Jph2 (Hcn4:shJph2). In vivo cardiac rhythm was monitored by surface ECG, implantable cardiac telemetry, and intracardiac electrophysiology studies. Intracellular Ca2+ imaging was performed using confocal-based line scans of isolated nodal cells loaded with fluorescent Ca2+ reporter Cal-520. Whole cell patch clamp was conducted on isolated nodal cells to determine action potential kinetics and sodium-calcium exchanger function. RESULTS: Hcn4:shJph2 mice demonstrated a 40% reduction in nodal Jph2 expression, resting sinus tachycardia, and impaired heart rate response to pharmacologic stress. In vivo intracardiac electrophysiology studies and ex vivo optical mapping demonstrated accelerated junctional rhythm originating from the atrioventricular node. Hcn4:shJph2 nodal cells demonstrated increased and irregular Ca2+ transient generation with increased Ca2+ spark frequency and Ca2+ leak from the sarcoplasmic reticulum. This was associated with increased nodal cell AP firing rate, faster diastolic repolarization rate, and reduced sodium-calcium exchanger activity during repolarized states compared to control. Phenome-wide association studies of the JPH2 locus identified an association with sinoatrial nodal disease and atrioventricular nodal block. CONCLUSIONS: Nodal-specific Jph2 knockdown causes increased nodal automaticity through increased Ca2+ leak from intracellular stores. Dysregulated intracellular Ca2+ underlies nodal arrhythmogenesis in this mouse model.

Telehealth use by mental health professionals during COVID-19.

OBJECTIVE: To examine and describe telehealth use and attitudes among mental health professionals in Australia and New Zealand during the initial stages of the COVID-19 pandemic. METHODS: Participants completed a brief online survey between May and July 2020. Participants were recruited via peak and professional organisations and through psychology-focused social media groups and networks. The survey examined frequency of telehealth use, reasons for non-use, telehealth modalities, prior use, attitudes towards use, plans for future use, and training, information or resource needs. RESULTS: A total of 528 professionals (85.2% female) participated in the survey, of which 98.9% reported using telehealth and 32.2% reported using telehealth exclusively. Respondents were less likely to use telehealth if they worked with clients experiencing complex issues (e.g. trauma), had more hours of weekly client contact, had a choice about whether to use telehealth or felt less positive about using technology. Respondents were more likely to hold positive views towards telehealth if they were female, had used online programmes with clients previously, were frequent telehealth users and were comfortable using technology. Participants expressed mixed views on client safety and the impact of telehealth on therapeutic process and effectiveness. CONCLUSION: Telehealth has a clear and ongoing role within mental healthcare and there is a need for strong guidance for professionals on how to manage client risk, privacy, security and adapt therapy for delivery via telehealth. In particular, there is a need for individual-, organisational-, professional- and policy-level responses to ensure that telehealth remains a viable and effective healthcare medium into the future.

Genetic inhibition of nuclear factor of activated T-cell c2 prevents atrial fibrillation in CREM transgenic mice.

AIMS: Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. METHODS AND RESULTS: Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2-/- mice. Programmed electrical stimulation revealed that CREM:NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription. CONCLUSION: Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.

Physical and Mental Health Effects of Bushfire and Smoke in the Australian Capital Territory 2019-20.

The 2019-20 bushfire season in south-eastern Australia was one of the most severe in recorded history. Bushfire smoke-related air pollution reached hazardous levels in major metropolitan areas, including the Australian Capital Territory (ACT), for prolonged periods of time. Bushfire smoke directly challenges human health through effects on respiratory and cardiac function, but can also indirectly affect health, wellbeing and quality of life. Few studies have examined the specific health effects of bushfire smoke, separate from direct effects of fire, and looked beyond physical health symptoms to consider effects on mental health and lifestyle in Australian communities. This paper describes an assessment of the health impacts of this prolonged exposure to hazardous levels of bushfire smoke in the ACT and surrounding area during the 2019-20 bushfire season. An online survey captured information on demographics, health (physical and mental health, sleep) and medical advice seeking from 2,084 adult participants (40% male, median age 45 years). Almost all participants (97%) experienced at least one physical health symptom that they attributed to smoke, most commonly eye or throat irritation, and cough. Over half of responders self-reported symptoms of anxiety and/or feeling depressed and approximately half reported poorer sleep. Women reported all symptoms more frequently than men. Participants with existing medical conditions or poorer self-rated health, parents and those directly affected by fire (in either the current or previous fire seasons) also experienced poorer physical, mental health and/or sleep symptoms. Approximately 17% of people sought advice from a medical health practitioner, most commonly a general practitioner, to manage their symptoms. This study demonstrated that prolonged exposure to bushfire smoke can have substantial effects on health. Holistic approaches to understanding, preventing and mitigating the effects of smoke, not just on physical health but on mental health, and the intersection of these, is important. Improved public health messaging is needed to address uncertainty about how individuals can protect their and their families health for future events. This should be informed by identifying subgroups of the population, such as those with existing health conditions, parents, or those directly exposed to fire who may be at a greater risk.

Video-consulting to address mental health needs after traumatic brain injury: evaluation of a training workshop to build capacity among psychologists.

BACKGROUND: Traumatic brain injury (TBI) can lead to significant psychological distress, but few psychologists in Australia are trained in working with this complex clinical group. Despite government funding to provide video-consulting (VC) services in Australia, uptake before COVID-19 was limited. OBJECTIVE: This mixed methods study evaluated whether training in eHealth and evidence based TBI psychological therapies increased provider uptake of VC in clinical practice, and delivery of mental health services to individuals with TBI. METHODS: Mental health professionals completed a range of self-report measures before (n = 50), after (n = 48), and four months following (n = 30) a one-day workshop. Participants' TBI knowledge, client-base and levels of access, confidence, motivation and attitudes toward VC were assessed. Knowledge did not increase after training but participants had significant increases in their confidence and motivation to using VC at follow up. Significant reductions in pragmatic barriers to using VC were reported post training and at follow up, all barrier categories indicated significant reductions. There was no significant change in clinical practice of the participants. CONCLUSIONS: Training to increase TBI knowledge requires specific assessment tools and although training appears to reduce barriers to using VC, uptake in clinical practice may require additional supervision and warrants further research.

An Ecological Momentary Intervention for people with social anxiety: A descriptive case study.

This study describes the development and pilot evaluation of a smartphone- delivered Ecological Momentary Intervention (EMI) for people with social anxiety symptoms. Using a software engineering framework (agile modeling, model-driven development, bottom-up development), mental health experts and software developers collaborated to develop a 4-module EMI app designed to reduce social anxiety in real-time. Fifty-five participants with social anxiety were randomly allocated to the EMI or a wait-list control arm. App downloads, usage and user satisfaction data were collected and mental health outcomes assessed at baseline and post-intervention. Software development practices allowed mental health experts to distil core elements of a psychological intervention into discrete software components but there were challenges in engaging mental health experts in the process. Relative to control there was no significant reduction in social anxiety among the EMI participants in the pilot trial. However, post-test data were available for only 4 intervention and 10 control participants and only 2 (4.0%) of the EMI participants downloaded the app. The two participants who both accessed the app and completed the post-test reported being satisfied with the intervention. Future research should address managing resources and providing additional training to support ongoing engagement with key stakeholders.

Calmodulin kinase II regulates atrial myocyte late sodium current, calcium handling, and atrial arrhythmia.

BACKGROUND: Atrial fibrillation (AF) is the most common type of arrhythmia. Abnormal atrial myocyte Ca2+ handling promotes aberrant membrane excitability and remodeling that are important for atrial arrhythmogenesis. The sequence of molecular events leading to loss of normal atrial myocyte Ca2+ homeostasis is not established. Late Na+ current (INa,L) is increased in atrial myocytes from AF patients together with an increase in activity of Ca2+/calmodulin-dependent kinase II (CaMKII). OBJECTIVE: The purpose of this study was to determine whether CaMKII-dependent phosphorylation at Ser571 on NaV1.5 increases atrial INa,L, leading to aberrant atrial Ca2+ cycling, altered electrophysiology, and increased AF risk. METHODS: Atrial myocyte electrophysiology, Ca2+ handling, and arrhythmia susceptibility were studied in wild-type and Scn5a knock-in mice expressing phosphomimetic (S571E) or phosphoresistant (S571A) NaV1.5 at Ser571. RESULTS: Atrial myocytes from S571E but not S571A mice displayed an increase in INa,L and action potential duration, and with adrenergic stress have increased delayed afterdepolarizations. Frequency of Ca2+ sparks and waves was increased in S571E atrial myocytes compared to wild type. S571E mice showed an increase in atrial events induced by adrenergic stress and AF inducibility in vivo. Isolated S571E atria were more susceptible to spontaneous atrial events, which were abrogated by inhibiting sarcoplasmic reticulum Ca2+ release, CaMKII, or the Na+/Ca2+ exchanger. Expression of phospho-NaV1.5 at Ser571 and autophosphorylated CaMKII were increased in atrial samples from human AF patients. CONCLUSION: This study identified CaMKII-dependent regulation of NaV1.5 as an important upstream event in Ca2+ handling defects and abnormal impulse generation in the setting of AF.

Stakeholder perspectives on evidence for digital mental health interventions: Implications for accreditation systems.

BACKGROUND: Digital mental health interventions can be effective for treating mental health problems, but uptake by consumers and clinicians is not optimal. The lack of an accreditation pathway for digital mental health interventions is a barrier to their uptake among clinicians and consumers. However, there are a number of factors that may contribute to whether a digital intervention is suitable for recommendation to the public. The aim of this study was to identify the types of evidence that would support the accreditation of digital interventions. METHOD: An expert workshop was convened, including researcher, clinician, consumer (people with lived experience of a mental health condition) and policymaker representatives. RESULTS: Existing methods for assessing the evidence for digital mental health interventions were discussed by the stakeholders present at the workshop. Empirical evidence from randomised controlled trials was identified as a key component for evaluating digital interventions. However, information on the safety of users, data security, user ratings, and fidelity to clinical guidelines, along with data from routine care including adherence, engagement and clinical outcomes, were also identified as important considerations when evaluating an intervention. There are considerable challenges in weighing the evidence for a digital mental health intervention. CONCLUSIONS: Empirical evidence should be the cornerstone of any accreditation system to identify appropriate digital mental health interventions. However, robust accreditation systems should also account for program and user safety, user engagement and experience, and fidelity to clinical treatment guidelines.

A Peer-Led Electronic Mental Health Recovery App in a Community-Based Public Mental Health Service: Pilot Trial.

BACKGROUND: There is an increasing need for peer workers (people with lived experience of mental health problems who support others) to work alongside consumers to improve recovery and outcomes. In addition, new forms of technology (tablet or mobile apps) can deliver services in an engaging and innovative way. However, there is a need to evaluate interventions in real-world settings. OBJECTIVE: This exploratory proof-of-concept study aimed to determine if a peer worker-led electronic mental health (e-mental health) recovery program is a feasible, acceptable, and effective adjunct to usual care for people with moderate-to-severe mental illness. METHODS: Overall, 6 consumers and 5 health service staff participated in the evaluation of a peer-led recovery app delivered at a community-based public mental health service. The peer worker and other health professional staff invited attendees at the drop-in medication clinics to participate in the trial during June to August 2017. Following the intervention period, participants were also invited by the peer worker to complete the evaluation in a separate room with the researcher. Consumers were explicitly informed that participation in the research evaluation was entirely voluntary. Consumer evaluation measures at postintervention included recovery and views on the acceptability of the program and its delivery. Interviews with staff focused on the acceptability and feasibility of the app itself and integrating a peer worker into the health care service. RESULTS: Consumer recruitment in the research component of the study (n=6) fell substantially short of the target number of participants (n=30). However, from those who participated, both staff and consumers were highly satisfied with the peer worker and somewhat satisfied with the app. Health care staff overall believed that the addition of the peer worker was highly beneficial to both the consumers and staff. CONCLUSIONS: The preliminary findings from this proof-of-concept pilot study suggest that a peer-led program may be a feasible and acceptable method of working on recovery in this population. However, the e-mental health program did not appear feasible in this setting. In addition, recruitment was challenging in this particular group, and it is important to note that these study findings may not be generalizable. Despite this, ensuring familiarity of technology in the target population before implementing e-mental health interventions is likely to be of benefit.

Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation.

BACKGROUND: Abnormal calcium (Ca2+) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN. METHODS: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln-knockout mice. Ppp1r3a-knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a-knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca2+ imaging. RESULTS: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF. CONCLUSIONS: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.

Service availability and capacity in rural mental health in Australia: Analysing gaps using an Integrated Mental Health Atlas.

OBJECTIVE: Access to services and workforce shortages are major challenges in rural areas worldwide. In order to improve access to mental health care, it is imperative to understand what services are available, what their capacity is and where existing funds might be spent to increase availability and accessibility. The aim of this study is to investigate mental health service provision in a selection of rural and remote areas across Australia by analysing service availability, placement capacity and diversity. METHOD: This research studies the health regions of Western New South Wales and Country Western Australia and their nine health areas. Service provision was analysed using the DESDE-LTC system for long-term care service description and classification that allows international comparison. Rates per 100,000 inhabitants were calculated to compare the care availability and placement capacity for children and adolescents, adults and older adults. RESULTS: The lowest diversity was found in northern Western Australia. Overall, Western New South Wales had a higher availability of non-acute outpatient services for adults, but hardly any acute outpatient services. In Country Western Australia, substantially fewer non-acute outpatient services were found, while acute services were much more common. Acute inpatient care services were more common in Western New South Wales, while sub-acute inpatient services and non-acute day care services were only found in Western New South Wales. CONCLUSION: The number and span of services in the two regions showed discrepancies both within and between regions, raising issues on the equity of access to mental health care in Australia. The standard description of the local pattern of rural mental health care and its comparison across jurisdictions is critical for evidence-informed policy planning and resource allocation.

Implementation of genotype-guided dosing of warfarin with point-of-care genetic testing in three UK clinics: a matched cohort study.

BACKGROUND: Warfarin is a widely used oral anticoagulant. Determining the correct dose required to maintain the international normalised ratio (INR) within a therapeutic range can be challenging. In a previous trial, we showed that a dosing algorithm incorporating point-of-care genotyping information ('POCT-GGD' approach) led to improved anticoagulation control. To determine whether this approach could translate into clinical practice, we undertook an implementation project using a matched cohort design. METHODS: At three clinics (implementation group; n = 119), initial doses were calculated using the POCT-GGD approach; at another three matched clinics (control group; n = 93), patients were dosed according to the clinic's routine practice. We also utilised data on 640 patients obtained from routinely collected data at comparable clinics. Primary outcome was percentage time in target INR range. Patients and staff from the implementation group also provided questionnaire feedback on POCT-GGD. RESULTS: Mean percentage time in INR target range was 55.25% in the control group and 62.74% in the implementation group; therefore, 7.49% (95% CI 3.41-11.57%) higher in the implementation group (p = 0.0004). Overall, patients and staff viewed POCT-GGD positively, suggesting minor adjustments to allow smooth implementation into practice. CONCLUSIONS: In the first demonstration of the implementation of genotype-guided dosing, we show that warfarin dosing determined using an algorithm incorporating genetic and clinical factors can be implemented smoothly into clinic, to ensure target INR range is reached sooner and maintained. The findings are like our previous randomised controlled trial, providing an alternative method for improving the risk-benefit of warfarin use in daily practice.

Alterations in cardiovascular function in an experimental model of lung fibrosis and pulmonary hypertension.

NEW FINDINGS: What is the central question of this study? We have evaluated changes in cardiovascular physiology using echocardiography in an experimental model of lung fibrosis. What is the main finding and its importance? Remarkably, we report changes in cardiovascular function as early as day 7, concomitant with evidence of vascular remodelling. We also report that isolated pulmonary arteries were hypercontractile in response to a thromboxane A2 agonist. These findings are significant because the development of pulmonary hypertension is one of the most significant predictors of mortality in patients with lung fibrosis, where there are no available therapies and a lack of animal models. ABSTRACT: Group III pulmonary hypertension is observed in patients with chronic lung diseases such as chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. Pulmonary hypertension (PH) develops as a result of extensive pulmonary vascular remodelling and resultant changes in vascular tone that can lead to right ventricle hypertrophy. This eventually leads to right heart failure, which is the leading indicator of mortality in patients with idiopathic pulmonary fibrosis. Treatments for group III PH are not available, in part owing to a lack of viable animal models. Here, we have evaluated the cardiovascular changes in a model of lung fibrosis and PH. Data obtained from this study indicated that structural alterations in the right heart, such as right ventricular wall hypertrophy, occurred as early as day 14, and similar increases in right ventricle chamber size were seen between days 21 and 28. These structural changes were correlated with decreases in the systolic function of the right ventricle and right ventricular cardiac output, which also occurred between the same time points. Characterization of pulmonary artery dynamics also highlighted that PH might be occurring as early as day 21, indicated by reductions in the velocity-time integral; however, evidence for PH is apparent as early as day 7, indicated by the significant reduction in pulmonary acceleration time values. These changes are consistent with evidence of vascular remodelling observed histologically starting on day 7. In addition, we report hyperactivity of bleomycin-exposed pulmonary arteries to a thromboxane A2 receptor (Tbxa2r) agonist.

Twik-2-/- mouse demonstrates pulmonary vascular heterogeneity in intracellular pathways for vasocontractility.

We have previously shown Twik-2-/- mice develop pulmonary hypertension and vascular remodeling. We hypothesized that distal pulmonary arteries (D-PAs) of the Twik-2-/- mice are hypercontractile under physiological venous conditions due to altered electrophysiologic properties between the conduit and resistance vessels in the pulmonary vascular bed. We measured resting membrane potential and intracellular calcium through Fura-2 in freshly digested pulmonary artery smooth muscles (PASMCs) from both the right main (RM-PA) and D-PA (distal) regions of pulmonary artery from WT and Twik-2-/- mice. Whole segments of RM-PAs and D-PAs from 20 to 24-week-old wildtype (WT) and Twik-2-/- mice were also pressurized between two glass micropipettes and bathed in buffer with either arterial or venous conditions. Abluminally-applied phenylephrine (PE) and U46619 were added to the buffer at log increments and vessel diameter was measured. All values were expressed as averages with ±SEM. Vasoconstrictor responses did not differ between WT and Twik-2-/- RM-PAs under arterial conditions. Under venous conditions, Twik-2-/- RM-PAs showed an increased sensitivity to PE with a lower EC50 (P = 0.02). Under venous conditions, Twik-2-/- D-PAs showed an increase maximal vasoconstrictor response to both phenylephrine and U46619 compared to the WT mice (P < 0.05). Isolated PASMCs from Twik-2 -/- D-PA were depolarized and had higher intracellular calcium levels compared to PASMCs from RM-PA of both WT and Twik-2-/- mice. These studies suggest that hypercontractile responses and electrophysiologic properties unique to the anatomic location of the D-PAs may contribute to pulmonary hypertensive vasculopathy.

Atrial-Specific Gene Delivery Using an Adeno-Associated Viral Vector.

RATIONALE: Somatic overexpression in mice using an adeno-associated virus (AAV) as gene transfer vectors has become a valuable tool to analyze the roles of specific genes in cardiac diseases. The lack of atrial-specific AAV vector has been a major obstacle for studies into the pathogenesis of atrial diseases. Moreover, gene therapy studies for atrial fibrillation would benefit from atrial-specific vectors. Atrial natriuretic factor (ANF) promoter drives gene expression specifically in atrial cardiomyocytes. OBJECTIVE: To establish the platform of atrial specific in vivo gene delivery by AAV-ANF. METHODS AND RESULTS: We constructed AAV vectors based on serotype 9 (AAV9) that are driven by the atrial-specific ANF promoter. Hearts from mice injected with AAV9-ANF-GFP (green fluorescent protein) exhibited strong and atrial-specific GFP expression without notable GFP in ventricular tissue. In contrast, similar vectors containing a cardiac troponin T promoter (AAV9-TNT4-GFP) showed GFP expression in all 4 chambers of the heart, while AAV9 with an enhanced chicken ß-actin promoter (AAV-enCB-GFP) caused ubiquitous GFP expression. Next, we used Rosa26mT/mG (membrane-targeted tandem dimer Tomato/membrane-targeted GFP), a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato before Cre-mediated excision, and membrane-targeted GFP after excision. AAV9-ANF-Cre led to highly efficient LoxP recombination in membrane-targeted tandem dimer Tomato/membrane-targeted green fluorescent protein mice with high specificity for the atria. We measured the frequency of transduced cardiomyocytes in atria by detecting Cre-dependent GFP expression from the Rosa26mT/mG allele. AAV9 dose was positively correlated with the number of GFP-positive atrial cardiomyocytes. Finally, we assessed whether the AAV9-ANF-Cre vector could be used to induce atrial-specific gene knockdown in proof-of-principle experiments using conditional JPH2 (junctophilin-2) knockdown mice. Four weeks after AAV9-ANF-Cre injection, a strong reduction in atrial expression of JPH2 protein was observed. Furthermore, there was evidence for abnormal Ca2+ handling in atrial myocytes isolated from mice with atrial-restricted JPH2 deficiency. CONCLUSIONS: AAV9-ANF vectors produce efficient, dose-dependent, and atrial-specific gene expression following a single-dose systemic delivery in mice. This vector is a novel reagent for both mechanistic and gene therapy studies on atrial diseases.

In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia.

RATIONALE: Autosomal-dominant mutations in ryanodine receptor type 2 ( RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia. OBJECTIVE: To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+). METHODS AND RESULTS: Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 ( Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients. CONCLUSIONS: AAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.