No abstract

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.

Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact. This combination can be applied to single rodent primary adult cardiomyocytes, and human stem cell derived cardiomyocytes, enabling contactless small molecule evaluation for inhibitors of sodium, potassium and calcium channels suggesting it may be useful for early toxicity work. In pancreatic beta-cells it reveals the effects of glucose and the KATP inhibitor gliclazide.

Background Transthyretin amyloidosis, or ATTR, is a progressive and debilitating rare proteopathy generally manifested as either transthyretin amyloid polyneuropathy (ATTR-PN) or transthyretin amyloid cardiomyopathy (ATTR-CM). Irrespective of the clinical presentation, affected patients manage a chronic and life-threatening condition that severely impacts their quality of life. Although the primary symptoms and diagnostic criteria for ATTR are increasingly being discussed in the medical literature, due in large part by continual advances in uncovering disease pathophysiology, there exists a surprising paucity of published data on the patient journey and family experience. In order to address this disparity, two focus groups, one for ATTR-CM and one for ATTR-PN, were convened and asked to describe the diagnostic process, symptoms, and impact on their own quality of life that was experienced from these rare and typically misdiagnosed illnesses. Results Patients in both ATTR groups often underwent a long and difficult diagnostic odyssey characterized by seemingly nonspecific physical manifestations resulting in mismanagement and suboptimal care, inadequate interventions, and delays in establishing the correct diagnosis, which was integral to determining the specialized treatment they needed. Collectively, patients with ATTR-CM and patients with ATTR-PN reported a similar number of symptoms, but the type of symptoms varied. The ATTR-CM group identified intolerance to activity, inability to exercise, insomnia and fatigue as the most challenging symptoms. The ATTR-PN group identified fatigue, diarrhea/constipation and sensory deficits as the most difficult symptoms. In general, ATTR was reported to be highly stressful for both patients and their families. Spouses of patients with ATTR-CM were often in a caregiver role and reported experiencing considerable anxiety. Patients with ATTR-PN were stressed not only by the physical consequences of their illness, but also by its effects on their parents and other relatives, as well as concerns about children and grandchildren inheriting the disease-causing mutations associated with ATTR. Despite such challenges, family members are identified as an important resource of coping, motivation, inspiration and support. Conclusions Several steps can be taken to reduce the challenges and burdens of living with ATTR, including increased education for primary care physicians and specialists who unknowingly encounter ATTR, increased access to and ready availability of mental health services and support, and increased engagement with support groups and advocacy organizations. Input from patients and their representatives should guide clinical trials, increase the availability of genetic testing, and generate natural history and qualitative studies detailing patients’ experience. Although each recommendation is impactful in itself, taken together they would jointly facilitate a shortened and ameliorated patient journey through more timely diagnosis and greater access to personalized medical care.

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescence-based sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction.