Search

Abstract Cellular stress and ageing involve an increase in crowding and aggregation of amylogenic proteins. We here investigate if crowding is the intrinsic cause of aggregation and utilise a previously established non-protein aggregation sensor, namely pseudoisocyanine chloride (PIC). PIC shows fibrillization in cells into a highly fluorescent J-aggregated state and is sensitive to crowding. Surprisingly, cell stress conditions stabilise the monomeric rather than the aggregated state of PIC both in the cytoplasm and in stress granules. Regarding the different physiochemical changes of the cytoplasm occurring upon cell stress, involving volume reduction, phase separation and solidification, the intrinsic crowding effect is not the key factor to drive associated self-assembly processes.

Pseudo isocyanine chloride (PIC) has been identified in a preceding work as a sensor suited to probe macromolecular crowding both in test tubes with solutions of synthetic crowding agents and in HeLa cells as a representative of living systems. The sensing is based on a delicate response of the self-assembly pattern of PIC towards a variation in macromolecular crowding. Based on a suitable selection of criteria established in the present study, four additional cyanine dyestuffs (TDBC, S071, S2275, and PCYN) were scrutinized for their ability to act as such a sensor, and the results were compared with the corresponding performance of PIC. UV-VIS and fluorescence spectroscopy were applied to investigate the photo-physical properties of the four candidates and, if possible, light scattering was used to characterize the self-assembly of the dyestuffs in solution. Finally, HeLa cells were exposed to solutions of the most promising candidates in order to analyze their ability to infiltrate the cells and to self-assemble therein. None of the dyestuff candidates turned out to be as similarly promising in probing crowding effects in cells as PIC turned out to be. S0271 and S2275 are at least stable enough and meet the photophysical requirements necessary to act as sensors responding to changes in macromolecular crowding.

Background: Whether patients with subclinical cardiomyopathy (CMP) are more prone to experience Takotsubo syndrome (TTS) than patients without CMP, is unknown. We present a patient with TTS as the initial manifestation of a hitherto unrecognized genetic CMP. Method: case report. Results: At age 55 after the unexpected death of her father, a now 61-year-old female had developed precordial pressure. Work-up revealed moderately reduced systolic function, dyskinesia of the interventricular septum, and indications for a TTS. Coronary angiography was normal but ventriculography showed TTS. Cardiac MRI confirmed reduced systolic function and TTS. TTS resolved without treatment and sequelae. At age 57 atrial fibrillation was recorded. After deterioration of systolic function at age 59 dilated CMP was diagnosed. Despite application of levosimendan, sacubitril, valsartan, and ivabradine, complete remission could not be achieved. Upon genetic work-up by means of a gene panel, the heterozygous mutation c.1489G > T (p. E497X) in exon 9 of the titin gene was detected and made responsible for the phenotype. Neurological work-up precluded involvement of the skeletal muscles. The further course was complicated by ventricular arrhythmias, requiring implantation of an implantable cardioverter defibrillator (ICD). Conclusions: previously subclinical CMP may initially manifest as TTS. Since patients with titin CMP are at risk of developing ventricular arrhythmias and thus to experience sudden cardiac death, appropriate anti-arrhythmic therapy needs to be established.

Abstract Protein aggregation is a hallmark of several severe neurodegenerative disorders such as Huntington's, Parkinson's, or Alzheimer's disease. Metal ions play a profound role in protein aggregation and altered metal‐ion homeostasis is associated with disease progression. Here we utilize μ‐X‐ray fluorescence imaging in combination with rapid freezing to resolve the elemental distribution of phosphorus, sulfur, potassium, and zinc in huntingtin exon‐1‐mYFP expressing HeLa cells. Using quantitative XRF analysis, we find a threefold increase in zinc and a 10‐fold enrichment of potassium that can be attributed to cellular stress response. While the averaged intracellular ion areal masses are significantly different in aggregate‐containing cells, a local intracellular analysis shows no different ion content at the location of intracellular inclusion bodies. The results are compared to corresponding experiments on HeLa cells forming pseudoisocyanine chloride aggregates. As those show similar results, changes in ion concentrations are not exclusively linked to huntingtin exon‐1 amyloid formation.