Separate extraction of human eccrine sweat gland activity and peripheral hemodynamics from high- and low-quality thermal imaging data.

Sweat gland activity and peripheral hemodynamics, which characterize the function of sympathetic cholinergic nerve fibers and the manifestation mechanisms of vascular tone regulation, respectively, can be detected via dynamic thermography of the skin. Thus, they are useful parameters for diagnosing various forms of neuropathy and functional circulatory disorders. Both parameters affect the dynamics of the skin temperature; therefore, for an adequate description of thermographic data, it is necessary to build models that consider both these coexisting components simultaneously. PURPOSE: The objective of this study was to determine the spatiotemporal and statistical features of dynamic thermograms of skin areas with sweat glands and to develop methods for the extraction of temperature components mediated by sweat gland activity (Tsweat) separately from hemodynamics (Tblood) based on thermograms of high and low temperature resolutions. METHODS: To separate the Tsweat and Tblood components, simultaneous thermographic and photoplethysmographic (PPG) measurements were performed in the area of the fingers during a deep inspiratory gasp (DIG). PPG data, which were obtained solely by hemodynamics, were converted into a temperature signal (Tblood) using the spectral filtering approach. By calculating the difference between the skin (Tskin) and blood (Tblood) temperature components, the Tsweat component was determined, which characterizes sweat gland activity and the integrity of the cholinergic sympathetic nerve fibers that innervate them. The Tsweat component was compared with the active sweat pore count curve, which was determined by the adaptive detection of local temperature minima. Thermographic and PPG measurements were performed for 3 min on a group of 15 volunteers during the DIG test. The skin temperature was measured using a cooled thermal imaging camera in the spectral range of 8-9 µm with a temperature sensitivity of 0.02 °C. PPG measurements were performed using a reflectance sensor with a central wavelength of 800 nm. Wavelet analysis with the Morlet basis function was used to preliminarily determine the spectrum of spontaneous temperature oscillations in an area of the skin with and without sweat pores. Statistical parameters of the histogram, such as the standard deviation and the statistical pore activation index (SPAI) - which is proposed in this paper were used in the DIG test to detect sweat gland activity with low-temperature resolution thermograms. The temporal dynamics of the statistical parameters were compared with the dynamics of the sweat pore count. RESULTS: The Tsweat component was correlated with the sweat pore count on the thermogram with a coefficient of 0.75, confirming the dependence of this temperature component on the sweat gland activity and the need for considering this activity in the analysis of the spatiotemporal dynamics of the human skin temperature. The use of the proposed SPAI and standard deviation allows the detection of sweat gland activity even with thermograms of a low temperature resolution. The use of an integrated map of the sweat gland activity will help the specialist to assess the degree of integrity of the innervation of skin areas in a single image. The primary assessment of the spectrum of temperature oscillations at rest indicated that spontaneous sweat gland activity is accompanied by high-frequency oscillations in the skin temperature localized in the area of sweat pores, within the frequency range of 0.07-0.3 Hz. This suggests the possibility of spectral separation of the temperature component mediated by sweat gland activity from the hemodynamic component, which dominates in the region of <0.1 Hz. The proposed two-component approach for the analysis of skin temperature dynamics allows separate assessment of sympathetic innervation and rhythms of hemodynamic regulation using dynamic thermograms.

Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations.

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell­derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9­generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.

Real-time technique for conversion of skin temperature into skin blood flow: human skin as a low-pass filter for thermal waves.

Monitoring of skin blood flow oscillations related with mechanical activity of vessels is a very useful modality during diagnosis of peripheral hemodynamic disorders. In this study, we developed a new model and technique for real-time conversion of skin temperature into skin blood flow oscillations, and vice versa. The technique is based on the analogy between the thermal properties of the human skin and electrical properties of the special low-pass filter. Analytical and approximated impulse response functions for the low- and high-pass filters are presented. The general algorithm for the reversible conversion of temperature into blood flow is described. The proposed technique was verified using simulated or experimental data of cold stress, deep inspiratory gasp, and post-occlusive reactive hyperaemia tests. The implementation of the described technique will enable to turn a temperature sensor into a blood flow sensor.

Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes.

The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases.

A novel native derived coronary artery tissue-flap model.

Although tissue-engineering approaches have led to significant progress in the quest of finding a viable substitute for dysfunctional myocardium, the vascularization of such bioartificial constructs still remains a major challenge. Hence, there is a need for model systems that allow us to study and better understand cardiac and vascular biology to overcome current limitations. Therefore, in this study, in toto decellularized rat hearts with a patent vessel system were processed into standardized coronary artery tissue flaps adherent to the ascending aorta. Protein diffusivity analysis and blood perfusion of the coronary arteries showed proper sealing of the de-endothelialized vessels. Retrograde aortic perfusion allowed for selective seeding of the coronary artery system, while surface seeding of the tissue flaps allowed for additional controlled coculture with cardiac cells. The coronary artery tissue-flap model offers a patent and perfusable coronary vascular architecture with a preserved cardiac extracellular matrix, therefore mimicking nature's input to the highest possible degree. This offers the possibility to study re-endothelialization and endothelial function of different donor cell types and their interaction with cardiac cells in a standardized biologically derived cardiac in vitro model, while establishing a platform that could be used for in vitro drug testing and stem cell differentiation studies.