De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

The effects of fentanyl-like opioids and hydromorphone on human 5-HT3A receptors.

BACKGROUND: 5-HT(3) receptors are involved in various physiologic functions, including the modulation of emesis. 5-HT(3) antagonists are clinically widely used as potent antiemetics. Emesis is also a side effect of opioid analgesics. Intriguingly, the natural opioid morphine shows specific interactions with human 5-HT(3) receptors at clinically relevant concentrations. In the present study, we investigated whether this is a general effect of opioids, even when they are structurally diverse. Therefore, another morphine (phenanthrene-type) derivative, hydromorphone, and fentanyl including its (4-anilinopiperidine-type) derivatives were tested. METHODS: Whole-cell patches from human embryonic kidney-293 cells, stably transfected with the human 5-HT(3A) receptor cDNA, were used to determine the opioid effects on the 5-HT (3 microM)-induced currents using the patch clamp technique (voltage-clamp). RESULTS: None of the fentanyl derivatives affected currents through the 5-HT(3A) receptor (3 microM 5-HT) significantly in the clinically relevant nanomolar concentration range (IC(50) values >30 microM). In contrast, hydromorphone was considerably more potent (IC(50) = 5.3 microM), slowing the current activation- and desensitization-kinetics significantly (at 3 microM by a factor of 1.9 and 2.4, respectively), similar to morphine. At concentrations much higher than clinically relevant, but within the range predicted from Meyer-Overton correlations for nonspecific interactions, the fentanyl derivatives all showed at least a tendency to suppress current amplitudes, but they had diverse effects on the activation- and desensitization-kinetics of 5-HT(3A) receptors. CONCLUSIONS: Only morphine and hydromorphone, but not the fentanyl derivatives, reduced 5-HT-induced current amplitudes and slowed current kinetics near clinically relevant concentrations. The high potencies of morphine and hydromorphone, when compared to their lipophilicities, suggest a specific interaction with 5-HT(3A) receptors. In contrast, the effects of fentanyl-type opioids appear to be of unspecific nature. Because the rank order of opioid potencies for human 5-HT(3A) receptors is opposite of that for opioid receptors, the site involved is structurally different from opioid receptor binding sites. In agreement with recent data on different phenols, a phenolic OH-group (which morphine and hydromorphone possess) may contribute to specific interactions of morphine and hydromorphone with the 5-HT(3A) receptor. Future clinical studies could test whether corresponding differences in emetogenicity between different classes of opioids will be found.

Aging adult skull remains through radiological density estimates: A comparison of different computed tomography systems and the use of computer simulations to judge the accuracy of results.

INTRODUCTION: The objective of this article was to explore age-at-death estimates in forensic medicine, which were methodically based on age-dependent, radiologically defined bone-density (HC) decay and which were investigated with a standard clinical computed tomography (CT) system. Such density decay was formerly discovered with a high-resolution flat-panel CT in the skulls of adult females. The development of a standard CT methodology for age estimations--with thousands of installations--would have the advantage of being applicable everywhere, whereas only few flat-panel prototype CT systems are in use worldwide. METHODS: A Multi-Slice CT scanner (MSCT) was used to obtain 22,773 images from 173 European human skulls (89 male, 84 female), taken from a population of patients from the Department of Neuroradiology at the University Hospital Giessen and Marburg during 2010 and 2011. An automated image analysis was carried out to evaluate HC of all images. The age dependence of HC was studied by correlation analysis. The prediction accuracy of age-at-death estimates was calculated. Computer simulations were carried out to explore the influence of noise on the accuracy of age predictions. RESULTS: Human skull HC values strongly scatter as a function of age for both sexes. Adult male skull bone-density remains constant during lifetime. Adult female HC decays during lifetime, as indicated by a correlation coefficient (CC) of -0.53. Prediction errors for age-at-death estimates for both of the used scanners are in the range of ±18 years at a 75% confidence interval (CI). Computer simulations indicate that this is the best that can be expected for such noisy data. CONCLUSIONS: Our results indicate that HC-decay is indeed present in adult females and that it can be demonstrated both by standard and by high-resolution CT methods, applied to different subject groups of an identical population. The weak correlation between HC and age found by both CT methods only enables a method to estimate age-at-death with limited practical relevance since the errors of the estimates are large. Computer simulations clearly indicate that data with less noise and CCs in the order of -0.97 or less would be necessary to enable age-at-death estimates with an accuracy of ±5 years at a 75% CI.