Monitoring mitochondrial translation in living cells

Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we devise an imaging approach to analyze mitochondrial translation within the context of single cells, by following the incorporation of clickable non-canonical amino acids. We apply this method to multiple cell types, including specialized cells such as cardiomyocytes and neurons, and monitor with spatial resolution mitochondrial translation in axons and dendrites. We also show that translation imaging allows to monitor mitochondrial protein expression in patient fibroblasts. Approaching mitochondrial translation with click chemistry opens new avenues to understand how mitochondrial biogenesis is integrated into the cellular context and can be used to assess mitochondrial gene expression in mitochondrial diseases

Proteomic mapping of atrial and ventricular heart tissue in patients with aortic valve stenosis

Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues. To overcome these issues this study used high resolution mass spectrometry in combination with a stem cell- derived cardiac specific protein quantification-standard to profile the proteomes of 17 atrial and 29 left ventricular myocardial FFPE human myocardial tissue samples from AVS-patients. In our proteomic analysis we quantified a median of 1980 (range 1495–2281) proteins in every single sample and identified significant upregulation of 239 proteins in atrial and 54 proteins in ventricular myocardium. We compared the proteins with published data. Well studied proteins reflect disease-related changes in AVS, such as cardiac hypertrophy, development of fibrosis, impairment of mitochondria and downregulated blood supply. In summary, we provide both a workflow for quantitative proteomics of human FFPE heart tissue and a comprehensive proteomic resource for AVS induced changes in the human myocardium

CRISPLD1: a novel conserved target in the transition to human heart failure

Heart failure is a major health problem worldwide with a significant morbidity and mortality rate. Although studied extensively in animal models, data from patients at the compensated disease stage are lacking. We sampled myocardium biopsies from aortic stenosis patients with compensated hypertrophy and moderate heart failure and used transcriptomics to study the transition to failure. Sequencing and comparative analysis of analogous samples of mice with transverse aortic constriction identified 25 candidate genes with similar regulation in response to pressure overload, reflecting highly conserved molecular processes. The gene cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is upregulated in the transition to failure in human and mouse and its function is unknown. Homology to ion channel regulatory toxins suggests a role in Ca(2+) cycling. CRISPR/Cas9-mediated loss-of-function leads to dysregulated Ca(2+) handling in human-induced pluripotent stem cell-derived cardiomyocytes. The downregulation of prohypertrophic, proapoptotic and Ca(2+)-signaling pathways upon CRISPLD1-KO and its upregulation in the transition to failure implicates a contribution to adverse remodeling. These findings provide new pathophysiological data on Ca(2+) regulation in the transition to failure and novel candidate genes with promising potential for therapeutic interventions

Combination of linear classifiers using score function -- analysis of possible combination strategies

In this work, we addressed the issue of combining linear classifiers using their score functions. The value of the scoring function depends on the distance from the decision boundary. Two score functions have been tested and four different combination strategies were investigated. During the experimental study, the proposed approach was applied to the heterogeneous ensemble and it was compared to two reference methods -- majority voting and model averaging respectively. The comparison was made in terms of seven different quality criteria. The result shows that combination strategies based on simple average, and trimmed average are the best combination strategies of the geometrical combination

Studying Brugada syndrome with an SCN1B variants in human-induced pluripotent stem cell-derived cardiomyocytes

BACKGROUND: Among rare channelopathies BrS patients are at high risk of sudden cardiac death (SCD). SCN5A mutations are found in a quarter of patients. Other rare gene mutations including SCN1B have been implicated to BrS. Studying the human cellular phenotype of BrS associated with rare gene mutation remains lacking. OBJECTIVES: We sought to study the cellular phenotype of BrS with the SCN1B gene variants using human-induced pluripotent stem cell (hiPSCs)–derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: A BrS patient suffering from recurrent syncope harboring a two variants (c.629T > C and c.637C > A) in SCN1B, which encodes the function-modifying sodium channel beta1 subunit, and three independent healthy subjects were recruited and their skin biopsies were used to generate hiPSCs, which were differentiated into cardiomyocytes (hiPSC-CMs) for studying the cellular electrophysiology. A significantly reduced peak and late sodium channel current (I(Na)) and a shift of activation curve to more positive potential as well as a shift of inactivation curve to more negative potential were detected in hiPSC-CMs of the BrS patient, indicating that the SCN1B variants impact the function of sodium channels in cardiomyocytes. The reduced I(Na) led to a reduction of amplitude (APA) and upstroke velocity (V(max)) of action potentials. Ajmaline, a sodium channel blocker, showed a stronger effect on APA and Vmax in BrS cells as compared to cells from healthy donors. Furthermore, carbachol was able to increase arrhythmia events and the beating frequency in BrS. CONCLUSION: Our hiPSC-CMs from a BrS-patient with two variants in SCN1B recapitulated some key phenotypic features of BrS and can provide a platform for studies on BrS with SCN1B variants

Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7-related osteopetrosis

Human induced pluripotent stem cells (hiPSCs) hold great potential for modelling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to nine weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behaviour of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudo-resorption. We used hiPSCs from an ARO patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl(-)/H(+) -exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, indicating a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO-hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases

A preclinical study on brugada syndrome with a CACNB2 variant using human cardiomyocytes from induced pluripotent stem cells

AIMS: Some gene variants in the sodium channels, as well as calcium channels, have been associated with Brugada syndrome (BrS). However, the investigation of the human cellular phenotype and the use of drugs for BrS in presence of variant in the calcium channel subunit is still lacking. OBJECTIVES: The objective of this study was to establish a cellular model of BrS in the presence of a CACNB2 variant of uncertain significance (c.425C > T/p.S142F) using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and test drug effects using this model. METHODS AND RESULTS: This study recruited cells from a patient with Brugada syndrome (BrS) and recurrent ventricular fibrillation carrying a missense variant in CACNB2 as well as from three healthy independent persons. These cells (hiPSC-CMs) generated from skin biopsies of healthy persons and the BrS patient (BrS-hiPSC-CMs) as well as CRISPR/Cas9 corrected cells (isogenic control, site-variant corrected) were used for this study. The hiPSC-CMs from the BrS patient showed a significantly reduced L-type calcium channel current (I(Ca-L)) compared with the healthy control hiPSC-CMs. The inactivation curve was shifted to a more positive potential and the recovery from inactivation was accelerated. The protein expression of CACNB2 of the hiPSC-CMs from the BrS-patient was significantly decreased compared with healthy hiPSC-CMs. Moreover, the correction of the CACNB2 site-variant rescued the changes seen in the hiPSC-CMs of the BrS patient to the normal state. These data indicate that the CACNB2 gene variant led to loss-of-function of L-type calcium channels in hiPSC-CMs from the BrS patient. Strikingly, arrhythmia events were more frequently detected in BrS-hiPSC-CMs. Bisoprolol (beta-blockers) at low concentration and quinidine decreased arrhythmic events. CONCLUSIONS: The CACNB2 variant (c.425C > T/p.S142F) causes a loss-of-function of L-type calcium channels and is pathogenic for this type of BrS. Bisoprolol and quinidine may be effective for treating BrS with this variant

Plasma levels of matrix metalloproteinase-2, -3, -10, and tissue inhibitor of metalloproteinase-1 are associated with vascular complications in patients with type 1 diabetes: The EURODIAB Prospective Complications Study

Impaired regulation of extracellular matrix remodeling by matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP) may contribute to vascular complications in patients with type 1 diabetes. We investigated associations between plasma MMP-1, -2, -3, -9, -10 and TIMP-1, and cardiovascular disease (CVD) or microvascular complications in type 1 diabetic patients. We also evaluated to which extent these associations could be explained by low-grade inflammation (LGI) or endothelial dysfunction (ED). Methods: 493 type 1 diabetes patients (39.5 ± 9.9 years old, 51% men) from the EURODIAB Prospective Complications Study were included. Linear regression analysis was applied to investigate differences in plasma levels of MMP-1, -2, -3, -9, -10, and TIMP-1 between patients with and without CVD, albuminuria or retinopathy. All analyses were adjusted for age, sex, duration of diabetes, Hba1c and additionally for other cardiovascular risk factors including LGI and ED. Results: Patients with CVD (n = 118) showed significantly higher levels of TIMP-1 [β = 0.32 SD (95%CI: 0.12; 0.52)], but not of MMPs, than patients without CVD (n = 375). Higher plasma levels of MMP-2, MMP-3, MMP-10 and TIMP-1 were associated with higher levels of albuminuria (p-trends were 0.028, 0.004, 0.005 and 0.001, respectively). Severity of retinopathy was significantly associated with higher levels of MMP-2 (p-trend = 0.017). These associations remained significant after further adjustment for markers of LGI and ED. Conclusions: These data support the hypothesis that impaired regulation of matrix remodeling by actions of MMP-2, -3 and-10 and TIMP-1 contributes to the pathogenesis of vascular complications in type 1 diabetes

Relationship Between Risk Factors and Mortality in Type 1 Diabetic Patients in Europe: The EURODIAB Prospective Complications Study (PCS)

OBJECTIVE—The purpose of this study was to examine risk factors for mortality in patients with type 1 diabetes