Targeting the High-Density Lipoprotein Proteome for the Treatment of Post-Acute Sequelae of SARS-CoV-2.

Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.

Genome-wide association analysis in dilated cardiomyopathy reveals two new players in systolic heart failure on chromosomes 3p25.1 and 22q11.23.

AIMS: Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure. METHODS AND RESULTS: We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 × 10-11 and 7.7 × 10-4 in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10-8 and 1.4 × 10-3 in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene. CONCLUSION: This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure.

Deficiency of Nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 reduces atherosclerosis.

Atherosclerosis is crucially fueled by inflammatory pathways including pattern recognition receptor (PRR)-related signaling of the innate immune system. Currently, the impact of the cytoplasmic PRRs nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2 is incompletely characterized. We, therefore, generated Nod1/Nod2 double knockout mice on a low-density lipoprotein receptor (Ldlr)-deficient background (= Ldlr-/-Nod1/2-/-) which were subsequently analyzed regarding experimental atherosclerosis, lipid metabolism, insulin resistance and gut microbiota composition. Compared to Ldlr-/- mice, Ldlr-/-Nod1/2-/- mice showed reduced plasma lipids and increased hepatic expression of the scavenger receptor LDL receptor-related protein 1 after feeding a high-fat diet for 12 weeks. Furthermore, intestinal cholesterol and its bacterial degradation product coprostanol were elevated in Ldlr-/-Nod1/2-/- mice, correlating with the increased abundance of Eubacterium coprostanoligenes as assessed by 3rd generation sequencing of the gut microbiota. Atherosclerotic plaques of Ldlr-/-Nod1/2-/- mice exhibited less lipid deposition and macrophage accumulation. Moreover, macrophages from Ldlr-/-Nod1/2-/- mice showed higher expression of the cholesterol efflux transporters Abca1 and Abcg1 and accordingly reduced foam cell formation. Deficiency of Nod1 and Nod2 led to reduced plaque lipid deposition and inflammatory cell infiltration in atherosclerotic plaques. This might be explained by diminished plasma lipid levels and foam cell formation due to altered expression of key regulators of the hepatic cholesterol pathway as well as differential intestinal cholesterol metabolism and microbiota composition.

Mitochondrial active and relaxed state respiration after heat shock mRNA response in the heart.

Induction of Heat Shock Proteins results in cytoprotection. Beneficial effect results from transcription and translational cellular components' involvement that defends metabolism and thus induce ischemic protection of the tissue. Mitochondrial respiration is also involved in stress- induced conditions. It is not a uniform process. Cytochrome c Oxidase (CytOx) representing complex IV of the Electron Transfer Chain (ETC) has a regulatory role for mitochondrial respiratory activity, which is tested in our study after hsp induction. Moreover, protein translation for mitochondrial components was probed by the detection of MT-CO1 for Subunit 1 of CytOx neosynthesis. Wistar rats were subjected to whole-body hyperthermia at 42.0-42.5 °C for 15 min followed by a normothermic recovery period. Heat shock response was monitored time dependent from LV biopsies of all control and heat treated animals with PCR-analysis for hsp 32, 60, 70.1, 70.2, 90 and MT-CO1 expression at 15, 30, 45, 60, 120 and 360 min recovery (n = 5 in each group), respectively. Enzymatic activity of CytOx were evaluated polarographically. High energy phosphates were detected by chromatographic analysis. The mRNA expression of MT-CO1 peaked at 60 min and was accompanied by hsp 32 (r = 0.457; p = 0.037) and hsp 70.2 (r = 0.615; p = 0.003) upregulation. With hsp induction, mitochondrial respiration was increased initially. Enzymatic activity reconciled from active into relaxed status wherein CytOx activity was completely inhibited by ATP. Myocardial ATP content increased from stress induced point i.e. < 1 µmol g-1 protein w/w to finally 1.5 ±â€¯0.53 µmol g-1 protein w/w at 120 min recovery interval. Hyperthermic, myocardial hsp- induction goes along with increased CytOx activity representing an increased "active" mitochondrial respiration. In parallel, de -novo holoenzyme assembly of CytOx begins as shown by MT-CO1 upregulation at 60 min recovery time crossing with a final return to the physiological "relaxed" state and ATP -inhibited respiration.

Influence of different aetiologies on clinical course and outcome in patients with dilated cardiomyopathy.

BACKGROUND: The clinical phenotype dilated cardiomyopathy is assumed to be the endstage of a multifactorial aetiopathogenetic pathophysiology which includes a not satisfactorily defined group of patients with inflammatory cardiomyopathy. METHODS: Within the German Competence Network Heart Failure patients with heart failure due to dilated cardiomyopathy of viral/inflammatory (DCMi/v) and nonviral/noninflammatory (DCM) aetiology were enrolled. After 1 year 237 patients (180 male/57 female) were re-examined including complete clinical work-up. The association of different clinical courses with the time from initial diagnosis of heart failure (newly: ≤ 1 year; late: > 1 year) was investigated. RESULTS: After 1-year-follow-up New York Heart Association (NYHA) class (by -0.48 in newly diagnosed DCM and -0.82 in newly diagnosed DCMi/v in addition to -0.24 in late diagnosed DCM and -0.17 in late diagnosed DCMi/v) as well as left ventricular ejection fraction (+14% in newly diagnosed DCM and DCMi/v and +6% in later diagnosed DCM and DCMi/v) were significantly improved in all patients. In patients with early diagnosed dilated cardiomyopathy a strong improvement of NYHA class could be demonstrated. CONCLUSIONS: This study demonstrates for the first time a significant interaction between duration of disease, NYHA class and left ventricular ejection fraction in patients with DCM. Our results clearly demonstrate that in patients with DCM an early diagnosis within 1 year after occurrence of clinical signs is associated with a strong improvement in the clinical course, whereas late diagnosis results in a loss of change in clinical course and outcome.

A genome-wide association study identifies 6p21 as novel risk locus for dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) is one of the leading causes for cardiac transplantations and accounts for up to one-third of all heart failure cases. Since extrinsic and monogenic causes explain only a fraction of all cases, common genetic variants are suspected to contribute to the pathogenesis of DCM, its age of onset, and clinical progression. By a large-scale case-control genome-wide association study we aimed here to identify novel genetic risk loci for DCM. METHODS AND RESULTS: Applying a three-staged study design, we analysed more than 4100 DCM cases and 7600 controls. We identified and successfully replicated multiple single nucleotide polymorphism on chromosome 6p21. In the combined analysis, the most significant association signal was obtained for rs9262636 (P = 4.90 × 10(-9)) located in HCG22, which could again be replicated in an independent cohort. Taking advantage of expression quantitative trait loci (eQTL) as molecular phenotypes, we identified rs9262636 as an eQTL for several closely located genes encoding class I and class II major histocompatibility complex heavy chain receptors. CONCLUSION: The present study reveals a novel genetic susceptibility locus that clearly underlines the role of genetically driven, inflammatory processes in the pathogenesis of idiopathic DCM.

Management of fulminant myocarditis: a diagnosis in search of its etiology but with therapeutic options.

Fulminant myocarditis is a clinical syndrome with signs of acute heart failure, cardiogenic shock, or life-threating rhythm disturbances in the context of suspected myocarditis. It is not an etiological diagnosis, but may have different underlying causes and pathogenetic processes - viral, bacterial, toxic, and autoreactive. Clinical management of the disease entity at the acute stage involves hemodynamic monitoring in an intensive care unit or similar setting. Rapid routine work-up is mandatory with serial EKGs, echocardiography, cardiac MRI, heart catheterization with endomyocardial biopsy for histology, immunohistology, and molecular analysis for the underlying infection and pathogenesis. Heart failure therapy is warranted in all cases according to current guidelines. For fulminant autoreactive myocarditis, immunosuppressive treatment is beneficial; for viral myocarditis, IVIg can resolve the inflammation, reduce the viral load, and even eradicate the microbial agent. ECMO, IABP, ventricular assist devices, LifeVest, or ICD implantation can bridge to recovery or to heart transplantation.

Influence of cyclical mechanical loading on osteogenic markers in an osteoblast-fibroblast co-culture in vitro: tendon-to-bone interface in anterior cruciate ligament reconstruction.

PURPOSE: We aimed to evaluate the influence of cyclical mechanical loading on osteoblasts and fibroblasts, and co-cultures of both in vitro, simulating the conditions of the tendon-to-bone interface in anterior cruciate ligament reconstruction. METHODS: Osteoblast-like cells (OBL) and tendon-derived rodent fibroblasts (TDF) were cultured alone or in co-culture to simulate the tendon-to-bone interface. Cyclical loading was applied for one hour twice a day for three days, with a frequency of 1 Hz and 3 % strain. Alkaline phosphatase (AP), osteocalcin (OC), collagen type 1 (COL1A1), and bone morphogenetic protein 2 (BMP-2) gene expression and protein deposition were detected by real-time polymerase chain reaction (qPCR) and immunocytochemical analysis. RESULTS: Mechanical loading significantly decreased AP, OC, and COL1A1 gene expression in both OBL and TDF, compared to non-loaded culture. However, mechanical load increased gene expression of the same marker genes including BMP-2 during co-culture. Immunocytochemistry demonstrated increased deposition of corresponding proteins in the same range, independent of culture conditions. Higher depositions of BMP-2 were shown under loading conditions for osteoblast and TDF monocultures. Prolongation of mechanical loading resulted in cell detachment and spheroid formation. CONCLUSION: Cyclical mechanical loading caused downregulation of genes involved in osteointegration and osteoinduction, such as OC, ALP, and COL1A1 in monocultures of osteoblasts and fibroblasts; co-cultures lacked this phenomenon. Immunocytochemistry and qPCR analysis showed slight upregulations of marker genes and corresponding proteins. This might be due to the potential stabilising effects of osteoblast-fibroblast cross talk in the co-culture environment, simulating fibrocartilage formation at the tendon-to-bone interface.

The ratio of cytochrome c oxidase subunit 4 isoform 4I1 and 4I2 mRNA is changed in permanent atrial fibrillation.

AIMS: The conditions of hypoxia are suggested to induce permanent atrial fibrillation (AF). The regulation of COX4I2 and COX4I1 depends on oxygen availability in tissues. A role of COX4I2 in the myocardium of AF patients is supposed for pathogenesis of AF and subsequent alterations in the electron transfer chain (ETC) under hypoxia. METHODS AND RESULTS: In vitro, influence of hypoxia on HeLa 53 cells was studied and elevated parts of COX 4I2 were confirmed. Myocardial biopsies were taken ex vivo from the patients' Right Atria with SR (n = 31) and AF (n = 11), respectively. RT- PCR for mRNA expresson, mitochondrial respiration by polarography and the protein content of cytochrome c oxidase (CytOx) subunit 4I1 and CytOx subunit 4I2 by ELISA were studied. Clinical data were correlated to the findings of gene expressions in parallel. Patients with permanent AF had a change in isoform 4I2/4I1 expression along with a decrease of isoform COX 4I1 expression. The 4I2/4I1 ratio of mRNA expression was increased from 0.630 to 1.058 in comparison. However, the protein content of CytOx subunit 4 was much lower in the AF group, whereas the respiration/units enzyme activity in both groups remained the same. CONCLUSIONS: This study describes a possible molecular correlate for the development of AF. Due to the known functional significance of COX 4I2, mitochondrial dysfunction can be assumed as a part of the pathogenesis of AF.

Trypomastigotes and amastigotes of Trypanosoma cruzi induce apoptosis and STAT3 activation in cardiomyocytes in vitro.

The haemoflagellate Trypanosoma cruzi is the causative agent of Chagas' disease that occurs in approximately 8 million people in Latin America. Patients infected with T. cruzi frequently suffer of cardiomegaly and may die of myocardial failure. Here we show that T. cruzi trypomastigotes (extracellular form) increased in vitro apoptosis of rat cardiomyocytes. Additionally, we demonstrated that amastigotes (intracellular form), for which a method for purification was established, were also able to induce cardiomyocyte apoptosis. Increase of apoptosis was associated with up-regulation of the apoptotic gene bax by trypomastigotes, while expression of the anti-apoptotic gene bcl-2 was down-regulated by amastigotes. The transcription factor STAT3 but not STAT1 was activated in cardiomyocytes by trypomastigotes. In addition, tlr7 gene expression was up-regulated in cardiomyocytes incubated with trypomastigotes, suggesting that this Toll-like receptor is involved in the intracellular recognition after host cell invasion by T. cruzi. Glycosylphosphatidylinositols purified from trypomastigotes did not induce cardiomyocyte apoptosis and STAT activation but down-regulated tlr7 gene expression. In conclusion, cardiomyopathy observed in Chagas' disease might be in part due to apoptosis of cardiomyocytes induced directly by the parasite.

Viral genomes in the pericardial fluid and in peri- and epicardial biopsies from a German cohort of patients with large to moderate pericardial effusions.

The aetiology of pericardial effusion has been generally assessed by clinical work-up only, which leaves a large cohort of patients with "idiopathic" effusions virtually undiagnosed. In accordance with the ESC guidelines, this contribution intends to change this attitude. After therapeutic or diagnostic pericardiocentesis of 259 patients with large to moderate pericardial effusions, pericardial fluid, epicardial and pericardial biopsies, and blood samples were analysed by PCR for cardiotropic microbial agents. Cytology, histology, immunohistology of tissue and fluids and laboratory tests were performed. Of the 259 patients, 35 % suffered from an autoreactive aetiology, 28 % suffered from a malignant and 14 % from an infectious cause. Investigating all samples by PCR, we identified viral genomes in 51 (19.7 %) patients, parvovirus B19 (B19 V) being identified in 25 and Epstein-Barr virus (EBV) in 19 cases. In patients with a sole infectious aetiology (n = 36), B19 V was detected in 21 and EBV in 10 cases. When differentiating with regard to the material investigated for the presence of cardiotropic viruses, parvovirus B19 was most often detected in the epicardium and EBV was most frequently detected in the pericardial fluid independent from the final diagnostic categorisation. Bacterial cultures including tests for tuberculosis were all negative. Molecular techniques improve sensitivity, specificity and diagnostic accuracy for the underlying aetiology in pericarditis patients with effusion. The identification of specific viral signatures will help to understand pathogenetic mechanisms in pericarditis and allow to tailor an adequate therapy beyond antiphlogistic treatment.

Comparative analysis of full-length 16s ribosomal RNA genome sequencing in human fecal samples using primer sets with different degrees of degeneracy.

Next-generation sequencing has revolutionized the field of microbiology research and greatly expanded our knowledge of complex bacterial communities. Nanopore sequencing provides distinct advantages, combining cost-effectiveness, ease of use, high throughput, and high taxonomic resolution through its ability to process long amplicons, such as the entire 16s rRNA genome. We examine the performance of the conventional 27F primer (27F-I) included in the 16S Barcoding Kit distributed by Oxford Nanopore Technologies (ONT) and that of a more degenerate 27F primer (27F-II) in the context of highly complex bacterial communities in 73 human fecal samples. The results show striking differences in both taxonomic diversity and relative abundance of a substantial number of taxa between the two primer sets. Primer 27F-I reveals a significantly lower biodiversity and, for example, at the taxonomic level of the phyla, a dominance of Firmicutes and Proteobacteria as determined by relative abundances, as well as an unusually high ratio of Firmicutes/Bacteriodetes when compared to the more degenerate primer set (27F-II). Considering the findings in the context of the gut microbiomes common in Western industrial societies, as reported in the American Gut Project, the more degenerate primer set (27F-II) reflects the composition and diversity of the fecal microbiome significantly better than the 27F-I primer. This study provides a fundamentally relevant comparative analysis of the in situ performance of two primer sets designed for sequencing of the entire 16s rRNA genome and suggests that the more degenerate primer set (27F-II) should be preferred for nanopore sequencing-based analyses of the human fecal microbiome.

Two glutamic acid residues in the DNA-binding domain are engaged in the release of STAT1 dimers from DNA.

BACKGROUND: In interferon-γ-stimulated cells, the dimeric transcription factor STAT1 (signal transducer and activator of transcription 1) recognizes semi-palindromic motifs in the promoter regions of cytokine-driven target genes termed GAS (gamma-activated sites). However, the molecular steps that facilitate GAS binding and the subsequent liberation of STAT1 homodimers from these promoter elements are not well understood. RESULTS: Using a mutational approach, we identified two critical glutamyl residues within the DNA-binding domain adjacent to the phosphodiester backbone of DNA which efficiently release phospho-STAT1 from DNA. The release of STAT1 dimers from DNA enhances transcriptional activity on both interferon-driven reporter and endogenous target genes. A substitution of either of the two glutamic acid residues broadens the repertoire of putative binding sites on DNA and enhances binding affinity to GAS sites. However, despite elevated levels of tyrosine phosphorylation and a prolonged nuclear accumulation period, the STAT1 DNA-binding mutants show a significantly reduced transcriptional activity upon stimulation of cells with interferon-γ. This reduced transcriptional response may be explained by the deposition of oligomerized STAT1 molecules outside GAS sites. CONCLUSIONS: Thus, two negatively charged amino acid residues in the DNA-binding domain are engaged in the liberation of STAT1 from DNA, resulting in a high dissociation rate from non-GAS sites as a key feature of STAT1 signal transduction, which positively regulates cytokine-dependent gene expression probably by preventing retention at transcriptionally inert sites.

A genome-wide association study identifies two loci associated with heart failure due to dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) is a major cause of heart failure with a high familial recurrence risk. So far, the genetics of DCM remains largely unresolved. We conducted the first genome-wide association study (GWAS) to identify loci contributing to sporadic DCM. METHODS AND RESULTS: One thousand one hundred and seventy-nine DCM patients and 1108 controls contributed to the discovery phase. Pools of DNA stratified on disease status, population, age, and gender were constituted and used for testing association of DCM with 517 382 single nucleotide polymorphisms (SNPs). Three DCM-associated SNPs were confirmed by individual genotyping (P < 5.0 10(-7)), and two of them, rs10927875 and rs2234962, were replicated in independent samples (1165 DCM patients and 1302 controls), with P-values of 0.002 and 0.009, respectively. rs10927875 maps to a region on chromosome 1p36.13 which encompasses several genes among which HSPB7 has been formerly suggested to be implicated in DCM. The second identified locus involves rs2234962, a non-synonymous SNP (c.T757C, p. C151R) located within the sequence of BAG3 on chromosome 10q26. To assess whether coding mutations of BAG3 might cause monogenic forms of the disease, we sequenced BAG3 exons in 168 independent index cases diagnosed with familial DCM and identified four truncating and two missense mutations. Each mutation was heterozygous, present in all genotyped relatives affected by the disease and absent in a control group of 347 healthy individuals, strongly suggesting that these mutations are causing the disease. CONCLUSION: This GWAS identified two loci involved in sporadic DCM, one of them probably implicates BAG3. Our results show that rare mutations in BAG3 contribute to monogenic forms of the disease, while common variant(s) in the same gene are implicated in sporadic DCM.

Cardiomyopathies: classification, diagnosis, and treatment.

This article comments on the recent classifications of cardiomyopathies by the American Heart Association and the European Society of Cardiology with respect to their clinical applicability. Taking them and the statement on the role of endomyocardial biopsies in different clinical scenarios together, the clinician is now able to identify genetic, autoimmune, and viral causative factors by using a thorough and logical approach to reach a diagnosis in patients with familial and nonfamilial forms of the underlying structural heart muscle diseases. In this overview, a special emphasis is also placed on the management of inflammatory and viral cardiomyopathies.

Fast and Easy Nanopore Sequencing Workflow for Rapid Genetic Testing of Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant lipid metabolism disorder characterized by severely elevated plasma low-density lipoprotein cholesterol levels. The disease is caused by mutations in 3 genes (LDLR, APOB and PCSK9) while over 90% of the mutations are located within the LDLR gene. Thus, genetic analysis of the LDLR gene is the first step in the genetic diagnosis of FH. However, conventional methods like Sanger and NextGen sequencing are still costly and time-consuming. In contrast, Oxford Nanopore technology sequencing is an emerging third-generation sequencing technology featured by easy operability, low cost, small size and the capability of parallel sample sequencing. Here, we present an easy Nanopore-sequencing-based workflow for the rapid genetic testing of FH taking only 3 days and costing less than $50 per sample without the requirement for deep bioinformatic knowledge. Using our workflow, we were able to identify the underlying pathogenic variants of 10 FH patients including one novel, not yet recorded pathogenic variants. Our workflow allows the rapid evaluation of the pathogenic variants by utilizing detailed variant information from Ensembl. Additionally, our workflow is not restricted to sequencing the LDLR gene alone but can be easily adapted to the other FH-causing genes and more importantly, to any desired gene contributing to any hereditary disease. Therefore, our workflow is an attractive opportunity for every diagnostic laboratory to offer fast and easy in-house genetic diagnostics.

Heat shock protein expression and change of cytochrome c oxidase activity: presence of two phylogenic old systems to protect tissues in ischemia and reperfusion.

Induction of heat shock proteins (hsp) has been shown to protect cells from ischemia by providing transient tolerance against myocardial injury and improving postischemic functional recovery. Attenuation of ATP depletion and earlier restoration of ATP content on reperfusion are thought to play a role in this scenario. Hsp induction is accompanied by altered enzyme activity of the respiratory chain, the major generator of ATP under physiological conditions. This report addresses the question whether processing and final assembly of the active holoenzyme cytochrome c oxidase (CcO, complex IV), member of the respiratory chain, is compromised under hypoxic conditions unless protected by stress proteins. Special focus is laid on function of the enzyme's subunits and importance of cellular energy availability and maintenance.

Genotype-specific effects on left ventricular function in parvovirus B19-positive patients with dilated cardiomyopathy.

Genotype-specific effects of parvovirus B19 (B19V) infections on left ventricular function in patients with dilated cardiomyopathy (DCM) have not been investigated so far. In this prospective clinical study, the prevalences of B19V genotypes in endomyocardial biopsies from patients presenting with inflammatory heart disease and DCM were determined. A total of 139 consecutive patients were included in the study; among them 53 patients were diagnosed as DCM. Among the total study cohort, B19V DNA was detected in 65 study participants (46.8%). Genotyping of the B19V genomes in the total cohort identified genotype 1 in 38 samples (27.3%), genotype 2 in 25 samples (18.0%), and genotype 3 in only two patients (1.4%). During an average follow-up period of 8 months left ventricular ejection fraction (LVEF) improved significantly both in B19V-positive (7.1 ± 13.8%, n = 17, P = 0.038) as well as B19V-negative patients with DCM (9.5 ± 13.9%, n = 20, P = 0.017). However, mean LVEF improved only in patients with genotype 1 (11.0 ± 14.4%, n = 7), whereas it even decreased in patients with genotype 2 (-6.2 ± 6.3%, n = 5, P = 0.033). These data from a small sample of patients diagnosed as DCM suggested that myocardial function during short-time follow-up differed between genetic variants of B19V. Patients with genotype 1 were on average younger than genotype 2 and appeared to be more prone to a beneficial course of left ventricular function than patients with genotype 2. Future studies with larger sample sizes and longer follow-up periods will be required to confirm this observation.

Mutations in the ANKRD1 gene encoding CARP are responsible for human dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) is familial in approximately 30% of cases, and mutations have been identified in several genes. However, in a majority of familial cases, the responsible genes are still to be discovered. The ANKRD1 gene is over-expressed in heart failure in human and animal models. The encoded protein CARP interacts with partners such as myopalladin or titin, previously shown to be involved in DCM. We hypothesized that mutations in ANKRD1 could be responsible for DCM. METHODS AND RESULTS: We sequenced the coding region of ANKRD1 from 231 independent DCM cases. We identified five missense mutations (three sporadic and two familial) absent from 400 controls and affecting highly conserved residues. Expression of the mutant CARP proteins after transfection in rat neonate cardiomyocytes indicated that most of them led to both significantly less repressor activity measured in a reporter gene assay and greater phenylephrin-induced hypertrophy, suggesting altered function of CARP mutant proteins. CONCLUSION: On the basis of genetic and functional analysis of CARP mutations, we have identified ANKRD1 as a new gene associated with DCM, accounting for approximately 2% of cases.