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Abstract Single cell RNA sequencing has provided unprecedented insights into the molecular cues and cellular heterogeneity underlying human disease. However, the high costs and complexity of single cell methods remain a major obstacle for generating large-scale human cohorts. Here, we compare current state-of-the-art single cell multiplexing technologies, and provide a widely applicable demultiplexing method, SoupLadle, that enables simple, yet robust high-throughput multiplexing leveraging genetic variability of patients.

Abstract The cardiac muscle consists of individual cardiomyocytes that are mechanically linked by desmosomes. Desmosomal adhesion is mediated by densely packed and organized cadherins which, in presence of Ca2+, stretch out their extracellular domains (EC) and dimerize with opposing binding partners by exchanging an N-terminal tryptophan. The strand-swap binding motif of cardiac cadherins like desmocollin 2 (Dsc2) (and desmoglein2 alike) is highly specific but of low affinity with average bond lifetimes in the range of approximately 0.3 s. Notably, despite this comparatively weak interaction, desmosomes mediate a stable, tensile-resistant bond. In addition, force mediated dissociation of strand-swap dimers exhibit a reduced bond lifetime as external forces increase (slip bond). Using atomic force microscopy based single molecule force spectroscopy (AFM-SMFS), we demonstrate that Dsc2 has two further binding modes that, in addition to strand-swap dimers, most likely play a significant role in the integrity of the cardiac muscle. At short interaction times, the Dsc2 monomers associate only loosely, as can be seen from short-lived force-independent bonds. These ideal bonds are a precursor state and probably stabilize the formation of the self-inhibiting strand-swap dimer. The addition of tryptophan in the measurement buffer acts as a competitive inhibitor, preventing the N-terminal strand exchange. Here, Dsc2 dimerizes as X-dimer which clearly shows a tri-phasic slip-catch-slip type of dissociation. Within the force-mediated transition (catch) regime, Dsc2 dimers switch between a rather brittle low force and a strengthened high force adhesion state. As a result, we can assume that desmosomal adhesion is mediated not only by strand-swap dimers (slip) but also by their precursor states (ideal bond) and force-activated X-dimers (catch bond).

TMEM43 (LUMA) is a ubiquitously expressed protein with unknown function. The protein is phylogenetically highly conserved and also found in Drosophila melanogaster (Klinke et al., 2022). TMEM43-p.S358L is a rare, fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). To understand the function of the ARVC5-associated mutation it is first important to understand the function of the TMEM43 protein. Therefore, a TMEM43 knockout induced pluripotent stem cell (iPSC) line was generated using the CRISPR/Cas9 genome editing system.The resulting cell line had a deficiency of TMEM43 and showed normal morphology and a stable karyotype. The colonies were positive for pluripotency markers and could be differentiated into the three germ layers.

Muscle contraction depends on strictly controlled calcium transients within myocytes. Here, the authors show that the endopeptidase Neprilysin 4 represents an essential regulator of these transients and, consequently, of proper heart function.

The aim of the study was to assess the occurrence of classic risk factors in the study group of patients with heart failure and to link them with the transcriptional activity of the examined genes: metalloproteinase 9 (MMP-9) and the tissue inhibitor of metalloproteinases 1 (TIMP-1). A total of 150 (100%) patients qualified for the study, including 80 (53.33%) patients with heart failure in the course of coronary artery disease, 40 (26.67%) with coronary artery disease without heart failure, and 30 (20.00%) in whom the presence of atherosclerotic changes in the coronary arteries was excluded. The material for molecular tests was peripheral blood collected from patients within the first 24 h of hospitalisation. A quantitative analysis of transcriptional activity was performed using the RT-qPCR technique. The most common classic risk factors among the patients in the study group were arterial hypertension (117; 78.00%) and overweight/obesity (102; 68%). In the group of patients with coronary artery disease and heart failure burdened with overweight/obesity, a significantly higher transcriptional activity of the metalloproteinase 9 (MMP-9) gene was found in comparison to patients who were not burdened with this risk factor. The analysis also showed the statistically significant higher transcriptional activity of the metalloproteinase 9 (MMP-9) gene in a group of patients with coronary artery disease and heart failure who smoked. The examined patients with heart failure due to myocardial ischemia were burdened with numerous cardiovascular risk factors, the most common of which were arterial hypertension, obesity/overweight, and hypercholesterolemia. A significant increase in the transcriptional activity of the metalloproteinase 9 (MMP-9) gene in the presence of risk factors (male sex, overweight/obesity, smoking) indicates another pathomechanism of their action and participation in the development and progression of heart failure during myocardial ischemia. There is a need for systematic information and educational activities promoting a healthy lifestyle with the elimination of modifiable risk factors for cardiovascular diseases.

BackgroundArrhythmogenic cardiomyopathy can be caused by genetic variants in desmosomal cadherins. Since cardiac desmosomal cadherins are crucial for cell-cell-adhesion, their correct localization at the plasma membrane is essential.MethodsNine desmocollin-2 variants at five positions from various public genetic databases (p.D30N, p.V52A/I, p.G77V/D/S, p.V79G, p.I96V/T) and three additional conserved positions (p.C32, p.C57, p.F71) within the prodomain were investigated in vitro using confocal microscopy. Model variants (p.C32A/S, p.V52G/L, p.C57A/S, p.F71Y/A/S, p.V79A/I/L, p.I96l/A) were generated to investigate the impact of specific amino acids.ResultsWe revealed that all analyzed positions in the prodomain are critical for the intracellular transport. However, the variants p.D30N, p.V52A/I and p.I96V listed in genetic databases do not disturb the intracellular transport revealing that the loss of these canonical sequences may be compensated.ConclusionAs disease-related homozygous truncating desmocollin-2 variants lacking the transmembrane domain are not localized at the plasma membrane, we predict that some of the investigated prodomain variants may be relevant in the context of arrhythmogenic cardiomyopathy due to disturbed intracellular transport.

An abnormal cardiac action potential underlies different types of cardiac arrhythmias. Here the authors show that microRNA-365 regulates the cardiac action potential by modulating key cardiac repolarizing channels.

Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-β1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.

In heart failure, increased CaMKII activity is decisively involved in arrhythmia formation. Here, the authors introduce the neuronal sodium channel NaV1.8 as a CaMKII downstream target as its specific knock-out reduces arrhythmias and improves survival in a CaMKII-overexpressing mouse model.

Summary: Recombinant SaCas9 is useful for a broad range of applications in the context of genome editing, especially when the specific protospacer adjacent motifs of other Cas9 derivatives are missing. Here, we describe a detailed protocol for the expression and purification of recombinant SaCas9. We detail the main steps for immobilized metal affinity chromatography and size exclusion chromatography. In addition, we present an assay for activity determination of SaCas9. Active SaCas9 can be purified in a week by using this protocol.

Myofibrillar myopathies (MFM) are a group of chronic muscle diseases pathophysiologically characterized by accumulation of protein aggregates and structural failure of muscle fibers. A subtype of MFM is caused by heterozygous mutations in the filamin C (FLNC) gene, exhibiting progressive muscle weakness, muscle structural alterations and intracellular protein accumulations. Here, we characterize in depth the pathogenicity of two novel truncating FLNc variants (p.Q1662X and p.Y2704X) and assess their distinct effect on FLNc stability and distribution as well as their impact on protein quality system (PQS) pathways. Both variants cause a slowly progressive myopathy with disease onset in adulthood, chronic myopathic alterations in muscle biopsy including the presence of intracellular protein aggregates. Our analyses revealed that p.Q1662X results in FLNc haploinsufficiency and p.Y2704X in a dominant-negative FLNc accumulation. Moreover, both protein-truncating variants cause different PQS alterations: p.Q1662X leads to an increase in expression of several genes involved in the ubiquitin-proteasome system (UPS) and the chaperone-assisted selective autophagy (CASA) system, whereas p.Y2704X results in increased abundance of proteins involved in UPS activation and autophagic buildup. We conclude that truncating FLNC variants might have different pathogenetic consequences and impair PQS function by diverse mechanisms and to varying extents. Further studies on a larger number of patients are necessary to confirm our observations.

Desmocollin‐2 (DSC2) is a desmosomal protein of the cadherin family. Desmosomes are multiprotein complexes, which are involved in cell adhesion of cardiomyocytes and of keratinocytes. The molecular structure of the complete extracellular domain (ECD) of DSC2 was recently described, revealing three disulfide bridges, four N‐glycosylation sites, and four O‐mannosylation sites. However, the functional relevance of these post‐translational modifications for the protein trafficking of DSC2 to the plasma membrane is still unknown. Here, we generated a set of DSC2 mutants, in which we systematically exchanged all N‐glycosylation sites, O‐mannosylation sites, and disulfide bridges within the ECD and investigated the resulting subcellular localization by confocal laser scanning microscopy. Of note, all single and double N‐glycosylation‐ deficient mutants were efficiently incorporated into the plasma membrane, indicating that the absence of these glycosylation sites has a minor effect on the protein trafficking of DSC2. However, the exchange of multiple N‐glycosylation sites resulted in intracellular accumulation. Colocalization analysis using cell compartment trackers revealed that N‐glycosylation‐ deficient DSC2 mutants were retained within the Golgi apparatus. In contrast, elimination of the four O‐mannosylation sites or the disulfide bridges in the ECD has no obvious effect on the intracellular protein processing of DSC2. These experiments underscore the importance of N‐glycosylation at multiple sites of DSC2 for efficient intracellular transport to the plasma membrane.

Desmin is the major intermediate filament protein of all three muscle cell types, and connects different cell organelles and multi-protein complexes such as the cardiac desmosomes. Several pathogenic mutations in the DES gene cause different skeletal and cardiac myopathies. However, the significance of the majority of DES missense variants is currently unknown, since functional data are lacking. To determine whether desmin missense mutations within the highly conserved 1A coil domain cause a filament assembly defect, we generated a set of variants with unknown significance and systematically analyzed the filament assembly using confocal microscopy in transfected SW-13, H9c2 cells and cardiomyocytes derived from induced pluripotent stem cells. We found that mutations in the N-terminal part of the 1A coil domain affect filament assembly, leading to cytoplasmic desmin aggregation. In contrast, mutant desmin in the C-terminal part of the 1A coil domain forms filamentous structures comparable to wild-type desmin. Our findings suggest that the N-terminal part of the 1A coil domain is a hot spot for pathogenic desmin mutations, which affect desmin filament assembly. This study may have relevance for the genetic counselling of patients carrying variants in the 1A coil domain of the DES gene.

Patients with refractory heart failure due to chronic progressive cardiac myopathy (CM) may require mechanical circulatory support as a bridge to transplantation. A few patients can be weaned from support devices if recovery can be achieved. The identification of these patients is of great importance as recovery may be missed if the heart is unloaded by the ventricular assist device (VAD). Testing the load-bearing capacity of the supported left ventricle (LV) by temporarily and gradually reducing mechanical support during cardiac exercise can help identify responders and potentially aid the recovery process. An exercise training protocol was used in 3 patients (8 months, 18 months and 8 years old) with histological CM findings and myocarditis. They were monitored regularly using clinical information and functional imaging with VAD support. Echocardiographic examination included both conventional real-time 3D echocardiography (RT3DE) and speckle tracking (ST). A daily temporary reduction in pump rate (phase A) was followed by a permanent reduction in rate (phase B). Finally, pump stops of up to 30 min were performed once a week (phase C). The final decision on explantation was based on at least three pump stops. Two patients were weaned and successfully removed from the VAD. One of them was diagnosed with acute viral myocarditis. The other had chronic myocarditis with dilated myopathy and mild interstitial fibrosis. The noninvasive assessment of cardiac output and strain under different loading conditions during VAD therapy is feasible and helps identify candidates for weaning despite severe histological findings. The presented protocol, which incorporates new echocardiographic techniques for determining volume and deformation, can be of great help in positively guiding the process of individual recovery, which may be essential for selecting and increasing the number of patients to be weaned from VAD.

Myocarditis is an inflammatory disease of the heart. Pediatric myocarditis with the dilated cardiomyopathy (DCM) phenotype may be caused by likely pathogenic or pathogenic genetic variants [(L)P] in cardiomyopathy (CMP) genes. Systematic analysis of immune disorder gene defects has not been performed so far. We analyzed 12 patients with biopsy-proven myocarditis and the DCM phenotype together with their parents using whole-exome sequencing (WES). The WES data were filtered for rare pathogenic variants in CMP (n = 89) and immune disorder genes (n = 631). Twelve children with a median age of 2.9 (1.0–6.8) years had a mean left ventricular ejection fraction of 28% (22–32%) and myocarditis was confirmed by endomyocardial biopsy. Patients with primary immunodeficiency were excluded from the study. Four patients underwent implantation of a ventricular assist device and subsequent heart transplantation. Genetic analysis of the 12 families revealed an (L)P variant in the CMP gene in 8/12 index patients explaining DCM. Screening of recessive immune disorder genes identified a heterozygous (L)P variant in 3/12 index patients. This study supports the genetic impact of CMP genes for pediatric myocarditis with the DCM phenotype. Piloting the idea that additional immune-related genetic defects promote myocarditis suggests that the presence of heterozygous variants in these genes needs further investigation. Altered cilium function might play an additional role in inducing inflammation in the context of CMP.

This case report describes the contributions of multimodality imaging, cardiac biopsy, and genetic sequencing to the diagnosis and management of heart transplant rejection in a 23-year old patient with dilated cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC-5) is a dominantly inherited cardiomyopathy caused by the mutation TMEM43-p.S358L. An induced pluripotent stem cell (iPSC) line (HDZi001-A) from an adult male mutation carrier was generated, using the CytoTune Sendai Kit. The resulting iPSCs carried the mutation TMEM43-p.S358L, had a normal morphology, a stable karyotype and were positive for the expression of pluripotency markers. This iPSC line can be differentiated into the three germ layers and might be a useful model for the characterization of ARVC-5 associated pathomechanism.

Myocardial tissue undergoes steady functional decline when cultured in vitro. Here, the authors report a protocol for culture of human cardiac slices that allows maintenance of contractility for up to four months, and show that the model is suitable for evaluation of drug safety, as exemplified for drugs interfering with cardiomyocyte repolarization.

Currently, little is known about the genetic background of restrictive cardiomyopathy (RCM). Herein, we screened an index patient with RCM in combination with atrial fibrillation using a next generation sequencing (NGS) approach and identified the heterozygous mutation DES-c.735G>C. As DES-c.735G>C affects the last base pair of exon-3, it is unknown whether putative missense or splice site mutations are caused. Therefore, we applied nanopore amplicon sequencing revealing the expression of a transcript without exon-3 in the explanted myocardial tissue of the index patient. Western blot analysis verified this finding at the protein level. In addition, we performed cell culture experiments revealing an abnormal cytoplasmic aggregation of the truncated desmin form (p.D214-E245del) but not of the missense variant (p.E245D). In conclusion, we show that DES-c.735G>C causes a splicing defect leading to exon-3 skipping of the DES gene. DES-c.735G>C can be classified as a pathogenic mutation associated with RCM and atrial fibrillation. In the future, this finding might have relevance for the genetic understanding of similar cases.

The normally positive cardiac force-frequency relationship (FFR) becomes flat or negative in chronic heart failure (HF). Here we explored if remodeling of the cardiomyocyte transverse tubular system (t-system) is associated with alterations in FFR and contractile kinetics in failing human myocardium. Left-ventricular myocardial slices from 13 failing human hearts were mounted into a biomimetic culture setup. Maximum twitch force (F), 90% contraction duration (CD90), time to peak force (TTP) and time to relaxation (TTR) were determined at 37°C and 0.2–2 Hz pacing frequency. F1Hz/F0.5Hz and F2Hz/F0.5Hz served as measures of FFR, intracellular cardiomyocyte t-tubule distance (ΔTT) as measure of t-system remodeling. Protein levels of SERCA2, NCX1, and PLB were quantified by immunoblotting. F1Hz/F0.5Hz (R2 = 0.82) and F2Hz/F0.5Hz (R2 = 0.5) correlated negatively with ΔTT, i.e., samples with severe t-system loss exhibited a negative FFR and reduced myocardial wall tension at high pacing rates. PLB levels also predicted F1Hz/F0.5Hz, but to a lesser degree (R2 = 0.49), whereas NCX1 was not correlated (R2 = 0.02). CD90 correlated positively with ΔTT (R2 = 0.39) and negatively with SERCA2/PLB (R2 = 0.42), indicating that both the t-system and SERCA activity are important for contraction kinetics. Surprisingly, ΔTT was not associated with TTP (R2 = 0) but rather with TTR (R2 = 0.5). This became even more pronounced when interaction with NCX1 expression was added to the model (R2 = 0.79), suggesting that t-system loss impairs myocardial relaxation especially when NCX1 expression is low. The degree of t-system remodeling predicts FFR inversion and contraction slowing in failing human myocardium. Moreover, together with NCX, the t-system may be important for myocardial relaxation.

Abstract Background Hypertrophic cardiomyopathy (HCM) is a genetic cardiomyopathy with a prevalence of about 1:200. It is characterized by left ventricular hypertrophy, diastolic dysfunction and interstitial fibrosis; HCM might lead to sudden cardiac death (SCD) especially in the young. Due to low autopsy frequencies of sudden unexplained deaths (SUD) the true prevalence of SCD and especially of HCM among SUD remains unclear. Even in cases of proven SCD genetic testing is not a routine procedure precluding appropriate risk stratification and counseling of relatives. Methods Here we report a case of SCD in a 19‐year‐old investigated by combined forensic and molecular autopsy. Results During autopsy of the index‐patient HCM was detected. As no other possible cause of death could be uncovered by forensic autopsy the event was classified as SCD. Molecular autopsy identified two (probably) pathogenic genetic variants in FHL1 and MYBPC3. The MYBPC3 variant had an incomplete penetrance. The FHL1 variant was a de novo mutation. We detected reduced FHL1 mRNA levels and no FHL1 protein in muscle samples suggesting nonsense‐mediated mRNA decay and/or degradation of the truncated protein in the SCD victim revealing a plausible disease mechanism. Conclusion The identification of the genetic cause of the SCD contributed to the rational counseling of the relatives and risk assessment within the family. Furthermore our study revealed evidences for the pathomechanism of FHL1 mutations.

Chronic heart failure (HF) is a clinical syndrome characterized by functional impairments of the myocardium. Metabolic and clinical changes develop with disease progression. In an advanced state, left ventricular assist devices (LVADs) are implanted for mechanical unloading. Our study aimed to assess the effects of LVAD implantation on the metabolic phenotypes and their potential to reverse the latter in patients with advanced HF. Plasma metabolites were analyzed by LC–MS/MS in 20 patients with ischemic cardiomyopathy (ICM), 20 patients with dilative cardiomyopathy (DCM), and 20 healthy controls. Samples were collected in HF patients before, 30 days after, and >100 days after LVAD implantation. Out of 188 measured metabolites, 63 were altered in HF. Only three metabolites returned to pre-LVAD concentrations 100 days after LVAD implantation. Pre-LVAD differences between DCM and ICM were mainly observed for amino acids and biogenic amines. This study shows a reversal of metabolite abnormalities in HF as a result of LVAD implantation. The etiology of the underlying disease plays an essential role in defining which specific metabolic parameter is altered in HF and reversed by LVAD implantation. Our findings provide a detailed insight into the disease pattern of ICM and DCM and the potential for reversibility of metabolic abnormalities in HF.

Thrombus formation and thromboembolic events play important roles in various cardiovascular pathologies. The key receptor involved in platelet aggregation is the fibrinogen receptor glycoprotein IIb/IIIa. [18F]GP1, a derivative of the GPIIb/IIIa antagonist elarofiban, is a specific 18F-labeled small-molecule radiotracer that binds with high affinity to GPIIb/IIIa receptors of activated platelets. An improved, robust and fully automated radiosynthesis of [18F]GP1 has been developed. [18F]GP1 has been synthesized with decay corrected radiochemical yields of 38 ± 6%, with a radiochemical concentration up to 1900 MBq/mL, molar activities of 952–9428 GBq/µmol and a radio-chemical purity >98%. After determination of the optimal reaction conditions, in particular for HPLC separation, adaption of the reaction conditions to PET center requirements, validation of the manufacturing process and the quality control methods, the synthesis of [18F]GP1 was successfully implemented to GMP standards and was available for clinical application. We describe the GMP-compliant synthesis of the novel radiotracer [18F]GP1. Moreover, we provide some proof-of-concept examples for clinical application in the cardiovascular field. PET/CT with the novel small-molecular radiotracer [18F]GP1 may serve as a novel highly sensitive tool for visualizing active platelet aggregation at the molecular level.

In terminal failing hearts ventricular assist devices (VAD) are implanted as a bridge to transplantation. Endothelin receptor (ETR) antagonists are used for treatment of secondary pulmonary hypertension in VAD patients. However, the cardiac ETR regulation in human heart failure and during VAD support is incompletely understood.In paired left ventricular samples of 12 dilated cardiomyopathy patients we investigated the density of endothelin A (ETA) and B (ETB) receptors before VAD implantation and after device removal. Left ventricular samples of 12 non-failing donor hearts served as control. Receptor quantification was performed by binding of [125I]-ET-1 in the presence of nonselective and ETA selective ETR ligands as competitors. Additionally, the ETR mRNA expression was analyzed using quantitative real-time-PCR.The mRNA of ETA but not ETB receptors was significantly elevated in heart failure, whereas total ETR density analyzed by radioligand binding was significantly reduced due to ETB receptor down regulation. ETA and ETB receptor density showed poor correlation to mRNA data (spearman correlation factor: 0.43 and 0.31, respectively). VAD support had no significant impact on the density of both receptors and on mRNA expression of ETA whereas ETB mRNA increased during VAD. A meta-analysis reveals that the ETA receptor regulation in human heart failure appears to depend on non-failing hearts.In deteriorating hearts of patients suffering from dilated cardiomyopathy the ETA receptor density is not changed whereas the ETB receptor is down regulated. The mRNA and the proteins of ETA and ETB show a weak correlation. Non-failing hearts might influence the interpretation of ETA receptor regulation. Mechanical unloading of the failing hearts has no impact on the myocardial ETR density.

Cardiomyopathies might lead to end-stage heart disease with the requirement of drastic treatments like bridging up to transplant or heart transplantation. A not precisely known proportion of these diseases are genetically determined. We genotyped 43 index-patients (30 DCM, 10 ARVC, 3 RCM) with advanced or end stage cardiomyopathy using a gene panel which covered 46 known cardiomyopathy disease genes. Fifty-three variants with possible impact on disease in 33 patients were identified. Of these 27 (51%) were classified as likely pathogenic or pathogenic in the MYH7, MYL2, MYL3, NEXN, TNNC1, TNNI3, DES, LMNA, PKP2, PLN, RBM20, TTN, and CRYAB genes. Fifty-six percent (n = 24) of index-patients carried a likely pathogenic or pathogenic mutation. Of these 75% (n = 18) were familial and 25% (n = 6) sporadic cases. However, severe cardiomyopathy seemed to be not characterized by a specific mutation profile. Remarkably, we identified a novel homozygous PKP2-missense variant in a large consanguineous family with sudden death in early childhood and several members with heart transplantation in adolescent age.

The original version of this Article incorrectly acknowledged Elisabeth Reiser and Rene Schramm as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article.

Calcium and phosphate are central for myocardial contractility and energy metabolism, and low levels of the calciotropic hormone 1,25-dihydroxyvitamin D (1,25(OH)2D), as well as high levels of the phosphaturic hormone fibroblast growth factor (FGF)-23, are independently associated with poor clinical outcome in heart failure (HF) patients. We therefore aimed to investigate the postoperative time course of the aforementioned hormones in HF patients supported with a left-ventricular assist device (LVAD) implant.For the present study, stored biobank plasma samples of 69 patients, collected before LVAD implantation (t0) and 12 days (t1), 30 days (t2), 83 days (t3), and 300 days (t4) post-intervention, were used to measure circulating FGF-23, parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD), 1,25(OH)2D, and kidney function.Most patients were male and had baseline INTERMACS levels and cardiac index values ≤ 3 and ≤ 2.7 L/min/m2, respectively. There were significant time effects on estimated glomerular filtration rate (eGFR), FGF-23 and 1,25(OH)2D, but not on PTH or 25OHD. Notably, eGFR values increased and FGF-23 levels decreased only transiently, whereas 1,25(OH)2D increased continuously until t4. The rise in 1,25(OH)2D was largely influenced by those patients who survived the first post-implant year, and was not seen in non-survivors. Variations in 1,25(OH)2D levels could only partly be explained by eGFR values or FGF-23, 25OHD, and PTH levels (multiple R2 = 0.305;P