Publications by authors named "Karl Toischer"

54 Publications

Epigenetic gene expression links heart failure to memory impairment.

EMBO Mol Med 2021 Mar 20;13(3):e11900. Epub 2021 Jan 20.

Department for Systems Medicine and Epigenetics, German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.

In current clinical practice, care of diseased patients is often restricted to separated disciplines. However, such an organ-centered approach is not always suitable. For example, cognitive dysfunction is a severe burden in heart failure patients. Moreover, these patients have an increased risk for age-associated dementias. The underlying molecular mechanisms are presently unknown, and thus, corresponding therapeutic strategies to improve cognition in heart failure patients are missing. Using mice as model organisms, we show that heart failure leads to specific changes in hippocampal gene expression, a brain region intimately linked to cognition. These changes reflect increased cellular stress pathways which eventually lead to loss of neuronal euchromatin and reduced expression of a hippocampal gene cluster essential for cognition. Consequently, mice suffering from heart failure exhibit impaired memory function. These pathological changes are ameliorated via the administration of a drug that promotes neuronal euchromatin formation. Our study provides first insight to the molecular processes by which heart failure contributes to neuronal dysfunction and point to novel therapeutic avenues to treat cognitive defects in heart failure patients.
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http://dx.doi.org/10.15252/emmm.201911900DOI Listing
March 2021

Perturbed differentiation of murine embryonic stem cells upon Pelota deletion due to dysregulated FOXO1/β-catenin signaling.

FEBS J 2020 Nov 27. Epub 2020 Nov 27.

Institute of Human Genetics, University Medical Centre, Göttingen, Germany.

Differentiation of the embryonic stem cells (ESCs) is regulated by a variety of different signaling pathways. Genetic depletion of murine Pelota gene (Pelo) leads to early embryonic lethality. Here, we aimed at determining the embryonic stage and deciphering the dysregulated signaling pathways affected upon Pelo deletion. We found that development of PELO-null embryos is perturbed between the embryonic day E4.5 and E5.5, at which first differentiation process of ESCs takes place. Molecular analysis revealed enhanced activity of phosphoinositide 3-kinase-protein kinase B/ AKT (PI3K-PKB/AKT) signaling, but nuclear accumulation of forkhead box O1 (FOXO1), and upregulation of the pluripotency-related gene, Oct4, in mutant ESCs cultured under differentiation condition. Despite increased levels of nuclear β-catenin in PELO-null ESCs as a result of decreased activity of glycogen synthase kinase-3β, the activity of the canonical Wingless (Wnt)/β-catenin/T cell factor (TCF) was significantly attenuated as judged by the promoter reporter assay, downregulated Wnt/β-catenin target genes, and impaired cell proliferation. Interestingly, we demonstrated an increased binding of β-catenin to FOXO1 in PELO-mutant ESCs cultured under differentiation condition that could explain, on one side, the nuclear accumulation of FOXO1 protein and hence persistent pluripotency of PELO-mutant ESCs, and on the other side, the dysregulated transcriptional activity of β-catenin/TCF and therefore attenuated PELO-null ESCs self-renewal. Taken together, our results strongly suggest that PELO deletion averts ESCs differentiation through promoting FOXO1/β-catenin binding with subsequent dysregulation of FOXO1 and canonical β-catenin/TCF signaling pathways.
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http://dx.doi.org/10.1111/febs.15643DOI Listing
November 2020

X-ray diffraction and second harmonic imaging reveal new insights into structural alterations caused by pressure-overload in murine hearts.

Sci Rep 2020 11 9;10(1):19317. Epub 2020 Nov 9.

Institute for X-Ray Physics, Georg-August-Universität Göttingen, Friedrich-Hund-Platz 1, 37077, Göttingen, Germany.

We demonstrate a label-free imaging approach to study cardiac remodeling of fibrotic and hypertrophic hearts, bridging scales from the whole organ down to the molecular level. To this end, we have used mice subjected to transverse aortic constriction and imaged adjacent cardiac tissue sections by microfocus X-ray diffraction and second harmonic generation (SHG) imaging. In this way, the acto-myosin structure was probed in a spatially resolved manner for entire heart sections. From the recorded diffraction data, spatial maps of diffraction intensity, anisotropy and orientation were obtained, and fully automated analysis depicted the acto-myosin filament spacing and direction. X-ray diffraction presented an overview of entire heart sections and revealed that in regions of severe cardiac remodeling the muscle mass is partly replaced by connective tissue and the acto-myosin lattice spacing is increased at these regions. SHG imaging revealed sub-cellular structure of cardiac tissue and complemented the findings from X-ray diffraction by revealing micro-level distortion of myofibrils, immune cell infiltration at regions of cardiac remodeling and the development of fibrosis down to the scale of a single collagen fibril. Overall, our results show that both X-ray diffraction and SHG imaging can be used for label-free and high-resolution visualization of cardiac remodeling and fibrosis progression at different stages in a cardiac pressure-overload mouse model that cannot be achieved by conventional histology.
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http://dx.doi.org/10.1038/s41598-020-76163-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653033PMC
November 2020

X-Ray Structural Analysis of Single Adult Cardiomyocytes: Tomographic Imaging and Microdiffraction.

Biophys J 2020 10 28;119(7):1309-1323. Epub 2020 Aug 28.

Institute for X-ray Physics, Göttingen, Germany. Electronic address:

We present a multiscale imaging approach to characterize the structure of isolated adult murine cardiomyocytes based on a combination of full-field three-dimensional coherent x-ray imaging and scanning x-ray diffraction. Using these modalities, we probe the structure from the molecular to the cellular scale. Holographic projection images on freeze-dried cells have been recorded using highly coherent and divergent x-ray waveguide radiation. Phase retrieval and tomographic reconstruction then yield the three-dimensional electron density distribution with a voxel size below 50 nm. In the reconstruction volume, myofibrils, sarcomeric organization, and mitochondria can be visualized and quantified within a single cell without sectioning. Next, we use microfocusing optics by compound refractive lenses to probe the diffraction signal of the actomyosin lattice. Comparison between recordings of chemically fixed and untreated, living cells indicate that the characteristic lattice distances shrink by ∼10% upon fixation.
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http://dx.doi.org/10.1016/j.bpj.2020.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567981PMC
October 2020

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx).

Proc Natl Acad Sci U S A 2020 09 14;117(39):24545-24556. Epub 2020 Sep 14.

Institute of Physiology II, University of Munster, 48149 Munster, Germany;

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.
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http://dx.doi.org/10.1073/pnas.2004900117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533878PMC
September 2020

Preclinical evidence for the therapeutic value of TBX5 normalization in arrhythmia control.

Cardiovasc Res 2020 Aug 10. Epub 2020 Aug 10.

Institute of Pharmacology and Toxicology, University Medical Center, Goettingen, Germany.

Aims: Arrhythmias and sudden cardiac death (SCD) occur commonly in patients with heart failure. We found T-box 5 (TBX5) dysregulated in ventricular myocardium from heart failure patients and thus we hypothesized that TBX5 reduction contributes to arrhythmia development in these patients. To understand the underlying mechanisms, we aimed to reveal the ventricular TBX5-dependent transcriptional network and further test the therapeutic potential of TBX5 level normalization in mice with documented arrhythmias.

Methods And Results: We used a mouse model of TBX5 conditional deletion in ventricular cardiomyocytes. Ventricular (v) TBX5 loss in mice resulted in mild cardiac dysfunction and arrhythmias and was associated with a high mortality rate (60%) due to SCD. Upon angiotensin stimulation, vTbx5KO mice showed exacerbated cardiac remodelling and dysfunction suggesting a cardioprotective role of TBX5. RNA sequencing of a ventricular specific TBX5KO mouse and TBX5 chromatin immunoprecipitation were used to dissect TBX5 transcriptional network in cardiac ventricular tissue. Overall, we identified 47 transcripts expressed under the control of TBX5, which may have contributed to the fatal arrhythmias in vTbx5KO mice. These included transcripts encoding for proteins implicated in cardiac conduction and contraction (Gja1, Kcnj5, Kcng2, Cacna1g, Chrm2), in cytoskeleton organization (Fstl4, Pdlim4, Emilin2, Cmya5), and cardiac protection upon stress (Fhl2, Gpr22, Fgf16). Interestingly, after TBX5 loss and arrhythmia development in vTbx5KO mice, TBX5 protein level normalization by systemic adeno-associated-virus (AAV) 9 application, re-established TBX5-dependent transcriptome. Consequently, cardiac dysfunction was ameliorated and the propensity of arrhythmia occurrence was reduced.

Conclusions: This study uncovers a novel cardioprotective role of TBX5 in the adult heart and provides preclinical evidence for the therapeutic value of TBX5 protein normalization in the control of arrhythmia.

Translational Perspective: Cardiovascular disease (CVD) is the number one cause of death worldwide (WHO factsheets 09/2016). Although more than 60% of CVD-related deaths are due to out-of-hospital sudden cardiac death (SCD), we have little insight in the mechanisms underlying SCD pathophysiology. Our data show a link between TBX5 dysregulation and arrhythmia occurrence in patients. To test the therapeutic potential of TBX5, we normalized TBX5 levels in a mouse model with TBX5 dysregulation, which developed arrhythmias and SCD. TBX5 normalization re-established TBX5 target gene expression and more importantly, rescued the arrhythmia phenotype. Altogether, we provide proof-of-concept for the therapeutic potential of TBX5 expression restoration against arrhythmia and SCD.
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http://dx.doi.org/10.1093/cvr/cvaa239DOI Listing
August 2020

Dysferlin links excitation-contraction coupling to structure and maintenance of the cardiac transverse-axial tubule system.

Europace 2020 07;22(7):1119-1131

Department of Child and Adolescent Health, University Medical Center Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany.

Aims: The multi-C2 domain protein dysferlin localizes to the T-Tubule system of skeletal and heart muscles. In skeletal muscle, dysferlin is known to play a role in membrane repair and in T-tubule biogenesis and maintenance. Dysferlin deficiency manifests as muscular dystrophy of proximal and distal muscles. Cardiomyopathies have been also reported, and some dysferlinopathy mouse models develop cardiac dysfunction under stress. Generally, the role and functional relevance of dysferlin in the heart is not clear. The aim of this study was to analyse the effect of dysferlin deficiency on the transverse-axial tubule system (TATS) structure and on Ca2+ homeostasis in the heart.

Methods And Results: We studied dysferlin localization in rat and mouse cardiomyocytes by immunofluorescence microscopy. In dysferlin-deficient ventricular mouse cardiomyocytes, we analysed the TATS by live staining and assessed Ca2+ handling by patch-clamp experiments and measurement of Ca2+ transients and Ca2+ sparks. We found increasing co-localization of dysferlin with the L-type Ca2+-channel during TATS development and show that dysferlin deficiency leads to pathological loss of transversal and increase in longitudinal elements (axialization). We detected reduced L-type Ca2+-current (ICa,L) in cardiomyocytes from dysferlin-deficient mice and increased frequency of spontaneous sarcoplasmic reticulum Ca2+ release events resulting in pro-arrhythmic contractions. Moreover, cardiomyocytes from dysferlin-deficient mice showed an impaired response to β-adrenergic receptor stimulation.

Conclusions: Dysferlin is required for TATS biogenesis and maintenance in the heart by controlling the ratio of transversal and axial membrane elements. Absence of dysferlin leads to defects in Ca2+ homeostasis which may contribute to contractile heart dysfunction in dysferlinopathy patients.
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http://dx.doi.org/10.1093/europace/euaa093DOI Listing
July 2020

Real-time cardiovascular magnetic resonance T1 and extracellular volume fraction mapping for tissue characterisation in aortic stenosis.

J Cardiovasc Magn Reson 2020 06 22;22(1):46. Epub 2020 Jun 22.

University Medical Center Göttingen, Department of Cardiology and Pneumology, Georg-August University, Robert-Koch-Str. 40, 37099, Göttingen, Germany.

Background: Myocardial fibrosis is a major determinant of outcome in aortic stenosis (AS). Novel fast real-time (RT) cardiovascular magnetic resonance (CMR) mapping techniques allow comprehensive quantification of fibrosis but have not yet been compared against standard techniques and histology.

Methods: Patients with severe AS underwent CMR before (n = 110) and left ventricular (LV) endomyocardial biopsy (n = 46) at transcatheter aortic valve replacement (TAVR). Midventricular short axis (SAX) native, post-contrast T1 and extracellular volume fraction (ECV) maps were generated using commercially available modified Look-Locker Inversion recovery (MOLLI) (native: 5(3)3, post-contrast: 4(1)3(1)2) and RT single-shot inversion recovery Fast Low-Angle Shot (FLASH) with radial undersampling. Focal late gadolinium enhancement was excluded from T1 and ECV regions of interest. ECV and LV mass were used to calculate LV matrix volumes. Variability and agreements were assessed between RT, MOLLI and histology using intraclass correlation coefficients, coefficients of variation and Bland Altman analyses.

Results: RT and MOLLI derived ECV were similar for midventricular SAX slice coverage (26.2 vs. 26.5, p = 0.073) and septal region of interest (26.2 vs. 26.5, p = 0.216). MOLLI native T1 time was in median 20 ms longer compared to RT (p < 0.001). Agreement between RT and MOLLI was best for ECV (ICC > 0.91), excellent for post-contrast T1 times (ICC > 0.81) and good for native T1 times (ICC > 0.62). Diffuse collagen volume fraction by biopsies was in median 7.8%. ECV (RT r = 0.345, p = 0.039; MOLLI r = 0.40, p = 0.010) and LV matrix volumes (RT r = 0.45, p = 0.005; MOLLI r = 0.43, p = 0.007) were the only parameters associated with histology.

Conclusions: RT mapping offers fast and sufficient ECV and LV matrix volume calculation in AS patients. ECV and LV matrix volume represent robust and universally comparable parameters with associations to histologically assessed fibrosis and may emerge as potential targets for clinical decision making.
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http://dx.doi.org/10.1186/s12968-020-00632-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310147PMC
June 2020

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

Basic Res Cardiol 2020 03 7;115(3):27. Epub 2020 Mar 7.

Laboratory of Experimental Cardiology, Clinic for Cardiology and Pneumology, Heart Research Center, University Medical Center Goettingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany.

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 cycling. CRISPR/Cas9-mediated loss-of-function leads to dysregulated Ca handling in human-induced pluripotent stem cell-derived cardiomyocytes. The downregulation of prohypertrophic, proapoptotic and Ca-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 regulation in the transition to failure and novel candidate genes with promising potential for therapeutic interventions.
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http://dx.doi.org/10.1007/s00395-020-0784-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060963PMC
March 2020

Impact of myocardial fibrosis on left ventricular remodelling, recovery, and outcome after transcatheter aortic valve implantation in different haemodynamic subtypes of severe aortic stenosis.

Eur Heart J 2020 05;41(20):1903-1914

Clinic of Cardiology and Pneumology, University Medical Center Göttingen, 37099 Göttingen, Germany.

Aims: Myocardial fibrosis (MF) might represent a key player in pathophysiology of heart failure in aortic stenosis (AS). We aimed to assess its impact on left ventricular (LV) remodelling, recovery, and mortality after transcatheter aortic valve implantation (TAVI) in different AS subtypes.

Methods And Results: One hundred patients with severe AS were prospectively characterized clinically and echocardiographically at baseline (BL), 6 months, 1 year, and 2 years following TAVI. Left ventricular biopsies were harvested after valve deployment. Myocardial fibrosis was assessed after Masson's trichrome staining, and fibrotic area was calculated as percentage of total tissue area. Patients were stratified according to MF above (MF+) or below (MF-) median percentage MF (≥11% or <11%). Myocardial fibrosis burden differed significantly between AS subtypes, with highest levels in low ejection fraction (EF), low-gradient AS and lowest levels in normal EF, high-gradient AS (29.5 ± 26.4% vs. 13.5 ± 16.1%, P = 0.003). In the entire cohort, MF+ was significantly associated with poorer LV function, higher extent of pathological LV remodelling, and more pronounced clinical heart failure at BL. After TAVI, MF+ was associated with a delay in normalization of LV geometry and function but not per se with absence of reverse remodelling and clinical improvement. However, 22 patients died during follow-up (mean, 11 months), and 14 deaths were classified as cardiovascular (CV) (n = 9 arrhythmia-associated). Importantly, 13 of 14 CV deaths occurred in MF+ patients (CV mortality 26.5% in MF+ vs. 2% in MF- patients, P = 0.0003). Multivariate analysis identified MF+ as independent predictor of CV mortality [hazard ratio (HR) 27.4 (2.0-369), P = 0.01].

Conclusion: Histological MF is associated with AS-related pathological LV remodelling and independently predicts CV mortality after TAVI.
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http://dx.doi.org/10.1093/eurheartj/ehaa033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242071PMC
May 2020

Changes in m6A RNA methylation contribute to heart failure progression by modulating translation.

Eur J Heart Fail 2020 01 17;22(1):54-66. Epub 2019 Dec 17.

Clinic for Cardiology and Pneumology, University Medical Center, Göttingen, Germany.

Aims: Deregulation of epigenetic processes and aberrant gene expression are important mechanisms in heart failure. Here we studied the potential relevance of m6A RNA methylation in heart failure development.

Methods And Results: We analysed m6A RNA methylation via next-generation sequencing. We found that approximately one quarter of the transcripts in the healthy mouse and human heart exhibit m6A RNA methylation. During progression to heart failure we observed that changes in m6A RNA methylation exceed changes in gene expression both in mouse and human. RNAs with altered m6A RNA methylation were mainly linked to metabolic and regulatory pathways, while changes in RNA expression level mainly represented changes in structural plasticity. Mechanistically, we could link m6A RNA methylation to altered RNA translation and protein production. Interestingly, differentially methylated but not differentially expressed RNAs showed differential polysomal occupancy, indicating transcription-independent modulation of translation. Furthermore, mice with a cardiomyocyte restricted knockout of the RNA demethylase Fto exhibited an impaired cardiac function compared to control mice.

Conclusions: We could show that m6A landscape is altered in heart hypertrophy and heart failure. m6A RNA methylation changes lead to changes in protein abundance, unconnected to mRNA levels. This uncovers a new transcription-independent mechanisms of translation regulation. Therefore, our data suggest that modulation of epitranscriptomic processes such as m6A methylation might be an interesting target for therapeutic interventions.
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http://dx.doi.org/10.1002/ejhf.1672DOI Listing
January 2020

NADPH oxidase-4 promotes eccentric cardiac hypertrophy in response to volume overload.

Cardiovasc Res 2021 Jan;117(1):178-187

King's College London British Heart Foundation Centre of Excellence, School of Cardiovascular Medicine & Sciences, The James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK.

Aims: Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling.

Methods And Results: We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4-/-) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4-/- mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P < 0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P < 0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 μm2, P < 0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4-/- mice (+29% vs. -21%, P < 0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4-/- mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A.

Conclusion: Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.
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http://dx.doi.org/10.1093/cvr/cvz331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797217PMC
January 2021

Cell Cycle-Mediated Cardiac Regeneration in the Mouse Heart.

Curr Cardiol Rep 2019 09 16;21(10):131. Epub 2019 Sep 16.

Krannert Institute of Cardiology and the Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut Street, Indianapolis, IN, 46202, USA.

Purpose Of Review: Many forms of heart disease result in the essentially irreversible loss of cardiomyocytes. The ability to promote cardiomyocyte renewal may be a promising approach to reverse injury in diseased hearts. The purpose of this review is to describe the impact of cardiomyocyte cell cycle activation on cardiac function and structure in several different models of myocardial disease.

Recent Findings: Transgenic mice expressing cyclin D2 (D2 mice) exhibit sustained cardiomyocyte renewal in the adult heart. Earlier studies demonstrated that D2 mice exhibited progressive myocardial regeneration in experimental models of myocardial infarction, and that cardiac function was normalized to values seen in sham-operated litter mates by 180 days post-injury. D2 mice also exhibited markedly improved atrial structure in a genetic model of atrial fibrosis. More recent studies revealed that D2 mice were remarkably resistant to heart failure induced by chronic elevated afterload as compared with their wild type (WT siblings), with a 6-fold increase in median survival as well as retention of relatively normal cardiac function. Finally, D2 mice exhibited a progressive recovery in cardiac function to normal levels and a concomitant reduction in adverse myocardial remodeling in an anthracycline cardiotoxicity model. The studies reviewed here make a strong case for the potential utility of inducing cardiomyocyte renewal as a means to treat injured hearts. Several challenges which must be met to develop a viable therapeutic intervention based on these observations are discussed.
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http://dx.doi.org/10.1007/s11886-019-1206-9DOI Listing
September 2019

Assessing the role of extracellular signal-regulated kinases 1 and 2 in volume overload-induced cardiac remodelling.

ESC Heart Fail 2019 10 19;6(5):1015-1026. Epub 2019 Jul 19.

Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany.

Aims: Volume overload (VO) and pressure overload (PO) induce differential cardiac remodelling responses including distinct signalling pathways. Extracellular signal-regulated kinases 1 and 2 (ERK1/2), key signalling components in the mitogen-activated protein kinase (MAPK) pathways, modulate cardiac remodelling during pressure overload (PO). This study aimed to assess their role in VO-induced cardiac remodelling as this was unknown.

Methods And Results: Aortocaval fistula (Shunt) surgery was performed in mice to induce cardiac VO. Two weeks of Shunt caused a significant reduction of cardiac ERK1/2 activation in wild type (WT) mice as indicated by decreased phosphorylation of the TEY (Thr-Glu-Tyr) motif (-28% as compared with Sham controls, P < 0.05). Phosphorylation of other MAPKs was unaffected. For further assessment, transgenic mice with cardiomyocyte-specific ERK2 overexpression (ERK2tg) were studied. At baseline, cardiac ERK1/2 phosphorylation in ERK2tg mice remained unchanged compared with WT littermates, and no overt cardiac phenotype was observed; however, cardiac expression of the atrial natriuretic peptide was increased on messenger RNA (3.6-fold, P < 0.05) and protein level (3.1-fold, P < 0.05). Following Shunt, left ventricular dilation and hypertrophy were similar in ERK2tg mice and WT littermates. Left ventricular function was maintained, and changes in gene expression indicated reactivation of the foetal gene program in both genotypes. No differences in cardiac fibrosis and kinase activation was found amongst all experimental groups, whereas apoptosis was similarly increased through Shunt in ERK2tg and WT mice.

Conclusions: VO-induced eccentric hypertrophy is associated with reduced cardiac ERK1/2 activation in vivo. Cardiomyocyte-specific overexpression of ERK2, however, does not alter cardiac remodelling during VO. Future studies need to define the pathophysiological relevance of decreased ERK1/2 signalling during VO.
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http://dx.doi.org/10.1002/ehf2.12497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816056PMC
October 2019

Control of p21Cip by BRCA1-associated protein is critical for cardiomyocyte cell cycle progression and survival.

Cardiovasc Res 2020 03;116(3):592-604

Department of Cardiology and Pulmonology, Georg-August University, Robert-Koch Str. 40, 37075 Göttingen, Germany.

Aims: Identifying the key components in cardiomyocyte cell cycle regulation is of relevance for the understanding of cardiac development and adaptive and maladaptive processes in the adult myocardium. BRCA1-associated protein (BRAP) has been suggested as a cytoplasmic retention factor for several proteins including Cyclin-dependent-kinase inhibitor p21Cip. We observed profound expressional changes of BRAP in early postnatal myocardium and investigated the impact of BRAP on cardiomyocyte cell cycle regulation.

Methods And Results: General knockout of Brap in mice evoked embryonic lethality associated with reduced myocardial wall thickness and lethal cardiac congestion suggesting a prominent role for BRAP in cardiomyocyte proliferation. αMHC-Cre driven cardiomyocyte-specific knockout of Brap also evoked lethal cardiac failure shortly after birth. Likewise, conditional cardiomyocyte-specific Brap deletion using tamoxifen-induced knockout in adult mice resulted in marked ventricular dilatation and heart failure 3 weeks after induction. Several lines of evidence suggest that Brap deletion evoked marked inhibition of DNA synthesis and cell cycle progression. In cardiomyocytes with proliferative capacity, this causes developmental arrest, whereas in adult hearts loss of BRAP-induced apoptosis. This is explained by altered signalling through p21Cip which we identify as the link between BRAP and cell cycle/apoptosis. BRAP deletion enhanced p21Cip expression, while BRAP overexpression in cardiomyocyte-specific transgenic mice impeded p21Cip expression. That was paralleled by enhanced nuclear Ki-67 expression and DNA synthesis.

Conclusion: By controlling p21Cip activity BRAP expression controls cell cycle activity and prevents developmental arrest in developing cardiomyocytes and apoptosis in adult cardiomyocytes.
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http://dx.doi.org/10.1093/cvr/cvz177DOI Listing
March 2020

Genetic deletion of calcium/calmodulin-dependent protein kinase type II delta does not mitigate adverse myocardial remodeling in volume-overloaded hearts.

Sci Rep 2019 07 8;9(1):9889. Epub 2019 Jul 8.

Department of Cardiology and Pneumology, Georg-August-University, Göttingen, Germany.

Calcium/calmodulin-dependent protein kinase type II delta (CaMKIIδ), the predominant CaMKII isoform expressed in the heart, has been implicated in the progression of myocardial infarction- and pressure overload-induced pathological remodeling. However, the role of CaMKIIδ in volume overload (VO) has not been explored. We have previously reported an activation of CaMKII during transition to HF in long-term VO. Here, we address whether CaMKIIδ is critically involved in the mortality, myocardial remodeling, and heart failure (HF) progression in response to VO. CaMKIIδ knockout (δ-KO) and wild-type (WT) littermates were exposed to aortocaval shunt-induced VO, and the progression of adverse myocardial remodeling was assessed by serial echocardiography, histological and molecular analyses. The mortality rates during 10 weeks of VO were similar in δ-KO and WT mice. Both genotypes displayed comparable eccentric myocardial hypertrophy, altered left ventricle geometry, perturbed systolic and diastolic functions after shunt. Additionally, cardiomyocytes hypertrophy, augmented myocyte apoptosis, and up-regulation of hypertrophic genes were also not significantly different in δ-KO versus WT hearts after shunt. Therefore, CaMKIIδ signaling seems to be dispensable for the progression of VO-induced maladaptive cardiac remodeling. Accordingly, we hypothesize that CaMKIIδ-inhibition as a therapeutic approach might not be helpful in the context of VO-triggered HF.
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http://dx.doi.org/10.1038/s41598-019-46332-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614357PMC
July 2019

Proteasome-Dependent Regulation of Distinct Metabolic States During Long-Term Culture of Human iPSC-Derived Cardiomyocytes.

Circ Res 2019 06 20;125(1):90-103. Epub 2019 May 20.

From the Stanford Cardiovascular Institute (A.E., Y.D., S.S., H.C., Y.L., P.G., J.C.W.), Stanford University School of Medicine, CA.

Rationale: The immature presentation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is currently a challenge for their application in disease modeling, drug screening, and regenerative medicine. Long-term culture is known to achieve partial maturation of iPSC-CMs. However, little is known about the molecular signaling circuitries that govern functional changes, metabolic output, and cellular homeostasis during long-term culture of iPSC-CMs.

Objective: We aimed to identify and characterize critical signaling events that control functional and metabolic transitions of cardiac cells during developmental progression, as recapitulated by long-term culture of iPSC-CMs.

Methods And Results: We combined transcriptomic sequencing with pathway network mapping in iPSC-CMs that were cultured until a late time point, day 200, in comparison to a medium time point, day 90, and an early time point, day 30. Transcriptomic landscapes of long-term cultured iPSC-CMs allowed mapping of distinct metabolic stages during development of maturing iPSC-CMs. Temporally divergent control of mitochondrial metabolism was found to be regulated by cAMP/PKA (protein kinase A)- and proteasome-dependent signaling events. The PKA/proteasome-dependent signaling cascade was mediated downstream by Hsp90 (heat shock protein 90), which in turn modulated mitochondrial respiratory chain proteins and their metabolic output. During long-term culture, this circuitry was found to initiate upregulation of iPSC-CM metabolism, resulting in increased cell contractility that reached a maximum at the day 200 time point.

Conclusions: Our results reveal a PKA/proteasome- and Hsp90-dependent signaling pathway that regulates mitochondrial respiratory chain proteins and determines cardiomyocyte energy production and functional output. These findings provide deeper insight into signaling circuitries governing metabolic homeostasis in iPSC-CMs during developmental progression.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613799PMC
June 2019

CD8-T Cells With Specificity for a Model Antigen in Cardiomyocytes Can Become Activated After Transverse Aortic Constriction but Do Not Accelerate Progression to Heart Failure.

Front Immunol 2018 15;9:2665. Epub 2018 Nov 15.

Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.

Heart failure due to pressure overload is frequently associated with inflammation. In addition to inflammatory responses of the innate immune system, autoimmune reactions of the adaptive immune system appear to be triggered in subgroups of patients with heart failure as demonstrated by the presence of autoantibodies against myocardial antigens. Moreover, T cell-deficient and T cell-depleted mice have been reported to be protected from heart failure induced by transverse aortic constriction (TAC) and we have shown recently that CD4-helper T cells with specificity for an antigen in cardiomyocytes accelerate TAC-induced heart failure. In this study, we set out to investigate the potential contribution of CD8-cytotoxic T cells with specificity to a model antigen (ovalbumin, OVA) in cardiomyocytes to pressure overload-induced heart failure. In 78% of cMy-mOVA mice with cardiomyocyte-specific OVA expression, a low-grade OVA-specific cellular cytotoxicity was detected after TAC. Adoptive transfer of OVA-specific CD8-T cells from T cell receptor transgenic OT-I mice before TAC did not increase the risk of OVA-specific autoimmunity in cMy-mOVA mice. After TAC, again 78% of the mice displayed an OVA-specific cytotoxicity with on average only a three-fold higher killing of OVA-expressing target cells. More CD8 cells were present after TAC in the myocardium of cMy-mOVA mice with OT-I T cells (on average 17.5/mm) than in mice that did not receive OVA-specific CD8-T cells (3.6/mm). However, the extent of fibrosis was similar in both groups. Functionally, as determined by echocardiography, the adoptive transfer of OVA-specific CD8-T cells did not significantly accelerate the progression from hypertrophy to heart failure in cMy-mOVA mice. These findings argue therefore against a major impact of cytotoxic T cells with specificity for autoantigens of cardiomyocytes in pressure overload-induced heart failure.
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http://dx.doi.org/10.3389/fimmu.2018.02665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249381PMC
October 2019

Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1α Expression in Hypoxia.

Cell Rep 2018 10;25(3):561-570.e6

Department of Cellular Biochemistry, University Medical Center Göttingen, GZMB, 37073 Göttingen, Germany.

Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs, integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin remodeling, upon loss of the cardiolipin acyl transferase tafazzin, decreases HIF-1α signaling in hypoxia. Tafazzin deficiency does not affect posttranslational HIF-1α regulation but rather HIF-1α gene expression, a dysfunction recapitulated in iPSC-derived cardiomyocytes from Barth syndrome patients with tafazzin deficiency. RNA-seq analyses confirmed drastically altered signaling in tafazzin mutant cells. In hypoxia, tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-κB activation and concomitantly HIF-1α gene expression. Tafazzin-deficient mice hearts display reduced HIF-1α levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin remodeling dampens HIF-1α signaling due to a lack of NF-κB activation through reduced mitochondrial ROS production, decreasing HIF-1α transcription.
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http://dx.doi.org/10.1016/j.celrep.2018.09.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205837PMC
October 2018

Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression.

Sci Transl Med 2018 09;10(458)

Department of Cardiology and Pneumology, Georg-August-University, 37075 Göttingen, Germany.

Increased sarcoplasmic reticulum (SR) Ca leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36- versus placebo-treated mice. Reduction of SR Ca leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca leak-independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca transient, SR Ca load, SR Ca adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.
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http://dx.doi.org/10.1126/scitranslmed.aan0724DOI Listing
September 2018

X-ray diffraction imaging of cardiac cells and tissue.

Prog Biophys Mol Biol 2019 07 18;144:151-165. Epub 2018 Jun 18.

Universität Göttingen, Institut für Röntgenphysik, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany. Electronic address:

With the development of advanced focusing optics for x-rays, we can now use x-ray beams with spot sizes in the micro- or nanometer range to scan cells and large areas of tissues and continuously record the diffraction signals. From this data, x-ray scattering maps or so-called x-ray darkfield images are computed showing how different types of cells or regions of tissues differ in their diffraction intensity. At the same time a diffraction pattern is available for each scan point which encodes the local nanostructure, averaged over many contributing constituents illuminated by the beam. In this work we have exploited these new capabilities of scanning x-ray diffraction to investigate cardiac muscle cells as well as cardiac tissue. We give examples of how cardiac cells, especially living, cultured cells, can be prepared to be compatible with the instrumentation constraints of nano- or micro-diffraction instruments. Furthermore, we show how the developmental stage, ranging from neonatal to adult cells, as well as the final preparation state of the cardiomyocytes influences the recorded scattering signal and how these diffraction signals compare to the structure of a fully developed cardiac muscle.
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http://dx.doi.org/10.1016/j.pbiomolbio.2018.05.012DOI Listing
July 2019

A context-specific cardiac β-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart.

Nucleic Acids Res 2018 04;46(6):2850-2867

Institute of Pharmacology and Toxicology, University Medical Center Goettingen, Georg-August University, Goettingen 37075, Germany.

Chromatin remodelling precedes transcriptional and structural changes in heart failure. A body of work suggests roles for the developmental Wnt signalling pathway in cardiac remodelling. Hitherto, there is no evidence supporting a direct role of Wnt nuclear components in regulating chromatin landscapes in this process. We show that transcriptionally active, nuclear, phosphorylated(p)Ser675-β-catenin and TCF7L2 are upregulated in diseased murine and human cardiac ventricles. We report that inducible cardiomyocytes (CM)-specific pSer675-β-catenin accumulation mimics the disease situation by triggering TCF7L2 expression. This enhances active chromatin, characterized by increased H3K27ac and TCF7L2 occupancies to cardiac developmental and remodelling genes in vivo. Accordingly, transcriptomic analysis of β-catenin stabilized hearts shows a strong recapitulation of cardiac developmental processes like cell cycling and cytoskeletal remodelling. Mechanistically, TCF7L2 co-occupies distal genomic regions with cardiac transcription factors NKX2-5 and GATA4 in stabilized-β-catenin hearts. Validation assays revealed a previously unrecognized function of GATA4 as a cardiac repressor of the TCF7L2/β-catenin complex in vivo, thereby defining a transcriptional switch controlling disease progression. Conversely, preventing β-catenin activation post-pressure-overload results in a downregulation of these novel TCF7L2-targets and rescues cardiac function. Thus, we present a novel role for TCF7L2/β-catenin in CMs-specific chromatin modulation, which could be exploited for manipulating the ubiquitous Wnt pathway.
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http://dx.doi.org/10.1093/nar/gky049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887416PMC
April 2018

T helper cells with specificity for an antigen in cardiomyocytes promote pressure overload-induced progression from hypertrophy to heart failure.

Sci Rep 2017 11 22;7(1):15998. Epub 2017 Nov 22.

Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.

We investigated whether CD4-T cells with specificity for an antigen in cardiomyocytes promote the progression from hypertrophy to heart failure in mice with increased pressure load due to transverse aortic constriction (TAC). OT-II mice expressing a transgenic T cell receptor (TCR) with specificity for ovalbumin (OVA) on CD4-T cells and cMy-mOVA mice expressing OVA on cardiomyocytes were crossed. The resulting cMy-mOVA-OT-II mice did not display signs of spontaneous autoimmunity despite the fact that their OVA-specific CD4-T cells were not anergic. After TAC, progression to heart failure was significantly accelerated in cMy-mOVA-OT-II compared to cMy-mOVA mice. No OVA-specific antibodies were induced in response to TAC in cMy-mOVA-OT-II mice, yet more CD3 T cells infiltrated their myocardium when compared with TAC-operated cMy-mOVA mice. Systemically, the proportion of activated CD4-T cells with a Th and Th cytokine profile was increased in cMy-mOVA-OT-II mice after TAC. Thus, T helper cells with specificity for an antigen in cardiomyocytes can directly promote the progression of heart failure in response to pressure overload independently of autoantibodies.
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http://dx.doi.org/10.1038/s41598-017-16147-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700082PMC
November 2017

The contractile adaption to preload depends on the amount of afterload.

ESC Heart Fail 2017 11 19;4(4):468-478. Epub 2017 Apr 19.

Department of Cardiology and Pneumology, Georg-August-University, Goettingen, Germany.

Aims: The Frank-Starling mechanism (rapid response (RR)) and the secondary slow response (SR) are known to contribute to increases contractile performance. The contractility of the heart muscle is influenced by pre-load and after-load. Because of the effect of pre-load vs. after-load on these mechanisms in not completely understood, we studied the effect in isolated muscle strips.

Methods And Results: Progressive stretch lead to an increase in shortening/force development under isotonic (only pre-load) and isometric conditions (pre- and after-load). Muscle length with maximal function was reached earlier under isotonic (L ) compared with isometric conditions (L ) in nonfailing rabbit, in human atrial and in failing ventricular muscles. Also, SR after stretch from slack to L was comparable under isotonic and isometric conditions (human: isotonic 10 ± 4%, isometric 10 ± 4%). Moreover, a switch from isotonic to isometric conditions at L showed no SR proving independence of after-load. To further analyse the degree of SR on the total contractile performance at higher pre-load muscles were stretched from slack to 98% L under isotonic conditions. Thereby, the SR was 60 ± 9% in rabbit and 51 ± 14% in human muscle strips.

Conclusions: This work shows that the acute contractile response largely depends on the degree and type of mechanical load. Increased filling of the heart elevates pre-load and prolongs the isotonic part of contraction. The reduction in shortening at higher levels of pre-load is thereby partially compensated by the pre-load-induced SR. After-load shifts the contractile curve to a better 'myofilament function' by probably influencing thin fibers and calcium sensitivity, but has no effect on the SR.
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http://dx.doi.org/10.1002/ehf2.12164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695189PMC
November 2017

Cardiomyocyte proliferation prevents failure in pressure overload but not volume overload.

J Clin Invest 2017 12 30;127(12):4285-4296. Epub 2017 Oct 30.

Department of Cardiology and Pneumology, Heart Center, Georg-August-University, Goettingen, Germany.

Induction of the cell cycle is emerging as an intervention to treat heart failure. Here, we tested the hypothesis that enhanced cardiomyocyte renewal in transgenic mice expressing cyclin D2 would be beneficial during hemodynamic overload. We induced pressure overload by transthoracic aortic constriction (TAC) or volume overload by aortocaval shunt in cyclin D2-expressing and WT mice. Although cyclin D2 expression dramatically improved survival following TAC, it did not confer a survival advantage to mice following aortocaval shunt. Cardiac function decreased following TAC in WT mice, but was preserved in cyclin D2-expressing mice. On the other hand, cardiac structure and function were compromised in response to aortocaval shunt in both WT and cyclin D2-expressing mice. The preserved function and improved survival in cyclin D2-expressing mice after TAC was associated with an approximately 50% increase in cardiomyocyte number and exaggerated cardiac hypertrophy, as indicated by increased septum thickness. Aortocaval shunt did not further impact cardiomyocyte number in mice expressing cyclin D2. Following TAC, cyclin D2 expression attenuated cardiomyocyte hypertrophy, reduced cardiomyocyte apoptosis, fibrosis, calcium/calmodulin-dependent protein kinase IIδ phosphorylation, brain natriuretic peptide expression, and sustained capillarization. Thus, we show that cyclin D2-induced cardiomyocyte renewal reduced myocardial remodeling and dysfunction after pressure overload but not after volume overload.
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http://dx.doi.org/10.1172/JCI81870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707145PMC
December 2017

Echocardiographic evaluation of diastolic function in mouse models of heart disease.

J Mol Cell Cardiol 2018 01 19;114:20-28. Epub 2017 Oct 19.

King's College London British Heart Foundation Centre of Excellence, Cardiovascular Division, London, United Kingdom. Electronic address:

Background: Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice.

Methods And Results: We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate.

Conclusion: These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.
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http://dx.doi.org/10.1016/j.yjmcc.2017.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807035PMC
January 2018

Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair.

Circulation 2017 May 6;135(19):1832-1847. Epub 2017 Feb 6.

From Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wettwer, W.-H.Z.); German Center for Cardiovascular Research (DZHK), partner site Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wingender, W.A.L., W.-H.Z.); Institute of Bioinformatics, University Medical Center Göttingen, Germany (S.Z., E. Wingender); Stanford Cardiovascular Institute (J.R., M.W., J.D.G., J.C.W.) and Department of Radiology (J.D.G., J.C.W.), Molecular Imaging Program, Stanford University School of Medicine, CA; The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Technion-Israel Institute of Technology, Haifa (I.K., L.G.); Institute of Pharmacology and Toxicology, Technical University Dresden, Germany (E. Wettwer, U.R.); University Medical Center Utrecht and Hubrecht Institute, The Netherlands (P.D., L.W.v.L.); Leiden University Medical Center, The Netherlands (M.J.G.); Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Germany (S.K., K.T., G.H., W.A.L.); Center for Applied Technology, Beckman Research Institute, City of Hope, Duarte, CA (L.A.C.); Department of Cardiovascular Physiology, Institute of Physiology, Ruhr University Bochum, Bochum, Germany (A.U., W.A.L.); New Laura and Isaac Perlmutter Cancer Center at New York University Langone (T.A., B.N.); and McEwen Centre for Regenerative Medicine, Toronto, Canada (G.K.). The current address for Dr Hudson is Laboratory for Cardiac Regeneration, School of Biomedical Sciences, The University of Queensland, Australia.

Background: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions.

Methods: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM.

Results: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β- and β-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair.

Conclusions: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.024145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501412PMC
May 2017

Enhanced cardiac TBC1D10C expression lowers heart rate and enhances exercise capacity and survival.

Sci Rep 2016 Sep 26;6:33853. Epub 2016 Sep 26.

Department of Cardiology and Pulmonology, Georg-August-University, Robert-Koch Str. 40, 37075 Göttingen, Germany.

TBC1D10C is a protein previously demonstrated to bind and inhibit Ras and Calcineurin. In cardiomyocytes, also CaMKII is inhibited and all three targeted enzymes are known to promote maladaptive cardiomyocyte hypertrophy. Here, in accordance with lack of Calcineurin inhibition in vivo, we did not observe a relevant anti-hypertrophic effect despite inhibition of Ras and CaMKII. However, cardiomyocyte-specific TBC1D10C overexpressing transgenic mice exhibited enhanced longevity. Ejection fraction and exercise capacity were enhanced in transgenic mice, but shortening of isolated cardiomyocytes was not increased. This suggests longevity resulted from enhanced cardiac performance but independent of cardiomyocyte contractile force. In further search for mechanisms, a transcriptome-wide analysis revealed expressional changes in several genes pertinent to control of heart rate (HR) including Hcn4, Scn10a, Sema3a and Cacna2d2. Indeed, telemetric holter recordings demonstrated slower atrial conduction and significantly lower HR. Pharmacological reduction of HR was previously demonstrated to enhance survival in mice. Thus, in addition to inhibition of stress signaling, TBC1D10C economizes generation of cardiac output via HR reduction, enhancing exercise capacity and survival. TBC1D10C may be a new target for HR reduction and longevity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036039PMC
http://dx.doi.org/10.1038/srep33853DOI Listing
September 2016

Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure.

Eur Heart J 2017 02;38(5):362-372

Division of Molecular and Translational Cardiology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany.

Aims: Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia.

Methods And Results: Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content.

Conclusions: ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.
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http://dx.doi.org/10.1093/eurheartj/ehw333DOI Listing
February 2017

Proteomic analysis of short-term preload-induced eccentric cardiac hypertrophy.

J Transl Med 2016 05 27;14(1):149. Epub 2016 May 27.

Department of Cardiology and Pneumology, University Medical Center, Goettingen, Germany.

Background: Hemodynamic load leads to cardiac hypertrophy and heart failure. While afterload (pressure overload) induces concentric hypertrophy, elevation of preload (volume overload) yields eccentric hypertrophy and is associated with a better outcome. Here we analysed the proteomic pattern of mice subjected to short-term preload.

Methods And Results: Female FVB/N mice were subjected to aortocaval shunt-induced volume overload that leads to an eccentric hypertrophy (left ventricular weight/tibia length +31 %) with sustained systolic heart function at 1 week after operation. Two-dimensional gel electrophoresis (2-DE) followed by mass spectrometric analysis showed alteration in the expression of 25 protein spots representing 21 different proteins. 64 % of these protein spots were up-regulated and 36 % of the protein spots were consistently down-regulated. Interestingly, α-1-antitrypsin was down-regulated, indicating higher elastin degradation and possibly contributing to the early dilatation. In addition to contractile and mitochondrial proteins, polymerase I and transcript release factor protein (PTRF) was also up-regulated, possibly contributing to the preload-induced signal transduction.

Conclusions: Our findings reveal the proteomic changes of early-stage eccentric myocardial remodeling after volume overload. Induced expression of some of the respiratory chain enzymes suggests a metabolic shift towards an oxidative phosphorylation that might contribute to the favorable remodeling seen in early VO. Down-regulation of α-1-antitrypsin might contribute to extracellular matrix remodeling and left ventricular dilatation. We also identified PTRF as a potential signaling regulator of volume overload-induced cardiac hypertrophy.
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http://dx.doi.org/10.1186/s12967-016-0898-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884361PMC
May 2016