Mirko Völkers

Identification of Cardiac Troponin I Sequence Motifs Leading to Heart Failure by Induction of Myocardial Inflammation and Fibrosis

Background— Despite the widespread use of cardiac troponins for diagnosis of myocyte injury and risk stratification in acute cardiac disorders, little is known about the long-term effects of the released troponins on cardiac function. Recently, we showed that an autoimmune response to cardiac troponin I (cTnI) induces severe inflammation and subsequent fibrosis in the myocardium. This autoimmune disorder predisposes to heart failure and cardiac death in mice. Methods and Results— To investigate the role of cTnI-specific T cells, T cells were isolated from splenocytes of mice immunized with murine cTnI (mcTnI). Wild-type mice that received mcTnI-specific T cells showed high mcTnI-specific antibody titers, increased production of the proinflammatory cytokines interleukin-1&bgr; and tumor necrosis factor-&agr;, severe inflammation and fibrosis in the myocardium, and reduced fractional shortening. To identify the antigenic determinants of troponin I responsible for the observed inflammation, fibrosis, and heart failure, 16 overlapping 16mer to 18mer peptides covering the entire amino acid sequence of mcTnI (211 residues) were synthesized. Only mice immunized with residues 105 to 122 of mcTnI developed significant inflammation and fibrosis in the myocardium, with increased expression of the inflammatory chemokines RANTES, monocyte chemotactic protein-1, macrophage inflammatory protein-1&agr;, macrophage inflammatory protein-1&bgr;, macrophage inflammatory protein-2, T-cell activation-3, and eotaxin and the chemokine receptors CCR1, CCR2, and CCR5. Mice immunized with the corresponding human cTnI residues 104 to 121 and the mcTnI residues 131 to 148 developed milder disease. Conclusions— Transfer of troponin I–specific T cells can induce inflammation and fibrosis in wild-type mice, which leads to deterioration of contractile function. Furthermore, 2 sequence motifs of cTnI that induce inflammation and fibrosis in the myocardium are characterized.

S100A1 is released from ischemic cardiomyocytes and signals myocardial damage via Toll-like receptor 4.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll‐like receptor 4 (TLR4)‐dependent activation of MAP kinases and NF‐κB. CFs exposed to S100A1 assumed an immunomodulatory and anti‐fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule‐1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody‐mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post‐MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI‐related inflammation and repair warrants further investigation.

Identification of circular RNAs with host gene-independent expression in human model systems for cardiac differentiation and disease

AIMSnCardiovascular disease, one of the most common causes of death in western populations, is characterized by changes in RNA splicing and expression. Circular RNAs (circRNA) originate from back-splicing events, which link a downstream 5 splice site to an upstream 3 splice site. Several back-splicing junctions (BSJ) have been described in heart biopsies from human, rat and mouse hearts (Werfel et al., 2016; Jakobi et al., 2016 ). Here, we use human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to identify circRNA and host gene dynamics in cardiac development and disease. In parallel, we explore candidate interactions of selected homologs in mouse and rat via RIP-seq experiments.nnnMETHODS AND RESULTSnDeep RNA sequencing of cardiomyocyte development and β-adrenergic stimulation uncovered 4518 circRNAs. The set of circular RNA host genes is enriched for chromatin modifiers and GTPase activity regulators. RNA-seq and qRT-PCR data showed that circular RNA expression is highly dynamic in the hiPSC-CM model with 320 circRNAs showing significant expression changes. Intriguingly, 82 circRNAs are independently regulated to their host genes. We validated the same circRNA dynamics for circRNAs from ATXN10, CHD7, DNAJC6 and SLC8A1 in biopsy material from human dilated cardiomyopathy (DCM) and control patients. Finally, we could show that rodent homologs of circMYOD, circSLC8A1, circATXN7 and circPHF21A interact with either the ribosome or Argonaute2 protein complexes.nnnCONCLUSIONnCircRNAs are dynamically expressed in a hiPSC-CM model of cardiac development and stress response. Some circRNAs show similar, host-gene independent expression dynamics in patient samples and may interact with the ribosome and RISC complex. In summary, the hiPSC-CM model uncovered a new signature of potentially disease relevant circRNAs which may serve as novel therapeutic targets.

S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction

Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1s cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and β-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.

Central role of PKCα in isoenzyme-selective regulation of cardiac transient outward current Ito and Kv4.3 channels

The transient outward current I(to) is an important determinant of the early repolarization phase. I(to) and its molecular basis Kv4.3 are regulated by adrenergic pathways including protein kinase C. However, the exact regulatory mechanisms have not been analyzed yet. We here analyzed isoenzyme specific regulation of Kv4.3 and I(to) by PKC. Kv4.3 channels were expressed in Xenopus oocytes and currents were measured with double electrode voltage clamp technique. Patch clamp experiments were performed in isolated rat cardiomyocytes. Unspecific PKC stimulation with PMA resulted in a reduction of Kv4.3 current. Similar effects could be observed after activation of conventional PKC isoforms by TMX. Both effects were reversible by pharmacological inhibition of the conventional PKC isoenzymes (Gö6976). In contrast, activation of the novel PKC isoforms (ingenol) did not significantly affect Kv4.3 current. Whereas TMX-induced PKC activation was not attenuated inhibition of PKCβ, inhibition of PKCα with HBDDE prevented inhibitory effects of both PMA and TMX. Accordingly, stimulatory effects of PMA and TMX could be mimicked by the α-isoenzyme selective PKC activator iripallidal. Further evidence for the central role of PKCα was provided with the use of siRNAs. We found that PKCα siRNA but not PKCβ siRNA abolished the TMX induced effect. In isolated rat cardiomyocytes, PMA dependent I(to) reduction could be completely abolished by pharmacologic inhibition of PKCα. In summary we show that PKCα plays a central role in protein kinase C dependent regulation of Kv4.3 current and native I(to). These results add to the current understanding of isoenzyme selective ion channel regulation by protein kinases.

A proteolytic fragment of histone deacetylase 4 protects the heart from failure by regulating the hexosamine biosynthetic pathway

The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

Genomic structural variations lead to dysregulation of important coding and non-coding RNA species in dilated cardiomyopathy

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non‐coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome‐wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non‐protein‐coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non‐coding RNAs and 1,756 protein‐coding transcripts. 65.3% of the SV‐eQTLs do not harbor a significant SNV‐eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein‐coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike.

Inhibition of Cardiac Kir Current (IK1) by Protein Kinase C Critically Depends on PKCβ and Kir2.2.

Background Cardiac inwardly rectifying Kir current (IK1) mediates terminal repolarisation and is critical for the stabilization of the diastolic membrane potential. Its predominant molecular basis in mammalian ventricle is heterotetrameric assembly of Kir2.1 and Kir2.2 channel subunits. It has been shown that PKC inhibition of IK1 promotes focal ventricular ectopy. However, the underlying molecular mechanism has not been fully elucidated to date. Methods and Results In the Xenopus oocyte expression system, we observed a pronounced PKC-induced inhibition of Kir2.2 but not Kir2.1 currents. The PKC regulation of Kir2.2 could be reproduced by an activator of conventional PKC isoforms and antagonized by pharmacological inhibition of PKCβ. In isolated ventricular cardiomyocytes (rat, mouse), pharmacological activation of conventional PKC isoforms induced a pronounced inhibition of IK1. The PKC effect in rat ventricular cardiomyocytes was markedly attenuated following co-application of a small molecule inhibitor of PKCβ. Underlining the critical role of PKCβ, the PKC-induced inhibition of IK1 was absent in homozygous PKCβ knockout-mice. After heterologous expression of Kir2.1-Kir2.2 concatemers in Xenopus oocytes, heteromeric Kir2.1/Kir2.2 currents were also inhibited following activation of PKC. Conclusion We conclude that inhibition of cardiac IK1 by PKC critically depends on the PKCβ isoform and Kir2.2 subunits. This regulation represents a potential novel target for the antiarrhythmic therapy of focal ventricular arrhythmias.