Elisabeth Krämer

Cardiac Myosin-Binding Protein C Is Required for Complete Relaxation in Intact Myocytes

The role of cardiac myosin-binding protein C (cMyBP-C) in cardiac contraction is still not fully resolved. Experimental ablation of cMyBP-C by various means resulted in inconsistent changes in Ca2+ sensitivity and increased velocity of force of skinned preparations. To evaluate how these effects are integrated in an intact, living myocyte context, we investigated consequences of cMyBP-C ablation in ventricular myocytes and left atria from cMyBP-C knock-out (KO) mice compared with wild-type (WT). At 6 weeks, KO myocytes exhibited mild hypertrophy that became more pronounced by 30 weeks. Isolated cells from KO exhibited markedly lower diastolic sarcomere length (SL) without change in diastolic Ca2+. The lower SL in KO was partly abolished by the actin-myosin ATPase inhibitors 2,3-butanedione monoxime or blebbistatin, indicating residual actin-myosin interaction in diastole. The relationship between cytosolic Ca2+ and SL showed that KO cells started to contract at lower Ca2+ without reaching a higher maximum, yielding a smaller area of the phase-plane diagram. Both sarcomere shortening and Ca2+ transient were prolonged in KO. Isolated KO left atria exhibited a marked increase in sensitivity to external Ca2+ and, in contrast to WT, continued to develop twitch force at low micromolar Ca2+. Taken together, the main consequence of cMyBP-C ablation was a defect in diastolic relaxation and a smaller dynamic range of cell shortening, both of which likely result from the increased myofilament Ca2+ sensitivity. Our findings indicate that cMyBP-C functions as a restraint on myosin-actin interaction at low Ca2+ and short SL to allow complete relaxation during diastole.

Nonsense-Mediated mRNA Decay and Ubiquitin–Proteasome System Regulate Cardiac Myosin-Binding Protein C Mutant Levels in Cardiomyopathic Mice

Rationale: Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive. Objective: The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. Methods and Results: We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype. Conclusions: These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin–proteasome system in lowering the level of mutant proteins.

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

AIMS Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. METHODS AND RESULTS Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. CONCLUSION These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca2+ sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca2+ sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca2+ transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E′/A′, and higher E/E′ ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca2+ sensitivity, faster Ca2+ transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca2+ sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca2+ transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.

Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice

Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.

The γH2AX Assay for Genotoxic and Nongenotoxic Agents: Comparison of H2AX Phosphorylation with Cell Death Response

DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if γH2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified γH2AX foci manually and automatically. In addition, total γH2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced γH2AX dose-dependently whereas nongenotoxic agents do not; (2) γH2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and γH2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to γH2AX; (3) manual scoring of γH2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that γH2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) γH2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of γH2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of γH2AX by genotoxicants is related to loss of viability and support γH2AX as a reliable bio-indicator for pretoxic DNA damage.

Repair of Mybpc3 mRNA by 5′-trans-splicing in a Mouse Model of Hypertrophic Cardiomyopathy

RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated. We evaluated the 5′-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5′-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM) carrying a FLAG-tagged wild-type (WT) Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV) for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i) the first evidence of successful 5′-trans-splicing in vivo and (ii) proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease.

A Minimal Structural Analogue of Cyclic ADP-ribose SYNTHESIS AND CALCIUM RELEASE ACTIVITY IN MAMMALIAN CELLS

Cyclic ADP-ribose (cADPR) is an endogenous Ca2+-mobilizing second messenger in many cell types and organisms. Although the biological activity of several modified analogues of cADPR has been analyzed, most of these structures were still very similar to the original molecule. Recently, we have introduced simplified analogues in which the northern ribose (N1-linked ribose) was replaced by an ether strand (Gu, X., Yang, Z., Zhang, L., Kunerth, S., Fliegert, R., Weber, K., Guse, A. H., and Zhang, L. (2004) J. Med. Chem. 47, 5674–5682). Here we also demonstrate that the southern ribose (N9-linked ribose) can be replaced by an ether strand resulting in N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthinecyclic pyrophosphate (cIDP-DE). This minimal structural analogue of cyclic ADP-ribose released Ca2+ from intracellular stores of permeabilized Jurkat T lymphocytes. In intact T lymphocytes initial subcellular Ca2+ release events, global Ca2+ release, and subsequent global Ca2+ entry were observed. Cardiac myocytes freshly prepared from mice responded to cIDP-DE by increased recruitment of localized Ca2+ signals and by global Ca2+ waves.

Differentiation of cardiomyocytes and generation of human engineered heart tissue

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)—in a strip format—that generate force under auxotonic stretch conditions. 10–15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

The E3 ubiquitin ligase Asb2β is downregulated in a mouse model of hypertrophic cardiomyopathy and targets desmin for proteasomal degradation

BACKGROUND Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease with mutations in genes encoding sarcomeric proteins. Previous findings suggest deregulation of the ubiquitin proteasome system (UPS) in HCM in humans and in a mouse model of HCM (Mybpc3-targeted knock-in (KI) mice). In this study we investigated transcript levels of several muscle-specific E3 ubiquitin ligases in KI mice and aimed at identifying novel protein targets. METHODS AND RESULTS Out of 9 muscle-specific E3 ligases, Asb2β was found with the lowest mRNA level in KI compared to wild-type (WT) mice. After adenoviral-mediated Asb2β transduction of WT neonatal mouse cardiomyocytes with either a WT or inactive Asb2β mutant, desmin was identified as a new target of Asb2β by mass spectrometry, co-immunoprecipitation and immunoblotting. Immunofluorescence analysis revealed a co-localization of desmin with Asb2β at the Z-disk of the sarcomere. Knock-down of Asb2β in cardiomyocytes resulted in higher desmin protein levels. Furthermore, desmin levels were higher in ventricular samples of HCM mice and patients than controls. CONCLUSIONS This study identifies desmin as a new Asb2β target for proteasomal degradation in cardiomyocytes and suggests that accumulation of desmin could contribute to UPS impairment in HCM mice and patients.