Actin binding GFP allows 4D in vivo imaging of myofilament dynamics in the zebrafish heart and the identification of Erbb2 signaling as a remodeling factor of myofibril architecture

Abstract

Rationale: Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date.

Objective: To image myocardial contractile filament dynamics in vivo and to assess potential causes of dilated cardiomyopathy in antineoplastic therapies targeting the epidermal growth factor receptor Erbb2.

Methods and results: We generated a transgenic zebrafish line expressing an actin-binding green fluorescent protein in cardiomyocytes, allowing an in vivo imaging of myofilaments. Analysis of this line revealed architectural differences in myofibrils of the distinct cardiomyocyte subtypes. We used this model to investigate the effects of Erbb2 signaling on myofibrillar organization because drugs targeting ERBB2 (HER2/NEU) signaling, a mainstay of breast cancer chemotherapy, cause dilated cardiomyopathy in many patients. High-resolution in vivo imaging revealed that Erbb2 signaling regulates a switch between a dense apical network of filamentous myofibrils and the assembly of basally localized myofibrils in ventricular cardiomyocytes.

Conclusions: Using this novel line, we compiled a reference for myofibrillar microarchitecture among myocardial subtypes in vivo and at different developmental stages, establishing this model as a tool to analyze in vivo cardiomyocyte contractility and remodeling for a broad range of cardiovascular questions. Furthermore, we applied this model to study Erbb2 signaling in cardiomyopathy. We show a direct link between Erbb2 activity and remodeling of myofibrils, revealing an unexpected mechanism with potentially important implications for prevention and treatment of cardiomyopathy.

Keywords: Erbb2 protein, human; cardiomyopathies; growth & development; heart contractility; myocardial contraction; myofibrils; sarcomeres.

Figure 1. Myofilament components are associated with inherited dilated cardiomyopathy and can be visualized using LifeAct-GFP expression in vivo

(A) Schematic representation of sarcomere units and components associated with familial cases of dilated cardiomyopathy (DCM).(B) Schematic representation of the constructs and primary sequence of the LifeAct-GFP used in this study.(C, D) Comparative imaging of skeletal muscle myofibrils fixed and stained with phalloidin (C) vs. in vivo LifeAct-GFP(D). Note that high-affinity phalloidin binds actin within the Z-disc while low-affinity LifeAct-GFP cannot access actin within the Z-disc. (E–H) Transgenic expression of LifeAct-GFP under the myocardial myl7 promoter does not cause any developmental defects. Scale bars indicate equal magnification (E,F;G,H).

Figure 2. Myofibrillar architecture changes during development

Myocardial expression of LifeAct-GFP allows the detailed in vivo analysis of myofibril architectural features at highresolution.(A–A‴) Early cardiac tube at 20 hpf, before the initiation of contractions. (A′–A‴) Optical sections through the cardiac tube reveal no signs of myofilament assembly. (B–B‴) Early cardiac tube at 22 hpf, after the initiation of contractions (individual cells outlined). (B′–B‴) Optical sections through the cardiac tube show the establishment of myofilaments starting from actin rich I-Z-I bodies (thin filament/Z-band precursors) in a highly branched fashion where I-Z-I bodies act as branch points (red arrowheads). This early contractile network strictly localizes to the basal (luminal) side of cardiomyocytes. (C) Overview of the heart during cardiac looping (36 hpf). Cardiomyocytes of the ventricular chamber (C′) exhibit cortical localization of actin-rich structures but no myofilaments are present within the center of the cell. This localization is in contrast to that observed in atrial cardiomyocytes (C″), where myofibrils span the cell in a highly organized fashion along the transverse axis of the heart. (D) 3-D reconstruction of the ventricular chamber at 4.5 dpf, i.e., after the onset of trabeculation (luminal view). See Online Movie I for entire 3-D reconstruction. (E) 3-D reconstruction of the atrial chamber (abluminal view) at 4.5 dpf. (F) Schematic of imaging procedure and mounting.

Figure 3. Clonal analysis of myofibril organization in individual myocardial subtypes at 4.5 dpf

The larval zebrafish heart contains three clearly distinguishable myocardial subtypes. (A) Clone of three atrial cardiomyocytes. (B) Abluminal surface scan of a single cardiomyocyte in the ventricular compact wall. (C) Cross-section through the ventricular lumen showing trabecular myofibers. (D) Cardiomyocytes of the ventricular wall during the process of trabeculation. While the apical (abluminal) side of ventricular wall cardiomyocytes is filled with a dense network of myofibrils (B, D′), the basal side contains thicker, cortically localized myofibrils which are clearly observable in a maximum intensity projection of the whole cell (D, white arrowhead) but not in a scan along the apical surface (D′, red arrowhead). (D″) Optical cross-sectioning through the cortical region of cardiomyocytes of the compact wall also show this polarized localization (dashed lines indicate cell-cell boundaries). Cellular protrusions (pseudocolored in red) which extend into the ventricular lumen appear entirely free of thin myofibrils (D). (E) Trabecular cardiomyocytes are tubular cells of variable length with cortically localized cell-spanning myofibrils and a central nucleus (red). At their respective ends, the myofibrils branch and connect to intercellular membranes of compact layer (CL) cardiomyocytes or adjacent trabecular cells. (F) Clonal analysis of ventricular wall cardiomyocytes, one of which (boxed) is in the process of leaving the ventricular wall. (F′) Closeup of the cardiomyocyte from (F); luminal protrusions (pseudocolored in red), the nucleus containing cell body residing in the wall (green). See Online Movie II for 3D reconstruction. (G–H) Schematic representation of individual myocardial subtypes and their typical myofibril organization and localization (nucleus in blue). (G) Atrial cardiomyocyte. (H) Ventricular wall cardiomyocyte. (I) Trabecular cardiomyocyte. These architectural features can also be observed in Online Movies IIIa and IIIb in beating hearts.

Figure 4. Myofibril organization inerbb2^−/− cardiomyocytes at different developmental stages

Confocal scans of erbb2^−/− and wild-type siblings reveal differences between density and size of myofibrils of compact wall cardiomyocytes. As described above, cardiomyocytes of the ventricular wall exhibit thin filamentous myofibrils on their apical side and more prominent myofibrils forming a cortical ring on their basal side. In erbb2^−/−, ventricular wall cardiomyocytes exhibit a reduction of thin filamentous myofibrils on their apical side while their basolateral myofibrils dramatically increase in size as can be seen in (A) and (B) at 3.5 and 4.5 dpf respectively. Surface scans at higher magnification reveal the reduction of the apical filamentous myofibrils (A′ and B′+B‴ red arrowheads) and thickening of the basolaterally located myofibrils (A″ and B″+B‴ white arrowheads) 5 μm below. (B‴) Sagital cross-sections through cells of the ventricular wall highlight this effect. (C, D) Schematic representation of myofibril localization and architecture in cardiomyocytes of the ventricular compact wall in (C) wild-type and (D) erbb2^−/−animals. Note: a comparison of a 4.5 dpf erbb2^−/− and a wild-type sibling is also available as full 4D reconstruction, 4D luminal view surface rendering and single plane imaging in Online Movies IVa+IVb, IVc+IVd and IVe+IVf respectively. (E) Measurements of ventricular fractional shortening in erbb2^−/− and sibling animals reveal a 25% decrease in cardiac performance in erbb2 mutants at 4.5 dpf.

Figure 5. Late-stage inhibition of Erbb2 signaling causes myofibril reorganization and bundling

(A) Tg(myl7:LifeAct-GFP);Tg(myl7:nDsRed) larvae where exposed to 10μM PD168393 from 4.5 to 6.5 dpf. (B–B′, C–C′) In ventricular wall cardiomyocytes, Erbb2 inhibition caused a dramatic increase inmyofibril diameter on their apical side. (D, E) Trabecular cardiomyocytes showed increased thickness of myofilament bundles and overall myofibril content.

Figure 6. Erbb2 acts cell-autonomously on myofibrillar architecture, and long-term inhibition of Erbb2 signaling causes DCM and dysregulation of DCM genes

To reduce Erbb2 signaling exclusively in the myocardium, we developed a cardiomyocyte-specific dominant negative strategy. (A–A″) Expression of the dnErbb2 receptor in clones (with coexpression of RFP) reveals cell-autonomous remodeling of myofibrillar architecture, leading to a reduction of thin myofibrils and an overrepresentation of large diameter myofibrils. This effect is most striking at the boundaries between normal and transgenic cells (arrowheads). We further created transgenic animals expressing dnErbb2 under the pan-myocardial myl7 promoter. The resulting transgenics exhibited severe edema (C; red arrowheads), decreased trabeculation (E, white arrowheads) and dilated cardiac morphology (G) compared to non-transgenic siblings (B, D, F). Using quantitative rtPCR, we tested the expression of genes associated with familial cases of DCM (H), and found a strong and consistent upregulation of several members of this gene set including those encoding components of the contractile machinery. The dashed line indicates the expression level in non-transgenic siblings.

Figure 7. Doxorubicin treatment leads to loss of myofibrils in vivo

Doxorubicin was injected into the pericardial cavity at 4.5 dpf and the larvae raised for another 24 hours. Subsequent confocal imaging revealed a significant effect on overall myofibril structure and density. While vehicle-injected control larvae showed no detectable alterations (A, A′), injection of 1 pL (4 mmol/L) doxorubicin entirely ablated fine myofibrils in compact wall cardiomyocytes and caused myofibrillar disarray in the thick myofibrils in trabecular cardiomyocytes (B, B′). A′ and B′ are also available as 3-D reconstructions (Online Movies Va and Vb respectively).