Hans M. Eppenberger

Modulation of Anthracycline-Induced Myofibrillar Disarray in Rat Ventricular Myocytes by Neuregulin-1β and Anti-erbB2 Potential Mechanism for Trastuzumab-Induced Cardiotoxicity

Background—There is an increased incidence of heart failure in patients treated concurrently with anthracyclines and the chemotherapeutic anti-erbB2 agent trastuzumab (Herceptin). On the basis of our previous studies with recombinant neuregulin-1&bgr; (NRG-1&bgr;), a ligand for the erbB2 receptor tyrosine kinase, we hypothesized that activation of erbB2 by anti-erbB2 versus NRG-1 would cause differential effects on myocyte intracellular signaling as well as anthracycline-induced myofibrillar injury and might potentially account for the clinical toxicity of trastuzumab in the setting of concurrent anthracycline therapy. Methods and Results—We tested this hypothesis using adult rat ventricular myocytes (ARVMs) in culture, assessing myofibrillar structure by immunostaining for myomesin and filamentous actin. Activation of erbB2, extracellular signal–regulated kinase 1/2 (Erk1/2), and Akt was assessed by use of antibodies to phosphorylated activated receptor or kinase detected by immunoblot. ARVMs treated with doxorubicin (0.1 to 0.5 &mgr;mol/L) showed a concentration-dependent increase in myofilament disarray. NRG-1&bgr; (10 ng/mL) activated erbB2, Erk1/2, and Akt in ARVMs and significantly reduced anthracycline-induced disarray. In contrast to NRG-1&bgr;, anti-erbB2 (1 &mgr;g/mL) caused rapid phosphorylation of erbB2 but not Erk1/2 or Akt, with downregulation of erbB2 by 24 hours. Concomitant treatment of myocytes with anti-erbB2 and doxorubicin caused a significant increase in myofibrillar disarray versus doxorubicin alone. Conclusions—NRG-1&bgr;/erbB signaling regulates anthracycline-induced myofilament injury. The increased susceptibility of myofilaments to doxorubicin in the presence of antibody to erbB2 may explain the contractile dysfunction seen in patients receiving concurrent trastuzumab and anthracyclines.

The ontogeny of creatine kinase isozymes

Abstract The ontogeny of creatine kinase isozymes in the rat brain, heart, and skeletal muscle and the chicken skeletal muscle has been studied by agar gel electrophoresis and visualization of the enzyme by an optical test. In adult animals three creatine kinase isozymes are observed: isozyme I (brain) migrating toward the anode, isozyme III (skeletal muscle and heart) migrating toward the cathode, and isozyme II (heart) with an intermediate electrophoretic mobility. In the early stages of ontogeny only isozyme I is present in all organs investigated. In skeletal muscle isozyme I slowly disappears and is replaced first by isozyme II and finally by isozyme III. In the intermediary stages, mixtures of isozymes are present. The adult pattern is reached about 90 days after birth in the rat and at hatching in the chicken. In heart muscle a similar shift is observed as in skeletal muscle, but the changes start earlier and remain stationary at the (isozyme II + isozyme III) stage. In the brain isozyme I remains the only isozyme throughout life. The changes in the isozyme pattern in skeletal muscle during ontogeny are correlated with simultaneous morphological, biochemical, and physiological events. Possibly, active creatine kinase is a dimer. Two types of monomers (B or brain type and M or muscle type) may combine to form three species of active enzymes: BB, BM, and MM.

Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 μmol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in ∼3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.

Various hypertrophic stimuli induce distinct phenotypes in cardiomyocytes.

Abstract Cardiac hypertrophy is characterized by an increase in cell size in the absence of cell division and is accompanied by a number of qualitative and quantitative changes in gene expression. Most forms of hypertrophy in vivo are compensatory or adaptative responses to increased workload resulting from various physiological and/or pathological etiologies. Until severe pathological alterations become apparent, myocytes show no drastic morphological changes. On the level of gene expression, upregulation of the so-called fetal genes, i.e., β-myosin heavy chain, α-skeletal and α-smooth muscle actin, and atrial natriuretic factor (ANF) may be observed concomitant with a downregulation of α-myosin heavy chain and the Ca pump of sarcoplasmic reticulum. The use of primary cell culture systems for cardiomyocytes as an in vitro model for the hypertrophic reaction has identified a number of different stimuli as mediators of cardiac myocyte hypertrophy. The molecular dissection of the different intracellular signaling pathways involved herein has uncovered a number of branching points to cytosolic and nuclear targets and has identified many interactions between these pathways. The individual administration of these hypertrophic stimuli, i.e., hormones, cytokines, growth factors, vasoactive peptides, and catecholamines, to cultured cardiomyocytes, reveals that each stimulus induces a distinct phenotype as characterized by gene expression pattern and cellular morphology. Surprisingly, triiodothyronine (T3) and basic fibroblast growth factor (bFGF) effect a similar cellular phenotype although they use completely different intracellular pathways. This phenotype is characterized by drastic inhibition of myofibrillar growth and by upregulation of α-smooth muscle actin. On the other hand, insulin-like growth factor (IGF) I, a factor promoting muscle cell differentiation, and bFGF, an inhibitor of differentiation, cause completely different cardiomyocyte phenotypes although both are known to signal via receptor tyrosine kinases and have been shown to activate the Ras-Raf-MEK-MAP kinase pathway. However, both IGF-I and bFGF depend on T3 to bring about their typical responses, i.e., T3 is permissive for the action of these two growth factors on the expression of α-smooth muscle actin and cell morphology. Most of the hypertrophic stimuli are balanced under normal circumstances in vivo. When this balance is disturbed, however, a pathological heart phenotype may become dominant. Thus the knowledge of signaling pathways and cellular responses triggered by hypertrophic stimuli may be essential for the implementation of therapeutic strategies in the treatment of cardiac hypertrophy.

Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells

Abstract Changes in the isoenzyme patterns and activities of the two enzymes creatine kinase (CPK) and fructose diphosphate aldolase have been followed during the course of differentiation of chick skeletal muscle cells in vitro. The characteristic isoenzyme transitions of both of these enzymes known to occur in developing muscle in situ can be demonstrated in extracts of cultured myogenic cells by cellulose polyacetate electrophoresis followed by specific enzymatic staining: MM-CPK replaces the embryonic BB-CPK, while aldolase isoenzymes containing A subunits replace the C-containing forms which predominate at earlier stages. The specific activities of both enzymes increase during in vitro differentiation. Although the major part of these concomitant changes occurs after myoblast fusion has reached a maximum level, analysis of their timing relative to the process of fusion indicates that the increases in the activities of both enzymes, as well as the accumulation of nuclei within myotubes, proceed exponentially from the beginning of the second day in culture. Fusion and enzyme accumulation are unaffected by addition of dibutyryl cyclic AMP (1 × 10−4M) to the medium. In calcium-deficient medium, or in media containing 5-bromodeoxyuridine (BrdUrd) at concentrations from 0.2 to 7 × 10−5M, fusion is almost completely blocked, while cell viability is maintained. The CPK and aldolase isoenzyme transitions fail to occur normally in both fusion-preventing media. This blockage of the normal differentiative changes is, however, less complete in the calcium-deficient cultures, which, in contrast to the BrdUrd containing cultures, contained a number of long bipolar cells thought to be able to differentiate without fusion. These results are interpreted as indicating that for most, but possibly not for all, myogenic cells in typical primary muscle cell cultures, fusion is a prerequisite for the parallel differentiative changes in CPK and aldolase isoenzymes. The possibility is discussed that a “cluster” of proteins, including CPK and aldolase, may be coordinately regulated during myogenesis.

Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle.

Purified, repeatedly washed, skeletal muscle myofibrils contain approx. 0.2 U of creatine kinase (CK) activity (equivalent to 2.5 micrograms CK) per milligram dry weight; this firmly bound CK activity is estimated to represent 3-5% of the total cellular CK. It had been shown previously that the myofibrillar CK, which can be quantitatively extracted at low ionic strength and purified to homogeneity, is very similar, if not identical, to the bulk MM-CK. It is shown that the two protein preparations also have the same peptide pattern after cyanogen bromide fractionation and very similar specific activities, confirming their identity. The earlier demonstration that the bound CK is specifically located at the M-lines of isolated myofibrils has been confirmed by immunofluorescence. Antibodies directed against purified MM- and BB-CK were used in the indirect fluorescent antibody technique to study the specificity of myofibril binding sites for different forms of CK. With myofibrils from adult muscle, which has only MM-CK, as well as from early developmental stages in which BB-CK is the predominant isoenzyme, M-type CK was localized exclusively at the M-line, while greater or lesser amounts of B-type CK were found at the Z-line. The data provide strong evidence that the MM-CK at the M-lines in skeletal myofibrils is not adventitiously bound but is rather an integral element in the M-line structure. The amount of CK bound is reasonably consistent with the earlier proposal that the CK molecules might be the transverse M-bridges and appears to be sufficient to regenerate all of the ATP hydrolyzed during muscle contraction.

Myosin types in human skeletal muscle fibers.

SummaryBy combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.

Reexpression of α-smooth muscle actin isoform in cultured adult rat cardiomyocytes☆

Abstract Expression of α-smooth muscle (sm) actin in regenerating adult cardiomyocytes in culture was investigated. No α-sm-actin could be detected in adult ventricular tissue or in newly dissociated rod-shaped cells, whereas a fraction of the polymorphic flattened out adult cardiac cells in culture did express the protein. Immunofluorescence studies revealed a characteristic staining pattern, suggesting the preferential presence of α-sm-actin in stress fiber-like structures, while newly formed myofibrils contained only little α-sm-actin isoprotein. Cell-cell contacts were resumed, but formation of new gap junctions, as revealed by microinjecting Lucifer yellow, was not dependent on α-sm-actin expression. The behavior corresponds to fetal cardiomyocytes either in tissue or as single cells in culture where expression of α-sm-actin can be observed. Such immunofluorescence staining patterns with corresponding immunoblot data can be expected when a return to a less differentiated, more fetal state of the adult cardiomyocyte in culture is assumed. The possible role of the α-sm-actin and α-sarcomeric actin isoforms during reformation of myofibrillar sarcomeres is discussed.

Muscle morphogenesis: Evidence for an organizing function of exogenous fibronectin.

Abstract Chicken myoblasts and myotubes attach to, and elongate upon, solid substrates only in the presence of exogenous fibronectin derived from, for example, serum or fibroblasts ( M. Chiquet, E. C. Puri, and D. C. Turner, 1979 , J. Biol. Chem.254, 5475–5482). We have sought answers to two questions: (1) whether myoblasts depend on exogenous fibronectin for attachment because they themselves synthesize none or too little of this glycoprotein; and (2) whether interaction of myogenic cells with fibroblast-derived fibronectin might guide muscle morphogenesis. Under conditions in which fibroblasts synthesized fibronectin and released it into the medium, myoblasts and myotubes synthesized and accumulated only small amounts of fibronectin. Although proliferating myogenic cells treated with 5-bromodeoxyuridine accumulate fibronectin on their surfaces, other evidence suggests that normal myogenic precursor cells, like myoblasts and myotubes, synthesize little fibronectin: (1) the accumulation of newly synthesized fibronectin in untreated primary myogenic cultures is attributable, at least in part, to the fibroblasts present; and (2) 1-week-old myogenic clones fail to form a fibronectin network, in contrast to fibrogenic clones of the same age and cell density. In cryosections of chick embryos, less fibronectin was found in the premuscle masses than in the surrounding mesenchyme. The splitting of the muscle anlagen in the limb bud was correlated with the appearance of fibronectin-accumulating cells in the cleavage furrows. In vitro, myoblasts displayed a marked tendency to align themselves along oriented fibrils or streaks of purified fibronectin that had been deposited on the substrate; this myoblast alignment influenced shape and branching of the developing myotubes. These results suggest that formation of myotubes in a certain spatial arrangement during muscle morphogenesis may be regulated by a fibronectin-containing matrix produced by connective tissue cells.

Mass Production of Embryoid Bodies in Microbeads

Abstract: Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types. Stem cell‐derived, differentiated cells are of increasing importance as a potential source for non‐proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications. Differentiation of ESC is initiated by the formation of embryoid bodies (EB). Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state. To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies. Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas. However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula‐like stage, so that no cystic EB can be formed within the beads. ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes. Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.