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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 mol/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 springlike domain of titin. Doxorubicin treatment for 1 h resulted in ϳ3-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.

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␤ (NRG-1␤), 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 anthracyclineinduced 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 mol/L) showed a concentrationdependent increase in myofilament disarray. NRG-1␤ (10 ng/mL) activated erbB2, Erk1/2, and Akt in ARVMs and significantly reduced anthracycline-induced disarray. In contrast to NRG-1␤, anti-erbB2 (1 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. Key Words: erbB2 Ⅲ cardiotoxicity Ⅲ neuregulins Ⅲ anthracyclines Ⅲ myocytes T reatment of metastatic breast cancer with anthracyclines and trastuzumab, a novel therapy derived from an antibody to the erbB2 receptor tyrosine kinase, results in a marked increase of left ventricular dysfunction and symptomatic heart failure. 1 ErbB2 is a member of the epidermal growth factor receptor family, and along with neuregulin and the erbB4 receptor, it plays an essential role in cardiac development. [2][3][4] We have shown that recombinant neuregulin 1␤ (NRG-1␤) activates both erbB2 and erbB4 receptor tyrosine kinase activity and promotes growth, myofilament organization, and survival of isolated cardiac myocytes. 5,6 The clinical observation of the cardiotoxicity of trastuzumab and anthracyclines suggests that the neuregulin/erbB system modulates the response of the myocardium to anthracyclines. Possible mechanisms for this toxicity are alterations in the structure, 7 gene expression, 8 and survival 9 of cardiac myocytes. The main purpose of this study was to test the hypothesis that trastuzumab alters the susceptibility of myocytes to anthracycline-induced myofibrillar disarray. We therefore characterized the effect of anthracyclines on myocyte myofibrillar structure in isolated adult rat cardiac myocytes in primary culture and examined the effect of NRG-1␤ and an antibody to erbB2 with properties similar to trastuzumab 10 on erbB2 signaling and anthracycline-induced changes in myofibrillar structure. Conclusions Methods Chemicals...

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain– containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.

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