Anthracyclines Induce Calpain-dependent Titin Proteolysis and Necrosis in Cardiomyocytes

Abstract

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.

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin

“…The mechanism of this type of cellular injury is likely multifactorial including the formation of reactive oxygen species, elevation of cytoplasmic Ca 2? concentration and activation of intracellular proteases as well as inhibition of the mTOR signaling pathway and the cardiac transcription factor GATA4 (Lim et al 2004;Pentassuglia et al 2009;Timolati et al 2006;Zhu et al 2009). We have used myofibrillar changes as a marker for cancer therapy-associated cardiotoxicity with different classes of drugs such as paclitaxel, anti-ErbB2 antibodies, MEK-inhibitors and small tyrosine kinase inhibitors for ErbB1/ErbB2 (Pentassuglia et al 2007(Pentassuglia et al , 2009Sawyer et al 2002).…”

“…A well-studied example of cardiotoxicity is cancer therapy with anthracyclines, for instance with doxorubicin (Doxo), which can be associated with early cardiomyopathy as well as late-onset ventricular dysfunction (Lipshultz et al 1991;Bristow et al 1978). Possible mechanisms of anthracycline-induced cardiotoxicity are alterations in cellular structure (Lim et al 2004), induction of apoptosis (Sawyer et al 1999), disruption of energy production (Tokarska-Schlattner et al 2005) and formation of reactive oxygen species (ROS) (Doroshow and Davies 1986). Anthracyclines have been reported to induce structural changes and cell death in the myocardium of treated patients, and the extent of structural damage has been correlated with early cardiac dysfunction .…”

“…Isolated ventricular cardiomyocytes or muscle strips from adult animals in serum-free conditions allow investigations on electric properties, contractility and other functions, but only for relatively short time. On the other hand, adult rat ventricular cardiomyocytes (ARVM) forming a monolayer of redifferentiated, contracting cells in serum-containing media can be maintained for several weeks and allow studies on cytoskeleton remodeling, metabolic changes and longterm effects of toxic agents and signaling modulators (Lim et al 2004;Pentassuglia et al 2009;Eppenberger-Eberhardt et al 1997;Suzuki et al 2001).…”

Rodent models of heart failure: an updated review

“…Other studies suggest that cardiotoxicity pathways include abnormalities in Ca 2? handling [50,54,285]; induction of mitochondrial DNA lesions [149]; degradation of myofilamental and cytoskeletal proteins, including titin [160] and dystrophin [40]; interference with various pro-survival kinases [224]; and changes in adrenergic and adenylate cyclase function [33,74]. These examples of a much larger set of proposed cardiotoxic mechanisms are not mutually exclusive: they may each contribute to cardiac cell damage, ultimately leading to myocyte death, by either necrosis or apoptosis [254].…”

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity

“…(3) inhibition of cardiac specific muscle gene transcription and translation, in combination with an increase in myofibril protein degradation, leading to loss of myofibrils (Lewis and Gonzalez, 1987;Ito et al, 1990;Kurabayashi et al, 1994;Toyoda et al, 1998;d'Anglemont de Tassigny et al, 2004;Lim et al, 2004b), and (4) disturbance of intracellular calcium homeostasis (De Beer et al, 2001;Wallace, 2003). The mechanism of doxorubicin-induced free radical generation and oxidative stress has been reviewed in other chapters of this book as well as comprehensive reviews in the field (Singal et al, 2000;Berthiaume and Wallace, 2007;Simunek et al, 2009).…”