Carlo G. Tocchetti

Doxorubicin-induced cardiomyopathy: From molecular mechanisms to therapeutic strategies

The utility of anthracycline antineoplastic agents in the clinic is compromised by the risk of cardiotoxicity. It has been calculated that approximately 10% of patients treated with doxorubicin or its derivatives will develop cardiac complications up to 10 years after the cessation of chemotherapy. Oxidative stress has been established as the primary cause of cardiotoxicity. However, interventions reducing oxidative stress have not been successful at reducing the incidence of cardiotoxicity in patients treated with doxorubicin. New insights into the cardiomyocyte response to oxidative stress demonstrate that underlying differences between in vitro and in vivo toxicities may modulate the response to superoxide radicals and related compounds. This has led to potentially new uses for pre-existing drugs and new avenues of exploration to find better pharmacotherapies and interventions for the prevention of cardiotoxicity. However, much work still must be done to validate the clinical utility of these new approaches and proposed mechanisms. In this review, the authors have reviewed the molecular mechanisms of the pathogenesis of acute and chronic doxorubicin-induced cardiotoxicity and propose potential pharmacological interventions and treatment options to prevent or reverse this specific type of heart failure.

Compartmentalized Phosphodiesterase-2 Activity Blunts β-Adrenergic Cardiac Inotropy via an NO/cGMP-Dependent Pathway

&bgr;-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving &bgr;3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by &bgr;-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the &bgr;-adrenergic pathway. In this, activation of &bgr;3-adrenergic receptors counteracts cAMP generation obtained via stimulation of &bgr;1/&bgr;2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Reversal of Cardiac Hypertrophy and Fibrosis From Pressure Overload by Tetrahydrobiopterin: Efficacy of Recoupling Nitric Oxide Synthase as a Therapeutic Strategy

Background— Sustained pressure overload induces pathological cardiac hypertrophy and dysfunction. Oxidative stress linked to nitric oxide synthase (NOS) uncoupling may play an important role. We tested whether tetrahydrobiopterin (BH4) can recouple NOS and reverse preestablished advanced hypertrophy, fibrosis, and dysfunction. Methods and Results— C57/Bl6 mice underwent transverse aortic constriction for 4 weeks, increasing cardiac mass (190%) and diastolic dimension (144%), lowering ejection fraction (−46%), and triggering NOS uncoupling and oxidative stress. Oral BH4 was then administered for 5 more weeks of pressure overload. Without reducing loading, BH4 reversed hypertrophy and fibrosis, recoupled endothelial NOS, lowered oxidant stress, and improved chamber and myocyte function, whereas untreated hearts worsened. If BH4 was started at the onset of pressure overload, it did not suppress hypertrophy over the first week when NOS activity remained preserved even in untreated transverse aortic constriction hearts. However, BH4 stopped subsequent remodeling when NOS activity was otherwise declining. A broad antioxidant, Tempol, also reduced oxidant stress yet did not recouple NOS or reverse worsened hypertrophy/fibrosis from sustained transverse aortic constriction. Microarray analysis revealed very different gene expression profiles for both treatments. BH4 did not enhance net protein kinase G activity. Finally, transgenic mice with enhanced BH4 synthesis confined to endothelial cells were unprotected against pressure overload, indicating that exogenous BH4 targeted myocytes and fibroblasts. Conclusions— NOS recoupling by exogenous BH4 ameliorates preexisting advanced cardiac hypertrophy/fibrosis and is more effective than a less targeted antioxidant approach (Tempol). These data highlight the importance of myocyte NOS uncoupling in hypertrophic heart disease and support BH4 as a potential new approach to treat this disorder.

Nitroxyl improves cellular heart function by directly enhancing cardiac sarcoplasmic reticulum Ca2+ cycling

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca2+. A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca2+ pump and the ryanodine receptor 2, leading to increased Ca2+ uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca2+-release channels and accelerates Ca2+ reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca2+ transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca2+ cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.

Myocardial Collagen Turnover in Hypertrophic Cardiomyopathy

Background—Myocardial interstitial fibrosis is a characteristic of hypertrophic cardiomyopathy (HCM). This study evaluates the collagen turnover in HCM and its impact on left ventricular (LV) diastolic function. Methods and Results—Thirty-six HCM patients and 14 sex- and age-matched controls were studied. Collagen turnover was assessed as follows. By radioimmunoassay, a byproduct of collagen III synthesis (PIIINP) and 3 peptides resulting from collagen I synthesis (PICP and PINP) and degradation (ICTP) were measured. By ELISA, matrix metalloproteinases (MMPs) were determined, as follows: active MMP-2; active MMP-9; and MMP-1 as active, free (as active MMP-1 plus its precursor), and total (as free MMP-1 plus MMP-1/tissue inhibitor complexes). Tissue inhibitor of metalloproteinases-1 (TIMP-1) was also assayed. All patients underwent echocardiography. The difference in duration between transmitral forward (A) and pulmonary venous retrograde (AR) waves (A−Ar) was considered an estimate of passive diastolic function. Furthermore, restrictive or pseudonormal LV filling patterns were considered to identify patients with passive diastolic dysfunction. Patients had higher levels of PIIINP, ICTP, MMP-2, MMP-9, and total TIMP-1 than did controls. PIIINP was inversely related to LV end-diastolic diameter. A−Ar was inversely related to PICP, PINP, and their differences with ICTP (estimates of collagen I buildup). Furthermore, A−Ar was directly related to MMP-1 and MMP-2. Conclusions—As compared with controls, collagen turnover is enhanced in HCM patients. As collagen I synthesis prevails over degradation and MMP-1 and MMP-2 are inhibited, passive diastolic dysfunction occurs in patients with HCM.

PED/PEA-15 gene controls glucose transport and is overexpressed in type 2 diabetes mellitus.

We have used differential display to identify genes whose expression is altered in type 2 diabetes thus contributing to its pathogenesis. One mRNA is overexpressed in fibroblasts from type 2 diabetics compared with non‐diabetic individuals, as well as in skeletal muscle and adipose tissues, two major sites of insulin resistance in type 2 diabetes. The levels of the protein encoded by this mRNA are also elevated in type 2 diabetic tissues; thus, we named it PED for phosphoprotein enriched in diabetes. PED cloning shows that it encodes a 15 kDa phosphoprotein identical to the protein kinase C (PKC) substrate PEA‐15. The PED gene maps on human chromosome 1q21–22. Transfection of PED/PEA‐15 in differentiating L6 skeletal muscle cells increases the content of Glut1 transporters on the plasma membrane and inhibits insulin‐stimulated glucose transport and cell‐surface recruitment of Glut4, the major insulin‐sensitive glucose transporter. These effects of PED overexpression are reversed by blocking PKC activity. Overexpression of the PED/PEA‐15 gene may contribute to insulin resistance in glucose uptake in type 2 diabetes.

Compartmentalization of Cardiac β-Adrenergic Inotropy Modulation by Phosphodiesterase Type 5

Background— Recent cell-based studies have found that cGMP synthesis and hydrolysis by phosphodiesterase (PDE) appear compartmentalized, with nitric oxide synthase–derived and/or PDE type 5 (PDE-5)–hydrolyzable cGMP undetected at the sarcolemmal membrane in contrast to cGMP stimulated by natriuretic peptide. In the present study, we determine the functional significance of such compartments with a comparison of &bgr;-adrenergic modulation by PDE-5 inhibition to that of natriuretic peptide stimulation in both cardiomyocytes and intact hearts. The potential role of differential cGMP and protein kinase G stimulation by these 2 modulators was also studied. Methods and Results— Intact C57/BL6 mouse hearts were studied with pressure-volume analysis, and adult isolated myocytes were studied with fluorescence microscopy. PDE-5 inhibition with 0.1 to 1 &mgr;mol/L sildenafil (SIL) suppressed isoproterenol (ISO)-stimulated contractility, whereas 10 &mgr;mol/L atrial natriuretic peptide (ANP) had no effect. ISO suppression by SIL was prevented in cells pretreated with a protein kinase G inhibitor. Surprisingly, myocardial cGMP changed little with SIL+ISO yet rose nearly 5-fold with ANP, whereas protein kinase G activation (vasodilator-stimulated protein phosphorylation; ELISA assay) displayed the opposite: increased with SIL+ISO but unaltered by ANP+ISO. PDE-5 and ANP compartments were functionally separated, as inhibition of nitric oxide synthase by Nw-nitro-L-arginine methyl ester eliminated antiadrenergic effects of SIL, yet this was not restorable by co-stimulation with ANP. Conclusions— Regulation of cardiac &bgr;-adrenergic response by cGMP is specifically linked to a nitric oxide–synthesis/PDE-5–hydrolyzed pool signaling via protein kinase G. Natriuretic peptide stimulation achieves greater detectable increases in cGMP but not protein kinase G activity and does not modulate &bgr;-adrenergic response. Such disparities likely contribute to differential cardiac regulation by drugs that modulate cGMP synthesis and hydrolysis.

Nitroxyl increases force development in rat cardiac muscle

Donors of nitroxyl (HNO), the reduced congener of nitric oxide (NO), exert positive cardiac inotropy/lusitropy in vivo and in vitro, due in part to their enhancement of Ca2+ cycling into and out of the sarcoplasmic reticulum. Here we tested whether the cardiac action of HNO further involves changes in myofilament–calcium interaction. Intact rat trabeculae from the right ventricle were mounted between a force transducer and a motor arm, superfused with Krebs–Henseleit (K‐H) solution (pH 7.4, room temperature) and loaded iontophoretically with fura‐2 to determine [Ca2+]i. Sarcomere length was set at 2.2–2.3 μm. HNO donated by Angelis salt (AS; Na2N2O3) dose‐dependently increased both twitch force and [Ca2+]i transients (from 50 to 1000 μm). Force increased more than [Ca2+]i transients, especially at higher doses (332 ± 33%versus 221 ± 27%, P < 0.01 at 1000 μm). AS/HNO (250 μm) increased developed force without changing Ca2+ transients at any given [Ca2+]o (0.5–2.0 mm). During steady‐state activation, AS/HNO (250 μm) increased maximal Ca2+‐activated force (Fmax, 106.8 ± 4.3 versus 86.7 ± 4.2 mN mm−2, n= 7–8, P < 0.01) without affecting Ca2+ required for 50% activation (Ca50, 0.44 ± 0.04 versus 0.52 ± 0.04 μm, not significant) or the Hill coefficient (4.75 ± 0.67 versus 5.02 ± 1.1, not significant). AS/HNO did not alter myofibrillar Mg‐ATPase activity, supporting an effect on the myofilaments themselves. The thiol reducing agent dithiothreitol (DTT, 5.0 mm) both prevented and reversed HNO action, confirming AS/HNO redox sensitivity. Lastly, NO (from DEA/NO) did not mimic AS/HNO cardiac effects. Thus, in addition to reported changes in Ca2+ cycling, HNO also acts as a cardiac Ca2+ sensitizer, augmenting maximal force without altering actomyosin ATPase activity. This is likely to be due to modulation of myofilament proteins that harbour reactive thiolate groups that are targets of HNO.

Phospholamban thiols play a central role in activation of the cardiac muscle sarcoplasmic reticulum calcium pump by nitroxyl.

Nitroxyl (HNO) donated by Angelis salt activates uptake of Ca(2+) by the cardiac SR Ca(2+) pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca(2+) pump.

Detection, monitoring, and management of trastuzumab-induced left ventricular dysfunction: an actual challenge.

The antibody trastuzumab, targeted to inhibit the signalling of ErbB2, a tyrosine kinase receptor overexpressed in 20–30% of breast cancers, improves the prognosis in women affected by this tumour, but produces cardiotoxicity, since ErbB2 is also involved in myocardial homeostasis. In this review, we discuss the pathophysiology of trastuzumab cardiomyopathy and the complex interplay between ErbB2 inhibition and anthracyclines, and we focus on the actual challenges of detecting, monitoring, and managing trastuzumab cardiotoxicity: the research of new, sensitive markers of early trastuzumab toxicity, before the ejection fraction is reduced, is an active field of research.