Curr Hypertens Rep (2010) 12:426432 DOI 10.

1007/s11906-010-0149-8

Mitochondrial Dysfunction and Oxidative Damage to Sarcomeric Proteins

Marina Bayeva & Hossein Ardehali

Published online: 24 September 2010 # Springer Science+Business Media, LLC 2010

Abstract Hypertension is an important risk factor for the development of heart failure. Increased production of reactive oxygen species (ROS) contributes to cardiac dysfunction by activating numerous pro-hypertrophic signaling cascades and damaging the mitochondria, thus setting off a vicious cycle of ROS generation. The way in which oxidative stress leads to exacerbation of systolic and diastolic dysfunction is still unclear, however. In skeletal muscle and ischemic myocardium, increased ROS production causes preferential oxidation of myofibrillar proteins and provides a mechanistic link between oxidative damage and impaired contractility through disruption of actin-myosin interactions, enzymatic functions, calcium sensitivity, and efficiency of cross-bridge cycling. In this review, we summarize recent findings in the fields of heart failure and sarcomere biology and speculate that oxidative damage to myofibrils may contribute to the development of heart failure. Keywords Hypertension . ROS . Hypertrophy . Heart failure . Diastolic dysfunction . Mitochondria . Sarcomere . Myofibrils . Contractile dysfunction

Introduction Hypertension is one of the most common reasons for adults in the United States to visit a physicians office [1].
M. Bayeva : H. Ardehali (*) Feinberg Cardiovascular Research Institute, Northwestern University, Feinberg School of Medicine, 303 East Chicago Avenue, Tarry 14-733, Chicago, IL 60611, USA e-mail: h-ardehali@northwestern.edu M. Bayeva e-mail: mabayeva@northwestern.edu

Although elevated blood pressure is a well-established risk factor for adverse cardiovascular outcomes, the underlying mechanisms are complex and not completely understood. Long-standing hypertension is an important cause of diastolic dysfunction, a condition characterized by stiffening of the myocardium and impaired relaxation phase with preservation of contractility and ejection fraction [2]. The development of left ventricular hypertrophy is another response to hypertension, and this condition may be found even in patients with borderline blood pressure. Though hypertrophy initially develops as an adaptive mechanism to maintain adequate tissue perfusion against increased outflow resistance, sustained hypertension leads to a gradual loss of contractile function and transition into heart failure. Because there is still no curative treatment for heart failure and the supportive therapies are often inadequate, it is necessary to understand the molecular events that induce hypertrophy and cardiac dysfunction in response to elevated blood pressure. Multiple mechanisms are activated in the pressureoverloaded heart, including alterations in the creatine kinase energy system, changes in glucose and fatty acid metabolism, activation of signaling cascades such as AMPactivated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK), a switch to the fetal isoforms of numerous contractile and regulatory proteins, and enhanced production of reactive oxygen species (ROS) [3]. The early hypertrophic changes include increase in myocyte size, ventricular wall thickening, and enhanced contractile function. Chronic pressure overload, however, leads to progressive interstitial fibrosis and stiffening of the heart, ventricular dilatation, loss of contractility, and transition into decompensated heart failure. The role of redoxsensitive pathways in hypertrophy and heart failure has been extensively studied, but the structural damage to the

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heart associated with increased ROS production has not been adequately addressed. The following sections summarize the evidence that oxidative damage to sarcomeric components may represent an important (but not well-characterized) pathway that contributes to the transition of hypertrophy to decompensated heart failure via reduction in contractility, relaxation, and calcium sensitivity (Fig. 1).

Hypertension and the Production of Reactive Oxygen Species The causative relationship between hypertension and ROS production is well established [3, 4]. In isolated cardiomyocytes, stimulation with angiotensin II (ATII) [5], alphaadrenergic agonists [6], or cyclic stretch [7] resulted in increased oxidative stress and correlated with increases in cell size, protein synthesis, and molecular markers of hypertrophy. The observed changes were reversible with antioxidant treatments. Progression of hypertrophy in animal models of pressure overload or salt-induced hypertrophy also correlated with increased ROS production and was attenuated by administration of antioxidants [8, 9, 10]. Moreover, increased indices of oxidative stress were observed in the hearts of spontaneously hypertensive rats [11]; again, the cardiac dysfunction was reversible with antioxidants. In humans, hypertension was also associated with increased ROS production [12] and decreased content and activity of antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase, and catalase [13], although the data are not as complete as in animal models. Hypertension increases ROS production via several mechanisms. Adrenergic stimulation, ATII treatment, mechanical stress, pressure overload, and various cytokines all increase the activity of NADPH oxidase, a cytoplasmic
Fig. 1 Mitochondrial and oxidative damage to sarcomeric proteins. Generation of reactive oxygen species (ROS) from both cytoplasm and mitochondria can cause oxidation of the sarcomeric proteins. These can lead to contractile dysfunction and cardiomyopathy. ETCelectron transport chain

enzyme whose primary function appears to be ROS generation. This leads to activation of multiple downstream redox signaling pathways such as MAPKs; protein kinases B, C, and D; nuclear factor kappa-B; HIF1; STATS; Erk1/2; and other receptors, ion channels, and matrix metalloproteases (MMPs), as reviewed by Murdoch et al. [14]. The importance of NADPH oxidase in cardiac remodeling was highlighted by the studies of gp91phox-knockout mice that lack the Nox2 subunit of the enzyme and are protected from ATII-induced hypertrophy [15]. However, these mice remain just as susceptible to hypertrophy induced by transverse aortic constriction (TAC) as their littermate controls, and in both groups the cardiomyopathy is reversible with antioxidants [16]. These observations support the importance of ROS in hypertension-induced hypertrophy and suggest that multiple sources of oxidative stress are involved. Xanthine oxidase (XO), an enzyme that catalyzes oxidation of hypoxanthine and xanthine to urate with the generation of superoxide, was shown to be activated by ATII and adrenergic stimuli, and its inhibition led to a reduction in cardiac hypertrophy without the corresponding decrease in blood pressure [17]. Mitochondria also contribute to ROS production in pressure-overload hypertrophy through uncoupling of the electron transport chain (ETC) and the leak of electrons from Complex I [18] and Complex II [19]. In vitro, mitochondria isolated from failing canine hearts were shown to generate more superoxide in the presence of NADH, supporting the notion that mitochondrial ETC may be an important source of ROS production in the pathophysiology of heart failure [18]. Importantly, cardiac muscle is highly dependent on oxidative phosphorylation and therefore contains a large number of mitochondria that form an extensive reticular network around cardiac sarcomeres. Because ROS act close to their site of formation, they may damage critical components of cardiac myofibrils

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and impair contractile function, as was shown for other disorders associated with mitochondrial dysfunction and oxidative stress, including ischemia-reperfusion (I/R) [20], exercise-induced muscle fatigue [21], and sarcopenia of aging [22]. Moreover, mitochondria themselves are major targets of oxidative insults such as accumulation of mutations and large-scale deletions in mitochondrial DNA (mtDNA), decreased transcription of mitochondrially encoded subunits of ETC, reduced capacity for oxidative phosphorylation, and further uncoupling of ETC complexes that fuel the vicious cycle of ROS generation [18, 19]. Importantly, mitochondria-targeted antioxidants were successful in decreasing both blood pressure and cardiac hypertrophy in the spontaneously hypertensive rat model, indicating that mitochondria-derived ROS may be important in the pathogenesis of both conditions [23].

The Transition From Hypertrophy to Heart Failure The transition from hypertrophy to decompensated heart failure is of great clinical significance because of its irreversible nature and the lack of effective medical therapies. Several excellent reviews have been published recently, summarizing the role of signaling cascades in cardiac remodeling [3, 4]. Many unanswered questions remain in this field, however, and it is important to characterize mechanisms underlying the pathology of heart failure. In particular, structural studies of myofibrillar proteins in skeletal muscle subjected to oxidative stress have revealed interesting connections between ROS, oxidative damage, sarcomeric function, and contractility; similar connections may also apply to the pathophysiology of heart failure. Changes Occur in the Sarcomeric Architecture

Mitochondrial Dysfunction and Hypertension Mitochondrial dysfunction has been observed in multiple models of hypertension-induced cardiac hypertrophy and heart failure. Impairment of oxygen consumption and energy production was shown to correlate with exacerbation of heart function in pulmonary arterial hypertension (PAH)induced right ventricular (RV) failure [24]. A more detailed examination of energetic dysfunction in a pressureoverloaded rat heart suggested that the transition into heart failure correlates closely with decreases in mitochondrial respiratory capacity [10]. Moreover, elevated ROS production by mitochondria was implicated in the transition from hypertrophy to overt RV heart failure [19], which was reversible with antioxidants [25]. Oxidative stress is a key contributor to cardiac dysfunction in mitochondrial mutator mice that lack an exonuclease-encoding domain of mitochondrial DNA polymerase. By 14 months of age, these mice display all attributes typical of heart failure, including hypertrophy, systolic and diastolic dysfunction, reduced contractility, and increased fibrosis. A genetic cross with mice overexpressing mitochondria-targeted catalase (mCAT) resulted in a significant improvement of heart function, restoration of mitochondrial oxidative capacity, and amelioration of oxidative stress [26], confirming that mitochondrial dysfunction and ROS production are important mechanisms in the transition from hypertrophy to heart failure. Heterozygous deletion of a crucial mitochondrial antioxidant enzyme, SOD2, resulted in a similar heart failure phenotype as well [27], whereas SOD2 transgenic mice were protected from oxidant-induced cardiac dysfunction [28]. Thus, mitochondrial dysfunction and the subsequent increase in oxidative stress are important contributors to the development of hypertrophy and the transition to heart failure.

Structural integrity of a sarcomere is essential for normal contractile function, as exemplified by various familial hypertrophic cardiomyopathies resulting from genetic mutations in myofibrillar proteins (as reviewed by Marian [29]). Although the damage to the contractile apparatus is clearly sufficient to induce myocardial dysfunction, the effects of heart failure on myofibrillar protein structure and function have not been fully characterized. Myocytes from failing dog hearts and human hearts exhibit numerous abnormalities in their contractile properties, including impaired calcium sensitivity, decreased contraction amplitude and force, and inefficient relaxation [30]. Moreover, an early study described significant distortion of the sarcomeric architecture with interruption of myofibrils, Z-disc irregularities, and myofilament disarray in septal biopsies from patients with chronic heart failure [31]. Thus, contractile units are clearly abnormal in failing hearts, but the details are only beginning to emerge. Many groups have observed dramatic shifts towards expression of fetal isoforms of contractile proteins, including myosin heavy and light chains, troponin T (TnT), and tropomyosin [32, 33]. These shifts greatly affect calcium sensitivity and contractile performance of the myocytes, but whether they contribute to the pathology or represent an adaptive mechanism is an ongoing debate. Redox-dependent regulation of various kinases and phosphatases and subsequent changes in the phosphorylation status of contractile proteins were also implicated in the reduction of contractility and the development of heart failure [33, 34]. However, the degree of oxidative damage to myofibrils and the functional consequences thereof have not been studied in heart failure. Sarcomeric Proteins Are Preferentially Oxidized Myofibrillar proteins are markedly susceptible to oxidative damage both in vitro and in vivo, and recent data suggest

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that oxidation of sarcomeric proteins is a significant contributor to dysfunction of skeletal and cardiac muscle. Proteomic analysis proved to be an invaluable tool for identifying preferentially oxidized proteins and the functional consequences of these modifications. Because the topic of oxidative damage to sarcomeric proteins has not been addressed in heart failure, the evidence summarized here regarding specific modifications to myofibrillar components and their contribution to observed defects comes from studies of other ROS-related conditions. Sarcomeric proteins undergo a variety of oxidative modifications, including irreversible carbonylation of lysine, arginine, and proline residues; tyrosine nitration; reversible thiol oxidation; and disulfide bond formation [35, 36, 37]. Costa et al. [38] found that structural proteins, including myosin light chain-2, myosin light polypeptide-3, alpha-actinin, and TnT, are altered following incubation with a ROS-generating xanthine + XO system. Preferential oxidation and increased degradation of myosin heavy chain was noted in the diaphragm and soleus muscle of hyperthyroid rats; these effects were reversed with the administration of carvedilol, a beta blocker with antioxidant properties [39]. Proteomic analyses of tissues subjected to I/R protocols found that actin, desmin, tropomyosin, and troponins were also targets of ROS [20, 40, 41]. One group recently adopted an x-ray irradiation model to study the effects of acute oxidative stress on posttranslational modifications and function of myofibrillar proteins, which yielded intriguing results [36]. Using a gel-based proteomics approach, these researchers identified a total of 38 carbonylated proteins at different times after irradiation. Of these proteins, 15 were components of the sarcomere, with myosin light chain and desmin oxidized within the first 3 h, and TnT isoforms displaying the highest carbonylation content 9 h after irradiation. The same group detected intramolecular and intermolecular disulfide bridges inside actin and various isoforms of myosin light chain [42]. Oxidative Damage Impairs Contractile Function The functional consequences of oxidative damage to myofibrillar proteins have been extensively investigated and are closely correlated with quantitative measures of muscle function. In vitro, treatment of permeabilized rabbit muscle fibers with H2O2 led to a reduction in muscle contractility, a decrease in enzymatic activity of myosin and actomyosin, and a shift towards rigor-like conformation of myosin heads even under low-calcium conditions. The latter finding would translate into impaired relaxation of the oxidized muscle, similar to that observed in diastolic dysfunction. The maximal isometric force of contraction was partly rescued by incubation with a reducing agent, dithiothreitol (DTT), suggesting the presence of both

reversible and irreversible modifications [43]. H2O2 treatment also resulted in oxidation of actin on two residues and alterations of structural coupling between actin and myosin [44]. Treatment of isolated rat myofibrillar components with peroxynitrite (ONOO) led to oxidation of cysteine residues and partial unfolding of the proteins. This resulted in over 80% decrease in the sliding velocity of thin filaments over myosin, as determined by the in vitro motility assay [37]. Importantly, even low levels of oxidative damage were found to severely impair myosin function. In vivo studies that directly test the functional consequences of oxidative damage are challenging, but a number of recent reports support the in vitro data. Detailed characterization of actin isolated from irradiated rat muscle showed that both polymerization of globular actin (G-actin) and activation of myosin ATP-ase activity decreased by about 60% within 9 h following the initiation of oxidative stress [45]. In addition, actin was extensively glutathionylated in rat hearts subjected to I/R [20]. Again, polymerization of modified G-actin and the affinity of tropomyosin for glutathionylated actin filaments were greatly reduced. These changes correlated with the decrease in forcegenerating capacity and fiber stiffness, consistent with a reduced number of cross-bridge attachments. Importantly, the ATP content was maintained constant in this experimental system, implicating that the contractile dysfunction was due to oxidative damage to sarcomeric proteins and not to a reduction in energy availability. Rao et al. [46] showed that I/R increased oxidative stress even in the remote areas of the heart and that cardiac myosin isolated from distal myocardium was less efficient in propelling thin filaments purified from either I/R or control mouse hearts. Both filament sliding velocity and actin-myosin binding were impaired, suggesting that posttranslational modifications of myosin play a role in the pathology of remote-zone left ventricular dysfunction. Lastly, de Paula Brotto et al. [41] showed that hypoxia-induced diaphragmatic dysfunction was associated with degradation of troponins I and C. Incubation of ischemic diaphragm with troponins isolated from normoxic control tissue resulted in a significant restoration of contractility. On the other hand, adding troponins from the hypoxic diaphragm greatly depressed the contractility of nonischemic control fibers, demonstrating the direct effect of oxidative damage on contractile performance. The ability of antioxidants to reverse structural damage to the sarcomere and preserve contractile function strongly supports the role of ROS in pathophysiology of muscle dysfunction. In rabbit hearts subjected to low-flow ischemia, treatment with hydroxyl radical scavenger N-(2mercaptopropionyl) glycine (MPG) prevented the degradation of myosin-regulatory light chain (MLC)-2 and preserved contractility [47]. In ischemic hearts from pigs and dogs, contractile dysfunction was associated with formation

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of disulfide cross-bridges in tropomyosin and was reversed by treatment with DTT and ascorbic acid [48]. Lastly, antioxidant agents such as N-acetylcysteine (NAC) were shown to delay exhaustive exercise-induced muscle fatigue [21], a condition associated with elevated ROS production, irreversible modifications of multiple proteins, impairment of actomyosin interactions, and decreased calcium sensitivity of myofibrillar components. Taken together, this evidence makes it clear that myofibrillar proteins are particularly sensitive to ROS-induced damage and are important players in certain conditions associated with muscle dysfunction. Does Sarcomere Damage Lead to Heart Failure? Given the pro-oxidant state of the failing myocardium, it is reasonable to hypothesize that myofibrillar protein damage may contribute to systolic and diastolic dysfunction in heart failure. After all, the failing heart exhibits the same qualitative defects as dysfunctional skeletal muscle, namely reduced force generation by individual myocytes, decreased calcium sensitivity, and impaired relaxation. In fact, all of these findings were noted by Prochniewicz et al. [43], who studied structural modifications of myosin heavy and light chains in permeabilized muscle strips subjected to H2O2 treatment. In particular, the structural basis for functional impairment of relaxation under oxidative stress was demonstrated by electron paramagnetic resonance (EPR) spectroscopy, which found a large proportion of myosin heads in a strong-binding, or rigor-like structural state even under relaxing (low-calcium) conditions. Do the same mechanisms operate in the failing heart? More than a decade ago, de Tombe et al. [49] suggested that reduced force development [in a right ventricular model of heart failure] is due, in part, to depressed function of the contractile filaments, and oxidative damage is a plausible mechanism that may contribute to myocardial dysfunction in heart failure. Until now (to our knowledge), no proteomic studies of the failing myocardium have addressed changes in protein carbonylation, nitrosylation, or other oxidative modifications, although the total protein carbonylation content is known to be increased in heart failure. Oxidation status of the most ROS-susceptible myofibrillar proteins has thus far not been carefully evaluated in animal models or patients with cardiac dysfunction. Few groups have looked at degradation of troponins in heart failure [50]. Interestingly, although the introduction of truncated cardiac troponin I into permeabilized cardiomyocytes from healthy human donors did not alter the maximal tension in the fibers, the relaxation kinetics, which underlies the pathology of diastolic dysfunction, was significantly impaired [51]. Moreover, increased oxidative stress is associated with enhanced activity of the proteasome, which is needed for the effective

clearance of damaged proteins [52]. Paradoxically, proteasomal activity is reduced in a failing heart [53], and accumulation of oxidized proteins is predicted to interfere with the function of intact components of the sarcomere, impairing force generation. Thus, whether oxidative damage to sarcomeric proteins plays a role in the pathology of heart failure remains to be determined through careful proteomic analyses and protein exchange experiments.

Conclusions Hypertension is a well-established risk factor for the development of diastolic dysfunction and cardiac hypertrophy with subsequent progression into systolic heart failure. Elevated blood pressure is associated with enhanced ROS production in endothelium, vascular smooth muscle, and cardiomyocytes. In turn, oxidative stress is a key mediator of hypertrophy and heart failure through activation of redox-sensitive pathways, direct regulation of kinases and ion channels, and impairment of mitochondrial function and energy homeostasis. Thus, there is a clear link between hypertension, ROS, and heart failure. A number of recent studies on ROS-induced striated muscle dysfunction identified myofibrillar proteins as preferential targets of oxidative damage and suggested their causative role in defects of myocyte contractility and relaxation. Interestingly, the deficiencies observed in the failing myocardium are very similar to those of oxidized skeletal muscle, suggesting that similar mechanisms may be at play. Thus, we believe it is important to examine the degree of oxidative damage to specific sarcomeric proteins in heart failure and to establish their contribution to systolic and diastolic dysfunction. In turn, thorough understanding of molecular pathways responsible for the transition to decompensated heart failure will guide the development of novel clinical treatments for this disease.

Disclosure No potential conflicts of interest relevant to this article were reported.

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