Thioimidate Bond Formation between Cardiac Troponin C and Nitrile-containing Compounds.

We have investigated the mechanism and reactivity of covalent bond formation between cysteine-84 of the regulatory domain of cardiac troponin C and compounds containing a nitrile moiety similar to the calcium sensitizer levosimendan. The results of modifications to the levosimendan framework ranged from a large increase in covalent bond formation to complete inactivity. We present the biological activity of one of the most potent compounds. Limitations, including compound solubility and degradation at acidic pH, have prevented thorough investigation of the potential of these compounds. Our studies reveal the efficacious nature of the malononitrile moiety in targeting cNTnC and its potential in future cardiotonic drug design.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.

Co-administration of resveratrol with doxorubicin in young mice attenuates detrimental late-occurring cardiovascular changes.

Aims: Doxorubicin (DOX) is among the most effective chemotherapies used in paediatric cancer patients. However, the clinical utility of DOX is offset by its well-known cardiotoxicity, which often does not appear until later in life. Since hypertension significantly increases the risk of late-onset heart failure in childhood cancer survivors, we investigated whether juvenile DOX exposure impairs the ability to adapt to angiotensin II (Ang II)-induced hypertension later in life and tested a treatment that could prevent this. Methods and results: Five-week-old male mice were administered a low dose of DOX (4 mg/kg) or saline once a week for 3 weeks and then allowed to recover for 5 weeks. Following the 5-week recovery period, mice were infused with Ang II or saline for 2 weeks. In another cohort, mice were fed chow containing 0.4% resveratrol 1 week before, during, and 1 week after the DOX administrations. One week after the last DOX administration, p38 mitogen-activated protein kinase (MAPK) was activated in hearts of DOX-treated mice demonstrating molecular signs of cardiac stress; yet, there was no change in cardiac function between groups. However, DOX-treated mice failed to develop compensatory cardiac hypertrophy in response to Ang II-induced hypertension later in life. Of importance, mice receiving DOX with resveratrol co-administration displayed normalization in p38 MAPK activation in the heart and a restored capacity for cardiac hypertrophy in response to Ang II-induced hypertension. Conclusion: We have developed a juvenile mouse model of DOX-induced cardiotoxicity that displays no immediate overt physiological dysfunction; but, leads to an impaired ability of the heart to adapt to hypertension later in life. We also show that co-administration of resveratrol during DOX treatment was sufficient to normalize molecular markers of cardiotoxicity and restore the ability of the heart to undergo adaptive remodelling in response to hypertension later in life.

Resveratrol improves exercise performance and skeletal muscle oxidative capacity in heart failure.

We investigated whether treatment of mice with established pressure overload-induced heart failure (HF) with the naturally occurring polyphenol resveratrol could improve functional symptoms of clinical HF such as fatigue and exercise intolerance. C57Bl/6N mice were subjected to either sham or transverse aortic constriction surgery to induce HF. Three weeks postsurgery, a cohort of mice with established HF (%ejection fraction <45) was administered resveratrol (~450 mg·kg-1·day-1) or vehicle for 2 wk. Although the percent ejection fraction was similar between both groups of HF mice, those mice treated with resveratrol had increased total physical activity levels and exercise capacity. Resveratrol treatment was associated with altered gut microbiota composition, increased skeletal muscle insulin sensitivity, a switch toward greater whole body glucose utilization, and increased basal metabolic rates. Although muscle mass and strength were not different between groups, mice with HF had significant declines in basal and ADP-stimulated O2 consumption in isolated skeletal muscle fibers compared with sham mice, which was completely normalized by resveratrol treatment. Overall, resveratrol treatment of mice with established HF enhances exercise performance, which is associated with alterations in whole body and skeletal muscle energy metabolism. Thus, our preclinical data suggest that resveratrol supplementation may effectively improve fatigue and exercise intolerance in HF patients.NEW & NOTEWORTHY Resveratrol treatment of mice with heart failure leads to enhanced exercise performance that is associated with altered gut microbiota composition, increased whole body glucose utilization, and enhanced skeletal muscle metabolism and function. Together, these preclinical data suggest that resveratrol supplementation may effectively improve fatigue and exercise intolerance in heart failure via these mechanisms.

Enhancing radiation tolerance by controlling defect mobility and migration pathways in multicomponent single-phase alloys.

A grand challenge in material science is to understand the correlation between intrinsic properties and defect dynamics. Radiation tolerant materials are in great demand for safe operation and advancement of nuclear and aerospace systems. Unlike traditional approaches that rely on microstructural and nanoscale features to mitigate radiation damage, this study demonstrates enhancement of radiation tolerance with the suppression of void formation by two orders magnitude at elevated temperatures in equiatomic single-phase concentrated solid solution alloys, and more importantly, reveals its controlling mechanism through a detailed analysis of the depth distribution of defect clusters and an atomistic computer simulation. The enhanced swelling resistance is attributed to the tailored interstitial defect cluster motion in the alloys from a long-range one-dimensional mode to a short-range three-dimensional mode, which leads to enhanced point defect recombination. The results suggest design criteria for next generation radiation tolerant structural alloys.

Reversible Covalent Binding to Cardiac Troponin C by the Ca2+-Sensitizer Levosimendan.

The binding of Ca2+ to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca2+, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca2+ (Ca2+-sensitizers) have therapeutic promise. The only Ca2+-sensitizer used regularly in clinical settings is levosimendan. While the primary target of levosimendan is thought to be cTnC, the molecular details of this interaction are not well understood. In this study, we used mass spectrometry, computational chemistry, and nuclear magnetic resonance spectroscopy to demonstrate that levosimendan reacts specifically with cysteine 84 of cTnC to form a reversible thioimidate bond. We also showed that levosimendan only reacts with the active, Ca2+-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca2+-sensitizing function of levosimendan is due to stabilization of the Ca2+-bound conformation of cTnC.

The structural and functional effects of the familial hypertrophic cardiomyopathy-linked cardiac troponin C mutation, L29Q.

Familial hypertrophic cardiomyopathy (FHC) is characterized by severe abnormal cardiac muscle growth. The traditional view of disease progression in FHC is that an increase in the Ca(2+)-sensitivity of cardiac muscle contraction ultimately leads to pathogenic myocardial remodeling, though recent studies suggest this may be an oversimplification. For example, FHC may be developed through altered signaling that prevents downstream regulation of contraction. The mutation L29Q, found in the Ca(2+)-binding regulatory protein in heart muscle, cardiac troponin C (cTnC), has been linked to cardiac hypertrophy. However, reports on the functional effects of this mutation are conflicting, and our goal was to combine in vitro and in situ structural and functional data to elucidate its mechanism of action. We used nuclear magnetic resonance and circular dichroism to solve the structure and characterize the backbone dynamics and stability of the regulatory domain of cTnC with the L29Q mutation. The overall structure and dynamics of cTnC were unperturbed, although a slight rearrangement of site 1, an increase in backbone flexibility, and a small decrease in protein stability were observed. The structure and function of cTnC was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. L29Q reduced the cooperativity of the Ca(2+)-dependent structural change in cTnC in trabeculae under basal conditions and abolished the effect of force-generating myosin cross-bridges on this structural change. These effects could contribute to the pathogenesis of this mutation.

Preclinical and clinical evidence for the role of resveratrol in the treatment of cardiovascular diseases.

Cardiovascular disease is the leading cause of death worldwide. Despite advancements in diagnosis and treatment of cardiovascular disease, the incidence of cardiovascular disease is still rising. Therefore, new lines of medications are needed to treat the growing population of patients with cardiovascular disease. Although the majority of the existing pharmacotherapies for cardiovascular disease are synthesized molecules, natural compounds, such as resveratrol, are also being tested. Resveratrol is a non-flavonoid polyphenolic compound, which has several biological effects. Preclinical studies have provided convincing evidence that resveratrol has beneficial effects in animal models of hypertension, atherosclerosis, stroke, ischemic heart disease, arrhythmia, chemotherapy-induced cardiotoxicity, diabetic cardiomyopathy, and heart failure. Although not fully delineated, some of the beneficial cardiovascular effects of resveratrol are mediated through activation of silent information regulator 1 (SIRT1), AMP-activated protein kinase (AMPK), and endogenous anti-oxidant enzymes. In addition to these pathways, the anti-inflammatory, anti-platelet, insulin-sensitizing, and lipid-lowering properties of resveratrol contribute to its beneficial cardiovascular effects. Despite the promise of resveratrol as a treatment for numerous cardiovascular diseases, the clinical studies for resveratrol are still limited. In addition, several conflicting results from trials have been reported, which demonstrates the challenges that face the translation of the exciting preclinical findings to humans. Herein, we will review much of the preclinical and clinical evidence for the role of resveratrol in the treatment of cardiovascular disease and provide information about the physiological and molecular signaling mechanisms involved. This article is part of a Special Issue entitled: Resveratrol: Challenges in translating pre-clinical findings to improved patient outcomes.

Solution structure of human cardiac troponin C in complex with the green tea polyphenol, (-)-epigallocatechin 3-gallate.

Heart muscle contraction is regulated by Ca(2+) binding to the thin filament protein troponin C. In cardiovascular disease, the myofilament response to Ca(2+) is often altered. Compounds that rectify this perturbation are of considerable interest as therapeutics. Plant flavonoids have been found to provide protection against a variety of human illnesses such as cancer, infection, and heart disease. (-)-Epigallocatechin gallate (EGCg), the prevalent flavonoid in green tea, modulates force generation in isolated guinea pig hearts (Hotta, Y., Huang, L., Muto, T., Yajima, M., Miyazeki, K., Ishikawa, N., Fukuzawa, Y., Wakida, Y., Tushima, H., Ando, H., and Nonogaki, T. (2006) Eur. J. Pharmacol. 552, 123-130) and in skinned cardiac muscle fibers (Liou, Y. M., Kuo, S. C., and Hsieh, S. R. (2008) Pflugers Arch. 456, 787-800; and Tadano, N., Yumoto, F., Tanokura, M., Ohtsuki, I., and Morimoto, S. (2005) Biophys. J. 88, 314a). In this study we describe the solution structure of the Ca(2+)-saturated C-terminal domain of troponin C in complex with EGCg. Moreover, we show that EGCg forms a ternary complex with the C-terminal domain of troponin C and the anchoring region of troponin I. The structural evidence indicates that the binding site of EGCg on the C-terminal domain of troponin C is in the hydrophobic pocket in the absence of troponin I, akin to EMD 57033. Based on chemical shift mapping, the binding of EGCg to the C-terminal domain of troponin C in the presence of troponin I may be to a new site formed by the troponin C.troponin I complex. This interaction of EGCg with the C-terminal domain of troponin C.troponin I complex has not been shown with other cardiotonic molecules and illustrates the potential mechanism by which EGCg modulates heart contraction.