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.

The Ca 2+ dependant interaction between troponin I (cTnI) and troponin C (cTnC) triggers contraction in heart muscle. Heart failure is characterized by a decrease in cardiac output, and compounds that increase the sensitivity of cardiac muscle to Ca 2+ have therapeutic potential. The Ca 2+ -sensitizer, levosimendan, targets cTnC; however, detailed understanding of its mechanism has been obscured by its instability. In order to understand how this class of positive inotropes function, we investigated the mode of action of two fluorine containing novel analogues of levosimendan; 2' ,4'-difluoro(1,1'-biphenyl)-4-yloxy acetic acid (dfbp-o) and 2' ,4'-difluoro(1,1'-biphenyl)-4-yl acetic acid (dfbp). The affinities of dfbp and dfbp-o for the regulatory domain of cTnC were measured in the absence and presence of cTnI by NMR spectroscopy, and dfbp-o was found to bind more strongly than dfbp. Dfbp-o also increased the affinity of cTnI for cTnC. Dfbp-o increased the Ca 2+ -sensitivity of demembranated cardiac trabeculae in a manner similar to levosimendan. The high resolution NMR solution structure of the cTnC-cTnI-dfbp-o ternary complex showed that dfbp-o bound at the hydrophobic interface formed by cTnC and cTnI making critical interactions with residues such as Arg147 of cTnI. In the absence of cTnI, docking localized dfbp-o to the same position in the hydrophobic groove of cTnC. The structural and functional data reveal that the levosimendan class of Ca 2+ -sensitizers work by binding to the regulatory domain of cTnC and stabilizing the pivotal cTnC-cTnI regulatory unit via a network of hydrophobic and electrostatic interactions, in contrast to the destabilizing effects of antagonists such as W7 at the same interface.

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Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca2+-sensitivity of the thin filament have been linked to disease. Tikunova and Davis have engineered a series of cNTnC mutations that altered Ca2+ binding properties and studied the effects on the Ca2+ sensitivity of the thin filament and contraction [Tikunova and Davis (2004) J Biol Chem279, 35341–35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in cNTnC Ca2+ binding affinity and Ca2+ sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca2+ sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca2+ and cTnI to cTnC. NMR chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca2+ sensitivity can do so by modulating Ca2+ and cTnI binding.

Over the forty years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca 2+ -dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca 2+ -sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca 2+ concentration is not perturbed, preserving the regulation of other Ca 2+ -based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C -troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.

The solution structure of Ca 2+ -bound regulatory domain of cardiac troponin C (cNTnC) in complex with the switch region of troponin I (cTnI [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] ) and the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfinamide (W7), has been determined by NMR spectroscopy. The structure reveals that the W7 naphthalene ring interacts with the terminal methyl groups of M47, M60, and M81 as well as aliphatic and aromatic side-chains of several other residues in the hydrophobic pocket of cNTnC. The H3 ring proton of W7 also contacts the methyl groups of I148 and M153 of cTnI [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] . The N-(6-aminohexyl) tail interacts primarily with the methyl groups of V64 and M81, which are located on the C-and D-helices of cNTnC. Compared to the structure of the cNTnC•Ca 2+ •W7 complex (Hoffman, R. M. B. and Sykes, B. D. (2009) Biochemistry 48, 5541-5552), the tail of W7 reorients slightly towards the surface of cNTnC while the ring remains in the hydrophobic pocket. The positively charged -NH 3 + group from the tail of W7 repels the positively charged R147 of cTnI [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] . As a result, the N-terminus of the peptide moves away from cNTnC and the helical content of cTnI [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] is diminished, when compared to the structure of cNTnC•Ca 2+ •cTnI [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] (Li, M. X., Spyracopoulos, L., and Sykes B. D. (1999) Biochemistry 38, 8289-8298). Thus the ternary structure cNTnC•Ca 2+ •W7•cTnI [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] reported in this study offers an explanation for the ∼13-fold affinity reduction of cTnI [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] for cNTnC•Ca 2+ in the presence of W7, and provides a structural basis for the inhibitory effect of W7 in cardiac muscle contraction. This generates molecular insight into structural features that are useful for the design of cTnC-specific Ca 2+ -desensitizing drugs. Keywordstroponin; structure; drugs; mechanism; inhibition A healthy human heart generates ∼3 billion contractile cycles over an average life span. Each contractile cycle involves systolic activation and diastolic relaxation, regulated by Ca 2+ association and dissociation from troponin in the cardiac myofilaments. Troponin is a *To whom correspondence should be addressed. Phone Number: (780) 492-5460, Fax Number: (780) 492-0886, brian.sykes@ualberta.ca. Data Deposition: The atomic coordinates have been deposited in the RCSB Protein Data Bank (PDB accession code: 2KRD) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early ve...

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.

The binding of Ca to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca (Ca-sensitizers) have therapeutic promise. The only Ca-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, Ca-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca-sensitizing function of levosimendan is due to stabilization of the Ca-bound conformation of cTnC.