Adriana V. Treuer

Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes

Altered Ca2+ homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via S-nitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S-nitrosylation, leading to altered Ca2+ homeostasis. Diastolic Ca2+ levels are elevated in NOS1−/− and NOS1/NOS3−/− but not NOS3−/− myocytes compared with wild-type (WT), suggesting diastolic Ca2+ leakage. Measured leak was increased in NOS1−/− and NOS1/NOS3−/− but not in NOS3−/− myocytes compared with WT. Importantly, NOS1−/− and NOS1/NOS3−/− myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1−/− hearts, RyR2 S-nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca2+ leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease.

Impaired S-Nitrosylation of the Ryanodine Receptor Caused by Xanthine Oxidase Activity Contributes to Calcium Leak in Heart Failure

S-Nitrosylation is a ubiquitous post-translational modification that regulates diverse biologic processes. In skeletal muscle, hypernitrosylation of the ryanodine receptor (RyR) causes sarcoplasmic reticulum (SR) calcium leak, but whether abnormalities of cardiac RyR nitrosylation contribute to dysfunction of cardiac excitation-contraction coupling remains controversial. In this study, we tested the hypothesis that cardiac RyR2 is hyponitrosylated in heart failure, because of nitroso-redox imbalance. We evaluated excitation-contraction coupling and nitroso-redox balance in spontaneously hypertensive heart failure rats with dilated cardiomyopathy and age-matched Wistar-Kyoto rats. Spontaneously hypertensive heart failure myocytes were characterized by depressed contractility, increased diastolic Ca2+ leak, hyponitrosylation of RyR2, and enhanced xanthine oxidase derived superoxide. Global S-nitrosylation was decreased in failing hearts compared with nonfailing. Xanthine oxidase inhibition restored global and RyR2 nitrosylation and reversed the diastolic SR Ca2+ leak, improving Ca2+ handling and contractility. Together these findings demonstrate that nitroso-redox imbalance causes RyR2 oxidation, hyponitrosylation, and SR Ca2+ leak, a hallmark of cardiac dysfunction. The reversal of this phenotype by inhibition of xanthine oxidase has important pathophysiologic and therapeutic implications.

S-nitrosylation of cardiac ion channels

Nitric oxide (NO) exerts ubiquitous signaling via posttranslational modification of cysteine residues, a reaction termed S-nitrosylation. Important substrates of S-nitrosylation that influence cardiac function include receptors, enzymes, ion channels, transcription factors, and structural proteins. Cardiac ion channels subserving excitation-contraction coupling are potentially regulated by S-nitrosylation. Specificity is achieved in part by spatial colocalization of ion channels with nitric oxide synthases (NOSs), enzymatic sources of NO in biologic systems, and by coupling of NOS activity to localized calcium/second messenger concentrations. Ion channels regulate cardiac excitability and contractility in millisecond timescales, raising the possibility that NO-related species modulate heart function on a beat-to-beat basis. This review focuses on recent advances in understanding of NO regulation of the cardiac action potential and of the calcium release channel ryanodine receptor, which is crucial for the generation of force. S-Nitrosylation signaling is disrupted in pathological states in which the redox state of the cell is dysregulated, including ischemia, heart failure, and atrial fibrillationS.

Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and β-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed β-adrenergic inotropic responses that were associated with impaired β-agonist–induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.

Increased Potency of Cardiac Stem Cells Compared with Bone Marrow Mesenchymal Stem Cells in Cardiac Repair

Whereas cardiac‐derived c‐kit+ stem cells (CSCs) and bone marrow‐derived mesenchymal stem cells (MSCs) are undergoing clinical trials testing safety and efficacy as a cell‐based therapy, the relative therapeutic and biologic efficacy of these two cell types is unknown. We hypothesized that human CSCs have greater ability than MSCs to engraft, differentiate, and improve cardiac function. We compared intramyocardial injection of human fetal CSCs (36,000) with two doses of adult MSCs (36,000 and 1,000,000) or control (phosphate buffered saline) in nonobese diabetic/severe combined immune deficiency mice after coronary artery ligation. The myocardial infarction‐induced enlargement in left ventricular chamber dimensions was ameliorated by CSCs (p < .05 for diastolic and systolic volumes), as was the decline in ejection fraction (EF; p < .05). Whereas 1 × 106 MSCs partially ameliorated ventricular remodeling and improved EF to a similar degree as CSCs, 36,000 MSCs did not influence chamber architecture or function. All cell therapies improved myocardial contractility, but CSCs preferentially reduced scar size and reduced vascular afterload. Engraftment and trilineage differentiation was substantially greater with CSCs than with MSCs. Adult‐cultured c‐kit+CSCs were less effective than fetal, but were still more potent than high‐dose MSCs. These data demonstrate enhanced CSC engraftment, differentiation, and improved cardiac remodeling and function in ischemic heart failure. MSCs required a 30‐fold greater dose than CSCs to improve cardiac function and anatomy. Together, these findings demonstrate a greater potency of CSCs than bone marrow MSCs in cardiac repair.

Activation of growth hormone releasing hormone (GHRH) receptor stimulates cardiac reverse remodeling after myocardial infarction (MI)

Both cardiac myocytes and cardiac stem cells (CSCs) express the receptor of growth hormone releasing hormone (GHRH), activation of which improves injury responses after myocardial infarction (MI). Here we show that a GHRH-agonist (GHRH-A; JI-38) reverses ventricular remodeling and enhances functional recovery in the setting of chronic MI. This response is mediated entirely by activation of GHRH receptor (GHRHR), as demonstrated by the use of a highly selective GHRH antagonist (MIA-602). One month after MI, animals were randomly assigned to receive: placebo, GHRH-A (JI-38), rat recombinant GH, MIA-602, or a combination of GHRH-A and MIA-602, for a 4-wk period. We assessed cardiac performance and hemodynamics by using echocardiography and micromanometry derived pressure-volume loops. Morphometric measurements were carried out to determine MI size and capillary density, and the expression of GHRHR was assessed by immunofluorescence and quantitative RT-PCR. GHRH-A markedly improved cardiac function as shown by echocardiographic and hemodynamic parameters. MI size was substantially reduced, whereas myocyte and nonmyocyte mitosis was markedly increased by GHRH-A. These effects occurred without increases in circulating levels of growth hormone and insulin-like growth factor I and were, at least partially, nullified by GHRH antagonism, confirming a receptor-mediated mechanism. GHRH-A stimulated CSCs proliferation ex vivo, in a manner offset by MIA-602. Collectively, our findings reveal the importance of the GHRH signaling pathway within the heart. Therapy with GHRH-A although initiated 1 mo after MI substantially improved cardiac performance and reduced infarct size, suggesting a regenerative process. Therefore, activation of GHRHR provides a unique therapeutic approach to reverse remodeling after MI.

Altered Renal Expression of Angiotensin II Receptors, Renin Receptor, and ACE-2 Precede the Development of Renal Fibrosis in Aging Rats

Background: The susceptibility to fibrosis and progression of renal disease is mitigated by inhibition of the renin-angiotensin system (RAS). We hypothesized that activation of the intrarenal RAS predisposes to renal fibrosis in aging. Methods: Intrarenal expression of angiotensin II type 1 (AT1R), type 2 (AT2R), and (pro)renin receptors, ACE and ACE-2, as well as pro- and antioxidant enzymes were measured in 3-month-old (young), 14-month-old (middle-aged), and 24-month-old (old) male Sprague-Dawley rats. Results: Old rats manifested glomerulosclerosis and severe tubulointerstitial fibrosis with increased fibronectin and TGF-β expression (7-fold). AT1R /AT2R ratios were increased in middle-aged (cortical 1.6-fold, medullary 5-fold) and old rats (cortical 2-fold, medullary 4-fold). Similarly, (pro)renin receptor expression was increased in middle-aged (cortical 2-fold, medullary 3-fold) and old (cortical 5-fold, medullary 3-fold) rats. Cortical ACE was increased (+35%) in old rats, whereas ACE-2 was decreased (–50%) in middle-aged and old rats. NADPH oxidase activity was increased (2-fold), whereas antioxidant capacity and expression of the mitochondrial enzyme manganese superoxide dismutase (cortical –40%, medullary –53%) and medullary endothelial nitric oxide synthase (–48%) were decreased in old rats. Conclusion: Age-related intrarenal activation of the RAS preceded the development of severe renal fibrosis, suggesting that it contributes to the increased susceptibility to renal injury observed in the elderly.

Effects of combination of proliferative agents and erythropoietin on left ventricular remodeling post-myocardial infarction.

Erythropoietin (EPO) has the potential to improve ischemic tissue by mobilizing endothelial progenitor cells and enhancing neovascularization. We hypothesized that combining EPO with human chorionic gonadotrophin (hCG) would improve post–myocardial infarction (MI) effects synergistically.

Neuronal nitric oxide synthase in heart mitochondria: a matter of life or death

Due to its high energy demand, cardiac muscle has the highest density of mitochondria of all mammalian organs. Mitochondria supply the cells with ATP generated from the electron transport chain by oxidative phosphorylation (0.5 μmol ATP (g wet wt)−1 s−1 at rest). Oxygen is the terminal electron acceptor in this chain. A side-effect of this high metabolic rate is an important production of oxygen radicals, which are an inevitable side product of the electron transport chain due to ‘electron leakage’ generated in the sequential donation of electrons to molecular oxygen, which normally ends in H2O production. n nMitochondria also generate nitric oxide (NO), another radical species. NO is generated by nitric oxide synthases (NOS). The specific isoform that is expressed in this organelle is still not clear. Nevertheless, in the heart, there is consistent evidence suggesting that neuronal NOS (nNOS) is the isoform that is found in mitochondria, based on pharmacological evidence, and on the fact that genetic deletion of nNOS abolishes NO production in mouse heart mitochondria (Kanai et al. 2001). The actual role of mitochondrial nitric oxide synthase (NOS) has remained elusive. n nThe importance of NOS in heart mitochondria is high, given the critical role of this organelle in energy production in such a metabolically active organ. Beside their role in energy production, mitochondria are deeply connected to the processes that lead to cell death. In the case of the heart, the impact of apoptosis and necrosis is clearly evidenced in myocardial infarction or after an episode of ischaemia–reperfusion. For instance, an episode of ischemia/reperfusion is followed by a burst of reactive oxygen species (ROS). In this phenomenon, mitochondria also play in important role generating these species. On the other hand, the protective effects of nitric oxide on cardiac disease are also established in the literature. n nIn a recent issue of The Journal of Physiology, Dedkova and Blatter explored the role of mitochondrial NO production and its relationship with ROS production and the mitochondrial permeability transition (Dedkova & Blatter, 2009). Using permeabilized cat cardiomyocytes as a model, the authors used pharmacologic probes, together with confocal microscopy that imaged a battery of pathway-specific fluorophores, to investigate some of the mechanisms by which NO modulates mitochondrial permeability. NO production was assessed using DAF-2, in the absence of functional caveolae and sarcoplasmic reticulum (disrupted by cyclodextrin and thapsigargin, respectively), to rule out the possible influence of eNOS and nNOS activity from those compartments. Although these manoeuvres may appear questionable, these results were also confirmed using specific inhibitors for NOS isoforms. More importantly, mitochondria-derived NO was abolished when mitochondria were uncoupled (blocking the respiratory chain or dissipating the mitochondrial membrane potential, Δψ), or when the mitochondrial Ca2+ uniporter was blocked. Since nNOS activity is Ca2+ dependent, dissipating the membrane potential that creates the driving force for Ca2+ influx or blocking the uniporter directly abolished NO production. These results confirmed the observations of Kanai et al. (2001) on isolated heart mitochondria using a NO-sensitive electrode. n nInterestingly, cytosolic [Ca2+] above 1 μm (a concentration observed during adrenergic stimulation or reperfusion, for instance) was necessary to activate mtNOS, and this Ca2+ requirement also included calmodulin. Since the cardiomyocytes were permeabilized, supplementation with l-arginine was necessary for mitochondrial NO synthesis. n nImportantly, part of the urea cycle in which l-arginine is produced and consumed takes place in the mitochondria. Arginase II, an enzyme that catabolizes arginine, is located in mitochondria and competes with mtNOS for substrate. In absence of l-arginine, ROS production was observed upon Ca2+ rise. The addition of arginine almost abolished ROS production and arginase inhibition decreased ROS production by 50% (without arginine supplementation). n nAnother target for NO assessed by the authors (and probably the most critical experiment) was the mitochondrial permeability transition pore (PTP). The permeability transition pore is a large conductance channel (about 1 nS) in the inner mitochondrial membrane that opens in response to high [Ca2+], low [ATP] and ROS. Opening of this channel causes a dramatic depolarization of the mitochondria followed by ATP depletion and cell death. The PTP opening (induced by high [Ca2+] and monitored by using calcein-loaded mitochondria) was prevented when ROS production was neutralized using a superoxide dismutase mimetic, or when l-arginine or tetrahydrobiopterin (BH4), a co-factor for NOS, was added as a supplement. Notably, supplementation with l-arginine nearly abolishes the pore opening, with an effect similar to cyclosporine A, a PTP inhibitor. These results suggest that mtNOS-derived NO inhibits the PTP opening when cytosolic [Ca2+] is high. It is not clear whether this effect is mediated directly by NO (for instance direct S-nitrosylation of reactive thiols) in the pore or is a result of neutralizing ROS production. On the contrary, in the presence of oxidative stress (like after reperfusion or during congestive heart failure) or in the absence of enough l-arginine, an increase in cytosolic [Ca2+] triggers opening of the permeability transition pore and, inevitably, leads to cell death. n nThese results strongly support a novel cardioprotective role for mtNOS. It seems likely that mtNOS is nNOS or a spliced variant of it (likely nNOSα or nNOSμ), as nNOS-deficient mice exhibit increased ROS production (Kinugawa et al. 2005) and after a myocardial infarction, they show increased myocardial damage and lower survival rates than wild-type animals (Saraiva et al. 2005). Consistent with this idea, heart-specific nNOS overexpression has been shown to be cardioprotective in a model of volume overload-hypertrophy conductive to heart failure (Loyer et al. 2008). These results have been attributed to nNOS located in the sarcolemma or in the sarcoplasmic reticulum, but not to nNOS in mitochondria. n nAn exciting question that arises from Dedkovas observations is whether the effects of NO are direct on the permeability transition pore, or indirect, based on modulation of another target that may prevent its opening, like the mitochondrial K+ channel (mitoKATP) or protein kinase Cɛ, both known as cardioprotective mediators that prevent PTP induction. Indeed, the PTP may be an interesting target for preventing myocardial damage. Recently, a pilot clinical study showed that treatment with cyclosporine A, a PTP inhibitor, decreased myocardial damage in patients who underwent percutaneous coronary intervention (reperfusion) after a myocardial infarction (Piot et al. 2008). This is encouraging for the search of other compounds that inhibit PTP. n nIn summary, the work by Dedkova and Blatter suggests that mtNOS, likely to be an nNOS, plays an important role in cardioprotection, especially under circumstances that produce ROS generation or high calcium load into the mitochondria such as reperfusion after a myocardial infarction.

Nitroso-Redox Crosstalk in Diabetic Cardiomyopathy

Diabetes mellitus is one of the most common chronic diseases worldwide. Diabetic cardiomyopathy (DM) is the deterioration of the myocardial function and morphology produced by the altered glucose metabolism imposed in diabetes. This process of cardiac deterioration involves the generation of oxidative species. In the diabetic heart, several sources contribute to the observed oxidative stress, such as xanthine oxidore‐ ductase (XOR), nicotinamide adenine dinucleotide phosphate (NADPH), nitrogen oxidases (NOX), mitochondria, and uncoupled nitric oxide synthases (NOS). A direct consequence of the increased production of reactive oxygen species (ROS) is NOS uncoupling. This is the aftermath of the oxidation of tetrahydrobioterin (BH4), an essential cofactor for NOS activity. When NOS is uncoupled, its activity is redirected toward the production of superoxide, instead of nitric oxide (NO), further contributing to the oxidative process. This nitroso‐redox disarrangement has a direct impact on the excitation‐contraction‐coupling machinery of the myocyte, in the mitochondrial stability impairing energy production and favoring apoptosis, myocardial fibrosis, ultimately reducing cardiac function. This review focuses on the impact of superoxide sources in the diabetic heart and the pharmacological approaches that are currently under investigation as possible therapeutic tools.