Daniel R. Gonzalez

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.

Molecular basis of calcium regulation in connexin-32 hemichannels

In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca2+ concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca2+ regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of ≈90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels (≈18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca2+ that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca2+ to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca2+ deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.

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.

Species specificity of mammalian connexin-26 to form open voltage-gated hemichannels

Mutations of connexin‐26 (Cx26) cause nonsyndromic hearing loss and other syndromes affecting ectoderm‐derived tissues. While the exact mechanisms underlying these diseases remain elusive, Cxs are generally considered to mediate cell‐to‐cell communication by forming gap junction channels. We show here that unlike rat Cx26, human and sheep Cx26 form voltage‐gated hemichannels when expressed in oocytes and Neuro2A cells. A single evolutionary amino acidic change at position 159 of the rodent protein, the replacement of aspartic acid with asparagine in the human and sheep proteins, accounts for this species specificity. At the resting potential and in normal millimolar extracellular calcium, open human Cx26 hemichannels can be detected both electrophysiologically and by dye uptake, although they did not affect cell viability. These hemichannels opened at ~ −50 mV and their activation increased by depolarization until they inactivate at positive membrane potentials. Single‐channel analysis revealed that activation and inactivation involved two distinct voltage gating mechanisms and that the fully open hemichannel displays a conductance twice that of the intercellular channel. The existence of a hemichannel that opens under physiological control of the membrane potential may have important implications for the normal and pathological activity of Cx26 in humans, particularly with respect to hearing and the epidermis.—González, D., Gómez‐Hernández, J. M., Barrio, L. C. Species specificity of mammalian connexin‐26 to form open voltage‐gated hemichannels. FASEB J. 20, 2329–2338 (2006)

Replacement of Connexin40 by Connexin45 in the Mouse: Impact on Cardiac Electrical Conduction

Abstract— Gap junction channels, required for the propagation of cardiac impulse, are intercellular structures composed of connexins (Cx). Cx43, Cx40, and Cx45 are synthesized in the cardiomyocytes, and each of them has a unique cardiac expression pattern. Cx40 knock-in Cx45 mice were generated to explore the ability of Cx45 to replace Cx40, and to assess the functional equivalence of these two Cxs that are both expressed in the conduction system. ECGs revealed that the consequences resulting from the biallelic replacement of Cx40 by Cx45 were an increased duration of the P wave, and a prolonged and fractionated QRS complex. Epicardial mapping indicated that the conduction velocities (CV) in the right atrium and the ventricular myocardium, as well as conduction through the AV node, were unaffected. The significant reduction of the CV in the left atrium would be the most likely cause of the P-wave lengthening. In the right ventricle, a changed and prolonged activation in sinus rhythm was found in homozygous mutant mice, which may explain the prolongation and splitting of the QRS complex. Electrical mapping of the His bundle branches revealed that this was due to slow conduction measured in the right branch. The CV in the left branch was unchanged. Therefore, in the absence of Cx40, the upregulation of Cx45 in the heart results in a normal impulse propagation in the right atrium, the AV node, and the left His bundle branch only.

Leptin repletion restores depressed β-adrenergic contractility in ob/ob mice independently of cardiac hypertrophy

Impaired leptin signalling in obesity is increasingly implicated in cardiovascular pathophysiology. To explore mechanisms for leptin activity in the heart, we hypothesized that physiological leptin signalling participates in maintaining cardiac β‐adrenergic regulation of excitation–contraction coupling. We studied 10‐week‐old (before development of cardiac hypertrophy) leptin‐deficient (ob/ob, n= 12) and C57Bl/6 (wild‐type (WT), n= 15) mice at baseline and after recombinant leptin infusion (0.3 mg kg−1 day−1 for 28 days, n= 6 in each group). Ob/ob‐isolated myocytes had attenuated sarcomere shortening and calcium transients ([Ca2+]i) versus WT (P < 0.01 for both) following stimulation of the β‐receptor (with isoproterenol (isoprenaline)) or at the post‐receptor level (with forskolin and dibutryl‐cAMP). In addition, sarcoplasmic reticulum (SR) Ca2+ stores were depressed. Leptin replenishment in ob/ob mice restored each of these abnormalities towards normal without affecting gross (wall thickness) or microscopic (cell size) measures of cardiac architecture. Immunoblots revealed alterations of several proteins involved in excitation–contraction coupling in the ob/ob mice, including decreased abundance of Gsα‐52 kDa, as well as alterations in the expression of Ca2+ cycling proteins (increased SR Ca2+‐ATPase, and depressed phosphorylated phospholamban). In addition, protein kinase A (PKA) activity in ob/ob mice was depressed at baseline and correctable towards the activity found in WT with leptin repletion, a finding that could account for impaired β‐adrenergic responsiveness. Taken together, these data reveal a novel link between the leptin signalling pathway and normal cardiac function and suggest a mechanism by which leptin deficiency or resistance may lead to cardiac depression.

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.

β2-adrenergic receptor-coupled phosphoinositide 3-kinase constrains cAMP-dependent increases in cardiac inotropy through phosphodiesterase 4 activation.

BACKGROUND:Emerging evidence suggests that phosphoinositide 3-kinase (PI3K) may modulate cardiac inotropy; however, the underlying mechanism remains elusive. We hypothesized that &bgr;2-adrenergic receptor (AR)-coupled PI3K constrains increases in cardiac inotropy through cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activation. METHODS:We tested the effects of PI3K and PDE4 inhibition on myocardial contractility by using isolated murine cardiac myocytes to study physiologic functions (sarcomere shortening [SS] and intracellular Ca+ transients), as well as cAMP and PDE activity. RESULTS:PI3K inhibition with the reversible inhibitor LY294002 (LY) resulted in a significant increase in SS and Ca2+ handling, indicating enhanced contractility. This response depended on Gi&agr; protein activity, because incubation with pertussis toxin (an irreversible Gi&agr; inhibitor) abolished the LY-induced hypercontractility. In addition, PI3K inhibition had no greater effect on SS than both a PDE3,4 inhibitor (milrinone) and LY combined. Furthermore, LY decreased PDE4 activity in a concentration-dependent manner (58.0% of PDE4 activity at LY concentrations of 10 &mgr;M). Notably, PI3K&ggr; coimmunoprecipitated with PDE4D. The &bgr;2-AR inverse agonist, ICI 118,551 (ICI), abolished induced increases in contractility. CONCLUSIONS:PI3K modulates myocardial contractility by a cAMP-dependent mechanism through the regulation of the catalytic activity of PDE4. Furthermore, basal agonist-independent activity of the &bgr;2-AR and its resultant cAMP production and enhancement of the catalytic activity of PDE4 through PI3K represents an example of integrative cellular signaling, which controls cAMP dynamics and thereby contractility in the cardiac myocyte. These results help to explain the mechanism by which milrinone is able to increase myocardial contractility in the absence of direct &bgr;-adrenergic stimulation and why it can further augment contractility in the presence of maximal &bgr;-adrenergic stimulation.