Inhibition of CYP2E1 attenuates myocardial dysfunction in a murine model of insulin resistance through NLRP3-mediated regulation of mitophagy

Insulin resistance leads to myocardial contractile dysfunction and deranged autophagy although the underlying mechanism or targeted therapeutic strategy is still lacking. This study was designed to examine the impact of inhibition of the cytochrome P450 2E1 (CYP2E1) enzyme on myocardial function and mitochondrial autophagy (mitophagy) in an Akt2 knockout model of insulin resistance. Adult wild-type (WT) and Akt2^?/? mice were treated with the CYP2E1 inhibitor diallyl sulfide (100?mg/kg/d, i.p.) for 4?weeks. Cardiac geometry and function were assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate autophagy, mitophagy, inducible NOS (iNOS), and the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex. Akt2 deletion triggered insulin resistance, compromised cardiac contractile and intracellular Ca²⁺ property, mitochondrial ultrastructural damage, elevated O2^– production, as well as suppressed autophagy and mitophagy, accompanied with elevated levels of NLRP3 and iNOS, the effects of which were significantly attenuated or ablated by diallyl sulfide. In vitro studies revealed that the NLRP3 activator nigericin nullified diallyl sulfide-offered benefit against Akt2 knockout on cardiomyocyte mechanical function and mitophagy (using Western blot and colocalization of GFP-LC3 and MitoTracker Red). Moreover, inhibition of iNOS but not mitochondrial ROS production attenuated Akt2 deletion-induced activation of NLRP3, substantiating a role for iNOS-mediated NLRP3 in insulin resistance-induced changes in mitophagy and cardiac dysfunction. In conclusion, these data depict that insulin resistance through CYP2E1 may contribute to the pathogenesis of myopathic changes including myocardial contractile dysfunction, oxidative stress and mitochondrial injury, possibly through activation of iNOS and NLRP3 signaling.     

Control and interaction of the cardiovascular and respiratory systems in anuran amphibians

In anuran amphibians, respiratory rhythm is generated within the central nervous system (CNS) and is modulated by chemo- and mechanoreceptors located in the vascular system and within the CNS. The site for central respiratory rhythmogenesis and the role of various neurotransmitters and neuromodulators is described. Ventilatory air flow is generated by a positive pressure, buccal force pump driven by efferent motor output from cranial nerves. The vagus (cranial nerve X) also controls heart rate and pulmocutaneous arterial resistance that, in turn, affect cardiac shunts within the undivided anuran ventricle; however, little is known about the control of central vagal motor outflow to the heart and pulmocutaneous artery. Anatomical evidence indicates a close proximity of the centers responsible for respiratory rhythmogenesis and the vagal motoneurons involved in cardiovascular regulation. Furthermore, anurans in which phasic feedback from chemo- and mechanoreceptors is prevented by artificial ventilation exhibit cardiorespiratory interactions that appear similar to those of conscious animals. These observations indicate interactions between respiratory and cardiovascular centers within the CNS. Thus, like mammals and other air-breathing vertebrates, the cardio-respiratory interactions in anurans result from both feedback and feed-forward mechanisms.     

Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle?

The majority of contractile calcium in cardiac muscle is released from stores in the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release (CICR) through ryanodine receptors. Because CICR is intrinsically self-reinforcing, the stability of and graded regulation of cardiac EC coupling appear paradoxical. It is now well established that this gradation results from the stochastic recruitment of varying numbers of elementary local release events, which may themselves be regenerative, and which can be directly observed as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices, and most calcium sparks are now believed to involve activation of multiple RyRs. This implies that local CICR is regenerative, requiring a mechanism to terminate it. It was initially assumed that this mechanism was inactivation of the RyR, but during the decade since the discovery of sparks, no sufficiently strong inactivation mechanism has been demonstrated in vitro and all empirically determined gating schemes for the RyR give unstable EC coupling in Monte Carlo simulations. We consider here possible release termination mechanisms. Stochastic attrition is the spontaneous decay of active clusters due to random channel closure; calculations show that it is much too slow unless assisted by another process. Calcium-dependent RyR inactivation involving third-party proteins remains a viable but speculative mechanism; current candidates include calmodulin and sorcin. Local depletion of SR release terminal calcium could terminate release, however calculations and measurements leave it uncertain whether a sufficient diffusion resistance exists within the SR to sustain such depletion. Depletion could be assisted by dependence of RyR activity on SR lumenal [Ca(2+)]. There is substantial evidence for such lumenal activation, but it is not clear if it is a strong enough effect to account for the robust termination of sparks. The existence of direct interactions among clustered RyRs might account for the discrepancy between the inactivation properties of isolated RyRs and intact clusters. Such coupled gating remains controversial. Determining the mechanism of release termination is the outstanding unsolved problem of cardiac EC coupling, and will probably require extensive genetic manipulation of the EC coupling apparatus in its native environment to unravel the solution.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres

C-reactive protein (CRP) is a positive, acute-phase protein. Plasma levels rise dramatically in response to tissue injury or inflammation and fall rapidly after recovery or treatment. Antibody-based human CRP test systems do not readily detect CRP from other animals due to the species specificity of antibodies directed against human CRP. Thus, generic systems for CRP detection, based solely on the interaction between CRP and phosphocholine (PC), have been developed. PC-bovine serum albumin (PC-BSA) conjugates were produced and either labeled with horseradish peroxidase to facilitate CRP detection in a CRP enzyme-linked sorbent assay (ELSA) or coupled to carboxy-modified microspheres to facilitate the nonenzymatic, turbidimetric detection of CRP. The CRP-ELSA is a competitive assay, where the total assay time is 45 min, the assay sensitivity is 1.06 mg/L CRP, and the dynamic range of the assay is 0-500 mg/L. When PC-BSA conjugate is covalently coupled to carboxylated microspheres, agglutination occurs in the presence of CRP, the extent of which depends on the quantity of CRP present in the sample. Total assay time is 5 min with a dynamic range of 25-500 mg/L. Both assay formats are capable of accurately detecting human CRP and the CRP-ELSA can detect canine CRP as a disease state indicator.     

Identification of carbonyl sulfide and sulfur dioxide in porcine coronary artery by gas chromatography/mass spectrometry, possible relevance to EDHF

Incubation of porcine coronary artery rings and cardiac muscle tissue in Krebs buffer followed by GC/MS analysis of the headspace gas revealed two gases, carbonyl sulfide (COS) and sulfur dioxide (SO(2)). The gases were identified by characteristic ions obtained by electron ionization, and by comparison of the retention time on a chromatographic column (GS GasPro) with standards of these gases. Stimulation of the arterial rings with acetylcholine and calcium ionophore A23187 increased the levels of SO(2) and COS in the vascular tissue. We also provide evidence that SO(2) could originate from disproportionation of a very unstable gas, sulfur monoxide (S=O). We suggest potential origins of these gases and discuss their relevance to endothelium-derived hyperpolarizing factor.     

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.