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The incidence of sudden cardiac death (SCD) in people living with HIV infection (PLWH), especially those with inadequate viral suppression, is high and the reasons for this remain incompletely characterized. The timely opening and closing of type 2 ryanodine receptor (RyR2) is critical for ensuring rhythmic cardiac contraction–relaxation cycles, and the disruption of these processes can elicit Ca2+ waves, ventricular arrhythmias, and SCD. Herein, we show that the HIV protein Tat (HIV-Tat: 0–52 ng/mL) and therapeutic levels of the antiretroviral drugs atazanavir (ATV: 0–25,344 ng/mL), efavirenz (EFV: 0–11,376 ng/mL), and ritonavir (RTV: 0–25,956 ng/mL) bind to and modulate the opening and closing of RyR2. Abacavir (0–14,315 ng/mL), bictegravir (0–22,469 ng/mL), Rilpivirine (0–14,360 ng/mL), and tenofovir disoproxil fumarate (0–18,321 ng/mL) did not alter [3H]ryanodine binding to RyR2. Pretreating RyR2 with low HIV-Tat (14 ng/mL) potentiated the abilities of ATV and RTV to bind to open RyR2 and enhanced their ability to bind to EFV to close RyR2. In silico molecular docking using a Schrodinger Prime protein–protein docking algorithm identified three thermodynamically favored interacting sites for HIV-Tat on RyR2. The most favored site resides between amino acids (AA) 1702–1963; the second favored site resides between AA 467–1465, and the third site resides between AA 201–1816. Collectively, these new data show that HIV-Tat, ATV, EFV, and RTV can bind to and modulate the activity of RyR2 and that HIV-Tat can exacerbate the actions of ATV, EFV, and RTV on RyR2. Whether the modulation of RyR2 by these agents increases the risk of arrhythmias and SCD remains to be explored. [ABSTRACT FROM AUTHOR]

Background and Purpose Ryanodine receptors ( Ry Rs) are Ca2+-release channels on the sarco(endo)plasmic reticulum that modulate a wide array of physiological functions. Three Ry R isoforms are present in cells: Ry R1, Ry R2 and Ry R3. To date, there are no reports on ligands that modulate Ry R in an isoform-selective manner. Such ligands are not only valuable research tools, but could serve as intermediates for development of therapeutics. Experimental approach Pyrrole-2-carboxylic acid and 1,3-dicyclohexylcarbodiimide were allowed to react in carbon tetrachloride for 24 h at low temperatures and pressures. The chemical structures of the two products isolated were elucidated using NMR spectrometry, mass spectrometry and elemental analyses. [3 H]-ryanodine binding, lipid bilayer and time-lapsed confocal imaging were used to determine their effects on Ry R isoforms. Key results The major product, 2-cyclohexyl-3-cyclohexylimino-2, 3, dihydro-pyrrolo[1,2- c]imidazol-1-one ( CCDI) dose-dependently potentiated Ca2+-dependent binding of [3 H]-ryanodine to Ry R1, with no significant effects on [3 H]-ryanodine binding to Ry R2 or Ry R3. CCDI also reversibly increased the open probability ( Po) of Ry R1 with minimal effects on Ry R2 and Ry R3. CCDI induced Ca2+ transients in C2 C12 skeletal myotubes, but not in rat ventricular myocytes. This effect was blocked by pretreating cells with ryanodine. The minor product 2-cyclohexyl-pyrrolo[1,2- c]imidazole-1,3-dione had no effect on either [3 H]-ryanodine binding or Po of Ry R1, Ry R2 and Ry R3. Conclusions and implications A new ligand that preferentially modulates Ry R1 was identified. In addition to being an important research tool, the pharmacophore of this small molecule could serve as a template for the synthesis of other isoform-selective modulators of Ry Rs. [ABSTRACT FROM AUTHOR]

Aims Ventricular myocytes isolated from hearts of streptozotocin (STZ)-diabetic rats exhibit increased spontaneous Ca2+ release. Studies attribute this defect to an enhancement in activity of type 2 ryanodine receptor (RyR2). To date, underlying reasons for RyR2 dysregulation remain undefined. This study assesses whether the responsiveness of RyR2 following stimulation by intrinsic ligands is being altered during experimental type 1 diabetes (T1D). Methods and results M-mode echocardiography established a cardiomyopathy in 8 weeks STZ-diabetic rats. Confocal microscopy confirmed an increase in the spontaneous Ca2+ release in isolated ventricular myocytes. Western blots revealed no significant change in steady-state levels of the RyR2 protein. When purified to homogeneity and incorporated into planar lipid bilayers, RyR2 from STZ-diabetic rats (dRyR2) exhibited reduced current amplitude at ±35 mV. dRyR2 was also more responsive to intrinsic cytoplasmic activators Ca2+, adenosine triphosphate, and cyclic adenosine diphosphate ribose and less responsive to the cytoplasmic deactivator Mg2+. Threshold for the activation of RyR2 by trans (luminal) Ca2+ was also reduced. These changes were independent of phosphorylation at Ser2808 and Ser2814. Two weeks of insulin treatment starting after 6 weeks of diabetes blunted the phenotype change, indicating that the gain of function is specific to the diabetes and not the result of STZ interacting directly with RyR2. Conclusion These data show, for the first time, that RyR2 is acquiring a gain-of-function phenotype independent of its phosphorylation status during T1D and provides new insights for the enhanced spontaneous Ca2+ release in myocytes from T1D rats. [ABSTRACT FROM AUTHOR]

Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca2+-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBDorg, were air-dried and studied further. In competition assays, HBDorg dose-dependently displaced [³H]ryanodine from binding sites on RyRl with an IC50 of 1.5 ± 0.1µg/ml (hi= 0.4± 0.0 µg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBDorg exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C2C12 myotubes, addition of HBD triggered a rise in intracellular Ca2+ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR 1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers. [ABSTRACT FROM AUTHOR]