No description available.

Standard local control theory, which describes Ca²⁺ release during excitation-contraction coupling (ECC), assumes that all Ryanodine Receptor (RyR) complexes are equivalent. Recent data from our laboratory has called this assumption into question. Specifically, we have shown that RyR complexes in ventricular myocytes differ depending on their location within the cell. This, and other data, has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labeled enzymatically-isolated, fixed myocytes to examine the distribution and colocalization of RyR, calsequestrin (CSQ), voltage-gated Ca²⁺ channels (Cav1.2), sodium-calcium exchangers (NCX), and caveolin-3 (cav-3). All images were acquired on a wide-field microscope, deconvolved, and subject to extensive analysis, including a novel method of measuring statistical significance of the recorded colocalization values. Overall, eight surface RyR populations were identified, depending on its binding partners. One of these groups, in which RyR, Cav1.2, and NCX colocalize, may provide the structural basis for ‘eager’ sites of Ca²⁺ release in atria, while other groups were defined based on their association with cav-3, and are therefore highly likely to be under the influence of other signaling molecules located within caveolae. Importantly, although a small portion of the surface RyR in atria do colocalize with NCX alone, the majority are tightly linked to Cav1.2 alone or Cav1.2 and NCX together. Therefore, it appears likely that Cav1.2-mediated calcium-induced calcium release (CICR) is the primary method of initiating Ca²⁺ release from the SR during EC coupling.

Standard local control theory, which describes Ca²⁺ release during excitation-contraction coupling (ECC), assumes that all Ryanodine Receptor (RyR) complexes are equivalent. Recent data from our laboratory has called this assumption into question. Specifically, we have shown that RyR complexes in ventricular myocytes differ depending on their location within the cell. This, and other data, has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labeled enzymatically-isolated, fixed myocytes to examine the distribution and colocalization of RyR, calsequestrin (CSQ), voltage-gated Ca²⁺ channels (Cav1.2), sodium-calcium exchangers (NCX), and caveolin-3 (cav-3). All images were acquired on a wide-field microscope, deconvolved, and subject to extensive analysis, including a novel method of measuring statistical significance of the recorded colocalization values. Overall, eight surface RyR populations were identified, depending on its binding partners. One of these groups, in which RyR, Cav1.2, and NCX colocalize, may provide the structural basis for ‘eager’ sites of Ca²⁺ release in atria, while other groups were defined based on their association with cav-3, and are therefore highly likely to be under the influence of other signaling molecules located within caveolae. Importantly, although a small portion of the surface RyR in atria do colocalize with NCX alone, the majority are tightly linked to Cav1.2 alone or Cav1.2 and NCX together. Therefore, it appears likely that Cav1.2-mediated calcium-induced calcium release (CICR) is the primary method of initiating Ca²⁺ release from the SR during EC coupling.

Standard local control theory, which describes Ca²⁺ release during excitation-contraction coupling (ECC), assumes that all Ryanodine Receptor (RyR) complexes are equivalent. Recent data from our laboratory has called this assumption into question. Specifically, we have shown that RyR complexes in ventricular myocytes differ depending on their location within the cell. This, and other data, has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labeled enzymatically-isolated, fixed myocytes to examine the distribution and colocalization of RyR, calsequestrin (CSQ), voltage-gated Ca²⁺ channels (Cav1.2), sodium-calcium exchangers (NCX), and caveolin-3 (cav-3). All images were acquired on a wide-field microscope, deconvolved, and subject to extensive analysis, including a novel method of measuring statistical significance of the recorded colocalization values. Overall, eight surface RyR populations were identified, depending on its binding partners. One of these groups, in which RyR, Cav1.2, and NCX colocalize, may provide the structural basis for ‘eager’ sites of Ca²⁺ release in atria, while other groups were defined based on their association with cav-3, and are therefore highly likely to be under the influence of other signaling molecules located within caveolae. Importantly, although a small portion of the surface RyR in atria do colocalize with NCX alone, the majority are tightly linked to Cav1.2 alone or Cav1.2 and NCX together. Therefore, it appears likely that Cav1.2-mediated calcium-induced calcium release (CICR) is the primary method of initiating Ca²⁺ release from the SR during EC coupling. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The cardiac ryanodine receptor (RyR2) plays a key role in excitation-contraction coupling (ECC). Mutations in RyR2 are known to be linked to the arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), a deadly disease which is characterized by a leak of calcium from sarcoplasmic reticulum and a decrease in calmodulin (CaM) binding. A novel drug, 84F2, shown to inhibit arrhythmias in RyR2-R176Q heterozygous CPVT mouse hearts (2.5 µg/kg), decrease spark frequency in cells derived from CPVT mice (IC50 = 35 nM), and inhibit RyR2 single channel activity at low nanomolar concentrations (IC50 = 8 nM). When CaM is added back to RyR2, 84F2's ability to inhibit channel activity is suppressed approximately 250 fold. A metabolite of 84F2, 78F3, is shown to also be active in the inhibition of RyR2. We propose that 84F2 decreases arrhythmias by binding to the CaM deficient RyR2, but does not affect normal ECC when CaM is present. This work characterizes for the first time a class of drugs whose inhibitory affects are dependent upon the removal of CaM from RyR2.

No description available.

During skeletal muscle stimulation, there is a summation of local events of Ca2+ release from the sarcoplasmic reticulum, known as Ca2+ sparks. Ca2+ sparks originate from groups of skeletal ryanodine receptors (RyR1) that activate and close in synchrony. This synchrony allows for the rapid and massive release of Ca2+ from the sarcoplasmic reticulum to initiate contraction and, more important, would provide a mechanism to terminate Ca2+ release under conditions where independent RyR1 are normally active. RyR1 mutations can result in abnormal intracellular Ca2+ signaling that is associated with numerous skeletal muscle disorders including malignant hyperthermia and central core disease. Therefore, investigating the mechanisms that control RyR1 function can help identify how these mutations cause deleterious Ca2+ handling. Currently, most published research on RyR1s gating utilizes single RyR1 reconstituted into planar lipid bilayers to test isolated RyR1. However, in vivo, arrays of RyR1 function in synchrony. Attempts to reconstitute RyR1s into planar lipid bilayers result in experiments that contain multiple channels, which under specific conditions may gate in synchrony, also known as coupled gating. Coupled RyR1 gating was first reported by A. Marks' laboratory and attributed to FK-506 binding protein 12 (FKBP12) associating with neighboring RyR1s the stabilization of RyR1-RyR1 interactions that promote coupled gating. Previous studies suggested that ATP is required for coupled RyR1 gating; however, the mechanism by which ATP promotes the coordinated activity of RyR1s has not been elucidated and is the focus of this thesis. Therefore, my hypothesis is that the agonist action of ATP and FKBP12 bound to RyR1 are required for coupled RyR1 gating. In addition, new pharmacological tools are required to better understand coupled RyR gating. Thus, an additional goal is to identify pharmacological agents that modulate RyR1s in an innovative manner, i.e., help to uncover novel aspects of RyR1 gating and conduction. This investigation suggests that the adenosine based nucleotides, ATP, ADP and AMP, are agonists of RyR1s and promote coupled RyR1 gating in planar lipid bilayers. However, ADP and AMP were unable to maintain coupled RyR1 gating with physiological levels of Mg2+. This suggests that coupled gating would be impaired when the levels ATP decrease, as in muscle fatigue. When ATP was compared to other nucleotides (GTP, ITP, and TTP), the results suggest that the nucleotide agonist action on RyR1s is dependent on the phosphate groups and amino group on the nucleobase. As ATP is the most efficient nucleotide for coupled gating, I also investigated the indirect action of ATP to act as a kinase substrate or alter the cytoskeletal network. The addition of kinases, phosphatases and cytoskeletal modulators did not produce a significant disruption of coupled RyR1 gating. I also tested the role of addition of exogenous FKBP12 to RyR1s that gated independently or had partial coupling, but coupled gating was never improved. Also, the addition of high doses of rapamycin to remove FKBP12 from coupled RyR1 failed to functionally uncouple the channels. Finally, I attempted to find pharmacological agents that could aid in the understanding of coupled RyR1. Some agents were found to modulate RyR1s; however, I did not find a probe that would affect kinetics/conductance of RyR1s and was suitable for comparing coupled gating in bilayers with Ca2+ sparks in cells. Overall, coupled RyR gating is dependent on the physiological modulators ATP and Mg2+. This thesis represents a step forward in identifying the requirements for coupled RyR1 gating and understanding how RyR1s function in cells. Until an understanding of how these receptors communicate in cells is obtained, how different mutations alter the Ca2+ leak will continue to be quite difficult to study.

Elevated levels in blood serum (≥10μmol/L) of the amino acid homocysteine is strongly correlated with the incidence of heart failure (HF). We present evidence that the cyclic thioester, homocysteine thiolactone (HTL), a metabolic product of homocysteine, irreversibly modifies proteins that regulate the contractile process in cardiac muscle. Two proteins found in the sarcoplasmic reticulum (SR), the Ca2+ pump (SERCA2), and the ryanodine receptor (RyR2), are responsible for controlling the cytosolic Ca2+ concentration and hence the contractile state of the heart. While both improper Ca2+ handling and elevated homocysteine levels have been considered bio-markers in HF, a direct connection between the two has not previously been made. We show that HTL reacts with lysine residues on RyR2, generating a Nε-homocysteine-protein, which results in carbonyl formation and a change in the Ca2+ sensitivity of RyR2. This is a new molecular mechanism linking elevated levels of Homocysteine, improper Ca2+ handling and heart failure. This work was supported by NIH 1 R41 HL105063-01 to J. Abramson and R. Strongin.

The cardiac ryanodine receptor (RyR2) is a Ca2+ ion channel found in the sarcoplasmic reticulum (SR), an intracellular membranous Ca2+ storage system. It is well known that a destabilization of RyR2 can lead to a Ca2+ flux out of the SR, which results in an overload of intracellular Ca2+; this can also lead to arrhythmias and heart failure. The catecholamines play a large role in the regulation of RyR2; stimulation of the Beta-adrenergic receptor on the cell membrane can lead to a hyperphosphorylation of RyR2, making it more leaky to Ca2+. We have previously shown that strong electron donors will inhibit RyR2. It is hypothesized that the catecholamines, sharing a similar structure with other proven inhibitors of RyR2, will also inhibit RyR2. Here we confirm this hypothesis and show for the first time that the catecholamines, isoproterenol and epinephrine, act as strong electron donors and inhibit RyR2 activity at the single channel level. This data suggests that the catecholamines can influence RyR2 activity at two levels. This offers promising insight into the potential development of a new class of drugs to treat heart failure and arrhythmia; ones that can both prevent the hyperphosphorylation of RyR2 by blocking the Beta;-adrenergic receptor, but can also directly inhibit the release of Ca2+ from RyR2.

The principle calcium regulator in the muscle cell is the calcium ion release channel (RyR). Improper calcium homeostasis in the muscle cell is the foundation of many pathological states and has been targeted as a contributing factor to ventricular tachycardia, which is known to precede sudden cardiac arrest. Numerous endogenous and exogenous compounds can affect the way RyR regulates calcium. In this study the anthocyanidin Pelargonidin (Pg), an important natural colorant and dietary antioxidant, is evaluated for its effect on regulating the transport of calcium through the RyR1 of skeletal muscle sarcoplasmic reticulum. Pelargonidin undergoes time dependent structural changes in aqueous solutions at physiological pH and a mixture of up to seven forms of Pelargonidin are present in solution simultaneously. Pelargonidin is a unique RyR1 modulator. It can both stimulate and inhibit the RyR1 depending on the experimental conditions. In addition, when Pelargonidin is irradiated with white light, its inhibition properties on the RyR1 are essentially nullified. Proposed mechanisms include excited state charge shift within RyR1-Pg complexes.