The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membraneous network that, through the cyclical release and re-uptake of Ca²⁺ into and from, respectively, the cytoplasmic space, regulates myofilament shortening and, therefore, muscle contraction. SR derived from the terminal cisternae (HSR) demonstrates the property of Ca²⁺-induced Ca²⁺ release. Upon attainment of a threshold intralumenal Ca²⁺ load, application of a small pulse of extralumenal Ca²⁺ stimulates the release of a pool of intralumenal Ca²⁺ via the ligand gated Ca²⁺ permeable pore of the Ca²⁺ release channel/ryanodine receptor complex. It was hypothesised that intralumenal Ca²⁺ regulates the opening of the release channel. HSR vesicles were purified from skeletal and cardiac muscle by a novel technique. Structural characterisation of these membranes demonstrated an enrichment of harvested fractions in the Ca²⁺ release channel and the intralumenal Ca²⁺ binding protein, calsequestrin. In radiometric studies, skeletal HSR vesicles were shown to bind ryanodine with high capacity at both low and high affinity sites, with 2 fold stimulation of Ca²⁺ accumulation by the polyorganic cation Ca²⁺ channel blocker, ruthenium red. HSR vesicles passively loaded Ca²⁺. Passive loading of HSR vesicles with Ca²⁺ was found to be non-linearly dependent upon the concentration of Ca²⁺ within the loading medium. This suggested the presence of 2 intralumenal Ca²⁺ binding sites with different affinities for Ca²⁺. A spectroscopic dual-wavelength assay of Ca²⁺ release was developed that took advantage of peculiar spectral properties of the metallochromic sensitive dye Antipyrylazo III. In the presence of mM MgATP and mM Mg2+ the initial fast phase of HSR Ca²⁺ was well resolved. Evidence was presented that initial rapid uptake was associated with high affinity binding to an intralumenal compartment. Ca²⁺ -induced Caz+ release was shown to occur with a threshold loading of intralumenal Ca²⁺. The intralumenal Ca²⁺ threshold for Ca²⁺-induced Ca²⁺ release was decreased in the presence of ryanodine. Ryanodine induced Ca²⁺ release was also dependent upon the amount of intralumenal Ca²⁺. Ryanodine was also shown to inhibit sustained Ca²⁺-induced Ca²⁺ release by apparent inhibition of the binding of Ca²⁺ to intralumenal sites. These results suggested that junctional state transitions of the Ca²⁺ channel and calsequestrin were interdependent. Purified mM and mM Ca²⁺ activated neutral protease isoforms selectively cleaved the Ca²⁺ channel into 410 and 150kDa peptides with limited proteolysis. This was demonstrated in both HSR vesicles and the purified Ca²⁺ release channel. A novel 88kDa protein was also shown to be fragmented by both CANP isoforms. The identity of this prominent HSR associated protein remains obscure. CANP fragmentation of HSR protein elevated passive and active 4^Ca²⁺ loading in vesicles. This indicated that selective structural modification of the cytoplasmic portion of the release channel modified the comformational states of a intralumenal Ca²⁺ binding compartment in HSR vesicles. In spectroscopic studies, CANP proteolysis of HSR proteins increased the sensitivity to Ca²⁺ and ryanodine-induced Ca²⁺ release through decreases in the required intralumenal Ca²⁺ threshold for release. These functional alterations coincided with apparent single site cleavage of the release channel. Further proteolysis of the initial 410 and 150kDa peptides was without further significant effect upon function. Based upon the hypothesis that primary sequences rich in proline (P), glutamate (E), aspartate (D), serine (S) and threonine (T) (PEST regions) are recognition sites for CANP binding to substrates, a search for PEST regions within the Ca²⁺ channel was undertaken. It was tentatively proposed that two PEST regions near the N-terminal of the Caz release channel may represent sites close to the CANP cleavage site. The results of this work were discussed in relation to a possible role of Ca²⁺-induced Ca²⁺ release in regulating the patterning of Ca²⁺ cytosolic transients. The frequency and amplitude of cytosolic Ca²⁺ transients appear to be important in regulating protein expression. The requirement of intralumenal Ca²⁺-induced Ca²⁺ release may be a means by which the cyclical uptake and release of Ca²⁺ during muscle relaxation and contraction can be coordinated. This coordination may define the patterning of cytosolic Ca²⁺ transients. The increased sensitivity to Ca²⁺-induced Ca²⁺ release by HSR after CANP treatment may represent a means by which the patterning of cytosolic Ca²⁺ transients can be altered to effect changes in protein synthesis. / Graduate and Postdoctoral Studies / Graduate

The production of reactive oxygen species (ROS) may be a contributor to the progression of sarcopenia. Sarcopenia is a generic term for the loss of skeletal muscle mass, quality and strength. ROS are usually produced by radiation, but are also the byproducts of aerobic metabolism. ROS have been found to mediate various pathological conditions in a variety of tissues, to cause oxidative damage to DNA, proteins, and lipids with advancing age, and is presumably a major factor contributing to changes associated with aging. The purpose of this investigation was to determine whether the sarcoplasmic reticulum (SR) of muscle from aged animals are more susceptible to the deleterious effects of ROS. Using isolated gastrocnemius SR vesicles extracted from adult (12m) and aged (27m) male Brown Norway-Fischer 344 hybrid rats, Ca2+ uptake and release measurements were obtained. The data showed that there was a 33% difference between aged and adult gastrocnemius mass. When gastrocnemius mass was corrected for body mass, the differences was ~20% between the two groups. A 20% decrease in SR Ca2+ uptake rate was noted in aged animals. HOCl also, decreased uptake by similar extents in both groups. This result suggest that the Ca2+ pump's response to ROS are similar in both groups. AgNO3 -induced and H2O2 -induced release in aged animals was 17.94 and 7.39 nmol/mg/min and in adult animals was 30.46 and 7.18 nmol/mg/min, respectively. H2O2-induced release, when expressed as a percent of AgNO3-induced release was increased in aged animals by 54%. The results suggest that the release channel of aged muscle appears to be more sensitive to ROS. In conclusion, the data support the theory that aged animal skeletal muscle is more susceptible to the adverse effects of ROS. / Master of Science

Isotopic and spectrophotometric assays show that micromolar concentrations of heavy metal ions (particularly Ag⁺ and Hg²⁺) stimulate ATPase activity but inhibit Ca²⁺ uptake in isolated SR vesicles. Both effects are caused by increased Ca²⁺ permeability of the membrane, apparently the result of activation of the Ca²⁺ release system of the vesicles due to heavy metal binding to a critical sulfhydryl group associated with the Ca²⁺ release channels. CU²⁺catalyzed co-oxidation of this sulfhydryl with exogenous cysteine to form a mixed disulfide also results in activation of the Ca²⁺ release system. The rate and extent of Cu²⁺/cysteine-induced release is maximal at physiological pH and is inhibited by local anaesthetics and Mg²⁺, suggesting that the redox state of this sulfhydryl may play a role in activation of the Ca²⁺ release system of intact muscle. Modification of the SR with the proteolytic enzyme trypsin also increases the Ca²⁺ permeability of the SR, and enhances the rate of Ca²⁺ release activated by cAMP, doxorubicin, Hg²⁺, and Cu²⁺/cysteine. The rates of release activated by all reagents are stimulated by a factor of five after five minutes exposure to trypsin. Hg²⁺- and Cu²⁺/cysteine-activated release are not stimulated further, while cAMP- and doxorubicin-activated release continue to increase up to maximum of 20-fold stimulation after 15 minutes exposure to trypsin. Inhibitors of the Ca²⁺ release system such as Mg²⁺ and ruthenium red still inhibit release from proteolytically modified SR, and the binding affinities of activators and inhibitors to their sites are not significantly altered by proteolysis; only the rates of Ca²⁺ transport are affected. The most probable mechanisms of tryptic stimulation of Ca²⁺ release are (1) removal of a regulatory protein or subunit of the Ca²⁺ release system, making more channels available for transport; (2) increasing the single channel unitary conductance; (3) increasing the open time of activated channels. The biphasic character of proteolytic stimulation of cAMP- and doxorubicin-activated release (as opposed to monophasic stimulation of Hg²⁺- and Cu²⁺/cysteine-activated release} suggests that more than one of the above parameters are involved in tryptic stimulation of the Ca²⁺ release system.

Muscle contraction and relaxation are controlled by the intracellular free Ca²⁺ concentration. The sarcoplasmic reticulum (SR) is an intracellular membrane system which regulates this internal free Ca²⁺ concentration. Responding to an electrical excitation of the cell surface membrane, the SR releases Ca²⁺ through a specific Ca²⁺ release channel, thus elevating the Ca²⁺ concentration inside muscle cell and causing the muscle to contract. Subsequent sequestration of Ca²⁺ by the SR Ca²⁺ pumps restores the resting state of the muscle cell. This research focuses on the Ca²⁺ release channel from skeletal muscle SR. The planar lipid bilayer technique was used to study the channel at the single channel level. The SR Ca²⁺ release channel was identified and isolated via its interaction with specific sulfhydryl oxidizing agents. This protein of a molecular mass of 106 kDa was then incorporated into a planar lipid bilayer membrane (BLM). In an asymmetrical Ca²⁺ solution, the channel protein demonstrates a single channel conductance of 107 ± 13 pS and a permeability ratio of Ca²⁺ versus Tris⁺ of 7.4 ± 3.3. In a symmetrical 250 mM NaCl solution, the channel protein displays a large single channel conductance of 400 ± 20 pS, and a weak voltage-dependence. The channel is activated by millimolar ATP and inhibited by micromolar ruthenium red. Nanomolar concentrations of ryanodine modify the channel by changing it from a rapidly gating full conductance state to a long-lived subconductance state. These results demonstrate that the isolated 106 kDa protein channel has properties similar to those observed following fusion of SR vesicles to a BLM. The bilayer system was also used to examine the effect of Ag⁺ on the SR Ca²⁺ release channel. Ag⁺ (0.2-1. 0 μM ) activates the SR Ca²⁺ release channel. Activation by Ag⁺ does not require the presence of Ca²⁺, Mg²⁺, or ATP. Ag⁺ activates the channel by increasing the open probability Po. Ag⁺ activation is always followed by a spontaneous inactivation. The channel is still sensitive to ruthenium red inhibition after exposure to Ag⁺. Isolated SR vesicles were fused to a BLM to study the effect of the photooxidizing dye, rose bengal, on the gating characteristics of the reconstituted SR Ca²⁺ release channel. Rose bengal activates the Ca²⁺ release channel in the presence of light by increasing the channel open probability and leaving the single channel conductance unchanged. This photoactivation is independent of the myoplasmic Ca²⁺ concentration, and can be achieved from either side of the membrane. In addition, the effect is inhibited by addition of 10-20 μM ruthenium red. When modified to its subconducting state by ryanodine, subsequent addition of rose Bengal reactivates the channel to a rapidly fluctuating full conducting state. These studies carried out at the single channel level utilizing the planar lipid bilayer technique have not only enhanced our understanding of the Ca²⁺ release mechanism of skeletal muscle SR, but also provided information about the toxic effects on biological membrane systems caused by heavy metals and oxidizing agents.

Calcium ions are important mediators in the mechanism of contraction and relaxation of muscle fibers. Depolarization of sarcolemma and transverse tubule causes an increase of myoplasmic ca2+ concentration which induces contraction of the myofibrils. In skeletal muscle fibers, the intracellular Ca2+ concentraton is regulated by an extensive membrane system, the sarcoplasmic reticulum (SR). Ca2+-release from SR is initiated by depolarization of the transverse tubule via a process referred to as excitation-contraction coupling. The Ca2+ - release channel located in the junctional SR plays an important role in this mechanism.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 183-216). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 183-216). Also available online.

A prominent finding in the diabetic rat heart is a decrease in the rate at which the ventricular muscle can contract and relax. Since cardiac sarcoplasmic reticulum is thought to be intimately involved in muscle contraction and relaxation we studied the ability of diabetic rat cardiac sarcoplasmic reticulum to transport Ca²⁺ . Hearts were obtained from female Wistar rats 7, 30, 42, and 120 days after the induction of diabetes by a single i.v. injection of either alloxan (65 mg/kg) or streptozotocin (60 mg/kg). At all Ca²⁺ concentrations tested (0.2 μM-5.0 μM free Ca²⁺) cardiac sarcoplasmic reticulum obtained from 42 and 120 day diabetic rats showed a significant decrease in the rate of ATP-dependent tns-oxalate facilitated ²⁺ransport. This was accompanied by a decrease in Ca²⁺ -ATPase activity. The levels of long chain acylcarnitines associated with the microsomal sarcoplasmic reticulum preparation from 42 and 120 day diabetic rats were significantly higher than those present in sarcoplasmic reticulum from control rats. Palmitylcarnitine, the most abundant of the long chain acylcarnitines, in concentrations < 7 μM was found to be a potent time-dependent inhibitor of both Ca²⁺ transport and Ca²⁺ -ATPase in both control and diabetic rat sarcoplasmic reticulum preparations; inhibition of Ca²⁺ transport was found to be more marked in the control preparations. This would indicate that a degree of inhibition produced by the high endogenous levels of palmitylcarnitine may already be present in the diabetic rat heart preparations. Cardiac sarcoplasmic reticulum prepared from acutely diabetic rats (7 days) did not show any decrease in Ca²⁺ transport ability. Levels of long chain acylcarnitines associated with the microsomal preparation enriched in sarcoplasmic reticulum were also unchanged. Insulin treatment of diabetic rats could significantly increase the ability of cardiac sarcoplasmic reticulum to transport Ca²⁺, although at the time period obtested (30 days) the SR Ca²⁺ transport activity was only slightly depressed as compared to control. Insulin treatment also resulted in a slight, but non-significant, lowering of the levels of long chain acylcarnitines associated with the sarcoplasmic reticulum microsomal preparations. These findings suggest that the alteration in sarcoplasmic reticulum function in chronically diabetic rats may be due to the buildup of cellular long chain acylcarnitines which inhibit sarcoplasmic reticulum Ca²⁺ transport. The absence of any significant change in Ca²⁺ transport activity or levels of long chain acylcarnitines at 7 and 30 days suggests that the alterations in 42 and 120 day diabetic rats must be of gradual onset. Cardiac sarcoplasmic reticulum is known to be regulated by a number of factors, among them calmodulin, cAMP-dependent protein kinase, and K⁺. Since Ca²⁺ transport activity in cardiac sarcoplasmic reticulum from chronically diabetic rats is depressed, the role that these regulators play was investigated. Calmodulin (0.61 μM), cAMP (10 μM) plus cAMP-dependent protein kinase (0.2 mg/0.5 ml), and K⁺ (0-110 mM) all stimulated Ca transport in both control and streptozotocin-treated diabetic rats to the same degree. This suggests that the depression observed in sarcoplasmic reticulum function from diabetic rats is not due to altered regulation by these putative mediators of Ca²⁺ uptake. A number of studies suggest that carnitine administration may lower myocardial levels of long chain acylcarnitines in the diabetic rat. Therefore, D,L-carnitine (1 g/kg/day, orally) was administered to 120 day diabetic rats for a 30 day period. The elevated levels of long chain acylcarnitines normally seen in diabetic rats were significantly reduced in the diabetic rats administered carnitine. Carnitine administration, however, could not reverse the previously noted depression in diabetic rat heart function, as measured on an isolated working heart apparatus. In an effort to prevent the onset of the diabetic cardiomyopathy D,L-carnitine was administered (3 g/kg/day, orally) 3 days after the induction of diabetes for a 42 day period. As previously mentioned, sarcoplasmic reticulum Ca²⁺ transport activity was depressed in diabetic rats, as compared to control rats, at all free Ca²⁺ concentrations tested (0.1 μM-3.5 μM). Similarly, sarcoplasmic reticulum levels of long chain acylcarnitines were significantly elevated in these diabetic rats. The diabetic rats treated with carnitine did not show any depression in Ca²⁺ transport activity; long chain acylcarnitine levels were also similar to control. The carnitine-treated diabetic rats, however, showed no improvement in heart function compared to untreated-diabetic rats. These data suggest that although the long chain acylcarnitines are inhibiting cardiac sarcoplasmic reticulum function in chronically diabetic rats other factors must also be contributing to the depression in heart function. / Pharmaceutical Sciences, Faculty of / Graduate

The sarcoplasmic reticulum Ca²⁺ -pumping ATPase is the primary system responsible for the removal of calcium from the sarcoplasm during relaxation of skeletal and cardiac muscles. Since the rat heart SR is used frequently in our laboratory to study the Ca²⁺ -transport defects in disease states, the Ca²⁺ - ATPase activity of this system was characterized. Calmodulin (CaM) and cAMP-dependent protein kinase (cAMP-PK) are known to regulate the dog cardiac SR Ca²⁺ -pump. The effects of these regulators on the rat heart SR Ca²⁺ -pump were studied. Studies were also carried out to investigate the effects of Triton X-100 on SR Ca²⁺ -ATPase activity and the regulation of this activity by CaM. The rat heart SR Ca²⁺-ATPase was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Magnesium produced a concentration-dependent increase in the basal ATPase activity without affecting the maximal ATPase activity. This appeared to result in a gradual disappearance of the Ca²⁺ dependency of the ATPase activity. Addition of 100µM CDTA (trans-1,2-diaminocyclo- hexane-N,N,N',N'-tetraacetic acid), in the absence of added magnesium, produced no effect on Ca²⁺ stimulation of ATPase activity. The results appear to indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase. At a constant Mg: ATP ratio, ATP simulated the SR Ca²⁺-ATPase activity in a concentration-dependent manner. Double-reciprocal plots of the data suggest that the true substrate for rat heart SR Ca²⁺-ATPase may be ATP and not Mg.ATP. In the crude SR, CaM did not stimulate total or Ca²⁺-stimulated ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations. CaM also failed to stimulate membrane phosphorylation over a range of Mg²⁺ concentrations. Furthermore, CaM did not produce a significant effect on calcium transport into SR vesicles. The catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating membrane phosphorylation and Ca²⁺ -ATPase activity. Two CaM antagonists, trifluperazine and compound 48/80, did not affect the rat heart SR ATPase activity. The ATPase activity in Triton-washed SR membranes appeared to be increased at low Triton concentrations. This effect was probably due to the removal of non-intrinsic proteins, leaky vesicles or altered membrane fluidity. At higher Triton X-100 concentrations, the ATPase activity was lost, probably due to loss of the phospholipid environment. When SR membranes phosphorylated under conditions similar to those used for the ATPase assay were analysed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) followed by autoradiography, a single phosphorylated protein of 7,500-9,000 dalton was observed. This protein may represent the monomeric form of phospholamban. CaM, however, appeared to have no effect on the phosphorylation of this 7,500-9,000 dalton protein in either untreated or Tritan-washed SR membranes. It is speculated that the rat heart SR contains tightly bound CaM which cannot be removed by treatment with Triton X-100. / Pharmaceutical Sciences, Faculty of / Graduate

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.