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We have previously shown that reduction of ischaemic cell swelling via enhanced cell volume regulation is a key mechanism of ischaemic preconditioning (IPC) in cardiomyocytes. We have also shown that pharmacological blockade of Cl(-) channels abolishes cardioprotection achieved by IPC in Langendorff-perfused hearts and freshly isolated cardiomyocytes, thus suggesting that Cl(-) plays a key role in IPC cardioprotection. However, direct evidence of Cl(-) channel activation resulting in transsarcolemmal Cl(-) efflux by IPC had been lacking. To address this issue, 24 h cultured rabbit cardiomyocytes were loaded with 5 mM 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), a specific fluorescence indicator that is quenched by Cl(-) so that cellular efflux of Cl(-) results in an increase in SPQ fluorescence.After stabilization for 10 min, cardiomyocytes were preconditioned either with 10 min simulated ischaemia/10 min simulated reperfusion or with 10 min treatment with 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA). IPC and APNEA significantly (P0.001) reduced the intracellular Cl(-) concentration ([Cl(-)](i)) to 31.9 +/- 3.2 mM (mean +/- SEM) and 32.5 +/- 2.8 mM, respectively, from an initial [Cl(-)](i) (pooled stabilization 61.5 +/- 7.1 mM). [Cl(-)](i) did not change in control (non-preconditioned) cardiomyocytes (control 58.1 +/- 1.9 mM and control + vehicle 62.6 +/- 4.9 mM, P = 0.98 and 0.99 vs. pooled pre-treatment baseline, respectively). Inhibition of Cl(-) channels with 50 microM indanyloxyacetic acid 94 completely blocked preconditioning-induced Cl(-) efflux. Thus, a net Cl(-) efflux of 29.6 and 29.0 mM was triggered by IPC and APNEA.These findings provide the first direct evidence of activation of sarcolemmal Cl(-) channels by ischaemic and pharmacological preconditioning in cardiomyocytes.

Volume regulatory Cl − channels are key regulators of ischemic preconditioning (IPC). Because Cl − efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that I K1 channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly ( P CM 18.0±2.1% versus control (C CM ) 48.3±1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPC CM +AdEGFPKir2.1DN 45.8±2.3% (mean±SEM) and IPC CM +AdEGFPKir2.2DN 47.9±1.4% versus IPC CM ; P CM + AdEGFPKir2.1DN 45.8±0.7% and C CM +AdEGFPKir2.2DN 46.2±1.3% versus C CM ; not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after I K1 blockade with 20 μmol/L BaCl 2 plus 1 μmol/L nifedipine (to prevent Ba 2+ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that I K1 channels play an important role in IPC protection.

Primary deficiency of β-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date. We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.

We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole hearts and in both fresh and cultured cardiomyocytes, as well as the improved regulatory volume decrease to hypoosmotic swelling in cardiomyocytes, is abrogated through Cl− channel blockade, pointing to a role for enhanced cell volume regulation in IPC. To further define this cardioprotective mechanism, cultured rabbit ventricular cardiomyocytes were preconditioned either by 10-min simulated ischemia (SI) followed by 10-min simulated reperfusion (SR), by 10-min exposure/10-min washout of remote IPC (rIPC) plasma dialysate (from rabbits subjected to repetitive limb ischemia), or by adenoviral transfection with the constitutively active PKC-ε gene. These interventions were done before cardiomyocytes were subjected to either 60- or 75-min SI/60-min SR to assess cell necrosis (by trypan blue staining), 30-min SI to assess ischemic cell swelling, or 30-min hypoosmotic (200 mosM) stress to assess cell volume regulation. Necrosis after SI/SR and both SI- and hypoosmotic stress-induced swelling was reduced in preconditioned cardiomyocytes compared with control cardiomyocytes (neither preconditioned nor transfected). These effects on necrosis and cell swelling were blocked by either Cl− channel blockade or dominant negative knockdown of inwardly rectifying K+ channels with adenoviruses, suggesting that Cl− and K+ movements across the sarcolemma are critical for cell volume regulation and, thereby, cell survival under hypoxic/ischemic conditions. Our results define enhanced cell volume regulation as a key common mechanism of cardioprotection by preconditioning in cardiomyocytes.

We have identified and characterized three missense mutations in a patient with type 1 G(M1) gangliosidosis, namely a substitution of G for A at nucleotide position 1044 (G1044-->A; in exon 10) on one allele, which converts Asp(332) into asparagine, and both a mutation (C492-->A in exon 4, leading to the amino acid change of Arg(148)-->Ser) and a polymorphism (A1644-->G in exon 15, leading to a change of Ser(532)-->Gly) on the other allele. This patient had less than 1% residual beta-galactosidase activity and minimally detectable levels of immunoreactive beta-galactosidase protein in fibroblasts. To account for the above findings, a series of expression and immunolocalization studies were undertaken to assess the impact of each mutation. Transient overexpression in COS-1 cells of cDNAs encoding Asp(332)Asn, Arg(148)Ser and Ser(532)Gly mutant beta-galactosidases produced abundant amounts of precursor beta-galactosidase, with activities of 0, 84 and 81% compared with the cDNA clone for wild-type beta-galactosidase (GP8). Since the level of vector-driven expression is much less in Chinese hamster ovary (CHO) cells than in COS-1 cells, and we knew that exogenous beta-galactosidase undergoes lysosomal processing when expressed in these cells, transient expression studies were performed of Arg(148)Ser and Ser(532)Gly, which yielded active forms of the enzyme. In this case, the Arg(148)Ser and Ser(532)Gly products gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg(148)Ser mutation introduced a major conformational change into the protein, and we anticipated that it would be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp(332)Asn mutation on the catalytic activity, we isolated CHO clones permanently transfected with the Asp(332)Asn and Asp(332)Glu constructs, purified the enzymes by substrate-analogue-affinity chromatography, and determined their kinetic parameters. The V(max) values of both mutant recombinant enzymes were markedly reduced (less than 0.9% of the control), and the K(m) values were unchanged compared with the corresponding wild-type enzyme isolated at the same time. Both the Arg(148)Ser beta-galactosidase in CHO cells and Asp(332)Asn beta-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal beta-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg(148)Ser mutation has no effect on catalytic activity, whereas the Asp(332)Asn mutation seriously reduces catalytic activity, suggesting that Asp(332) might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg(148)Ser and Asp(332)Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in G(M1) gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most G(M1) gangliosidosis patient fibroblasts.

Multiple initiatives are underway to harness the clinical benefits of remote ischemic preconditioning (rIPC) based on applying non-invasive, brief, intermittent limb ischemia/reperfusion using an external occluder. However, little is known about how rIPC induces protection in cardiomyocytes, particularly through G-protein coupled receptors. In these studies, we determined the role of opioid and adenosine receptors and their functional interactions in rIPC cardioprotection. In freshly isolated cardiomyocytes subjected to 45-min simulated ischemia followed by 60-min simulated reperfusion, we examined the ability of plasma dialysate (derived from blood obtained from rabbits remotely preconditioned by application of brief cycles of hind limb ischemia/reperfusion, rIPC dialysate) to protect cells against necrosis. rIPC dialysate and selective activation of either δ-opioid receptors or κ-opioid receptors significantly reduced the % of dead cells after simulated ischemia and simulated reperfusion. Inhibition of adenosine A1 receptors, but not adenosine A3 receptors, blocked the protection by rIPC dialysate, δ-opioid receptor and κ-opioid receptor activation. In HEK293 cells expressing either hemagglutinin A-tagged δ-opioid receptors or hemagglutinin A-tagged κ-opioid receptors, selective immunoprecipitation of adenosine A1 receptors pulled down both δ-opioid and κ-opioid receptors. This molecular association of adenosine A1 receptors with δ-opioid and κ-opioid receptors was confirmed by reverse pull-down assays. These findings strongly suggest that rIPC cardioprotection requires the activation of δ-opioid and κ-opioid receptors and relies on these receptors functionally interacting with adenosine A1 receptors.