Abstract-To evaluate the effect of sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad-sorcin). A ␤-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37°C) was significantly reduced in Ad-sorcin-transfected myocytes compared with control myocytes (2.10Ϯ0.05% [nϭ311] versus 2.42Ϯ0.06% [nϭ312], respectively; PϽ0.001). Action potential duration (at 20°C) was significantly less in the Ad-sorcin group (458Ϯ22 ms, nϭ11) compared with the control group (520Ϯ19 ms, nϭ10; PϽ0.05). In voltageclamped, fura 2-loaded myocytes (20°C), a reduced peak-systolic and end-diastolic [Ca 2ϩ ] i was observed after Ad-sorcin transfection. L-type Ca 2ϩ current amplitude and time course were unaffected. Caffeine-induced Ca 2ϩ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na ϩ -Ca 2ϩ exchanger (NCX) current revealed a significantly lower SR Ca 2ϩ content and faster Ca 2ϩ -extrusion kinetics in Ad-sorcin-transfected cells. Higher NCX activity after Ad-sorcin transfection was confirmed by measuring the NCX current-voltage relationship. ␤-Escin-permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on The cardiac RyR isoform (RyR2) forms a macromolecular complex with numerous regulatory proteins, 5 the functional effects of which are currently under investigation. One such protein is sorcin, a 22-kDa protein of the penta-EF hand Ca 2ϩ -binding protein family. 6 Recent work has shown that sorcin associates with both the LTCC 7 and RyR2. 8 In lipid bilayers, sorcin was found to decrease the open probability (P o ) of RyR2, 9 and on the basis of these findings, sorcin has been suggested to play a mechanistic role in the termination of RyR activation. 9,10 Information on the effect of sorcin on Ca 2ϩ handling in adult cardiomyocytes is not available. Materials and Methods Adenoviral Overexpression of Sorcin in Isolated Adult CardiomyocytesThe human sorcin gene in vector pCI-neo was cloned and ligated downstream from an immediate-early cytomegalovirus (CMV) promoter into vector pACCMV.pLpA using primers creating KpnI and HindIII sites. Recombination with pJM17 plasmid and production of replication-deficient adenovirus were carried out according to standard procedures. 11 Isolation and culture of adult rabbit ventricular cardiomyocytes were carried out as described. 12,13 Measurements of Protein Expression Levels and Cardiomyocyte ContractilityFor Western blot protocols, see online data supplement (available at www.circres.ahajournals.org). Contractility data were recorded via video edge detection as described, 12 with cardiomyocytes superfused with pH-equilibrated Krebs-Henseleit solution containing 1.75 mmol/L Ca 2ϩ and stimulated via parallel field electrodes at 1 Hz and 37°C. The shortening measurements were with the experimenter blinded to the group assignment. Electrophysiological and [Ca 2؉ ] i Mea...

This study investigated the function of FK506-binding protein (FKBP12.6) using adenoviralmediated gene transfer to over-express FKBP12.6 (Ad-FKBP12.6) in adult rabbit ventricular cardiomyocytes. Infection with a β-galactosidase-expressing adenovirus (Ad-LacZ) was used as a control. Peak-systolic intracellular [Ca 2+ ] (measured with Fura-2) was higher in the Ad-FKBP12.6 group compared to Ad-LacZ (1 Hz field stimulation at 37• C). ) is a member of a family of FKBP proteins expressed in a wide range of eukaryote cells including striated muscle cells (Marks, 1996). Previous work has suggested that FKBP12.6 modulates cardiac excitation-contraction (E-C) coupling by binding to the SR Ca 2+ release channel (ryanodine receptor type 2, RyR2). Generally, three to four FKBP12.6 proteins bind to each RyR2 tetramer . The physiological pathways that modulate FKBP12.6 occupancy of RyR2 are currently under study. One possibility is that A-kinase-mediated phosphorylation of RyR2 dissociates FKBP12.6 and alters the properties of the channel (Marx et al. 2000). But recent work has failed to find a link between A-kinase-mediated phosphorylation and FKBP12.6 occupancy (Jiang et al. 2002;Stange et al. 2003). Changes in isolated RyR2 channel kinetics have been observed on addition of FKBP12.6 (Marx et al. 2001) or removal of FKBP12.6 by FK506 or rapamycin (Kaftan et al. 1996;Xiao et al. 1997). FKBP12.6 is also thought to mediate the coupled gating observed in RyR2 channel clusters (Marx et al. 2001). A prolongation of Ca 2+ spark duration (Xiao et al. 1997), and increased spark frequency (McCall et al. 1996) were reported in response to FKBP sequestering drugs, but other studies have not shown any effects of FKBP12.6 added to isolated RyR2 (Timerman et al. 1996) (Xin et al. 2002). Similarly, larger Ca 2+ transients have also been noted on addition of FK506 or rapamycin to rat cardiomyocytes (McCall et al. 1996;Xiao et al. 1997). In apparent contradiction to these studies, over-expression of FKBP12.6 by up to six times the normal value increased twitch shortening in cultured adult rabbit cardiomyocytes (Prestle et al. 2001). In a recent study comparing mouse and rabbit cardiac myocytes, divergent effects of FK506 on Ca 2+ transient amplitude were noted and attributed to species differences in intracellular Na + levels (Su et al. 2003). The present study examines the effects of FKBP12.6 over-expression on Ca 2+ transient, Ca 2+ spark and Ca 2+ wave characteristics in cultured adult rabbit cardiomyocytes. Methods Single ventricular cardiomyocyte isolation from the rabbit heartNew Zealand White rabbits (2-2.5 kg) were killed by administration of an intravenous injection of 500 IU heparin together with an overdose of sodium pentobarbitone (100 mg kg −1 ). Single rabbit ventricular cardiomyocytes were then isolated as previously described (Loughrey et al. 2002. FK506-binding protein 12.6 over-expression within rabbit cardiomyocytesRecombinant adenoviruses were generated by standard procedures using full-length cDNA of the human FKBP12.6...

Journal of Physiologyventricle in rabbit heart 8-9 weeks after an experimentally induced apical infarct, and compares these values with equivalent measurements from sham-operated (sham) hearts.

CSQ over-expression increased SR Ca2+ content but reduced the gain of E-C coupling in rabbit cardiomyocytes.

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