Online-Only Supplemental

Online-Only Supplemental MATERIALS AND METHODS Mutagenesis Strategy

Full length rat wild-type ssTnI and cTnI cDNAs (kind gifts of A. Murphy) were used to generate the N-card/slow-C TnI chimera. To begin construction of the chimeric TnI, cTnI was mutagenized to form cTnI-Sac I as described by Westfall et al. (1) A 390 bp EcoRI-SacI fragment isolated from pGEM-3ZcTnI-SacI was ligated with an EcoR1-SacI digest of pGEM-

3ZssTnI to form pGEM3ZN-card/slow-C TnI chimera. The complete nucleotide sequence of N- card/slow-C TnI was confirmed by DNA sequencing, and a fragment of N-card/slow-C TnI was subcloned into an adenovirus shuttle vector plasmid using EcoRI and BamHI sites to form pAdCMVN-card/slow-C TnI (1,2). Chimeric TnI protein production was verified in HEK 293 cells prior to making adenovirus (1).

Generation of adenoviral vectors

Recombinant adenovirus vectors were constructed by cotransfecting shuttle plasmids containing TnI cDNAs (cTnI, ssTnI, N-card/slow-C TnI) and pJM17 into HEK 293 cells as described in detail previously (2). The expression cassette within each shuttle plasmid

(pAdCMVxTnI, x denotes isoform/chimera of TnI) includes a CMV promoter, the coding sequence for one of the TnI cDNAs, and the SV40 polyadenylation signal. The resulting recombinant adenoviruses (AdCMVxTnI) are capable of being packaged but replication defective in cardiac myocytes. High titers of plaque-purified adenoviral stocks were prepared as described previously (2) and aliquots of virus were stored at -80oC. Recombinant adenovirus genomic structure was verified by Southern blot analysis (1,3).

Primary cultures of rat ventricular myocytes

Ventricular myocytes were isolated from adult female rats as described in detail by

Westfall et al. (2). An aliquot of Ca2+ tolerant myocytes (2 x 104 myocytes) was then plated on laminin-coated coverslips and incubated at 37oC in Dulbecco’s modified Eagle’s medium

(DMEM) containing 5% FBS, 50 U/ml pencillin (Pen) and 50 g/ml streptomycin (S) for 2 hrs.

Cells were then incubated with recombinant adenovirus in DMEM + Pen/S for 1 hr and then

DMEM + Pen/S was added to each coverslip. Serum-free medium was changed the day after adding virus and then every 2-3 days for up to 8 days of culture.

Analysis of protein composition by gel electrophoresis and Western blots

Gel electrophoresis. Approximately ten ventricular myocytes were collected on the tip of a glass micropipet and transferred to microcentrifuge tubes containing 10 l of sample buffer for analysis by gel electrophoresis (3,4). Fiber segments of soleus muscles were collected as described previously (5). Samples were sonicated for 10 min and briefly centrifuged prior to analysis by gel electrophoresis. Gels for SDS-PAGE were prepared and stained as described previously (4,6). Stained gels were scanned and analyzed with Multi-Analyst software

(BioRad).

Western blot analysis. Cultured ventricular myocytes from coverslips were collected in sample buffer 4 to 7 days after plating, separated by gel electrophoresis as described above, and then transblotted onto PVDF membrane as previously described in detail (6). Protein expression in HEK 293 cells was identified using a similar protocol. Permeabilized myocytes were prepared by transferring cells to relaxing solution (see composition below) containing 0.1%

Triton X-100 for 1 min and rinsed three times in relaxing solution alone prior to collection in sample buffer. After proteins were separated by SDS-PAGE, immunodetection was carried out as described in Westfall et al. (6). TnI isoform/chimera composition was determined using the primary anti-TnI mAb, MAB 1691 (1:4000), which recognizes all striated muscle isoforms of

TnI from rat and the N-card/slow-C TnI chimera. Expression of N-card/slow-C TnI also was screened with the anti-cTnI mAb, TI-1 (1:1000; kind gift of S. Schiaffino; ref. 7) and this mAb did not recognize the expressed TnI chimera (results not shown), which indicates TI-1 interacts with the carboxyl-terminus of cTnI. The thin filament proteins TnT and Tm were detected with the 1F2 (1:105) and TM311 (1:106) mAbs, respectively.

Indirect immunohistochemistry in single cardiac myocytes

Indirect immunofluorescence with a dual mAb protocol (2,6) was used to determine the extent of thin filament remodeling resulting from ectopic N-card/slow-C TnI expression within single cardiac myocytes in primary culture. The primary mAbs used to evaluate N-card/slow-C

TnI expression and incorporation into the myofilament were: 1) anti-cTnI TI-1 mAb (ref. 7;

1:1000) and; 2) MAB1691 (1:1000). Goat anti-mouse IgG antibodies conjugated to Texas Red

(TR) or fluorescein isothiocyanate (FITC) were used to detect cTnI-specific and TnI binding, respectively (2,5). Immunofluorescence was examined on a Zeiss microscope and representative cells were photographed on a Noran OZ laser scanning confocal microscope.

Measurement of Ca2+-activated tension in single cardiac myocytes at pH 7.0 and 6.2.

Solutions and preparation of samples for mechanical studies. Complete details of the relaxing and activating solutions used, experimental chamber and attachment procedure for mounting single, rod-shaped cardiac myocytes has been reported elsewhere (5). The relaxing and activating solutions used for experiments contained 7 mM EGTA, 20 mM imidazole, 1mM free Mg2+, 14.5 mM creatine phosphate, and 4 mM MgATP with sufficient KCl to yield a total ionic strength of 180 mM. Solution pH was adjusted to 7.00 or 6.20 with KOH/HCl. The relaxing solution had a pCa (-log[Ca2+]) of 9.0 while the pCa of the solution for maximal activation was 4.0. The computer program of A. Fabiato (8) was used to calculate the final concentrations of each metal, ligand and metal-ligand complex, employing the stability constants listed by Godt and Lindley (9). Cultured cardiac myocytes, briefly treated with 0.2% TX-100, were washed repeatedly in relaxing solution and then attached to a force transducer (model 403A; Cambridge Technology

Inc., Watertown, MA) and a high performance moving coil galvanometer (model 6350;

Cambridge Technology) via glass micropipets. Sarcomere length was set at 2.1 m and the experimental temperature was set at 15oC to allow comparison to earlier work (1,3), and because preparation viability decreases and sarcomere length non-uniformity increases more rapidly at higher temperatures.

Measurement of steady-state isometric tension-pCa relationship. Ca2+-activated tension was measured in single myocytes, as described in detail elsewhere (5). Tension-pCa relationships were constructed by expressing tension (P) at various submaximal Ca2+ concentrations as a fraction of tension at maximal activation (Po, pCa 4.0). Every third or fourth activation was carried out at pCa 4.0. The Marquardt-Levenberg nonlinear least squares fitting

2+ algorithm was used to derive values for the Hill coefficient (nH) and Ca required for half maximal activation (pCa50) from the tension-pCa relationship, using the Hill equation P =

2+ nH nH 2+ nH [Ca ] /(K + [Ca ] ) where P is the fraction of maximum tension (Po), the pCa50 is used as an

indicator of K, and nH is the Hill coefficient.

Statistics

Values for each group are expressed as mean + SEM. An analysis of variance (ANOVA) was used to test for significant differences (p<0.05) between groups, with a post-hoc Student-

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