All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of The University of Vermont College of Medicine and complied with the Guide for the Use and Care of Laboratory Animals published by the National Institutes of Health. Sixteen male Wistar rats (Charles River Laboratories) were fed an iodine-deficient 0.15% 6-n-propyl-2-thiouracil (PTU) diet (Harlan Teklan) for at least 12 weeks. After 6 weeks on the diet, eight rats received tail vein injection of 50 mg/g STZ prepared in sodium citrate solution. The remaining rats received injection of similar volume of vehicle. At least 6 weeks after induction of HG by STZ, rats were anesthetized with 1.5-3% inhaled isoflurane, and short-axis view M-mode echocardiography was performed using Acuson Sequoia C256 (Seimens Medical Solutions) fitted with a 15 MHz probe suitable for resolving dimensions of the rat heart. Hearts were removed, and subjects died of exsanguination. Papillary muscles were then taken for myofilament performance assessment, apexes were harvested for elemental analysis and cardiomyocytes were isolated from the remaining LV.
Measures of heart size and function included heart rate, left ventricular posterior wall thickness (LVPWT), left ventricular diameter at end-diastole (LVD dias) and end-systole (LVD sys), fractional shortening = (LVD dias - LVD sys)/LVD dias, ejection fraction = (LVD dias3 - LVD sys3)/LVD dias3, ejection time taken between QRS complex and peak shortening, and circumferential velocity = fractional shortening/ejection time.
Solutions for skinned myocardial strips
Solution concentrations (mmol/L) were formulated by solving equations describing ionic equilibria . Relaxing solution: pCa 8.0, 5 EGTA, 5 MgATP, 1 Mg2+, 35 phosphocreatine (PCr), 300 U/mL creatine kinase (CK), ionic strength 200, pH 7.0. Activation solution: same as relaxing with pCa 4.0. Storage solution: same as relaxing with 10 μg/mL leupeptin and 50% glycerol wt/vol.
Skinned myocardial strips
Skinned myocardial strips were studied as previously described . Papillary muscles from the LV were dissected to yield at least two thin strips (~140 μm diameter, ~800 μm length) with longitudinally oriented parallel fibers, skinned for 2 hr at 22°C, and stored at −20°C. Aluminum T-clips were attached to the ends of a strip ~300 μm apart. The strip was mounted between a piezoelectric motor (Physik Instrumente, Auburn, MA) and a strain gauge (SensorNor, Horten, Norway), lowered into a 30 μL droplet of relaxing solution (pCa 8.0) maintained at 37°C, and incrementally stretched to 2.2 μm sarcomere length measured by digital Fourier Transform (IonOptix Corp, Milton, MA). Strips were calcium activated from pCa 8.0 to pCa 4.8.
Recorded forces were normalized to cross-sectional area to provide isometric tension (T). Individual recordings of T minus relaxed tension (Tmin
) were then normalized to maximum developed tension (Tmax
) and fit to the Hill equation:
where [Ca2+]50 = calcium concentration at half activation, pCa50 = −log [Ca2+]50, and n = Hill coefficient using a nonlinear least squares algorithm (Sigma Plot 8.0, SPSS, Chicago, IL).
The force-clamp technique was applied at maximal calcium activation. Various mechanical loads were expressed as a fraction of maximum absolute tension (Tmax). Force was maintained constant over a designated period of time using software-based feedback control, and the length change was continuously monitored. The tension-velocity (T-V) relationship was generated from these data.
The relationship was fit to a hyperbolic Hill equation normalized to Tmax
where T'= T/Tmax
, a'= a/Tmax
, and a and b are the parameters of the non-normalized hyperbolic Hill equation using a nonlinear least squares algorithm (Sigma Plot 8.0, SPSS, Chicago, IL). The physiological characteristics maximum unloaded shortening velocity (Vmax
), velocity at maximum power (Vopt
), tension at maximum power (T`opt
) and maximum power production (Pmax
) were calculated from a' and b, as follows [31
Native thin filament isolation
Intact native thin filaments were isolated from ventricular tissue as described previously . In brief, myofibrils were isolated from flash frozen muscle tissue and subsequently homogenized in the following solution (in mmol/L): 100 NaCl, 5 MgCl2, 1 NaN3, 1 ethylene glycol tetraacetic acid (EGTA), 5 MgATP, 10 Na3PO4 and 2 μg/ml leupeptin at a ratio 40 ml/gm of myofibril (pH 6.5). Debris and thick filament were pelleted with centrifugation 137,000 × g for 20 minutes. Native thin filaments were then collected with centrifugation (137,000 x g for 180 minutes) with the pellet being raised in low salt buffer (in mmol/L): 25 KCl, 25 imidazole, 1 EGTA, 5 MgCl2, 10 DTT. Protein concentration was determined for native thin filaments with a protein assay (Bio-Rad; Hercules, CA, USA) using bovine serum albumin as the standard. Chicken skeletal myosin was isolated as previously described. Chicken skeletal myosin was used as the motility substrate in the in vitro motility assay due to its temporal stability, creating consistency between experiments . Native thin filaments were labeled with rhodamine-phalloidin (1:1 molar ratio) prior to use in the motility assay.
In vitro motility assay
Native thin filament contractile performance was assessed using the in vitro motility as described previously . In brief, myosin (100 μg/ml, unless otherwise noted) was applied for one minute to a nitrocellulose coated coverslip in a high salt buffer (in mmol/L): 300 KCl, 25 imidazole, 5 MgCl2, 2 EGTA and 10 DTT. The surface was then washed with bovine serum albumin (0.5 mg/ml) in low salt buffer. Employing epifluorescent microscopy, rhodamine-labeled thin filaments were observed moving across the myosin coated surface in the presence of MgATP (2 mmol/L) in a buffered motility solution (in mmol/L unless otherwise noted): 25 KCl, 25 imidazole, 5 MgCl2, 2 EGTA and 10 DTT, glucose oxidase 0.1 mg/ml, catalase 1.8 μg/ml, glucose 2.3 mg/ml, and 0.38% methylcellulose. Free calcium was varied in the motility solution (pCa 10–4.0). Thin filament motility was recorded on videotape, and subsequently analyzed with the motion analysis system VP110 (Motion Analysis Corporation, Santa Rosa, CA). Typically >300 individual filament velocities were averaged to determine the mean velocity of each experiment. All motility experiments were performed at 30°C.
Isolated cardiomyocyte function
Rat LV cardiomyocytes were isolated using retrograde perfusion of collagenase solution according to methods described elsewhere . Cardiomyocytes were observed with a 40× objective on an inverted microscope (Nikon Diaphot) while bathed in normal Tyrode’s solution containing in mmol/L: 137 NaCl, 5.4 KCl, 1.2 CaCl2, 0.5 MgCl2, 10 HEPES, 5.5 glucose, 0.5 probenecid at 37°C (pH 7.4). Cardiomyocytes were electrically paced at 2, 4 and 6 Hz for at least 5 minutes at each frequency. Half of the cardiomyocytes were exposed to Tyrode’s with 32 μM Zn-acetate. Dynamic sarcomere length was detected by Fourier Transform of the digital image.
A subset of cardiomyocytes from each heart was loaded with Fura-2 AM (Invitrogen, Carlsbad, CA) and excited at 365 nm and 400 nm. Emission at 510 nm was used to detect Fura-2 fluorescence at each pacing frequency, although not with zinc exposure. We did not measure intracellular Ca2+ during Zn2+ exposure, because Fura-2 is sensitive to both physiological Zn2+ and Ca2+ and we would not have been able to differentiate the two ions. Background fluorescence was detected after lysing cardiomyocytes with 4 μM digitonin. Parameters of fluorescence ratio 365/400 (R) dynamics were used to represent those of calcium dynamics .
Myosin isoform determination and western blots
Ventricular myosin isoform content of HG and nonHG rats was determined by the method of Reiser and Kline  with the use of Fluormax-2 Imaging analysis (Bio-Rad, Hercules, CA). Protein phosphorylation stain (Pro-Q diamond, Invitrogen) and Western blots for myosin regulatory light chain (MLC2) content (cat. ab92721, Abcam, San Francisco, CA), MLC2 phosphorylation (MLC2-P) at Serine-19 (cat. ab2480, Abcam), RyR (cat. MA3-916, Thermo Scientific), RYR phosphorylation at Serine-2808/9 (cat. A010-30AP, Badrilla), O-linked N-Acetylglucosamine (cat. ab2739, Abcam), lysine acetylation (cat. #9441, Cell Signaling Technologies) and pentosidine (PEN012, Biologo) were performed using tissue homogenates prepared in (mmol/L) 300 KCl, 25 Imidazole, 5 MgCl2, 2 EGTA, 10 DTT and pH 7.4. In some cases, myofilament fraction was extracted as the pellet after centrifugation and non-myofilament fraction as the supernatant.
Cardiac element content measurements
Polypropylene conical tubes of 15 mL volume were soaked in 0.4 mM EGTA overnight, then rinsed four times with ddH2O and allowed to drip dry according to published recommendations . Between 40–80 mg of each LV sample was placed in its own tube and lyophilized by 90 min of vacuum freeze drying. Samples were digested in 10 μL nitric acid (15.8 Normal) per mg tissue wet weight. Gallium was added in the amount 0.2 μL of 1 g/L Ga standard per mg tissue wet weight. Finally, ddH2O was added to bring total volume to 200× dilution of original tissue wet weight including final concentrations of 0.8 N nitric acid and 1 ppm Ga. Standards were prepared for elements Ca, Cu, Fe, K, Mg, Na, P, S and Zn over ranges 0–0.4 ppm, 0–4 ppm or 0–40 ppm depending on the element and all including 0.8 N nitric acid and 1 ppm Ga. Detection of element content was performed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) performed by the ULTIMA2C operated in Poly-mode (Horiba Scientific, Edison, NJ). Element content was detected in triplicate for each sample and normalized against the Ga internal standard to provide a measure of element content in ppm relative to tissue wet weight.
mRNA analysis and quantitative PCR
Total RNA was extracted from the rat hearts using Qiagen RNeasy kit and cDNA was synthesized using Invitrogen Superscript III first-strand synthesis system according to manufacturer instructions. Taqman primer sets were purchased from Applied Biosystems (Life Technologies): reference numbers HPRT Rn01527840, MT1a Rn00821759, ZnT2 Rn00563633 and γ-GCSh Rn00689046. Cycle threshold (Ct) values from quantitative RT-PCR were obtained from ABI Prism 7900HT sequence detection system. The mRNA expression levels measured as Ct were normalized to that of the endogenous control, HPRT, as ΔCt. The average ΔCt of the nonHG controls was used as the normalization factor to determine ΔΔCt for the HG group. Relative fold difference (RQ) in transcription levels between the HG and nonHG groups was calculated as RQ = 2-ΔΔCt.
Analysis was performed using PASW Statistics 18.0 (SPSS). Multiple measurements of myofilament performance from the same heart were averaged to provide a single measure for that heart. Tension and actin velocity were normalized to values at maximal calcium activation and fit to the Hill equation using a non-linear least squares algorithm (Sigma Plot 8.0, SPSS, Chicago, IL). Repeated-measures ANOVA, using stimulation frequency within the same cardiomyocyte and zinc exposure within the same heart as repeated measures, was used to detect the relative effects of frequency and zinc exposure between HG and nonHG controls. Data are presented as mean ± SEM. For function analyses, n = 8 in each group. Significance by statistical tests are reported at the P<0.05, P<0.01 and P<0.001 levels. Trends at P<0.1 are also reported if in support of other statistically significant results.