Novel Small-Molecule Troponin Activator Increases Cardiac Contractile Function Without Negative Impact on Energetics

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Compound was then added and 1/3 of media in the test chamber was gently pipetted in the test chamber to mix. Measurements were taken after a 15-minute incubation with compound or vehicle. Video microscopy was then used to monitor deflection of poly(octamethylene maleate (anhydride) citrate) (POMaC) wires; images of autofluorescent POMaC wires were acquired at 100 fps with a 10x objective in the blue channel (λex = 350 nm, λem = 470 nm). This procedure was then repeated for escalating compound dosages. Videos were then analyzed using a custom MATLAB (MathWorks, Inc., Natick, MA, USA) algorithm to extract maximum twitch amplitude from raw force traces.
Skinned papillary fiber force mechanics -Papillary fibers were carefully dissected from the left ventricles of 12-14 weeks old C57BL6 mice (n=4). Papillary fibers were skinned over night at 4 0 C in 1% Triton X-100 in relaxing buffer [55.74 mM potassium propionate, 7 mM ethylene glycol bis(2-aminoethyl)tetraacetic acid, 100 mM N,N-bis(2-hydroxyethyl)-2-amino ethanesulfonic acid, 0.02 mM CaCl2, 5.5 mM MgCl2, 5 mM dithithreitol, 15 mM creatine phosphate and 4.7 mM ATP. Skinned papillary fibers were sequentially exposed to increasing calcium solutions made by mixing relaxing buffer and activating solution [relaxing buffer with the addition of 7 mM CaCl2] at room temperature. For the measurement of maximal force and calcium sensitivity, a single fiber bundle was cycled through all calcium solutions three times, first with vehicle (DMSO) followed by increasing concentrations of TA1. Resulting curves where fit to a modified Hill Equation in GraphPad Prism.

Evaluation of TA1 in normal healthy ratsin vivo rodent pharmacodynamics
All animal care and use was in compliance with the Amgen IACUC protocol, appropriate guidelines of the test facility, and animal welfare regulations. Normal, healthy, male CD rats aged 9-10 weeks, were obtained from Charles River, Hollister, CA, USA. On the day of study, rats were implanted with a jugular vein catheter under isoflurane anesthesia for intravenous (IV) test article delivery. Infusion of vehicle or a fixed concentration of TA1 was then initiated with the aid of a programmable syringe pump. The dose delivered was escalated by increasing the infusion rate in a stepwise manner at 15-minute intervals.
Time-matched controls were generated by dosing rats designated for the "vehicle" group with vehicle only. Samples of whole blood (5uL; expressed from a small nick in the tail) were collected at 5 min intervals for subsequent bioanalytical determination of test article exposure. In this manner both pharmacodynamics (PD; echo measures) and pharmacokinetic (PK; exposure) were determined in the same animal.
An ultrasound probe (Vevo2100) was positioned to acquire M-Mode images at the midventricular level as indicated by the presence of papillary muscles on the short axis view and fractional shortening (FS) was acquired. Baseline-corrected, vehicle-subtracted FS was plotted against logarithmically-transformed measured total whole blood concentrations of TA1. A 4-parameter nonlinear fit (bottom constrained to zero, variable slope; GraphPad Prism) was performed.

Simultaneous measurement of LV contractile function and high-energy phosphate concentrations (HEP) by 31 P NMR spectroscopy
Isolated hearts of twenty-six rats were used to simultaneously measure LV contractile function and HEP by 31 P NMR spectroscopy. There were two groups of hearts: i) perfused with increasing concentrations of dobutamine (60 nM, 120 nM, and 240 nM for 20 min each, n=11), and ii) perfused with increasing concentrations of TA1 (15 nM, 30 nM, and 60 nM for 20 min each, n=15) ( Figure 2A). Left ventricular contractile function was assessed in an isolated retrograde-perfused Langendorff heart preparation as described previously. [16][17][18] Rats were heparinized (4U IP per g of body weight) and anesthetized with isoflurane. The heart was excised and perfused at a constant pressure of 80 mm Hg. The perfusate (modified Krebs-Henseleit buffer) consisted of the following (in mmol/L): NaCl 118, NaHCO3 25, KCl 5.3, CaCl2 2, MgSO4 1.2, EDTA 0.5, glucose 10, palmitate 0.4 bound to 1% albumin equilibrated with 95% O2 and 5% CO2 (pH 7.4). Dobutamine and TA1 were added to the perfusate in incremental concentrations as per study protocol ( Figure 2).
To assess left ventricular (LV) contractile function, a water-filled balloon was inserted into the LV. After stabilization, balloon volume was adjusted to achieve an LV end-diastolic pressure (LVEDP) of 8 to 9 mmHg and held constant during the protocol. LV-developed pressure (DevP) was calculated as: DevP = LV systolic pressure (LVSP) -LVEDP. The rate pressure product (RPP = DevP x heart rate) was calculated to estimate the work performed by LV. The perfused hearts were placed in a 20 mm glass tube in a 9.4 T vertical bore magnet and maintained at 37°C allowing for simultaneous measurement of LV contractile function and HEP. [16][17][18] The left ventricular wall stress was determined by using the law of Laplace: wall stress = (left ventricular systolic pressure x ventricular radius)/(2 x ventricular wall thickness), as described previously. 15 HEP were measured by 31 P NMR spectroscopy using a Varian VNMRS spectrometer (9.4T,161.4 MHz). 16,18 Each 31 P-NMR spectrum resulted from the average of 240 free induction decay signals over 10 min. Fully relaxed spectra acquired with a recycle time of 20 s were used to determine saturation factors. Intracellular pH was calculated from the chemical shift of intracellular inorganic phosphate (Pi) relative to PCr. 16,18,19 At the end of each experiment, beating hearts were freeze-clamped using Wollenberger tongs pre-cooled in liquid nitrogen and stored at −80 °C for subsequent analysis of total creatine concentration by high-performance liquid chromatography (HPLC

Magnetization transfer experiments
As per study protocol ( Figure 2B

Supplemental Tables
Supplemental Table I. Fitted data for Figure 1B.  Supplemental Table III