cent of total PDH that was active. Protein concentration was measured using a PierceTM BCA protein assay kit. Cytosolic calcium imaging Cytosolic Ca2+ transients were measured before and after administering DCA or pyruvate. Hearts were excised and Langendorff perfused as described above. The Ca2+ sensitive dye, Rhod-2AM, was mixed with 1 mL of 37 C perfusate and injected into the bubble trap directly above the heart. Blebbistatin, an actin-myosin ATPase inhibitor, was added to both perfusate reservoirs to prevent motion artifact while imaging Rhod-2AM fluorescence. Rhod-2AM was excited using an LED spotlight with a peak wavelength of 530 nm and a 54520-nm excitation filter. Emitted light passed through a lens, was band pass filtered at 60535 nm, and imaged at >600 frames per second using a CCD camera. Hearts were monitored for 10 min with baseline KH after administration of blebbistatin and Rhod-2AM. Perfusate was then switched to a solution that was either baseline KH or contained 5 mM pyruvate or 5 mM DCA. Ca2+ transients were imaged at 2-min intervals during baseline perfusion and after the perfusate switch. When Pflugers Arch. Author manuscript; available in PMC 2016 January 06. Jaimes et al. Page 6 imaging, hearts were sequentially paced at cycle lengths of 240 and 180 ms. Electrodes were placed in the superfusion AMI-1 web chamber to record electrical activity throughout the studies and to monitor for pacing capture. Average Ca2+ transients were computed from Rhod-2AM images using pixels from a small ROI having a diameter of 5 mm on the LV epicardium. Average transients were then analyzed using custom MATLAB algorithms to measure the following parameters: duration from activation time to peak fluorescence, duration from activation time to 30 % and 80 % relaxation, and decay time constant. The activation time was defined as the maximum of the second derivative, at the first detection of SR Ca2+ release. The return to baseline was fitted as a monoexponential from 70 % height of the transient to the baseline to determine the decay time constant as described by Laurita et al.. Average Ca2+ transient parameters were taken at 30 min after a perfusate switch to ensure the response had reached a steady-state. We conducted parallel studies, as described above; with the exception that fNADH PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19854301 instead of Rhod-2AM was imaged. The objective was to determine if the inhibition of the actinmyosin ATPase with 4.75 M of blebbistatin affected the dynamics of fNADH after administering DCA or pyruvate. Measurement of SR calcium load The effect of DCA and pyruvate on SR Ca2+ load was measured using neonatal myocyte monolayers and a caffeine surge protocol. Neonatal rat ventricular myocytes were isolated and plated from a heterogeneous population of 
hearts, as previously described. Intracellular Ca2+ transients were imaged using Fluo-4 and confocal fluorescence microscopy. Cells were field stimulated at 0.2 Hz for 30 s, followed by an injection of 20 mM caffeine to induce total SR calcium release. The injection of caffeine was supplemented with 20 mM KCl and 1 mM verapamil to prevent rapid contractions. The average area under the curve of three baseline transients was compared to the area of the large transient induced by the caffeine surge. Arrhythmia scoring Pressure and electrogram signals were examined to identify premature ventricular contractions and episodes of non-sustained ventricular tachycardia. Arrhythmias were scored using a modified method from Jin et