F 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 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 buy 1235481-90-9 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 al., where hearts having 20 or less PVCs received a score of 0 and hearts having more than 20 PVCs or one episode of NSVT for less than 2 s received a score of 1. We did not observe any heart to have NSVT longer than 2 s. We also did not observe VF or other significant arrhythmias, so scores beyond 
1 were not necessary. All hearts received arrhythmia scores of either 0 or 1. Statistics Statistical analyses were performed in R. Data are presented as meanstandard error of mean. Significance was defined by p<0.05, unless noted as p<0.01. One-way ANOVAs with Tukey post hoc tests were used to identify significant differences between groups. A threeway ANOVA with Tukey post hoc tests were used to compare calcium transient Author Manuscript Author Manuscript Author Manuscript Author Manuscript Pflugers Arch. Author manuscript; available in PMC 2016 January 06. Jaimes et al. Page 7 characteristics between baseline and treatments, pacing rates, and between treatments. All data were determined to be normal using the ShapiroWilk test. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Results Control studies with identical KH solutions in each side of the dual perfusion apparatus confirmed that perfusate switching did not cause artifacts or alter heart function. Perfusate switching introduced a maximum temperature variation of less than 1 C and a heart rate change of less than 5 %. No other changes in heart function were detected. HR changes of less than 5 % were also measured when administering DCA or pyruvate. LVDP and nNADH signals in all contracting heart studies consisted of three phases: a baseline phase, a transient phase, and a steady-state phase. The BP was the 10 min of baseline perfusion before a perfusate switch occurred. The TP was the period from the perfusate switch to when changes in LVDP or nNADH subsided. The SSP began when a given variable reached steady-state and corresponded to the time from the end of the TP to the end MedChemExpress Vorapaxar 22102576?report=abstract” title=View Abstract(s)”>PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850718,22102576 of study. Measureme.F 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 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 al., where hearts having 20 or less PVCs received a score of 0 and hearts having more than 20 PVCs or one episode of NSVT for less than 2 s received a score of 1. We did not observe any heart to have NSVT longer than 2 s. We also did not observe VF or other significant arrhythmias, so scores beyond 1 were not necessary. All hearts received arrhythmia scores of either 0 or 1. Statistics Statistical analyses were performed in R. Data are presented as meanstandard error of mean. Significance was defined by p<0.05, unless noted as p<0.01. One-way ANOVAs with Tukey post hoc tests were used to identify significant differences between groups. A threeway ANOVA with Tukey post hoc tests were used to compare calcium transient Author Manuscript Author Manuscript Author Manuscript Author Manuscript Pflugers Arch. Author manuscript; available in PMC 2016 January 06. Jaimes et al. Page 7 characteristics between baseline and treatments, pacing rates, and between treatments. All data were determined to be normal using the ShapiroWilk test. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Results Control studies with identical KH solutions in each side of the dual perfusion apparatus confirmed that perfusate switching did not cause artifacts or alter heart function. Perfusate switching introduced a maximum temperature variation of less than 1 C and a heart rate change of less than 5 %. No other changes in heart function were detected. HR changes of less than 5 % were also measured when administering DCA or pyruvate. LVDP and nNADH signals in all contracting heart studies consisted of three phases: a baseline phase, a transient phase, and a steady-state phase. The BP was the 10 min of baseline perfusion before a perfusate switch occurred. The TP was the period from the perfusate switch to when changes in LVDP or nNADH subsided. The SSP began when a given variable reached steady-state and corresponded to the time from the end of the TP to the end PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850718,22102576 of study. Measureme.