Be an intertwined result of increases in NADH production and lower NADH consumption due to the initially reduced LVDP in the TP. Maximum nNADH with 40 mM DCA was not significantly greater than that with 5 mM DCA, despite the transient reduction in LVDP being more significant. These results support the hypothesis that reduction in LVDP is due to cytosolic acidosis. The increase in nNADH is due not only to the decrease in LVDP, but increased metabolism as well. Subsequently, with both 5 and 40 mM DCA, nNADH begins to decline Pflugers Arch. Author manuscript; available in PMC 2016 January 06. Jaimes et 
al. Page 11 as LVDP increases. The decline of nNADH is attributed to increased work output as reductions in LVDP are abolished. This interpretation is further supported by the absence of any decrease in nNADH when the actin-myosin ATPase is inhibited with blebbistatin. With pyruvate, nNADH stabilizes at a level substantially higher than baseline, MedChemExpress BAY-41-2272 consistent with previous studies. This is also consistent with lower FAD+, as observed by Zima et al. after the introduction of Vorapaxar web pyruvate to individual ventricular myocytes. Others have shown that increased pyruvate oxidation increases cytosolic phosphorylation potential during normoxia and post-ischemic recovery, to which increased LVDP and contractility have been attributed. Although we did not measure /, cytosolic phosphorylation potential can be inferred from changes in mitochondrial NADH when glucose-only perfusate is supplemented with pyruvate. Altogether, our SSP results are consistent with previous observations of higher cytosolic phosphorylation potential in hearts perfused with pyruvate. Our hypothesis that the administration of DCA to a normoxic heart would lower mitochondrial steady-state NADH when glucose is the only exogenous supplied fuel is supported by the reduction of NADH to a level lower than baseline. Since DCA terminates regulation of PDH via negative feedback loops, leaving PDH in an active state despite low levels of pyruvate, we surmise that DCA depletes endogenous pyruvate if only glucose is available. Indeed, it has been shown DCA reduces cytosolic pyruvate in the heart. After administering DCA with only glucose, the only available source of pyruvate is that of which is glycolytically produced, on the order of 67 M. Furthermore, increased workload increases NADH consumption rate and decreases mitochondrial NADH. Our results showing DCA simultaneously increases LVDP and decreases nNADH are consistent with the concept that in the absence of abundant substrate increased work output is associated with a decrease in steady-state mitochondrial NADH. Cytosolic calcium transient kinetics We show for the first time the effect of DCA and pyruvate on the kinetics of cytosolic calcium transients in isolated perfused hearts. With both compounds, we found reductions in Ca2+ TTP and CaD30. We also found that CaD80 remained unchanged: a combined effect of shortened CaD30 with lengthening of. These results are consistent with increased SR Ca2+ load as a result of increased cytosolic phosphorylation potential. Indeed, studies in isolated cardiac myocytes found that pyruvate increases SR Ca2+ load due to increased cytosolic phosphorylation potential, which result in increased Ca2+ transient amplitude and increased systolic contractile force. Although nNADH dropped below baseline in contracting hearts with DCA, nNADH rose steadily above baseline with DCA when the conditions PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850718,22102576 of the calcium meas.Be an intertwined result of increases in NADH production and lower NADH consumption due to the initially reduced LVDP in the TP. Maximum nNADH with 40 mM DCA was not significantly greater than that with 5 mM DCA, despite the transient reduction in LVDP being more significant. These results support the hypothesis that reduction in LVDP is due to cytosolic acidosis. The increase in nNADH is due not only to the decrease in LVDP, but increased metabolism as well. Subsequently, with both 5 and 40 mM DCA, nNADH begins to decline Pflugers Arch. Author manuscript; available in PMC 2016 January 06. Jaimes et al. Page 11 as LVDP increases. The decline of nNADH is attributed to increased work output as reductions in LVDP are abolished. This interpretation is further supported by the absence of any decrease in nNADH when the actin-myosin ATPase is inhibited with blebbistatin. With pyruvate, nNADH stabilizes at a level substantially higher than baseline, consistent with previous studies. This is also consistent with lower FAD+, as observed by Zima et al. after the introduction of pyruvate 
to individual ventricular myocytes. Others have shown that increased pyruvate oxidation increases cytosolic phosphorylation potential during normoxia and post-ischemic recovery, to which increased LVDP and contractility have been attributed. Although we did not measure /, cytosolic phosphorylation potential can be inferred from changes in mitochondrial NADH when glucose-only perfusate is supplemented with pyruvate. Altogether, our SSP results are consistent with previous observations of higher cytosolic phosphorylation potential in hearts perfused with pyruvate. Our hypothesis that the administration of DCA to a normoxic heart would lower mitochondrial steady-state NADH when glucose is the only exogenous supplied fuel is supported by the reduction of NADH to a level lower than baseline. Since DCA terminates regulation of PDH via negative feedback loops, leaving PDH in an active state despite low levels of pyruvate, we surmise that DCA depletes endogenous pyruvate if only glucose is available. Indeed, it has been shown DCA reduces cytosolic pyruvate in the heart. After administering DCA with only glucose, the only available source of pyruvate is that of which is glycolytically produced, on the order of 67 M. Furthermore, increased workload increases NADH consumption rate and decreases mitochondrial NADH. Our results showing DCA simultaneously increases LVDP and decreases nNADH are consistent with the concept that in the absence of abundant substrate increased work output is associated with a decrease in steady-state mitochondrial NADH. Cytosolic calcium transient kinetics We show for the first time the effect of DCA and pyruvate on the kinetics of cytosolic calcium transients in isolated perfused hearts. With both compounds, we found reductions in Ca2+ TTP and CaD30. We also found that CaD80 remained unchanged: a combined effect of shortened CaD30 with lengthening of. These results are consistent with increased SR Ca2+ load as a result of increased cytosolic phosphorylation potential. Indeed, studies in isolated cardiac myocytes found that pyruvate increases SR Ca2+ load due to increased cytosolic phosphorylation potential, which result in increased Ca2+ transient amplitude and increased systolic contractile force. Although nNADH dropped below baseline in contracting hearts with DCA, nNADH rose steadily above baseline with DCA when the conditions PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850718,22102576 of the calcium meas.