D with pyruvate. Altogether, our SSP results are consistent with previous

D with pyruvate. Altogether, our SSP results are consistent with previous observations of higher K-858 web 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 of the calcium measurements were reproduced by inhibiting the actin-myosin ATPase with blebbistatin. This result supports the evidence that elevated cytosolic phosphorylation potential with both DCA and pyruvate was the primary mechanism of the observed changes in Ca2+ kinetics. Our finding of reduced TTP corresponds to increased PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19854301 RyR synchronization, despite a possible reduction in RyR open probability by the presence of pyruvate. Higher Pflugers Arch. SCH58261 site Author manuscript; available in PMC 2016 January 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Jaimes et al. Page 12 cytosolic phosphorylation potential should enable the SERCA pump to maintain higher trans-SR Ca2+ gradients. However, longer indicates that SERCA requires more time to resequester the larger amount of Ca2+ released, particularly in the later part of the Ca2+ uptake phase. This interpretation is aligned with results of Torres et al. that show increases in relaxation time in trabeculae muscle after administering pyruvate. Our finding of reductions in CaD30 is evidence that SERCA activity is increased in the early portion of the calcium transient. This would also explain the narrowing of the transient peak observed with DCA and pyruvate in Fig. 5b, c. DCA caused more significant shortening of CaD30 than pyruvate. This may reflect that more Ca2+ is released from the SR with pyruvate, as demonstrated by our myocyte SR load experiments which show pyruvate causes more significant elevation of SR load tha.D 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 of the calcium measurements were reproduced by inhibiting the actin-myosin ATPase with blebbistatin. This result supports the evidence that elevated cytosolic phosphorylation potential with both DCA and pyruvate was the primary mechanism of the observed changes in Ca2+ kinetics. Our finding of reduced TTP corresponds to increased PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19854301 RyR synchronization, despite a possible reduction in RyR open probability by the presence of pyruvate. Higher Pflugers Arch. Author manuscript; available in PMC 2016 January 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Jaimes et al. Page 12 cytosolic phosphorylation potential should enable the SERCA pump to maintain higher trans-SR Ca2+ gradients. However, longer indicates that SERCA requires more time to resequester the larger amount of Ca2+ released, particularly in the later part of the Ca2+ uptake phase. This interpretation is aligned with results of Torres et al. that show increases in relaxation time in trabeculae muscle after administering pyruvate. Our finding of reductions in CaD30 is evidence that SERCA activity is increased in the early portion of the calcium transient. This would also explain the narrowing of the transient peak observed with DCA and pyruvate in Fig. 5b, c. DCA caused more significant shortening of CaD30 than pyruvate. This may reflect that more Ca2+ is released from the SR with pyruvate, as demonstrated by our myocyte SR load experiments which show pyruvate causes more significant elevation of SR load tha.