INTRODUCTION: Although some properties of the fluorescent sodium indicator "SBFI" are known, there is no accepted method by which the SBFI signal might be calibrated for intracellular Na (Nai) in cardiac cells. The first aim of this study was to characterize the loading and compartmentalization of this indicator in single cardiac myocytes. The second aim was, from experimental observation, to develop a rational calibration method for SBFI. The third aim was to use this Na indicator to study the dependence of tonic contraction on Nai in voltage-clamped ventricular myocytes. METHODS AND RESULTS: SBFI was incorporated into myocytes by incubating with the acetoxymethyl ester (10 microM) for 2 hours. This led to an intracellular concentration of SBFI free acid of 122 +/- 17 microM. We considered a number of issues with respect to compartmentalization of indicator and, under our conditions, we found the majority (71%) of indicator was situated in the cytoplasm. Therefore, SBFI indicates mainly changes of cytoplasmic Na. Calibration of the indicator for Nai was performed by equilibrating internal and external Na. We investigated the conditions required for optimum transmembrane Na equilibration and found it necessary to use a calibration solution free of both Ca and magnesium (Mg). The Na ionophores gramicidin D and monensin were both required, and it was also necessary to inhibit the Na/K pump for optimum Na equilibration. Using these conditions, the Nai concentration in quiescent rat ventricular myocytes was 10.9 +/- 0.74 mM (mean +/- SEM, n = 40; equivalent to an Na activity of 8.3 mM). The concentration of Nai was significantly lower in quiescent rabbit myocytes: 3.8 +/- 0.23 mM (n = 24; equivalent to an Na activity of 2.9 mM). In voltage-clamped rabbit myocytes, inhibition of the Na/K pump caused a rise of Nai; there were also marked effects on the tonic shortening elicited by ramp depolarizations. As Nai rose, the magnitude of tonic shortening increased and its voltage dependence also changed. CONCLUSION: These results confirm the suitability of SBFI for measuring Nai in cardiac cells. Provided that steps are taken to optimize transmembrane Na equilibration, the indicator can be calibrated for Nai. The different Nai of rat and rabbit myocytes has implications for the function of sarcolemmal Na/Ca exchange in each cell type. An Nai of 10.9 mM in rat myocytes gives a calculated reversal potential for the exchange of -35 mV. In comparison, an Nai of 3.8 mM in rabbit myocytes leads to a reversal potential for the exchange +45 mV. Therefore, relatively small changes of Nai can shift the reversal potential of the exchange to values that are substantially more positive or negative than zero. The behavior of voltage-dependent tonic contraction in rabbit myocytes was consistent with the Na/Ca exchange reversal potential being more positive than zero in these cells.
INTRODUCTION: Although some properties of the fluorescent sodium indicator "SBFI" are known, there is no accepted method by which the SBFI signal might be calibrated for intracellular Na (Nai) in cardiac cells. The first aim of this study was to characterize the loading and compartmentalization of this indicator in single cardiac myocytes. The second aim was, from experimental observation, to develop a rational calibration method for SBFI. The third aim was to use this Na indicator to study the dependence of tonic contraction on Nai in voltage-clamped ventricular myocytes. METHODS AND RESULTS:SBFI was incorporated into myocytes by incubating with the acetoxymethyl ester (10 microM) for 2 hours. This led to an intracellular concentration of SBFI free acid of 122 +/- 17 microM. We considered a number of issues with respect to compartmentalization of indicator and, under our conditions, we found the majority (71%) of indicator was situated in the cytoplasm. Therefore, SBFI indicates mainly changes of cytoplasmic Na. Calibration of the indicator for Nai was performed by equilibrating internal and external Na. We investigated the conditions required for optimum transmembrane Na equilibration and found it necessary to use a calibration solution free of both Ca and magnesium (Mg). The Na ionophores gramicidin D and monensin were both required, and it was also necessary to inhibit the Na/K pump for optimum Na equilibration. Using these conditions, the Nai concentration in quiescent rat ventricular myocytes was 10.9 +/- 0.74 mM (mean +/- SEM, n = 40; equivalent to an Na activity of 8.3 mM). The concentration of Nai was significantly lower in quiescent rabbit myocytes: 3.8 +/- 0.23 mM (n = 24; equivalent to an Na activity of 2.9 mM). In voltage-clamped rabbit myocytes, inhibition of the Na/K pump caused a rise of Nai; there were also marked effects on the tonic shortening elicited by ramp depolarizations. As Nai rose, the magnitude of tonic shortening increased and its voltage dependence also changed. CONCLUSION: These results confirm the suitability of SBFI for measuring Nai in cardiac cells. Provided that steps are taken to optimize transmembrane Na equilibration, the indicator can be calibrated for Nai. The different Nai of rat and rabbit myocytes has implications for the function of sarcolemmal Na/Ca exchange in each cell type. An Nai of 10.9 mM in rat myocytes gives a calculated reversal potential for the exchange of -35 mV. In comparison, an Nai of 3.8 mM in rabbit myocytes leads to a reversal potential for the exchange +45 mV. Therefore, relatively small changes of Nai can shift the reversal potential of the exchange to values that are substantially more positive or negative than zero. The behavior of voltage-dependent tonic contraction in rabbit myocytes was consistent with the Na/Ca exchange reversal potential being more positive than zero in these cells.
Authors: Sara Gattoni; Åsmund Treu Røe; Michael Frisk; William E Louch; Steven A Niederer; Nicolas P Smith Journal: J Physiol Date: 2016-06-26 Impact factor: 5.182
Authors: Miguel A Mondragão; Hartmut Schmidt; Christian Kleinhans; Julia Langer; Karl W Kafitz; Christine R Rose Journal: J Physiol Date: 2016-05-27 Impact factor: 5.182