Naohisa Noda

Cytosolic Ca2+ concentration and pH of diabetic rat myocytes during metabolic inhibition.

To investigate the role of Ca2+ metabolism and pH in diabetic cardiomyopathy, intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) of isolated myocytes were measured simultaneously using fura-2 and BCECF. We used diabetic (D.M.) rats at 8 weeks after the injection of streptozotocin (45 mg/kg, i.v.). (1) [Ca2+]i of D.M. myocytes was lower than that of controls (53 +/- 3 and 75 +/- 5 nM, mean +/- S.E., P less than 0.01). There was no difference in pHi (7.06 +/- 0.02 in D. M., 7.07 +/- 0.02 in control). There was no difference in the percentage of non-rounded cells at 30 min after the perfusion of glucose-free solution which contained 2 mM sodium cyanide (NaCN) between D.M. and controls (53% and 52%). When cells were rounded, the value of [Ca2+]i was significantly lower in D.M. myocytes than that in controls (172 +/- 21 and 421 +/- 106 nM, P less than 0.05). (2) When the cells were shortened or rounded in the high [Ca2+]o solution (24.5 mM), [Ca2+]i of D.M. rats was significantly lower than that of control rats. (3) The percentage of non-rounded cells at 30 min after the perfusion of NaCN increased in controls by 50 mM glucose (95%, P less than 0.01), but not in D.M. (47%). Insulin (25 mU/ml) and glucose (15 mM) increased the percentage of non-rounded cells in D.M. after 30 min perfusion with NaCN (88%, P less than 0.01 v.s. 53% without glucose nor insulin). It is suggested that there are disturbances of Ca2+ metabolism in D.M. myocytes, and that there is a close relation between cell injury and glucose utilization during metabolic inhibition.

Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.

QUANTIFICATION OF INTRACELLULAR FREE SODIUM IONS BY USING A NEW FLUORESCENT INDICATOR, SODIUM-BINDING BENZOFURAN ISOPHTHALATE IN GUINEA PIG MYOCYTES

Isolated guinea pig myocytes were loaded with the Na(+)-sensitive fluorescent probe, sodium-binding benzofuran isophthalate (SBFI). The 340/380 nm fluorescence ratios were measured with fluorescence microscopy. The distribution of intracellular Na+ concentration ([Na+]i) was homogenous, and the mean resting [Na+]i was 8.4 +/- 0.5 mM. There was a significant relationship (r = 0.66, p less than 0.001) between elevation of [Na+]i and shortening of longitudinal length of the cells, during the perfusion of 100 microM strophanthidin. It is concluded that this method is suitable for measuring [Na+]i in isolated myocytes.

Cytosolic Ca2+ concentration decreases in diabetic rat myocytes.

Time for primary review 24 days. Diabetes mellitus has been shown to be associated with heart failure of unknown origin, which is termed ‘diabetic cardiomyopathy’ [1]. Regan et al. [2]have shown that the small vessel lesions in diabetes mellitus have little or no relation to diabetic cardiomyopathy, suggesting that the contractile function of the diabetic cardiomyopathy was primarily defective. Although many studies have been performed to reveal cellular and subcellular derangement in diabetic cardiomyopathy [3–6], the precise mechanism is still unknown. It has been reported that Ca2+ metabolism in diabetic myocardium has several abnormalities, including a decreased activity of Ca2+-ATPase in sarcoplasmic reticulum (SR) [7, 8]and sarcolemma [9, 10]. It has also been reported that the activity of Na+/Ca2+ exchange is lower in diabetic myocardium [10]. These reports have suggested that Ca2+ overload may be involved in the pathogenesis of diabetic cardiomyopathy [3]. Although it has been shown that certain mechanical and electrophysiological features in diabetic rats correlate well with conditions of intracellular Ca2+ overload [11, 12], direct measurement of the cytosolic Ca2+ concentration ([Ca2+]i) has not been made previously. The most popular method for measuring [Ca2+]i uses fluorescent probes such as fura-2 and indo-1 [13]. We have reported that [Ca2+]i of unstimulated rat myocytes isolated from insulin-dependent diabetes mellitus (IDDM) was lower than that of normal myocytes using fura-2 (53±3 nM in diabetic myocytes and 75±5 nM in normal myocytes, mean±s.e.; P <0.01) [14]. Recently, Lagadic-Gossmann et al. [15]also reported that basal [Ca2+]i level was lower in diabetic myocytes using indo-1. Horackova and Murphy [16]have reported that the Ca2+ content is decreased in diabetic rat … * Corresponding author. Tel. +81 53 435-2391; Fax +81 53 435-2394.

Changes in Cell Morphology, [Ca2+]i and pHi During Metabolic Inhibition in Isolated Myocytes of Diabetic Rats Using Dual-Loading of Fura-2 and BCECF

Diabetes Mellitus (DM) has been shown to be associated with heart failure in the absence of atherosclerosis (1,2), suggesting a diabetic cardiomyopathy (3,4). The mechanism of heart failure due to the diabetic cardiomyopathy remains to be elucidated. The small vessel disease (5) and the abnormalities of subcellular mechanisms such as myosin ATPase (6) and myosin isoenzymes (7), have been reported in DM myocardium. Recently, abnormalities of Ca2+ metabolism have been reported in DM myocardium, which showed decreased Ca2+-ATPase of sarcoplasmic reticulum (SR) (8,9) and sarcolemma (10). It has also been reported that the activity of Na+/Ca2+-exchange was lower in DM myocardium (11). Previous reports have suggested the possibility of the Ca2+ overload in diabetic cardiomyopathy (11).