Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Effects of cold cardioplegia on pH, Na, and Ca in newborn rabbit hearts

Many studies suggest myocardial ischemia-reperfusion (I/R) injury results largely from cytosolic proton (H(i))-stimulated increases in cytosolic Na (Na(i)), which cause Na/Ca exchange-mediated increases in cytosolic Ca concentration ([Ca]i). Because cold, crystalloid cardioplegia (CCC) limits [H]i, we tested the hypothesis that in newborn hearts, CCC diminishes H(i), Na(i), and Ca(i) accumulation during I/R to limit injury. NMR measured intracellular pH (pH(i)), Na(i), [Ca]i, and ATP in isolated Langendorff-perfused newborn rabbit hearts. The control ischemia protocol was 30 min for baseline perfusion, 40 min for global ischemia, and 40 min for reperfusion, all at 37 degrees C. CCC protocols were the same, except that ice-cold CCC was infused for 5 min before ischemia and heart temperature was lowered to 12 degrees C during ischemia. Normal potassium CCC solution (NKCCC) was identical to the control perfusate, except for temperature; the high potassium (HKCCC) was identical to NKCCC, except that an additional 11 mmol/l KCl was substituted isosmotically for NaCl. NKCCC and HKCCC were not significantly different for any measurement. The following were different (P < 0.05). End-ischemia pH(i) was higher in the CCC than in the control group. Similarly, CCC limited increases in Na(i) during I/R. End-ischemia Na(i) values (in meq/kg dry wt) were 115 +/- 16 in the control group, 49 +/- 13 in the NKCCC group, and 37 +/- 12 in the HKCCC group. CCC also improved [Ca]i recovery during reperfusion. After 40 min of reperfusion, [Ca](i) values (in nmol/l) were 302 +/- 50 in the control group, 145 +/- 13 in the NKCCC group, and 182 +/- 19 in the HKCCC group. CCC limited ATP depletion during ischemia and improved recovery of ATP and left ventricular developed pressure and decreased creatine kinase release during reperfusion. Surprisingly, CCC did not significantly limit [Ca]i during ischemia. The latter is explained as the result of Ca release from intracellular buffers on cooling.     

The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast

In S. cerevisiae the induction of heat-shock protein (HSP) synthesis is accompanied by a decrease in the cytoplasmic and vacuolar pH as determined by means of [31P]NMR spectroscopy. The relationship of HSP synthesis and acidification of the cytoplasmic pH is dose-dependent under a variety of treatments (temperature increases (23-32 degrees C), addition of 2,4-dinitrophenol (greater than 1 mM), sodium arsenite (greater than 3.75 X 10(-5) M) or sodium cyanide (greater than 10 mM]. Changes in the intracellular pH occur within 5 min after treatment, attain a maximum within 30 min and are subsequently stable. HSPs 98, 85 and 70 show maximum synthesis rates 1-2 h after a 40 degrees C heat shock. The synthesis rates then decline. HSPs 56, 44 and 33 reveal a smaller and slower increase and almost no decrease in the synthesis rate within 4 h at 40 degrees C. The similar dose dependencies of HSP synthesis and cytoplasmic pH. as well as the immediate response of the pH, can also be demonstrated in the mitochondrial mutant of S. cerevisiae (Q0). This result indicates that the heat-shock response is mainly independent of intact oxidative phosphorylation. No correlation was observed between HSP synthesis rate and total intracellular ATP content.     

Free fatty acids, but not ketone bodies, protect diabetic rat hearts during low-flow ischemia

To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pH(i)) by using (31)P NMR spectroscopy plus [2-(3)H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM beta-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml x g wet wt(-1) x min(-1)) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 +/- 17 to 52 +/- 12 micromol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 +/- 5 micromol/g wet wt. Recovery of contractile function was unrelated to pH(i) during ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pH(i) values that were significantly different at 6.36 +/- 0.04 and 6.60 +/- 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pH(i) was 6.49 +/- 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.     

Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO

Recent studies suggest a role for phospholamban phosphorylation during ischemia and reperfusion. The role of phospholamban in ischemia was studied by subjecting hearts from male and female wild-type (MWT/FWT) and phospholamban-knockout (MKO/FKO) mice to 20 min of ischemia-40 min of reperfusion while (31)P NMR spectra were acquired. ATP and pH values fell lower during ischemia, and postischemic contractility was less, in MKO and FKO versus WT hearts. After shorter ischemia (15 min), recoveries of contraction, ATP, and pH were greater in FKO than MKO hearts. To examine the role of nitric oxide (NO) synthases (NOS) in the protection in FKO versus MKO hearts, we utilized 1 microM l-NAME, a NOS inhibitor, or 100 microM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor. Recoveries of function, ATP, and pH were less in l-NAME-treated FKO than untreated FKO hearts and greater in SNAP-treated MKO than untreated MKO hearts. In conclusion, phospholamban ablation increased ischemic injury in both males and females; however, female hearts were less susceptible than male hearts. Protection in females was decreased by a NOS inhibitor and mimicked in males by an NO donor, implying that protection was NOS mediated.     

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).     

An alteration in molecular form associated with activation of human heat shock factor

In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a 32P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.     

The 31P NMR visibility of ATP in perfused rat liver remains about 90%, unaffected by changes of metabolic state.

The 31P NMR visibility of ATP of the perfused rat liver was tested over a wide range of metabolic conditions, including normoxic and hypoxic perfusions, fructose loads, and various intervals of normothermic ischemia, for both ad libitum fed and 24-h fasted rats. The 31P NMR signal of ATP was compared to the concentration of ATP determined by enzymatic assays on liver biopsies performed at the end of NMR acquisition. In a first series of experiments, the NMR resonance of intracellular ATP was quantitated in absolute terms by applying the 1H NMR water signal as internal reference: during normoxic and hypoxic perfusions, a constant amount of ATP (0.43 +/- 0.19 mM, mean +/- SD), approximately 12% of the cellular ATP, is not detected by NMR. Nevertheless, there is a high correlation (slope = 0.96 +/- 0.09; r2 = 0.93) between the measurements of ATP by 31P NMR spectroscopy and by biochemical analysis. In a second series of experiments, there was a highly significant correlation between the NMR and analytical biochemical measurements of ATP for whole range of metabolic states, i.e., fructose loads (1.0-10 mM) and various intervals of normothermic ischemia (ranging from 2 to 12 min), indicating unchanged ATP visibility. Thus, as opposed to the studies of Murphy et al. [Murphy, E., et al. (1988) Biochemistry 27, 526-528], it is concluded that ATP at 37 degrees C remains almost entirely visible in the perfused rat liver, also during ischemia.