James V. Haist

Exercise Improves Postischemic Cardiac Function in Males but Not Females Consequences of a Novel Sex-Specific Heat Shock Protein 70 Response

Exercise is a physiological inducer of the cardioprotective heat shock protein, Hsp70. The putative biological events involved in signaling this response exhibit sexual dimorphism. Thus, it was hypothesized that exercise-mediated induction of Hsp70 would demonstrate sex specificity. After treadmill running, male rats exhibited 2-fold greater levels of cardiac Hsp70 relative to the levels in gonadally intact female rats (P <0.001). Ovariectomized female rats exhibited exercise-mediated induction of Hsp70 similar to that observed for male rats, and estrogen treatment in these female rats reversed this effect (P <0.001). Attenuation of Hsp70 signaling by estrogen was non–receptor-mediated, possibly involving a cellular membrane–stabilizing mechanism of action. The physiological importance of this sex-specific hormone-mediated stress response is underscored by the disparity in functional adaptation in response to exercise between male rats and female rats. Exercise markedly improved postischemic left ventricular developed pressure, the maximal rate of contraction, and maximal rate of relaxation, and it reduced left ventricular end-diastolic pressure in male rats (P <0.001). No such benefit of exercise was observed in intact female rats. A causal role for Hsp70 in this sex-specific cardioprotective adaptation was indicated, inasmuch as ablation of Hsp70 induction with antisense oligonucleotides designed against Hsp70 transcripts attenuated improvement in the recovery of cardiac function in exercised male rats (P <0.05). Thus, the sex-specific hormone-mediated Hsp70 response to exercise results in cardioprotective adaptation, preferentially in male rats relative to female rats. These findings suggest that exercise may be more important for males than for females in defending against the effects of heart disease and offer a novel manner by which males may reduce the sex gap in susceptibility to adverse cardiac events.

Orally administered NHE1 inhibitor cariporide reduces acute responses to coronary occlusion and reperfusion

Na+/H+exchange (NHE) mediates myocardial ischemic and reperfusion injury. We examined the effects of dietary administration of the potent and selective NHE1 inhibitor cariporide on acute responses to coronary artery ligation and reperfusion in the anesthetized rat. Male Sprague-Dawley rats received control rat chow or an identical diet containing 3 parts per million of cariporide for 1 wk before 225 min of occlusion of the left main coronary artery or 45 min of occlusion followed by 180 min of reperfusion. Hearts were excised and divided into left ventricle, right ventricle, and interventricular septum for analysis of NHE1 mRNA expression and apoptosis by staining with terminal deoxynucleotidyl transferase-mediated nick end labeling. Ischemia and reperfusion were associated with a threefold elevation in NHE1 mRNA expression in control animals that was significantly reduced in cariporide-fed rats. Cariporide reduced mortality from 26% of animals to 0%. The incidence of all arrhythmias was significantly reduced, including ventricular fibrillation (from 42 to 0%) and ventricular tachycardia (from 81 to 15%), as well as the number of ventricular premature beats (from 70 ± 12 to 17 ± 6). Cariporide moderately reduced apoptosis only in the reperfused left ventricle to values not significantly greater than those in sham-operated animals, and this was associated with a significantly higher ratio of Bcl-2 to Bax. This study suggests that NHE inhibition with dietary cariporide represents an effective management of acute postinfarction responses.Na+/H+ exchange (NHE) mediates myocardial ischemic and reperfusion injury. We examined the effects of dietary administration of the potent and selective NHE1 inhibitor cariporide on acute responses to coronary artery ligation and reperfusion in the anesthetized rat. Male Sprague-Dawley rats received control rat chow or an identical diet containing 3 parts per million of cariporide for 1 wk before 225 min of occlusion of the left main coronary artery or 45 min of occlusion followed by 180 min of reperfusion. Hearts were excised and divided into left ventricle, right ventricle, and interventricular septum for analysis of NHE1 mRNA expression and apoptosis by staining with terminal deoxynucleotidyl transferase-mediated nick end labeling. Ischemia and reperfusion were associated with a threefold elevation in NHE1 mRNA expression in control animals that was significantly reduced in cariporide-fed rats. Cariporide reduced mortality from 26% of animals to 0%. The incidence of all arrhythmias was significantly reduced, including ventricular fibrillation (from 42 to 0%) and ventricular tachycardia (from 81 to 15%), as well as the number of ventricular premature beats (from 70 +/- 12 to 17 +/- 6). Cariporide moderately reduced apoptosis only in the reperfused left ventricle to values not significantly greater than those in sham-operated animals, and this was associated with a significantly higher ratio of Bcl-2 to Bax. This study suggests that NHE inhibition with dietary cariporide represents an effective management of acute postinfarction responses.

Comparative effects of Na+/H+ exchange inhibitors against cardiac injury produced by ischemia/reperfusion, hypoxia/reoxygenation, and the calcium paradox.

To examine the role of Na + /H+ exchange in cardiac injury, we compared the effect of amiloride (174 μM) with the markedly more specific and potent inhibitor 5-(N,N-hexamethylene) amiloride (HMA, 1 μ.M) against cardiac injury produced by reperfusion, reoxygenation, and the calcium paradox. Reperfusion after 15-min ischemia resulted in a 55 ± 4% recovery in contractility, whereas in the presence of amiloride or HMA, recovery was increased to 82 ± 5.8 and 72 ± 7.8%, respectively (p < 0.05 from control), with HMA showing particular efficacy in accelerating recovery. The rapid restoration of function with HMA was also evident in hearts reoxygenated for 1 min after 12-min hypoxia (control 35 ± 3.2%, HMA 66 ± 4.1%, p < 0.05) although the protective effect gradually reversed with continued reoxygenation. On the other hand, with addition of amiloride, the protective effect persisted so that after 30-min reoxygenation values were significantly higher than control (65 ± 4.1 vs. 47 ± 3.1%, p < 0.05). Resting tension increases after either reperfusion or reoxygenation were moderate: 124 ± 8 and 119 ± 6%, respectively (p > 0.05), but no increases were observed with amiloride or HMA. Bepridil (10 μ. M), a purported Na+/Ca2+ exchange inhibitor, exerted a salutary effect against reperfusion dysfunction identical to that of amiloride and HMA, whereas in reoxygenated hearts the effects were identical to those observed with HMA. The protective effects of the drugs were not related to improved energy metabolic status. None of the pharmacologic interventions exerted beneficial effects against the calcium paradox. The present results support the concept of Na+/H+ exchange-mediated injury, possibly linked to Na+/Ca2+ exchange activation, in reperfused and reoxygenated myocardium but not in hearts subjected to the calcium paradox.

Na+-H+ Exchange Inhibition Protects Against Mechanical, Ultrastructural, and Biochemical Impairment Induced by Low Concentrations of Lysophosphatidylcholine in Isolated Rat Hearts

Lysophophatidylcholine (LysoPC) accumulates rapidly in the ischemic myocardium and is an important mediator of ischemia-induced cell injury. Na(+)-H+ exchange (NHE) inhibition has been demonstrated to protect the ischemic and reperfused myocardium. We determined whether NHE inhibition can also modulate cardiotoxicity produced by LysoPC (3 and 5 mumol/L) in isolated rat hearts. At 3 mumol/L, LysoPC produced a depression in left ventricular developed pressure (LVDP) and elevation in left ventricular end-diastolic pressure (LVEDP), which were 19 +/- 7% and 1290 +/- 205% of pre-LysoPC values, respectively, after 30 minutes of treatment. In the presence of the NHE inhibitor 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642, 5 mumol/L), LVDP was reduced to only 80.8 +/- 8.6%, and LVEDP increased to 270 +/- 32% (P < .05 for both parameters). LysoPC significantly depressed tissue ATP, creatine phosphate, and glycogen contents and increased lactate levels, all of which were significantly attenuated by HOE 642. Moreover, marked LysoPC-induced ultrastructural abnormalities, including mitochondrial and myofibrillar disruption, were totally prevented by HOE 642. This protection was mimicked by another NHE inhibitor, methylisobutylamiloride (5 mumol/L). HOE 642 was also effective against injury produced by 5 mumol/L LysoPC although, generally, the protection was less marked than that observed against 3 mumol/L; LVDP depression after 30 minutes was 10.1 +/- 4.3% and 41.4 +/- 10.4% of pre-LysoPC values in control and HOE 642-treated hearts, respectively (P < .05), whereas corresponding LVEDP elevations were 1629 +/- 393% and 990 +/- 144% (P > .05). In myocytes superfused with bicarbonate-free buffer subjected to acid loading by NH4Cl pulsing, pH recovery (as measured by acid flux) was significantly stimulated by 3 mumol/L LysoPC, indicative of NHE activation. Our study shows that cardiac injury produced by low concentrations of LysoPC can be effectively attenuated by NHE inhibition. The results also suggest that the beneficial effects of NHE inhibitors on the ischemic myocardium may be, at least partially, mediated by inhibiting the deleterious effects of LysoPC.

Ginseng Inhibits Cardiomyocyte Hypertrophy and Heart Failure via NHE-1 Inhibition and Attenuation of Calcineurin Activation

Background—Ginseng is a medicinal plant used widely in Asia that has gained popularity in the West during the past decade. Increasing evidence suggests a therapeutic role for ginseng in the cardiovascular system. The pharmacological properties of ginseng are mainly attributed to ginsenosides, the principal bioactive constituents in ginseng. The present study was carried out to determine whether ginseng exerts a direct antihypertrophic effect in cultured cardiomyocytes and whether it modifies the heart failure process in vivo. Moreover, we determined the potential underlying mechanisms for these actions. Methods and Results—Experiments were performed on cultured neonatal rat ventricular myocytes as well as adult rats subjected to coronary artery ligation (CAL). Treatment of cardiomyocytes with the &agr;1 adrenoceptor agonist phenylephrine (PE) for 24 hours produced a marked hypertrophic effect as evidenced by significantly increased cell surface area and ANP gene expression. These effects were attenuated by ginseng in a concentration-dependent manner with a complete inhibition of hypertrophy at a concentration of 10 &mgr;g/mL. Phenylephrine-induced hypertrophy was associated with increased gene and protein expression of the Na+-H+ exchanger 1 (NHE-1), increased NHE-1 activity, increased intracellular concentrations of Na+ and Ca2+, enhanced calcineurin activity, increased translocation of NFAT3 into nuclei, and GATA-4 activation, all of which were significantly inhibited by ginseng. Upregulation of these systems was also evident in rats subjected to 4 weeks of CAL. However, animals treated with ginseng demonstrated markedly reduced hemodynamic and hypertrophic responses, which were accompanied by attenuation of upregulation of NHE-1 and calcineurin activity. Conclusions—Taken together, our results demonstrate a robust antihypertrophic and antiremodeling effect of ginseng, which is mediated by inhibition of NHE-1–dependent calcineurin activation.

Probiotic Administration Attenuates Myocardial Hypertrophy and Heart Failure After Myocardial Infarction in the Rat

Background—Probiotics are extensively used to promote gastrointestinal health, and emerging evidence suggests that their beneficial properties can extend beyond the local environment of the gut. Here, we determined whether oral probiotic administration can alter the progression of postinfarction heart failure. Methods and Results—Rats were subjected to 6 weeks of sustained coronary artery occlusion and administered the probiotic Lactobacillus rhamnosus GR-1 or placebo in the drinking water ad libitum. Culture and 16s rRNA sequencing showed no evidence of GR-1 colonization or a significant shift in the composition of the cecal microbiome. However, animals administered GR-1 exhibited a significant attenuation of left ventricular hypertrophy based on tissue weight assessment and gene expression of atrial natriuretic peptide. Moreover, these animals demonstrated improved hemodynamic parameters reflecting both improved systolic and diastolic left ventricular function. Serial echocardiography revealed significantly improved left ventricular parameters throughout the 6-week follow-up period including a marked preservation of left ventricular ejection fraction and fractional shortening. Beneficial effects of GR-1 were still evident in those animals in which GR-1 was withdrawn at 4 weeks, suggesting persistence of the GR-1 effects after cessation of therapy. Investigation of mechanisms showed a significant increase in the leptin:adiponectin plasma concentration ratio in rats subjected to coronary ligation, which was abrogated by GR-1. Metabonomic analysis showed differences between sham control and coronary artery ligated hearts particularly with respect to preservation of myocardial taurine levels. Conclusions—The study suggests that probiotics offer promise as a potential therapy for the attenuation of heart failure.

Interrelationships between iron and lead absorption in iron-deficient mice.

Mice were made iron-deficient by feeding a semisynthetic low-iron diet, by bleeding, or by a combination of both methods. Dietary-induced iron deficiency, by itself, enhanced iron absorption from an. intragastric dose to a much greater extent than did bleeding mice fed an iron-supplemented diet. Enhanced lead absorption was seen in the former but not in the latter group. Bleeding was effective, however, in raising lead absorption when mice were fed the iron-deficient diet. Additionally, the kinetics of lead absorption were studied by using an openended duodenal perfusion technique. When lead was perfused in solutions containing either 0.15 M sodium chloride or 0.15 M sodium ascorbate, irondeficient animals took up more lead into the mucosa and transferred more to the body than mice fed an iron-supplemented diet. Intestinal uptake of lead from both perfusates in iron-sufficient animals appeared to occur by a process that was primarily dependent on the luminal lead concentration. Lead uptake from the saline perfusate in iron-deficient mice was also directly related to the lead concentration of the perfusate, whereas uptake from the ascorbate perfusate was proportionally greater from lowerthan from higher-lead concentrations, giving rise to saturation-type kinetics. Lead transfer to the body from both types of perfusate also displayed saturation kinetics in both iron-sufficient and iron-deficient animals. When iron and lead were perfused together in 0.15 M sodium ascorbate, iron inhibited lead up take and transfer at Fe/Pb molar ratios as low as 0.5:1, whereas lead inhibited iron uptake and transfer at a Pb/Fe ratio of 5:1. These mutually inhibitory effects were not as easily demonstrated with a saline perfusate. Our results suggest that lead absorption occurs by two simultaneous processes: the first is a diffusion-type mechanism, and the second is a carrier-mediated process, which is enhanced by feeding a low-iron diet. Lead appeared to have less affinity than iron for the latter mechanism.

Cadmium-induced enteropathy: comparative toxicity of cadmium chloride and cadmium-thionein.

In protecting the body against the noxious effects of dietary cadmium ions, cadmium is bound to metallothionein in the proximal intestine, and subsequently excreted into the lumen with desquamation of the epithelium. The purpose of this study was to determine the extent to which cadmium in the form of intestinal cadmium-thionein is absorbed from the intestinal lumen and to appraise the toxicity of cadmium-thionein on the intestinal mucosa. With open-ended duodenal perfusion, equivalent amounts of cadmium administered as CdCl2 or cadmium-thionein entered the mucosa, but significantly less cadmium from the perfusate of cadmium-thionein passed into the body. Exposure of the mucosa to CdCl2 for 1 hr led to minor abnormalities in the form of broadening of villi with pseudostratification of epithelium, and swelling of mitochondria, whereas cadmium-thionein produced extensive necrosis of absorptive cells. The results suggest that cadmium-thionein may play a paradoxical role, providing protection against the cadmium ion in the intracellular milieu, but promoting cadmium toxicity when it is present in sufficient amounts in the lumen of the intestine.

Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification

Dehydroascorbic acid (DHAA) enters cells via Na(+)-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na(+) removal inhibited [(14)C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2-12 min at 10-200 microM DHAA in either the presence of 134 mM Na(+) or in its absence (N-methyl D-glucamine, choline or sucrose replaced Na(+)). This inhibition of DHAA uptake by Na(+) removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [(14)C]DHAA into [(14)C]ascorbate upon intracellular acidosis caused by the Na(+) removal. Consistent with this hypothesis: (i) the Na(+)/H(+)-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na(+) removal and Na(+)/H(+)-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12 min than in 2 min, and (iii) less of the cellular (14)C was present as ascorbate (determined by HPLC) in cells in Na(+)-free buffer than in those in Na(+)-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion.

The obesity-related peptide leptin sensitizes cardiac mitochondria to calcium-induced permeability transition pore opening and apoptosis.

The obesity-related 16 kDa peptide leptin is synthesized primarily in white adipocytes although its production has been reported in other tissues including the heart. There is emerging evidence that leptin may contribute to cardiac pathology especially that related to myocardial remodelling and heart failure. In view of the importance of mitochondria to these processes, the goal of the present study is to determine the effect of leptin on mitochondria permeability transition pore opening and the potential consequence in terms of development of apoptosis. Experiments were performed using neonatal rat ventricular myocytes exposed to 3.1 nM (50 ng/ml) leptin for 24 hours. Mitochondrial transition pore opening was analyzed as the capacity of mitochondria to retain the dye calcein-AM in presence of 200 µM CaCl2. Leptin significantly increased pore opening although the effect was markedly more pronounced in digitonin-permeabilized myocytes in the presence of calcium with both effects prevented by the transition pore inhibitor sanglifehrin A. These effects were associated with increased apoptosis as evidenced by increased TUNEL staining and caspase 3 activity, both of which were prevented by the transition pore inhibitor sanglifehrin A. Leptin enhanced Stat3 activation whereas a Stat 3 inhibitor peptide prevented leptin-induced mitochondrial transition pore opening as well as the hypertrophic and pro-apoptotic effects of the peptide. Inhibition of the RhoA/ROCK pathway prevented the hypertrophic response to leptin but had no effect on increased pore opening following leptin administration. We conclude that leptin can enhance calcium-mediated, Stat3-dependent pro-apoptotic effects as a result of increased mitochondrial transition pore opening and independently of its hypertrophic actions. Leptin may therefore contribute to mitochondrial dysfunction and the development of apoptosis in the diseased myocardium particularly under conditions of excessive intracellular calcium accumulation.