Reduced coronary reserve in response to short-term ischaemia and vasoactive drugs in ex vivo hearts from diabetic mice.

AIM: The aim of the present study was to compare the coronary flow (CF) reserve of ex vivo perfused hearts from type 2 diabetic (db/db) and non-diabetic (db/+) mice. METHODS: The hearts were perfused in the Langendorff mode with Krebs-Henseleit bicarbonate buffer (37 degrees C, pH 7.4) containing 11 mmol L(-1) glucose as energy substrate. The coronary reserve was measured in response to three different interventions: (1) administration of nitroprusside (a nitric oxide donor), (2) administration of adenosine and (3) production of reactive hyperaemia by short-term ischaemia. RESULTS: Basal CF was approximately 15% lower in diabetic when compared with non-diabetic hearts (2.1 +/- 0.1 vs. 2.6 +/- 0.2 mL min(-1)). The maximum increase in CF rate in response to sodium nitroprusside and adenosine was significantly lower in diabetic (0.6 +/- 0.1 and 0.9 +/- 0.1 mL min(-1) respectively) than in non-diabetic hearts (1.2 +/- 0.1 and 1.4 +/- 0.1 mL min(-1) respectively). Also, there was a clear difference in the rate of return to basal CF following short-term ischaemia between diabetic and non-diabetic hearts. Thus, basal tone was restored 1-2 min after the peak hyperaemic response in non-diabetic hearts, whereas it took approximately 5 min in diabetic hearts. CONCLUSION: These results show that basal CF, as well as the CF reserve, is impaired in hearts from type 2 diabetic mice. As diabetic and non-diabetic hearts were exposed to the same (maximum) concentrations of NO or adenosine, it is suggested that the lower coronary reserve in type 2 diabetic hearts is, in part, because of a defect in the intracellular pathways mediating smooth muscle relaxation.

Measurement of coronary flow reserve in isolated hearts from mice.

AIM: Langendorff-perfused murine hearts are increasingly used in cardiovascular research, but coronary cardiovascular haemodynamics vary considerably from one research group to another. The aim of this study was to establish an isolated, retrogradely perfused mouse heart preparation for the simultaneous measurement of left ventricular haemodynamics and of coronary flow (CF). METHODS: Heart rate was controlled by right atrial pacing (480 beats min(-1)) and heart temperature was kept constant. Accurate flow values of <0.5 mL min(-1) could be determined, and this methodology was then used to study the stability of this preparation, as well as coronary response to vasoactive drugs and to short-term ischaemia. RESULTS: The CF and maximum systolic pressure were well maintained over a 2-h perfusion period, both showing a 10% decline per hour. Sodium-nitroprusside (endothelium-independent) and adenosine (endothelium-dependent) increased CF relatively modest (30-50% above baseline values). Short-term no-flow ischaemia caused a transient 40-50% increase in CF on reperfusion. Peak reflow occurred approximately 15 s after start of reperfusion and flow returned to baseline during the following 1-2 min. Increased coronary blood flow following infusion of vasoactive drugs (nitroprusside or adenosine) or short-term ischaemia were associated with minor changes in ventricular pressure development. CONCLUSIONS: Blood flow and haemodynamics can readily be determined in this isolated perfused mouse heart model, but CF reserve is relatively small, compared with blood-perfused organs.

Different tolerance to hypothermia and rewarming of isolated rat and guinea pig hearts.

We examined the effect of hypothermia and rewarming on myocardial function and calcium control in Langendorff-perfused hearts from rat and guinea pig. Both rat and guinea pig hearts demonstrated a rise in myocardial calcium ([Ca]total) in response to hypothermic perfusion (40 min, 10 degrees C), which was accompanied by an increase in left ventricular end diastolic pressure (LVEDP). The elevation in [Ca]total was severalfold higher in guinea pig than in rat hearts, reaching 12.9 +/- 0.8 and 3.1 +/- 0.6 micromol.g dry wt-1, respectively. The rise in LVEDP, however, was comparable in the two species: 62.5 +/- 2.5 (guinea pig) and 52.5 +/- 5.1 mm Hg (rat). Following rewarming, [Ca]total remained elevated in guinea pig, whereas a moderate decline in [Ca]total was observed in the rat (13.6 +/- 1.9 and 2.2 +/- 0.3 micromol.g dry wt-1, respectively). Posthypothermic values of LVEDP were also significantly higher in guinea pig compared to rat hearts (42.5 +/- 6.8 vs 20.5 +/- 5.1 mm Hg, P < 0.027). Furthermore, whereas rat hearts demonstrated a 78 +/- 7% recovery of left ventricular developed pressure, there was only a 15 +/- 7% recovery in guinea pig hearts. Measurements of tissue levels of high energy phosphates and glycogen utilization indicated a higher metabolic requirement in guinea pig than in rat hearts in order to oppose the hypothermia-induced calcium load. Thus, we conclude that isolated guinea pig hearts are more sensitive to a hypothermic insult than rat hearts.

Stimulation of carbohydrate metabolism reduces hypothermia-induced calcium load in fatty acid-perfused rat hearts.

In the present study we examined the impact of glycolysis and glucose oxidation on myocardial calcium control and mechanical function of fatty acid-perfused rat hearts subjected to hypothermia rewarming. One group (control) was given glucose (11.1 mM) and palmitate (1.2 mM) as energy substrates. In a second group glycolysis was inhibited by iodoacetate (IAA, 100 microM) and replacement of glucose with pyruvate (5 mM), whereas in the third group glucose oxidation was stimulated by administration of dichloroacetate (DCA, 1 mM) and insulin (500 microU/ml). All groups showed a rise in myocardial calcium ([Ca]total in response to hypothermia (10 degrees C). However, [Ca]total was significantly lower both in IAA- and DCA-treated hearts, as compared to controls (2.20 +/- 0.22 and 2.94 +/- 0.20 v 3.83 +/- 0.29 nmol/mg dry wt., P < 0.025). The reduced calcium load in the treated hearts was correlated with higher levels of high energy phosphates. Following rewarming control and DCA-treated hearts still showed elevated [Ca]total, whereas IAA-treated hearts [Ca]total was not different from the pre-hypothermic value. All groups showed a reduction in cardiac output following rewarming. Furthermore, the control group, in contrast to both IAA- and DCA-treated hearts, showed a significant reduction in systolic pressure. These results show that hypothermia-induced calcium uptake in glucose and fatty acid-perfused rat hearts was reduced by two different metabolic approaches: (1) inhibition of glycolysis by IAA while simultaneously by-passing the glycolytic pathway by exogenous pyruvate: and (2) stimulation of glucose oxidation by DCA. Thus, glycolytic ATP is not an essential regulator of sarcolemmal calcium transport under the present experimental conditions. Instead, we suggest that a change in oxidative substrate utilization in favour of carbohydrates may improve myocardial calcium homeostasis during hypothermia and rewarming.

Effects of fatty acids on myocardial calcium control during hypothermic perfusion.

Although hypothermia is regarded as providing protection of the myocardium during cardiac operations, rapid cooling of the myocardium in the nonarrested state may have detrimental effects on the function of the myocardial cell membrane as a permeability barrier. We therefore measured total cellular calcium in isolated working rat hearts, receiving either glucose (11.1 mmol/L) or glucose plus palmitate (1.2 mmol/L), before, during, and after a 40-minute hypothermic arrest (10 degrees C, Langendorff perfusion). In both groups a rise in total cellular calcium, measured by 45Ca2+ technique, was observed during hypothermia, followed by a decline on rewarming. However, the rise in total cellular calcium during hypothermia was significantly (p < 0.05) higher in hearts perfused with palmitate (from 1.0 +/- 0.2 to 3.5 +/- 0.2 nmol/mg dry weight) compared with that in glucose-perfused hearts (from 1.1 +/- 0.13 to 2.6 +/- 0.2 nmol/mg dry weight). Palmitate-perfused, but not glucose-perfused, hearts showed arrhythmias and delayed pressure development 1 to 2 minutes after rewarming. In addition cardiac output of these hearts was significantly lower (p < 0.025) than that of glucose-perfused hearts 5 to 10 minutes after rewarming. These data show that hypothermia per se causes a net calcium uptake in isolated rat hearts and that this effect is aggravated by high concentrations of fatty acids. Thus the impaired recovery of myocardial function in palmitate-perfused hearts can possibly be related to a distorted calcium handling.