Jay Jayakumar

Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: Implications for myocardial protection

OBJECTIVES Induction of the 70 kd heat shock protein in the heart is known to exert a protective effect against postischemic mechanical and endothelial dysfunction. However, the exact site of induction and the mechanisms involved remain unknown. The aim of this study was to investigate the relative capacity of endothelial and myocardial cells to express the 70 kd heat shock protein in response to heat stress, as well as their significance. METHODS (1) Postischemic recovery of cardiac mechanical and endothelial function was studied in isolated rat hearts with and without endothelial denudation with saponin. (2) Semiquantitative determination of induction of 70 kd heat shock protein by Western immunoblotting was performed in the whole cardiac homogenate, in isolated cardiac myocytes, and in coronary endothelial cells. (3) Immunocytochemistry was used to visualize the distribution of induction of 70 kd heat shock protein in both cell types. RESULTS Postischemic recovery (percent preischemic value +/- standard error of the mean) of cardiac output in hearts from heat-stressed animals was significantly improved (66.7 +/- 6.9 vs 44.5 +/- 4.5 in the control group, p < 0.01). In heat-stressed hearts treated with saponin no improvement in the recovery of cardiac output was noted (44.7 +/- 6.9 in heat-stressed hearts vs 38.0 +/- 4.0 in heat-stressed, saponin-treated hearts, p = not significant). Endothelial function (as assessed by the vasodilatory response to the endothelium-dependent vasodilator 5-hydroxytryptamine) improved from 31.0 +/- 5.2 in the control group to 65.8 +/- 7.1 in heat-stressed hearts (p < 0.02 vs control) and dropped to -1.9 +/- 3.8 in heat-stressed hearts treated with saponin. Immunocytochemistry showed that only sections of hearts from heat-treated rats showed a strong specific reaction with heat shock protein antibody. The positive staining was seen in endothelial cells. Induction of 70 kd heat shock protein content in the whole cardiac homogenate from heat-stressed rats as measured by Western immunoblotting was 5.2 +/- 1.9 (vs 0.0 in non-heat-stressed rats, p < 0.0001) and dropped to 0.0 in heat-stressed hearts treated with saponin. The tentative amount of 70 kd heat shock protein was 18.1 +/- 7.8 in isolated endothelial cells from heat-stressed hearts and 2.3 +/- 2.3 in isolated cardiac myocytes (p < 0.01 vs endothelial cells). CONCLUSIONS Coronary endothelial cells are the main site of induction of 70 kd heat shock protein in the heart and appear to contribute to the protective effects of heat stress on the recovery of mechanical and endothelial function.

Coronary vasoconstriction to endothelin-1 increases with age before and after ischaemia and reperfusion

OBJECTIVE Ageing is known to be associated with changes within the heart. We investigated whether the coronary response to endothelin-1 (ET) and sarafotoxin S6c (S6c) is altered with increasing age, before and after cardioplegic arrest. METHODS Using an isolated rat heart model, increasing concentrations of ET and S6c were administered to rats of different ages (group I = one month; group II = five months; group III = 21 months). An identical series of experiments was performed following the addition of indomethacin and NG-nitro-L-arginine methyl ester (L-NAME) to the Krebs perfusion fluid. In a third series of experiments, increasing doses of ET-1 were added to hearts following 4 h of cardioplegic arrest at 4 degrees C. RESULTS Coronary flows are expressed as a percentage of initial coronary flow +/- SEM. There was a greater decrease in coronary flow in the older rats for all doses of ET-1. ET-1 (10(-9) M) reduced coronary flows to 72.8 +/- 3.7, 53.2 +/- 6.7 and 56.5 +/- 10.7% for groups I-III respectively (P = 0.01 I vs. II; P = 0.1 I vs. III). A similar response to ET-1 was seen in hearts perfused with indomethacin and L-NAME when compared to those perfused without (P = NS). Perfusion with ET-1 (10(-9) M) following 4 h of cardioplegic arrest reduced coronary flows to 40.5 +/- 4.9, 26.8 +/- 4.8 and 24.1 +/- 3.9%, respectively (P = 0.08 I vs. II; P = 0.03 I vs. III). Perfusion with S6c (10(-10) M) produced coronary flows of 93.3 +/- 5.5, 77.0 +/- 3.5 and 73.9 +/- 3.9% for groups I-III, respectively (P = 0.03 I vs. II; P = 0.01 I vs. III). Perfusion with S6c (10(-9) M) in the presence of L-NAME and indomethacin reduced coronary flows to 85.7 +/- 3.0, 81.6 +/- 2.2 and 74.6 +/- 3.6% (P = NS I vs. II; P = 0.03 I vs. III). CONCLUSIONS The coronary vasoconstrictor response to ET-1 and S6c increases with age. The increased vasoconstriction in response to ET-1 is independent of the decrease in NO release seen with ageing.

Role of endogenous endothelin in the regulation of basal coronary tone in the rat

1 Coronary vascular tone is a vital factor that regulates the delivery of oxygen to cardiac muscle. We tested the hypothesis that basal coronary tone may depend on the release of an endogenous vasoconstrictor peptide, endothelin (ET). 2 Using an isolated, Krebs solution‐perfused rat heart we measured the changes in coronary flow following the administration over a 30 min period of the ET antagonists Ro61‐0612 (mixed ETA/ETB), PD155080 (ETA) and BQ788 (ETB). 3 In a second series of experiments, hearts were randomly assigned to perfusion with plain Krebs solution, or with Krebs solution to which L‐NAME and/or indomethacin had been added. The effect on coronary flow following the addition of Ro61‐0612 was then measured. 4 Perfusion with Ro61‐0612 (10−4 M) alone increased coronary flow by 57.8 %vs. control (P= 0.00001). PD155080 (10−4 M) increased coronary flow by 28.9 % (P= 0.009), whereas BQ788 had no effect on coronary flow. 5 In the second series of experiments, Ro61‐0612 increased coronary flow by 6.6 ± 0.8 ml min−1 in hearts perfused with plain Krebs solution, by 3.8 ± 0.8 ml min−1 in hearts to which both L‐NAME and indomethacin had been added, by 3.3 ± 0.7 ml min−1 in hearts to which L‐NAME had been added, and by 6.9 ± 0.5 ml min−1 in hearts to which indomethacin had been added to the Krebs buffer. 6 In hearts perfused with Krebs solution alone, nitric oxide (NO) release into the coronary sinus increased from 219.8 to 544.9 pmol min−1 g−1 following the addition of Ro61‐0612 (P= 0.06). There was no detectable release of NO from hearts perfused with L‐NAME alone or in combination with indomethacin either before or after the addition of Ro61‐0612. 7 We conclude that endogenous ET plays a role in coronary tone mediated via ETA receptors. This vasodilatation is partially due to an increase in endogenous NO release. However, a significant vasodilatation is still seen following the inhibition of NO synthesis. We propose that basal coronary tone depends on a balance between the endogenous release of vasodilators such as NO and vasoconstrictors such as ET.

Differential effects of endothelin-1 on isolated working rat hearts before and after ischaemia and reperfusion.

Endothelin (ET) may have both detrimental (reduced coronary flow) and beneficial effects (positive inotrope, reduced arrhythmogenesis) following ischaemia. We examined the effects of ET on cardiac function during reperfusion following prolonged hypothermic cardioplegic arrest in a protocol mimicking cardiac transplantation. Isolated working rat hearts were perfused with Krebs buffer to which increasing concentrations of ET-1 or sarafotoxin S6c had been added. Identical experiments were performed after 4 h of cardioplegic arrest at 4 degrees C. Under pre-ischaemic conditions ET-1 caused a dose-dependent decrease in cardiac function compared with controls. In contrast, following ischaemia low doses of ET-1 (10(-10) M) caused a significant and beneficial increase in cardiac output (109.1% versus 81.3%), dP/dt i.e. the rate of change of pressure with time (94.7% versus 75.6%) and stroke volume (100.3% versus 77.5%) compared with controls (P<0.05). At higher doses of ET-1 there was a detrimental effect on cardiac output, dP/dt and stroke volume similar to that seen prior to ischaemia. Sarafotoxin S6c had no significant effect pre or post ischaemia on any of the parameters measured compared with controls (P=not significant). ET-1 at low concentrations during reperfusion can improve the recovery of cardiac function mediated via ET(A) receptors. ET may play an important physiological role in the recovery of cardiac function following prolonged ischaemia.

High-Energy Phosphate Changes in the Normal and Hypertrophied Heart During Cardioplegic Arrest and Ischemia

The profile of metabolic changes in the ischemic heart following repeated infusions of cardioplegic solution has not been well characterized in cardiac hypertrophy. In this study we evaluated the effect of mild cardiac hypertrophy on the changes in phosphocreatine (PCr) and ATP throughout a cardioplegic arrest/ischemia/reperfusion experimental protocol (see Fig. 1). In addition to metabolic status, mechanical recovery of systolic and diastolic function was evaluated in the same hearts.