Gram positive bacteria reduce viability and contraction of isolated cardiomyocytes: role of endothelin-1

Trupti A. Patel, Sian E. Harding, *Timothy D. Warner and Jane A. Mitchell, Myocardial Systems Biology and Cardiothoracic Pharmacology, Critical Care, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK. *The William Harvey Research Institute, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London EC1M 6BQ, UK.

The pathophysiology of myocardial contractile dysfunction in sepsis is incompletely understood. The aim of this study was to characterise the morphology and contractility changes induced by direct exposure of cardiomyocytes to whole heat-killed Gram positive, Staphylococcus aureus (SA), bacteria in vitro. The role of endothelin in the changes seen was investigated since this is one of the key mediators suspected to play a key role in septic myocardial depression (Spiers et al., 2001; Hirata et al., 2002).

Ventricular myocytes were isolated from adult male Sprague Dawley rats (250-350g) and cultured for 24 to 48h with SA (108 c.f.u./mL) or increasing concentrations of ET-1 or ET-3, with and without the selective ET A receptor antagonist ABT-627. For cell morphology and viability experiments, proportions of viable, (rod-shaped or hypercontracted (shorter lengths and sarcomeres in disarray)) and non-viable (rounded) myocytes were calculated. Both rod-shaped and hypercontracted myocytes were capable of contraction upon electrical stimulation (0.5Hz, 1mM Ca 2+) and proportions of contracting, non-contracting and arrhythmic cells were counted. A contraction was detectable when shortening with each beat was ≥ 0.25%.

At 24h, SA caused a significant reduction in the percentage of rod-shaped myocytes compared to control (Con 43.8 ± 3.4%, SA 22.9 ± 4.5%, P<0.05, n=5), with concomitant increases in both hypercontracted and non-viable myocytes. SA reduced the proportion of viable myocytes that had contractile activity from 56.9 ± 2.6% to 40.6 ± 2.9% (P<0.001, n=33) at 24h and from 43.5 ± 6.9% to 23.5 ± 4.5% (P<0.001, n=9) at 48h. Contraction amplitude of contracting myocytes was not significantly different between groups. After culturing with the selective ET A receptor antagonist ABT-627 (10-8M), the proportion of contracting cells was no longer significantly different between groups at either 24h (Con 78.1 ± 3.5%, SA 74.1 ± 2.7%, n=7) or 48h (Con 57.2 ± 8.9%, SA 50.9 ± 8.3%, n=7). All concentrations of ET-1 and ET-3 (10-9M, 10 -8M and 10-7M) resulted in a reduced population of viable myocytes. The reduction in the proportion of contracting cells after ET-1 (10-8M) exposure was not significant at 24h, but was at 48h (33.6 ± 5.7%, n=9, P<0.05 vs. Con). However, the reduction was not as great as observed with SA in the same experiments (P<0.001). ET-3 (10-8M) had no significant effect on the proportion of contracting myocytes at 24 or 48h.

In conclusion, this study is the first to identify how direct contact with bacteria damages the cardiac myocyte, and to show that the effect is, in part, mediated by ET-1 acting through ET A receptors. This data has implications for our understanding of infection as a risk factor for cardiovascular death.

Hirata et al. (2002) Clin.Sci.(Lond.), 103 S48, 332-335.
Spiers et al. (2001) J.Cardiovasc.Pharmacol., 38(2), 259-67.