Involvement of the IP 3 cascade in the damage to guinea-pig ventricular myocytes induced by cytotoxic T lymphocytes

Abstract

We have shown previously that the interaction between cytotoxic T lymphocytes (CTL) and ventricular myocytes, an in vitro model for heart transplant rejection, results in electrophysiological and morphological alterations indicative of overload of the intracellular [Ca2+] ([Ca2+]i). Since these deleterious effects cannot be accounted for by increased L-type Ca2+ current (ICa,L), we hypothesize that [Ca2+]i overload due to Ca2+ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inositol trisphosphate (IP3) cascade. Patch-clamp and fura-2-fluorescence techniques were utilized to record transmembrane potentials and [Ca2+]i from ventricular myocytes bound to peritoneal exudate CTL (PEL). In ventricular myocyte-PEL conjugates (after 60 min), resting potential was reduced (compared with the nonconjugated state) from -80.9 +/- 0.7 to -59.9 +/- 2.5 mV, action potential amplitude from 139.5 +/- 1.4 to 80.6 +/- 1.7 mV and action potential duration to 50% repolarization (APD50) from 797 +/- 97 to 52 +/- 12 ms. The ratio of fluorescence at 340 and 380 nm (R340/380) increased from a control value (in nonconjugated myocytes) of 0.71 +/- 0.02 to 2.07 +/- 0.03, 30 min, after conjugate formation, and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 micrograms/ml), an antagonist of IP3-induced Ca2+ release from SR channels, or U-73122 (2 microM), a phospholipase C (PLC) inhibitor (drugs were included in the pipette solution), prevented PEL-induced morphological and electrophysiological alterations. Accordingly, heparin attenuated the PEL-induced increase in [Ca2+]i; after 60 min of PEL-myocyte interaction, R340/380 was 1.15 +/- 0.09 (compared with approximately 4.0 in the absence of heparin). The results indicate that CTL-mediated damage to ventricular myocytes is, at least partially, mediated by PLC activation and IP3-induced Ca2+ release from intracellular stores. Pharmacological targeting of IP3 in heart transplant rejection is thus suggested.

Inositol 1,4,5-Trisphosphate Directs Ca2+Flow between Mitochondria and the Endoplasmic/Sarcoplasmic Reticulum: A Role in Regulating Cardiac Autonomic Ca2+Spiking

“…Although IP 3 has been implicated in cardiac arrhythmias (Jacobsen et al, 1996), heart failure (Marks, 1997), and graft rejection (Felzen et al, 1997), the significance of IP 3 in cardiac function has remained unresolved, in part, because of confounding effects of abundant RyRs. We here used neonatal rat cardiomyocytes in culture, which feature an immature SR and express a low density of RyRs (Fitzgerald et al, 1994).…”

Adult rat cardiomyocytes exhibit capacitative calcium entry

“…The efflux from the SR that mediates excitation-contraction coupling occurs through Ca 2ϩ -activated Ca 2ϩ channels termed ryanodine receptors (RyRs). Working in parallel with this efflux pathway are SR Ca 2ϩ channels activated by the second messenger inositol (1,4,5)-trisphosphate (InsP 3 ). InsP 3 receptors are about 50-fold less abundant than RyRs in these cells (19) and are localized to particular portions of the SR (13).…”

Inhibition of Inositol(1,4,5)Trisphosphate Generation by Endothelin-1 During Postischemic Reperfusion

“…Although our studies have implicated IP 3 in the genesis of arrhythmias under ischemic 22 and reperfusion conditions, 9,10 recent studies have extended the pathological role of IP 3 to inflammatory heart diseases by showing that inhibitors of the IP 3 pathway prevent the development of electrophysiological disturbances in cardiomyocytes caused by the presence of activated lymphocytes. 11,23 Responses to combinations of thrombin and ET-1 or norepinephrine and ET-1 together are different from the responses to either effector alone, and this applies to InsP generation as well as arrhythmogenesis. In the in vivo situation, all of these factors are likely to be present in the ischemic myocardium.…”