Mechanical and electrophysiological effects of protamine on isolated ventricular myocardium: evidence for calcium overload.
OBJECTIVE
The aim was to examine in vitro the cellular cardiac effects of protamine, the cationic polypeptide employed to reverse heparin anticoagulation, and to define its mechanisms of action.


METHODS
Isometric contractile force and action potential characteristics after rest and at frequencies up to 3 Hz were recorded in guinea pig ventricular papillary muscle. The actions of protamine (10-300 micrograms.ml-1) were compared to those of heparin (10, 30 units.ml-1), and to heparin (10 units.ml-1) neutralised with equivalent (100 micrograms.ml-1) or excess (200 micrograms.ml-1) protamine. The effects of protamine were also examined using (1) muscle rapid cooling contractures (RCC) to assess intracellular Ca2+ stores and (2) the whole cell voltage clamp method to evaluate K+ and Ca2+ currents of isolated ventricular myocytes.


RESULTS
Protamine (100-300 micrograms.ml-1) depressed contractions by 35-65% at 3 Hz, whereas contractions were enhanced by 150-500% at lower rates (resting state, 0.5 Hz), with a concomitant rise in resting force. Protamine caused a resting depolarisation from -90 to -76 mV and depressed action potential amplitude. In contrast, heparin altered contractile or action potential characteristics minimally. In 26 mM K+ solution with 0.1 microM isoprenaline, 30-300 micrograms.ml-1 protamine caused dose dependent depression of late peaking force development and slow action potential prolongation. After 15 min rest, when RCC were not normally elicited, rest RCC became prominent in 100-300 micrograms.ml-1 protamine. Effects of heparin with 100 micrograms.ml-1 excess protamine were similar to those of 100 micrograms.ml-1 protamine alone. Voltage clamp of isolated myocytes revealed that 10 micrograms.ml-1 protamine irreversibly decreased current through inwardly rectifying K+ channels (IK1), increased leakage current, and decreased inward Ca2+ current (ICa).


CONCLUSIONS
The loss of the normal force-frequency relation, partial depolarisation, rise in resting tension, and appearance of rested state rapid cooling contractures suggest that unbound protamine can lead to excess intracellular Ca2+, mediated by an alteration in membrane ionic conductances.
Park
W K
WK
Department of Anaesthesiology, Yonsei University School of Medicine, Seoul, Korea.
Pancrazio
J J
JJ
Lynch
C
C
D000200:Action Potentials / Q000187:drug effects*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D046508:Culture Techniques; D005260:Female; D006168:Guinea Pigs; D006493:Heparin / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D011479:Protamines / Q000494:pharmacology*; D013268:Stimulation, Chemical
D011479:Protamines; C030101:protamine heparin aggregate; D006493:Heparin; D002118:Calcium