© 1996 American Heart Association, Inc.
Articles |
Binding of Cytosolic Proteins to Myofibrils in Ischemic Rat Hearts
Correspondence to Prof Fabio Di Lisa, Dipartimento di Chimica Biologica, Via Trieste, 75, 35121 Padova, Italy. E-mail dilisa@civ.bio.unipd.it.
Abstract Myofibrillar proteins (MPs) were extracted from
isolated and perfused rat hearts subjected to different periods of
ischemia to investigate the occurrence of protein degradation
and/or the association of cytosolic proteins with the myofibrillar
pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of
ischemia, with its density gradually increasing to a plateau
after 20 minutes. Longer periods of ischemia were associated
with the appearance of a 39-kD band. Irrespective of the duration of
ischemia, both these bands persisted during reperfusion. A
partial proteolytic degradation of troponin T (TnT) and troponin I
(TnI) has been claimed to be responsible for the generation of these
peptides. However, the N-terminal sequence of the 39-kD band was
identical to that of GAPDH, whereas Edman sequencing after pepsin
digestion showed that the 23 kD is 
B-crystallin. The binding of the
two cytosolic proteins to myofibrils was confirmed by
immunofluorescence analysis on cryosections
of ischemic hearts. In vitro studies showed that acidosis was
sufficient to induce the binding of B-crystallin, whereas the
inhibition of ATP depletion prevented the binding of GAPDH. Thiol
oxidation is unlikely to promote GAPDH binding, since perfusion with
iodoacetate under aerobic conditions or treatment of
homogenates with N-ethylmaleimide or diamide
failed to induce GAPDH association with the myofibrils. These changes
of the myofibrillar proteins could be considered as intracellular
markers of the evolution of the ischemic damage. In addition,
the binding of the 23-kD peptide might be involved in alterations of
contractility.
Key Words: GAPDH • B-crystallin • acidosis • troponin • ischemia
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