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Binding of cytosolic proteins to myofibrils in ischemic rat hearts
R Barbato1, R Menabò, P Dainese
1Dipartimento di Chimica Biologica e Centro per lo Studio delle Biomembrane, Università di Padova, Italy.
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 alpha 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 alpha 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.
Insights
Ischemia in rat hearts causes specific cytosolic proteins, alpha B-crystallin and GAPDH, to bind to myofibrillar proteins. These changes may serve as markers for ischemic damage and affect heart contractility.
Area of Science:
- Cardiovascular Biology
- Protein Biochemistry
- Cellular Pathology
Background:
- Myofibrillar proteins (MPs) undergo changes during cardiac ischemia.
- Cytosolic protein association with MPs during ischemia is not fully understood.
- Potential degradation or binding events may impact cardiac function.
Purpose of the Study:
- To investigate protein degradation and cytosolic protein association with MPs in rat hearts during ischemia.
- To identify specific peptides appearing in MPs under ischemic conditions.
- To elucidate the mechanisms and implications of these protein changes.
Main Methods:
- Isolation and perfusion of rat hearts subjected to varying ischemia durations.
- SDS-PAGE and Edman sequencing to identify protein bands.
- Immunofluorescence analysis to confirm protein localization.
- In vitro studies to assess binding mechanisms (acidosis, ATP depletion).
Main Results:
- A 23-kD band (alpha B-crystallin) and a 39-kD band (GAPDH) were identified in MPs after ischemia.
- These bands persisted during reperfusion.
- Alpha B-crystallin binding was induced by acidosis; GAPDH binding was prevented by inhibiting ATP depletion.
- Thiol oxidation did not induce GAPDH binding.
Conclusions:
- Ischemia induces specific cytosolic protein binding (alpha B-crystallin, GAPDH) to myofibrils in rat hearts.
- These protein alterations can serve as intracellular markers of ischemic damage evolution.
- The binding of alpha B-crystallin may contribute to altered cardiac contractility.