A Fast Signal-Induced Activation of PARP-1 in
Cardiomyocytes: A Novel Mechanism Regulating
the Activity of DNA-Binding Proteins.

Cohen-Armon M.
Department of Cardiology, Shebah Medical Center

We found that depolarizing stimulation of brain cortical neurons rapidly modulates the activity of nuclear proteins that regulates DNA repair and transcription by polyADP-ribosylation mediated by activation of PLC and IP3-gated Ca⁺² release into the nucleoplasm. Cardiac myocytes undergo constant electrical stimulation, it is unknown if this can influences nuclear proteins in a manner similar to that found in brain cortical neurons. Furthermore, recent data highlight the role of PLC activation and IP₃-gated Ca⁺² release in inflammatory heart disease, myocardial ischemia and reperfusion, heart failure and apoptosis. Thus, the possibility of unveiling a mechanism in which membrane depolarization, receptor stimulation and intracellular Ca⁺² release are linked to nuclear protein activity may be especially important in cardiac myocytes.

Induction of polyADP- ribosylation in high frequency palpitating myocytes:
The effect of norepinephrine.
Adrenergic stimulation profoundly alters the fluxes of Ca⁺² in cardiomyocytes and thereby significantly affects contraction and relaxation. We examined the effect of adrenergic activation on the activity of PARP-1 in cardiomyocytes. Addition of NE to the culture resulted immediately an increase in the spontaneous contractility of the cultured cardiomyocytes. Simultanously, an extended activity of PARP-1 was measured in their isolated nuclei. Adding the antagonist propranolol together with NE abolished PARP-1 activation by NE indicating the role of adrenergic receptors in modulating the activity of PARP-1.

Extranuclear Ca⁺² Promotes Activation of PARP-1 in cardiomyocytes
We examined the effect of extranuclear [Ca⁺²] on [³²P]PolyADP-ribosylation of nuclear proteins in isolated nuclei of cardiomyocyte. Increasing the extranuclear [Ca⁺²] during [32P]Poly-ADP-ribosylation immediately enhanced dose-dependantly the [³²P]PolyADP-ribosylation of PARP-1. The maximal effect was at [Ca⁺²] = 5 µM, while normal free [Ca⁺²] in the cytoplasm is 100 nM.

A possible role of stimulation-induced polyADP-ribosylation in modification of the myocard: Induction of PolyADP-ribosylation by growth factors
In cortical neurons, PARP-1 has been shown to act as a downstream target of PLC, being activated by IP3-gated release of Ca⁺². We examined the possibility that PARP-1 activation is mediated by activation of PLC in cardiomyocytes as well. Since phsphorylation of RTKs promotes PLC activation we examined the effect of receptor tyrosine kinase phosphorylation following their binding to growth factors on the activity of PARP-1. Induction of polyADP-ribosylation by growth factors was examined by adding insulin and epidermal growth factors (L-IGF and L-EGF) to cultured myocytes. After being exposed to the growth factor for 5 min, cardiomyocytes were lysed and their nuclei were isolated. [³²P]polyADP-ribosylation was conducted in the isolated nuclei. revealed activation of PARP-1 in cardiomyocytes exposed to L-IGF or L-EGF.

Perspectives
In the next phase of the study we intend to examine alteration of this signal transduction pathway in models of disease states as ischemia-reperfusion and myocardial hibernation, cardiac hypertrophy, heart failure and myocyte apoptosis.