Pretreatment with d-myo-Inositol Trisphosphate Reduces Infarct Size in Rabbit Hearts: Role of Inositol Trisphosphate Receptors and Gap Junctions in Triggering Protection

Abstract

Pretreatment with D-myo-inositol-1,4,5-trisphosphate hexasodium (D-myo-IP 3 ), the sodium salt of the second messenger inositol 1,4,5-trisphosphate (IP 3 ), is cardioprotective and triggers a reduction of infarct size comparable in magnitude to that obtained with ischemic preconditioning. However, this observation is enigmatic; whereas IP 3 signaling is conventionally initiated by receptor binding, IP 3 receptors are typically considered to be intracellular, and D-myo-IP 3 is membrane-impermeable. We propose that this paradox is explained by the presence of poorly characterized external IP 3 receptors and hypothesize that: 1) infarct size reduction with D-myo-IP 3 is receptor-mediated; and 2) communication via gap junctions and/or hemichannels is required to initiate this protection. To investigate the role of receptor binding, isolated buffer-perfused rabbit hearts underwent 30 min of coronary occlusion (CO) and 2 h of reflow. Prior to CO, hearts received no treatment (controls), D-myo-IP 3 , L-myo-IP 3 (enantiomer not recognized by the IP 3 receptor), D-myo-IP 3 ϩ the IP 3 receptor inhibitor xestospongin C (XeC), or XeC alone. Infarct size, assessed by tetrazolium staining, was reduced with D-myo-IP 3 treatment, whereas hearts that received L-myo-IP 3 or D-myo-IP 3 ϩ XeC showed no protection. To evaluate the contribution of gap junctions/hemichannels, additional control and D-myo-IP 3 -treated cohorts received a 5-min infusion of heptanol or Gap 27, two structurally distinct gap junction inhibitors, administered at doses confirmed to attenuate intercellular transmission of a gap junction-permeable fluorescent dye. There was no infarct-sparing effect of D-myo-IP 3 in inhibitor-treated hearts. These data support the concepts that infarct size reduction with D-myo-IP 3 is triggered by receptor binding and that communication via gap junctions/hemichannels is involved in initiating this protection.Inositol 1,4,5-trisphosphate (IP 3 ) is a ubiquitous second messenger generated in parallel with diacylglycerol in response to activation of G-protein-coupled receptors. Previous studies from our group have shown that prophylactic administration of D-myo-IP 3 hexasodium, the sodium salt of IP 3 , renders the heart resistant to a subsequent sustained ischemic insult and triggers a reduction of infarct size that is comparable in magnitude to the cardioprotection achieved with ischemic preconditioning (Gysembergh et al., 1999). However, this observation is enigmatic; i.e., whereas IP 3 (and presumably D-myo-IP 3 ) signaling is conventionally initiated by receptor binding, IP 3 receptors have historically been considered to be intracellular (specifically located on the endoplasmic reticulum or in the perinuclear region), and D-myo-IP 3 is well known to be membrane-impermeable (Berridge, 1993(Berridge, , 2002Taylor and Broad, 1998;Wilcox et al., 1998;Gysembergh et al., 1999;Ibarra et al., 2004;Vermassen et al., 2004).One potential explanation for this apparent paradox is that reduction of infarct size triggered by exog...

Reduction of infarct size with d-myo-inositol trisphosphate: role of PI3-kinase and mitochondrial KATP channels

“…Experiments were conducted by using the isolated buffer-perfused rabbit heart model of regional ischemia (2,6,10,24). In brief, New Zealand White rabbits weighing 2.5-3.5 kg were anesthetized with an intramuscular injection of ketamine plus xylazine (150 mg and 100 mg, respectively).…”

“…This was preceded by an intervention period, during which hearts were allocated into 15 treatment groups (n ϭ 6 -8 hearts/group). Five groups of hearts were randomly assigned to receive D-myo-Ins(1,4,5)P3 (CalBiochem, La Jolla, CA), dissolved in 5 ml buffer and administered over 1 min, beginning 25 min before the onset of coronary artery occlusion, via a sidearm located immediately proximal to the heart (final concentration in perfusate was 6 M) (10,24). This dose and timing of D-myoIns(1,4,5)P 3 treatment was shown in early pilot studies to trigger optimal protection in this model (data not shown).…”

“…The heart was immediately removed from the apparatus, sliced into 5-7 transverse slices, illuminated under ultraviolet light, and digitally photographed. To distinguish necrotic from viable myocardium, the heart slices were then incubated in triphenyltetrazolium chloride for 15 min at 37°C, rephotographed, and stored in formalin (2,6,10,24).…”

“…Previous studies (10,24) have shown that prophylactic administration of exogenous D-myo-Ins(1,4,5)P 3 hexasodium, the sodium salt of Ins(1,4,5)P 3 , renders the rabbit heart resistant to a subsequent, sustained ischemic insult and initiates a reduction of infarct size similar in magnitude to that achieved with ischemic preconditioning (PC), the current "gold standard" of experimental cardioprotection. The mechanisms responsible for the infarct-sparing effect of exogenous D-myo-Ins(1,4,5)P 3 are, however, largely unknown; although recent evidence has identified Ins(1,4,5)P 3 -receptor binding and communication via gap junctions/hemichannels as elements of the trigger phase of D-myo-Ins(1,4,5)P 3 -induced protection (24), the distal signaling pathways contributing to the increased resistance to ischemia afforded by D-myo-Ins(1,4,5)P 3 have, to date, not been investigated. Four classic cellular mediators [PKC (in particular, the ⑀-isoform of the kinase), phosphatidylinositol-3-kinase (PI3-kinase), as well as sarcolemmal and mitochondrial ATPsensitive K ϩ (K ATP ) channels] have emerged as pivotal mechanistic components in multiple cardioprotective strategies, including, most notably, PC (9,18,31).…”

Effect of heptanol on noradrenaline-induced contractions in rat vas deferens

“…However, in a recent detailed characterization of its effects on vascular smooth muscle cells (Matchkov et al, 2004), it was shown that heptanol, over the range of concentrations used in our study (0.5-2.0 mM), potently increased the input resistance of the cells and desynchronized their [Ca 2+ ]i oscillations, indicating that it powerfully uncoupled the cells. Indeed, in several recent studies carried out on a range of cell / tissue preparations, including mouse heart (Knapp et al, 2005), rabbit heart (Przyklenk et al, 2005), bovine oocytes (Atef et al, 2005), ciliary body (Do et al, 2004), and human HeLa cells (Bader and Weingart, 2004), heptanol has been either shown (Knapp et al, 2005;Atef et al, 2005) or assumed (Do et al, 2004;Bader and Weingart, 2004) to act as a specific gap junction blocker over a range of concentrations (≤ 10 mM), substantially larger than those used in our study (≤ 2 mM). In our own studies on mammalian vas deferens, the concentrations of heptanol employed to study the effects of uncoupling have not been found to affect resting potential (guinea-pig vas deferens: Manchanda and Venkateswarlu, 1997; rat vas deferens: Palani and Manchanda, unpublished observations), indicating a lack of effect on membrane ion channels.…”