Rs are intracellular ligand-gated Ca2+
release channels that are activated by IP3
binding where they function downstream of growth factors and GPCR signaling events 6
. The physiological role of Ca2+
release from the IP3
R in ventricular myocytes has been a controversial issue, with some reports suggesting no effect on ECC, while others have observed a small but significant effect on spontaneous Ca2+
release in the form of sparks and enhanced Ca2+
transients 15, 32-34
. In atrial myocytes, IP3
release appears to be a more prominent event that modulates ECC and SR Ca2+
release 35 36
, likely because of higher endogenous IP3
R expression levels 34 15
. Consistent with these reports, transgene-directed overexpression of IP3
R2 in ventricular myocytes had a prominent effect on ECC and even arrhythmia upon ET-1 stimulation, further suggesting that IP3
Rs are positioned within the junctional SR where they can affect ryanodine receptor (RyR) activity.
To affect ECC, the IP3
Rs need to be positioned within the proper functional domains of the SR. Previous analysis of this issue demonstrated that IP3
R2 is enriched at the nuclear envelop, which is contiguous with the SR/ER network in adult cardiac myocytes 12 37 38
. However, localization to the nuclear envelope should not affect ECC, but instead appears to control nuclear Ca2+
and a local pool of CaMKII 5
. In addition to the nuclear envelope, the IP3
Rs are prominently localized to the SR, in similar regions as the RyR 16
. Indeed, immunohistochemistry of adult myocytes from IP3
R2 DTG overexpressing mice versus tTA controls unequivocally showed IP3
R2 localization within the entire expanse of the SR and around the nucleus.
Another aspect of the controversy surrounding a functional role for IP3
Rs in cardiac myocytes is that generation of IP3
appears to be relatively weak compared with other celltypes 40 41
. For example, cardiomyocytes from mouse or human display only a 1.5- to 2-fold activation of PLC in response to α1-adrenergic receptor stimulation, achieving a level of approximately 30 nM 42 43 44 45
. However, phosphorylation of IP3
R2 by PKA sensitizes the channel and enhances IP3
release at lower IP3
. In addition to this mechanism, Iso stimulation in cultured cardiomyocytes enhances IP3
generation through an ET-1 paracrine/autocrine signaling circuit 38
. β-adrenergic stimulation also enhances SR Ca2+
levels and sensitizes the RyR, together leading to augmented Ca2+
release. In support of this contention, low levels of ET-1, which did not change Ca2+
release or induce arrhythmia, did synergistically increase arrhythmia when forskolin was also used to elevate cAMP. We also believe that physiologic exercise-induced cardiac hypertrophy, which appears to elicit a strong fear response in mice, augmented IP3
release through an associated β-adrenergic co-stimulation effect. These concepts are also consistent with the vast array of neurohumoral mediators that underlie pathologic cardiac hypertrophy and failure, where multiple Gαq-coupled receptor agonists likely synergize with cAMP elevation afforded by β-receptor signaling to induce pathology.
Interestingly, only the high IP3
R2 DTG line showed augmented cardiac hypertrophy with pressure overload stimulation. In contrast, the low overexpressors and the IP3
-sponge mice each failed to show an effect with pressure overload stimulation. The simplest interpretation of these results is that a requirement for endogenous IP3
Rs is easily bypassed with extreme hypertrophic stimulation, such as afforded by pressure overload. Indeed, TRPC channels are also activated in pressure-overloaded hearts where they could easily compensate and provide local Ca2+
entry in the absence of IP3
R signaling to maintain calcineurin activation 46
. In contrast, Iso infusion engages a more restricted set of signaling pathways, such as PKA activation through elevated cAMP. As discussed earlier, β-adrenergic/PKA stimulation uniquely primes IP3
R2 activation and Ca2+
release, possibly explaining why Iso infusion in low expressing DTG mice and in IP3
-sponge mice demonstrated a positive effect versus tTA control mice.
The increase in Ca2+
release mediated by IP3
Rs at the level of the SR could induce the hypertrophic response through a number of different signaling mechanisms. Our working hypothesis is that IP3
release generates a local Ca2+
signaling effect at the level of the T-tubular-SR junctional complex. Indeed, calcineurin is anchored at the Z-lines to calsarcin and α-actinin, in immediate proximity to the SR junctions 47
. Calcineurin was also shown to respond to IP3
release from a perinuclear/nuclear location in neonatal cardiomyocytes to induce hypertrophy 38
. Consistent with these results, we observed prominent NFAT activation in IP3
R2 DTG hearts and that deletion of CnAβ−/−
blocked the ability of Iso to enhance hypertrophy through the IP3
R2 transgene. This IP3
R-dependent release of Ca2+
at the T-tubular-SR junctions could also stimulate RyR Ca2+
leak, providing additional regional Ca2+
elevations to affect calcineurin. Thus, antagonism of the IP3
R may be a clinically relevant target, as it might reduce both arrhythmia and hypertrophic growth propensity.
Novelty and Significance
What Is Known
- IP3R signaling has been implicated in regulating cardiac hypertrophy.
- IP3R mediated calcium release has been implicated in regulating excitation contraction-coupling in the heart.
- IP3R signaling has been implicated in regulating calcineurin-NFAT signaling.
What New Information Does this Article Contribute
- The first description of IP3R2 overexpression in the heart of transgenic mice, which definitively shows that it can regulate cardiac hypertrophy.
- The first description of IP3 inhibition in the mouse heart and the observation that this mediator is required for isoproterenol induced hypertrophy.
- The first proof that calcineurin/NFAT signaling mediate IP3R2 signaling in vivo to control cardiac hypertrophy.
This study was designed to evaluate the importance of IP3R2 calcium release and signaling in mediating the cardiac hypertrophic response in vivo. We examined both pressure overload induced hypertrophy and neurohormonal-regulated hypertrophy in IP3R2 TG mice and mice expressing the IP3-sponge to block this calcium release pathway. Our results definitely show that IP3R2 mediated calcium release in response to neurohormonal stimulation can underlie the cardiac hypertrophic response, in part, by activating calcineurin/NFAT signaling.