The Ca/calmodulin-dependent protein kinase II (CaMKII) is regulated by and involved in control of Ca cycling in the myocardium and has been implicated in transcriptional regulation leading to cardiac hypertrophy. CaMKIIdelta is the predominant cardiac isoform and splice variants lacking (delta-c) or containing (delta-B) a nuclear localization signal are both expressed in cardiomyocytes. The functional significance of the existence of both cytoplasmic (delta-c) and nuclear (delta-B) CaMKII subunits in the heart, and the nature of the Ca signals that activate them, remain unknown. The Ca involved in E-C coupling is released from the sarcopiasmic reticulum (SR), but cardiomyocytes also contain inositol 1,4,5-triphosphate (InsP3) receptor regulated Ca stores. Ligands for receptors that activate phospholipase C (PLC)-mediated InsP3 formation are established activators of hypertrophy, but a signaling role for InsP3 has not been demonstrated. We hypothesize that local Ca release from perinuclear InsP3 sensitive stores activates CaMKII and regulates gene expression and hypertrophy through effects on HDAC/MEF2 or other transcriptional pathways. Studies proposed in Aim #1 examine the effects of deletion of the type 2 and type 3 InsP3 receptor (InsP3Rs) or CaMKIIdelta on the development of physiological and pathological hypertrophy and failure induced by transverse aortic constriction (TAG) or agonist infusion, assessing changes in echocardiographic parameters, left ventricle to body weight ratio, ventricular myocyte size, gene expression and protein phosphorylation.
In Aim #2 we determine whether localized Ca release from InsP3 sensitive Ca stores preferentially activates nuclear CaMKIIdelta while stimuli that lead to global increases in cytosolic Ca via release from the SR activate the cytoplasmic CaMKIIdelta. CaMKII activation in these compartments and the activation of delta-B versus delta-c CaMKII will be examined by cell fractionation and immunoblotting, as well as by development of a FRET based CaMKII activity reporter (CaMKAR).
In Aim #3 we examine a further corollary to this hypothesis i.e, that the nuclear vs. cytosolic localization of CaMKIIdelta targets different substrates. Proposed studies are directed at establishing whether there are distinct roles for nuclear and cytoplasmic CaMKII in activating MEF2/SRF gene expression, HDAC kinase activity, HDAC phosphorylation and cardiomyocyte hypertrophy, versus in phosphorylation of RyR and other SR proteins. This hypothesis is also tested in vivo using mice in which delta-B and delta-c subunits are individually targeted for knockout.
Aim #4 considers an intriguing but unexplored possibility for feedback regulation i.e, in the ability of CaMKII to regulate both an InsP3 kinase and an InsP3 5'phosphatase involved in metabolism of InsP3. The possibility that CaMKII regulates these enzymes in cardiomyocytes will be examined by direct expression and phosphorylation assays, and by measuring InsP3 accumulation. Biochemical assays and a FRET based InsP3 sensor will be utilized to examine changes in InsP3 metabolism.
Aim #4 also uses these methods to compare agonist induced InsP3 formation in atrial versus ventricular cardiomyocytes and alterations that accompany heart failure, and considers the question of whether and how InsP3 levels increase in the nuclear compartment.
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