Calcium-calmodulin dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac electrophysiology, calcium (Ca) balance, contraction, transcription, arrhythmias and progression to heart failure. CaMKII is readily activated at mouths of dyadic cleft Ca channels, but because of its low Ca-calmodulin affinity and presumed immobility it is less clear how CaMKII gets activated near other known, extra-dyad targets. CaMKII is typically considered to be anchored in cardiomyocytes, but while untested, mobility of active CaMKII could provide a mechanism for broader target phosphorylation in cardiomyocytes. We therefore tested CaMKII mobility and how this is affected by kinase activation in adult rabbit cardiomyocytes. We measured translocation of both endogenous and fluorescence-tagged CaMKII using immunocytochemistry, fluorescence recovery after photobleach (FRAP) and photoactivation of fluorescence. In contrast to the prevailing view that CaMKII is anchored near its myocyte targets, we found CaMKII to be highly mobile in resting myocytes, which was slowed by Ca chelation and accelerated by pacing. At low [Ca], CaMKII was concentrated at Z-lines near the dyad but spread throughout the sarcomere upon pacing. Nuclear exchange of CaMKII was also enhanced upon pacing- and heart failure-induced chronic activation. This mobilization of active CaMKII and its intrinsic memory may allow CaMKII to be activated in high [Ca] regions and then move towards more distant myocyte target sites.

Background and aimsFasting insulin (FI), fasting glucose (FG), systolic blood pressure (SBP), high density lipoproteins (HDL), triacylglycerides (TAG), and body mass index (BMI) are well-known risk factors for type 2 diabetes. Reliable estimates of lifestyle intervention effects on these factors allow diabetes risk to be predicted accurately. The present meta-analyses were conducted to quantitatively summarize effects of diet and exercise intervention programs on FI, FG, SBP, HDL, TAG and BMI in adults without diabetes.Materials and methodsMEDLINE and EMBASE were searched to find studies involving diet plus exercise interventions. Studies were required to use adults not diagnosed with type 2 diabetes, involve both dietary and exercise counseling, and include changes in diabetes risk factors as outcome measures. Data from 18, 24, 23, 30, 29 and 29 studies were used for the analyses of FI, FG, SBP, HDL, TAG and BMI, respectively. About 60% of the studies included exclusively overweight or obese adults. Mean age and BMI of participants at baseline were 48 years and 30.1 kg/m2. Heterogeneity of intervention effects was first estimated using random-effect models and explained further with mixed-effects models.ResultsAdults receiving diet and exercise education for approximately one year experienced significant (P <0.001) reductions in FI (-2.56 ± 0.58 mU/L), FG (-0.18 ± 0.04 mmol/L), SBP (-2.77 ± 0.56 mm Hg), TAG (-0.258 ± 0.037 mmol/L) and BMI (-1.61 ± 0.13 kg/m2). These risk factor changes were related to a mean calorie intake reduction of 273 kcal/d, a mean total fat intake reduction of 6.3%, and 40 minutes of moderate intensity aerobic exercise four times a week. Lifestyle intervention did not have an impact on HDL. More than 99% of total variability in the intervention effects was due to heterogeneity. Variability in calorie and fat intake restrictions, exercise type and duration, length of the intervention period, and the presence or absence of glucose, insulin, or lipid abnormalities explained 23-63% of the heterogeneity.ConclusionsCalorie and total fat intake restrictions coupled with moderate intensity aerobic exercises significantly improved diabetes risk factors in healthy normoglycemic adults although normoglycemic adults with glucose, insulin, and lipid abnormalities appear to benefit more.

Persistent over-activation of CaMKII (Calcium/Calmodulin-dependent protein Kinase II) in the heart is implicated in arrhythmias, heart failure, pathological remodeling, and other cardiovascular diseases. Several posttranslational modifications (PTMs)-including autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation-have been shown to trap CaMKII in an autonomously active state. The molecular mechanisms by which these PTMs regulate calmodulin (CaM) binding to CaMKIIδ-the primary cardiac isoform-has not been well-studied particularly in its native myocyte environment.Typically, CaMKII activates upon Ca-CaM binding during locally elevated [Ca] free and deactivates upon Ca-CaM dissociation when [Ca] free returns to basal levels. To assess the effects of CaMKIIδ PTMs on CaM binding, we developed a novel FRET (Förster resonance energy transfer) approach to directly measure CaM binding to and dissociation from CaMKIIδ in live cardiac myocytes. We demonstrate that autophosphorylation of CaMKIIδ increases affinity for CaM in its native environment and that this increase is dependent on [Ca] free . This leads to a 3-fold slowing of CaM dissociation from CaMKIIδ (time constant slows from ~0.5 to 1.5 s) when [Ca] free is reduced with physiological kinetics. Moreover, oxidation further slows CaM dissociation from CaMKIIδ T287D (phosphomimetic) upon rapid [Ca] free chelation and increases FRET between CaM and CaMKIIδ T287A (phosphoresistant).The CaM dissociation kinetics-measured here in myocytes-are similar to the interval between heartbeats, and integrative memory would be expected as a function of heart rate. Furthermore, the PTM-induced slowing of dissociation between beats would greatly promote persistent CaMKIIδ activity in the heart. Together, these findings suggest a significant role of PTM-induced changes in CaMKIIδ affinity for CaM and memory under physiological and pathophysiological processes in the heart.

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CaMKII is a central regulator of cardiac Ca handling, electrophysiology and transcription. Its persistent over-activation has been implicated in arrhythmias, heart failure, and other diseases. Typically, CaMKII activates upon Ca-CaM binding during elevated [Ca] free and inactivates upon Ca-CaM dissociation when [Ca] free drops. Several post-translational-modifications (PTMs) also trap CaMKII in an autonomously activated state even after [Ca] i declines. The PTMs include autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation. Exactly how these PTMs regulate CaM binding to CaM-KIId-the predominant cardiac isoform-is unknown. Schulman's group showed that autophosphorylation of CaMKIIa (neuronal isoform) increases its CaM affinity by 1,000-fold in lysates. CaMKII activity that persists during low [Ca] free creates a form of molecular memory. Here we perform novel tests of changes in CaM affinity (or trapping) for CaMKIId by known PTMs. Importantly, these are measured in adult rabbit ventricular myocytes, CaMKIId's native environment. Briefly, we express CaMKIId-GFP in isolated ventricular myocytes and load fluorescent-CaM after saponin-permeabilization of the myocytes. We use FRET as a readout for CaM dissociation upon the addition of unlabeled CaM or upon rapidly dropping [Ca] free with BAPTA. In myocytes, expressing the phosphomimetic CaMKIId (T287D) caused a dramatic slowing of CaM dissociation (>5 fold) upon abrupt reduction of [Ca] i (<50 nM) when compared to the non-phosphorylatable mutant (T287A). These differences provide a quantitative, mechanistic basis for PTM-dependent regulation of CaM-KIId in cardiac myocytes and its potential role in disease. We extended this analysis with CaMKIId-GFP mutants and corresponding agonists for other PTMs: e.g. 100uM H 2 O 2 -WT vs. M281/282V and analogous for the S-nitrosylation sites (C273S, C290A) and the O-GlcNAcylation site (S280A). This allows us to test which of these PTMs creates the longest CaM-binding state which will influence its relative impact on cardiac CaMKII memory and contributions to disease.