Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular rate/contractility. We investigated whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Caffeine treatment of adult cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ -/- cardiomyocytes showed significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Abrogation of PLN phosphorylation in the PI3Kγ -/- cardiomyocytes shows that the loss in the steady-state phosphorylation of PLN leads to increased inhibition of SERCA. This inhibition is reflected by the slow reuptake of calcium by the SR in the PI3Kγ -/- cardiomyocytes. Concomitantly, significant interaction was observed between SERCA and PLN in the PI3Kγ -/- hearts compared to the controls. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ -/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in the global PI3Kγ -/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. These studies bring-to-fore an unrecognized kinase-independent regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.

Insulin impairs β2-adrenergic receptor (β2AR) function via trans-phosphorylation through G protein-coupled receptor kinase 2 (GRK2). However, less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A) during this insulin-β2AR cross-talk. Pharmacologic or genetic inhibition of phosphoinositide 3-kinase γ (PI3Kγ) unexpectedly resulted in significant reduction of insulin-mediated β2AR phosphorylation. Interestingly, β2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3Kγ showing negative regulation of PP2A by PI3Kγ. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3Kγ form a complex and could be recruited to β2ARs as GRK2 interacts with insulin receptor substrate (IRS) following insulin treatment. Further, co-immunoprecipitation studies showed that PI3Kγ directly interacted with both IRS-1 and IRS-2 but only IRS-2 interaction with PI3Kγ significantly increased following insulin stimulation. These results indicated that PI3Kγ could also be directly recruited to the receptor complex by IRS-2. Consistently, β-blocker pretreatment did not reduce insulin-mediated β2AR phosphorylation indicating agonist- and Gβγ-independent non-canonical regulation of receptor function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating β2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-β2AR cross-talk.

Introduction: Circulating blood troponin complexes and free fractions remain poorly characterised in different conditions where troponin is detectable in blood Hypothesis: The aim of the study was to compare the differences in troponin-I(TnI) complexes/free-forms in end stage renal disease (ESRD) compared to acute myocardial infarction(AMI) Methods: Blood was collected from patients with AMI(n=7) or ESRD(n=4) at two time points (a)As early as possible after AMI or at initial contact with ESRD patients and (b)24-48 hours later. Western blotting was carried out with HyTest cTnI-560cc antibody on plasma extracted from whole blood. Densitometry analysis was performed and evaluated using the independent samples T-test and paired T-test as appropriate Results: Prominent bands were noted at ~45,~37 and ~25 kDa respectively representing low molecular weight(LMW) TnI-TnT-TnC complex, binary TnI-TnC complex and free-TnI. At time-point (a), there was no difference in these bands between STEMI and CKD patients. Interestingly, at time-point (b), AMI patients had significantly lower intensity of the 45kDa and 37kDa bands compared to CKD patients(for 45 kDa band mean difference was 54.3±19.4 AU, p=0.02; for 37 kDa band mean difference was 27.7±10.5 AU, p=0.03) as well as compared to the initial STEMI samples taken at time-point (a)(for 45 kDa band mean difference was 41.4±8.1 AU, p=0.002; for 37 kDa band mean difference was 16.7±6.3 AU, p=0.002) ,however there was no difference in the 25kDa band Conclusions: AMI patients had progressively lesser quantities of circulating LMW-ITC and binary IC complexes following AMI compared to ESRD patients, but similar quantities of circulating free TnI. This indicates a constant release of LMW-ITC and binary-IC complexes from the myocardium or reduced glomerular filtration of these complexes in ESRD while in the AMI patients, the LMW-ITC and binary I-C complexes appear to be progressively eliminated from plasma after the initial release