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.

Objective: To determine the underlying pathways that promote cardiotoxicity following anti-ERBB2 cancer therapeutics.Background: Epidermal growth factor receptor 2 (EGFR2/ERBB2) is a member of EGFR (ERBB) family represented by ERBB 1 through 4 that either homo- or hetero-dimerize to signal. ERBB2 has constitutively active confirmation but explicitly needs to hetero-dimerize with either ERBB1 or ERBB4 to mediate signaling and cardiac hypertrophy. Less is known about the mechanisms through which ERBB2 mediates the adaptive cardiac hypertrophy given that anti-ERBB2 therapeutics results in cardiac dilation of the otherwise healthy heart.Methods: We generated transgenic (Tg) mice with cardiomyocyte-specific overexpression of microRNA-7 (miRNA-7) that targets ERBB2. Echocardiography, immunohistochemistry and β-adrenergic receptor (βAR) density were assessed upon aging and transverse aortic constriction (TAC). To determine whether inhibition of ERBB2 has similar outcomes, C57Bl/6 mice were administered ERBB2-specific inhibitor AG825. Also, βAR density was assessed in end-stage human heart failure patient samples with Adriamycin cardiotoxicity.Results: Loss in ERBB2 expression due to miRNA-7 targeting or ERRB2 inhibition by AG 825 results in cardiac dilation. Loss of ERBB2 in miRNA-7 Tg mice leads to increased fibrosis post-TAC. Selective significant increase in β1AR density is observed in miRNA-7 Tg mice or in mice following ERBB2 inhibition by AG825 and in the human heart failure patient samples due to Adriamycin cardiotoxicity.Conclusions: Cardiomyocyte ERBB2 expression and function is required for maintaining the normal cardiac function. Targeting ERBB2 leads to selective and significant increase in β1ARs that may promote cardiac dilation and heart failure.

Objective To understand the underlying pathways that promote cardiotoxicity following chemotherapy. Background Anthracyclines are associated with cardiotoxicity which could be potentiated with use of complementary agents (like anti-ERBB2 inhibitors) which together afford robust anti-neoplastic effects. Anthracyclines lead to oxidative stress and thought to induce cardiotoxicity. However, interventions reducing oxidative stress in patients have been unsuccessful suggesting mechanisms beyond oxidative stress. Despite β-adrenergic receptors (βARs) being key regulators of cardiac function, nothing is known about their role in chemotherapy-mediated cardiotoxicity. Methods β1 and/or β2-AR density was assessed in end-stage human heart failure patient samples either due to anthracycline cardiotoxicity or non-anthracycline dilated cardiomyopathy (DCM). Since ERBB2 inhibition is integral to overall chemotherapeutic arsenal, we assessed β1- and/or β2-AR density, cardiac function by echocardiography and immunohistochemistry in mice following ERBB2-specific inhibitor AG825. Results Selective increase in cardiac β1AR density is observed in end-stage human heart failure patient samples due to anthracycline cardiotoxicity as well as in ERBB2 inhibitor-treated mice. Conclusions Elevated β1AR density may be the key common underlying mechanism which is altered in response to chemotherapy promoting cardiac dilation of otherwise healthy hearts. Highlights In contrast to downregulation of β1-adrenergic receptors (β1AR) in end-stage human heart failure, anthracycline cardiotoxicity-mediated failure is associated with selective increase in β1AR density. ERBB2 inhibitor (AG825) treatment in mice results in cardiac dilation and selective rise in β1AR density showing that increased β1AR density in the heart could be a common mechanism underlying cardiotoxicity. ### Competing Interest Statement The authors have declared no competing interest. * EGFR : Epidermal growth factor receptor ERBB2 : Erythroblastic oncogene B DCM : Dilated Cardiomyopathy βAR : beta-adrenergic receptor

Epithelial ovarian carcinoma (EOC) is the most prevalent form of ovarian cancer in the United States, representing approximately 85% of all cases and causing more deaths than any other gynecologic malignancy. We propose that optimized control of EOC requires the incorporation of a vaccine capable of inducing safe and effective preemptive immunity in cancer-free women. In addition, we hypothesize that ovarian-specific self-proteins that are “retired” from autoimmune-inducing expression levels as ovaries age but are expressed at high levels in emerging EOC may serve as vaccine targets for mediating safe and effective primary immunoprevention. Here, we show that expression of the extracellular domain of anti-Müllerian hormone receptor II (AMHR2-ED) in normal tissues is confined exclusively to the human ovary, drops to nonautoimmune inducing levels in postmenopausal ovaries, and is at high levels in approximately 90% of human EOC. We found that AMHR2-ED vaccination significantly inhibits growth of murine EOC and enhances overall survival without inducing oophoritis in aged female mice. The observed inhibition of EOC growth was mediated substantially by induction of AMHR2-ED–specific IgG antibodies that agonize receptor signaling of a Bax/caspase-3–dependent proapoptotic cascade. Our results indicate that AMHR2-ED vaccination may be particularly useful in providing safe and effective preemptive immunity against EOC in women at high genetic or familial risk who have the greatest need for a preventive vaccine and ultimately in cancer-free postmenopausal women who account for 75% of all EOC cases. Cancer Prev Res; 10(11); 612–24. ©2017 AACR. See related editorial by Shoemaker et al., p. 607

Phosphoinositide 3 Kinase γ (PI3Kγ) is a lipid kinase that regulates downstream anti-apoptotic Akt signaling. Thus, pressure overload in PI3Kγ null (PI3Kγ -/- ) mice leads to significant cardiac fibrosis, a key underlying cause of fatal heart failure. Classical hallmark of tissue fibrosis is differentiation of fibroblasts to myofibroblasts characterized by smooth muscle α-actin (αSMA) overexpression. However, less is known about the role of PI3Kγ in cardiac myofibroblast differentiation. Assessment of αSMA expression in cardiac lysates from WT and PI3Kγ -/- showed significant baseline upregulation in PI3Kγ -/- showing that loss of PI3Kγ predisposes the hearts towards fibrosis. To directly confirm that PI3Kγ -/- cardiac fibroblasts (CF) exhibit a myofibroblast phenotype, CF were isolated from hearts of WT and PI3Kγ -/- and assessed by immunostaining for αSMA in stress fibers. Greater number of CF from PI3Kγ -/- exhibited αSMA in stress fibers than CF from WT. Correspondingly, immunoblotting showed significantly higher expression of αSMA in PI3Kγ -/- CF compared to WT showing enhanced myofibroblast differentiation by PI3Kγ -/- fibroblasts. Surprisingly, abundance of αSMA protein is significantly reduced in the hearts of mice with cardiomyocyte-specific expression of kinase-dead PI3Kγ (PI3Kγ inact ) in the PI3Kγ -/- (PI3Kγ inact /PI3Kγ -/- ) suggesting that myocytes derived factors responsible for myofibroblast differentiation are regulated by kinase-independent function of PI3Kγ. To directly evaluate the PI3Kγ-dependent cardiomyocyte derived factors responsible for myofibroblast differentiation; fibroblasts were treated with conditioned media derived from primary adult cardiomyocytes from WT, PI3Kγ -/- and PI3Kγ inact /PI3Kγ -/- mice. Conditioned media derived from PI3Kγ -/- showed pro-fibrotic effects, while that from PI3Kγ inact /PI3Kγ -/- showed fibrosis protective biological activity compared to WT. These findings reveal that kinase-independent function of PI3Kγ is a key regulator of the myocyte-initiated pathway that ultimately drives myofibroblast conversion. Proteomic analysis of conditioned media identified several pro-fibrotic factors that are regulated by PI3Kγ, the results of which will be discussed.

Studies in cancer have established clearly the role of epidermal growth factors receptors as targets of microRNA-7 (miR-7) but there are no studies so far to elucidate the role and function of miR-7 in human heart. Our studies show that ERBB2 (a member of epidermal growth factors receptor family) is targeted by miR-7 in human heart failure as well as in mice hearts undergoing stress. In order to understand the role of miR-7 in the heart, we generated miR-7 transgenic (Tg) mice with cardiac myocyte-specific overexpression of miR-7. miR-7 Tg mice have age dependent deterioration in cardiac dysfunction associated with cardiac dilation as measured by echocardiography (3 months - 60% FS, 6 month -52% FS and 12 months - 24%FS) and yet, they survive well for more than a year. To investigate whether pathological stress would accelerate the deterioration in cardiac function, miR-7 Tg mice were subjected to transverse aortic constriction (TAC) for two weeks. In contrast to the wild type littermates which undergo hypertrophic response following TAC, miR-7 Tg mice have accelerated cardiac dysfunction and dilation within two weeks. Histological analysis shows increased fibrosis in miR-7 Tg mice hearts as shown by Picro Sirius red and Meson Trichome staining which is further accelerated after TAC stress in hearts of the miR-7 Tg mice as compared to their sham controls. We also find that there is a difference in the mitochondrial morphology and structure in miR-7 Tg mice as compared to Wildtype controls as seen by transmission Electron Microscopy (TEM) explaining the reason for deteriorated cardiac function in terms of energy generation. Our study will discuss the mechanism underlying the deteriorated cardiac function in the miR-7 Tg mice after pathological stress as compared to wildtype littermates.

We are interested in understanding mechanisms that govern the protective role of exercise against lipid-induced insulin resistance, a key driver of type 2 diabetes. In this context, cell culture models provide a level of abstraction that aid in our understanding of cellular physiology. Here we describe the development of an in vitro myotube contraction system that provides this protective effect, and which we have harnessed to investigate lipid-induced insulin resistance. C2C12 myocytes were differentiated into contractile myotubes. A custom manufactured platinum electrode system and pulse stimulator, with polarity switching, provided an electrical pulse stimulus (EPS) (1 Hz, 6-ms pulse width, 1.5 V/mm, 16 h). Contractility was assessed by optical flow flied spot noise mapping and inhibited by application of ammonium acetate. Following EPS, myotubes were challenged with 0.5 mM palmitate for 4 h. Cells were then treated with or without insulin for glucose uptake (30 min), secondary insulin signaling activation (10 min), and phosphoinositide 3-kinase-α (PI3Kα) activity (5 min). Prolonged EPS increased non-insulin-stimulated glucose uptake (83%, P = 0.002), Akt (Thr308) phosphorylation (P = 0.005), and insulin receptor substrate-1 (IRS-1)-associated PI3Kα activity (P = 0.048). Palmitate reduced insulin-specific action on glucose uptake (-49%, P < 0.001) and inhibited insulin-stimulated Akt phosphorylation (P = 0.049) and whole cell PI3Kα activity (P = 0.009). The inhibitory effects of palmitate were completely absent with EPS pretreatment at the levels of glucose uptake, insulin responsiveness, Akt phosphorylation, and whole cell PI3Kα activity. This model suggests that muscle contraction alone is a sufficient stimulus to protect against lipid-induced insulin resistance as evidenced by changes in the proximal canonical insulin-signaling pathway.

Beta adrenergic receptors (βARs) are one of the most powerful regulators of cardiac function, and βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization by G-protein coupled receptor kinase phosphorylation leading to βAR internalization. Desensitized βARs are resensitized by dephosphorylation in the endosomes by PP2A; however, less is known about role of resensitization in heart failure. Although we have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on in silico predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also tested whether resensitization as a mechanism is altered in endstage human heart failure samples. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of βAR function. Significant PI3Kγ activity was observed to increase in heart failure samples while AC activity was reduced in plasma membranes. Consistent with the reduced recovery in βAR function in heart failure samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of human heart failure samples compared to non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in heart failure samples versus controls suggesting inhibited resensitization occurs in human heart failure. Together our studies suggest that resensitization is inhibited in human heart failure and targeting I2PP2A may provide preservation of receptor function and beneficial cardiac remodeling.

Catestatin (CST), an endogenous antihypertensive/antiadrenergic peptide, is a novel regulator of cardiovascular physiology. Here, we report case-control studies in 2 geographically/ethnically distinct Indian populations (n approximate to 4000) that showed association of the naturally-occurring human CST-Gly364Ser variant with increased risk for hypertension (age-adjusted odds ratios: 1.483; P=0.009 and 2.951; P=0.005). Consistently, 364Ser allele carriers displayed elevated systolic (up to approximate to 8 mm Hg; P=0.004) and diastolic (up to approximate to 6 mm Hg; P=0.001) blood pressure. The variant allele was also found to be in linkage disequilibrium with other functional single-nucleotide polymorphisms in the CHGA promoter and nearby coding region. Functional characterization of the Gly364Ser variant was performed using cellular/molecular biological experiments (viz peptide-receptor binding assays, nitric oxide [NO], phosphorylated extracellular regulated kinase, and phosphorylated endothelial NO synthase estimations) and computational approaches (molecular dynamics simulations for structural analysis of wild-type [CST-WT] and variant [CST-364Ser] peptides and docking of peptide/ligand with beta-adrenergic receptors [ADRB1/2]). CST-WT and CST-364Ser peptides differed profoundly in their secondary structures and showed differential interactions with ADRB2; although CST-WT displaced the ligand bound to ADRB2, CST-364Ser failed to do the same. Furthermore, CST-WT significantly inhibited ADRB2-stimulated extracellular regulated kinase activation, suggesting an antagonistic role towards ADRB2 unlike CST-364Ser. Consequently, CST-WT was more potent in NO production in human umbilical vein endothelial cells as compared with CST-364Ser. This NO-producing ability of CST-WT was abrogated by ADRB2 antagonist ICI 118551. In conclusion, CST-364Ser allele enhanced the risk for hypertension in human populations, possibly via diminished endothelial NO production because of altered interactions of CST-364Ser peptide with ADRB2 as compared with CST-WT.

MicroRNA-7 (miR-7) is known to target epidermal growth factor receptors (EGFRs) in oncogenic cells however, less is known about its role and function in the heart. Our studies have identified reciprocal expression pattern for ERBB2 (a member of EGFR family) in the context of miR-7 expression in human heart failure and in mice post-TAC (Transverse Aortic Constriction). To directly determine the role of miR-7 in the heart, we generated transgenic (Tg) mice with cardiomyocyte-specific overexpression of miR-7 (miR-7 Tg). miR-7 Tg mice are characterized by marked loss in ERBB2 expression compared to their wildtype littermate controls. Also, miR-7 Tg mice have age-dependent deterioration in cardiac dysfunction and is associated with dilation as measured by echocardiography (3 months - 60% FS, 6 month -52% FS and 12 months - 24%FS) and yet, they survive over 18 months as assessed by Kaplan-Meier curves. To investigate whether pathological stress would accelerate the deterioration in cardiac function, miR-7 Tg mice were subjected to TAC for two weeks. In contrast to the wild type littermates that showed significant hypertrophic response, miR-7 Tg mice have accelerated cardiac dysfunction and dilation following two weeks of TAC. Histological analysis shows increased collagen deposition in miR-7 Tg mice which is further accelerated following after TAC compared to their sham controls. These observations suggest that ERBB2 expression in the cardiomyocytes may play a critical role in delaying the pro-fibrotic response post-TAC by supporting an adaptive hypertrophic response in response to stress. Our study will discuss the mechanisms of increased fibrosis observed in miR-7 Tg mice subjected to TAC.

Hypertension is the most common cardiovascular disease, afflicting >30% of adults. The cause of hypertension in most individuals remains unknown, suggesting that additional contributing factors have yet to be discovered. Corin is a serine protease that activates the natriuretic peptides, thereby regulating blood pressure. It is synthesized as a zymogen that is activated by proteolytic cleavage. CORIN variants and mutations impairing corin activation have been identified in people with hypertension and pre-eclampsia. To date, however, the identity of the protease that activates corin remains elusive. Here we show that proprotein convertase subtilisin/kexin-6 (PCSK6, also named PACE4; ref. 10) cleaves and activates corin. In cultured cells, we found that corin activation was inhibited by inhibitors of PCSK family proteases and by small interfering RNAs blocking PCSK6 expression. Conversely, PCSK6 overexpression enhanced corin activation. In addition, purified PCSK6 cleaved wild-type corin but not the R801A variant that lacks the conserved activation site. Pcsk6-knockout mice developed salt-sensitive hypertension, and corin activation and pro-atrial natriuretic peptide processing activity were undetectable in these mice. Moreover, we found that CORIN variants in individuals with hypertension and pre-eclampsia were defective in PCSK6-mediated activation. We also identified a PCSK6 mutation that impaired corin activation activity in a hypertensive patient. Our results indicate that PCSK6 is the long-sought corin activator and is important for sodium homeostasis and normal blood pressure.

Beta-adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Agonist occupied βAR undergo desensitization through phosphorylation by G-protein coupled receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by protein phosphatase 2A (PP2A) in the endosomes and we have shown previously shown that PP2A activity is regulated by phosphoinositide3-kinaseγ (PI3Kγ). Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and an integral contributor to heart failure. To test whether resensitization mechanisms are altered in heart failure and could play a role in recovery of βAR function post-LVAD, we used paired pre- and post-LVAD human heart samples along with non-failing hearts. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from these human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of recovery in βAR function by G-protein coupling. Significant PI3Kγ activity was observed in the endosomes of pre-LVAD compared post-LVAD and non-failing human heart samples while plasma membrane PI3K activity remained similar across all samples. Similarly, AC activity was markedly reduced in plasma membranes and endosomes of pre-LVAD samples compared to post-LVAD and non-failing showing reduced ability of receptors to couple to G-proteins indicating reduced recovery of receptor function in post-LVAD heart samples. Consistent with the reduced recovery in βAR function in pre-LVAD samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of pre-LVAD compared to post-LVAD and non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in the pre-LVAD samples which was remarkably reversed in post-LVAD samples similar to the activity in non-failing. These studies suggest that resensitization is inhibited in end-stage human heart failure and may critically contribute to cardiac remodeling and hypertrophic response upon cardiac stress.

Beta-Adrenergic receptors (bARs) play a key role in regulating cardiac function. Loss of surface receptors and desensitization (impaired G-protein coupling) of bARs are hallmarks of a failing heart. Desensitization occurs by phosphorylation of bARs. The bARs are resensitized by protein phosphatase 2A (PP2A) mediated dephosphorylation in the endosomes before recycling to the plasma membrane. While mechanisms of desensitization are well understood, little is known about mechanisms regulating resensitization. Our previous work has shown that PI3Kg phosphorylates an endogenous inhibitor of PP2A (I2PP2A) on serine 9 & 93, which then robustly binds to PP2A inhibiting bAR resensitization. Since it is not known whether resensitization is altered in response to cardiac stress or whether altered bAR resensitization contributes to cardiac hypertrophy and failure, we generated transgenic mice with cardiomyocyte specific overexpression of wild type I2PP2A (WT I2PP2A Tg), I2PP2A phospho-mimetic mutants S9, 93D and mutants with constitutively dephosphorylated S9, 93A state. To test whether resensitization is critical in the development of bAR dysfunction during cardiac hypertrophy, WT I2PP2A Tg mice were subjected to transverse aortic constriction (TAC) for 8 weeks. Echocardiographic analysis post-TAC showed that WT I2PP2A Tg mice had accelerated cardiac dysfunction compared to their littermate controls [HW (mg)/BW(g): Sham: WT - 4.83, WT I2PP2A Tg - 4.82, TAC: WT- 6.47, WT I2PP2A Tg - 7.61; %EF: Sham: WT - 83.53, WT I2PP2A Tg - 74.72, TAC: WT - 70.47, WT I2PP2A Tg - 49.62]. To directly test whether resensitization mechanisms are altered, plasma membranes and endosomes were isolated and in vitro Adenylyl Cyclase activity assessed. Our studies show that compared to littermate controls, WT I2PP2A Tg had altered in vitro adenylyl cyclase activity showing that resensitization mechanisms in the endosomes may in part, contribute to cardiac dysfunction. Mechanistic underpinnings of the resensitization pathways using the I2PP2A S9, 93A and S9, 93D will be presented showing that bAR resensitization a process considered passive is altered in conditions of cardiac stress that in part may contribute to bAR dysfunction leading to cardiac hypertrophy and heart failure.

miRNA-7 is known to target epidermal growth factors receptor 1(EGFR1) in cancer cells. EGFR family members (EGFR 1, 2, 3 and 4) are known to form homo- and/or hetero-dimers to mediate downstream signals . Our previous study in human heart failure (Naga Prasad etal., JBC, 2009) showed that EGFR2 (ERBB2) was targeted by miRNA-7. Based on this study, we developed transgenic (Tg) mice with cardiomyocyte-specific overexpression of miRNA-7. miRNA-7 Tg mice have age dependent deterioration in cardiac dysfunction and is associated with cardiac dilation as measured by echocardiography (3 months - 60% FS, 6 month -52% FS &12 months - 24%FS) and yet, they survive well for more than a year. To investigate whether pathological stress would accelerate the deterioration in cardiac function, miRNA-7 Tg mice were subjected to transverse aortic constriction (TAC) for two weeks. In contrast to the wild type littermates which undergo hypertrophic response following TAC, miRNA-7 Tg mice have accelerated cardiac dysfunction and dilation within two weeks. Biochemical analysis interestingly showed differential switching of dimeric EGFR partners in hearts of the miRNA-7 Tg mice compared to their sham controls. Our presentation will discuss the differential downstream signaling induced by changing of EGFR dimeric partners induced by pathological stress like TAC in the wildtype and miRNA-7 Tg mice.

Glycogen synthase kinase-3 (GSK-3) is a known negative regulator of cardiac hypertrophic response and much remains unknown about its dephosphorylation process that activates GSK-3. Since GSK-3 function is regulated by PI3K, age based assessment of cardiac hypertrophic response was measured in PI3Kγ knockout (PI3Kγ KO) mice. Interestingly, we observed marked reduction in heart size in PI3Kγ KO mice compared to littermate controls. Consistent with the reduced size, we observed elevated PP2A activity and dephosphorylation mediated activation of GSK-3 in PI3Kγ-KO hearts. Mechanistically, we found that higher PP2A activity in PI3Kγ-KO was due to PP2A methylation mediated by elevated PP2A methyl transferase (PPMT-1) activity. To test in vivo whether PI3K activity regulates GSK-3 dephosphorylation mediated cardiac growth, we generated mice with cardiac specific overexpression of inactive PI3Kγ (PI3Kγinact) and constitutively active PI3Kγ (myrPI3Kγ) in the PI3Kγ-KO background. Age based hypertrophic response and cardiac function was assessed by heart weight/body weight ratios and echocardiography. The heart size was significantly increased in PI3Kγinact (LVEDD- 3.41+/- 0.12) and myrPI3Kγ (LVEDD- 3.73 +/- 0.14) overexpressing mice, when compared to WT (LVEDD- 3.00 +/- 0.15) mice at 18 months age, indicating that kinase activity of PI3Kγ is inconsequential for age-dependent regulation of cardiac growth. Similar cardiac growth due to overexpression of active and inactive form of PI3Kγ suggests that the kinase independent function potentially overrides the kinase activity of PI3Kγ. Correspondingly, PP2A activity was normalized and was associated with decreased GSK-3 dephosphorylation with overexpression of PI3Kγinact. This led to normalization of heart size in these mice compared to PI3Kγ-KO littermates correlating with the decreased GSK-3 dephosphorylation and consequent inhibition of GSK-3 activity. Regulation of PP2A mediated dephosphorylation of GSK-3 by PI3Kγ combined with similar level of increased cardiac growth in PI3Kγ-KO mice with cardiac overexpression of PI3Kγinact or myrPI3Kγ, suggests a non-canonical role of PI3Kγ in hypertrophy of the heart due to age related increase in mechanical demand.

βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization through phosphorylation by G-protein coupled receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by PP2A in the endosomes. In contrast to this paradigm our recent studies have shown that resensitization of βARs can occur at the plasma membrane through the inhibition of PP2A activity by I2PP2A. We generated a stable HEK cell line expressing short hairpin RNA targeting I2PP2A (shRNA-I2PP2A). Confocal microscopy studies show cells expressing shRNA-I2PP2A resulted in significant loss of receptor phosphorylation despite the presence of the agonist. Radioligand binding and confocal imaging also showed marked inhibition of receptor internalization upon depletion of I2PP2A with significant PP2A activation. We also observed preservation of βAR function despite the presence of agonist measured by cAMP generation and adenylyl cyclase activity. To further dissect the interaction of I2PP2A-PP2A we generated mutants of PP2A from amino acids 263-309. Over expression of these PP2A mutant peptides significantly reversed receptor phosphorylation upon isoproterenol (ISO) stimulation compared to full length PP2A. Also, expression of PP2A mutant showed marked increase in adenylyl cyclase activity in response ISO stimulation suggesting that this mutant I2PP2A competes out inhibitory I2PP2A interaction with PP2A. To test whether alteration in βAR resensitization contributes to cardiac dysfunction with stress we generated transgenic (Tg) mice with cardiac specific over expression of wt I2PP2A and mutant I2PP2A (phopspho- and dephospho-). ISO treatment of wt I2PP2A Tg and littermate controls showed that wt I2PP2A Tg mice had significant βAR dysfunction and cardiac hypertrophy. Assessment of age dependent cardiac function of these mice showed that wt I2PP2A Tg and phospho-I2PP2A Tg mice have cardiac hypertrophic response followed by dilated cardiomyopathy; expression of dephospho-I2PP2A mutant reversed this phenotype. These studies suggest targeting I2PP2A alters receptor function and may have implications in cardiac remodeling and hypertrophy with cardiac stress.

βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization through phosphorylation by G-protein coupled receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by PP2A in the endosome, a paradigm in receptor resensitization. Our recent studies have suggested that resensitization of βARs can occur on the plasma membrane through the inhibition of PP2A activity by I2PP2A. To test whether I2PP2A regulates βAR and cardiac function we generated transgenic mice with cardiac specific over expression of I2PP2A. Assessment of age dependent cardiac function of these mice showed that transgenic mice have cardiac hypertrophic response followed by dilated cardiomyopathy. These observations are further supported by immunohistochemistry. Since we have observed cardiac dysfunction and βAR desensitization in transgenic mice, we tested whether targeting I2PP2A in cells would allow for plasma membrane resensitization. We generated a stable HEK (Human Embryonic Kidney) cell line expressing short hairpin RNA targeting I2PP2A (shRNA-I2PP2A). Confocal microscopy studies show that cells expressing shRNA-I2PP2A resulted in significant loss of receptor phosphorylation despite the presence of the agonist. Furthermore, radioligand binding and confocal imaging showed marked inhibition of receptor internalization upon depletion of I2PP2A with significant PP2A activation. Consistently, we also observed preservation of βAR function despite the presence of agonist as measured by cAMP generation and adenylyl cyclase activity. Together these studies suggest that targeting I2PP2A may lead to plasma membrane receptor resensitization demonstrating that modulating PP2A function can potentially change desensitization and resensitization dynamics of the receptors on the plasma membrane.

mouse models. In vitro studies in β 2 AR-overexpressing human embryonic kidney 293 cells showed significant β AR desensitization, GRK2 upregulation, and recruitment to the β AR complex following TNF α . Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated β AR phosphorylation and GRK2 recruitment on TNF α . Furthermore, TNF α -mediated β AR phosphorylation was not blocked with β AR antagonist propranolol. Additionally, TNF α administration in transgenic mice with cardiac overexpression of G βγ -sequestering peptide β ARK-ct could not prevent β AR desensitization or cardiac dysfunction showing that GRK2 recruitment to the β AR is G βγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNF α -mediated β AR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNF α -mediated loss in contractility, showing that TNF α -induced β AR desensitization is GRK2 dependent. Conclusions — TNF α -induced β AR desensitization is mediated by GRK2 and is independent of G βγ , uncovering a hitherto unknown cross-talk between TNF α and β AR function, providing the underpinnings of inflammation-mediated cardiac dysfunction. ( Circulation . 2013;128:377-387.)