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.

BACKGROUND: Impaired beta-adrenergic receptor (β1 and β2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3Kγ feedback mechanism in regulating hypoxia-mediated β1 and β2AR dysfunction and cardiac remodeling. METHODS: Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for β1 and β2AR density and function, PI3Kγ activity and β1 and β2AR-associated PP2A activity. Metabolic labeling was performed to assess β1 and β2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. RESULTS: Hypoxia increased β1 and β2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated β2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased β1 and β2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal β1- and β2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased β1 and β2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with β1AR-selective agonist dobutamine post-hypoxia. CONCLUSIONS: Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in β1- and β2AR phosphorylation and amelioration of cardiac dysfunction.

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Hypoxia to heart or brain is a primary cause of heart failure or stroke. Studies have shown hypoxia increases the beta-adrenergic receptors (βARs) phosphorylation and dysfunction (Cheong et. al., 2016). These observations provide evidence that βARs can directly be regulated by hypoxia but less is known about the underlying mechanisms. We postulated that hypoxia shifts the homeostasis between kinase and phosphatase driven mechanisms may underlie βAR dysfunction. β2AR HEK 293 cells were exposed to hypoxia (2% O 2 ) and assessed the mechanisms underlying desensitization (G-protein coupled receptor kinases, GRKs) and resensitization (Protein phosphatase 2A, PP2A). Six hours of hypoxia treatment resulted in increase of βAR phosphorylation and GRK2 expression. Assessment of βAR phosphorylation in the plasma membrane and endosomal fractions surprisingly, showed marked increase in β2AR phosphorylation in the endosomal fraction. Furthermore, we also observed that receptor associated PP2A activity was inhibited in the endosomes following hypoxia with minimal changes of activity at the plasma membranes. Similarly subjecting normal mice to 20 hours of hypoxia resulted in significant cardiac dysfunction (% FS: Normoxia 38.83% vs. Hypoxia 32.38%, P=0.0055; % EF: Normoxia 69.71% vs. Hypoxia 60.76%,P=0.0105) and was associated with significant increase in β2AR phosphorylation associated with significant loss in βAR function as measured by G-protein coupling adenylyl cyclase activity. Given that β-blockers confer beneficial effects, we tested whether β-blocker (propranolol) would prevent βARs phosphorylation under hypoxia or normoxia. Consistently, β-blocker treatment in normoxia results in increased β2AR phosphorylation however, remarkably β-blocker treatment in hypoxia results in loss of β2AR phosphorylation, reduction in GRK2 expression and increase in βAR-associated PP2A. These studies show that agonist-independent hypoxia-driven β2AR dysfunction can be ameliorated by β-blockers and the underlying mechanisms for this expected findings will be discussed in the presentation. These findings have significant clinical implications as understanding these mechanisms could provide novel insights into the benefits provided by β-blockers.

Ischemia/hypoxia is major underlying cause for heart failure and stroke. Although beta-adrenergic receptor (βAR) is phosphorylated in response to hypoxia, less is known about the underlying mechanisms. Hypoxia results in robust GRK2-mediated β2AR phosphorylation but does not cause receptor internalization. However, hypoxia leads to significant endosomal-β2AR phosphorylation accompanied by inhibition of β2AR-associated protein phosphatase 2A (PP2A) activity impairing resensitization. Phosphoinositide 3-kinase γ (PI3Kγ) impedes resensitization by phosphorylating endogenous inhibitor of protein phosphatase 2A, I2PP2A that inhibits PP2A activity. Hypoxia increased PI3Kγ activity leading to significant phosphorylation of I2PP2A resulting in inhibition of PP2A and consequently resensitization. Surprisingly, β-blocker abrogated hypoxia-mediated β2AR phosphorylation instead of phosphorylation in normoxia. Subjecting mice to hypoxia leads to significant cardiac dysfunction and β2AR phosphorylation showing conservation of non-canonical hypoxia-mediated pathway in vivo. These findings provide mechanistic insights on hypoxia-mediated βAR dysfunction which is rescued by β-blocker and will have significant implications in heart failure and stroke.