Chastity L. Healy

Omega-3 Fatty Acids Prevent Pressure Overload–Induced Cardiac Fibrosis Through Activation of Cyclic GMP/Protein Kinase G Signaling in Cardiac Fibroblasts

Background— Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis. Methods and Results— We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction–induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-&bgr;1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-&bgr;1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. Conclusion— Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-&bgr;1–induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.

Nuclear α1-Adrenergic Receptors Signal Activated ERK Localization to Caveolae in Adult Cardiac Myocytes

We previously identified an α1-AR-ERK (α1A-adrenergic receptor–extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of α1-AR subtypes (α1A and α1B) and how their localization influences α1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent α1-AR antagonist, we localized endogenous α1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify α1 subtype localization, we reconstituted α1 signaling in cultured α1A- and α1B-AR double knockout cardiac myocytes using α1-AR–green fluorescent protein (GFP) fusion proteins. Similar to endogenous α1-ARs and α1A- and α1B-GFP colocalized with LAP2 at the nuclear membrane. α1-AR nuclear localization was confirmed in vivo using α1-AR-GFP transgenic mice. The α1-signaling partners Gαq and phospholipase Cβ1 also colocalized with α1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that α1-mediated activation of ERK was not inhibited by a membrane impermeant α1-blocker, suggesting α1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe α1-AR localization to caveolae, but we found that α1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear α1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.

An Association Between Gene Expression and Better Survival in Female Mice Following Myocardial Infarction

Following myocardial infarction, the prognosis for females is better than males. Estrogen is thought to be protective, but clinical trials with hormone replacement failed to show protection. Here, we sought to identify novel mechanisms that might explain this sex-based difference. By diverging from the traditional focus on sex hormones, we employed a conceptually novel approach to this question by using a non-biased approach to measure global changes in gene expression following infarction. We hypothesized that specific gene programs are initiated in the heart following infarction that might account for this sex-based difference. We induced small, medium, and large infarcts in male and female mice and measured changes in gene expression by microarray following infarction. Regardless of infarct size, survival was better in females, while mortality occurred 3-10 days following infarction in males. Two days following infarction, males developed significant ventricular dilation, the best predictor of mortality in humans. Three days following infarction, we measured gene expression by microarray, comparing male versus female and sham versus surgery/infarction. In general, our results indicate a higher relative level of gene induction in females versus males and identified programs for angiogenesis, extracellular matrix remodeling, and immune response. This pattern of gene expression was linked to less pathologic remodeling in female hearts, including increased capillary density and decreased fibrosis. In summary, our results suggest an association between improved survival and less pathologic remodeling and the relative induction of gene expression in females following myocardial infarction.

Nuclear Localization of α1A-Adrenergic Receptors Is Required for Signaling in Cardiac Myocytes: An “Inside-Out” α1-AR Signaling Pathway

Background Recent studies indicate that α1‐adrenergic receptors (α1‐ARs) are cardioprotective by preventing cardiac myocyte death and augmenting contractility in heart failure. Although G‐protein‐coupled receptors are assumed to localize to and signal at the plasma membrane, we previously demonstrated that endogenous α1‐ARs localize to the nuclei in adult cardiac myocytes. However, the functional consequence of this nuclear localization remains unclear. Here, we attempted to reconcile nuclear localization of α1‐ARs with their physiologic function by examining α1‐AR‐induced contractility in adult cardiac myocytes. Methods and Results By measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the α1A‐subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an α1A‐subtype nuclear localization mutant in cardiac myocytes lacking α1‐ARs failed to rescue nuclear α1A‐mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked α1A‐mediated phosphorylation of cTnI. These data indicate that α1‐AR signaling originates in the nucleus. Consistent with these observations, we localized the α1A‐subtype to the inner nuclear membrane, identified PKCα, δ, and ε in the nucleus, and found that α1‐ARs activate PKCδ in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCδ nuclear localization mutant blunted α1‐induced phosphorylation of cTnI. Conclusions Together, our data identify a novel, “inside‐out” nuclear α1A‐subtype/PKCδ/cTnI‐signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of α1‐ARs and α1‐AR‐mediated physiologic function.

GATA4 is a survival factor in adult cardiac myocytes but is not required for α1A-adrenergic receptor survival signaling

Recently, we defined an alpha1A-adrenergic receptor-ERK (alpha1A-AR-ERK) survival signaling pathway in adult cardiac myocytes. Previous studies in neonatal cardiac myocytes indicated that the cardiac-specific transcription factor GATA4 is a downstream mediator of alpha1-ERK signaling and that phosphorylation of GATA4 by ERK increases DNA binding and transcriptional activity. Therefore, we examined GATA4 as a potential downstream effector of alpha1A-ERK survival signaling in adult cardiac myocytes. We measured norepinephrine (NE)-induced cell death in cultured cardiac myocytes lacking alpha1-ARs (cultured from alpha1A/B-AR double-knockout mice, alpha1ABKO mice) that are susceptible to cell death induced by several proapoptotic stimuli, including NE. Our results show that overexpression of GATA4 is sufficient to protect alpha1ABKO cardiac myocytes from NE-induced cell death. However, we found that the alpha1A-subtype did not induce phosphorylation or increase the activity of GATA4 in adult mouse cardiac myocytes in culture or in vivo. Furthermore, we examined the effect of siRNA-mediated knockdown of GATA4 on alpha1A-survival signaling. In alpha1B-knockout cardiac myocytes, which express only the alpha1A-subtype and are protected from NE-induced cell death, GATA4 knockdown did not reverse alpha1A-survival signaling in response to NE. In summary, we found that GATA4 acted as a survival factor by preventing cell death in alpha1ABKO cardiac myocytes, but GATA4 was not activated by alpha1-AR stimulation and was not required for alpha1A-survival signaling in adult cardiac myocytes. This also identifies an important mechanistic difference in alpha1-signaling between adult and neonatal cardiac myocytes.

EPA, not DHA, prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4.

Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor β1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.

Omega-3 Fatty Acids Prevent Pressure Overload–Induced Cardiac Fibrosis Through Activation of Cyclic GMP/Protein Kinase G Signaling in Cardiac FibroblastsClinical Perspective

Background— Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis. Methods and Results— We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction–induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-&bgr;1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-&bgr;1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. Conclusion— Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-&bgr;1–induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.

Subcellular Compartmentalization of proximal Gαq-receptor signaling produces unique hypertrophic phenotypes in adult cardiac myocytes

G protein–coupled receptors that signal through Gαq (Gq receptors), such as α1-adrenergic receptors (α1-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cβ1 (PLCβ1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Despite these common proximal signaling mechanisms, Gq receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, Gq receptors are thought to induce myocyte hypertrophy through a mechanism termed excitation–transcription coupling, which provides a mechanistic basis for compartmentalization of calcium required for contraction versus IP3-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of Gq-receptor signaling as a mechanistic basis for unique Gq receptor–induced hypertrophic phenotypes in cardiac myocytes. We show that α1-ARs co-localize with PLCβ1 and PIP2 at the nuclear membrane. Further, nuclear α1-ARs induced intranuclear PLCβ1 activity, leading to histone deacetylase 5 (HDAC5) export and a robust transcriptional response (i.e. significant up- or down-regulation of 806 genes). Conversely, we found that angiotensin receptors localize to the sarcolemma and induce sarcolemmal PLCβ1 activity, but fail to promote HDAC5 nuclear export, while producing a transcriptional response that is mostly a subset of α1-AR–induced transcription. In summary, these results link Gq-receptor compartmentalization in cardiac myocytes to unique hypertrophic transcription. They suggest a new model of excitation–transcription coupling in adult cardiac myocytes that accounts for differential Gq-receptor localization and better explains distinct physiological functions of Gq receptors.

ω-3 fatty acids prevent pressure overload-induced cardiac fibrosis through activation of cGMP/PKG signaling in cardiac fibroblasts

Background— Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis. Methods and Results— We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction–induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-&bgr;1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-&bgr;1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. Conclusion— Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-&bgr;1–induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.