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A surge in the prevalence of obesity and metabolic syndrome, which promote systemic inflammation, underlies an increase in cardiometabolic disease. Free fatty acid receptor 4 is a nutrient sensor for long-chain fatty acids, like ω3-polyunsaturated fatty acids (ω3-PUFAs), that attenuates metabolic disease and resolves inflammation. Clinical trials indicate ω3-PUFAs are cardioprotective, and this review discusses the mechanistic links between ω3-PUFAs, free fatty acid receptor 4, and attenuation of cardiometabolic disease.

Aims Free fatty acid receptor 4 (Ffar4) is a G-protein-coupled receptor for endogenous medium-/long-chain fatty acids that attenuates metabolic disease and inflammation. However, the function of Ffar4 in the heart is unclear. Given its putative beneficial role, we hypothesized that Ffar4 would protect the heart from pathologic stress. Methods and results In mice lacking Ffar4 (Ffar4KO), we found that Ffar4 is required for an adaptive response to pressure overload induced by transverse aortic constriction (TAC), identifying a novel cardioprotective function for Ffar4. Following TAC, remodelling was worsened in Ffar4KO hearts, with greater hypertrophy and contractile dysfunction. Transcriptome analysis 3-day post-TAC identified transcriptional deficits in genes associated with cytoplasmic phospholipase A2α signalling and oxylipin synthesis and the reduction of oxidative stress in Ffar4KO myocytes. In cultured adult cardiac myocytes, Ffar4 induced the production of the eicosapentaenoic acid (EPA)-derived, pro-resolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE). Furthermore, the activation of Ffar4 attenuated cardiac myocyte death from oxidative stress, while 18-HEPE rescued Ffar4KO myocytes. Systemically, Ffar4 maintained pro-resolving oxylipins and attenuated autoxidation basally, and increased pro-inflammatory and pro-resolving oxylipins, including 18-HEPE, in high-density lipoproteins post-TAC. In humans, Ffar4 expression decreased in heart failure, while the signalling-deficient Ffar4 R270H polymorphism correlated with eccentric remodelling in a large clinical cohort paralleling changes observed in Ffar4KO mice post-TAC. Conclusion Our data indicate that Ffar4 in cardiac myocytes responds to endogenous fatty acids, reducing oxidative injury, and protecting the heart from pathologic stress, with significant translational implications for targeting Ffar4 in cardiovascular disease.

Patients with well-controlled low-density lipoprotein cholesterol levels, but persistent high triglycerides, remain at increased risk for cardiovascular events as evidenced by multiple genetic and epidemiologic studies, as well as recent clinical outcome trials. While many trials of low-dose ω3-polyunsaturated fatty acids (ω3-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) have shown mixed results to reduce cardiovascular events, recent trials with high-dose ω3-PUFAs have reignited interest in ω3-PUFAs, particularly EPA, in cardiovascular disease (CVD). REDUCE-IT demonstrated that high-dose EPA (4 g/day icosapent-ethyl) reduced a composite of clinical events by 25% in statin-treated patients with established CVD or diabetes and other cardiovascular risk factors. Outcome trials in similar statin-treated patients using DHA-containing high-dose ω3 formulations have not yet shown the benefits of EPA alone. However, there are data to show that high-dose ω3-PUFAs in patients with acute myocardial infarction had reduced left ventricular remodelling, non-infarct myocardial fibrosis, and systemic inflammation. ω3-polyunsaturated fatty acids, along with their metabolites, such as oxylipins and other lipid mediators, have complex effects on the cardiovascular system. Together they target free fatty acid receptors and peroxisome proliferator-activated receptors in various tissues to modulate inflammation and lipid metabolism. Here, we review these multifactorial mechanisms of ω3-PUFAs in view of recent clinical findings. These findings indicate physico-chemical and biological diversity among ω3-PUFAs that influence tissue distributions as well as disparate effects on membrane organization, rates of lipid oxidation, as well as various receptor-mediated signal transduction pathways and effects on gene expression.

Atrial natriuretic peptide (NP) and BNP increase cGMP, which reduces blood pressure and cardiac hypertrophy by activating guanylyl cyclase (GC)-A, also known as NPR-A or Npr1. Although GC-A is highly phosphorylated, and dephosphorylation inactivates the enzyme, the significance of GC-A phosphorylation to heart structure and function is unknown. To identify in vivo processes that are regulated by GC-A phosphorylation, we substituted glutamates for known phosphorylation sites to make GC-A(8E/8E) mice that express an enzyme that cannot be inactivated by dephosphorylation. GC-A activity, but not protein, was increased in heart and kidney membranes from GC-A(8E/8E) mice. Activities were 3-fold higher in female compared to male cardiac ventricles. Plasma cGMP and testosterone were elevated in male and female GC-A(8E/8E) mice, but aldosterone was only increased in mutant male mice. Plasma and urinary creatinine concentrations were decreased and increased, respectively, but blood pressure and heart rate were unchanged in male GC-A(8E/8E) mice. Heart weight to body weight ratios for GC-A(8E/8E) male, but not female, mice were 12% lower with a 14% reduction in cardiomyocyte cross sectional area. Subcutaneous injection of fsANP, a long-lived ANP analog, increased plasma cGMP and decreased aldosterone in male GC-A(WT/WT) and GC-A(8E/8E) mice at 15 minutes, but only GC-A(8E/8E) mice had elevated levels of plasma cGMP and aldosterone at 60 minutes. fsANP reduced ventricular ERK1/2 phosphorylation to a greater extent and for a longer time in the male mutant compared to WT mice. Finally, ejection fractions were increased in male but not female hearts from GC-A(8E/8E) mice. We conclude that increased phosphorylation-dependent GC-A activity decreases cardiac ERK activity, which results in smaller male hearts with improved systolic function.

Objectives The aim of this study was to determine if plasma eicosapentaenoic acid (EPA) abundance (%EPA) is associated with reduced hazard for primary heart failure (HF) events in the MESA (Multi-Ethnic Study of Atherosclerosis) trial. Background Clinical trials suggest that omega-3 polyunsaturated fatty acids (ω3 PUFAs) prevent sudden death in coronary heart disease and HF, but this is controversial. In mice, the authors demonstrated that the ω3 PUFA EPA prevents contractile dysfunction and fibrosis in an HF model, but whether this extends to humans is unclear. Methods In the MESA cohort, the authors tested if plasma phospholipid EPA predicts primary HF incidence, including HF with reduced ejection fraction (EF) (EF Results A total of 6,562 participants 45 to 84 years of age had EPA measured at baseline (1,794 black, 794 Chinese, 1,442 Hispanic, and 2,532 white; 52% women). Over a median follow-up period of 13.0 years, 292 HF events occurred: 128 HF with reduced EF, 110 HF with preserved EF, and 54 with unknown EF status. %EPA in HF-free participants was 0.76% (0.75% to 0.77%) but was lower in participants with HF at 0.69% (0.64% to 0.74%) (p = 0.005). Log %EPA was associated with lower HF incidence (hazard ratio: 0.73 [95% confidence interval: 0.60 to 0.91] per log-unit difference in %EPA; p = 0.001). Adjusting for age, sex, race, body mass index, smoking, diabetes mellitus, blood pressure, lipids and lipid-lowering drugs, albuminuria, and the lead fatty acid for each cluster did not change this relationship. Sensitivity analyses showed no dependence on HF type. Conclusions Higher plasma EPA was significantly associated with reduced risk for HF, with both reduced and preserved EF. (Multi-Ethnic Study of Atherosclerosis [MESA]; NCT00005487)

BackgroundNon-­resolving activation of immune responses is central to the pathogenesis of heart failure (HF). Free fatty acid receptor 4 (Ffar4) is a G-protein coupled receptor (GPR) for medium-and long-chain fatty acids (FA) that regulates metabolism and attenuates inflammation in diabetes and obesity. Here, we tested the hypothesis that Ffar4 functions as a cardioprotective nutrient sensor that resolves inflammation to maintain cardiac homeostasis.MethodsMice with systemic deletion of Ffar4 (Ffar4KO) were subjected to pressure overload by transverse aortic constriction (TAC). Transcriptome analysis of cardiac myocytes was performed three days post-TAC. Additionally, Ffar4-mediated effects on inflammatory oxylipin production in cardiac myocytes and oxylipin composition in plasma lipoproteins were evaluated.ResultsIn Ffar4KO mice, TAC induced more severe remodeling, identifying an entirely novel cardioprotective role for Ffar4 in the heart. Transcriptome analysis 3-days post-TAC indicated a failure to induce cell death and inflammatory genes in Ffar4KO cardiac myocytes, as well as a specific failure to induce cytoplasmic phospholipase A2α (cPLA2α) signaling genes. In cardiac myocytes, Ffar4 signaling through cPLA2α-cytochrome p450 ω/ω-1 hydroxylase induced production of the EPA-derived anti-inflammatory oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE). Systemically, loss of Ffar4 altered oxylipin content in circulating plasma lipoproteins consistent with a loss of anti-inflammatory oxylipins at baseline, and inability to produce both pro-inflammatory and pro-resolving oxylipins following TAC. Finally, we confirmed that Ffar4 is expressed in human heart and down-regulated in HF.ConclusionsOur results identify a novel function for Ffar4 in the heart as a FA nutrient sensor that resolves inflammation to maintain cardiac homeostasis.

Free fatty acid receptor 4 (Ffar4, GPR120) is a GPCR for long chain fatty acids, including omega-3 polyunsaturated fatty acids (ω3-PUFAs). Previously, we demonstrated that eicosapentaenoic acid (EPA), an ω3-PUFA, prevents interstitial fibrosis and contractile dysfunction in pressure overload heart failure (transverse aortic constriction, TAC), but EPA was not incorporated into cardiac myocytes or fibroblasts, the traditional mechanism of action. Therefore, we hypothesized that Ffar4 is necessary for the anti-fibrotic and cardioprotective effects of EPA. In fact, we previously found that in primary cardiac fibroblasts, Ffar4 was sufficient and necessary to prevent TGFβ1-induced fibrosis. Here, we were surprised to find that Ffar4 also has important ω3-independent effects in cardiac myocytes in vivo . In mice with systemic deletion of Ffar4 (Ffar4KO mice) on a standard, corn oil diet (no EPA supplementation), TAC induced significantly more cardiac hypertrophy and more systolic (ejection fraction) and diastolic dysfunction (E/A ratio), but without excess fibrosis after 4 weeks. Mechanistically, transcriptome analysis in cardiac myocytes 3 days post-TAC indicated a deficiency of transcription in Ffar4KO myocytes. Sorting based on gene ontology indicated that genes functionally categorized as cell death and inflammation, including ilrn1 , hmox1 , Il33 , and ptgs2 (cox2) were not induced in the Ffar4KO myocytes. Ffar4 also activates cytoplasmic phospholipase A2 (cPLA2) inducing the production of oxylipins, and systemic deletion of cPLA2 induces exaggerated hypertrophy during development and following TAC. TAC increased cPLA2 expression in WT, but not Ffar4KO cardiac myocytes and oxylipin production was reduced in Ffar4KO mice. Finally, we confirmed that Ffar4 is expressed in human heart and down-regulated in heart failure. In summary, our data identify a novel, ω3-independent cardioprotective role for Ffar4 in cardiac myocytes.

Background: Our previous results indicate that eicosapentaenoic acid (EPA) prevents interstitial fibrosis and pathologic remodeling in the transverse aortic constriction (TAC) model of pressure overload induced heart failure in mice. However, EPA supplementation did not lead to incorporation into cardiac myocytes or fibroblasts in vivo , a traditional mechanism of action. Interestingly, we found that free fatty acid receptor 4 (Ffar4), a G-protein coupled receptor for long-chain fatty acids, was expressed in cardiac myocytes and fibroblasts. In primary cultures of cardiac fibroblasts, Ffar4 was sufficient and required to prevent TGFβ1-induced fibrosis, suggesting Ffar4 might mediate EPA cardioprotection. Methods: To understand the function of Ffar4 in the heart in vivo , we examined the response to TAC in male and female Ffar4 knockout mice (Ffar4KO) under basal conditions without EPA supplementation. Results: In male Ffar4KO mice, TAC induced a worsened dilated cardiomyopathy relative to wild-type (WT) mice. Specifically, in male Ffar4KO mice, TAC produced more hypertrophy (Heart weight: WT: 175±8 mg, n=14; Ffar4KO: 223±14 mg, n=10; P P P ns ). While TAC worsened outcomes in male Ffar4KO mice, TAC in Ffar4 KO female mice produced no significant differences in the degree of hypertrophy or contractile dysfunction relative to WT mice. Discussion: Our results indicate that Ffar4 is required for an adaptive response to pathologic pressure overload in male mice. The lack of an exaggerated fibrotic response was surprising given our previous results indicating that in cardiac fibroblasts, Ffar4 prevents TGFβ1-induced fibrosis. This might indicate that Ffar4 signaling is minimal in fibroblasts under conditions of low omega-3 fatty acid levels. This result also suggests that the worsened contractile dysfunction in Ffar4KO mice is unrelated to fibrosis, but involves a direct effect in cardiac myocytes.

G protein–coupled receptors that signal through Gα(q) (G(q) receptors), such as α(1)-adrenergic receptors (α(1)-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cβ1 (PLCβ1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP(2)) to produce inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol. Despite these common proximal signaling mechanisms, G(q) receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, G(q) 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 IP(3)-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of G(q)-receptor signaling as a mechanistic basis for unique G(q) receptor–induced hypertrophic phenotypes in cardiac myocytes. We show that α(1)-ARs co-localize with PLCβ1 and PIP(2) at the nuclear membrane. Further, nuclear α(1)-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 α(1)-AR–induced transcription. In summary, these results link G(q)-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 G(q)-receptor localization and better explains distinct physiological functions of G(q) receptors.

Introduction: In animal models of afterload stress, eicosapentaenoic acid (EPA) prevents interstitial myocardial fibrosis and preserves diastolic dysfunction. Hypothesis: We hypothesized that EPA is similarly protective in humans. Methods: In the Multi-Ethnic Study of Atherosclerosis, we tested for an effect of plasma phospholipid EPA percent of total fatty acids on primary heart failure incidence across heart failure types including, heart failure with reduced ejection fraction (HFrEF; Results: A total of 6566 subjects had measured baseline EPA, including 1797 black, 794 Chinese, 1444 Hispanic, and 2531 white participants; 52% were female. Over a median follow-up of 13.0 years, 293 heart failure events occurred in subjects with measured EPA: 129 had HFrEF, 110 had HFpEF, and the remaining 54 had unknown ejection fraction status. Mean EPA in HF-free subjects was 0.77% (0.76 - 0.79), and was lower in heart failure subjects 0.70% (0.65 - 0.74), p=0.002. EPA was associated with lower incidence of HF, having a hazard ratio of 0.73 (0.60 - 0.89) per unit change in percentage of total fatty acids, p=0.001. Adjusting for age, sex, race, BMI, smoking, diabetes mellitus, blood pressure, lipids and lipid-lowering drugs, and albuminuria did not change this relationship. Sensitivity analysis showed no dependence on heart failure type. Adjusting for other fatty acids with clustering did not change hazards. In animals, >2.5% EPA is required for prevention of HF, between 2.5% and 1% is marginal, and Conclusion: High abundance of EPA is robustly associated with reduced risk for heart failure, independent of established risk factors and regardless of ejection fraction status.

To study molecular and cellular mechanisms for normal cardiac function and cardiac pathology, high-quality isolated adult cardiomyocytes are essential. Isolated cardiomyocytes can be used directly for cellular function studies such as measuring myocyte contraction, cellular electrophysiology, calcium imaging, myofilament calcium sensitivity, cytoskeletal function, drug effects, and metabolism. Isolated myocytes can also be cultured for signaling studies with less contamination from nonmyocytes. In addition, the isolated cells are also essential for studying the morphology and structure of cardiomyocytes. Furthermore, the function of nonmyocytes including cardiac fibroblasts, smooth muscle cells, infiltrated inflammatory cells, and endothelial cells in the heart is also an important topic in cardiac biology, and there is an increasing appreciation of the structure and function of these cells in cardiac pathology. Therefore, the development of techniques for heart cell isolation and harvest dates back to ≈40 years ago. In the current issue of Circulation Research , Ackers-Johnson et al1 describe a simple approach for the isolation and culture of adult mouse cardiac myocytes and fibroblasts without the traditional Langendorff perfusion system. Article, see p 909 Although techniques for isolation of adult ventricular myocytes from many species including rat,2 rabbit,3 and canine4 have been available for ≈40 years, there remains no clear consensus on the optimal protocol to isolate and culture viable adult cardiac myocytes. However, at their core, these protocols generally all rely on digestion of the heart with an enzymatic solution.5 All myocyte isolation techniques can be essentially categorized into either chunk (a small piece of tissue) digestion in an enzymatic solution or coronary artery perfusion with enzymatic solution. The chunk digestion technique basically allows the enzymes (collagenases supplemented with other proteases) to chew the extracellular matrix directly and dissociate cardiomyocytes and nonmyocytes from small pieces (normally

Background— Induced pluripotent stem cells (iPSCs) can be differentiated into potentially unlimited lineages of cell types for use in autologous cell therapy. However, the efficiency of the differentiation procedure and subsequent function of the iPSC-derived cells may be influenced by epigenetic factors that the iPSCs retain from their tissues of origin; thus, iPSC-derived cells may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells, rather than dermal fibroblasts. Methods and Results— We show that human cardiac iPSCs (hciPSCs) can be generated from cardiac fibroblasts and subsequently differentiated into exceptionally pure (>92%) sheets of cardiomyocytes (CMs). The hciPSCs passed through all the normal stages of differentiation before assuming a CM identity. When using the fibrin gel–enhanced delivery of hciPSC-CM sheets at the site of injury in infarcted mouse hearts, the engraftment rate was 31.91%±5.75% at Day 28 post transplantation. The hciPSC-CM in the sheet also appeared to develop a more mature, structurally aligned phenotype 28 days after transplantation and was associated with significant improvements in cardiac function, vascularity, and reduction in apoptosis. Conclusions— These data strongly support the potential of hciPSC-CM sheet transplantation for the treatment of heart with acute myocardial infarction.

Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate "inside-out" signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure.

Heart failure (HF) affects 5.7 million in the U.S., and despite well-established pharmacologic therapy, the 5-year mortality rate remains near 50%. Furthermore, the mortality rate for HF has not declined in years, highlighting the need for new therapeutic options. Omega-3 polyunsaturated fatty acids (ω3-PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are important regulators of cardiovascular health. However, questions of efficacy and mechanism of action have made the use of ω3-PUFAs in all cardiovascular disease (CVD) controversial. Here, we review recent studies in animal models of HF indicating that ω3-PUFAs, particularly EPA, are cardioprotective, with the results indicating a threshold for efficacy. We also examine clinical studies suggesting that ω3-PUFAs improve outcomes in patients with HF. Due to the relatively small number of clinical studies of ω3-PUFAs in HF, we discuss EPA concentration-dependency on outcomes in clinical trials of CVD to gain insight into the perceived questionable efficacy of ω3-PUFAs clinically, with the results again indicating a threshold for efficacy. Ultimately, we suggest that the main failing of ω3-PUFAs in clinical trials might be a failure to reach a therapeutically effective concentration. We also examine mechanistic studies suggesting that ω3-PUFAs signal through free fatty acid receptor 4 (Ffar4), a G-protein coupled receptor (GPR) for long-chain fatty acids (FA), thereby identifying an entirely novel mechanism of action for ω3-PUFA mediated cardioprotection. Finally, based on mechanistic animal studies suggesting that EPA prevents interstitial fibrosis and diastolic dysfunction, we speculate about a potential benefit for EPA-Ffar4 signaling in heart failure preserved with ejection fraction.

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCRs oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.

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 ω-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of ω-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-β1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-β1 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-β1–induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.

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.

Background— In α1-AR knockout (α1ABKO) mice that lacked cardiac myocyte α1-adrenergic receptor (α1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte α1-ARs and whether the α1A, α1B, or both subtypes mediated protection. Therefore, we investigated α1A and α1B subtype–specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of α1-ARs in cardiac myocytes. Methods and Results— We cultured α1ABKO myocytes and reconstituted α1-AR signaling with adenoviruses expressing α1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H 2 O 2 and was measured by annexin V/propidium iodide staining. In α1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the α1A subtype, but not the α1B, rescued α1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined α1-AR activation of extracellular signal-regulated kinase (ERK). In α1ABKO hearts, aortic constriction failed to activate ERK, and in α1ABKO myocytes, expression of a constitutively active MEK1 rescued α1ABKO myocytes from norepinephrine-induced death. In addition, only the α1A-AR activated ERK in α1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked α1A survival signaling in α1ABKO myocytes. Conclusions— Our results demonstrate a direct protective effect of the α1A subtype in cardiac myocytes and define an α1A-ERK signaling pathway that is required for myocyte survival. Absence of the α1A-ERK pathway can explain the failure to activate ERK after aortic constriction in α1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.

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.

Heart failure with preserved ejection fraction (HFpEF) is ~50% of all heart failure (HF) diagnoses, however, standard HF therapies show no efficacy in HFpEF. Omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), improve outcomes in coronary heart disease and HF with reduced ejection fraction, but whether this benefit extends to HFpEF is unclear. Here, we examined the effect of EPA and DHA on remodeling following transverse aortic constriction (TAC) in mice. TAC induced concentric hypertrophy, interstitial fibrosis, and diastolic and systolic dysfunction similar, in several respects, to remodeling in HFpEF. Diastolic dysfunction correlated with the extent of interstitial fibrosis and hypertrophy after TAC (R2=0.39, P=0.02; R2 = 0.34, P=0.04, respectively), but systolic dysfunction did not (R2=0.19, P = 0.14; R2=0.07, P=0.40). Interestingly, blood levels of EPA (n=58, P=0.04), but not DHA (n=58, P=0.63), inversely correlated with interstitial fibrosis after TAC. Ho...

Catecholamines and alpha(1)-adrenergic receptors (alpha(1)-ARs) cause cardiac hypertrophy in cultured myocytes and transgenic mice, but heart size is normal in single KOs of the main alpha(1)-AR subtypes, alpha(1A/C) and alpha(1B). Here we tested whether alpha(1)-ARs are required for developmental cardiac hypertrophy by generating alpha(1A/C) and alpha(1B) double KO (ABKO) mice, which had no cardiac alpha(1)-AR binding. In male ABKO mice, heart growth after weaning was 40% less than in WT, and the smaller heart was due to smaller myocytes. Body and other organ weights were unchanged, indicating a specific effect on the heart. Blood pressure in ABKO mice was the same as in WT, showing that the smaller heart was not due to decreased load. Contractile function was normal by echocardiography in awake mice, but the smaller heart and a slower heart rate reduced cardiac output. alpha(1)-AR stimulation did not activate extracellular signal-regulated kinase (Erk) and downstream kinases in ABKO myocytes, and basal Erk activity was lower in the intact ABKO heart. In female ABKO mice, heart size was normal, even after ovariectomy. Male ABKO mice had reduced exercise capacity and increased mortality with pressure overload. Thus, alpha(1)-ARs in male mice are required for the physiological hypertrophy of normal postnatal cardiac development and for an adaptive response to cardiac stress.

Two functional α1-adrenergic receptor (AR) subtypes (α1Aand α1B) have been identified in the mouse heart. However, it is unclear whether the third known subtype, α1D-AR, is also present. To investigate this, we determined whether there were α1-AR responses in hearts from a novel mouse model lacking α1A- and α1B-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, α1-ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 ± 2% and 86 ± 4% of initial, respectively, n = 11, P < 0.01). These effects were blocked by prazosin and 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione} dihydrochloride, suggesting that α1D-AR-mediated responses were present. In contrast, right ventricular trabeculae from ABKO hearts did not respond to phenylephrine, suggesting that in ABKO perfused hearts, the effects of phenylephrine were not mediated by direct actions on cardiomyocytes. A novel finding was that α1-AR stimulation caused positive inotropy in the wild-type mouse heart, in contrast to negative inotropy observed in mouse cardiac muscle strips. We conclude that mouse hearts lacking α1A- and α1B-ARs retain functional α1-AR responses involving decreases of coronary flow and ventricular pressure that reflect α1D-AR-mediated vasoconstriction. Furthermore, α1-AR inotropic responses depend critically on the experimental conditions.

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.

It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKCalpha, PKCbeta, PKCbetaI, and PKCbetaII on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)2D3 (20 nM for 48 h) was inhibited similarly in cells treated with PKCbeta antisense (30 microM) 24 h prior to or at the same time as hormone treatment (86 +/- 9% inhibition; n = 4 versus 82 +/- 8% inhibition; n = 4 (mean +/- S.E.), respectively). In contrast, cells treated with PKCbeta antisense 24 h after 1, 25-(OH)2D3 were unaffected and fully differentiated. PKCalpha antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCbetaI- and PKCbetaII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCbetaII antisense (30 microM) completely blocked CD14 expression induced by 1, 25-(OH)2D3, whereas PKCbetaI antisense had little effect. Interestingly, PKCbetaII antisense blocked differentiation by 87 +/- 7% (n = 2, mean +/- S.D.) but had no effect on 1,25-(OH)2D3 inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)2D3 on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCbetaII expression.

Heart failure (HF) secondary to hypertensive heart disease typically presents as heart failure with preserved ejection fraction (HFPEF), which now accounts for nearly half of all HF diagnoses. HFPEF pathology is characterized by interstitial fibrosis leading to ventricular stiffness, diastolic dysfunction, and ultimately HF. Clinically HFPEF is a significant problem, as standard HF therapies, which target heart failure with reduced ejection fraction, are ineffective in HFPEF. A major challenge to developing new therapies for HFPEF is the lack of an animal model. In mice, although transverse aortic constriction (TAC) induces significant systolic dysfunction, it produces ventricular remodeling similar to chronic, untreated hypertension, which is associated with concentric hypertrophy, interstitial fibrosis, and diastolic dysfunction. Here, we examined pathologic remodeling and ventricular dysfunction following TAC in mice to determine what factors drive remodeling. TAC mice developed significant hypertrophy after 4 weeks (HW/BW, TAC: 7.2±0.4, n=13; Sham: 4.5±0.2, n=3; P

In the heart, α1-adrenergic receptors (α1-ARs) regulate physiologic hypertrophy, cardiac myocyte survival, and contractility. Conventionally, G protein-coupled receptors such as α1-ARs are thought to localize and signal at the plasma membrane. However, we demonstrated that endogenous α1-ARs localize to the nucleus in wild-type (WT) adult cardiac myocytes, and identified nuclear localization sequences (NLS) in the α1A- and α1B-subtypes. Mutation of the NLS in each subtype resulted in mis-localization of the receptors and loss of signaling. Here, we examined how nuclear α1-ARs induce inotropic responses through regulation of cardiac troponin I (cTnI). In unloaded WT cardiac myocytes, the α1-agonist phenylephrine (PE) reduced sarcomeric shortening and increased cTnI phosphorylation at the protein kinase C (PKC) site threonine 144 (Thr144). PE had no effect in cardiac myocytes lacking both α1A- and α1B-subtypes (α1ABKO), but reconstitution of the α1A-subtype restored PE effects. Conversely, reconstitution of an NLS-mutant-α1A-subtype blunted the effects of PE on shortening and cTnI phosphorylation. Furthermore, in WT cardiac myocytes, the nuclear export inhibitor leptomycin B also blunted the effects of PE. With nuclei isolated from WT cardiac myocytes, we found high levels of PKCδ with lower levels of PKCα and ε, and that PE induced PKCδ activation in isolated nuclei. We also examined endogenous PKC translocation induced by PE in membrane, cytosolic, nuclear, and myofilament fractions, and found that PKCδ translocated from the nuclear to the myofilament fraction. Finally, expression of a dominant-negative PKCδ in WT myocytes blunted PE-induced effects on shortening and cTnI phosphorylation. Our data show that α1A-subtype nuclear localization is required for activation of PKCδ in the nucleus, subsequent phosphorylation of cTnI at Thr144 at the sarcomere to regulate myocyte contractility.

Cardiac myocytes have a complex network of signals that regulates their essential role in the rhythmic pumping of the heart. This network is an appealing model system in which to study the basic principles underlying cellular signalling mechanisms. Progress in this effort has come through the establishment of standardized myocyte isolation and culture procedures and characterization of important signalling responses.

We previously demonstrated that α1-adrenergic receptors (α1-ARs) in the heart are required for physiologic hypertrophy during development and prevent a maladaptive response to pathologic stress. We have also shown that the major subtypes, α1A and α1B, both localize to the nucleus in adult cardiac myocytes. Importantly, we have defined a nuclear α1A-ERK survival and an α1A-PKCδ-cTnI inotropic signaling pathway that both originate at the nucleus and are transduced to cytosolic targets, suggesting that the α1A is required for cardiac myocyte survival and contractility. However, less is known about the molecular function of the α1B. In the current study, we examined the role of the α1B-subtype in hypertrophic signaling. First, we identified a bi-partite nuclear localization sequence (NLS) in the carboxy-terminal tail of the receptor. Mutation of the NLS (α1B-NLSmut) disrupted its localization to the nucleus when expressed in adult cardiac myocytes. We then compared hypertrophic signaling of the wild-type α1B- to the mutated receptor by reconstitution in cardiac myocytes lacking endogenous α1B (α1BKO) receptors. Activation of the wild-type receptor by the α1-agonist phenylephrine in α1BKO myocytes restored hypertrophic signaling, as we observed increased phosphorylation of protein kinase C (PKC) isoforms δ and ε, and histone deacetylases (HDAC) 4 and 5. We also observed increased expression of the hypertrophic gene marker, atrial natriuretic factor (ANF). Expression of the α1B-NLSmut failed to activate hypertrophic signaling in α1BKO cardiac myocytes despite phenylephrine stimulation. Together, our data show that nuclear localization of the α1B-subtype is required for hypertrophic signaling and overall further suggest that α1-adrenergic receptors are functional only at the nucleus in cardiac myocytes.

The enhanced stimulation and outflow of catecholamines has long been recognized as an important mechanism in the progression of heart failure [(1)][1]. In 1984, Cohn et al. [(2)][2] established that higher plasma concentrations of norepinephrine predicted worse survival in patients with heart

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. The transcription factor GATA4 is essential for cardiac homeostasis, and its protein levels are dramatically reduced in the heart in response to diverse pathologic stresses. In this study, we investigated if hyperglycemia affects GATA4 expression in cardiomyocytes and if enhancing GATA4 signaling could attenuate hyperglycemia-induced cardiomyocyte injury. In cultured rat cardiomyocytes, high glucose (HG, 25 or 40 mm) markedly reduced GATA4 protein levels as compared with normal glucose (NG, 5.5 mm). Equal amount of mannitol did not affect GATA4 protein expression (NG, 100 ± 12%; mannitol, 97 ± 8%, versus HG, 43 ± 16%, p < 0.05). The GATA4 mRNA content, either steady-state or polysome-associated, remained unchanged. HG-induced GATA4 reduction was reversed by MG262, a specific proteasome inhibitor. HG did not activate the ubiquitin proteasome system (UPS) in cardiomyocytes as indicated by a UPS reporter, nor did it increase the peptidase activities or protein expression of the proteasomal subunits. However, the mRNA levels of ubiquitin-protein isopeptide ligase (E3) carboxyl terminus of Hsp70-interacting protein (CHIP) were markedly increased in HG-treated cardiomyocytes. CHIP overexpression promoted GATA4 protein degradation, whereas small interfering RNA-mediated CHIP knockdown prevented HG-induced GATA4 depletion. Moreover, overexpression of GATA4 blocked HG-induced cardiomyocyte death. Also, GATA4 protein levels were diminished in the hearts of streptozotocin and db/db diabetic mice (44 ± 7% and 67 ± 13% of control, p < 0.05), which correlated with increased CHIP mRNA abundance. In summary, increased GATA4 protein degradation may be an important mechanism that contributes to hyperglycemic cardiotoxicity.

Thyroid hormones (THs) have many effects on the cardiovascular system including cardiac hypertrophy. Although THs induce cardiac hypertrophy, the mechanism through which they exert this effect is unknown. We previously found that THs activate signaling related to increased protein synthesis [mammalian target of rapamycin (mTOR) and p70 S6 kinase] in the heart. It is unknown whether this activation contributes to TH-induced hypertrophy or whether it is merely incidental. In this study, we used rapamycin to inhibit mTOR function in mice and neonatal cardiomyocyte cultures treated with THs to test whether mTOR/S6 kinase signaling is involved in TH-mediated cardiac hypertrophy. C57 mice were treated with T4 for 3 d, 1 wk, 2 wk, or 1 month with either placebo, T4 (50 μg/100 g body weight·d), rapamycin (200 μg/100 g body weight·d) or T4/rapamycin by sc slow-release pellets. At the end of the treatment period, hemodynamics and physical data were collected and hearts were frozen for Western blot analysis or myocytes were isolated. The effects of T3 and rapamycin were also investigated using neonatal cardiomyocytes. THs activated specific components of the AKT signaling pathway in vivo and in vitro. THs induced cardiac hypertrophy, which was completely inhibited by rapamycin. Our results suggest that TH-induced hypertrophy is mediated by AKT/mTOR/S6 kinase signaling, which is important in the regulation of protein synthesis, a hallmark of cardiac hypertrophy.

An alpha1-adrenergic receptor (alpha1-AR) antagonist increased heart failure in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), but it is unknown whether this adverse result was due to alpha1-AR inhibition or a nonspecific drug effect. We studied cardiac pressure overload in mice with double KO of the 2 main alpha1-AR subtypes in the heart, alpha 1A (Adra1a) and alpha 1B (Adra1b). At 2 weeks after transverse aortic constriction (TAC), KO mouse survival was only 60% of WT, and surviving KO mice had lower ejection fractions and larger end-diastolic volumes than WT mice. Mechanistically, final heart weight and myocyte cross-sectional area were the same after TAC in KO and WT mice. However, KO hearts after TAC had increased interstitial fibrosis, increased apoptosis, and failed induction of the fetal hypertrophic genes. Before TAC, isolated KO myocytes were more susceptible to apoptosis after oxidative and beta-AR stimulation, and beta-ARs were desensitized. Thus, alpha1-AR deletion worsens dilated cardiomyopathy after pressure overload, by multiple mechanisms, indicating that alpha1-signaling is required for cardiac adaptation. These results suggest that the adverse cardiac effects of alpha1-antagonists in clinical trials are due to loss of alpha1-signaling in myocytes, emphasizing concern about clinical use of alpha1-antagonists, and point to a revised perspective on sympathetic activation in heart failure.

Two functional alpha(1)-adrenergic receptor (AR) subtypes (alpha(1A) and alpha(1B)) have been identified in the mouse heart. However, it is unclear whether the third known subtype, alpha(1D)-AR, is also present. To investigate this, we determined whether there were alpha(1)-AR responses in hearts from a novel mouse model lacking alpha(1A)- and alpha(1B)-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, alpha(1)-ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 +/- 2% and 86 +/- 4% of initial, respectively, n = 11, P0.01). These effects were blocked by prazosin and 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione] dihydrochloride, suggesting that alpha(1D)-AR-mediated responses were present. In contrast, right ventricular trabeculae from ABKO hearts did not respond to phenylephrine, suggesting that in ABKO perfused hearts, the effects of phenylephrine were not mediated by direct actions on cardiomyocytes. A novel finding was that alpha(1)-AR stimulation caused positive inotropy in the wild-type mouse heart, in contrast to negative inotropy observed in mouse cardiac muscle strips. We conclude that mouse hearts lacking alpha(1A)- and alpha(1B)-ARs retain functional alpha(1)-AR responses involving decreases of coronary flow and ventricular pressure that reflect alpha(1D)-AR-mediated vasoconstriction. Furthermore, alpha(1)-AR inotropic responses depend critically on the experimental conditions.

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

To the Editor: Many patients with an implantable cardioverter-defibrillator (ICD) require concomitant antiarrhythmic drug (AAD) therapy at some point. Sotalol and especially amiodarone have been shown to reduce the number of ventricular tachycardia (VT)/ventricular fibrillation (VF) episodes and

The steroid hormone 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] induces maturation of many cells, including HL-60 promyelocytic leukemia cells that are differentiated to monocytes/macrophages. This process involves changes in transcription of genes such as c-myc. We investigated the effects of 1,25-(OH)2D3 on nuclear protein binding to the c-myc intron element (MIE), a region of DNA within the c-myc gene. A mutation in this MIE sequence has been shown to be associated with uncontrolled expression of the c-myc gene in various cell lines. In this report, we demonstrate for the first time that 1,25-(OH)2D3 induces increased binding of nuclear proteins to this MIE in HL-60 cells. The major MIE-binding proteins were approximately 32 kDa in size. Interestingly, phorbol 12-myristate 13-acetate induced a similar increase in binding of the 32-kDa doublet protein to the MIE. In addition, we showed that the level of 138-kDa MIE-binding protein was increased by 1,25-(OH)2D3 and phorbol 12-myristate 13-acetate. However, the extent of MIE binding by the 138-kDa protein is significantly less than that of the 32-kDa doublet binding species. MIE binding by the 32-kDa doublet protein was significantly increased within 12 h of 1,25-(OH)2D3 treatment. The time course of this increase was similar to that of 1,25-(OH)2D3-induced inhibition of c-myc gene transcription. We also demonstrated that dephosphorylation of the 32-kDa doublet protein inhibited its binding to the MIE. Thus, this study shows that the mechanism employed by 1,25-(OH)2D3 for regulation of c-myc expression may involve an increase in protein binding to the MIE, which has been shown to be the site for control of c-myc gene expression.

A number of recent clinical observations suggest that vitamin D3 plays an important role in maintaining normal cardiovascular function, either directly through its receptor in cardiac muscle, or indirectly through its influence on circulating levels of calcium or on other regulatory factors. By using an antibody directed against the recombinant vitamin D3 receptor, we have identified the receptor protein for 1,25(OH)2D3 in tissue from two human hearts. The identification of the 1,25(OH)2D3 receptor in human heart lends credence to the hypothesis that 1,25(OH)2D3 directly effects the human heart and may be involved in several clinically relevant pathological conditions involving the vitamin D3 endocrine system.