Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice.

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

Thymosin ß4 Prevents Angiotensin II-Induced Cardiomyocyte Growth by Regulating Wnt/WISP Signaling.

Thymosin beta-4 (Tß4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tß4 in cardiomyocyte hypertrophy is currently unknown. The purpose of this study was to determine the cardio-protective effect of Tß4 in angiotensin II (Ang II)-induced cardiomyocyte growth. Neonatal rat ventricular cardiomyocytes (NRVM) were pretreated with Tß4 followed by Ang II stimulation. Cell size, hypertrophy marker gene expression and Wnt signaling components, ß-catenin, and Wnt-induced secreted protein-1 (WISP-1) were evaluated by quantitative real-time PCR, Western blotting and fluorescent microscopy. Pre-treatment of Tß4 resulted in reduction of cell size, hypertrophy marker genes and Wnt-associated gene expression, and protein levels; induced by Ang II in cardiomyocyte. WISP-1 was overexpressed in NRVM and, the effect of Tß4 in Ang II-induced cardiomyocyte growth was evaluated. WISP-1 overexpression promoted cardiomyocytes growth and was reversed by pretreatment with Tß4. This is the first report which demonstrates that Tß4 targets Wnt/WISP-1 to protect Ang II-induced cardiomyocyte growth. J. Cell. Physiol. 231: 1737-1744, 2016. © 2015 Wiley Periodicals, Inc.

Cardiac-specific suppression of NF-κB signaling prevents diabetic cardiomyopathy via inhibition of the renin-angiotensin system.

Activation of NF-κB signaling in the heart may be protective or deleterious depending on the pathological context. In diabetes, the role of NF-κB in cardiac dysfunction has been investigated using pharmacological approaches that have a limitation of being nonspecific. Furthermore, the specific cellular pathways by which NF-κB modulates heart function in diabetes have not been identified. To address these questions, we used a transgenic mouse line expressing mutated IκB-α in the heart (3M mice), which prevented activation of canonical NF-κB signaling. Diabetes was developed by streptozotocin injections in wild-type (WT) and 3M mice. Diabetic WT mice developed systolic and diastolic cardiac dysfunction by the 12th week, as measured by echocardiography. In contrast, cardiac function was preserved in 3M mice up to 24 wk of diabetes. Diabetes induced an elevation in cardiac oxidative stress in diabetic WT mice but not 3M mice compared with nondiabetic control mice. In diabetic WT mice, an increase in the phospholamban/sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 ratio and decrease in ryanodine receptor expression were observed, whereas diabetic 3M mice showed an opposite effect on these parameters of Ca(2+) handling. Significantly, renin-angiotensin system activity was suppressed in diabetic 3M mice compared with an increase in WT animals. In conclusion, these results demonstrate that inhibition of NF-κB signaling in the heart prevents diabetes-induced cardiac dysfunction through preserved Ca(2+) handling and inhibition of the cardiac renin-angiotensin system.

Activation of retinoid receptor-mediated signaling ameliorates diabetes-induced cardiac dysfunction in Zucker diabetic rats.

Diabetic cardiomyopathy (DCM) is a significant contributor to the morbidity and mortality associated with diabetes and metabolic syndrome. Retinoids, through activation of retinoic acid receptor (RAR) and retinoid x receptor (RXR), have been linked to control glucose and lipid homeostasis, with effects on obesity and diabetes. However, the functional role of RAR and RXR in the development of DCM remains unclear. Zucker diabetic fatty (ZDF) and lean rats were treated with Am580 (RARα agonist) or LGD1069 (RXR agonist) for 16 weeks, and cardiac function and metabolic alterations were determined. Hyperglycemia, hyperlipidemia and insulin resistance were observed in ZDF rats. Diabetic cardiomyopathy was characterized in ZDF rats by increased oxidative stress, apoptosis, fibrosis, inflammation, activation of MAP kinases and NF-κB signaling and diminished Akt phosphorylation, along with decreased glucose transport and increased cardiac lipid accumulation, and ultimately diastolic dysfunction. Am580 and LGD1069 attenuated diabetes-induced cardiac dysfunction and the pathological alterations, by improving glucose tolerance and insulin resistance; facilitating Akt activation and glucose utilization, and attenuating oxidative stress and interrelated MAP kinase and NF-κB signaling pathways. Am580 inhibited body weight gain, attenuated the increased cardiac fatty acid uptake, ß-oxidation and lipid accumulation in the hearts of ZDF rats. However, LGD1069 promoted body weight gain, hyperlipidemia and cardiac lipid accumulation. In conclusion, our data suggest that activation of RAR and RXR may have therapeutic potential in the treatment of diabetic cardiomyopathy. However, further studies are necessary to clarify the role of RAR and RXR in the regulation of lipid metabolism and homeostasis.

Retinoic acid protects cardiomyocytes from high glucose-induced apoptosis through inhibition of NF-κB signaling pathway.

We have previously shown that retinoic acid (RA) has protective effects on high glucose (HG)-induced cardiomyocyte apoptosis. To further elucidate the molecular mechanisms of RA effects, we determined the interaction between nuclear factor (NF)-κB and RA signaling. HG induced a sustained phosphorylation of IKK/IκBα and transcriptional activation of NF-κB in cardiomyocytes. Activated NF-κB signaling has an important role in HG-induced cardiomyocyte apoptosis and gene expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1). All-trans RA (ATRA) and LGD1069, through activation of RAR/RXR-mediated signaling, inhibited the HG-mediated effects in cardiomyocytes. The inhibitory effect of RA on NF-κB activation was mediated through inhibition of IKK/IκBα phosphorylation. ATRA and LGD1069 treatment promoted protein phosphatase 2A (PP2A) activity, which was significantly suppressed by HG stimulation. The RA effects on IKK and IκBα were blocked by okadaic acid or silencing the expression of PP2Ac-subunit, indicating that the inhibitory effect of RA on NF-κB is regulated through activation of PP2A and subsequent dephosphorylation of IKK/IκBα. Moreover, ATRA and LGD1069 reversed the decreased PP2A activity and inhibited the activation of IKK/IκBα and gene expression of MCP-1, IL-6, and TNF-α in the hearts of Zucker diabetic fatty rats. In summary, our findings suggest that the suppressed activation of PP2A contributed to sustained activation of NF-κB in HG-stimulated cardiomyocytes; and that the protective effect of RA on hyperglycemia-induced cardiomyocyte apoptosis and inflammatory responses is partially regulated through activation of PP2A and suppression of NF-κB-mediated signaling and downstream targets.

Angiotensin type 1a receptor-deficient mice develop diabetes-induced cardiac dysfunction, which is prevented by renin-angiotensin system inhibitors.

BACKGROUND: Diabetes-induced organ damage is significantly associated with the activation of the renin-angiotensin system (RAS). Recently, several studies have demonstrated a change in the RAS from an extracellular to an intracellular system, in several cell types, in response to high ambient glucose levels. In cardiac myocytes, intracellular angiotensin (ANG) II synthesis and actions are ACE and AT1 independent, respectively. However, a role of this system in diabetes-induced organ damage is not clear. METHODS: To determine a role of the intracellular ANG II in diabetic cardiomyopathy, we induced diabetes using streptozotocin in AT1a receptor deficient (AT1a-KO) mice to exclude any effects of extracellular ANG II. Further, diabetic animals were treated with a renin inhibitor aliskiren, an ACE inhibitor benazeprilat, and an AT1 receptor blocker valsartan. RESULTS: AT1a-KO mice developed significant diastolic and systolic dysfunction following 10 wks of diabetes, as determined by echocardiography. All three drugs prevented the development of cardiac dysfunction in these animals, without affecting blood pressure or glucose levels. A significant down regulation of components of the kallikrein-kinin system (KKS) was observed in diabetic animals, which was largely prevented by benazeprilat and valsartan, while aliskiren normalized kininogen expression. CONCLUSIONS: These data indicated that the AT1a receptor, thus extracellular ANG II, are not required for the development of diabetic cardiomyopathy. The KKS might contribute to the beneficial effects of benazeprilat and valsartan in diabetic cardiomyopathy. A role of intracellular ANG II is suggested by the inhibitory effects of aliskiren, which needs confirmation in future studies.

Direct renin inhibition prevents cardiac dysfunction in a diabetic mouse model: comparison with an angiotensin receptor antagonist and angiotensin-converting enzyme inhibitor.

Hyperglycaemia up-regulates intracellular AngII (angiotensin II) production in cardiac myocytes, effects of which are blocked more effectively by renin inhibition than ARBs (angiotensin receptor blockers) or ACEis (angiotensin-converting enzyme inhibitors). In the present study, we determined whether renin inhibition is more effective at preventing diabetic cardiomyopathy than an ARB or ACEi. Diabetes was induced in adult mice for 10 weeks by STZ (streptozotocin). Diabetic mice were treated with insulin, aliskiren (a renin inhibitor), benazeprilat (an ACEi) or valsartan (an ARB) via subcutaneous mini-pumps. Significant impairment in diastolic and systolic cardiac functions was observed in diabetic mice, which was completely prevented by all three RAS (renin-angiotensin system) inhibitors. Hyperglycaemia significantly increased cardiac oxidative stress and circulating inflammatory cytokines, which were blocked by aliskiren and benazeprilat, whereas valsartan was partially effective. Diabetes increased cardiac PRR (prorenin receptor) expression and nuclear translocation of PLZF (promyelocytic zinc finger protein), which was completely prevented by aliskiren and valsartan, and partially by benazeprilat. Renin inhibition provided similar protection of cardiac function to ARBs and ACEis. Activation of PLZF by PRR represented a novel mechanism in diabetic cardiomyopathy. Differential effects of the three agents on oxidative stress, cytokines and PRR expression suggested subtle differences in their mechanisms of action.

High glucose-induced repression of RAR/RXR in cardiomyocytes is mediated through oxidative stress/JNK signaling.

The biological actions of retinoids are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We have recently reported that decreased expression of RARα and RXRα has an important role in high glucose (HG)-induced cardiomyocyte apoptosis. However, the regulatory mechanisms of HG effects on RARα and RXRα remain unclear. Using neonatal cardiomyocytes, we found that ligand-induced promoter activity of RAR and RXR was significantly suppressed by HG. HG promoted protein destabilization and serine-phosphorylation of RARα and RXRα. Proteasome inhibitor MG132 blocked the inhibitory effect of HG on RARα and RXRα. Inhibition of intracellular reactive oxidative species (ROS) abolished the HG effect. In contrast, H(2)O(2) stimulation suppressed the expression and ligand-induced promoter activity of RARα and RXRα. HG promoted phosphorylation of ERK1/2, JNK and p38 MAP kinases, which was abrogated by an ROS inhibitor. Inhibition of JNK, but not ERK and p38 activity, reversed HG effects on RARα and RXRα. Activation of JNK by over expressing MKK7 and MEKK1, resulted in significant downregulation of RARα and RXRα. Ligand-induced promoter activity of RARα and RXRα was also suppressed by overexpression of MEKK1. HG-induced cardiomyocyte apoptosis was potentiated by activation of JNK, and prevented by all-trans retinoic acid and inhibition of JNK. Silencing the expression of RARα and RXRα activated the JNK pathway. In conclusion, HG-induced oxidative stress and activation of the JNK pathway negatively regulated expression/activation of RAR and RXR. The impaired RAR/RXR signaling and oxidative stress/JNK pathway forms a vicious circle, which significantly contributes to hyperglycemia induced cardiomyocyte apoptosis.

Cardiac-specific genetic inhibition of nuclear factor-κB prevents right ventricular hypertrophy induced by monocrotaline.

Uncontrolled pulmonary arterial hypertension (PAH) results in right ventricular (RV) hypertrophy (RVH), progressive RV failure, and low cardiac output leading to increased morbidity and mortality (McLaughlin VV, Archer SL, Badesch DB, Barst RJ, Farber HW, Lindner JR, Mathier MA, McGoon MD, Park MH, Rosenson RS, Rubin LJ, Tapson VF, Varga J. J Am Coll Cardiol 53: 1573-1619, 2009). Although the exact figures of its prevalence are difficult to obtain because of the diversity of identifiable causes, it is estimated that the incidence of pulmonary hypertension is seven to nine cases per million persons in the general population and is most prevalent in the age group of 20-40, occurring more commonly in women than in men (ratio: 1.7 to 1; Rubin LJ. N Engl J Med 336: 111-117, 1997). PAH is characterized by dyspnea, chest pain, and syncope. Unfortunately, there is no cure for this disease and medical regimens are limited (Simon MA. Curr Opin Crit Care 16: 237-243, 2010). PAH leads to adverse remodeling that results in RVH, progressive right heart failure, low cardiac output, and ultimately death if left untreated (Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, Christman BW, Weir EK, Eickelberg O, Voelkel NF, Rabinovitch M. J Am Coll Cardiol 43: 13S-24S, 2004; Humbert M, Sitbon O, Simonneau G. N Engl J Med 351: 1425-1436, 2004. LaRaia AV, Waxman AB. South Med J 100: 393-399, 2007). As there are no direct tools to assess the onset and progression of PAH and RVH, the disease is often detected in later stages marked by full-blown RVH, with the outcome predominantly determined by the level of increased afterload (D'Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring RM, Groves BM, Kernis JT, et al. Ann Intern Med 115: 343-349, 1991; Sandoval J, Bauerle O, Palomar A, Gomez A, Martinez-Guerra ML, Beltran M, Guerrero ML. Validation of a prognostic equation Circulation 89: 1733-1744, 1994). Various studies have been performed to assess the genetic, biochemical, and morphological components that contribute to PAH. Despite major advances in the understanding of the pathogenesis of PAH, the molecular mechanism(s) by which PAH promotes RVH and cardiac failure still remains elusive. Of all the mechanisms involved in the pathogenesis, inflammation and oxidative stress remain the core of the etiology of PAH that leads to development of RVH (Dorfmüller P, Perros F, Balabanian K, Humbert M. Eur Respir J 22: 358-363, 2003).

Intracardiac intracellular angiotensin system in diabetes.

The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appears to differ from the circulating and the local RAS, in terms of components and the mechanism of action. These differences may alter treatment strategies that target the RAS in several pathological conditions. Recent work from our laboratory has demonstrated significant upregulation of the cardiac, intracellular RAS in diabetes, which is associated with cardiac dysfunction. Here, we have reviewed evidence supporting an intracellular RAS in different cell types, ANG II's actions in cardiac cells, and its mechanism of action, focusing on the intracellular cardiac RAS in diabetes. We have discussed the significance of an intracellular RAS in cardiac pathophysiology and implications for potential therapies.

The intracrine renin-angiotensin system.

The RAS (renin-angiotensin system) is one of the earliest and most extensively studied hormonal systems. The RAS is an atypical hormonal system in several ways. The major bioactive peptide of the system, AngII (angiotensin II), is neither synthesized in nor targets one specific organ. New research has identified additional peptides with important physiological and pathological roles. More peptides also mean newer enzymatic cascades that generate these peptides and more receptors that mediate their function. In addition, completely different roles of components that constitute the RAS have been uncovered, such as that for prorenin via the prorenin receptor. Complexity of the RAS is enhanced further by the presence of sub-systems in tissues, which act in an autocrine/paracrine manner independent of the endocrine system. The RAS seems relevant at the cellular level, wherein individual cells have a complete system, termed the intracellular RAS. Thus, from cells to tissues to the entire organism, the RAS exhibits continuity while maintaining independent control at different levels. The intracellular RAS is a relatively new concept for the RAS. The present review provides a synopsis of the literature on this system in different tissues.

Retinoic acid receptor-mediated signaling protects cardiomyocytes from hyperglycemia induced apoptosis: role of the renin-angiotensin system.

Diabetes mellitus (DM) is a primary risk factor for cardiovascular diseases and heart failure. Activation of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) has an anti-diabetic effect; but, a role in diabetic cardiomyopathy remains unclear. Using neonatal and adult cardiomyocytes, we determined the role of RAR and RXR in hyperglycemia-induced apoptosis and expression of renin-angiotensin system (RAS) components. Decreased nuclear expression of RARα and RXRα, activation of apoptotic signaling and cell apoptosis was observed in high glucose (HG) treated neonatal and adult cardiomyocytes and diabetic hearts in Zucker diabetic fatty (ZDF) rats. HG-induced apoptosis and reactive oxygen species (ROS) generation was prevented by both RAR and RXR agonists. Silencing expression of RARα and RXRα, by small interference RNA, promoted apoptosis under normal conditions and significantly enhanced HG-induced apoptosis, indicating that RARα and RXRα are required in regulating cell apoptotic signaling. Blocking angiotensin type 1 receptor (AT(1) R); but, not AT(2) R, attenuated HG-induced apoptosis and ROS generation. Moreover, HG induced gene expression of angiotensinogen, renin, AT(1) R, and angiotensin II (Ang II) synthesis were inhibited by RARα agonists and promoted by silencing RARα. Activation of RXRα, downregulated the expression of AT(1) R; and RXRα silencing accelerated HG induced expression of angiotensinogen and Ang II synthesis, whereas there was no significant effect on renin gene expression. These results indicate that reduction in the expression of RARα and RXRα has an important role in hyperglycemia mediated apoptosis and expression of RAS components. Activation of RAR/RXR signaling protects cardiomyocytes from hyperglycemia, by reducing oxidative stress and inhibition of the RAS.

The intracellular renin-angiotensin system in the heart.

Recently, several novel aspects of the renin-angiotensin system (RAS) were described, which potentially may change the therapeutic strategy to treat cardiovascular disease, in addition to enhancing understanding of this system's mechanism of action. Most notably, identification of a functional intracellular RAS may address several unanswered questions regarding a direct role of angiotensin (Ang) II in cardiac remodeling and incomplete efficacy of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers or superiority of a renin inhibitor in cardiovascular disorders. We describe the physiology of the intracellular RAS, potential pathologic roles of intracellular Ang II, and the relevance of the intracellular system in view of recent clinical trials involving various RAS inhibitors.

All-trans retinoic acid prevents angiotensin II- and mechanical stretch-induced reactive oxygen species generation and cardiomyocyte apoptosis.

Cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodeling to heart failure. All-trans retinoic acid (RA), a bioactive vitamin A derivative, prevents stretch- and angiotensin II (Ang II)-induced cardiac hypertrophy. However, the anti-apoptotic potential of RA in the heart remains unexplored. Here, we demonstrate that stretch- and Ang II-induced apoptosis is prevented by RA in neonatal cardiomyocytes. RA improved mitochondrial function by inhibiting the stretch- and Ang II-induced reduction in mitochondrial membrane potential, cytochrome c release and by increasing the Bcl2/Bax ratio. RA inhibited stretch- and Ang II-induced intracellular reactive oxygen species (ROS) generation and upregulated the SOD2 level. Hydrogen peroxide-induced increases in the number of TUNEL-positive cells and percentage of Annexin V positive cells, were dose-dependently inhibited by RA. The thiol antioxidant, N-acetyl cysteine (NAC), completely inhibited stretch- and Ang II-induced apoptosis. Using diazoxide (mitochondrial ATP-sensitive K(+) channel opener) and SDS (NADPH oxidase activator), we confirmed that RA suppressed both mitochondrial- and NADPH oxidase-derived ROS. We also observed that both RAR and RXR were involved in preventing Ang II- and stretch-induced ROS production and apoptosis, by using selective retinoid receptor agonists and antagonists. Our data provide the first evidence that RA prevents Ang II and stretch induced apoptosis, by inhibiting ROS generation and increasing the anti-oxidant defense system, suggesting that RA-mediated signaling may provide a new therapeutic target for the prevention of the cardiac remodeling process.

The intracellular renin-angiotensin system: a new paradigm.

More than a century after its discovery, the physiological implications of the renin-angiotensin system (RAS) continue to expand, with the identification of new components, functions and subsystems. These advancements have led to better management and understanding of a broad range of cardiovascular and metabolic disorders. The RAS has traditionally been viewed as a circulatory system, involved in the short-term regulation of volume and blood pressure homeostasis. Recently, local RASs have been described as regulators of chronic tissue effects. Most recently, studies have provided evidence of a complete, functional RAS within cells, described as an 'intracrine' or intracellular system. A more comprehensive understanding of the intracellular RAS provides for new strategies in system regulation and a more efficacious approach to the management of RAS-related diseases.

Gastrin-mediated activation of cyclin D1 transcription involves beta-catenin and CREB pathways in gastric cancer cells.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G(1)-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho beta-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of beta-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of beta-catenin and CREB pathways.

Activation of protein kinase A by atrial natriuretic peptide in neonatal rat cardiac fibroblasts: role in regulation of the local renin-angiotensin system.

There is an inverse relationship between renin and atrial natriuretic peptide (ANP) levels in the plasma. Since both the ANP and renin-angiotensin system (RAS) are upregulated in development and cardiac hypertrophy, we tested whether ANP differentially regulates RAS in cardiac cells. Cardiac fibroblasts isolated from neonatal rats were treated with ANP(1-28), a biologically active fragment of ANP. Renin and angiotensinogen (Ao) mRNA levels were measured by quantitative multiplex RT-PCR and protein levels determined by Western blot analysis. ANP(1-28) increased renin and Ao mRNA levels (737+/-131% and 178+/-51.3%) with EC50 values of 4.12+/-0.3 and 8.67+/-0.22 nmol/L, respectively. At the protein level, secretion of renin and Ao was significantly enhanced resulting in approximately 4-fold increase in ANG II level in the medium. The effect of ANP(1-28) on renin and Ao mRNA expression were reproduced by 8-bromo-cyclic GMP. Inhibition of protein kinase G (PKG) with KT5823 blunted ANP(1-28)-induced upregulation of renin, but not Ao mRNA, while inhibition of protein kinase A (PKA) with KT5720 attenuated the upregulation of both renin and Ao mRNA. These findings suggest that unlike in plasma, ANP positively regulates the RAS in cardiac fibroblasts, which may have a significant role in development of the fetal heart.

Activation of Foxo1 by insulin resistance promotes cardiac dysfunction and ß-myosin heavy chain gene expression.

BACKGROUND: Heart failure is a leading cause of morbidity and mortality in the USA and is closely associated with diabetes mellitus. The molecular link between diabetes mellitus and heart failure is incompletely understood. We recently demonstrated that insulin receptor substrates 1, 2 (IRS1, 2) are key components of insulin signaling and loss of IRS1 and IRS2 mediates insulin resistance, resulting in metabolic dysregulation and heart failure, which is associated with downstream Akt inactivation and in turn activation of the forkhead transcription factor Foxo1. METHODS AND RESULTS: To determine the role of Foxo1 in control of heart failure in insulin resistance and diabetes mellitus, we generated mice lacking Foxo1 gene specifically in the heart. Mice lacking both IRS1 and IRS2 in adult hearts exhibited severe heart failure and a remarkable increase in the ß-isoform of myosin heavy chain (ß-MHC) gene expression, whereas deletion of cardiac Foxo1 gene largely prevented the heart failure and resulted in a decrease in ß-MHC expression. The effect of Foxo1 deficiency on rescuing cardiac dysfunction was also observed in db/db mice and high-fat diet mice. Using cultures of primary ventricular cardiomyocytes, we found that Foxo1 interacts with the promoter region of ß-MHC and stimulates gene expression, mediating an effect of insulin that suppresses ß-MHC expression. CONCLUSIONS: Our study suggests that Foxo1 has important roles in promoting diabetic cardiomyopathy and controls ß-MHC expression in the development of cardiac dysfunction. Targeting Foxo1 and its regulation will provide novel strategies in preventing metabolic and myocardial dysfunction and influencing MHC plasticity in diabetes mellitus.

Phosphorylation of cardiac Myosin-binding protein-C is a critical mediator of diastolic function.

BACKGROUND: Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for ≈50% of all cases of HF and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status. Thus, we hypothesize that phosphorylated cMyBP-C accelerates the rate of cross-bridge detachment, thereby enhancing relaxation to mediate diastolic function. METHODS AND RESULTS: We compared mouse models expressing phosphorylation-deficient cMyBP-C(S273A/S282A/S302A)-cMyBP-C(t3SA), phosphomimetic cMyBP-C(S273D/S282D/S302D)-cMyBP-C(t3SD), and wild-type-control cMyBP-C(tWT) to elucidate the functional effects of cMyBP-C phosphorylation. Decreased voluntary running distances, increased lung/body weight ratios, and increased brain natriuretic peptide levels in cMyBP-C(t3SA) mice demonstrate that phosphorylation deficiency is associated with signs of HF. Echocardiography (ejection fraction and myocardial relaxation velocity) and pressure/volume measurements (-dP/dtmin, pressure decay time constant τ-Glantz, and passive filling stiffness) show that cMyBP-C phosphorylation enhances myocardial relaxation in cMyBP-C(t3SD) mice, whereas deficient cMyBP-C phosphorylation causes diastolic dysfunction with HFpEF in cMyBP-C(t3SA) mice. Simultaneous force and [Ca(2+)]i measurements on intact papillary muscles show that enhancement of relaxation in cMyBP-C(t3SD) mice and impairment of relaxation in cMyBP-C(t3SA) mice are not because of altered [Ca(2+)]i handling, implicating that altered cross-bridge detachment rates mediate these changes in relaxation rates. CONCLUSIONS: cMyBP-C phosphorylation enhances relaxation, whereas deficient phosphorylation causes diastolic dysfunction and phenotypes resembling HFpEF. Thus, cMyBP-C is a potential target for treatment of HFpEF.

Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy.

Angiotensin II (Ang II) has a significant role in regulating cardiac homeostasis through humoral, autocrine and paracrine pathways, via binding to the plasma membrane AT1 receptor. Recent literature has provided evidence for intracrine growth effects of Ang II in some cell lines, which does not involve interaction with the plasma membrane receptor. We hypothesized that such intracrine mechanisms are operative in the heart and likely participate in the cardiac hypertrophy induced by Ang II. Adenoviral and plasmid vectors were constructed to express Ang II peptide intracellularly. Neonatal rat ventricular myocytes (NRVMs) infected with the adenoviral vector showed significant hypertrophic growth as determined by cell size, protein synthesis and enhanced cytoskeletal arrangement. Adult mice injected with the plasmid vector developed significant cardiac hypertrophy after 48 h, without an increase in blood pressure or plasma Ang II levels. This was accompanied by increased transcription of transforming growth factor-beta (TGF-beta) and insulin-like growth factor-1 (IGF-1) genes. Losartan did not block the growth effects, excluding the involvement of extracellular Ang II and the plasma membrane AT1 receptor. These data demonstrate a previously unknown growth mechanism of Ang II in the heart, which should be considered when designing therapeutic strategies to block Ang II actions.