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.

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.

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.

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.

Do multiple nuclear factor kappa B activation mechanisms explain its varied effects in the heart?

BACKGROUND: Multiple studies have demonstrated the important role of the nuclear factor kappa B (NF-κB) in cardiac pathology. However, these studies' conclusions differ regarding whether NF-κB is protective or detrimental for heart function. This disagreement is not surprising considering the complexity of NF-κB signaling that involves multiple components and regulation at several steps. Furthermore, NF-κB is a pleiotropic transcription factor that receives signals from multiple pathways, including the renin-angiotensin system (RAS) and cytokines, 2 important modulators of cardiac remodeling. METHODS: In this article, we review studies related to the role and mechanisms of NF-κB activation in the heart, particularly with regard to the RAS, inflammation, and diabetes. The objective of this review is to consolidate multiple, often contradictory, findings to develop a clear understanding of NF-κB signaling in the heart. CONCLUSIONS: The studies we review demonstrate that NF-κB effects in the heart are mechanism specific and that NF-κB signaling is cyclical. Consequently, the timing of NF-κB measurement is critical, and studies focused on temporal changes in the NF-κB mechanism would help clarify its multiple roles in cardiac pathophysiology.

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.

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.

Lipid rafts, caveolae and GPI-linked proteins.

Lipid rafts and caveolae are specialized membrane microdomains enriched in sphingolipids and cholesterol. They function in a variety of cellular processes including but not limited to endocytosis, transcytosis, signal transduction and receptor recycling. Here, we outline the similarities and differences between lipid rafts and caveolae as well as discuss important components and functions of each.

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.

Smooth muscle cell alpha2delta-1 subunits are essential for vasoregulation by CaV1.2 channels.

RATIONALE: Voltage-dependent L-type (Ca(V)1.2) Ca(2+) channels are a heteromeric complex formed from pore-forming alpha(1) and auxiliary alpha(2)delta and beta subunits. Ca(V)1.2 channels are the principal Ca(2+) influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte Ca(V)1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of alpha(2)delta subunits. OBJECTIVE: We investigated the functional significance of alpha(2)delta subunits in myocytes of resistance-size (100 to 200 mum diameter) cerebral arteries. METHODS AND RESULTS: alpha(2)delta-1 was the only alpha(2)delta isoform expressed in cerebral artery myocytes. Pregabalin, an alpha(2)delta-1/-2 ligand, and an alpha(2)delta-1 antibody, inhibited Ca(V)1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of Ca(V)1.2 alpha(1) and alpha(2)delta-1 subunits were present in the arterial myocyte plasma membrane. Alpha(2)delta-1 knockdown using short hairpin RNA reduced plasma membrane-localized Ca(V)1.2 alpha(1) subunits, caused a corresponding elevation in cytosolic Ca(V)1.2 alpha(1) subunits, decreased intracellular Ca(2+) concentration, inhibited pressure-induced vasoconstriction ("myogenic tone"), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total alpha(2)delta-1 or Ca(V)1.2 alpha(1) proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone. CONCLUSIONS: alpha(2)delta-1 is essential for plasma membrane expression of arterial myocyte Ca(V)1.2 alpha(1) subunits. alpha(2)delta-1 targeting can block Ca(V)1.2 channels directly and inhibit surface expression of Ca(V)1.2 alpha(1) subunits, leading to vasodilation. These data identify alpha(2)delta-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.

Caveolae structure and function.

Studies on the structure and function of caveolae have revealed how this versatile subcellular organelle can influence numerous signalling pathways. This brief review will discuss a few of the key features of caveolae as it relates to signalling and disease processes.

Gender as a regulator of atherosclerosis in murine models.

The risk of development and progression of atherosclerosis is different between males and females. Premenopausal women have a lower risk of developing atherosclerosis and cardiovascular disease than men. However, after the onset of menopause the protection associated with gender is lost and the risk of women developing atherosclerosis gradually approaches that of men. In an effort to treat the elevated risk of cardiovascular disease in postmenopausal women, hormone replacement therapy has been used. However, the results of the randomized trials of the Women's Health Initiative indicated that hormone replacement therapy may not be cardioprotective. The use of mouse models have aided in the understanding of atherosclerosis for many years. These models along with the gender effects attributed to sex hormones are being used to generate a more complete understanding of the development of atherosclerosis. Mice lacking one or both of the genes for estrogen receptors have highlighted the role of estrogen in atherosclerosis. In addition to estrogen, the effects of testosterone have been researched in many animal models and several mechanisms incorporating its role in cholesterol homeostasis have emerged. Our understanding of the pathways involved in gender effects on cardiovascular disease is incomplete, however, a plethora of animal models offer the opportunity to dissect the molecular mechanisms involved.

How HIV protease inhibitors promote atherosclerotic lesion formation.

PURPOSE OF REVIEW: One of the aims of this review is to summarize recent clinical approaches used to determine the role of HIV protease inhibitors in the development of cardiovascular disease. Another aim is to discuss possible molecular mechanisms whereby HIV protease inhibitors may promote atherogenesis. RECENT FINDINGS: Several clinical studies have recently used ultrasonography to demonstrate increased intimal medial thickness and alterations in the structural characteristics of epi-aortic lesions in patients receiving HIV protease inhibitors. Molecular studies have indicated that several mechanisms are likely involved in mediating the effects of protease inhibitors. Possible mechanisms include inhibition of the proteasome, increased CD36 expression in macrophage, inhibition of lipoprotein lipase-mediated lipolysis, decreased adiponectin levels, and dysregulation of the NF-kappaB pathway. SUMMARY: The currently available data strongly suggest that HIV protease inhibitors negatively impact the cardiovascular system. As is often the case with complex diseases like atherosclerosis it appears that HIV protease inhibitors affect the cardiovascular system through several distinct mechanisms by affecting various components of the arterial wall directly or indirectly by influencing lipoprotein and glucose metabolism of the body.