New Perspectives in cAMP-Signaling Modulation

Cyclic adenosine 3′,5′-monophosphate (cAMP) mediates the biological effects of various hormones and neurotransmitters. Stimulation of cardiac β-adrenergic receptors (β-AR) via catecholamines leads to activation of adenylyl cyclases and increases cAMP production to enhance myocardial function. Because many other receptors signaling through cAMP generation exist in cardiac myocytes, a central question is how different hormones induce distinct cellular responses through the same second messenger. A large body of evidence suggests that the localization and compartmentalization of β-AR/cAMP signaling affects the net outcome of biological functions. Spatiotemporal dynamics of cAMP action is achieved by various proteins, including protein kinase A (PKA), phosphodiesterases, and scaffolding proteins such as A-kinase–anchoring proteins. In addition, the discovery of the cAMP target Epac (exchange proteins directly activated by cAMP), which functions in a PKA-independent manner, represents a novel mechanism for governing cAMP-signaling specificity. Aberrant cAMP signaling through dysregulation of β-AR/cAMP compartmentalization may contribute to cardiac remodeling and heart failure.     

Electrical Remodeling in Dyssynchrony and Resynchronization

Heart failure (HF) is associated with anatomic and functional remodeling of cardiac tissues in both animal models and humans, which alters Ca²⁺ homeostasis, protein phosphorylation, excitation–contraction coupling, results in arrhythmias. Indeed, the electrophysiological hallmark of cells and tissues isolated from failing hearts is prolongation of action potential duration (APD) and conduction slowing. The changes in cellular and tissue function are regionally heterogenous particularly in the dyssynchronously contracting heart. Cardiac resynchronization therapy (CRT) is widely applied in patients with HF and dyssynchronous left ventricular (LV) contraction (DHF), but the electrophysiological consequences of CRT are not fully understood. We demonstrated the molecular and cellular basis of excitability, conduction, and electrical remodeling in DHF and its restoration by CRT using a canine tachypacing HF model. CRT partially reversed the DHF-induced downregulation of K⁺ current and improved Na⁺ channel gating and abbreviated persistent (late) Na⁺ current. CRT reduced Ca²⁺/calmodulin protein kinase II activity and restored transverse tubular system and spatial distribution of ryanodine receptor, thus it significantly improved Ca²⁺ homeostasis especially in myocytes from late-activated, lateral wall and restored the DHF-induced blunted β-adrenergic receptor responsiveness. CRT abbreviated DHF-induced prolongation of APD in the lateral wall myocytes and reduced the LV regional gradient of APD and suppressed the development of early afterdepolarizations. In conclusion, CRT partially restores the DHF-induced ion channel remodeling, abnormal Ca²⁺ homeostasis, blunted β-adrenergic response, and regional heterogeneity of APD, thus it may suppress ventricular arrhythmias and contribute to the mortality benefit of CRT as well as improve mechanical performance of the heart.     

Modulation of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> cycling in systolic and diastolic heart failure associated with aging

Hypertension, atherosclerosis, and resultant chronic heart failure (HF) reach epidemic proportions among older persons, and the clinical manifestations and the prognoses of these worsen with increasing age. Thus, age per se is the major risk factor for cardiovascular disease. Changes in cardiac cell phenotype that occur with normal aging, as well as in HF associated with aging, include deficits in ß-adrenergic receptor (ß-AR) signaling, increased generation of reactive oxygen species (ROS), and altered excitation–contraction (EC) coupling that involves prolongation of the action potential (AP), intracellular Ca²⁺ (Ca _i ²⁺ ) transient and contraction, and blunted force- and relaxation-frequency responses. Evidence suggests that altered sarcoplasmic reticulum (SR) Ca²⁺ uptake, storage, and release play central role in these changes, which also involve sarcolemmal L-type Ca²⁺ channel (LCC), Na⁺–Ca²⁺ exchanger (NCX), and K⁺ channels. We review the age-associated changes in the expression and function of Ca²⁺ transporting proteins, and functional consequences of these changes at the cardiac myocyte and organ levels. We also review sexual dimorphism and self-renewal of the heart in the context of cardiac aging and HF.     

Chronic akt activation accentuates aging-induced cardiac hypertrophy and myocardial contractile dysfunction: role of autophagy

Aging is often accompanied with geometric and functional changes in the heart, although the underlying mechanisms remain unclear. Recent evidence has described a potential role of Akt and autophagy in aging-associated organ deterioration. This study was to examine the impact of cardiac-specific Akt activation on aging-induced cardiac geometric and functional changes and underlying mechanisms involved. Cardiac geometry, contractile and intracellular Ca²⁺ properties were evaluated using echocardiography, edge-detection and fura-2 techniques. Level of insulin signaling and autophagy was evaluated by western blot. Our results revealed cardiac hypertrophy (enlarged chamber size, wall thickness, myocyte cross-sectional area), fibrosis, decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca²⁺ release and clearance in aged (24–26 month-old) mice compared with young (3–4 month-old) mice, the effects of which were accentuated by chronic Akt activation. Aging enhanced Akt and mTOR phosphorylation while reducing that of PTEN, AMPK and ACC with a more pronounced response in Akt transgenic mice. GSK3β phosphorylation and eNOS levels were unaffected by aging or Akt overexpression. Levels of beclin-1, Atg5 and LC3-II-to-LC3-I ratio were decreased in aged hearts, the effect of which with the exception of Atg 5 was exacerbated by Akt overactivation. Levels of p62 were significantly enhanced in aged mice with a more pronounced increase in Akt mice. Neither aging nor Akt altered β-glucuronidase activity and cathepsin B although aging reduced LAMP1 level. In addition, rapamycin reduced aging-induced cardiomyocyte contractile and intracellular Ca²⁺ dysfunction while Akt activation suppressed autophagy in young but not aged cardiomyocytes. In conclusion, our data suggest that Akt may accentuate aging-induced cardiac geometric and contractile defects through a loss of autophagic regulation.     

Kappa and delta opioid receptor signaling is augmented in the failing heart

The opioidergic system, an endogenous stress pathway, modulates cardiac function. Furthermore, opioid peptide and receptor expression is altered in a number of cardiac pathologies. However, whether the response of myocardial opioid receptor signaling is altered in heart failure progression is currently unknown. Elucidating possible alterations in and effects of opioidergic signaling in the failing myocardium is of critical importance as opioids are commonly used for pain management, including in patients at risk for cardiovascular disease. A hamster model of cardiomyopathy and heart failure (Bio14.6) was used to investigate cardiac opioidergic signaling in heart failure development. This study found an augmented negative inotropic and lusitropic response to administration of agonists selective for the kappa opioid receptor and delta opioid receptor in the failing heart that was mediated by a pertussis toxin-sensitive G-protein. The augmented decrease in cardiac function was manifested by increased inhibition of cAMP accumulation and the amplitude of the systolic Ca²⁺ transient. Furthermore, increased depression of cardiac function and of two important second messengers, cAMP and intracellular Ca²⁺, were independent of changes in cardiac opioid peptide or receptor expression. Thus, the cardiomyopathy-induced failing heart experiences increased cardiac depressant effects following opioid receptor stimulation which could exacerbate diminished cardiac function in end-stage heart failure. As cardiac function is already depressed in heart failure patients, administration of opioids could exacerbate the degree of cardiac dysfunction and worsen disease progression.     

Heart failure. Excitation-contraction coupling and novel therapeutic options

Heart failure (HF) is a disease with an increasing prevalence and results in both reduced quality of life and decreased lifespan for patients. Despite improved therapy mortality remains very high. HF is induced by events that lead to reduced function of the heart, e.g. myocardial infarction and increased chronic afterload through arterial hypertension. For compensation to occur, neurohumoral mechanisms temporarily maintain cardiac function. Over time this results in left ventricular remodelling and, by means of a vicious circle, compensated HF becomes symptomatic HF. The myocardium of patients with HF is characterised by a dysfunction in excitation-contraction coupling (ECC), which causes reduced cell contractility due to reduced Ca²⁺ transients and SR Ca²⁺ load. The Ca²⁺/calmodulin-dependent protein kinase II?? (CaMKII??) plays an important role in the onset of HF. CaMKII?? phosphorylates several functional key proteins, leads to reduced SR Ca²⁺ load and Ca²⁺-transients in HF, acts as an arrhythmogenic protein by increasing late I_Na, and contributes to diastolic dysfunction by accumulation of intracellular Ca²⁺. CaMKII?? also plays an important role in atrial fibrillation. Interestingly, with regard to increased cardiac load, CaMKII?? is activated in increased afterload but not in preload. The important role of CaMKII?? in HF implies new therapeutic options to improve HF therapy in the future.     

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.     

Nitroxyl (HNO) for Treatment of Acute Heart Failure

The loss of contractile function is a hallmark of heart failure. Although increasing intracellular Ca²⁺ is a possible strategy for improving contraction, current inotropic agents that achieve this by raising intracellular cAMP levels, such as β-agonists and phosphodiesterase inhibitors, are generally deleterious when administered as long-term therapy due to arrhythmia and myocardial damage. Nitroxyl donors have been shown to improve cardiac function in normal and failing dogs, and in isolated cardiomyocytes they increase fractional shortening and Ca²⁺ transients, independently from cAMP/PKA or cGMP/PKG signaling. Instead, nitroxyl targets cysteines in the EC-coupling machinery and myofilament proteins, reversibly modifying them to enhance Ca²⁺ handling and myofilament Ca²⁺ sensitivity. Phase I–IIa trials with CXL-1020, a novel pure HNO donor, reported declines in left and right heart filling pressures and systemic vascular resistance, and increased cardiac output and stroke volume index. These findings support the concept of nitroxyl donors as attractive agents for the treatment of acute decompensated heart failure.     

Mechanisms of subcellular remodeling in heart failure due to diabetes

Diabetic cardiomyopathy is not only associated with heart failure but there also occurs a loss of the positive inotropic effect of different agents. It is now becoming clear that cardiac dysfunction in chronic diabetes is intimately involved with Ca²⁺-handling abnormalities, metabolic defects and impaired sensitivity of myofibrils to Ca²⁺ in cardiomyocytes. On the other hand, loss of the inotropic effect in diabetic myocardium is elicited by changes in signal transduction mechanisms involving hormone receptors and depressions in phosphorylation of various membrane proteins. Ca²⁺-handling abnormalities in the diabetic heart occur mainly due to defects in sarcolemmal Na⁺–K⁺ ATPase, Na⁺–Ca²⁺ exchange, Na⁺–H⁺ exchange, Ca²⁺-channels and Ca²⁺-pump activities as well as changes in sarcoplasmic reticular Ca²⁺-uptake and Ca²⁺-release processes; these alterations may lead to the occurrence of intracellular Ca²⁺ overload. Metabolic defects due to insulin deficiency or ineffectiveness as well as hormone imbalance in diabetes are primarily associated with a shift in substrate utilization and changes in the oxidation of fatty acids in cardiomyocytes. Mitochondria initially seem to play an adaptive role in serving as a Ca²⁺ sink, but the excessive utilization of long-chain fatty acids for a prolonged period results in the generation of oxidative stress and impairment of their function in the diabetic heart. In view of the activation of sympathetic nervous system and renin-angiotensin system as well as platelet aggregation, endothelial dysfunction and generation of oxidative stress in diabetes and blockade of their effects have been shown to attenuate subcellular remodeling, metabolic derangements and signal transduction abnormalities in the diabetic heart. On the basis of these observations, it is suggested that oxidative stress and subcellular remodeling due to hormonal imbalance and metabolic defects play a critical role in the genesis of heart failure during the development of diabetic cardiomyopathy.     

Cardiac hypertrophy induced by sustained β-adrenoreceptor activation: pathophysiological aspects

Cardiac hypertrophy is promoted by adrenergic over-activation and represents an independent risk factor for cardiovascular morbidity and mortality. The basic knowledge about mechanisms by which sustained adrenergic activation promotes myocardial growth, as well as understanding how structural changes in hypertrophied myocardium could affect myocardial function has been acquired from studies using an animal model of chronic systemic β-adrenoreceptor agonist administration. Sustained β-adrenoreceptor activation was shown to enhance the synthesis of myocardial proteins, an effect mediated via stimulation of myocardial growth factors, up-regulation of nuclear proto-oncogenes, induction of cardiac oxidative stress, as well as activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase. Sustained β-adrenoreceptor activation contributes to impaired cardiac autonomic regulation as evidenced by blunted parasympathetically-mediated cardiovascular reflexes as well as abnormal storage of myocardial catecholamines. Catecholamine-induced cardiac hypertrophy is associated with reduced contractile responses to adrenergic agonists, an effect attributed to downregulation of myocardial β-adrenoreceptors, uncoupling of β-adrenoreceptors and adenylate cyclase, as well as modifications of downstream cAMP-mediated signaling. In compensated cardiac hypertrophy, these changes are associated with preserved or even enhanced basal ventricular systolic function due to increased sarcoplasmic reticulum Ca²⁺ content and Ca²⁺-induced sarcoplasmic reticulum Ca²⁺ release. The increased availability of Ca²⁺ to maintain cardiomyocyte contraction is attributed to prolongation of the action potential due to inhibition of the transient outward potassium current as well as stimulation of the reverse mode of the Na⁺–Ca²⁺ exchange. Further progression of cardiac hypertrophy towards heart failure is due to abnormalities in Ca²⁺ handling, necrotic myocardial injury, and increased myocardial stiffness due to interstitial fibrosis.     

The cAMP binding protein Epac regulates cardiac myofilament function

In the heart, cAMP is a key regulator of excitation–contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT) reduced Ca²⁺ transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca²⁺ sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca²⁺ sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca²⁺ sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca²⁺ calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.     

Cyclic nucleotide phosphodiesterase 1A: a key regulator of cardiac fibroblast activation and extracellular matrix remodeling in the heart

Cardiac fibroblasts become activated and differentiate to smooth muscle-like myofibroblasts in response to hypertension and myocardial infarction (MI), resulting in extracellular matrix (ECM) remodeling, scar formation and impaired cardiac function. cAMP and cGMP-dependent signaling have been implicated in cardiac fibroblast activation and ECM synthesis. Dysregulation of cyclic nucleotide phosphodiesterase (PDE) activity/expression is also associated with various diseases and several PDE inhibitors are currently available or in development for treating these pathological conditions. The objective of this study is to define and characterize the specific PDE isoform that is altered during cardiac fibroblast activation and functionally important for regulating myofibroblast activation and ECM synthesis. We have found that Ca²⁺/calmodulin-stimulated PDE1A isoform is specifically induced in activated cardiac myofibroblasts stimulated by Ang II and TGF-β in vitro as well as in vivo within fibrotic regions of mouse, rat, and human diseased hearts. Inhibition of PDE1A function via PDE1-selective inhibitor or PDE1A shRNA significantly reduced Ang II or TGF-β-induced myofibroblast activation, ECM synthesis, and pro-fibrotic gene expression in rat cardiac fibroblasts. Moreover, the PDE1 inhibitor attenuated isoproterenol-induced interstitial fibrosis in mice. Mechanistic studies revealed that PDE1A modulates unique pools of cAMP and cGMP, predominantly in perinuclear and nuclear regions of cardiac fibroblasts. Further, both cAMP-Epac-Rap1 and cGMP-PKG signaling was involved in PDE1A-mediated regulation of collagen synthesis. These results suggest that induction of PDE1A plays a critical role in cardiac fibroblast activation and cardiac fibrosis, and targeting PDE1A may lead to regression of the adverse cardiac remodeling associated with various cardiac diseases.     

Mitochondrial dynamics and cell death in heart failure

The highly regulated processes of mitochondrial fusion (joining), fission (division) and trafficking, collectively called mitochondrial dynamics, determine cell-type specific morphology, intracellular distribution and activity of these critical organelles. Mitochondria are critical for cardiac function, while their structural and functional abnormalities contribute to several common cardiovascular diseases, including heart failure (HF). The tightly balanced mitochondrial fusion and fission determine number, morphology and activity of these multifunctional organelles. Although the intracellular architecture of mature cardiomyocytes greatly restricts mitochondrial dynamics, this process occurs in the adult human heart. Fusion and fission modulate multiple mitochondrial functions, ranging from energy and reactive oxygen species production to Ca²⁺ homeostasis and cell death, allowing the heart to respond properly to body demands. Tightly controlled balance between fusion and fission is of utmost importance in the high energy-demanding cardiomyocytes. A shift toward fission leads to mitochondrial fragmentation, while a shift toward fusion results in the formation of enlarged mitochondria and in the fusion of damaged mitochondria with healthy organelles. Mfn1, Mfn2 and OPA1 constitute the core machinery promoting mitochondrial fusion, whereas Drp1, Fis1, Mff and MiD49/51 are the core components of fission machinery. Growing evidence suggests that fusion/fission factors in adult cardiomyocytes play essential noncanonical roles in cardiac development, Ca²⁺ signaling, mitochondrial quality control and cell death. Impairment of this complex circuit causes cardiomyocyte dysfunction and death contributing to heart injury culminating in HF. Pharmacological targeting of components of this intricate network may be a novel therapeutic modality for HF treatment.     

Update on antiarrhythmic drug pharmacology

Cardiac arrhythmias constitute a major public health problem. Pharmacological intervention remains mainstay to their clinical management. This, in turn, depends upon systematic drug classification schemes relating their molecular, cellular, and systems effects to clinical indications and therapeutic actions. This approach was first pioneered in the 1960s Vaughan‐Williams classification. Subsequent progress in cardiac electrophysiological understanding led to a lag between the fundamental science and its clinical translation, partly addressed by The working group of the European Society of Cardiology (1991), which, however, did not emerge with formal classifications. We here utilize the recent Revised Oxford Classification Scheme to review antiarrhythmic drug pharmacology. We survey drugs and therapeutic targets offered by the more recently characterized ion channels, transporters, receptors, intracellular Ca²⁺ handling, and cell signaling molecules. These are organized into their strategic roles in cardiac electrophysiological function. Following analysis of the arrhythmic process itself, we consider (a) pharmacological agents directly targeting membrane function, particularly the Na⁺ and K⁺ ion channels underlying depolarizing and repolarizing events in the cardiac action potential. (b) We also consider agents that modify autonomic activity that, in turn, affects both the membrane and (c) the Ca²⁺ homeostatic and excitation‐contraction coupling processes linking membrane excitation to contractile activation. Finally, we consider (d) drugs acting on more upstream energetic and structural remodeling processes currently the subject of clinical trials. Such systematic correlations of drug actions and arrhythmic mechanisms at different molecular to systems levels of cardiac function will facilitate current and future antiarrhythmic therapy.     

A CaMKII/PDE4D negative feedback regulates cAMP signaling

cAMP production and protein kinase A (PKA) are the most widely studied steps in β-adrenergic receptor (βAR) signaling in the heart; however, the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is also activated in response to βAR stimulation and is involved in the regulation of cardiac excitation-contraction coupling. Its activity and expression are increased during cardiac hypertrophy, in heart failure, and under conditions that promote arrhythmias both in animal models and in the human heart, underscoring the clinical relevance of CaMKII in cardiac pathophysiology. Both CaMKII and PKA phosphorylate a number of protein targets critical for Ca²⁺ handling and contraction with similar, but not always identical, functional consequences. How these two pathways communicate with each other remains incompletely understood, however. To maintain homeostasis, cyclic nucleotide levels are regulated by phosphodiesterases (PDEs), with PDE4s predominantly responsible for cAMP degradation in the rodent heart. Here we have reassessed the interaction between cAMP/PKA and Ca²⁺/CaMKII signaling. We demonstrate that CaMKII activity constrains basal and βAR-activated cAMP levels. Moreover, we show that these effects are mediated, at least in part, by CaMKII regulation of PDE4D. This regulation establishes a negative feedback loop necessary to maintain cAMP/CaMKII homeostasis, revealing a previously unidentified function for PDE4D as a critical integrator of cAMP/PKA and Ca²⁺/CaMKII signaling.During cardiac excitation-contraction coupling (ECC), Ca²⁺ elevation throughout the cell promotes myofilament sliding, which generates contractile force. This process is highly regulated by positive and negative regulatory circuits, the most critical being the sympathetic nervous system that acts via activation of the β-adrenergic (βAR)/cAMP/PKA signaling pathway. During βAR stimulation, PKA phosphorylates and activates key proteins involved in ECC and Ca²⁺ handling. These proteins include L-type Ca²⁺ channels and ryanodine receptors (RyR), leading to enhanced Ca²⁺ influx and consequent sarcoplasmic reticulum (SR) Ca²⁺ release; phospholamban (PLB), increasing SR Ca²⁺ uptake by the Ca²⁺ ATPase (SERCA), thereby accelerating cardiac relaxation; and contractile proteins, increasing cell contraction. Collectively, these events produce the typical inotropic and lusitropic effects of βAR stimulation (1).This βAR/cAMP/PKA pathway is only one of the components involved in regulating cardiac function, however. Data accumulated in the last decade have revealed that Ca²⁺/calmodulin-dependent kinase II (CaMKII) is equally important to the regulation of cardiac function under physiological and pathological conditions (2–7). Most of the functions distal to cAMP and PKA are regulated by CaMKII as well (8). Proteins critical for ECC are substrates for both PKA and CaMKII, and phosphorylation at different sites often produces similar changes in protein function. Whereas CaMKII is directly regulated by Ca²⁺, its activity is indirectly regulated by cAMP (9). In response to βAR stimulation, Epac (Exchange Protein directly Activated by cAMP) activates CaMKII (10–14). This regulation is critical in pathophysiological conditions in which CaMKII expression and activation may be elevated, such as hypertrophy, heart failure, and arrhythmias (2, 3). Despite the wealth of data available, the exact mechanisms integrating CaMKII activity with cAMP/PKA signaling remain unclear.An established concept in cell signaling is that freely diffusible cAMP is not distributed uniformly throughout the cell, but is compartmentalized to generate specificity and to allow PKA regulation in distinct subdomains (15). Phosphodiesterases (PDEs), the enzymes that degrade cAMP, have emerged as ubiquitous and important modulators of cAMP/PKA signaling in specific cellular compartments, including cardiac myocytes (16, 17). They are part of macromolecular complexes that include PKAs and A-kinase anchoring proteins (AKAPs). In contrast to the large body of data linking PDEs to PKA regulation, whether PDEs regulate CaMKII is unclear. We and others have shown that genetic ablation of PDE4s, the isoform responsible for cAMP degradation in the heart, disrupts ECC via PKA-mediated alteration in Ca²⁺ handling (18–20); however, the possibility that some of the effects are mediated by CaMKII has not been investigated.For proper homeostasis, cAMP signals are constrained by feedback mechanisms required to tightly regulate cyclic nucleotide levels under basal or stimulated conditions, with the feedback regulation of PDE4s a preeminent example. PDE4s are activated by PKA phosphorylation, providing a negative feedback mechanism by which cAMP regulates its own level (21, 22). Given the cAMP-dependent, Epac-mediated CaMKII activation, we surmise that feedback mechanisms linking cAMP and CaMKII must be operating in cardiac myocytes as well. In the present study, we reassessed the interaction between cAMP signaling and CaMKII. We demonstrate that CaMKII activity constrains basal and βAR-activated cAMP signaling. These effects are mediated, at least in part, by CaMKII regulation of PDE4D, revealing an additional function of this enzyme as a critical integrator of cAMP/PKA and Ca²⁺/CaMKII signaling.     

Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

Phospholamban (PLN) plays a central role in Ca²⁺ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 2A (SERCA2A) Ca²⁺ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.Cardiovascular diseases (CVDs) leading to systolic/diastolic heart failure (HF), such as hypertensive/diabetic heart disease, stroke, and vascular atherosclerosis, are leading causes of death in the developed world (1). Many CVDs are associated with genetic predispositions. For example, in humans, the arginine to cysteine (R9C) substitution in phospholamban (PLN) has been shown to result in dilated cardiomyopathy (DCM) presenting in adolescence, leading to rapid deterioration of heart function and premature death (2). However, the etiology and molecular mechanisms of progression of DCM and other CVDs leading to HF are complex and still poorly understood, further complicating clinical assessment and management. From a biological and clinical perspective, the identification and characterization of clinically relevant, potentially druggable, pathways driving the maladaptive response in affected heart tissue are key challenges to improved diagnostic and therapeutic tools for earlier detection and preventative treatment of both inherited and chronic CVDs.Cardiac muscle contraction is controlled by Ca²⁺ flux and signaling relays, which are perturbed in HF. Internal stores of Ca²⁺ required for the proper functioning of cardiomyocytes (CMs) are normally maintained through the function of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 2 (SERCA2) (3), which is responsible for the sequestration of Ca²⁺ resulting in muscle relaxation. SERCA2 activity is regulated through a reversible inhibitory interaction with PLN, which can be relieved by phosphorylation of PLN by protein kinase A (PKA) or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (3).Proteomic analyses have revealed changes in the abundance of other effector proteins in diverse biochemical pathways in DCM. Notably, shotgun proteomic analysis of membrane protein expression dynamics in heart microsomes isolated from mice overexpressing a superinhibitory (I40A) mutant of PLN revealed changes in G protein-coupled receptor-mediated pathways leading to activation of protein kinase C (PKC) (4). We previously reported quantitative changes in protein and cognate mRNA expression levels in cardiac ventricular tissue at different time points in the development of DCM in R9C-PLN mice representing clear clinical stages in the progression to HF (5). We showed that the latter maladaptive response was driven by elevated activity of MAPK signaling by the protein kinases p38 and JNK, in part through down-regulation of prosurvival microRNAs (6). However, the underlying upstream and downstream signaling events preceding HF were not fully explored.In the present study, we report a systematic, large-scale quantitative phosphoproteomic analysis of dysregulated protein phosphorylation-dependent signaling occurring at the early symptomatic stages of DCM progression in whole hearts from R9C mutant mice compared with wild-type littermates.     

Overexpression of junctate induces cardiac hypertrophy and arrhythmia via altered calcium handling

Junctate-1 is a newly identified integral endoplasmic/sarcoplasmic reticulum Ca²⁺ binding protein. However, its functional role in the heart is unknown. In the present study, the consequences of constitutively overexpressed junctate in cardiomyocytes were investigated using transgenic (TG) mice overexpressing junctate-1. TG mice (8 weeks old) showed cardiac remodeling such as marked bi-atrial enlargement with intra-atrial thrombus and biventricular hypertrophy. The TG mice also showed bradycardia with atrial fibrillation, reduced amplitude and elongated decay time of Ca²⁺ transients, increased L-type Ca²⁺ current and prolonged action potential durations. Time-course study (2-8 weeks) showed an initially reduced SR function due to down-regulation of SERCA2 and calsequestrin followed by sarcolemmal protein expression and cardiac hypertrophy at later age. These sequential changes could well be correlated with the physiological changes. Adrenergic agonist treatment and subsequent biochemical study showed that junctate-1 TG mice (8 weeks old) were under local PKA signaling that could cause increased L-type Ca²⁺ current and reduced SR function. Junctate-1 in the heart is closely linked to the homeostasis of E-C coupling proteins and a sustained increase of junctate-1 expression leads to a severe cardiac remodeling and arrhythmias.     

Inhibition of Na/K-ATPase promotes myocardial tumor necrosis factor-alpha protein expression and cardiac dysfunction via calcium/mTOR signaling in endotoxemia

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that causes cardiac dysfunction during sepsis. Na/K-ATPase regulates intracellular Ca²⁺, which activates mammalian target of rapamycin (mTOR), a regulator of protein synthesis. The aim of this study was to investigate the role of Na/K-ATPase/mTOR signaling in myocardial TNF-α expression during endotoxemia. Results showed that treatment with LPS decreased Na/K-ATPase activity in the myocardium in vivo and in cultured neonatal cardiomyocytes. Inhibition of Na/K-ATPase by ouabain enhanced LPS-induced myocardial TNF-α protein production, but had no effect on TNF-α mRNA expression. More importantly, ouabain further decreased in vivo cardiac function in endotoxemic mice, which was blocked by etanercept, a TNF-α antagonist. LPS-induced reduction in Na/K-ATPase activity was prevented by inhibition of PI3K, Rac1 and NADPH oxidase using LY294002, a dominant-negative Rac1 adenovirus (Ad-Rac1N17) and apocynin, respectively. To assess the role of Rac1 in Ca²⁺ handling, Ca²⁺ transients in adult cardiomyocytes from cardiomyocyte-specific Rac1 knockout (Rac1^CKO) and wild-type (WT) mice were determined. LPS increased intracellular Ca²⁺ in WT but not in Rac1^CKO cardiomyocytes. Furthermore, LPS rapidly increased mTOR phosphorylation in cardiomyocytes, which was blocked by Rac1N17 and an inhibitor of calmodulin-dependent protein kinases (CaMKs) KN93, but enhanced by ouabain. Rapamycin, an inhibitor of mTOR suppressed TNF-α protein levels without any significant effect on its mRNA expression or global protein synthesis. In conclusion, myocardial Na/K-ATPase activity is inhibited during endotoxemia via PI3K/Rac1/NADPH oxidase activation. Inhibition of Na/K-ATPase activates Ca²⁺/CaMK/mTOR signaling, which promotes myocardial TNF-α protein production and cardiac dysfunction during endotoxemia.     

Myocardial Phosphodiesterases and Regulation of Cardiac Contractility in Health and Cardiac Disease

Phosphodiesterase (PDE) inhibitors are potent cardiotonic agents used for parenteral inotropic support in heart failure. Contractile effects of these agents are mediated through cAMP-protein kinase A-induced stimulation of I _Ca2+ which ultimately results in increased Ca²⁺-induced sarcoplasmic reticulum Ca²⁺ release. A number of additional effects such as increases in sarcoplasmic reticulum Ca²⁺ stores, stimulation of reverse mode Na⁺–Ca²⁺ exchange, direct or cAMP-mediated effects on sarcoplasmic reticulum ryanodine receptor, stimulation of the voltage-sensitive sarcoplasmic reticulum Ca²⁺ release mechanism, as well as A₁ adenosine receptor blockade could contribute to positive inotropic responses to PDE inhibitors. Moreover, some PDE inhibitors exhibit Ca²⁺ sensitizer properties as they could increase the affinity of troponin C Ca²⁺-binding sites as well as reduce Ca²⁺ threshold for thin myofilament sliding and facilitate cross-bridge cycling. Inotropic responses to PDE inhibitors are significantly reduced in cardiac disease, an effect largely attributed to downregulation of cAMP-mediated signalling due to sustained sympathetic activation. Four PDE isoenzymes (PDE1, PDE2, PDE3 and PDE4) are present in myocardial tissue of various mammalian species, of which PDE3 and PDE4 are particularly involved in regulation of cardiac myocyte contraction. PDE cAMP-hydrolysing activity is preserved in compensated cardiac hypertrophy but significantly reduced in animal models of heart failure. However, clinical studies have not revealed any changes in distribution profile as well as kinetic and regulatory properties of myocardial PDEs in failing human hearts. A reduction of PDE inhibitors-induced contractile responses in heart failure has therefore been ascribed to reduced cAMP synthesis due to uncoupling of adenylyl cyclase from β-adrenoreceptor. In cardiac myocytes, PDEs are targeted to distinct subcellular compartments by scaffolding proteins such as myomegalin, mAKAP and β-arrestins. Over subcellular microdomains, cAMP hydrolysis by PDE3 and PDE4 allows to control the activity of local pools of protein kinase A and therefore the extent of protein kinase A-mediated phosphorylation of cellular proteins.