Arresting developments in the cardiac myocyte cell cycle: Role of cyclin-dependent kinase inhibitors

Like most other cells in the body, foetal and neonatal cardiac myocytes are able to divide and proliferate. However, the ability of these cells to undergo cell division decreases progressively during development such that adult myocytes are unable to divide. A major problem arising from this inability of adult cardiac myocytes to proliferate is that the mature heart is unable to regenerate new myocardial tissue following severe injury, e.g. infarction, which can lead to compromised cardiac pump function and even death. Studies in proliferating cells have identified a group of genes and proteins that controls cell division. These proteins include cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs), which interact with each other to form complexes that are essential for controlling normal cell cycle progression. A variety of other proteins, e.g. the retinoblastoma protein (pRb) and members of the E2F family of transcription factors, also can interact with, and modulate the activities of, these complexes. Despite the major role that these proteins play in other cell types, little was known until recently about their existence and activities in immature (proliferating) or mature (non-proliferating) cardiac myocytes. The reason(s) why cardiac myocytes lose their ability to divide during development remains unknown, but if strategies were developed to understand the mechanisms underlying cardiac myocyte growth, it could open up new avenues for the treatment of cardiovascular disease. In this article, we shall review the function of the cell cycle machinery and outline some of our recent findings pertaining to the involvement of the cell cycle in modulating cardiac myocyte growth and hypertrophy.

Lipoma preferred partner is a mechanosensitive protein regulated by nitric oxide in the heart

Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats (p < 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes (p < 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions (p < 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load.

Increasing doxorubicin activity against breast cancer cells using PPARγ-ligands and by exploiting circadian rhythms

Doxorubicin is effective against breast cancer, but its major side effect is cardiotoxicity. The aim of this study was to determine whether the efficacy of doxorubicin on cancer cells could be increased in combination with PPARγ agonists or chrono-optimization by exploiting the diurnal cycle. We determined cell toxicity using MCF-7 cancer cells, neonatal rat cardiac myocytes and fibroblasts in this study. Doxorubicin damages the contractile filaments of cardiac myocytes and affects cardiac fibroblasts by significantly inhibiting collagen production and proliferation at the level of the cell cycle. Cyclin D1 protein levels decreased significantly following doxorubicin treatment indicative of a G1 /S arrest. PPARγ agonists with doxorubicin increased the toxicity to MCF-7 cancer cells without affecting cardiac cells. Rosiglitazone and ciglitazone both enhanced anti-cancer activity when combined with doxorubicin (e.g. 50% cell death for doxorubicin at 0.1 μM compared to 80% cell death when combined with rosiglitazone). Thus, the therapeutic dose of doxorubicin could be reduced by 20-fold through combination with the PPARγ agonists, thereby reducing adverse effects on the heart. The presence of melatonin also significantly increased doxorubicin toxicity, in cardiac fibroblasts (1 μM melatonin) but not in MCF-7 cells. Our data show, for the first time, that circadian rhythms play an important role in doxorubicin toxicity in the myocardium; doxorubicin should be administered mid-morning, when circulating levels of melatonin are low, and in combination with rosiglitazone to increase therapeutic efficacy in cancer cells while reducing the toxic effects on the heart.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signalling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors which convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain, 1Hz for 48 hours to mimic in vivo mechanical stress. Cells were also treated with and without L-NAME, a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres which was rescued by LNAME (P<0.05, n≥5 cultures). We hypothesized that the mechanism was through NOinduced alteration of myocyte gene expression. L-NAME up-regulated the mechanosensing proteins Muscle LIM protein (MLP (by 100%, p<0.05, n=4 cultures)) and lipoma preferred partner, a novel cardiac protein (LPP (by 80%, p<0.05, n=4 cultures)). L-NAME also significantly altered the subcellular localisation of LPP and MLP in a manner that favoured growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between iNOS and nNOS isoforms regulate gene expression. LPP knockdown by siRNA led to formation of α-actinin aggregates and Z-bodies showing that myofibrillogenesis was impaired. There was an up-regulation of E3 ubiquitin ligase (MUL1) by 75% (P<0.05, n=5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure. Keywords: Mechanotransduction, heart failure, stretch, heart, hypertrophy

Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.

Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) detected >400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly upregulated or downregulated (>2-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified >180 cardiomyocyte protein kinases, with high relative expression of mitogen-activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly-investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were upregulated in AVMs vs NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were upregulated (e.g. NRK, JAK2, STK38L) or downregulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts. Conclusions. This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets.

Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.

The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.

Activation of the mitogen-activated protein kinase cascade by pertussis toxin-sensitive and -insensitive pathways in cultured ventricular cardiomyocytes.

The involvement of pertussis toxin (PTX)-sensitive and -insensitive pathways in the activation of the mitogen-activated protein kinase (MAPK) cascade was examined in ventricular cardiomyocytes cultured from neonatal rats. A number of agonists that activate heterotrimeric G-protein-coupled receptors stimulated MAPK activity after exposure for 5 min. These included foetal calf serum (FCS), endothelin-1 (these two being the most effective of the agonists examined), phenylephrine, endothelin-3, lysophosphatidic acid, carbachol, isoprenaline and angiotensin II. Activation of MAPK and MAPK kinase (MEK) by carbachol returned to control levels within 30-60 min, whereas activation by FCS was more sustained. FPLC on Mono Q showed that carbachol and FCS activated two peaks of MEK and two peaks of MAPK (p42MAPK and p44MAPK). Pretreatment of cells with PTX for 24 h inhibited the activation of MAPK by carbachol, FCS and lysophosphatidic acid, but not that by endothelin-1, phenylephrine or isoprenaline. Involvement of G-proteins in the activation of the cardiac MAPK cascade was demonstrated by the sustained (PTX-insensitive) activation of MAPK (and MEK) after exposure of cells to AlF4-. AlF4- activated PtdIns hydrolysis, as did endothelin-1, endothelin-3, phenylephrine and FCS. In contrast, the effect of lysophosphatidic acid on PtdIns hydrolysis was small and carbachol was without significant effect even after prolonged exposure. We conclude that PTX-sensitive (i.e. Gi/G(o)-linked) and PTX-insensitive (i.e. Gq/Gs-linked) pathways of MAPK activation exist in neonatal ventricular myocytes. FCS may stimulate the MAPK cascade through both pathways.

Expression of protein kinase C isoforms during cardiac ventricular development.

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.

Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart.

We investigated the ability of phenylephrine (PE), an alpha-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 microM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4-5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 microM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2-3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 microM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).

Regulation of mitogen-activated protein kinase cascades in the heart.

Using primary cultures of neonatal rat ventricular myocytes and isolated adult rat hearts as models, we have characterized extensively the regulation of MAPKs in the heart. The ERKs are activated primarily by GPCR agonists acting through PKC. These agonists can also activate the JNKs although the mechanism is unclear. Cellular stresses stimulate strong activation of the JNKs, but also cause some stimulation of ERKs. Activation of p38-MAPK has so far only been demonstrated in intact adult hearts subjected to stresses and probably leads to activation of MAPKAPK2. Both cellular stresses and GPCR agonists induce phosphorylation of c-Jun, but only the latter causes upregulation of c-Jun protein.

Cellular mechanisms of cardiac hypertrophy.

Hypertrophy of myocytes in the heart ventricles is an important adaptation that in vivo occurs in response to a requirement for increased contractile power. It involves changes at the level of gene transcription, stimulation of the rate of protein synthesis (translation), and increased assembly of myofibrils. There is mounting evidence of the involvement of reversible protein phosphorylation and dephosphorylation in most of these processes. Protein kinase C, mitogen-activated protein kinases, and transcription factors have been implicated in the modulation of the transcriptional changes. Activation of translation may also be mediated through protein phosphorylation/dephosphorylation, although this has not been clearly established in the heart. Here we provide a critical overview of the signalling pathways involved in the hypertrophic response and provide a scheme to account for many of its features.

PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits.

A conscious rabbit model was used to study the effect of ischemic preconditioning (PC) on stress-activated kinases [c-Jun NH(2)-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK)] in an environment free of surgical trauma and attending external stress. Ischemic PC (6 cycles of 4-min ischemia/4-min reperfusion) induced significant activation of protein kinase C (PKC)-epsilon in the particulate fraction, which was associated with activation of p46 JNK in the nuclear fraction and p54 JNK in the cytosolic fraction; all of these changes were completely abolised by the PKC inhibitor chelerythrine. Selective enhancement of PKC-epsilon activity in adult rabbit cardiac myocytes resulted in enhanced activity of p46/p54 JNKs, providing direct in vitro evidence that PKC-epsilon is coupled to both kinases. Studies in rabbits showed that the activation of p46 JNK occurred during ischemia, whereas that of p54 JNK occurred after reperfusion. A single 4-min period of ischemia induced a robust activation of the p38 MAPK cascade, which, however, was attenuated after 5 min of reperfusion and disappeared after six cycles of 4-min ischemia/reperfusion. Overexpression of PKC-epsilon in cardiac myocytes failed to increase the p38 MAPK activity. These results demonstrate that ischemic PC activates p46 and p54 JNKs via a PKC-epsilon-dependent signaling pathway and that there are important differences between p46 and p54 JNKs with respect to the subcellular compartment (cytosolic vs. nuclear) and the mechanism (ischemia vs. reperfusion) of their activation after ischemic PC.

Small guanine nucleotide-binding proteins and myocardial hypertrophy.

The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.