Burns C. Blaxall

Cardiac Fibrosis: The Fibroblast Awakens

Myocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Cardiac fibroblasts comprise an essential cell type in the heart that is responsible for the homeostasis of the extracellular matrix; however, upon injury, these cells transform to a myofibroblast phenotype and contribute to cardiac fibrosis. This remodeling involves pathological changes that include chamber dilation, cardiomyocyte hypertrophy and apoptosis, and ultimately leads to the progression to heart failure. Despite the critical importance of fibrosis in cardiovascular disease, our limited understanding of the cardiac fibroblast impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression. This review summarizes current knowledge regarding the origins and roles of fibroblasts, mediators and signaling pathways known to influence fibroblast function after myocardial injury, as well as novel therapeutic strategies under investigation to attenuate cardiac fibrosis.

Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II.

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)ϵ that plays a critical role in maximal β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCϵ phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCϵ-stimulated Rap1 GEF activity are both required for PLCϵ-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCϵ significantly enhances Rap activation in response to βAR stimulation in the heart. Downstream of PLCϵ hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both βAR- and Epac-stimulated increases in CICR in PLCϵ+/+ myocytes but had no effect in PLCϵ–/– myocytes. βAR and Epac activation caused membrane translocation of PKCϵ in PLCϵ+/+ but not PLCϵ–/– myocytes and small interfering RNA-mediated PKCϵ knockdown significantly inhibited both βAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited βAR- and Epac-mediated CICR in PLCϵ+/+ but not PLCϵ–/– myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and βAR-stimulated peak L-type current density are similar in PLCϵ+/+ and PLCϵ–/– myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCϵ, PKCϵ, and CamKII downstream of βAR activation.

Functional Role of Phosphodiesterase 3 in Cardiomyocyte Apoptosis Implication in Heart Failure

Background—Myocyte apoptosis plays an important role in pathological cardiac remodeling and the progression of heart failure. cAMP signaling is crucial in the regulation of myocyte apoptosis and cardiac remodeling. Multiple cAMP-hydrolyzing phosphodiesterases (PDEs), such as PDE3 and PDE4, coexist in cardiomyocytes and elicit differential temporal/spatial regulation of cAMP signaling. However, the role of PDE3 and PDE4 in the regulation of cardiomyocyte apoptosis remains unclear. Although chronic treatment with PDE3 inhibitors increases mortality in patients with heart failure, the contribution of PDE3 expression/activity in heart failure is not well known. Methods and Results—In this study we report that PDE3A expression and activity were significantly reduced in human failing hearts as well as mouse hearts with chronic pressure overload. In primary cultured cardiomyocytes, chronic inhibition of PDE3 but not PDE4 activity by pharmacological agents or adenovirus-delivered antisense PDE3A promoted cardiomyocyte apoptosis. Both angiotensin II (Ang II) and the β-adrenergic receptor agonist isoproterenol selectively induced a sustained downregulation of PDE3A expression and induced cardiomyocyte apoptosis. Restoring PDE3A via adenovirus-delivered expression of wild-type PDE3A1 completely blocked Ang II– and isoproterenol-induced cardiomyocyte apoptosis, suggesting the critical role of PDE3A reduction in cardiomyocyte apoptosis. Moreover, we defined a crucial role for inducible cAMP early repressor expression in PDE3A reduction–mediated cardiomyocyte apoptosis. Conclusions—Our results suggest that PDE3A reduction and consequent inducible cAMP early repressor induction are critical events in Ang II– and isoproterenol-induced cardiomyocyte apoptosis and may contribute to the development of heart failure. Drugs that maintain PDE3A function may represent an attractive therapeutic approach to treat heart failure.

Epac-mediated activation of phospholipase C∈ plays a critical role in β-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes

Recently we demonstrated that PLCϵ plays an important role in β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of βAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLCϵ regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2′-O-methyladenosine-3′,5′-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca2+ transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLCϵ-/- cardiac myocytes but rescued by transduction with PLCϵ, indicating that Epac is upstream of PLCϵ. Furthermore, transduction of PLCϵ+/+ cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that βAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLCϵ pathway. We also show that Epac/PLCϵ-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca2+ clearance mechanisms. These data define a novel endogenous PKA-independent βAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLCϵ that enhances intracellular Ca2+ release in cardiac myocytes.

Role of Ca2+/Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase 1 in Mediating Cardiomyocyte Hypertrophy

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca2+/calmodulin (CaM)–activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca2+/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca2+/CaM–stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca2+/CaM–stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG–dependent mechanism, thereby demonstrating Ca2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Phospholipase C ε Modulates β-Adrenergic Receptor Dependent Cardiac Contraction and Inhibits Cardiac Hypertrophy

Phospholipase C (PLC) &egr; is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, G&agr;12/13, and G&bgr;&ggr; with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLC&egr; mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLC&egr; protein expression or PLC&egr; RNA. To identify a role for PLC&egr; in cardiac function and pathology, a PLC&egr;-deficient mouse strain was created. Echocardiography indicated PLC&egr;−/− mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLC&egr;−/− mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLC&egr;−/− mice had decreased &bgr;-adrenergic receptor (&bgr;AR)-dependent increases in Ca2+ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the &bgr;AR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLC&egr;−/− mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLC&egr;−/− mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of &bgr;AR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLC&egr; signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.

Differential Expression and Localization of the mRNA Binding Proteins, AU‐Rich Element mRNA Binding Protein (AUF1) and Hu Antigen R (HuR), in Neoplastic Lung Tissue

Modulation of gene expression at the level of mRNA stability has emerged as an important regulatory paradigm. In this context, differential expression of numerous mRNAs in normal versus neoplastic tissues has been described. Altered expression of these genes, at least in part, has been demonstrated to be at the level of mRNA stability. Two ubiquitously expressed mRNA binding proteins have recently been implicated in the stabilization (Hu antigen R/HuR) or destabilization (AU‐rich element mRNA binding protein [AUF1]/heterogeneous nuclear ribonucleoprotein D) of target mRNAs. Further, their functional activity appears to require cytoplasmic localization. In the present study, we demonstrate a strong correlation between increased cytoplasmic expression of both AUF1 and HuR with urethane‐induced neoplasia and with butylated hydroxytoluene–induced compensatory hyperplasia in mouse lung tissue. In addition, when compared with slower growing cells, rapidly growing neoplastic lung epithelial cell lines expressed a consistently higher abundance of both AUF1 and HuR proteins. Moreover, in nontumorigenic cell lines, both AUF1 and HuR protein abundance decreased with confluence and growth arrest. In contrast, in spontaneous transformants, AUF1 and HuR abundance was unaffected by changes in cell density. We suggest that growth‐regulated alterations in AUF1 and HuR abundance may have pleiotropic effects on the expression of a number of highly regulated mRNAs and that this significantly impacts the onset, maintenance, and progression of the neoplastic phenotype. Mol. Carcinog. 28:76–83, 2000.

Nuclear RNA Foci in the Heart in Myotonic Dystrophy

The disease mechanism underlying myotonic dystrophy type 1 (DM1) pathogenesis in skeletal muscle may involve sequestration of RNA binding proteins in nuclear foci of expanded poly(CUG) RNA. Here we report evidence for a parallel mechanism in the heart. Accumulation of expanded poly(CUG) RNA in nuclear foci is associated with sequestration of muscleblind proteins and abnormal regulation of alternative splicing in DM1 cardiac muscle. A toxic effect of RNA with an expanded repeat may contribute to cardiac disease in DM1.

Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy

Background— Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. Methods and Results— We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1−/− mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. Conclusions— Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Serotonin and Angiotensin Receptors in Cardiac Fibroblasts Coregulate Adrenergic-Dependent Cardiac Hypertrophy

By mimicking sympathetic stimulation in vivo, we previously reported that mice globally lacking serotonin 5-HT2B receptors did not develop isoproterenol-induced left ventricular hypertrophy. However, the exact cardiac cell type(s) expressing 5-HT2B receptors (cardiomyocytes versus noncardiomyocytes) involved in pathological heart hypertrophy was never addressed in vivo. We report here that mice expressing the 5-HT2B receptor solely in cardiomyocytes, like global 5-HT2B receptor–null mice, are resistant to isoproterenol-induced cardiac hypertrophy and dysfunction, as well as to isoproterenol-induced increases in cytokine plasma-levels. These data reveal a key role of noncardiomyocytes in isoproterenol-induced hypertrophy in vivo. Interestingly, we show that primary cultures of angiotensinogen null adult cardiac fibroblasts are releasing cytokines on stimulation with either angiotensin II or serotonin, but not in response to isoproterenol stimulation, demonstrating a critical role of angiotensinogen in adrenergic-dependent cytokine production. We then show a functional interdependence between AT1Rs and 5-HT2B receptors in fibroblasts by revealing a transinhibition mechanism that may involve heterodimeric receptor complexes. Both serotonin- and angiotensin II–dependent cytokine production occur via a Src/heparin-binding epidermal growth factor–dependent transactivation of epidermal growth factor receptors in cardiac fibroblasts, supporting a common signaling pathway. Finally, we demonstrate that 5-HT2B receptors are overexpressed in hearts from patients with congestive heart failure, this overexpression being positively correlated with cytokine and norepinephrine plasma levels. Collectively, these results reveal for the first time that interactions between AT1 and 5-HT2B receptors coexpressed by noncardiomyocytes are limiting key events in adrenergic agonist-induced, angiotensin-dependent cardiac hypertrophy. Accordingly, antagonists of 5-HT2B receptors might represent novel therapeutics for sympathetic overstimulation-dependent heart failure.