A review of the molecular mechanisms underlying the development and progression of cardiac remodeling

Pathological molecular mechanisms involved in myocardial remodeling contribute to alter the existing structure of the heart, leading to cardiac dysfunction. Among the complex signaling network that characterizes myocardial remodeling, the distinct processes are myocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, defective autophagy, metabolic abnormalities, and mitochondrial dysfunction. Several pathophysiological stimuli, such as pressure and volume overload, trigger the remodeling cascade, a process that initially confers protection to the heart as a compensatory mechanism. Yet chronic inflammation after myocardial infarction also leads to cardiac remodeling that, when prolonged, leads to heart failure progression. Here we review the molecular pathways involved in cardiac remodeling, with particular emphasis on those associated with myocardial infarction. A better understanding of cell signaling involved in cardiac remodeling may support the development of new therapeutic strategies towards the treatment of heart failure and reduction of cardiac complications. We will also discuss data derived from gene therapy approaches for modulating key mediators of cardiac remodeling

Critical Roles of STAT3 in β-Adrenergic Functions in the Heart

BACKGROUND: β-Adrenergic receptors (βARs) play paradoxical roles in the heart. On one hand, βARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of βARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac βAR-mediated signaling and function. METHODS AND RESULTS: We observed that STAT3 can be directly activated in cardiomyocytes by β-adrenergic agonists. To follow up this finding, we analyzed βAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute βAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic β-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for βAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of βAR pathway, including β1AR, protein kinase A, and T-type Ca(2+) channels. CONCLUSIONS: Our data demonstrate for the first time that STAT3 has a fundamental role in βAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for βAR-mediated cardiac stress adaption, pathological remodeling, and heart failure

ADENYLYL CYCLASE TYPE 9: REGULATION AND CARDIAC FUNCTION

Abnormalities in cardiac stress signaling underlie a number of cardiovascular diseases (e.g. arrhythmias and heart failure). Cardiac stress signaling pathways normally integrate signals from the sympathetic nervous system to promote efficient contraction and relaxation under stress. Sympathetic control through β-adrenergic stimulation is propagated by adenylyl cyclase (AC). AC synthesizes cyclic AMP (cAMP), an important second messenger that initiates signaling pathways to modulate physiological and pathophysiological functions of the heart, including the activation of PKA and subsequent phosphorylation of ion channels, contractile machinery, and stress response proteins that enhance cardiac function. Alterations of cAMP signaling occur in the failing heart and contribute to impaired function. Of the AC isoforms present in adult cardiomyocytes (AC 4, 5, 6, and 9), AC9 is the most divergent in sequence and understudied. The work presented in this dissertation sought to evaluate the direct regulatory properties of AC9 and explores roles for AC9 in heart. To clarify conflicting reports for AC9 regulation, proposed regulators were systematically evaluated, including G-proteins, protein kinases, and forskolin utilizing in vitro and cell based assays. Overall, I conclude that most G-proteins or protein kinases do not directly regulate AC9, except Gαs, in vitro. Although AC9 is forskolin insensitive alone, weak activation by forskolin in the presence of Gαs is possible. AC9 shows significant homodimerization and modest heterodimerization with AC5/6, which may account for the conflicting reports surrounding the regulation of this AC isoform. viii To study the role of AC9 in heart, a mouse model of AC9 genetic deletion was utilized. Although deletion of AC9 reduces less than 3% of total AC activity in heart, Yotiao-associated AC activity is eliminated. AC9-/- mice exhibit no structural abnormalities but show a significant bradycardia and alterations in Doppler echocardiography indicative of grade 1 diastolic dysfunction with preserved ejection fraction. Identification of novel AC9 binding partners, including the small heat shock protein 20 (Hsp20) and Popeye domain containing (Popdc) proteins may contribute to the underlying mechanisms of AC9-/- phenotypes. Collectively, this work suggests that AC9 forms distinct macromolecular complexes that contribute to local cAMP pools important for driving physiological function of the heart

Myocardial MiR-30 downregulation triggered by doxorubicin drives alterations in β-adrenergic signaling and enhances apoptosis

The use of anthracyclines such as doxorubicin (DOX) has improved outcome in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity is frequently irreversible and typically progresses to heart failure (HF) but our understanding of molecular mechanisms underlying this and essential for development of cardioprotective strategies remains largely obscure. As microRNAs (miRNAs) have been shown to play potent regulatory roles in both cardiovascular disease and cancer, we investigated miRNA changes in DOX-induced HF and the alteration of cellular processes downstream. Myocardial miRNA profiling was performed after DOX-induced injury, either via acute application to isolated cardiomyocytes or via chronic exposure in vivo, and compared with miRNA profiles from remodeled hearts following myocardial infarction. The miR-30 family was downregulated in all three models. We describe here that miR-30 act regulating the β-adrenergic pathway, where preferential β1- and β2-adrenoceptor (β1AR and β2AR) direct inhibition is combined with Giα-2 targeting for fine-tuning. Importantly, we show that miR-30 also target the pro-apoptotic gene BNIP3L/NIX. In aggregate, we demonstrate that high miR-30 levels are protective against DOX toxicity and correlate this in turn with lower reactive oxygen species generation. In addition, we identify GATA-6 as a mediator of DOX-associated reductions in miR-30 expression. In conclusion, we describe that DOX causes acute and sustained miR-30 downregulation in cardiomyocytes via GATA-6. miR-30 overexpression protects cardiac cells from DOX-induced apoptosis, and its maintenance represents a potential cardioprotective and anti-tumorigenic strategy for anthracyclines

Diabetic mouse angiopathy is linked to progressive sympathetic receptor deletion coupled to an enhanced caveolin-1 expression.

OBJECTIVE: Clinical studies have demonstrated that hyperglycaemia represents a major risk factor in the development of the endothelial impairment in diabetes, which is the first step in vascular dysfunction. Using non-obese diabetic mice, we have evaluated the role of the adrenergic system and eNOS on progression of the disease METHODS AND RESULTS: When glycosuria is high (20 to 500 mg/dL), there is a selective reduction in the response to alpha1 and beta2 agonists but not to dopamine or serotonin. When glycosuria is severe (500 to 1000 mg/dL), there is a complete ablation of the contracture response to the alpha1 receptor agonist stimulation and a marked reduced response to beta2 agonist stimulation. This effect is coupled with a reduced expression of alpha1 and beta2 receptors, which is caused by an inhibition at transcriptional level as demonstrated by RT-PCR. In the severe glycosuria (500 to 1000 mg/dL), although eNOS expression is unchanged, caveolin-1 expression is significantly enhanced, indicating that high glucose plasma levels cause an upregulation of the eNOS endogenous inhibitory tone. These latter results correlate with functional data showing that in severe glycosuria, there is a significant reduction in acetylcholine-induced vasodilatation. CONCLUSIONS: Our results show that in diabetes development, there is a progressive selective downregulation of the alpha1 and beta2 receptors. At the same time, there is an increased expression of caveolin-1, the endogenous eNOS inhibitory protein. Thus, caveolin-1 could represent a new possible therapeutic target in vascular impairment associated with diabetes

G protein-coupled receptor kinase 2 (GRK2) as a potential therapeutic target in cardiovascular and metabolic diseases

G protein-coupled receptor kinase 2 (GRK2) is a central signaling node involved in the modulation of many G protein-coupled receptors (GPCRs) and also displaying regulatory functions in other cell signaling routes. GRK2 levels and activity have been reported to be enhanced in patients or in preclinical models of several relevant pathological situations, such as heart failure, cardiac hypertrophy, hypertension, obesity and insulin resistance conditions, or non-alcoholic fatty liver disease (NAFLD), and to contribute to disease progression by a variety of mechanisms related to its multifunctional roles. Therefore, targeting GRK2 by different strategies emerges as a potentially relevant approach to treat cardiovascular disease, obesity, type 2 diabetes, or NAFLD, pathological conditions which are frequently interconnected and present as co-morbidities.Our laboratories are supported by Ministerio de Economía; Industria y Competitividad (MINECO) of Spain (grant SAF2017- 84125-R to FM and CM and SAF2016-80305-P to MS and AMB), CIBERCV-Instituto de Salud Carlos III, Spain (grants CB16/11/00278 and CB16/11/00286 to FM and MS, respectively, co-funded with European FEDER contribution), and Programa de Actividades en Biomedicina de la Comunidad de Madrid (grants B2017/BMD-3671-INFLAMUNE to FM and B2017/ BMD-3676-AORTASANA to MS)

The role of Follistatin-like 3 (Fstl3) in cardiac hypertrophy and remodelling

Follistatins are extracellular inhibitors of TGF-β family ligands like activin A, myostatin and bone morphogenetic proteins. Follistatin-like 3 (Fstl3) is a potent inhibitor of activin signalling and antagonises the cardioprotective role of activin A in the heart. The expression of Fstl3 is elevated in patients with heart failure and upregulated by hypertrophic stimuli in cardiomyocytes. However its role in cardiac remodelling is largely unknown. The aim of this thesis was to analyse the function of Fstl3 in the myocardium in response to hypertrophic stimuli using both in vivo and in vitro approaches. To explore the role of Fstl3 in cardiac hypertrophy, cardiac-specific Fstl3 knock-out mice (Fstl3 KO) were subjected to pressure overload induced by trans-aortic constriction. They showed attenuated cardiac hypertrophy and improved cardiac function compared to wild type littermate control mice. Knock-out of Fstl3 specifically from cardiomyocytes was sufficient to reduce the total expression of Fstl3 in the heart following pressure overload, implying that cardiomyocytes are the major source of Fstl3 in the heart after TAC. Fstl3 KO mice also showed reduced expression of typical hypertrophic markers including ANP, BNP, α-skeletal actin and β-MHC. Similarly, treatment of neonatal rat cardiomyocytes with Fstl3 resulted in hypertrophy as measured by increased cell size and protein synthesis. This was also accompanied by activation of p38 and JNK signalling pathways. Microarray analysis of ventricular samples from these mice indicated reduced expression of genes involved in protein binding and extracellular matrix. Verification by quantitative real-time RT-PCR revealed reduced expression of collagens and TGF- β1 in the myocardium, indicating reduced fibrosis. This was supported by histological analysis showing reduced interstitial fibrosis. As an in vitro model of pressure overload, cardiomyocytes were subjected to mechanical stretch, which elevated the expression of Fstl3. In order to explore the role of cardiomyocyte-derived Fstl3 in modulating fibroblast function, cardiac fibroblasts were treated with the conditioned medium from stretched cardiomyocytes and collagen synthesis was measured. Fstl3 was found to be necessary for conditioned medium to induce an increase in collagen synthesis in fibroblasts. Fstl3 was also shown to induce low levels of cell death in cardiac fibroblasts. In order to identify stretched cardiomyocyte derived factors necessary for Fstl3 action on fibroblast collagen synthesis, a yeast two hybrid analysis was undertaken. Results indicated that Fstl3 may act, at least in part, through binding to proteins of the extracellular matrix as well as pro-fibrotic factors, including connective tissue growth factor (CTGF). While CTGF did not affect fibroblast collagen synthesis in the presence of Fstl3, it inhibited Fstl3 induced cell death, indicating a protective role. In summary, data presented in this thesis demonstrates that Fstl3 is upregulated after cardiac injury and functions by activating the pro-hypertrophic and pro-fibrotic responses in the myocardium, making it an attractive therapeutic target for intervention of cardiac pathologies

Isoform-specific Modulation of Ventricular L-type Calcium Channels by Gαi Proteins in a Murine Model of Heart Failure

Background: Inhibitory G protein α-subunit is upregulated in heart failure (HF). In murine dilated cardiomyopathy induced by overexpression of the β1-adrenoceptor in transgenic mice (β1-tg), we have shown that Gαi2 deficiency aggravated the cardiac dysfunction, while the absence of the closely related Gαi3 had the opposite effect. The exact roles of these two Gi isoforms in failing cardiomyocytes are still unknown. As the L-type Ca2+ current (ICa,L) is altered in heart failure, this study aimed to investigate the effect of either Gαi-isoform deficiency on modulation of ventricular ICa,L in β1-tg mice. Methods: Ventricular ICa,L was measured by whole-cell patch-clamp under basal conditions or after incubation with isoproterenol (Iso) in cardiomyocytes from male β1-tg mice with or without specific Gαi expression. Absence of Gαi2 (β1-tg/Gαi2-/-) was studied at 4-5 months of age, while the absence of Gαi3 (β1-tg/Gαi3-/-) was studied at 10-11 months of age. Results: Basal peak ICa,L density was significantly reduced in β1-tg myocytes (-5.5±1.5 pA/pF) compared to wild-type (-8.1±1.6 pA/pF). In β1-tg/Gαi3-/-, ICa,L was raised towards wild-type levels (-7.5± 1.6 pA/pF). The voltage of half maximal ICaL activation was positively shifted in β1-tg myocytes compared to wildtypes (V0.5: -7.7±2.8 mV vs. -11.3±2.5 mV, p=01), whereas it was close to normal in β1-tg/Gαi3-/- myocytes (-10.6±4.3 mV). Iso (1µM) significantly increased ICa,L density in wild-type (to +169±65%, p<.001) and caused a leftward shift of activation voltage (V0.5 to -17.1±4.0 mV, p<.001). Both effects were attenuated in β1-tg (133±34%, p<.01 and -9.7±4.6 mV, p=ns) and in β1-tg/Gαi3-/- myocytes (+127±48%, p=<.05 and -14.2±6.7 mV, p=ns). In young β1-tg mice similar reduction in basal ICa,L activity was observed, but an additional lack of Gαi2 had no effect on this. However, there was a positive shift in the slope factor and voltage of basal steady-state of inactivation in β1-tg/Gαi2-/- myocytes (kinact: -3.3±0.8 mV, V0.5_inact -21.4±1.7 mV) versus wild-type (-5.3±0.8 mV, -28.8±3.7 mV, p<.001) and β1-tg (-5.1±1.1mV, -26.4±3.6 mV, p<.001). The ICa,L recovery kinetics were also significantly enhanced. Interestingly, Iso-mediated stimulatory effects were augmented in β1-tg/Gαi2-/- myocytes (e.g., ICa,L density was increased to +209±50%, p<.001). Conclusions: These findings suggest isoform-specific modulation of ventricular ICa,L by Gαi protein during cardiomyopathy progression. Gαi3 deficiency is cardioprotective, likely due to the restoration of basal ICa,L function and attenuation of adrenergic stimulatory effects. Conversely, Gαi2 deficiency can be detrimental as it fails to restore ICa,L or protect against intense β-adrenergic stimulation. It is also associated with unfavorable changes in ICa,L inactivation and recovery gating. Pharmacological intervention in the Gi-dependent signaling pathway shows promise for developing cardioprotective therapeutics