Marina M. López-Olañeta

Calcineurin Splicing Variant Calcineurin Aβ1 Improves Cardiac Function After Myocardial Infarction Without Inducing Hypertrophy

Background— Calcineurin is a calcium-regulated phosphatase that plays a major role in cardiac hypertrophy. We previously described that alternative splicing of the calcineurin A&bgr; (CnA&bgr;) gene generates the CnA&bgr;1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnA&bgr;1 is necessary for myoblast proliferation and stimulates regeneration, reducing fibrosis and accelerating the resolution of inflammation. Its role in the heart is currently unknown. Methods and Results— We generated transgenic mice overexpressing CnA&bgr;1 in postnatal cardiomyocytes under the control of the &agr;-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnA&bgr;1 overexpression did not induce cardiac hypertrophy. Moreover, transgenic mice showed improved cardiac function and reduced scar formation after myocardial infarction, with reduced neutrophil and macrophage infiltration and decreased expression of proinflammatory cytokines. Immunoprecipitation and Western blot analysis showed interaction of CnA&bgr;1 with the mTOR complex 2 and activation of the Akt/SGK cardioprotective pathway in a PI3K-independent manner. In addition, gene expression profiling revealed that CnA&bgr;1 activated the transcription factor ATF4 downstream of the Akt/mTOR pathway to promote the amino acid biosynthesis program, to reduce protein catabolism, and to induce the antifibrotic and antiinflammatory factor growth differentiation factor 15, which protects the heart through Akt activation. Conclusions— Calcineurin A&bgr;1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnA&bgr;1 an attractive candidate for the development of future therapeutic approaches.

Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction

AIMS After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross-links. This study investigates the contribution of LOX family members to the heart response to MI. METHODS AND RESULTS Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI, and this response was accompanied by a significant accumulation of mature collagen fibres in the infarcted area. LOX expression was observed in areas of extensive remodelling, partially overlapping with α-smooth muscle actin-expressing myofibroblasts. Tumour growth factor-β as well as hypoxia-activated pathways contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor β-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also resulted in reduced ventricular dilatation and improved cardiac function. CONCLUSION LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery.

Follistatin-Like 3 Mediates Paracrine Fibroblast Activation by Cardiomyocytes

Follistatins are extracellular inhibitors of the TGF-β family ligands including 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. FSTL3 expression is elevated in patients with heart failure and is upregulated in cardiomyocytes by hypertrophic stimuli, but its role in cardiac remodelling is largely unknown. Here, we show that the production of FSTL3 by cardiomyocytes contributes to the paracrine activation of cardiac fibroblasts, inducing changes in cell adhesion, promoting proliferation and increasing collagen production. We found that FSTL3 is necessary for this response and for the induction of cardiac fibrosis. However, full activation requires additional factors, and we identify connective tissue growth factor as a FSTL3 binding partner in this process. Together, our data unveil a novel mechanism of paracrine communication between cardiomyocytes and fibroblasts that may provide potential as a therapeutic target in heart remodelling.

Induction of the calcineurin variant CnAβ1 after myocardial infarction reduces post-infarction ventricular remodelling by promoting infarct vascularization.

AIMS Ventricular remodelling following myocardial infarction progressively leads to loss of contractile capacity and heart failure. Although calcineurin promotes maladaptive cardiac hypertrophy, we recently showed that the calcineurin splicing variant, CnAβ1, has beneficial effects on the infarcted heart. However, whether this variant limits necrosis or improves remodelling is still unknown, precluding translation to the clinical arena. Here, we explored the effects and therapeutic potential of CnAβ1 overexpression post-infarction. METHODS AND RESULTS Double transgenic mice with inducible cardiomyocyte-specific overexpression of CnAβ1 underwent left coronary artery ligation followed by reperfusion. Echocardiographic analysis showed depressed cardiac function in all infarcted mice 3 days post-infarction. Induction of CnAβ1 overexpression 1 week after infarction improved function and reduced ventricular dilatation. CnAβ1-overexpressing mice showed shorter, thicker scars, and reduced infarct expansion, accompanied by reduced myocardial remodelling. CnAβ1 induced vascular endothelial growth factor (VEGF) expression in cardiomyocytes, which resulted in increased infarct vascularization. This paracrine angiogenic effect of CnAβ1 was mediated by activation of the Akt/mammalian target of rapamycin pathway and VEGF. CONCLUSIONS Our results indicate that CnAβ1 exerts beneficial effects on the infarcted heart by promoting infarct vascularization and preventing infarct expansion. These findings emphasize the translational potential of CnAβ1 for gene-based therapies.

ZBTB17 (MIZ1) Is Important for the Cardiac Stress Response and a Novel Candidate Gene for Cardiomyopathy and Heart Failure.

Background—Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. Methods and Results—We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte–specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner. Conclusions—We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene.

H-ras deletion protects against angiotensin II–induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H‐ras gene deletion produces mice hypotension via a soluble guanylate cyclase‐protein kinase G (PKG)–dependent mechanism. In this study, we analyzed the consequences of H‐ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross‐sectional area were similar between AngII‐treated H‐Ras knockout (H‐ras−/−) and control wild‐type (H‐ras+/+) mice, as were extracellular matrix protein expression. Increased cardiac PKG‐Iβ protein expression in H‐ras−/− mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII‐induced cardiac hypertrophy and fibrosis markers in H‐ras−/− mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H‐Ras down‐regulates the B‐RAF/MEK/ERK pathway, which induces the glycogen synthase kinase‐3β‐dependent activation of the transcription factor, cAMP response element‐binding protein, which is responsible for PKG‐Iβ overexpression in H‐ras−/− mouse embryonic fibroblasts. This study demonstrates that H‐ras deletion protects against AngII‐induced cardiac remodeling, possibly via a mechanism in which PKG‐Iβ overexpression could play a partial role, and points to H‐Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.—Martín‐Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López‐Olañeta, M., Chiloeches, A., Lara‐Pezzi, E., de Frutos, S., Rodríguez‐Puyol, M., Calleros, L., Rodriíguez‐Puyol, D. H‐ras deletion protects against angiotensin II–induced arterial hypertension and cardiac remodeling through protein kinase G‐Iβ pathway activation. FASEB J. 32, 920–934 (2018). www.fasebj.org

Lung ultrasound as a translational approach for non-invasive assessment of heart failure with reduced or preserved ejection fraction in mice

Aims Heart failure (HF) has become an epidemic and constitutes a major medical, social, and economic problem worldwide. Despite advances in medical treatment, HF prognosis remains poor. The development of efficient therapies is hampered by the lack of appropriate animal models in which HF can be reliably determined, particularly in mice. The development of HF in mice is often assumed based on the presence of cardiac dysfunction, but HF itself is seldom proved. Lung ultrasound (LUS) has become a helpful tool for lung congestion assessment in patients at all stages of HF. We aimed to apply this non-invasive imaging tool to evaluate HF in mouse models of both systolic and diastolic dysfunction. Methods and results We used LUS to study HF in a mouse model of systolic dysfunction, dilated cardiomyopathy, and in a mouse model of diastolic dysfunction, diabetic cardiomyopathy. LUS proved to be a reliable and reproducible tool to detect pulmonary congestion in mice. The combination of LUS and echocardiography allowed discriminating those mice that develop HF from those that do not, even in the presence of evident cardiac dysfunction. The study showed that LUS can be used to identify the onset of HF decompensation and to evaluate the efficacy of therapies for this syndrome. Conclusions This novel approach in mouse models of cardiac disease enables for the first time to adequately diagnose HF non-invasively in mice with preserved or reduced ejection fraction, and will pave the way to a better understanding of HF and to the development of new therapeutic approaches.