Dasatinib targets c-Src kinase in cardiotoxicity.

Dasatinib is a multitargeted kinase inhibitor used for treatment of chronic myeloid leukemia and acute lymphoblastic leukemia. Unfortunately, treatment of cancer patients with some kinase inhibitors has been associated with cardiotoxicity. Cancer treatment with dasatinib has been reported to be associated with cardiotoxic side effects such as left ventricular dysfunction, heart failure, pericardial effusion and pulmonary hypertension. Here we aimed to investigate the molecular mechanisms underlying the cardiotoxicity of dasatinib. We found that among the resident cardiac cell types, cardiomyocytes were most sensitive to dasatinib-induced cell death. Exposure of cardiomyocytes to dasatinib attenuated the activity of extracellular signal-regulated kinase (ERK), which is a downstream target of dasatinib target kinase c-Src. Similar to dasatinib, c-Src depletion in cardiomyocytes compromised cardiomyocyte viability. Overexpression of dasatinib-resistant mutant of c-Src rescued the toxicity of dasatinib on cardiomyocytes, whereas forced expression of wild type c-Src did not have protective effect. Collectively, our results show that c-Src is a key target of dasatinib mediating the toxicity of dasatinib to cardiomyocytes. These findings may influence future drug design and suggest closer monitoring of patients treated with agents targeting c-Src for possible adverse cardiac effects.

Wnt11 in regulation of physiological and pathological cardiac growth.

Wnt11 regulates early cardiac development and left ventricular compaction in the heart, but it is not known how Wnt11 regulates postnatal cardiac maturation and response to cardiac stress in the adult heart. We studied cell proliferation/maturation in postnatal and adolescent Wnt11 deficient (Wnt11-/-) heart and subjected adult mice with partial (Wnt11+/-) and complete Wnt11 (Wnt11-/-) deficiency to cardiac pressure overload. In addition, we subjected primary cardiomyocytes to recombinant Wnt proteins to study their effect on cardiomyocyte growth. Wnt11 deficiency did not affect cardiomyocyte proliferation or maturation in the postnatal or adolescent heart. However, Wnt11 deficiency led to enlarged heart phenotype that was not accompanied by significant hypertrophy of individual cardiomyocytes. Analysis of stressed adult hearts from wild-type mice showed a progressive decrease in Wnt11 expression in response to pressure overload. When studied in experimental cardiac pressure overload, Wnt11 deficiency did not exacerbate cardiac hypertrophy or remodeling and cardiac function remained identical between the genotypes. When subjecting cardiomyocytes to hypertrophic stimulus, the presence of recombinant Wnt11 together with Wnt5a reduced protein synthesis. In conclusion, Wnt11 deficiency does not affect postnatal cardiomyocyte proliferation but leads to cardiac growth. Interestingly, Wnt11 deficiency alone does not substantially modulate hypertrophic response to pressure overload in vivo. Wnt11 may require cooperation with other noncanonical Wnt proteins to regulate hypertrophic response under stress.

GSK3ß Serine 389 Phosphorylation Modulates Cardiomyocyte Hypertrophy and Ischemic Injury.

Prior studies show that glycogen synthase kinase 3ß (GSK3ß) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3ß is constitutionally active and phosphorylation of GSK3ß at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3ß is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3ß S389 phosphorylation in diseased hearts and utilized overexpression of GSK3ß carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3ß in primary cardiomyocytes. We found that phosphorylation of GSK3ß at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3ß S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia-reoxygenation. Overexpression of double GSK3ß mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3ß S389A or GSK3ß S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3ß S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3ß at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy.

Vezf1 regulates cardiac structure and contractile function.

BACKGROUND: Vascular endothelial zinc finger 1 (Vezf1) is a transcription factor previously shown to regulate vasculogenesis and angiogenesis. We aimed to investigate the role of Vezf1 in the postnatal heart. METHODS: The role of Vezf1 in regulating cardiac growth and contractile function was studied in zebrafish and in primary cardiomyocytes. FINDINGS: We find that expression of Vezf1 is decreased in diseased human myocardium and mouse hearts. Our experimental data shows that knockdown of zebrafish Vezf1 reduces cardiac growth and results in impaired ventricular contractile response to ß-adrenergic stimuli. However, Vezf1 knockdown is not associated with dysregulation of cardiomyocyte Ca2+ transient kinetics. Gene ontology enrichment analysis indicates that Vezf1 regulates cardiac muscle contraction and dilated cardiomyopathy related genes and we identify cardiomyocyte Myh7/ß-MHC as key target for Vezf1. We further identify a key role for an MCAT binding site in the Myh7 promoter regulating the response to Vezf1 knockdown and show that TEAD-1 is a binding partner of Vezf1. INTERPRETATION: We demonstrate a role for Vezf1 in regulation of compensatory cardiac growth and cardiomyocyte contractile function, which may be relevant in human cardiac disease.

Connective Tissue Growth Factor Inhibition Enhances Cardiac Repair and Limits Fibrosis After Myocardial Infarction.

Myocardial infarction (MI)-induced cardiac fibrosis attenuates cardiac contractile function, and predisposes to arrhythmias and sudden cardiac death. Expression of connective tissue growth factor (CTGF) is elevated in affected organs in virtually every fibrotic disorder and in the diseased human myocardium. Mice were subjected to treatment with a CTGF monoclonal antibody (mAb) during infarct repair, post-MI left ventricular (LV) remodeling, or acute ischemia-reperfusion injury. CTGF mAb therapy during infarct repair improved survival and reduced LV dysfunction, and reduced post-MI LV hypertrophy and fibrosis. Mechanistically, CTGF mAb therapy induced expression of cardiac developmental and/or repair genes and attenuated expression of inflammatory and/or fibrotic genes.

Systemic Blockade of ACVR2B Ligands Protects Myocardium from Acute Ischemia-Reperfusion Injury.

Activin A and myostatin, members of the transforming growth factor (TGF)-ß superfamily of secreted factors, are potent negative regulators of muscle growth, but their contribution to myocardial ischemia-reperfusion (IR) injury is not known. The aim of this study was to investigate if activin 2B (ACVR2B) receptor ligands contribute to myocardial IR injury. Mice were treated with soluble ACVR2B decoy receptor (ACVR2B-Fc) and subjected to myocardial ischemia followed by reperfusion for 6 or 24 h. Systemic blockade of ACVR2B ligands by ACVR2B-Fc was protective against cardiac IR injury, as evidenced by reduced infarcted area, apoptosis, and autophagy and better preserved LV systolic function following IR. ACVR2B-Fc modified cardiac metabolism, LV mitochondrial respiration, as well as cardiac phenotype toward physiological hypertrophy. Similar to its protective role in IR injury in vivo, ACVR2B-Fc antagonized SMAD2 signaling and cell death in cardiomyocytes that were subjected to hypoxic stress. ACVR2B ligand myostatin was found to exacerbate hypoxic stress. In addition to acute cardioprotection in ischemia, ACVR2B-Fc provided beneficial effects on cardiac function in prolonged cardiac stress in cardiotoxicity model. By blocking myostatin, ACVR2B-Fc potentially reduces cardiomyocyte death and modifies cardiomyocyte metabolism for hypoxic conditions to protect the heart from IR injury.

Inhibition of cardiomyocyte Sprouty1 protects from cardiac ischemia-reperfusion injury.

Sprouty1 (Spry1) is a negative modulator of receptor tyrosine kinase signaling, but its role in cardiomyocyte survival has not been elucidated. The aim of this study was to investigate the potential role of cardiomyocyte Spry1 in cardiac ischemia-reperfusion (I/R) injury. Infarct areas of mouse hearts showed an increase in Spry1 protein expression, which localized to cardiomyocytes. To investigate if cardiomyocyte Spry1 regulates I/R injury, 8-week-old inducible cardiomyocyte Spry1 knockout (Spry1 cKO) mice and control mice were subjected to cardiac I/R injury. Spry1 cKO mice showed reduction in release of cardiac troponin I and reduced infarct size after I/R injury compared to control mice. Similar to Spry1 knockdown in cardiomyocytes in vivo, RNAi-mediated Spry1 silencing in isolated cardiomyocytes improved cardiomyocyte survival following simulated ischemia injury. Mechanistically, Spry1 knockdown induced cardiomyocyte extracellular signal-regulated kinase (ERK) phosphorylation in healthy hearts and isolated cardiomyocytes, and enhanced ERK phosphorylation after I/R injury. Spry1-deficient cardiomyocytes showed better preserved mitochondrial membrane potential following ischemic injury and an increase in levels of phosphorylated ERK and phosphorylated glycogen synthase kinase-3ß (GSK-3ß) in mitochondria of hypoxic cardiomyocytes. Overexpression of constitutively active GSK-3ß abrogated the protective effect of Spry1 knockdown. Moreover, pharmacological inhibition of GSK-3ß protected wild-type cardiomyocytes from cell death, but did not further protect Spry1-silenced cardiomyocytes from hypoxia-induced injury. Cardiomyocyte Spry1 knockdown promotes ERK phosphorylation and offers protection from I/R injury. Our findings indicate that Spry1 is an important regulator of cardiomyocyte viability during ischemia-reperfusion injury.

Characterization of apela, a novel endogenous ligand of apelin receptor, in the adult heart.

The G protein-coupled apelin receptor regulates important processes of the cardiovascular homeostasis, including cardiac development, cardiac contractility, and vascular tone. Most recently, a novel endogenous peptide ligand for the apelin receptor was identified in zebrafish, and it was named apela/elabela/toddler. The peptide was originally considered as an exclusively embryonic regulator, and so far its function in the adult organism remains elusive. We show here that apela is predominantly expressed in the non-cardiomyocyte fraction in the adult rodent heart. We also provide evidence that apela binds to apelin receptors in the heart. Using isolated adult rat hearts, we demonstrate, that just like the fellow receptor agonist apelin, apela increases cardiac contractility and induces coronary vasodilation already in the nanomolar level. The inotropic effect, as revealed by Western blot analysis, is accompanied by a significant increase in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Pharmacological inhibition of ERK1/2 activation markedly attenuates the apela-induced inotropy. Analysis of samples from infarcted mouse hearts showed that expression of both apela and apelin receptor is induced in failing mouse hearts and correlate with left ventricular ejection fraction. Hence, we conclude that apela is present in the adult heart, is upregulated in post-infarction cardiac remodeling, and increases cardiac contractility in an ERK1/2-dependent manner.

Connective tissue growth factor inhibition attenuates left ventricular remodeling and dysfunction in pressure overload-induced heart failure.

Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.

p38α regulates SERCA2a function.

cAMP-dependent protein kinase (PKA) regulates the L-type calcium channel, the ryanodine receptor, and phospholamban (PLB) thereby increasing inotropy. Cardiac contractility is also regulated by p38 MAPK, which is a negative regulator of cardiac contractile function. The aim of this study was to identify the mechanism mediating the positive inotropic effect of p38 inhibition. Isolated adult and neonatal cardiomyocytes and perfused rat hearts were utilized to investigate the molecular mechanisms regulated by p38. PLB phosphorylation was enhanced in cardiomyocytes by chemical p38 inhibition, by overexpression of dominant negative p38α and by p38α RNAi, but not with dominant negative p38ß. Treatment of cardiomyocytes with dominant negative p38α significantly decreased Ca(2+)-transient decay time indicating enhanced sarco/endoplasmic reticulum Ca(2+)-ATPase function and increased cardiomyocyte contractility. Analysis of signaling mechanisms involved showed that inhibition of p38 decreased the activity of protein phosphatase 2A, which renders protein phosphatase inhibitor-1 phosphorylated and thereby inhibits PP1. In conclusion, inhibition of p38α enhances PLB phosphorylation and diastolic Ca(2+) uptake. Our findings provide evidence for novel mechanism regulating cardiac contractility upon p38 inhibition.

Neuronostatin, a novel peptide encoded by somatostatin gene, regulates cardiac contractile function and cardiomyocyte survival.

Neuronostatin, a recently discovered peptide encoded by somatostatin gene, is involved in regulation of neuronal function, blood pressure, food intake, and drinking behavior. However, the biological effects of neuronostatin on cardiac myocytes are not known, and the intracellular signaling mechanisms induced by neuronostatin remain unidentified. We analyzed the effect of neuronostatin in isolated perfused rat hearts and in cultured primary cardiomyocytes. Neuronostatin infusion alone had no effect on left ventricular (LV) contractile function or on isoprenaline- or preload-induced increase in cardiac contractility. However, infusion of neuronostatin significantly decreased the positive inotropic response to endothelin-1 (ET-1). This was associated with an increase in phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK). Treatment of both neonatal and adult cardiomyocytes with neuronostatin resulted in reduced cardiomyocyte viability. Inhibition of JNK further increased the neuronostatin-induced cell death. We conclude that neuronostatin regulates cardiac contractile function and cardiomyocyte survival. Receptors for neuronostatin need to be identified to further characterize the biological functions of the peptide.