MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

BACKGROUND: Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. METHODS: We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. RESULTS: We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. CONCLUSIONS: Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response.

Raf1/c-Raf is a well-characterized serine/threonine-protein kinase that links Ras family members with the MAPK/ERK signaling cascade. We have identified a novel splice isoform of human Raf1 that causes protein truncation and loss of the C-terminal kinase domain (Raf1-tr). We found that Raf1-tr has increased nuclear localization compared with full-length Raf1, and this finding was secondary to reduced binding of Raf1-tr to the cytoplasmic chaperone FK506 binding protein 5. We show that Raf1-tr has increased binding to DNA-dependent protein kinase (DNA-PK), which inhibits DNA-PK function and causes amplification of irradiation- and bleomycin-induced DNA damage. We found that the human colorectal cancer cell line, HCT-116, displayed reduced expression of Raf1-tr, and reintroduction of Raf1-tr sensitized the cells to bleomycin-induced apoptosis. Furthermore, we identified differential Raf1-tr expression in breast cancer cell lines and showed that breast cancer cells with increased Raf1-tr expression become sensitized to bleomycin-induced apoptosis. Collectively, these results demonstrate a novel Raf1 isoform in humans that has a unique noncanonical role in regulating the double-stranded DNA damage response pathway through modulation of DNA-PK function.-Nixon, B. R., Sebag, S. C., Glennon, M. S., Hall, E. J., Kounlavong, E. S., Freeman, M. L., Becker, J. R. Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response.

Pseudomonas aeruginosa responds to exogenous polyunsaturated fatty acids (PUFAs) by modifying phospholipid composition, membrane permeability, and phenotypes associated with virulence.

BACKGROUND: Pseudomonas aeruginosa, a common opportunistic pathogen, is known to cause infections in a variety of compromised human tissues. An emerging mechanism for microbial survival is the incorporation of exogenous fatty acids to alter the cell's membrane phospholipid profile. With these findings, we show that exogenous fatty acid exposure leads to changes in bacterial membrane phospholipid structure, membrane permeability, virulence phenotypes and consequent stress responses that may influence survival and persistence of Pseudomonas aeruginosa. RESULTS: Thin-layer chromatography and ultra performance liquid chromatography / ESI-mass spectrometry indicated alteration of bacterial phospholipid profiles following growth in the presence of polyunsaturated fatty acids (PUFAs) (ranging in carbon length and unsaturation). The exogenously supplied fatty acids were incorporated into the major bacterial phospholipids phosphatidylethanolamine and phosphatidylglycerol. The incorporation of fatty acids increased membrane permeability as judged by both accumulation and exclusion of ethidium bromide. Individual fatty acids were identified as modifying resistance to the cyclic peptide antibiotics polymyxin B and colistin, but not the beta-lactam imipenem. Biofilm formation was increased by several PUFAs and significant fluctuations in swimming motility were observed. CONCLUSIONS: Our results emphasize the relevance and complexity of exogenous fatty acids in the membrane physiology and pathobiology of a medically important pathogen. P. aeruginosa exhibits versatility with regard to utilization of and response to exogenous fatty acids, perhaps revealing potential strategies for prevention and control of infection.

Cardiac natriuretic peptide deficiency sensitizes the heart to stress-induced ventricular arrhythmias via impaired CREB signalling.

AIMS: The cardiac natriuretic peptides [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities. METHODS AND RESULTS: We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP-deficient (Nppa-/-) or BNP-deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human-derived induced pluripotent stem cell cardiomyocytes (iPS-CMs). In the unstressed state, both ANP- and BNP-deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodelling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress-induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild-type hearts to stress-induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen-activated protein kinase (p38 MAPK) signalling cascade. CONCLUSIONS: Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signalling which sensitizes the heart to stress-induced ventricular arrhythmias.

Replication Stress Response Modifies Sarcomeric Cardiomyopathy Remodeling.

Background Sarcomere gene mutations lead to cardiomyocyte hypertrophy and pathological myocardial remodeling. However, there is considerable phenotypic heterogeneity at both the cellular and the organ level, suggesting modifiers regulate the effects of these mutations. We hypothesized that sarcomere dysfunction leads to cardiomyocyte genotoxic stress, and this modifies pathological ventricular remodeling. Methods and Results Using a murine model deficient in the sarcomere protein, Mybpc3-/- (cardiac myosin-binding protein 3), we discovered that there was a surge in cardiomyocyte nuclear DNA damage during the earliest stages of cardiomyopathy. This was accompanied by a selective increase in ataxia telangiectasia and rad3-related phosphorylation and increased p53 protein accumulation. The cause of the DNA damage and DNA damage pathway activation was dysregulated cardiomyocyte DNA synthesis, leading to replication stress. We discovered that selective inhibition of ataxia telangiectasia and rad3 related or cardiomyocyte deletion of p53 reduced pathological left ventricular remodeling and cardiomyocyte hypertrophy in Mybpc3-/- animals. Mice and humans harboring other types of sarcomere gene mutations also had evidence of activation of the replication stress response, and this was associated with cardiomyocyte aneuploidy in all models studied. Conclusions Collectively, our results show that sarcomere mutations lead to activation of the cardiomyocyte replication stress response, which modifies pathological myocardial remodeling in sarcomeric cardiomyopathy.

Ponatinib-induced cardiotoxicity: delineating the signalling mechanisms and potential rescue strategies.

AIMS: Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukaemia (CML). However, cardiotoxicity of these agents remains a serious concern. The underlying mechanism of these adverse cardiac effects is largely unknown. Delineation of the underlying mechanisms of TKIs associated cardiac dysfunction could guide potential prevention strategies, rescue approaches, and future drug design. This study aimed to determine the cardiotoxic potential of approved CML TKIs, define the associated signalling mechanism and identify potential alternatives. METHODS AND RESULTS: In this study, we employed a zebrafish transgenic BNP reporter line that expresses luciferase under control of the nppb promoter (nppb:F-Luciferase) to assess the cardiotoxicity of all approved CML TKIs. Our in vivo screen identified ponatinib as the most cardiotoxic agent among the approved CML TKIs. Then using a combination of zebrafish and isolated neonatal rat cardiomyocytes, we delineated the signalling mechanism of ponatinib-induced cardiotoxicity by demonstrating that ponatinib inhibits cardiac prosurvival signalling pathways AKT and extra-cellular-signal-regulated kinase (ERK), and induces cardiomyocyte apoptosis. As a proof of concept, we augmented AKT and ERK signalling by administration of Neuregulin-1ß (NRG-1ß), and this prevented ponatinib-induced cardiomyocyte apoptosis. We also demonstrate that ponatinib-induced cardiotoxicity is not mediated by inhibition of fibroblast growth factor signalling, a well-known target of ponatinib. Finally, our comparative profiling for the cardiotoxic potential of CML approved TKIs, identified asciminib (ABL001) as a potentially much less cardiotoxic treatment option for CML patients with the T315I mutation. CONCLUSION: Herein, we used a combination of in vivo and in vitro methods to systematically screen CML TKIs for cardiotoxicity, identify novel molecular mechanisms for TKI cardiotoxicity, and identify less cardiotoxic alternatives.

Alterations in sarcomere function modify the hyperplastic to hypertrophic transition phase of mammalian cardiomyocyte development.

It remains unclear how perturbations in cardiomyocyte sarcomere function alter postnatal heart development. We utilized murine models that allowed manipulation of cardiac myosin-binding protein C (MYBPC3) expression at critical stages of cardiac ontogeny to study the response of the postnatal heart to disrupted sarcomere function. We discovered that the hyperplastic to hypertrophic transition phase of mammalian heart development was altered in mice lacking MYBPC3 and this was the critical period for subsequent development of cardiomyopathy. Specifically, MYBPC3-null hearts developed evidence of increased cardiomyocyte endoreplication, which was accompanied by enhanced expression of cell cycle stimulatory cyclins and increased phosphorylation of retinoblastoma protein. Interestingly, this response was self-limited at later developmental time points by an upregulation of the cyclin-dependent kinase inhibitor p21. These results provide valuable insights into how alterations in sarcomere protein function modify postnatal heart development and highlight the potential for targeting cell cycle regulatory pathways to counteract cardiomyopathic stimuli.