Jean-Francois Desjardins

BRCA2 Protein Deficiency Exaggerates Doxorubicin-induced Cardiomyocyte Apoptosis and Cardiac Failure

Background: BRCA2 is widely implicated in breast and ovarian cancers, but the role of BRCA2 in the heart is unknown. Results: Loss of BRCA2 in the heart resulted in increased doxorubicin-induced DNA damage, apoptosis, and cardiac dysfunction. Conclusion: BRCA2 is a novel regulator of cardiomyocyte genomic integrity, survival, and function. Significance: BRCA2 mutation carriers may be at a heightened risk of anthracycline-induced cardiac failure. The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2−/−) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2−/− mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2−/− mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2−/− mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2−/− mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2−/− mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2−/− mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.

Lack of S100A1 in mice confers a gender-dependent hypertensive phenotype and increased mortality after myocardial infarction

S100A1 is a small Ca(2+)-binding protein expressed in the myocardium and blood vessels that is downregulated in the diseased heart and plays a role in the regulation of cardiac muscle Ca(2+) homeostasis and contractility. To understand its physiological role under basal conditions and after myocardial infarction (MI), we used a mouse strain with targeted deletion of the S100A1 gene [S100A1 knockout (KO) mice]. We compared 49 wild-type (WT) and 56 S100A1 KO mice (6-8 wk old) over 28 days after MI with sham-operated controls. We also examined the effect of S100A1 deficiency on vascular function of isolated blood vessels. S100A1 KO mice demonstrated worse survival compared with WT mice (21% vs. 69%, respectively, P < 0.001). Hemodynamic evaluation revealed a higher mean arterial pressure (MAP) in sham-operated KO animals compared with WT animals (99 +/- 4 vs. 77 +/- 3 mmHg, respectively, P < 0.001) that persisted in both groups after MI (86 +/- 2 vs. 66 +/- 4 mmHg, respectively, P < 0.001). Sham-operated male S100A1 KO mice had higher MAP than female KO mice (122 +/- 5 vs. 93 +/- 3 mmHg, respectively P < 0.05) and reduced survival after MI (4% vs. 27%, respectively, P < 0.05). In isolated aortas and mesenteric arteries, ACh-evoked vasodilatation in KO mice was significantly reduced compared with WT mice (P < 0.05). Nitric oxide production was reduced in endothelial cells isolated from KO mice. Thus, absence of S100A1 results in acute functional impairment and high mortality after MI associated with a gender-specific hypertensive phenotype. S100A1 appears to play a role in the endothelium-dependent regulation of blood pressure.

Dipeptidyl peptidase‐4 inhibition improves cardiac function in experimental myocardial infarction: Role of stromal cell‐derived factor‐1α

In addition to degrading glucagon‐like peptide‐1 (GLP‐1), dipeptidyl peptidase‐4 (DPP‐4) inactivates several chemokines, including stromal cell‐derived factor‐1α (SDF‐1α), a pro‐angiogenic and cardiomyocyte protective protein. We hypothesized that DPP‐4 inhibition may confer benefit following myocardial infarction (MI) in the diabetic setting as a consequence of enhanced SDF‐1α availability rather than potentiating GLP‐1. To test this we compared the effects of saxagliptin with those of liraglutide and used the SDF‐1α receptor (CXCR4) antagonist plerixafor.

Therapeutic hypercapnia prevents inhaled nitric oxide-induced right-ventricular systolic dysfunction in juvenile rats.

Chronic pulmonary hypertension in the neonate and infant frequently presents with right-ventricular (RV) failure. Current clinical management may include protracted treatment with inhaled nitric oxide (iNO), with the goal of reducing RV afterload. We have previously reported that prolonged exposure to iNO causes RV systolic dysfunction in the chronic hypoxia-exposed juvenile rat, which was prevented by a peroxynitrite decomposition catalyst. Given that inhalation of CO2 (therapeutic hypercapnia) may limit oxidative stress and upregulated cytokine expression in the lung and other organs, we hypothesized that therapeutic hypercapnia would attenuate cytokine-mediated nitric oxide synthase (NOS) upregulation, thus limiting peroxynitrite generation. Sprague-Dawley rat pups were exposed to chronic hypoxia (13% O2) from postnatal day 1 to 21, while receiving iNO (20 ppm) from day 14 to 21, with or without therapeutic hypercapnia (10% CO2). Therapeutic hypercapnia completely normalized RV systolic function, RV hypertrophy, and remodeling of pulmonary resistance arteries in animals exposed to iNO. Inhaled nitric oxide-mediated increases in RV peroxynitrite, apoptosis, and contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and NOS-2 were all attenuated by therapeutic hypercapnia. Inhibition of NOS-2 activity with 1400 W (1 mg/kg/day) prevented iNO-mediated upregulation of peroxynitrite and led to improved RV systolic function. Blockade of IL-1 receptor signaling with anakinra (500 mg/kg/day) decreased NOS-2 content and had similar effects compared to NOS-2 inhibition on iNO-mediated effects, whereas blockade of TNF-α signaling with etanercept (0.4 mg/kg on alternate days) had no effects on these parameters. We conclude that therapeutic hypercapnia prevents the adverse effects of sustained exposure to iNO on RV systolic function by limiting IL-1-mediated NOS-2 upregulation and consequent nitration. Therapeutic hypercapnia also acts synergistically with iNO in normalizing RV hypertrophy, vascular remodeling, and raised pulmonary vascular resistance secondary to chronic hypoxia.

Conditional Cardiac Overexpression of S100A6 Attenuates Myocyte Hypertrophy and Apoptosis Following Myocardial Infarction

S100A6, a 20 kDa, Ca2+ - binding dimer with low basal cardiac expression, is upregulated in the rat heart following infarction and forced expression of S100A6 in rat neonatal cardiac myocyte cultures, inhibited the induction of β myosin heavy chain (MHC), skeletal α actin (skACT) and myocyte apoptosis in response to diverse stimuli including tumor necrosis factor α. To define a role for S100A6 in vivo, we generated cardiac myocyte-specific transgenic mice by placing the human S100A6 cDNA downstream of a promoter responsive to a doxycycline (DOX)-regulated transcriptional activator (tTA) and breeding this line with one harboring cardiac myocyte-restricted (αMHC) expression of tTA (αMHC-tTA). We compared S100A6-αMHC-tTA mice 35 days post-myocardial infarction (MI) produced by coronary artery ligation with similar matched sham-operated controls on (S100A6 transgene overexpressed) or off (S100A6 transgene silenced) DOX. There were no differences between the sham groups on or off DOX. Thirty five days post-MI, myocardial S100A6 levels increased 12.5-fold in S100A6-α-MHC-tTA mice off DOX compared with S100A6-α-MHC-tTA mice on DOX. Hemodynamic studies, echocardiography and postmortem examination indicated that S100A6-αMHC-tTA mice on DOX 35 days post-MI mounted a hypertrophic response (20-22.5 % increase) accompanied by a program of fetal gene re-expression, fibrosis and myocardial apoptosis. Whereas the S100A6-α-MHC-tTA mice off DOX showed an attenuated myocyte hypertrophic response, less fibrosis and apoptosis which was beneficial to preservation of cardiac function. Therefore, S100A6 is a potential therapeutic target for modulation of adverse left ventricular remodeling in the early post infarct period.

The α11 integrin mediates fibroblast-extracellular matrix-cardiomyocyte interactions in health and disease.

Excessive cardiac interstitial fibrosis impairs normal cardiac function. We have shown that the α11β1 (α11) integrin mediates fibrotic responses to glycated collagen in rat myocardium by a pathway involving transforming growth factor-β. Little is known of the role of the α11 integrin in the developing mammalian heart. Therefore, we examined the impact of deletion of the α11 integrin in wild-type mice and in mice treated with streptozotocin (STZ) to elucidate the role of the α11 integrin in normal cardiac homeostasis and in the pathogenesis of diabetes-related fibrosis. As anticipated, cardiac fibrosis was reduced in α11 integrin knockout mice (α11(-/-); C57BL/6 background) treated with STZ compared with STZ-treated wild-type mice (P < 0.05). Unexpectedly, diastolic function was impaired in both vehicle and STZ-treated α11(-/-) mice, as shown by the decreased minimum rate of pressure change and prolonged time constant of relaxation in association with increased end-diastolic pressure (all P < 0.05 compared with wild-type mice). Accordingly, we examined the phenotype of untreated α11(-/-) mice, which demonstrated a reduced cardiomyocyte cross-sectional cell area and myofibril thickness (all P < 0.05 compared with wild-type mice) and impaired myofibril arrangement. Immunostaining for desmin and connexin 43 showed abnormal intermediate filament organization at intercalated disks and impaired gap-junction development. Overall, deletion of the α11 integrin attenuates cardiac fibrosis in the mammalian mouse heart and reduces ECM formation as a result of diabetes. Furthermore, α11 integrin deletion impairs cardiac function and alters cardiomyocyte morphology. These findings shed further light on the poorly understood interaction between the fibroblast-cardiomyocyte and the ECM.

Absence of the calcium binding protein, S100A1, confers pulmonary hypertension in mice associated with endothelial dysfunction and apoptosis

AIMS S100A1, a 10-kDa, Ca(2+)-binding protein, is expressed in endothelial cells (ECs) and binds eNOS. Its absence is associated with impaired production of nitric oxide (NO) and mild systemic hypertension. As endothelial dysfunction contributes to clinical and experimental pulmonary hypertension (PH), we investigated the impact of deleting S100A1 in mice, on pulmonary haemodynamics, endothelial function, NO production, associated signalling pathways, and apoptosis. METHODS AND RESULTS Compared with wild-type (WT), S100A1-knock-out mice (KO) exhibited increased right ventricular (RV) weight/body weight ratio and elevated RV pressure in the absence of altered left ventricular filling pressures, accompanied by increase in wall thickness of muscularized pulmonary arteries and a reduction in microvascular perfusion. In isolated lung preparations, KO revealed reduced basal NO, blunted dose-responsiveness to acetylcholine, and augmented basal and angiotensin (AII)-induced pulmonary vascular resistance (R₀) compared with WT. Pre-treatment of KO lungs with S100A1 attenuated the AII-induced increase in pulmonary arterial pressure and R₀. S100A1-induced phosphorylation of eNOS, Akt, and ERK1/2 is attenuated in pulmonary EC of KO compared with WT. Basal and TNF-α-induced EC apoptosis is greater in KO vs. WT, and cell survival is enhanced by S100A1 treatment. CONCLUSION Our data demonstrate that the absence of S100A1 results in PH by disruption of its normal capacity to (i) enhance pulmonary EC function by induction of eNOS activity and NO levels via Akt/ERK1/2 pathways and (ii) promote EC survival. The ability of exogenously administered S100A1 to rescue this phenotype makes it an attractive therapeutic target in the treatment of PH.

Rho Kinase Mediates Right Ventricular Systolic Dysfunction in Rats with Chronic Neonatal Pulmonary Hypertension

Chronic neonatal pulmonary hypertension frequently culminates in right ventricular (RV) failure and death. In juvenile rats, RV systolic dysfunction secondary to chronic hypoxia is rescued by systemic treatment with a Rho kinase (ROCK) inhibitor. To explore the relationship between ROCK inhibitor-mediated decreases in pulmonary vascular resistance and pressure, RV hypertrophy, and systolic dysfunction, we compared the effects of systemically administered to inhaled (pulmonary-selective) ROCK inhibitor on RV systolic function. Rat pups were exposed to air or hypoxia (13% O2) from Postnatal Days 1 to 21 and received rescue treatment with aerosolized fasudil (200 mM) for 15 minutes three times daily or intraperitoneal Y27632 (15 mg/kg twice daily) from Days 14 to 21. Chronic hypoxia differentially increased RhoA and ROCK activity in the right, but not left, cardiac ventricle. Inhaled ROCK inhibitor normalized pulmonary vascular resistance and caused regression of RV hypertrophy and pulmonary arterial wall remodeling but did not improve RV systolic dysfunction (decreased stroke volume and tricuspid annular plane systolic excursion). Systemic, but not inhaled, ROCK inhibitor normalized up-regulated ROCK and phosphodiesterase 5 activities in the right ventricle. Treatment with sildenafil (100 mg/kg/d intraperitoneally from Days 14 to 21) improved RV systolic function. Collectively, these data indicate that pressure unloading and regressed arterial and cardiac remodeling did not lead to recovery of systolic function while right ventricular ROCK activity remained increased. Right ventricle-specific up-regulation of RhoA/ROCK activity is critical to hypoxia-mediated systolic dysfunction, in part by regulating the activity of phosphodiesterase 5.

S100B: Role in cardiac remodeling and function following myocardial infarction in diabetes

AIM S100B plays a role in cardiac remodeling following myocardial infarction (MI) and in diabetic vascular complications but not examined in diabetic myocardium. We thus examined the effects of targeted deletion of S100B gene on post-MI hearts. MAIN METHODS Coronary artery ligation or sham was performed 15 weeks after streptozotocin (STZ) or vehicle injection in wild-type (WT) and S100B knock-out (BKO) mice. Left ventricular (LV) structural and functional remodeling was studied 35 days after induction of MI. KEY FINDINGS In diabetes, post-MI remodeling exhibited an attenuated increase in LV mass, dilation, and myocyte hypertrophy in association with increased apoptosis and fibrosis and reduced matrix metalloproteinase-2 (MMP-2) activity. Despite reduced LV dilation, impairment of cardiac function was similar to non-diabetic controls. Both diabetes and MI alone induced myocardial S100B and its canonical receptor for advanced glycation end product (RAGE) expression. By contrast, in post-MI diabetic myocardium, S100B expression was attenuated. Diabetic BKO, following MI demonstrated increased ventricular dilation compared to WT, in association with greater impairment of cardiac function, GLUT4 expression and systemic AGE levels. SIGNIFICANCE These data suggest that S100B expression may serve to modulate cardiac metabolism and adverse consequences of AGE in diabetic post-MI remodeling and function.

S100A1: A pluripotent regulator of cardiac and vascular function

S100A1, a small EF-hand Ca(2+)-binding protein with intracellular and extracellular functions, is predominantly expressed in cardiac muscle where it plays a crucial role as a modulator of Ca(2+) homeostasis, energy metabolism and contractile performance. Essentially, its beneficial effects on heart function have been attributed to its direct interaction with, and effects on, sarcoplasmic reticulum calcium handling proteins sarco(endo) plasmic reticulum Ca(2+) ATPase and the ryanodine receptor. Downregulated levels of S100A1 in cardiomyocytes postmyocardial infarction have been linked to diminished cardiac reserve and contribute to the development of heart failure. Interestingly, S100A1 expression has recently been described in endothelial cells where it is downregulated in heart failure and has been shown to modulate intracellular Ca(2+) levels and nitric oxide production. Absence of the Ca(2+) sensor protein in endothelial cells is associated with endothelial dysfunction and hypertension. Thus, S100A1 is emerging as a potential therapeutic target for diverse cardiovascular conditions.