Jane E Love

Cardiac-Specific IGF-1 Receptor Transgenic Expression Protects Against Cardiac Fibrosis and Diastolic Dysfunction in a Mouse Model of Diabetic Cardiomyopathy

OBJECTIVE Compelling epidemiological and clinical evidence has identified a specific cardiomyopathy in diabetes, characterized by early diastolic dysfunction and adverse structural remodeling. Activation of the insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) promotes physiological cardiac growth and enhances contractile function. The aim of the present study was to examine whether cardiac-specific overexpression of IGF-1R prevents diabetes-induced myocardial remodeling and dysfunction associated with a murine model of diabetes. RESEARCH DESIGN AND METHODS Type 1 diabetes was induced in 7-week-old male IGF-1R transgenic mice using streptozotocin and followed for 8 weeks. Diastolic and systolic function was assessed using Doppler and M-mode echocardiography, respectively, in addition to cardiac catheterization. Cardiac fibrosis and cardiomyocyte width, heart weight index, gene expression, Akt activity, and IGF-1R protein content were also assessed. RESULTS Nontransgenic (Ntg) diabetic mice had reduced initial (E)-to-second (A) blood flow velocity ratio (E:A ratio) and prolonged deceleration times on Doppler echocardiography compared with nondiabetic counterparts, indicative markers of diastolic dysfunction. Diabetes also increased cardiomyocyte width, collagen deposition, and prohypertrophic and profibrotic gene expression compared with Ntg nondiabetic littermates. Overexpression of the IGF-1R transgene markedly reduced collagen deposition, accompanied by a reduction in the incidence of diastolic dysfunction. Akt phosphorylation was elevated ∼15-fold in IGF-1R nondiabetic mice compared with Ntg, and this was maintained in a setting of diabetes. CONCLUSIONS The current study suggests that cardiac overexpression of IGF-1R prevented diabetes-induced cardiac fibrosis and diastolic dysfunction. Targeting IGF-1R–Akt signaling may represent a therapeutic target for the treatment of diabetic cardiac disease.

Targeting the upregulation of reactive oxygen species subsequent to hyperglycemia prevents type 1 diabetic cardiomyopathy in mice

Cardiac oxidative stress is an early event associated with diabetic cardiomyopathy, triggered by hyperglycemia. We tested the hypothesis that targeting left-ventricular (LV) reactive oxygen species (ROS) upregulation subsequent to hyperglycemia attenuates type 1 diabetes-induced LV remodeling and dysfunction, accompanied by attenuated proinflammatory markers and cardiomyocyte apoptosis. Male 6-week-old mice received either streptozotocin (55mg/kg/day for 5 days), to induce type 1 diabetes, or citrate buffer vehicle. After 4 weeks of hyperglycemia, the mice were allocated to coenzyme Q10 supplementation (10mg/kg/day), treatment with the angiotensin-converting-enzyme inhibitor (ACE-I) ramipril (3mg/kg/day), treatment with olive oil vehicle, or no treatment for 8 weeks. Type 1 diabetes upregulated LV NADPH oxidase (Nox2, p22(phox), p47(phox) and superoxide production), LV uncoupling protein UCP3 expression, and both LV and systemic oxidative stress (LV 3-nitrotyrosine and plasma lipid peroxidation). All of these were significantly attenuated by coenzyme Q10. Coenzyme Q10 substantially limited type 1 diabetes-induced impairments in LV diastolic function (E:A ratio and deceleration time by echocardiography, LV end-diastolic pressure, and LV -dP/dt by micromanometry), LV remodeling (cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis), and LV expression of proinflammatory mediators (tumor necrosis factor-α, with a similar trend for interleukin IL-1β). Coenzyme Q10s actions were independent of glycemic control, body mass, and blood pressure. Coenzyme Q10 compared favorably to improvements observed with ramipril. In summary, these data suggest that coenzyme Q10 effectively targets LV ROS upregulation to limit type 1 diabetic cardiomyopathy. Coenzyme Q10 supplementation may thus represent an effective alternative to ACE-Is for the treatment of cardiac complications in type 1 diabetic patients.

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation.

Background New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated. Methods Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angelis salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. Results We now demonstrate that Angelis salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angelis salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angelis salt were mimicked by BNP. We also demonstrate that the effects of Angelis salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP). Conclusions Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.

The Soluble Guanylyl Cyclase Activator Bay 58-2667 Selectively Limits Cardiomyocyte Hypertrophy

Background Although evidence now suggests cGMP is a negative regulator of cardiac hypertrophy, the direct consequences of the soluble guanylyl cyclase (sGC) activator BAY 58-2667 on cardiac remodeling, independent of changes in hemodynamic load, has not been investigated. In the present study, we tested the hypothesis that the NO•-independent sGC activator BAY 58-2667 inhibits cardiomyocyte hypertrophy in vitro. Concomitant impact of BAY 58-2667 on cardiac fibroblast proliferation, and insights into potential mechanisms of action, were also sought. Results were compared to the sGC stimulator BAY 41-2272. Methods Neonatal rat cardiomyocytes were incubated with endothelin-1 (ET1, 60nmol/L) in the presence and absence of BAY 41-2272 and BAY 58-2667 (0.01–0.3 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. The impact of both sGC ligands on basal and stimulated cardiac fibroblast proliferation in vitro was also determined. Results We now demonstrate that BAY 58-2667 (0.01–0.3 µmol/L) elicited concentration-dependent antihypertrophic actions, inhibiting ET1-mediated increases in cardiomyocyte 2D area and de novo protein synthesis, as well as suppressing ET1-induced cardiomyocyte superoxide generation. This was accompanied by potent increases in cardiomyocyte cGMP accumulation and activity of its downstream signal, vasodilator-stimulated phosphoprotein (VASP), without elevating cardiomyocyte cAMP. In contrast, submicromolar concentrations of BAY 58-2667 had no effect on basal or stimulated cardiac fibroblast proliferation. Indeed, only at concentrations ≥10 µmol/L was inhibition of cardiac fibrosis seen in vitro. The effects of BAY 58-2667 in both cell types were mimicked by BAY 41-2272. Conclusions Our results demonstrate that BAY 58-2667 elicits protective, cardiomyocyte-selective effects in vitro. These actions are associated with sGC activation and are evident in the absence of confounding hemodynamic factors, at low (submicromolar) concentrations. Thus this distinctive sGC ligand may potentially represent an alternative therapeutic approach for limiting myocardial hypertrophy.

Reperfusion‐induced myocardial dysfunction is prevented by endogenous annexin‐A1 and its N‐terminal‐derived peptide Ac‐ANX‐A12‐26

Annexin‐A1 (ANX‐A1) is an endogenous, glucocorticoid‐regulated anti‐inflammatory protein. The N‐terminal‐derived peptide Ac‐ANX‐A12–26 preserves cardiomyocyte viability, but the impact of ANX‐A1‐peptides on cardiac contractility is unknown. We now test the hypothesis that ANX‐A1 preserves post‐ischaemic recovery of left ventricular (LV) function.

HNO/cGMP-dependent antihypertrophic actions of isopropylamine-NONOate in neonatal rat cardiomyocytes: potential therapeutic advantages of HNO over NO˙

Nitroxyl (HNO) is a redox congener of NO. We now directly compare the antihypertrophic efficacy of HNO and NO donors in neonatal rat cardiomyocytes and compare their contributing mechanisms of actions in this setting. Isopropylamine-NONOate (IPA-NO) elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by l-cysteine (HNO scavenger), Rp-8-pCTP-cGMPS (cGMP-dependent protein kinase inhibitor), and 1-H-(1,2,4)-oxodiazolo-quinxaline-1-one [ODQ; to target soluble guanylyl cyclase (sGC)] but were unaffected by carboxy-PTIO (NO scavenger) or CGRP8-37 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an l-cysteine-sensitive effect) and stimulated sGC activity threefold, without detectable NO release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO donor diethylamine-NONOate (DEA-NO) reproduced these IPA-NO actions but was sensitive to carboxy-PTIO rather than l-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor was attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to pressure overload in the intact heart. Together these data strongly suggest that IPA-NO protection against cardiomyocyte hypertrophy is independent of both NO and CGRP but rather utilizes novel HNO activation of cGMP signaling. Thus HNO acutely limits hypertrophy independently of NO, even under conditions of elevated superoxide. Development of longer-acting HNO donors may thus represent an attractive new strategy for the treatment of cardiac hypertrophy, as stand-alone and/or add-on therapy to standard care.

Earlier onset of diabesity-Induced adverse cardiac remodeling in female compared to male mice: Adverse Cardiac Remodeling indb/dbMice

Emerging evidence suggests female type 2 diabetes (T2DM) patients may fare worse than males with respect to cardiovascular complications. Hence the impact of sex on relative progression of left ventricular (LV) remodeling in obese db/db mice was characterized.

The nitroxyl donor isopropylamine-NONOate elicits soluble guanylyl cyclase-dependent antihypertrophic actions: comparison of the potential therapeutic advantages of HNO over NO•

Results IPA-NO elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in neonatal rat cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by L-cysteine (HNO scavenger), Rp-8-pCTPcGMPS (cGMP-dependent protein kinase inhibitor), and ODQ (to target soluble guanylyl cyclase, sGC), but were unaffected by carboxy-PTIO (NO• scavenger) or CGRP837 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an L-cysteine-sensitive effect), and stimulated sGC activity 3-fold, without detectable NO• release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO• donor, DEA-NO, reproduced these IPA-NO actions, but were sensitive to carboxy-PTIO rather than L-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor were attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to acute pressureoverload in the intact heart, at doses that did not affect coronary perfusion pressure or LV function.

The novel NO redox sibling, nitroxyl (HNO), prevents cardiomyocyte hypertrophy and superoxide generation via cGMP

We have previously shown that NO•/cGMP signalling is an important antihyper-trophic mechanism in the heart [1-3]. HNO is the one electron reduction of NO•, thought to elicit cardiovascular actions via cGMP and/or calcitonin gene-related peptide (CGRP) [4]; we have recently shown that the HNO donor Angelis salt inhibits cardiomyocyte hypertrophy and superoxide generation [5]. We now test the hypothesis that isopropylamine/NO (IPA/NO) elicits concentration-dependent anti-hypertrophic and antioxidant actions via HNO/sGC/cGMP-dependent signalling. IPA/NO (0.1–3 μM, replenished 3×/day over 48 h) elicited concentration-dependent inhibition of endothelin-1 (ET1, 60 nM)-stimulated neonatal rat cardiomyocyte (NRCM) hypertrophy (on two dimensional area of live cells). At 3 μM, IPA/NO decreased cell size from 255 ± 28% to 96 ± 27% of paired control (n = 4, p < 0.001). This antihypertrophic action of IPA/NO was significantly attenuated in the presence of the HNO scavenger L-cysteine (3 mM) or the cGMP-dependent protein kinase inhibitor Rp-8 PCTP cGMPS (10 μM, both n = 4, p < 0.05), but was unaffected by the NO scavenger carboxy-PTIO (200 μM) or the CGRP antagonist, CGRP8–37 (1 μM, both n = 4). For comparison, the NO• donor DEA/NO elicited similar concentration-dependent inhibition of ET1-induced cardiomyocyte hypertrophy; this was inhibited by carboxy-PTIO and Rp-8 PCTP cGMPS (10 μM, both n = 4, p < 0.05), but was unaffected by L-cysteine. Both IPA/NO and DEA/NO also blocked ET1-induced cardiomyocyte superoxide generation (both n = 4, p < 0.001, on NADPH-driven lucigenin-enhanced chemiluminescence), a key trigger of hypertrophy [3]. IPA-NO stimulated purified cell-free sGC activity by 3.2 ± 0.6-fold, and elevated NRCM cGMP content by 3.5 ± 0.4-fold (both n = 5, p < 0.05 via cGMP ELISA, as previously described [2,3]. None of these agents alone, or their respective vehicles, elicited any effect on NRCM. Finally, using an NO•-sensing electrode, we demonstrated that IPA/NO (in contrast to DEA/NO), does not release NO• under these conditions, even at supra-pharmacological concentrations. In conclusion, these results provide convincing evidence that IPA/NO prevents cardiomyocyte hypertrophy via HNO activation of sGC. Although the antihypertrophic and antioxidant efficacy of IPA/NO was comparable to NO•, there is no role for extracellular oxidation of HNO to NO• or CGRP-mediated signalling in these IPA/NO actions. These studies may ultimately facilitate the development of HNO donors such as IPA/NO as novel antihypertrophic therapy for patients at risk of heart failure.

Antihypertrophic actions of NO-independent soluble guanylyl cyclase (sGC) ligands BAY 41-2272 and BAY 58-2667 in vitro

Background Over the last decade, we have shown that cGMP, derived from bradykinin, nitric oxide (NO•, both from endogenous and exogenous sources) or natriuretic peptides, is a potent inhibitor of cardiac hypertrophy, across isolated cardiomyocytes and intact hearts both ex vivo and in vivo. However, NO• bioavailability is reduced due to scavenging by ROS; furthermore, oxidation of sGC may result in sGC dysfunction (including loss of responsiveness to NO•). In the present study, we tested the hypothesis that the NO•-independent sGC stimulator BAY 41-2272 and the NO-independent sGC activator BAY 58-2667 elicit powerful antihypertrophic actions.