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The Activation of Natural Killer T Cells Ameliorates Post-Infarct Heart Failure in Mice
The 14th Annual Scientific Meeting Pharmacy, Ft. Lauderdale, FL; 2Cardiology Division, Fondazione ‘‘Salvatore Maugeri’’, IRCCS, Telese Terme (BN), Italy; 3Dept. of Pharmacy, University of Patras, Patras, Greece; 4Dept. of Medicine, Thomas Jefferson University, Philadelphia, PA We showed recently that activation of the scaffolding protein b-arrestin-1 (barr1) by the angiotensin II (AngII) type 1 receptor (AT1R) mediates AngII-induced aldosterone production in vitro and physiologically in vivo. Herein, we tested the relative potency of various AT1R antagonist drugs (sartans) at inhibiting barr vs. G-protein activation and hence aldosterone production in vitro and in vivo. Additionally, we tested novel AngII ‘‘biased’’ agonist (i.e. that only stimulate barrs and not Gproteins) analogs at stimulating barr activity and aldosterone production. Finally, we investigated the alterations in plasma aldosterone levels conferred by these agents and their impact on cardiac function of post-myocardial infarction (MI) rats. Methods: For in vitro tests, the adrenocortical H295R cell line was used, which produces aldosterone in response to AngII. For in vivo studies, post-MI rats overexpressing barr1 in their adrenals received 7-day-long treatments with the agents of interest. Results: Candesartan and valsartan were the most potent barr activation and barr-dependent aldosterone production inhibitors in vitro, as well as the most ‘‘biased’’ antagonists towards barr inhibition. Conversely, losartan and irbesartan were the least potent and the least ‘‘biased’’. These in vitro findings were corroborated in vivo, since candesartan and valsartan, contrary to irbesartan, significantly reduced plasma aldosterone in post-MI rats. Accordingly, ejection fraction (EF) and contractility were significantly augmented in candesartan- and valsartan-treated rats (EF: 41.1 6 1% and 40 6 1% respectively, vs. 35 6 0.3% for saline-treated), but further deteriorated in irbesartan-treated rats (EF: 32 6 1%, n 5 7 rats/group). Finally, a novel AT1R biased agonist has been identified, CORET ([Sar1,Cys(Et)5, Leu8]-AngII), far more potent at stimulating barr than SII, the standard AT1R biased agonist. Conclusions: Candesartan and valsartan are the most potent sartans at lowering aldosterone and thus ameliorating cardiac function post-MI. Losartan and irbesartan appear the least potent sartans at doing so. CORET is a novel, very potent AT1R-barr biased agonist.
089 Differential Anti-Fibrotic Effects of Vardenafil and Fasudil in the Heart and Kidney of Diabetic Rats Brenda K. Huntley1, Syed Ameenuddin1, Horng H. Chen1; 1Cardiorenal Research, Mayo Clinic, Rochester, MN Introduction: Diabetes is a major risk factor for left ventricular dysfunction with cardiac and renal fibrosis. Fasudil is a Rho kinase inhibitor shown to prevent the development of nephropathy in diabetic (DM) rats. Vardenafil, a phosphodiesterase-5 (PDE V) inhibitor, also impacts the RhoA/Rho Kinase pathway and has been shown to improve cardiovascular dysfunction in DM rats. We examined the differential effects of these treatments on heart and kidney fibrosis and associated Rho Kinase pathway involvement. Hypothesis: We hypothesized that Fasudil and Vardenafil would both affect RhoA/Rho Kinase signaling in DM rats, but at different pathway levels with distinct heart and kidney profiles. Methods: DM was induced in male Wistar rats with Streptozotocin (65 mg/Kg). Four weeks after establishing DM, rats were divided into groups; one received no treatment (n-6), one (n 5 5) received daily Fasudil for two months at 100mg/kg/day and one received Vardenafil for two months at 5mg/kg/day. A non-diabetic control (n 5 6) was also examined. Myocardial and kidney snap frozen tissue were used for quantitative PCR and protein expression using Western Blot (WB) analysis. Tissue sections were stained for picrosirius red and collagen III. Results: DM rats developed fibrosis in left ventricle (LV) and kidney medulla (KM) compared to non-diabetic controls. Both Fasudil and Vardenafil reduced fibrosis in LV and KM. Fasudil was more effective in the LV, while Vardenafil was more effective in the KM. Importantly, this differential effect on fibrosis resulted in a greater improvement of diastolic function by Fasudil and a greater improvement of renal function by Vardenafil. To assess Rho kinase pathway involvement, full length Rho-associated, coiled-coil containing protein kinase (ROCK 1) and active C-terminal ROCK 1 were assess by WB and the ratio of full length to active forms determined. Fasudil significantly reduced the ROCK 1 ratio in LV tissue, but not in KM. Conversely, Vardenafil did not affect ROCK 1 ratio in LV, but was significantly reduced in KM. Conclusion: We demonstrate that diabetes induces fibrosis in LV and KM. This fibrosis was more greatly attenuated by Fasudil in LV with a greater improvement in diastolic function, and by Vardenafil in KM with a greater improvement in renal function. Our studies suggest that the fibrosis induced in LV by diabetes is different from the fibrosis induced in KM and responds differently to two different treatments. Both may involve the RhoA/ Rho kinase pathway but at distinct pathway levels.
090 The Activation of Natural Killer T Cells Ameliorates Post-Infarct Heart Failure in Mice Mochamad Ali Sobirin1, Shintaro Kinugawa1, Taisuke Ono1, Kagami Hirabayashi1, Tadashi Suga1, Takashi Yokota1, Naoki Ishimori1, Kazuya Iwabuchi2, Hiroyuki Tsutsui1; 1Cardiovascular Medicine, Hokkaido University Graduate School of
HFSA
S31
Medicine, Sapporo, Hokkaido, Japan; 2Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan Background: Chronic inflammation has been reported to be involved in the development of left ventricular (LV) remodeling and failure after myocardial infarction (MI). Natural killer T (NKT) cells have capacity to produce various inflammatory cytokines and orchestrate tissue inflammation. However, no previous studies have examined the pathophysiological role of NKT cells in post-MI heart failure. Methods and Results: MI or sham operation was performed in mice. Mice received the injection of either a-galactosylceramide (a-GalCer), which specifically activates NKT cells, or vehicle 1 and 4 days after surgery, and were observed during 28 days. Survival rate was significantly higher in MI + a-GalCer than MI (59.3% (16/27) vs 32.3% (10/31), P ! 0.05). LV end-diastolic dimension was smaller (5.0 6 0.1 vs 5.4 6 0.1 mm, p ! 0.01), and LV fractional shortening was greater (18.8 6 0.6 vs 16.5 6 0.6%, p ! 0.05) in MI + a-GalCer than MI, with no significant changes in infarct size and aortic pressure between groups. LV end-diastolic pressure and lung weight/body weight in MI + a-GalCer were lower than those in MI. The injection of a-GalCer to sham mice did not affect cardiac function and structure. Improvement of LV function in MI + a-GalCer was accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis. NKT cell receptor mRNA expression was increased during the early phase in non-infarcted LV from MI and a-GalCer injection further enhanced its expression. It also enhanced LV interleukin-10 mRNA at 7 days, which persisted until 28 days. Conclusions: NKT cells may play a protective role against post-MI LV remodeling and failure through the enhanced expression of cardioprotective cytokines such as interleukin-10.
091 Plasma Renin Activity (PRA) on Admission Predicts Diuretic Resistance and Mortality in Decompensated Heart Failure (HF) Patients with Renal Dysfunction (RD) Tomohito Ohtani1, Selma F. Mohammed1, Kalkidan G. Bishu1, Horng H. Chen1, John C. Burnett, Jr.1, Margaret M. Redfield1; 1Cardiovascular Diseases, Mayo Clinic and Foundation, Rochester, MN Introduction: Identification of patients at increased risk for diuretic resistance and mortality upon admission for decompensated HF may allow early use of more advanced therapies. We hypothesized that increased PRA may reflect sodium avidity and HF severity and thus predict diuretic resistance and mortality in decompensated HF patients with RD. Methods: Patients (n 5 70) with decompensated HF and RD (20!GFR!60 ml/min) were enrolled and humoral function was
High PRA
Low PRA
Age (yrs) Ejection Fraction (%) GFR (mL/min/1.73m2) BNP (pg/ml) ACEI or ARB medication (%) PRA (ng/ml/hr) Aldosterone (pg/ml) Angiotensin II (pg/ml)
6 6 6 6 60 16.0 6 17.9 6 12.9 6
8 17 14 392
10 18 18 595
9.8 26.5 15.2
73 6 33 6 35 6 677 6 74 0.8 6 9.8 6 3.5 6
0.9 9.2 4.5
0.11 0.88 0.75 0.32 0.20 na 0.09 !0.01
Cumulative Cumulative Cumulative Cumulative
299 2.1 456 3.1
6 6 6 6
118 1.8 202 2.1
231 4.0 318 4.7
6 6 6 6
96 2.5 164 3.4
0.01 !0.001 !0.001 0.02
Furosemide - 48hours (mg) Fluid loss - 48 hours (L) Furosemide - 72hours (mg) Fluid Loss - 72 hours (L)
77 34 34 556
p
089 Differential Anti-Fibrotic Effects of Vardenafil and Fasudil in the Heart and Kidney of Diabetic Rats Brenda K. Huntley1, Syed Ameenuddin1, Horng H. Chen1; 1Cardiorenal Research, Mayo Clinic, Rochester, MN Introduction: Diabetes is a major risk factor for left ventricular dysfunction with cardiac and renal fibrosis. Fasudil is a Rho kinase inhibitor shown to prevent the development of nephropathy in diabetic (DM) rats. Vardenafil, a phosphodiesterase-5 (PDE V) inhibitor, also impacts the RhoA/Rho Kinase pathway and has been shown to improve cardiovascular dysfunction in DM rats. We examined the differential effects of these treatments on heart and kidney fibrosis and associated Rho Kinase pathway involvement. Hypothesis: We hypothesized that Fasudil and Vardenafil would both affect RhoA/Rho Kinase signaling in DM rats, but at different pathway levels with distinct heart and kidney profiles. Methods: DM was induced in male Wistar rats with Streptozotocin (65 mg/Kg). Four weeks after establishing DM, rats were divided into groups; one received no treatment (n-6), one (n 5 5) received daily Fasudil for two months at 100mg/kg/day and one received Vardenafil for two months at 5mg/kg/day. A non-diabetic control (n 5 6) was also examined. Myocardial and kidney snap frozen tissue were used for quantitative PCR and protein expression using Western Blot (WB) analysis. Tissue sections were stained for picrosirius red and collagen III. Results: DM rats developed fibrosis in left ventricle (LV) and kidney medulla (KM) compared to non-diabetic controls. Both Fasudil and Vardenafil reduced fibrosis in LV and KM. Fasudil was more effective in the LV, while Vardenafil was more effective in the KM. Importantly, this differential effect on fibrosis resulted in a greater improvement of diastolic function by Fasudil and a greater improvement of renal function by Vardenafil. To assess Rho kinase pathway involvement, full length Rho-associated, coiled-coil containing protein kinase (ROCK 1) and active C-terminal ROCK 1 were assess by WB and the ratio of full length to active forms determined. Fasudil significantly reduced the ROCK 1 ratio in LV tissue, but not in KM. Conversely, Vardenafil did not affect ROCK 1 ratio in LV, but was significantly reduced in KM. Conclusion: We demonstrate that diabetes induces fibrosis in LV and KM. This fibrosis was more greatly attenuated by Fasudil in LV with a greater improvement in diastolic function, and by Vardenafil in KM with a greater improvement in renal function. Our studies suggest that the fibrosis induced in LV by diabetes is different from the fibrosis induced in KM and responds differently to two different treatments. Both may involve the RhoA/ Rho kinase pathway but at distinct pathway levels.
090 The Activation of Natural Killer T Cells Ameliorates Post-Infarct Heart Failure in Mice Mochamad Ali Sobirin1, Shintaro Kinugawa1, Taisuke Ono1, Kagami Hirabayashi1, Tadashi Suga1, Takashi Yokota1, Naoki Ishimori1, Kazuya Iwabuchi2, Hiroyuki Tsutsui1; 1Cardiovascular Medicine, Hokkaido University Graduate School of
HFSA
S31
Medicine, Sapporo, Hokkaido, Japan; 2Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan Background: Chronic inflammation has been reported to be involved in the development of left ventricular (LV) remodeling and failure after myocardial infarction (MI). Natural killer T (NKT) cells have capacity to produce various inflammatory cytokines and orchestrate tissue inflammation. However, no previous studies have examined the pathophysiological role of NKT cells in post-MI heart failure. Methods and Results: MI or sham operation was performed in mice. Mice received the injection of either a-galactosylceramide (a-GalCer), which specifically activates NKT cells, or vehicle 1 and 4 days after surgery, and were observed during 28 days. Survival rate was significantly higher in MI + a-GalCer than MI (59.3% (16/27) vs 32.3% (10/31), P ! 0.05). LV end-diastolic dimension was smaller (5.0 6 0.1 vs 5.4 6 0.1 mm, p ! 0.01), and LV fractional shortening was greater (18.8 6 0.6 vs 16.5 6 0.6%, p ! 0.05) in MI + a-GalCer than MI, with no significant changes in infarct size and aortic pressure between groups. LV end-diastolic pressure and lung weight/body weight in MI + a-GalCer were lower than those in MI. The injection of a-GalCer to sham mice did not affect cardiac function and structure. Improvement of LV function in MI + a-GalCer was accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis. NKT cell receptor mRNA expression was increased during the early phase in non-infarcted LV from MI and a-GalCer injection further enhanced its expression. It also enhanced LV interleukin-10 mRNA at 7 days, which persisted until 28 days. Conclusions: NKT cells may play a protective role against post-MI LV remodeling and failure through the enhanced expression of cardioprotective cytokines such as interleukin-10.
091 Plasma Renin Activity (PRA) on Admission Predicts Diuretic Resistance and Mortality in Decompensated Heart Failure (HF) Patients with Renal Dysfunction (RD) Tomohito Ohtani1, Selma F. Mohammed1, Kalkidan G. Bishu1, Horng H. Chen1, John C. Burnett, Jr.1, Margaret M. Redfield1; 1Cardiovascular Diseases, Mayo Clinic and Foundation, Rochester, MN Introduction: Identification of patients at increased risk for diuretic resistance and mortality upon admission for decompensated HF may allow early use of more advanced therapies. We hypothesized that increased PRA may reflect sodium avidity and HF severity and thus predict diuretic resistance and mortality in decompensated HF patients with RD. Methods: Patients (n 5 70) with decompensated HF and RD (20!GFR!60 ml/min) were enrolled and humoral function was
High PRA
Low PRA
Age (yrs) Ejection Fraction (%) GFR (mL/min/1.73m2) BNP (pg/ml) ACEI or ARB medication (%) PRA (ng/ml/hr) Aldosterone (pg/ml) Angiotensin II (pg/ml)
6 6 6 6 60 16.0 6 17.9 6 12.9 6
8 17 14 392
10 18 18 595
9.8 26.5 15.2
73 6 33 6 35 6 677 6 74 0.8 6 9.8 6 3.5 6
0.9 9.2 4.5
0.11 0.88 0.75 0.32 0.20 na 0.09 !0.01
Cumulative Cumulative Cumulative Cumulative
299 2.1 456 3.1
6 6 6 6
118 1.8 202 2.1
231 4.0 318 4.7
6 6 6 6
96 2.5 164 3.4
0.01 !0.001 !0.001 0.02
Furosemide - 48hours (mg) Fluid loss - 48 hours (L) Furosemide - 72hours (mg) Fluid Loss - 72 hours (L)
77 34 34 556
p