Altered acylcarnitine metabolism and inflexible mitochondrial fuel utilization characterize the loss of neonatal myocardial regeneration capacity.

Myocardial regeneration capacity declines during the first week after birth, and this decline is linked to adaptation to oxidative metabolism. Utilizing this regenerative window, we characterized the metabolic changes in myocardial injury in 1-day-old regeneration-competent and 7-day-old regeneration-compromised mice. The mice were either sham-operated or received left anterior descending coronary artery ligation to induce myocardial infarction (MI) and acute ischemic heart failure. Myocardial samples were collected 21 days after operations for metabolomic, transcriptomic and proteomic analyses. Phenotypic characterizations were carried out using echocardiography, histology and mitochondrial structural and functional assessments. In both groups, MI induced an early decline in cardiac function that persisted in the regeneration-compromised mice over time. By integrating the findings from metabolomic, transcriptomic and proteomic examinations, we linked regeneration failure to the accumulation of long-chain acylcarnitines and insufficient metabolic capacity for fatty acid beta-oxidation. Decreased expression of the redox-sensitive mitochondrial Slc25a20 carnitine-acylcarnitine translocase together with a decreased reduced:oxidized glutathione ratio in the myocardium in the regeneration-compromised mice pointed to a defect in the redox-sensitive acylcarnitine transport to the mitochondrial matrix. Rather than a forced shift from the preferred adult myocardial oxidative fuel source, our results suggest the facilitation of mitochondrial fatty acid transport and improvement of the beta-oxidation pathway as a means to overcome the metabolic barrier for repair and regeneration in adult mammals after MI and heart failure.

Caloric restriction ameliorates kidney ischaemia/reperfusion injury through PGC-1α-eNOS pathway and enhanced autophagy.

AIM: We investigated whether preconditioning with caloric restriction (CR) ameliorates kidney ischaemia/reperfusion (I/R) injury and whether the salutary effects of CR are mediated through enhanced autophagy and/or activation of key metabolic sensors SIRT1, AMP-kinase and PGC-1α. METHODS: Six- to seven-week-old Wistar rats were divided into three groups: (i) sham-operated group; (ii) I/R group (40-min ischaemia followed by 24 h of reperfusion); and (iii) I/R group kept under CR (energy intake 70%) for 2 weeks before surgery. In additional experiments, sirtinol and 3-methyladenine (3-MA) were used as inhibitors of SIRT1 and autophagy respectively. Renal function was measured, and acute tubular damage and nitrotyrosine expression were scored. Kidney adenosine monophosphate-activated kinase (AMPK), SIRT1, eNOS, PGC-1α and LC-3B expressions were measured. RESULTS: Caloric restriction improved renal function, protected against the development of acute tubular necrosis and attenuated I/R-induced nitrosative stress. Kidney I/R injury decreased eNOS and PGC-1α expression, inhibit autophagy and increased SIRT1 and AMPK expressions by 2.6- and fourfold respectively. However, phosphorylation level of AMPK was decreased. As compared with I/R injury group, CR further increased kidney SIRT1 expression by 1.8-fold, promoted autophagy and counteracted I/R-induced decreases in the expression of eNOS and PGC-1α. 3-MA abolished the renoprotective effects of CR, whereas sirtinol did not influence renal function in CR rats with I/R injury. CONCLUSIONS: Caloric restriction ameliorates acute kidney I/R injury through enhanced autophagy and counteraction of I/R-induced decreases in the renal expression of eNOS and PGC-1α.

AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney.

BACKGROUND AND PURPOSE Renal ischaemia/reperfusion (RI/R) injury is a major cause of acute kidney injury (AKI) and an important determinant of long-term kidney dysfunction. AMP-kinase and histone deacetylase sirtuin 1 (SIRT1) regulate cellular metabolism and are activated during hypoxia. We investigated whether AMP-kinase activator AICAR (5-amino-4-imidazolecarboxamide riboside-1-ß-D-ribofuranoside) ameliorates RI/R injury and whether SIRT1 is involved in the pathogenesis. EXPERIMENTAL APPROACH Eight-week-old Sprague Dawley rats were divided into five groups: (i) sham-operated group; (ii) I/R group (40 min bilateral ischaemia followed by 24 h of reperfusion; (iii) I/R group + AICAR 50 mg·kg(-1) i.v. given 60 min before operation; (iv). I/R group + AICAR 160 mg·kg(-1) i.v; (v) I/R group + AICAR 500 mg·kg(-1) i.v. Serum creatinine and urea levels were measured. Acute tubular necrosis (ATN), monocyte/macrophage infiltration and nitrotyrosine expression were scored. Kidney AMP-activated protein kinase (AMPK) and SIRT1 expressions were measured. KEY RESULTS Highest dose of AICAR decreased serum creatinine and urea levels, attenuated I/R injury-induced nitrosative stress and monocyte/macrophage infiltration, and ameliorated the development of ATN. Kidney I/R injury was associated with decreased AMPK phosphorylation and a fivefold increase in kidney SIRT1 expression. AICAR increased pAMPK/AMPK ratio and prevented the I/R-induced increase in renal SIRT1 expression. CONCLUSIONS AND IMPLICATIONS AICAR protects against the development of ATN after kidney I/R injury. Activators of kidney AMP kinase may thus represent a novel therapeutic approach to patients susceptible to AKI and to those undergoing kidney transplantation. The present study also suggests a role for SIRT1 in the pathogenesis of RI/R injury.

Cardiovascular effects of the combination of levosimendan and valsartan in hypertensive Dahl/Rapp rats.

Hypertension is the main risk factor for left ventricular hypertrophy and development of diastolic heart failure. There is no yet treatment, which can effectively reduce mortality in patients suffering from heart failure with preserved systolic function. We tested whether the calcium sensitizer levosimendan and the AT1-receptor antagonist valsartan could protect from salt-induced hypertension, cardiovascular mortality and heart failure in Dahl/Rapp salt-sensitive rats fed for 7 weeks with a high salt diet (8% NaCl). Levosimendan (1 mg/kg/day via drinking water) and valsartan (30 mg/kg in the food) monotherapies and their combination prevented mortality in Dahl/Rapp rats. The drug combination evoked an additive effect on blood pressure, cardiac hypertrophy, cardiomyocyte cross-sectional area, target organ damage and myocardial ANP mRNA expression. There was a close correlation between systolic blood pressure and cardiac hypertrophy, cardiac and renal damage. As compared to Dahl/Rapp controls kept on low-salt diet (NaCl 0.3%). The high salt rats exhibited impaired diastolic relaxation as assessed by isovolumic relaxation time. Levosimendan alone and in combination with valsartan, improved diastolic relaxation without significantly improving systolic function. Our findings are evidence for an additive effect between levosimendan and valsartan on blood pressure and a blood pressure-dependent protection against the development of salt-induced target organ damage. The present study also demonstrates that levosimendan, alone or in combination with valsartan, can correct diastolic dysfunction induced by salt-dependent hypertension.

Effects of calcium sensitizer OR-1986 on a cardiovascular mortality and myocardial remodelling in hypertensive Dahl/Rapp rats.

Calcium-sensitizing agents have been shown to improve cardiac function in patients suffering from acute decompensated heart failure, however, their long-term effects on cardiac remodeling and cardiovascular mortality are still largely unknown. In the present study we tested the hypothesis whether OR-1896, an active and long-lasting metabolite of calcium sensitizer levosimendan, prevents cardiovascular mortality and hypertension-induced myocardial remodelling in salt-sensitive Dahl/Rapp rats. OR-1896 was given orally to Dahl/Rapp SS rats on high-salt diet (NaCl 7% w/w) for 7 weeks at two different doses (0.5 and 0.05 mg/kg). OR-1896 prevented salt-induced cardiovascular mortality (survival rate 75 % in OR-1896 treated groups vs 38 % in untreated controls, p<0.01), ameliorated cardiac hypertrophy and improved systolic functions of the heart without major influence on systemic blood pressure. OR-1896 also ameliorated salt-induced increase in cardiac ANP mRNA expression and plasma BNP level. Salt-induced cardiac remodelling was associated with 4-fold increase in cardiac p16(INK4a) mRNA expression, a marker of cellular senescence. OR-1896 dose-dependently ameliorated cardiomyocyte senescence. Our findings suggest a therapeutic role for OR-1896 in the prevention of cardiac remodelling in salt-sensitive forms of hypertension. The present study also underscores the importance of cellular senescence in the pathogenesis of salt-induced hypertensive heart disease.

Effects of levosimendan on cardiac remodeling and cardiomyocyte apoptosis in hypertensive Dahl/Rapp rats.

BACKGROUND AND PURPOSE: Progression of heart failure in hypertensive Dahl rats is associated with cardiac remodeling and increased cardiomyocyte apoptosis. This study was conducted to study whether treatment with a novel inotropic vasodilator compound, levosimendan, could prevent hypertension-induced cardiac remodeling and cardiomyocyte apoptosis. EXPERIMENTAL APPROACH: 6-week-old salt-sensitive Dahl/Rapp rats received levosimendan (0.3 mg kg(-1) and 3 mg kg(-1) via drinking fluid) and high salt diet (NaCl 7%) for 7 weeks, Dahl/Rapp rats on low-salt diet served as controls. Blood pressure, cardiac functions by echocardiography, cardiomyocyte apoptosis by TUNEL technique, tissue morphology, myocardial expression of calcium cycling proteins, and markers of neurohumoral activation were determined. KEY RESULTS: Untreated Dahl/Rapp rats on high salt diet developed severe hypertension, cardiac hypertrophy and moderate systolic dysfunction. 38% of Dahl/Rapp rats (9/24) survived the 7-week-follow-up period. Cardiomyocyte apoptosis was increased by 6-fold during high salt diet. Levosimendan improved survival (survival rates in low- and high-dose levosimendan groups 12/12 and 9/12, p<0.001 and p=0.05, respectively), increased cardiac function, and ameliorated cardiac hypertrophy. Levosimendan dose-dependently prevented cardiomyocyte apoptosis. Levosimendan normalized salt-induced increased expression of natriuretic peptide, and decreased urinary noradrenaline excretion. Levosimendan also corrected salt-induced decreases in myocardial SERCA2a protein expression and myocardial SERCA2a/NCX-ratio. CONCLUSIONS AND IMPLICATIONS: Improved survival by the novel inotropic vasodilator levosimendan in hypertensive Dahl/Rapp rats is mediated, at least in part, by amelioration of hypertension-induced cardiac remodeling and cardiomyocyte apoptosis.

Sustained cardiomyocyte apoptosis and left ventricular remodelling after myocardial infarction in experimental diabetes.

AIMS/HYPOTHESIS: Diabetes is known to reduce survival after myocardial infarction. Our aim was to examine whether diabetes is associated with enhanced cardiomyocyte apoptosis and thus interferes with the post-infarction remodelling process in myocardium in rat. METHODS: Four weeks after intravenous streptozotocin (diabetic groups) or citrate buffer (controls) injection, myocardial infarction was produced by ligation of left descending coronary artery. Level of cardiomyocyte apoptosis was quantified by TUNEL and caspase-3 methods. Collagen volume fraction and connective tissue growth factor were determined under microscope. Left ventricular dimensions were evaluated by echocardiography and planimetry. RESULTS: The number of apoptotic cardiomyocytes was equally high in diabetic and non-diabetic rats after 1 week from infarction. At 12 weeks after infarction the number of apoptotic cells was higher in the diabetic as compared to non-diabetic rats both in the border zone of infarction and in non-infarcted area. Correspondingly, left ventricular end diastolic diameter, relative cardiac weight, connective tissue growth factor-expression and fibrosis were increased in diabetic compared with non-diabetic rats with myocardial infarction. CONCLUSION/INTERPRETATION: Sustained cardiomyocyte apoptosis, left ventricular enlargement, increased cardiac fibrosis and enhanced profibrogenic connective tissue growth factor expression were detected after myocardial infarction in experimental diabetes. Apoptotic myocyte loss could be an important mechanism contributing to progressive dilatation of the heart and poor prognosis after myocardial infarction in diabetes.

Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet.

BACKGROUND: The introduction of cyclosporine (CsA) has led to an improvement in the prognosis of solid organ transplantation. However, drug-induced hypertension and nephrotoxicity, associated with the development of atherosclerosis and coronary heart disease, still worsen the long-term outcome of CsA-treated patients. Whether the CsA-induced myocardial changes are associated with the induction of connective tissue growth factor (CTGF), a recently found polypeptide implicated in extracellular matrix synthesis, is not known. METHODS: Spontaneously hypertensive rats (8-9 weeks old) were treated with CsA (5 mg x kg(-1) x d(-1) subcutaneously) for 6 weeks. The influence of angiotensin-converting enzyme inhibition (enalapril 30 mg x kg(-1) x d(-1) orally) and angiotensin-1 receptor blockade (valsartan 3 and 30 mg x kg(-1) x d(-1) orally) on CsA toxicity was also investigated. Myocardial morphology was examined, and vascular lesions were scored. Localization and the quantitative expression of CTGF, as well as collagen I and collagen III, mRNA were evaluated by in situ hybridization and Northern blot. RESULTS: CsA-induced hypertension and nephrotoxicity were associated with myocardial infarcts and vasculopathy of the coronary arteries. CsA increased myocardial CTGF, collagen I, and collagen III mRNA expressions by 91%, 198%, and 151%, respectively. CTGF mRNA expression colocalized with the myocardial lesions. Blockade of the renin-angiotensin system prevented vascular damage and the CsA-induced CTGF, collagen I, and collagen III mRNA overexpressions in the heart. CONCLUSIONS: CsA increases CTGF, collagen I, and collagen III mRNA expressions in the heart. The induction of CTGF gene is mediated, at least in part, by angiotensin II.

Vascular changes in cyclosporine A-induced hypertension and nephrotoxicity in spontaneously hypertensive rats on high-sodium diet.

Functional and morphological changes of blood vessels in cyclosporine A (CsA)-induced hypertension and nephrotoxicity were studied in spontaneously hypertensive rats (SHR). The role of the L-arginine-nitric oxide (NO) pathway and the importance of oxidative stress in CsA toxicity were also assessed. SHR (7-8 week old) on a high-sodium diet were treated with CsA (5 mg kg(-1) d(-1) s.c.) for 6 weeks. A proportion of the rats were treated concomitantly with the NO precursor L-arginine (1.7 g kg(-1)d(-1) p.o.). CsA elevated blood pressure and caused renal dysfunction and morphological nephrotoxicity. CsA also impaired mesenteric and renal arterial function and caused structural damage to intrarenal and extrarenal small arteries and arterioles. Medial atrophy of the mesenteric resistance vessels and decreased viability of smooth muscle cells of the thoracic aorta were observed. Renal and arterial damage was associated with the presence of inflammatory cells. CsA did not affect markers of the L-arginine-NO pathway (urinary cyclic GMP excretion or endothelial or inducible NO synthase expression in kidney, aorta or heart) or oxidative stress (urinary excretion of 8-isoprostaglandin F2alpha, plasma urate concentration or total radical trapping capacity). Concomitant L-arginine treatment did not affect CsA-induced changes in blood pressure or histological findings but tended to alleviate the arterial dysfunction. The renal and cardiovascular toxicity of CsA was associated with arterial dysfunction and morphological changes in small arteries and arterioles in SHR on a high-sodium diet. The findings did not support the role of oxidative stress or a defect in the L-arginine-NO pathway.

Long-term intake of milk peptides attenuates development of hypertension in spontaneously hypertensive rats.

Effect of long-term intake of isoleucine-proline-proline (IPP) and valine-proline-proline (VPP), or a sour milk product containing these peptides on development of hypertension was investigated in spontaneously hypertensive rats (SHR). Six-week-old SHR were given: 1) water (control group), 2) IPP and VPP dissolved in water (peptide group) or 3) sour milk containing IPP and VPP (sour milk group) for 12 weeks. Systolic blood pressure (SBP) was measured by tail-cuff method. Development of hypertension was attenuated in the groups receiving tripeptides or sour milk as compared to the control group. At the end of treatment period, SBP was 176 +/- 1 mmHg in sour milk group, 181 +/- 2 mmHg in peptide group, and 193 +/- 1 mmHg in control group (P < 0.001). After treatment withdrawal, SBP rose gradually reaching the level of control group within four weeks' follow-up. In functional bioassay of ACE inhibitory activity, effect of the tripeptides on angiotensin I or angiotensin II-induced contraction in rat mesenteric arteries was evaluated. IPP inhibited the angiotensin I-induced contraction, whereas the angiotensin II-induced contraction remained unaltered. In conclusion, long-term intake of IPP and VPP, or a sour milk containing these tripeptides attenuated the development of hypertension in SHR. One possible mechanism underlying this effect is ACE inhibition.

Comparison of enalapril and valsartan in cyclosporine A-induced hypertension and nephrotoxicity in spontaneously hypertensive rats on high-sodium diet.

1. We compared the effects of the angiotensin converting enzyme (ACE) inhibitor enalapril and the angiotensin AT(1) receptor antagonist valsartan in cyclosporine A (CsA)-induced hypertension and nephrotoxicity in spontaneously hypertensive rats (SHR). 2. SHR (8 - 9 weeks old) on high-sodium diet were given CsA (5 mg kg(-1)d (-1) s.c. ) for 6 weeks. The rats were treated concomitantly either with enalapril (30 mg kg(-1)d (-1) p.o.) or valsartan (3 or 30 mg kg(-1) d (-1) p.o.). To evaluate the role of bradykinin in the action of enalapril, some rats received a bradykinin B(2) receptor antagonist icatibant (HOE 140, 500 microg kg(-1) d (-1) s.c.) during the last 2 weeks of enalapril treatment. 3. Blood pressure was recorded every second week by tail cuff method. Renal function was measured by serum creatinine, creatinine clearance and urinary excretion of proteins at the end of the experiment. The activity of the renal kallikrein-kinin system was estimated by urinary kallikrein excretion. 4. CsA caused hypertension, impaired renal function and induced morphological nephrotoxicity with glomerular damage and interstitial fibrosis. Enalapril and the lower dose of valsartan attenuated the CsA-induced hypertension to the same extent, while the higher dose of valsartan totally abolished it. Icatibant did not reduce the antihypertensive effect of enalapril. Urinary kallikrein excretion was similar in all groups. 5. Enalapril and valsartan equally prevented the CsA-induced deterioration of kidney function and morphology. 6. The renin-angiotensin but not the kallikrein-kinin system plays a crucial role in CsA-toxicity during high intake of sodium in SHR.

Enalapril and valsartan improve cyclosporine A-induced vascular dysfunction in spontaneously hypertensive rats.

Cyclosporine A causes hypertension and nephrotoxicity in spontaneously hypertensive rats (SHR). In the present study, arterial function was investigated using in vitro vascular preparations after long-term treatment with cyclosporine A. SHR received cyclosporine A (5 mg kg(-1) day(-1) s.c.) and high-Na(+) diet for 6 weeks during the developmental phase of hypertension. Part of the rats were treated concomitantly either with the angiotensin converting enzyme inhibitor enalapril (30 mg kg(-1) day(-1) p.o.) or with an angiotensin AT(1) receptor antagonist valsartan (3 or 30 mg kg(-1) day(-1) p.o.). In renal arteries, contractile responses to noradrenaline and angiotensin II, as well as relaxation responses to acetylcholine (endothelium-dependent) and to sodium nitroprusside (endothelium-independent), were severely impaired by cyclosporine A-treatment. There was also a trend for the dysfunction of the mesenteric arteries, but the impairment did not reach statistical difference. Enalapril and valsartan improved the impaired renal arterial functions. Cyclosporine A-induced hypertension and nephrotoxicity seem to be associated with renal arterial dysfunction in SHR on high-Na(+) diet. Antagonism of the renin-angiotensin system protects from vascular toxicity of cyclosporine A.