Masahiro Sugano

Chronic inhibition of Rho kinase blunts the process of left ventricular hypertrophy leading to cardiac contractile dysfunction in hypertension-induced heart failure

The Gq-RhoA-Rho kinase pathway, activated by neurohormonal factors such as angiotensin II (Ang II), has been proposed to be one of the important signaling pathways involved in the progression of left ventricular (LV) hypertrophy to heart failure. We tested the hypothesis that chronic inhibition of Rho kinase prevents this process. Heart failure was induced in Dahl salt-sensitive (DS) rats fed an 8% NaCl diet from 8 until 17 weeks of age. Y-27632 (5 mg/kg per day), a selective Rho kinase inhibitor, was applied orally to DS rats starting at 10 weeks of age for 7 weeks (DS/Y+). DS rats without Y-27632 (DS/Y-) and Dahl salt-resistant (DR) rats fed the 8% NaCl diet were regarded as non-therapeutic and normotensive controls, respectively. At 17 weeks of age, there was no significant difference in the blood pressure of DS/Y- and DS/Y+ rats. DS/Y- rats exhibited: (1) increases in LV mass, cross-sectional area (CSA) of cardiomyocytes, and interstitial fibrosis; (2) contractile dysfunction, i.e. decreases in LV ejection fraction and % fractional shortening, and prolongation of time to peak tension as well as to 50% relaxation in the twitch contraction of isolated papillary muscle; and (3) increases in the protein expression of Galphaq and Rho kinase in the myocardial membrane fraction. In DS/Y+ rats, the degree of myocardial hypertrophy was significantly inhibited in association with improved contractile function, without a decrease in the degree of interstitial fibrosis. Our results suggest the possibility that the Gq-Rho kinase pathway plays an important role in the process of hypertension-induced LV hypertrophy leading to contractile dysfunction.

Interaction of mRNAs for Angiotensin II Type 1 and Type 2 Receptors to Vascular Remodeling in Spontaneously Hypertensive Rats

We administered angiotensin II (Ang II) receptor type 1 (AT1) blockade (losartan, 40 mg x kg-1 x d-1), type II receptor (AT2) blockade (PD123319, 100 mg x kg-1 x d-1), or angiotensin-converting enzyme (ACE) inhibitor (enalapril, 30 mg x kg-1 x d-1) to spontaneously hypertensive rats (SHR) from 10 to 20 weeks of age. Control SHR and Wister-Kyoto rats (WKY) received a placebo for the same period. At the end of treatment, losartan and enalapril were both found to have significantly reduced the arterial systolic blood pressure and the collagen concentration to the level of WKY, whereas PD123319 had no effect. Enalapril and PD123319 significantly reduced the media cross-sectional area of the aorta in comparison to that of untreated SHR, which was still larger than that of the WKY; however, losartan did not change it. Using reverse transcription-polymerase chain reaction, we next examined the mRNA expressions for ACE, AT1 receptor, and AT2 receptor in experimental animals. We observed significantly enhanced mRNA expression for AT1 and AT2 receptors and ACE in untreated SHR compared with WKY. The AT1 mRNA level was also significantly decreased in the SHR treated with either losartan or enalapril, whereas the AT2 mRNA level was significantly decreased in the SHR treated with either PD123319 or enalapril in comparison to untreated SHR. The level of ACE mRNA was significantly decreased only in the SHR treated with enalapril. These results indicate that AT1 receptor, but not AT2 receptor, plays a crucial role in the remodeling of matrix tissue, while AT2 receptor may play a role in the development of hypertrophy of smooth muscle in aorta in SHR, and that the reduction of hypertrophy of smooth muscle does not fully account for the suppression of hypertension.

Transient receptor potential (TRP) protein 7 acts as a G protein-activated Ca2+ channel mediating angiotensin II-induced myocardial apoptosis

Transient receptor potential (TRP) proteins have been identified as cation channels that are activated by agonist–receptor coupling and mediate various cellular functions. TRPC7, a homologue of TRP channels, has been shown to act as a Ca2+ channel activated by G protein-coupled stimulation and to be abundantly expressed in the heart with an as-yet-unknown function. We studied the role of TRPC7 in G protein-activated signaling in HEK293 cells and cultured cardiomyocytes in vitro transfected with FLAG-tagged TRPC7 cDNA and in Dahl salt-sensitive rats with heart failure in vivo. TRPC7-transfected HEK293 cells showed an augmentation of carbachol-induced intracellular Ca2+ transient, which was attenuated under a Ca2+-free condition or in the presence of SK&F96365 (a Ca2+-permeable channel blocker). Upon stimulation with angiotensin II (Ang II), cultured neonatal rat cardiomyocytes transfected with TRPC7 exhibited a significant increase in apoptosis detected by TUNEL staining, accompanied with a decrease in the expression of atrial natriuretic factor and destruction of actin fibers, as compared with non-transfected cardiomyocytes. Ang II-induced apoptosis was inhibited by CV-11974 (Candesartan; Ang II type 1 [AT1] receptor blocker), SK&F96365, and FK506 (calcineurin inhibitor). In Dahl salt-sensitive rats, apoptosis and TRPC7 expression were increased in the failing myocardium, and a long-term treatment with temocapril, an angiotensin-converting enzyme inhibitor, suppressed both. Our findings suggest that TRPC7 could act as a Ca2+ channel activated by AT1 receptors, leading to myocardial apoptosis possibly via a calcineurin-dependent pathway. TRPC7 might be a key initiator linking AT1-activation to myocardial apoptosis, and thereby contributing to the process of heart failure.

In vivo transfer of soluble TNF-alpha receptor 1 gene improves cardiac function and reduces infarct size after myocardial infarction in rats

Increased circulating and cardiac TNF‐alpha levels during myocardial ischemia have been found in both experimental animals and patients with ischemic heart disease and advanced heart failure. Soluble TNF‐alpha receptor 1 (sTNFR1) is an antagonist to TNF‐alpha. In the present study, we examined whether sTNFR1 improves cardiac function in rats after myocardial infarction. Male Wistar rats were subjected to left coronary artery (LCA) ligation. Immediately after the ligation, a total of 200 μg of either the sTNFR1 or LacZ plasmid was injected into three different sites in the left ventricular wall. From 1 to 21 days after LCA ligation, TNF‐alpha bioactivity in the heart was higher in rats receiving LacZ plasmid than in sham‐operated rats, whereas sTNFR1 plasmid significantly suppressed the increase. The LV diastolic dimension was significantly lower, and the fractional shortening was significantly higher in rats treated with the sTNFR1 plasmid than in those treated with the LacZ plasmid. At 21 days after LCA ligation, the LV end‐diastolic pressure was also significantly lower in the rats treated with the sTNFR1 plasmid. In addition, the sTNFR1 expression plasmid had significantly reduced the infarct size. In conclusion, TNF‐alpha bioactivity in the heart increased during the early stage of infarction and remained elevated. This elevation seemed partially responsible for the impairment of LV function and the increased infarct size. Suppression of TNF‐alpha bioactivity from the early stage of infarction with the sTNFR1 plasmid improved cardiac function and reduced infarct size.

Change in the telomere length distribution with age in the Japanese population

Telomeres play a role in cellular aging and they may also contribute to the genetic basis of human aging and longevity. A gradual loss of the telomeric repeat sequences has been reported in adult tissue specimens. This study determined the percentage of telomere restriction fragment in various molecular-sized regions in addition to measuring the average telomere length. Mean telomere restriction fragment (TRF) length was determined by Southern blot analysis using a longer telomeric repeat probe with higher sensitivity. A significant decrease in longer telomere fragments and a quick increase in the shortest fragments were observed, especially in male subjects. There was a tendency that the age-adjusted telomere length was longer in females than that observed in males, while males lose the telomeric sequence faster than females. These data indicated that the percentage of longer telomeres fragments decreased, while the shortest fragments increased quickly with age. In addition, the longest telomere fragments decreased and the short fragments increased with a relatively stable frequency with age. There was also a significant difference in the longest telomere fragment percentage between males and female in their 40s and 50s, whereas no difference was observed in the mean TRF length. Interestingly, the changing rate of the longest and the shortest range group of TRF percentage associated with aging seemed quite different between before and after 50-year old with a gender-related contrast. This contrast implies a drastic change around the age of 50 of unknown factors that affect telomere attrition.

Molecular Mechanism of Angiotensin II Type I and Type II Receptors in Cardiac Hypertrophy of Spontaneously Hypertensive Rats

We administered angiotensin (Ang) II receptor type 1 (AT1) blockade (losartan; 10 or 40 mg/kg per day), type II receptor (AT2) blockades (PD123319; 100 mg/kg per day), or angiotensin-converting enzyme (ACE) inhibitor (enalapril; 30 mg/kg per day) to spontaneously hypertensive rats (SHR) from 10 to 20 weeks of age. At the end of the treatment, high doses of losartan and enalapril significantly reduced the arterial systolic blood pressure compared with the untreated SHR to the level of WKY rats. But low doses of losartan and PD123319 were without effect. High doses of losartan and enalapril also significantly reduced both the left ventricular (LV) weight and the ratio of LV to body weight compared with the untreated SHR, which were still larger than that of WKY rats. However, the collagen concentration of SHR treated with high doses of losartan or enalapril was completely reduced to the level of WKY rats. Using reverse transcription polymerase chain reaction, we examined the mRNA expression for ACE, AT1, and AT2 in experimental animals. The enhanced AT1 mRNA expression was significantly decreased in the SHR treated with a high dose of losartan or PD123319 compared with the untreated SHR. The level of ACE mRNA was also decreased in the SHR treated with a high dose of losartan or enalapril. The level of AT2 mRNA was not significantly different between the Wistar-Kyoto rats and the SHR; however, this expression was decreased significantly after the treatment with a high dose of losartan or PD123319. These results indicate that AT1 receptor and ACE, but not AT2 receptor, play a crucial role in the remodeling of matrix tissue but a smaller role in the development of the hypertrophy of LV myocyte in SHR and that the LV/body weight changes do not fully account for the complete suppression of hypertension.

Intravenous Injection With Antisense Oligodeoxynucleotides Against Angiotensinogen Decreases Blood Pressure in Spontaneously Hypertensive Rats

In the renin-angiotensin system, renin is known to cleave angiotensinogen to generate angiotensin I, which is the precursor of angiotensin II. Angiotensin II is a vasoactive peptide that plays an important role in blood pressure. On the other hand, the liver is the major organ responsible for the production of angiotensinogen in spontaneously hypertensive rats (SHR). To test the hypothesis that a reduction of angiotensinogen mRNA in the liver by antisense oligodeoxynucleotides (ODNs) may affect both plasma angiotensinogen and angiotensin II levels, as well as blood pressure, we intravenously injected antisense ODNs against rat angiotensinogen coupled to asialoglycoprotein carrier molecules, which serve as an important regulator of liver gene expression, into SHR via the tail vein. The SHR used in the present study were studied at 20 weeks of age and were fed a standard diet throughout the experiment. Plasma angiotensinogen, angiotensin II concentrations, and blood pressure all decreased from the next day until up to 5 days after the injection of antisense ODNs. These concentrations thereafter returned to baseline by 7 days after injection. A reduction in the level of hepatic angiotensinogen mRNA was also observed from the day after injection until 5 days after injection with antisense ODNs. However, in the SHR injected with sense ODNs, plasma angiotensinogen, angiotensin II concentrations, and blood pressure, as well as hepatic angiotensinogen mRNA, did not significantly change throughout the experimental period. Although the exact role of angiotensinogen in hypertension still remains to be clarified, these findings showed that intravenous injection with antisense ODNs against angiotensinogen coupled to asialoglycoprotein carrier molecules targeted to the liver could thus inhibit plasma angiotensinogen levels and, as a result, induce a decrease in blood pressure in SHR.

Changes in Plasma Lipoprotein Cholesterol Levels by Antisense Oligodeoxynucleotides against Cholesteryl Ester Transfer Protein in Cholesterol-fed Rabbits

Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl ester from high density lipoprotein (HDL) to apoB-containing lipoproteins and also affects the low density lipoprotein metabolism. On the other hand, the liver is the major tissue responsible for the production of CETP (CETP mRNA) in rabbits. To test the hypothesis that a reduction of CETP mRNA in the liver by antisense oligodeoxynucleotides (ODNs) may affect the plasma lipoprotein cholesterol levels, we intravenously injected antisense ODNs against rabbit CETP coupled with asialoglycoprotein carrier molecules, which serve as an important method to regulate liver gene expression, to cholesterol-fed rabbits via their ear veins. All rabbits were fed a standard rabbit chow supplement with 0.1% cholesterol for 10 weeks before and throughout the experiment. After injecting rabbits with antisense ODNs, the plasma total cholesterol concentrations and plasma CETP activities all decreased at 24, 48, and 96 h, whereas the plasma HDL cholesterol concentrations increased at 48 h. A reduction in the hepatic CETP mRNA was also observed at 6, 24, and 48 h after the injection with antisense ODNs. However, in the rabbits injected with sense ODNs, the plasma total and HDL cholesterol concentrations and the plasma CETP activities did not significantly change, and the hepatic CETP mRNA did not change either throughout the experimental period. Although the exact role of CETP in the development of atherosclerosis remains to be clarified, these findings showed for the first time that the intravenous injection with antisense ODNs against CETP coupled to asialoglycoprotein carrier molecules targeted to the liver could thus inhibit plasma CETP activity and, as a result, could induce a decrease in the plasma low density lipoprotein and very low density lipoprotein cholesterol and an increase in the plasma HDL cholesterol in cholesterol-fed rabbits.

Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats.

Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 μg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion (Mol Cell Biochem 266: 127–132, 2004)

RNA interference targeting SHP-1 attenuates myocardial infarction in rats

The Src homology domain 2 (SH2)‐containing tyrosine phosphatase‐1 (SHP‐1) plays a key role in apoptosis and decreases phosphorylation of Akt. Apoptosis of cardiomyocytes is thought to contribute to the increased area of acute myocardial infarction (AMI), and Akt activation exerts a powerful cardioprotective effect after ischemia. Thus, a therapeutic strategy designed to inhibit expression of SHP‐1 would be beneficial in AMI. Here we report that siRNA targeting SHP‐1 reduced infarct size in a rat model of AMI. Upon injection into the ischemic left ventricular wall, the vector‐based siRNA significantly suppressed the increase in the SHP‐1 mRNA and the SHP‐1 protein levels. The siRNA vector also significantly reduced the SHP‐1 that bound to Fas‐R. The SHP‐1 siRNA vector increased phospho‐Akt and reduced DNA fragmentation and caspase activity compared with the scramble siRNA vector. Finally, the area of myocardial infarction was significantly smaller with the SHP‐1 siRNA vector than with the scramble siRNA vector at 2 days after LCA ligation. In conclusion, SHP‐1 in the heart increased from the early stage of AMI, and this increase was thought to contribute to the increased area of myocardial infarction. Suppression of SHP‐1 with the SHP‐1 siRNA vector markedly reduced the infarct size in AMI.