Age-related up-regulation of β3-adrenergic receptor in heart-failure rats

Stimulation of β1- and β2-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the β3-AR is also expressed in the heart and that its stimulation leads to negative inotropic effects. The aim of this study was to investigate the expression of β3-AR in age-related heart-failure rats and its relevance to left ventricular dysfunction. Aging male Wistar rats were divided into young and aging groups according to age, and each group included sham-operation and heart-failure subgroups. Left ventricular end-diastolic pressure (LVEDP) and the ratio of left ventricular weight to body weight (LV/BW) were significantly higher for the aging heart-failure versus young heart-failure and the heart-failure versus sham-operation groups (P?<?0.01, respectively). However, the left ventricular end-systolic pressure (LVESP) and the maximal rate of rise or fall of left ventricular pressure were all significantly lower for the aging heart-failure versus young heart-failure and the heart-failure versus sham-operation groups (P?<?0.01, respectively). β3-AR protein levels increased significantly when heart failure worsened in aging rats. These results suggest that β3-AR expression in age-related heart-failure rats and left ventricular function were highly correlated.     

The valosin‐containing protein is a novel repressor of cardiomyocyte hypertrophy induced by pressure overload

Hypertension‐induced left ventricular hypertrophy (LVH) is an independent risk factor for heart failure. Regression of LVH has emerged as a major goal in the treatment of hypertensive patients. Here, we tested our hypothesis that the valosin‐containing protein (VCP), an ATPase associate protein, is a novel repressor of cardiomyocyte hypertrophy under the pressure overload stress. Left ventricular hypertrophy (LVH) was determined by echocardiography in 4‐month male spontaneously hypertensive rats (SHRs) vs. age‐matched normotensive Wistar Kyoto (WKY) rats. VCP expression was found to be significantly downregulated in the left ventricle (LV) tissues from SHRs vs. WKY rats. Pressure overload was induced by transverse aortic constriction (TAC) in wild‐type (WT) mice. At the end of 2 weeks, mice with TAC developed significant LVH whereas the cardiac function remained unchanged. A significant reduction of VCP at both the mRNA and protein levels in hypertrophic LV tissue was found in TAC WT mice compared to sham controls. Valosin‐containing protein VCP expression was also observed to be time‐ and dose‐dependently reduced in vitro in isolated neonatal rat cardiomyocytes upon the treatment of angiotensin II. Conversely, transgenic (TG) mice with cardiac‐specific overexpression of VCP showed a significant repression in TAC‐induced LVH vs. litter‐matched WT controls upon 2‐week TAC. TAC‐induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling observed in WT mice LVs was also significantly blunted in VCP TG mice. In conclusion, VCP acts as a novel repressor that is able to prevent cardiomyocyte hypertrophy from pressure overload by modulating the mTORC1 signaling pathway.     

Left ventricular hypertrophy induced by abdominal aortic banding and its prevention by angiotensin receptor blocker telmisartan—a proteomic analysis

Cardiac hypertrophy is frequently caused by pressure overload (i.e., high blood pressure or hypertension) and can lead to heart failure. The major objective of the present study was to investigate the proteomic changes in response to the development of left ventricular hypertrophy (LVH) induced by abdominal aortic banding (AB) and its prevention by antihypertensive treatment with angiotensin II receptor blocker (ARB) telmisartan. One week after AB and Sham surgery, rats were assigned into three groups: SHAM–control, aortic banding without treatment (AB–Ctrl) and aortic banding with telmisartan treatment (AB–Telmi; 5mg/kg/day for 8 weeks). Echocardiography, hemodynamics, and pathology were performed to assess LVH. Left ventricular myocardium was sampled. The analysis of proteomic proteins from myocardium was performed by two-dimensional gel electrophoresis and MALDI–TOF–MS. In AB–Ctrl, heart rate, systolic arterial blood pressure, diastolic blood pressure, left ventricular end systolic pressure, interventricular septal thickness at diastole, posterior wall thickness in diastole, heart weight (HW) and HW/body weight (BW) were increased, indicating that both hypertension and LVH developed. Telmisartan prevented hypertension and LVH. Concurrently, among numerous proteins, there were 17 that were differentially expressed among hypertrophic hearts, normal hearts, and the hearts where hypertrophic response was suppressed by ARB treatment. Primarily, proteins involved in cell structure, metabolism, stress and signal transduction exhibited up-regulations in LVH, providing cellular and molecular mechanism for hypertrophic development. These changes were prevented or greatly attenuated by telmisartan regimen. Interestingly, antioxidative-related heat shock protein 2 was detected neither in SHAM–Ctrl nor in AB–Ctrl, but in AB–Telmi. LVH is accompanied by series changes of protein expression. Both LVH and proteomic changes can be prevented by blockade of renin–angiotensin system with telmisartan. These protein alterations may constitute mechanistic pathways leading to hypertrophy development and experimental targets for novel therapeutic strategy.     

Differential expression of TRPC channels in the left ventricle of spontaneously hypertensive rats

This study was designed to identify the differential expression of the canonical transient receptor potential (TRPC) channels in the left ventricle of spontaneously hypertensive rats (SHR). Echocardiography studies were performed to compare the left ventricular function in SHR vs. Wistar-Kyoto rats (WKY), and the mRNA level of the TRPC channels was determined by quantitative real-time RT-PCR (qRT-PCR). Western blots were performed to examine whether the mRNA expression corresponded with the protein expression. Compared with the WKY, the mRNA expression of TRPC4 and TRPC5 was significantly increased in the 10-week-old SHR (P = 0.032 for TRPC4 and P = 0.043 for TRPC5), so did the TRPC4/5 protein content. The midwall fractional shortening (mFS) of SHR was lower than WKY (P = 0.016). Furthermore, increased expression of TRPC4/5 was correlated with both increased blood pressure and decreased mFS. These findings suggest that TRPC4 and 5 seem to be the main subtypes expressed in the heart of the SHR at the beginning period of hypertension. Theses channels may participate in the development of left ventricular systolic dysfunction.     

心脏组织特异性表达 Neuregulin -2转基因小鼠的建立及心脏功能分析

目的：神经调节蛋白2（ neuregulin-2, NRG2）可促进神经系统发育,基因缺失表现早期生长延迟, NRG2在心脏中也有表达,但其在心脏发育尤其是病理刺激时对心脏结构及功能的影响尚未见报道。本文目的是建立心脏组织特异性表达NRG2转基因小鼠,分析其在正常及压力负荷刺激时对心脏结构及功能的影响。方法将人NRG2基因插入到心脏特异性启动子α-MHC下游,构建转基因表达载体,显微注射法建立NRG2转基因小鼠,PCR鉴定转基因小鼠基因型,western blot鉴定NRG2蛋白在心脏中的表达并筛选高表达的转基因品系,主动脉缩窄术（ transverse aortic constriction , TAC）制备压力负荷诱导的心肌肥厚小鼠模型。利用超声影像分析和病理学观察小鼠心脏结构和功能改变。结果建立了心脏组织特异性高表达NRG2转基因小鼠品系。与同窝阴性转基因小鼠相比,转基因小鼠左心室舒张末期后壁厚度（LVPWD）明显增加,3月龄时可达15．6％（P＜0．05）,经压力负荷刺激后,NRG2转基因手术小鼠心室壁增厚程度显著下降,心室腔增大,同时心肌排列紊乱程度和纤维化程度明显比NTG手术小鼠严重。结论在压力负荷下,转基因表达NRG2缩短了肥厚过程,同时加速了心衰进程。     

Effect of atorvastatin (Lipitor) on myocardial apoptosis and caspase-8 activation following coronary microembolization

We determined the effect of atorvastatin on myocardial apoptosis and caspase-8 activation following coronary microembolization (CME) in a rat model. For this, 50 rats were randomly and equally divided into CME; sham-operated (control); atorvastatin lavage; gastric lavage control; and caspase-8 inhibitor (CHO) groups. In CME animals, a microembolization ball was injected through the left ventricle. Sham animals were injected with normal saline (NS). Atorvastatin group received atorvastatin gastric lavage once-a-day, 1 week before surgery. Gastric lavage controls had similar lavage with NS. CHO group was i.p-injected (CHO: 10 mg/kg) 30 min before surgery. Cardiac indices in each group were determined by echocardiography 6-h postoperatively. TUNEL assay and western blot were used for myocardial apoptosis and expression of caspases-3/-8, respectively. Echocardiography data show that left ventricular ejection fraction (LVEF) in CME group was significantly decreased (P < 0.05) compared with sham controls. Besides, left ventricular fractional shortening (FS) and cardiac output (CO) were also decreased with an increase in left ventricular end-diastolic dimension (LVEDd). Atorvastatin and CHO animals had significantly improved (P < 0.05) cardiac function compared with CME group. Myocardial apoptosis and activation levels of caspases-3/-8 were significantly increased (P < 0.05) compared with sham; myocardial apoptosis and activation levels of caspases-3/-8 were significantly decreased (P < 0.05) in atorvastatin and CHO groups compared with CME group. In conclusion, atorvastatin pretreatment suppressed post-CME myocardial apoptosis and improved cardiac function through the blockade of a myocardial death receptor-mediated apoptotic pathway.     

Time course of sympathovagal imbalance and left ventricular dysfunction in conscious dogs with heart failure

To elucidate thetime course of sympathovagal balance and its relationship to leftventricular function in heart failure, we serially evaluated leftventricular contractility and relaxation and autonomic tone in 11 conscious dogs with tachycardia-induced heart failure. We determined adynamic map of sympathetic and parasympathetic modulation by powerspectral analysis of heart rate variability. The left ventricular peak+dP/dt substantially fell from 3,364 ± 338 to 1,959 ± 318 mmHg/s (P < 0.05) on the third day and declined gradually to 1,783 ± 312 mmHg/s at 2 wk of rapid ventricular pacing. In contrast, the timeconstant of left ventricular pressure decay and end-diastolic pressureincreased gradually from 25 ± 4 to 47 ± 5 ms(P < 0.05) and from 10 ± 2 to21 ± 3 mmHg (P < 0.05), respectively, at 2 wk of pacing. The high-frequency component(0.15-1.0 Hz), a marker of parasympathetic modulation, decreasedfrom 1,928 ± 1,914 to 62 ± 68 × 10³ms²(P < 0.05) on the third day andfurther to 9 ± 12 × 10³ms²(P < 0.05) at 2 wk. Similar to thetime course of left ventricular diastolic dysfunction, plasmanorepinephrine levels and the ratio of low (0.05- to 0.15-Hz)- tohigh-frequency component increased progressively from 135 ± 50 to 532 ± 186 pg/ml (P < 0.05) and from 0.06 ± 0.06 to 1.12 ± 1.01 (P < 0.05), respectively, at 2 wk ofpacing. These cardiac and autonomic dysfunctions recovered graduallytoward the normal values at 2 wk after cessation of pacing. Thus aparallel decline in left ventricular contractility with parasympatheticinfluence and a parallel progression in left ventricular diastolicdysfunction with sympathoexcitation suggest a close relationshipbetween cardiac dysfunction and autonomic dysregulation duringdevelopment of heart failure.

     

Detrimental effect of hypothermia during acute normovolaemic haemodilution in anaesthetized cats

 Haemodynamic responses to hypothermia were studied at normal haematocrit and following the induction of acute normovolaemic haemodilution. Experiments were performed on 20 cats anaesthetized with a mixture of chloralose and urethane in two groups. In one group (n=10) the effects of hypothermia on various haemodynamic variables were studied at normal haematocrit (41.0±1.7%) and in the second group of cats (n=10) the effects of hypothermia on various haemodynamic variables were studied after the induction of acute normovolaemic haemodilution (14.0±1.0%). The haemodynamic variables left ventricular pressure, left ventricular contractility, arterial blood pressure, heart rate and right atrial pressure were recorded on a polygraph. Cardiac output was measured using a cardiac output computer. In both groups hypothermia was induced by surface cooling with the help of ice. Cardiovascular variables were recorded at each 1° C fall in body temperature. Hypothermia produced a significant (P<0.05) drop in heart rate, cardiac output, arterial blood pressure and left ventricular contractility in both groups. However, the percentage decrease in these variables in response to hypothermia was significantly (P<0.05) higher in cats with low haematocrit than in those with normal haematocrit. The severity of hypothermia – induced cardiovascular effects is evident from the drastic decrease in heart rate, cardiac output, arterial blood pressure and myocardial contractility in cats with low haematocrit, indicating a higher risk of circulatory failure under anaemic conditions at low temperatures. Received: 21 October 1996 / Revised: 20 April 1997 / Accepted 21 May 1997     

Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.     

Beneficial effect of ACE inhibitor in congestive heart failure

The effects of captopril, an angiotensin-converting enzyme inhibitor, on congestive heart failure (CHF) were investigated in animal and clinical studies. Congestive heart failure was induced in rats by a combination of pressure and volume overload. Cardiac pressure overload was induced by constricting one renal artery (Goldblatt rat) and volume overload was induced by aorto-caval fistula. Captopril (100 mg/kg/day) was then administered for 14 weeks. Isometric contraction was assessed using isolated left ventricular papillary muscles. The maximum developed tension and the maximum rate of increase in tension (dT/dtmax) were decreased in untreated rats with CHF and improved in captopriltreated rats. The left ventricular myosin isoenzyme pattern was shifted towards V3 in untreated rats with CHF, and was shifted back towards V1 in the captopril-treated rats. In the clinical study, captopril (37.5–75 mg/day) was administered to patients with cardiomyopathy for 12 months. There was no effect on left ventricular mass in hypertrophic cardiomyopathy, although systolic anterior motion of the mitral valve disappeared in one patient. In dilated cardiomyopathy, however, left ventricular mass tended to decrease. These results indicate that captopril has a beneficial effect in congestive heart failure.     

Accelerated onset of heart failure in mice during pressure overload with chronically decreased SERCA2 calcium pump activity

We recently developed a mouse model with a single functional allele of Serca2 (Serca2+/-) that shows impaired cardiac contractility and relaxation without overt heart disease. The goal of this study was to test the hypothesis that chronic reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2 levels in combination with an increased hemodynamic load will result in an accelerated pathway to heart failure. Age-matched wild-type and Serca2+/- mice were subjected to 10 wk of pressure overload via transverse aortic coarctation surgery. Cardiac hypertrophy and heart failure were assessed by echocardiography, gravimetry/histology, hemodynamics, and Western blotting analyses. Our results showed that approximately 64% of coarcted Serca2+/- mice were in heart failure compared with 0% of coarcted wild-type mice (P < 0.05). Overall, morbidity and mortality were greatly increased in Serca2+/- mice under pressure overload. Echocardiography assessment revealed a significant increase in left ventricular (LV) mass, and LV hypertrophy in coarcted Serca2+/- mice converted from a concentric to an eccentric pattern, similar to that seen in human heart failure. Coarcted Serca2+/- mice had decreased contractile/systolic and relaxation/diastolic performance and/or function compared with coarcted wild-type mice (P < 0.05), despite a similar duration and degree of pressure overload. SERCA2a protein levels were significantly reduced (>50%) in coarcted Serca2+/- mice compared with noncoarcted and coarcted wild-type mice. Our findings suggest that reduction in SERCA2 levels in combination with an increased hemodynamic load results in an accelerated pathway to heart failure.     

Higher mortality in heterozygous neuropilin-1 mice after cardiac pressure overload

We previously identified that neuropilin-1 (NP-1) was a co-receptor of vascular endothelial growth factor receptor 2 (VEGFR2) and confirmed that NP-1 knockout mice were embryonic lethal due to impairment of vascular development, while VEGF was reported to be involved in the progression of heart failure. However, it is unknown whether NP-1 has any influence on cardiac function, and it also remains poor understood concerning cardiac expression of NP-1 and its interaction with other VEGF receptors in the heart. Here, we first showed that NP-1 heterozygous mice had significantly higher mortality due to either acute or chronic heart failure in response to left ventricular pressure overload. We also observed that NP-1 mRNA and protein were expressed in both neonatal rat cardiomyocytes and adult murine heart. Furthermore, we found that NP-1 formed complexes with VEGFR1 and VEGFR2, respectively, in cardiomyocytes. These findings suggest that NP-1 should play beneficial role in heart failure.     

Disease severity–related alterations of cardiac microRNAs in experimental pulmonary hypertension

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH). We hypothesized that heart‐relevant microRNAs, that is myomiRs (miR‐1, miR‐133a, miR‐208, miR‐499) and miR‐214, can have a role in the right ventricle in the development of PAH. To mimic PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.); control group received vehicle. MCT rats were divided into two groups, based on the clinical presentation: MCT group terminated 4 weeks after MCT administration and prematurely terminated group (ptMCT) displaying signs of terminal disease. Myocardial damage genes and candidate microRNAs expressions were determined by RT‐qPCR. Reduced blood oxygen saturation, breathing disturbances, RV enlargement as well as elevated levels of markers of myocardial damage confirmed PH in MCT animals and were more pronounced in ptMCT. MyomiRs (miR‐1/miR‐133a/miR‐208a/miR‐499) were decreased and the expression of miR‐214 was increased only in ptMCT group (P < 0.05). The myomiRs negatively correlated with Fulton index as a measure of RV hypertrophy in MCT group (P < 0.05), whereas miR‐214 showed a positive correlation (P < 0.05). We conclude that the expression of determined microRNAs mirrored the disease severity and targeting their pathways might represent potential future therapeutic approach in PAH.     

iPSC‐derived human mesenchymal stem cells improve myocardial strain of infarcted myocardium

We investigated global and regional effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)‐derived mesenchymal stem cells (iMSCs) in infarcted myocardium. Acute myocardial infarction (MI) was induced by ligation of left coronary artery of severe combined immunodeficient mice before 2 × 10⁵ iMSCs or cell‐free saline were injected into peri‐infarcted anterior free wall. Sham‐operated animals received no injection. Global and regional myocardial function was assessed serially at 1‐week and 8‐week by segmental strain analysis by using two dimensional (2D) speckle tracking echocardiography. Early myocardial remodelling was observed at 1‐week and persisted to 8‐week with global contractility of ejection fraction and fractional area change in saline‐ (32.96 ± 14.23%; 21.50 ± 10.07%) and iMSC‐injected (32.95 ± 10.31%; 21.00 ± 7.11%) groups significantly depressed as compared to sham control (51.17 ± 11.69%, P < 0.05; 34.86 ± 9.82%, P < 0.05). However, myocardial dilatation was observed in saline‐injected animals (4.40 ± 0.62 mm, P < 0.05), but not iMSCs (4.29 ± 0.57 mm), when compared to sham control (3.74 ± 0.32 mm). Furthermore, strain analysis showed significant improved basal anterior wall strain (28.86 ± 8.16%, P < 0.05) in the iMSC group, but not saline‐injected (15.81 ± 13.92%), when compared to sham control (22.18 ± 4.13%). This was corroborated by multi‐segments deterioration of radial strain only in saline‐injected (21.50 ± 5.31%, P < 0.05), but not iMSC (25.67 ± 12.53%), when compared to sham control (34.88 ± 5.77%). Improvements of the myocardial strain coincided with the presence of interconnecting telocytes in interstitial space of the infarcted anterior segment of the heart. Our results show that localized injection of iMSCs alleviates ventricular remodelling, sustains global and regional myocardial strain by paracrine‐driven effect on neoangiogenesis and myocardial deformation/compliance via parenchymal and interstitial cell interactions in the infarcted myocardium.     

Pulse pressure response to the strain of the Valsalva maneuver in humans with preserved systolic function

Arterial pulsepressure response during the strain phase of the Valsalva maneuver hasbeen proposed as a clinical tool for the diagnosis of left heartfailure, whereas responses of subjects with preserved systolic functionhave been poorly documented. We studied the relationship between theaortic pulse amplitude ratio (i.e., minimum/maximum pulse pressure)during the strain phase of the Valsalva maneuver and cardiachemodynamics at baseline in 20 adults (42 ± 14 yr) undergoingroutine right and left heart catheterization. They were normal subjects(n = 5) and patients withvarious forms of cardiac diseases(n = 15), and all had a leftventricular ejection fraction 40%. High-fidelity pressures wererecorded in the right atrium and the left ventricle at baseline and atthe aortic root throughout the Valsalva maneuver. Aortic pulseamplitude ratio 1) did not correlatewith baseline left ventricular end-diastolic pressure, cardiac index(thermodilution), or left ventricular ejection fraction(cineangiography) and 2) waspositively related to total arterial compliance (area method) (r = 0.59) and to basal mean rightatrial pressure (r = 0.57) (eachP < 0.01). Aortic pulse pressureresponses to the strain were not related to heart rate responses duringthe maneuver. In subjects with preserved systolic function, the aorticpulse amplitude ratio during the strain phase of the Valsalva maneuver relates to baseline total arterial compliance and right heart fillingpressures but not to left ventricular function.

     

Time course of changes in the expression of DHPR, RyR2, and SERCA2 after myocardial infarction in the rat left ventricle

Postinfarction left ventricular remodeling leads to the functional decline of the left ventricle (LV). Since dihydropyridine receptor (DHPR), ryanodine receptor (RyR₂), and sarco-endoplasmic reticulum (SR) Ca²⁺-ATPase2 (SERCA2a) play a major role in the contractility of the heart, the aim of our study was to evaluate the time course of changes in the expression of these proteins 1 day, 2 weeks and 4 weeks after myocardial infarction (MI). Myocardial infarction was produced by ligation of left anterior descending coronary artery of the rat. Transthoracic echocardiography was performed to characterize structural and functional changes after MI. To evaluate protein mRNA levels and the relative amount of proteins, real-time quantitative RT-PCR and Western blotting were used. LV ejection fraction and fractional shortening decreased significantly during the 4-week follow-up period (P < 0.001). Typical features of LV remodeling after MI were seen, with a decrease in anterior wall thickness (P < 0.001) and dilatation of the LV (P < 0.001). Expression of DHPR and RyR₂ mRNAs decreased and Serca2a mRNA tended to decrease 1 day after MI (P < 0.001, P < 0.01 and P = 0.06, respectively), followed by recovery of the expression during the next 4 weeks. In the infarcted hearts the quantities of SERCA2 proteins in the LV were significantly decreased at the time of 4 weeks. In conclusion, MI was associated with transient decrease in the expression of the DHPR and RyR₂ mRNAs and a reduced quantity of SERCA2 proteins in the LV. Since they have a key role in the contraction of the heart, changes in the expression of these proteins may be important regulators of LV systolic function after MI.