Basal and ethanol-induced cardiac contractile response in lean and obese zucker rat hearts

Obesity plays a pivotal role in metabolic and cardiovascular diseases. Certain types of obesity may be related to alcohol ingestion, which itself leads to impaired cardiac function. This study analyzed basal and ethanol-induced cardiac contractile response using left-ventricular papillary muscles and myocytes from lean and obese Zucker rats. Contractile properties analyzed include: peak tension development (PTD), peak shortening amplitude (PS), time to PTD/PS (TPT/TPS), time to 90% relaxation/relengthening (RT(90)/TR(90)) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT and +/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes and fluorescence decay time (FDT). In papillary muscles from obese rats, the baseline TPT and RT(90) were significantly prolonged accompanied with low to normal PTD and +/-VT compared to those in lean rats. Muscles from obese hearts also exhibited reduced responsiveness to postrest potentiation, increase in extracellular Ca(2+) concentration, and norepinephrine. By contrast, in isolated myocytes, obesity reduced PS associated with a significant prolonged TR(90), normal TPS and +/-dL/dt. Intracellular Ca(2+) recording revealed decreased resting Ca(2+) levels and prolonged FDT. Acute ethanol exposure (80-640 mg/dl) caused comparable concentration-dependent inhibitions of PTD/PS and DeltaFFI, associated with reduced +/-VT in both groups. Collectively, these results suggest altered cardiac contractile function and unchanged ethanol-induced depression in obesity.     

Acetaldehyde depresses myocardial contraction and cardiac myocyte shortening in spontaneously hypertensive rats: role of intracellular Ca2+.

Acetaldehyde (ACA), the major metabolite of ethanol, exerts both stimulatory and depressive actions on myocardial tissue. We have recently shown that ACA depresses myocardial contraction, cardiac myocyte shortening and intracellular Ca2+ transients in normal rat heart. The purpose of the present study was to determine the influence of hypertension on ACA-induced myocardial actions. Mechanical properties of left ventricular papillary muscles and ventricular myocytes isolated from both 25-week-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were evaluated using force-transducer and video edge-detection, respectively. Papillary muscles and cardiac myocytes were electrically stimulated to contract at 0.5 Hz. Contractile properties analyzed include: peak tension development (PTD), peak twitch amplitude (PTA), time-to-PTD/PTA (TPT/TPS), time-to-90% relaxation/relengthening (RT90/TR90) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT/+/-dL/dt). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity (FFI) changes. ACA (1-30 mM) depressed PTD without affecting other mechanical indices in both WKY and SHR myocardium, with maximal inhibition of 64 and 69%, respectively. SHR myocytes exhibited increased cell dimension, baseline PTA and resting intracellular Ca2+ levels, compared to WKY counterparts. ACA (0.03-30 mM) depressed PTA without affecting TPT, TR90 and +/-dL/dt. The maximal inhibitions were 31 and 36% in WKY and SHR groups, respectively. Interestingly, ACA exerted a biphasic effect on FFI, displaying potentiation at lower doses (<3 mM) and inhibition at higher doses (>3 mM). The maximal increase in FFI changes were 19 and 22% at 0.3 mM and the maximal decreases were 37 and 29% at 30 mM ACA, in WKY and SHR myocytes, respectively. Neither resting intracellular Ca2+ levels (FFI) nor fluorescence decay time (FDT) were affected by ACA. The increase in FFI was attenuated by propranolol (1 microM), whereas the decrease in FFI was reversed by BayK 8644 (1 microM). These results suggest that hypertension does not appear to alter ACA-induced myocardial depression. The mechanism underlying ACA-induced myocardial actions may involve increased beta-adrenergic activity at low doses and reduced Ca2+ entry and/or release at high doses.     

Influence of age on inotropic response to insulin and insulin-like growth factor I in spontaneously hypertensive rats: role of nitric oxide.

Insulin-like growth factor-1 (IGF-1) and insulin stimulate cardiac growth and contractility. Recent evidence suggests a relationship between essential hypertension, left ventricular hypertrophy, and circulating IGF-1 levels. Advanced age alters cardiac function in a manner similar to hypertension. The aim of this investigation was to evaluate the effects of IGF-1 and insulin on the force generating capacity of cardiac muscle in hypertension and the influence of age on this response. Contractile responses to IGF-1 and insulin were examined using papillary muscles from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) at 10 and 25 weeks of age. Muscles were electrically stimulated at 0.5 Hz, and contractile properties, including peak tension development (PTD), time-to-peak tension, time-to-90% relaxation, and the maximal velocities of contraction and relaxation, were evaluated. PTD was similar in WKY and SHR myocardium at both age groups. At 10 weeks of age, IGF-1 (1-500 ng/ml) caused a dose-dependent increase in PTD in WKY but not SHR myocardium, whereas insulin (1-500 nM) had no effect on PTD in either group. At 25 weeks of age, the positive inotropic effect of IGF-1 was attenuated in the WKY group, and IGF-1 exerted no inotropic action in the SHR group. Pretreatment with the nitric oxide synthase inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME, 100 microM), did not alter the IGF-1-induced positive inotropic response in 10-week-old WKY myocardium, whereas it unmasked a positive inotropic action in muscles from age-matched SHR animals. At 25 weeks of age, L-NAME abolished IGF-1-induced a positive inotropic response in WKY myocardium, and did not unmask an IGF-induced inotropic response in SHR myocardium. Our results suggest that alterations in nitric oxide modulation of IGF-1-induced contraction may underlie resistance to this inotropic peptide with advancing age, and/or hypertension.     

Modulation of stimulation frequency responses and calcium dependency of functional parameters in hyperthyroid rat ventricular papillary muscles

The effects of stimulation frequency (0.2-1.5 Hz) and extracellular calcium concentration ([Ca2+]o) (0.6-15.0 mM) on the contractile function of thin papillary muscles of euthyroid and hyperthyroid rats were studied. Hyperthyroidism led to a decrease in developed tension (DT) and time to peak tension (TPT), but it exhibited no influence on the maximal rates of contraction (+dT/dt) and relaxation (-dT/dt). Also, the mean rates of contraction were similar in euthyroid and hyperthyroid muscle groups. The increase in stimulation frequency brought about a marked decrease in DT, +dT/dt, and -dT/dt of euthyroid papillary muscles at lower frequencies in comparison to papillary muscles in the hyperthyroid group. At stimulation frequencies above 1.0 Hz, the absolute and relative levels of DT and -dT/dt of hyperthyroid myocardium were elevated over euthyroid preparations. At the same time, TPT was unchanged in any of the muscle groups. Hyperthyroidism modulated the relationships between contractile parameters and [Ca2+]o. At a [Ca2+]o of 1.0-4.0 mM, the DT of hyperthyroid papillary muscles was lower than in euthyroid muscle. At 4.0 and 8.0 mM of [Ca2+]o, the equal values of maximal DT were registered for euthyroid and hyperthyroid papillary muscles, respectively. An increase in the [Ca2+]o in the range of 1.0-15.0 mM was accompanied by an increase in TPT of both muscle groups, but to a greater extent in hyperthyroid myocardium. In conclusion, the myocardium of hyperthyroid rat appeared to exhibit decreased sensitivity to calcium as well as to the negative inotropic effect of enhanced stimulation frequency. Alterations of the processes of transsarcolemmal movement and intracellular recycling of Ca2 may be implicated.     

Negative inotropic effect of insulin in papillary muscles from control and diabetic rats

The inotropic effects of insulin in the rat heart are still incompletely understood. In this study, the effects of insulin on cardiac contraction were studied in right ventricular papillary muscles from both control rats and rats with chronic diabetes (lasting 16 weeks). Diabetes was induced by the application of streptozotocin (STZ) and the development of diabetes was documented by increased levels of blood glucose, by reduction in body weight and by decreased plasma concentrations of insulin. The contraction was significantly smaller in diabetic rats. Insulin (80 IU/l) reduced the contraction force in both control and diabetic groups. The post-rest potentiation of contraction was not influenced by insulin in control rats, but insulin increased it in diabetic rats. The negative inotropic effect of insulin was preserved in the presence of cyclopiazonic acid (3 micromol/l), a blocker of sarcoplasmic reticulum (SR) Ca2+ pump, in both control and diabetic groups. In contrast, the negative inotropic effect of insulin was completely prevented in the presence of nifedipine (3 micromol/l), a blocker of L-type Ca2+ current. We conclude that insulin exerts a significant negative inotropic effect in rat myocardium, both control and diabetic. This effect is probably related to processes of SR Ca2+ release triggering, whereas SR Ca2+ loading is not involved.     

Preserved contractile function despite atrophic remodeling in unloaded rat hearts

The present study was designed to determine whether myocardial atrophy is necessarily associated with changes in cardiac contractility. Myocardial unloading of normal hearts was produced via heterotopic transplantation in rats. Contractions of isolated myocytes (1.2 mM Ca2+; 37 degrees C) were assessed during field stimulation (0.5, 1.0, and 2.0 Hz), and papillary muscle contractions were assessed during direct stimulation (2.0 mM Ca2+; 37 degrees C; 0.5 Hz). Hemodynamic unloading was associated with a 41% decrease in median myocyte volume and proportional decreases in myocyte length and width. Nevertheless, atrophic myocytes had normal fractional shortening, time to peak contraction, and relaxation times. Despite decreases in absolute maximal force generation (F(max)), there were no differences in F(max)/ area in papillary muscles isolated from unloaded transplanted hearts. Therefore, atrophic remodeling after unloading is associated with intact contractile function in isolated myocytes and papillary muscles when contractile indexes are normalized to account for reductions in cell length and cross-sectional area, respectively. Nevertheless, in the absence of compensatory increases in contractile function, reductions in myocardial mass will lead to impaired overall work capacity.     

Mesenteric lymph from rats with trauma-hemorrhagic shock causes abnormal cardiac myocyte function and induces myocardial contractile dysfunction

Myocardial contractile dysfunction develops following trauma-hemorrhagic shock (T/HS). We have previously shown that, in a rat fixed pressure model of T/HS (mean arterial pressure of 30-35 mmHg for 90 min), mesenteric lymph duct ligation before T/HS prevented T/HS-induced myocardial contractile depression. To determine whether T/HS lymph directly alters myocardial contractility, we examined the functional effects of physiologically relevant concentrations of mesenteric lymph collected from rats undergoing trauma-sham shock (T/SS) or T/HS on both isolated cardiac myocytes and Langendorff-perfused whole hearts. Acute application of T/HS lymph (0.1-2%), but not T/SS lymph, induced dual inotropic effects on myocytes with an immediate increase in the amplitude of cell shortening (1.4 ± 0.1-fold) followed by a complete block of contraction. Similarly, T/HS lymph caused dual, positive and negative effects on cellular Ca2? transients. These effects were associated with changes in the electrophysiological properties of cardiac myocytes; T/HS lymph initially prolonged the action potential duration (action potential duration at 90% repolarization, 3.3 ± 0.4-fold), and this was followed by a decrease in the plateau potential and membrane depolarization. Furthermore, intravenous infusion of T/HS lymph, but not T/SS lymph, caused myocardial contractile dysfunction at 24 h after injection, which mimicked actual T/HS-induced changes; left ventricular developed pressure (LVDP) and the maximal rate of LVDP rise and fall (±dP/dt(max)) were decreased and inotropic response to Ca2? was blunted. However, the contractile responsiveness to β-adrenergic receptor stimulation in the T/HS lymph-infused hearts remained unchanged. These results suggest that T/HS lymph directly causes negative inotropic effects on the myocardium and that T/HS lymph-induced changes in myocyte function are likely to contribute to the development of T/HS-induced myocardial dysfunction.     

心肌α1—肾上腺素受体激动对豚鼠心室乳头肌的影响

The alpha-adrenoceptor agonist phenylephrine (5.0 x 10(-6) mol/L) was used to stimulate myocardial alpha-adrenoceptors of the guinea-pig ventricular papillary muscle, and changes of transmembrane action potential and contractile force of the muscle were observed. The alpha 1-adrenoceptor blocker prazosin (5.0 x 10(-7) mol/L) and the alpha 2-adrenoceptor blocker yohimbine (5.0 x 10(-7) mol/L) were used to determine which subtype of alpha-adrenoceptor is responsible for the effects. The beta-adrenoceptor blocker propranolol (1.0 x 10(-6) mol/L) was used throughout the experiment. The results show that the myocardial alpha 1-adrenoceptor stimulation (1) increases the contractile force of the guinea-pig ventricular papillary muscle, (2) prolongs the time to peak contractile force while the duration of relaxation is not altered, (3) prolongs the fast response action potential duration, and (4) increases the maximal rate of depolarization during the phase 0 of the slow response action potential. It is suggested that the electrophysiological and positive inotropic effects of myocardial alpha 1-adrenoceptor stimulation might be due to the activation of the slow inward current and an increase in Ca2+ influx.     

Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction

ANG II type 2 receptor (AT(2)) is upregulated in failing hearts, but its effect on myocyte contractile function is not known. We measured fractional cell shortening and intracellular Ca(2+) concentration transients in left ventricular myocytes derived from transgenic mice in which ventricle-specific expression of AT(2) was driven by the myosin light chain 2v promoter. Confocal microscopy studies confirmed upregulation of AT(2) in the ventricular myocytes and partial colocalization of AT(2) with AT(1). Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/l extracellular Ca(2+) concentration, 25 degrees C) and study of contractile reserve at faster pacing rates (1-5 Hz) revealed Ca(2+)-dependent contractile dysfunction in myocytes from AT(2) transgenic mice. Comparison of two transgenic lines suggested a dose-dependent relationship between magnitude of contractile dysfunction and level of AT(2) expression. Second, activity of the Na(+)/H(+) exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to beta-adrenergic versus ANG II stimulation differed. Both lines showed impaired contractile response to beta-adrenergic stimulation. ANG II elicited an increase in contractility and intracellular Ca(2+) in wild-type myocytes but caused a negative inotropic effect in myocytes from AT(2) transgenic mice. In contrast with beta-adrenergic response, the depressed response to ANG II was related to level of AT(2) overexpression. The depressed response to ANG II was also present in myocytes from young transgenic mice before development of heart failure. Thus chronic overexpression of AT(2) has the potential to cause Ca(2+)- and pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to ANG II.     

白藜芦醇甙对大鼠心室乳头状肌动作电位的影响及其离子机制(英文)

有研究表明白藜芦醇甙(polydatin)具有抗缺血性心律失常作用,但其电生理学机制尚未明了。本研究旨在应用细胞内记录和全细胞膜片钳方法,探讨白藜芦醇甙对大鼠心室乳头状肌动作电位的影响及其离子机制。结果显示:(1)白藜芦醇甙(50和100μmol/L)可剂量依赖性地缩短正常乳头状肌动作电位复极化50%时间(APD50)和90%时间(APD90)(P<0.01)。白藜芦醇甙对正常乳头状肌静息电位(resting potential,RP)、动作电位幅值(amplitude of action potential,APA)、超射值(overshoot,OS)和0期最大上升速度(Vmax)无影响(P>0.05)。(2)对部分去极化的乳头状肌,白藜芦醇甙(50μmol/L)不但缩短APD50和APD90,而且还降低动作电位OS、APA和Vmax(P<0.05)。(3)ATP敏感钾通道阻断剂格列本脲(10μmol/L)可部分阻断白藜芦醇甙(50μmol/L)的电生理效应。(4)一氧化氮合酶抑制剂L-NAME(1 mmol/L)对白藜芦醇甙的上述效应无影响。(5)白藜芦醇甙(25、50、75、100μmol/L)可浓度依...     

Mibefradil improves beta-adrenergic responsiveness and intracellular Ca(2+) handling in hypertrophied rat myocardium

The present study investigated the effects of mibefradil, a novel T-type channel blocker, on ventricular function and intracellular Ca(2+) handling in normal and hypertrophied rat myocardium. Ca(2+) transient was measured with the bioluminescent protein, aequorin. Mibefradil (2 microM) produced nonsignificant changes in isometric contraction and peak systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) in normal rat myocardium. Hypertrophied papillary muscles isolated from aortic-banded rats 10 weeks after operation demonstrated a prolonged duration of isometric contraction, as well as decreased amplitudes of developed tension and peak Ca(2+) transient compared with the sham-operated group. Additionally, diastolic [Ca(2+)](i) increased in hypertrophied rat myocardium. The positive inotropic effect of isoproterenol stimulation was blunted in hypertrophied muscles despite a large increase in Ca(2+) transient amplitude. Afterglimmers and corresponding aftercontractions were provoked with isoproterenol (10(-5) and 10(-4) M) stimulation in 4 out of 16 hypertrophied muscles, but were eliminated in the presence of mibefradil (2 microM). In addition, hypertrophied muscles in the presence of mibefradil had a significant improvement of contractile response to isoproterenol stimulation and a reduced diastolic [Ca(2+)](I), although a mild decrease of peak Ca(2+)-transient was also shown. However, verapamil (2 microM) did not restore the inotropic and Ca(2+) modulating effects of isoproterenol in hypertrophied myocardium. Mibefradil partly restores the positive inotropic response to beta-adrenergic stimulation in hypertrophied myocardium from aortic-banded rats, an effect that might be useful in hypertrophied myocardium with impaired [Ca(2+)](i) homeostasis.     

Metallothionein antagonizes aging-induced cardiac contractile dysfunction: role of PTP1B, insulin receptor tyrosine phosphorylation and Akt

Aging is often accompanied by reduced insulin sensitivity and cardiac dysfunction. However, the causal relationship between the two remains poorly understood. This study was designed to determine the impact of cardiac-specific overexpression of antioxidant metallothionein (MT) on aging-associated cardiac dysfunction and impaired insulin signaling. Contractile and intracellular Ca(2+) properties were evaluated in left ventricular myocytes including peak shortening (PS), maximal velocity of shortening/relengthening (+/- dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR(90)), fura-2 fluorescence intensity change (DeltaFFI) and intracellular Ca(2+) decay rate. Expression of insulin receptor, protein-tyrosine phosphatase 1B (PTP1B), phosphorylation of insulin receptor (Tyr1146) and Akt were evaluated by Western blot analysis. Aged wild-type FVB and MT transgenic mice (26-28 months old) displayed glucose intolerance and hyperinsulinemia. Cardiomyocytes from aged FVB mice exhibited prolonged TR(90) and intracellular Ca(2+) decay associated with normal PS, +/- dL/dt, TPS and DeltaFFI compared with those from young (2-3 months old) mice. Western blot analysis revealed reduced Akt expression and insulin (5 mU g(-1))-stimulated Akt phosphorylation, elevated PTP1B expression and diminished basal insulin receptor tyrosine phosphorylation associated with comparable insulin receptor expression in aged FVB mouse hearts. All of these aging-related defects in cardiac contractile function and insulin signaling (although not hyperinsulinemia and glucose intolerance) were significantly attenuated or ablated by MT transgene. These data indicate that enhanced antioxidant defense is beneficial for aging-induced cardiac contractile dysfunction and alteration in insulin signaling.     

Effect of insulin on characteristics of contractile responses of fast and slow skeletal muscles of rats with acute streptozotocin-induced diabetes

Comparison of amplitude-time characteristics of fast extensor digitorum longus muscles (m. EDL) isolated from control rats and rats with model of acute streptozotocin-induced diabetes mellitus (DM) 12 and 30 days after treatment with streptozotozin did not reveal significant changes of strength of single normalized contractile responses as compared with control. In slow (m. Soleus) muscles of rats with the 30-day long SD, essential changes of the amplitude-time characteristics of such contractile responses were observed: a decrease of their amplitude and an increase of duration. In the diabetic rats treated with insulin there develops resistance of skeletal muscles of both types to action of exogenous insulin. Both in control and in diabetic animals the exhausting stimulation of m. EDL with trains from 5 impulses did not reveal significant differences at early (up to 3 min) terms of development of fatigue. Under similar conditions, fatigue of m. Soleus in rats of the both diabetic groups developed significantly faster as compared with control (already in 30 s after the beginning of stimulation). Insulin at a concentration of 0.5–1 nM produced a dose-dependent decrease of amplitude of single contractile responses in fast and slow muscles of rats with the acute SD model (the negative inotropic action). Earlier, we demonstrated in healthy rats the similar action of insulin, but at the higher concentrations [1]. Insulin at a concentration of 10 nM did not produce an essential effect on dynamics of depression of responses in the course of development of fatigue at tetanical stimulation of m. EDL and m. Soleus both in control and in diabetic rats, but affected essentially the dynamics of change of duration of the half-decay (T_hd) of their tetanical responses. The presence of insulin in the washing solution led to stabilization of the period of muscle relaxation in the course of development of fatigue in all studied animal groups.     

增强Na+-Ca2+交换对大鼠心脏的正性变力作用及对哇巴因效应的加强

本文采用大鼠乳头肌张力测定及离体心脏灌流技术,研究大鼠心肌Na -Ca2 交换对乳头肌及离体灌流心肌变力性的影响。采用大鼠特异性Na -Ca2 交换激动剂E-4031能剂量依赖性地增加大鼠乳头肌的发展张力(P<0.05,n=6)及离体心脏的心泵功能(P<0.05,n=4);特异性Na -Ca2 交换抑制剂KB-R7943具有相反的效应,并可完全消除E-4031引起的正性变力作用。哇巴因(ouabain,0.5μmol/L)与E-4031(3μmol/L)联合使用,可使乳头肌发展张力由单独使用哇巴因时的0.25±0.03 g升高至0.29±0.04g(P<0.05,n=6);联合用药对大鼠离体心脏心泵功能的影响也强于哇巴因单独作用的效果。本研究结果证实,E-4031通过增强心肌Na -Ca2 交换,对大鼠乳头肌和离体心脏产生正性变力作用;与哇巴因合用时,它们的正性变力作用有相加作用。     

IGF-I attenuates diabetes-induced cardiac contractile dysfunction in ventricular myocytes

Diabetic cardiomyopathy is characterized by impaired ventricular contraction and altered function of insulin-like growth factor I (IGF-I), a key factor for cardiac growth and function. Endogenous IGF-I has been shown to alleviate diabetic cardiomyopathy. This study was designed to evaluate exogenous IGF-I treatment on the development of diabetic cardiomyopathy. Adult rats were divided into four groups: control, control + IGF-I, diabetic, and diabetic + IGF-I. Streptozotocin (STZ; 55 mg/kg) was used to induce experimental diabetes immediately followed by a 7-wk IGF-I (3 mg. kg(-1). day(-1) ip) treatment. Mechanical properties were assessed in ventricular myocytes including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were evaluated as Ca(2+)-induced Ca(2+) release and Ca(2+) clearing constant. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and glucose transporter (GLUT4) were assessed by Western blot. STZ caused significant weight loss and elevated blood glucose, demonstrating the diabetic status. The diabetic state is associated with reduced serum IGF-I levels, which were restored by IGF-I treatment. Diabetic myocytes showed reduced PS and +/-dL/dt as well as prolonged TPS, TR(90), and intracellular Ca(2+) clearing compared with control. IGF-I treatment prevented the diabetes-induced abnormalities in PS, +/-dL/dt, TR(90), and Ca(2+) clearing but not TPS. The levels of SERCA and GLUT4, but not PLB, were significantly reduced in diabetic hearts compared with controls. IGF-I treatment restored the diabetes-induced decline in SERCA, whereas it had no effect on GLUT4 and PLB levels. These results suggest that exogenous IGF-I treatment may ameliorate contractile disturbances in cardiomyocytes from diabetic animals and could provide therapeutic potential in the treatment of diabetic cardiomyopathy.     

Single adult rabbit and rat cardiac myocytes retain the Ca2+- and species-dependent systolic and diastolic contractile properties of intact muscle

The systolic and diastolic properties of single myocytes and intact papillary muscles isolated from hearts of adult rats and rabbits were examined at 37 degrees C over a range of stimulation frequencies and bathing [Ca2+]o (Cao). In both rabbit myocytes and intact muscles bathed in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted in a positive staircase of twitch performance. During stimulation at 2 min-1, twitch performance also increased with increases in Cao up to 20 mM. In the absence of stimulation, both rabbit myocytes and muscles were completely quiescent in less than 15 mM Cao. Further increases in Cao caused the appearance of spontaneous asynchronous contractile waves in myocytes and in intact muscles caused scattered light intensity fluctuations (SLIF), which were previously demonstrated to be caused by Ca2+-dependent spontaneous contractile waves. In contrast to rabbit preparations, intact rat papillary muscles exhibited SLIF in 1.0 mM Cao. Two populations of rat myocytes were observed in 1 mM Cao: approximately 85% of unstimulated cells exhibited low-frequency (3-4 min-1) spontaneous contractile waves, whereas 15%, during a 1-min observation period, were quiescent. In a given Cao, the contractile wave frequency in myocytes and SLIF in intact muscles were constant for long periods of time. In both intact rat muscles and myocytes with spontaneous waves, in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted, on the average, in a 65% reduction in steady state twitch amplitude. Of the rat myocytes that did not manifest waves, some had a positive, some had a flat, and some had a negative staircase; the average steady state twitch amplitude of these cells during stimulation at 120 min-1 was 30% greater than that at 6 min-1. In contrast to rabbit preparations, twitch performance during stimulation at 2 min-1 saturated at 1.5 mM Cao in both intact rat muscles and in the myocytes with spontaneous waves. We conclude that the widely divergent, Ca2+-dependent systolic and diastolic properties of intact rat and rabbit cardiac muscle are retained with a high degree of fidelity in the majority of viable single myocytes isolated from the myocardium of these species, and that these myocytes are thus a valid model for studies of Ca2+-dependent excitation-contraction mechanisms in the heart.     

High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus.

Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg.kg(-1).day(-1) ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (tau). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged tau associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and tau, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.     

Cellular distribution of GPR14 and the positive inotropic role of urotensin II in the myocardium in adult rat.

Urotensin II is a cyclic neuropeptide recently shown to play a role via its receptor GPR14 in regulating vascular tone in the mammalian cardiovascular system. The existence of GPR14 in rat heart has been validated by ligand binding assay and RT-PCR. In the present study, we investigated the cellular distribution of GPR14 protein in rat heart by using immunohistochemistry and confocal microscopic immunofluorescence double staining with antipeptide polyclonal antibodies against GPR14 and cell type markers for myocytes and endothelial cells. The direct effect of urotensin II on left ventricular contractility was further evaluated in isolated left ventricular papillary muscles of the rat. In paraffin-embedded heart sections, positive immunohistochemical staining was observed in the left ventricle but not in the right ventricle and atria. Immunofluorescence double staining revealed the cardiac myocyte as the only cell type expressing GPR14 protein in frozen heart sections as well as in isolated cardiac myocytes. There was no visible signal for GPR14 in intramyocardial coronary arteries and capillaries. The existence of GPR14 protein in rat heart was further validated by immunoprecipitation and Western blot analysis. In isolated rat left ventricular papillary muscle preparations, urotensin II induced an increase in active contractile force. GPR14 mRNA was also detected in rat heart by RT-PCR. These data provide the first direct evidence for the cellular localization of GPR14 receptor protein and a positive inotropic effect of urotensin II in normal rat heart.     

Pharmacological characterization of an Endogenous Negative Inotrophic Factor (ENIF) from porcine heart

Recently we have been successful in isolating an endogenous negative inotropic factor (ENIF) from porcine left ventricular tissue. In this study, we have characterized its pharmacological properties. The results of the study demonstrated that ENIF produces a concentration-dependent negative inotropic response on both guinea pig left atria and right ventricular trabeculae. The maximal reduction in contractile force produced by 300 ul of ENIF (5 ml bath) on atria and trabeculae were 90.0 ± 0.8% and 77.5 ± 6%. Atria, however, was significantly more sensitive to ENIF than trabeculae. The ED 50 of ENIF for atria was found to be 38 ul as opposed to ED 50 of 100 ul of ENIF for trabeculae.Acetylcholine (ACh), a muscarinic receptor agonist, decreased the contractile force of guinea pig atria in a dose-dependent manner with a maximal decline in the contractile force of 90%. However, none of the concentration of ACh used affected the contractile function of the trabeculae. Atropine (1 uM) completely blocked the negative inotropic response on atria of all the doses of ACh used. The same dose of atropine, however, was unable to influence the negative inotropic effect of any of the doses of ENIF used on either the atria or trabeculae preparations in our study. The maximal decline in the contractile force of atria was e.g. 94 and 95% in the presence and absence of atropine respectively. These data demonstrate that the myocardial negative inotropic effect of ENIF is not mediated via the cholinegic receptor mechanism.