Positive Inotropic Interventions Research Articles

Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy.

AimsDilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome—decreased ventricular function. Titin (TTN)—a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS).Methods and resultsiPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to β-adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells.ConclusionsThese findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.

Positive Inotropic Effects of Akrinor® in Human Atrial Tissue: Relevant PDE Inhibition at Clinically Used Concentration

Objectives: Akrinor® is a widely used drug to treat intraoperative hypotension. The drug consists of three different compounds: norepinephrine, norephedrine and theophylline. Norepinephrine and norephedrine are expected to exert sympathomimetic actions. Theophylline should inhibit PDE and therefore sensitize for cAMP-dependent positive inotropic interventions, like catecholamines. However, theophylline in Akrinor® is covalently linked to norepinephrine and norephedrine. It is not known if the potency of theophylline to inhibit PDE is preserved in such a formulation. Therefore, we have measured whether clinically used concentrations of Akrinor® inhibit PDE in human heart muscle.

A single resistance exercise session improves myocardial contractility in spontaneously hypertensive rats.

Resistance training evokes myocardial adaptation; however, the effects of a single resistance exercise session on cardiac performance are poorly understood or investigated. This study aimed to investigate the effects of a single resistance exercise session on the myocardial contractility of spontaneously hypertensive rats (SHRs). Male 3-month-old SHRs were divided into two groups: control (Ct) and exercise (Ex). Control animals were submitted to sham exercise. Blood pressure was measured in conscious rats before the exercise session to confirm the presence of arterial hypertension. Ten minutes after the exercise session, the animals were anesthetized and killed, and the hearts were removed. Cardiac contractility was evaluated in the whole heart by the Langendorff technique and by isometric contractions of isolated left ventricular papillary muscles. SERCA2a, phospholamban (PLB), and phosphorylated PLB expression were investigated by Western blot. Exercise increased force development of isolated papillary muscles (Ex=1.0±0.1 g/mg vs Ct=0.63±0.2 g/mg, P<0.05). Post-rest contraction was greater in the exercised animals (Ex=4.1±0.4% vs Ct=1.7±0.2%, P<0.05). Papillary muscles of exercised animals developed greater force under increasing isoproterenol concentrations (P<0.05). In the isolated heart, exercise increased left ventricular isovolumetric systolic pressure (LVISP; Δ +39 mmHg; P<0.05) from baseline conditions. Hearts from the exercised rats presented a greater response to increasing diastolic pressure. Positive inotropic intervention to calcium and isoproterenol resulted in greater LVISP in exercised animals (P<0.05). The results demonstrated that a single resistance exercise session improved myocardial contractility in SHRs.

Low-dose ouabain administration increases Na+,K+-ATPase activity and reduces cardiac force development in rats

BackgroundOuabain is a digitalis compound that inhibits the Na+,K+-ATPase (NKA) activity inducing increment in cardiac force. However, this effect seems to be dose dependent. At low concentration, ouabain can induce an increase of NKA activity. MethodsWe investigated the effects of ouabain administration (25μg/kg/day) for 15 days on cardiac contractility and NKA activity. Blood pressure and left ventricular papillary muscle contraction from placebo and ouabain-treated rats for 15 (OUA15) days were evaluated. Isometric force, post-rest potentiation, positive inotropic intervention produced by isoproterenol, and tetanic tension were measured. The activity and protein expression levels of α1 and α2 isoforms of NKA, sodium calcium exchanger (NCX), sarcoplasmic reticulum calcium ATPase (SERCA2a) and phospholamban (PLB) were also measured. ResultsSystolic and diastolic blood pressures increased after treatment with ouabain. However, isometric tension was reduced in the ouabain treated group. Post-rest potentiation, time parameters, inotropic interventions by isoproterenol and tetanic tension did not change. In the ouabain treated group, NKA activity was increased (Oua 406.16±70.6 vs. CT 282.80±80.5) while protein expression of the α1 isoform of NKA was reduced (Oua 0.97±0.06 vs. CT 0.76±0.05). No changes were observed in protein expression of α2 isoform of NKA, NCX, SERCA2a and PLB. Therefore, although 15-day ouabain treatment increases blood pressure (Oua: 116.4±3 vs. CT: 99.9±3), treatment also reduces isometric tension development (Oua: 0.34±0.14 vs. CT: 0.56±0.22). ConclusionWe suggest that the effects induced by ouabain in the isolated cardiac muscle could be related at least in part, to changes in NKA activity.

Moderate hypothermia for severe cardiogenic shock (COOL Shock Study I & II)

Aim of the studyHypothermia exerts profound protection from neurological damage and death after resuscitation from circulatory arrest. Its application during concomitant cardiogenic shock has been discussed controversially, and still hypothermia is used with reserve when haemodynamic parameters are impaired. On the other hand hypothermia improves force development in isolated human myocardium. Thus, we hypothesized that hypothermia could beneficially affect cardiac function in patients during cardiogenic shock. Methods14 Patients, admitted to Intensive Care Unit for cardiogenic shock under inotropic support, were enrolled and moderate hypothermia (33°C) was induced for either one (n=5, short-term) or twenty-four (n=9, mid-term) hours. Results12 patients suffered from ischaemic cardiomyopathy, 2 were female, and 6 were included after cardiac arrest and resuscitation. Body temperature was controlled by an intravascular cooling device. Short-term hypothermia consistently decreased heart rate, and increased stroke volume, cardiac index and cardiac power output. Metabolic and electrocardiographic parameters remained constant during cooling. Improved cardiac function persisted during mid-term hypothermia, but was reversed during re-warming. No severe or persistent adverse effects of hypothermia were observed. ConclusionModerate Hypothermia is safe and feasable in patients during cardiogenic shock. Moreover, hypothermia improved parameters of cardiac function, suggesting that hypothermia might be considered as a positive inotropic intervention rather than a risk for patients during cardiogenic shock.

Pacing stress echocardiography

BackgroundHigh-rate pacing is a valid stress test to be used in conjunction with echocardiography; it is independent of physical exercise and does not require drug administration. There are two main applications of pacing stress in the echo lab: the noninvasive detection of coronary artery disease through induction of a regional transient dysfunction; and the assessment of contractile reserve through peak systolic pressure/ end-systolic volume relationship at increasing heart rates to assess global left ventricular contractility.MethodsThe pathophysiologic rationale of pacing stress for noninvasive detection of coronary artery disease is obvious, with the stress determined by a controlled increase in heart rate, which is a major determinant of myocardial oxygen demand, and thereby tachycardia may exceed a fixed coronary flow reserve in the presence of hemodynamically significant coronary artery disease. The use of pacing stress echo to assess left ventricular contractile reserve is less established, but promising. Positive inotropic interventions are mirrored by smaller end-systolic volumes and higher end-systolic pressures. An increased heart rate progressively increases the force of ventricular contraction (Bowditch treppe or staircase phenomenon). To build the force-frequency relationship, the force is determined at different heart rate steps as the ratio of the systolic pressure (cuff sphygmomanometer)/end-systolic volume index (biplane Simpson rule). The heart rate is determined from ECG.ConclusionTwo-dimensional echocardiography during pacing is a useful tool in the detection of coronary artery disease. Because of its safety and ease of repeatability noninvasive pacing stress echo can be the first-line stress test in patients with permanent pacemaker.The force-frequency can be defined as up- sloping (normal) when the peak stress pacing systolic pressure/end-systolic volume index is higher than baseline and intermediate stress values, biphasic with an initial up- sloping followed by a later down-sloping trend, or flat or negative when peak stress pacing systolic pressure/end-systolic volume index is equal or lower than baseline stress values. This approach is certainly highly feasible and allows a conceptually immaculate definition of contractility with prognostic usefulness, but its therapeutic implications remains to be established. Bowditch treppe, assessed with pacing stress, can be used to assess the optimal stimulation frequency and to optimise the patient's chronotropic response in programming rate-adaptive pacemakers.

Operative contractility: A functional concept of the inotropic state

1. Initial unsuccessful attempts to evaluate ventricular function in terms of the 'heart as a pump' led to focusing on the 'heart as a muscle' and to the concept of myocardial contractility. However, no clinically ideal index exists to assess the contractile state. The aim of the present study was to develop a mathematical model to assess cardiac contractility. 2. A tri-axial system was conceived for preload (PL), afterload (AL) and contractility, where stroke volume (SV) was represented as the volume of the tetrahedron. Based on this model, 'operative' contractility ('OperCon') was calculated from the readily measured values of PL, AL and SV. The model was tested retrospectively under a variety of different experimental and clinical conditions, in 71 studies in humans and 29 studies in dogs. A prospective echocardiographic study was performed in 143 consecutive subjects to evaluate the ability of the model to assess contractility when SV and PL were measured volumetrically (mL) or dimensionally (cm). 3. With inotropic interventions, OperCon changes were comparable to those of ejection fraction (EF), velocity of shortening (Vcf) and dP/dt-max. Only with positive inotropic interventions did elastance (Ees) show significantly larger changes. With load manipulations, OperCon showed significantly smaller changes than EF and Ees and comparable changes to Vcf and dP/dt-max. Values of OperCon were similar when AL was represented by systolic blood pressure or wall stress and when volumetric or dimensional values were used. 4. Operative contractility is a reliable, simple and versatile method to assess cardiac contractility.

Myocardial contractility in the echo lab: molecular, cellular and pathophysiological basis

In the standard accepted concept, contractility is the intrinsic ability of heart muscle to generate force and to shorten, independently of changes in the preload or afterload with fixed heart rates. At molecular level the crux of the contractile process lies in the changing concentrations of Ca2+ ions in the myocardial cytosol. Ca2+ ions enter through the calcium channel that opens in response to the wave of depolarization that travels along the sarcolemma. These Ca2+ ions "trigger" the release of more calcium from the sarcoplasmic reticulum (SR) and thereby initiate a contraction-relaxation cycle.In the past, several attempts were made to transfer the pure physiological concept of contractility, expressed in the isolated myocardial fiber by the maximal velocity of contraction of unloaded muscle fiber (Vmax), to the in vivo beating heart. Suga and Sagawa achieved this aim by measuring pressure/volume loops in the intact heart: during a positive inotropic intervention, the pressure volume loop reflects a smaller end-systolic volume and a higher end-systolic pressure, so that the slope of the pressure volume relationship moves upward and to the left. The pressure volume relationship is the most reliable index for assessing myocardial contractility in the intact circulation and is almost insensitive to changes in preload and after load. This is widely used in animal studies and occasionally clinically. The limit of the pressure volume relationship is that it fails to take into account the frequency-dependent regulation of contractility: the frequency-dependent control of transmembrane Ca2+ entry via voltage-gated Ca2+ channels provides cardiac cells with a highly sophisticated short-term system for the regulation of intracellular Ca2+ homeostasis. An increased stimulation rate increases the force of contraction: the explanation is repetitive Ca2+ entry with each depolarization and, hence, an accumulation of cytosolic calcium. As the heart fails, there is a change in the gene expression from the normal adult pattern to that of fetal life with an inversion of the normal positive slope of the force-frequency relation: systolic calcium release and diastolic calcium reuptake process is lowered at the basal state and, instead of accelerating for increasing heart rates, slows down. Since the force-frequency relation uncovers initial alteration of contractility, as an intermediate step between normal and abnormal contractility at rest, a practical index to measure it is mandatory.Measuring end-systolic elastance for increasing heart rates is impractical: increasing heart rates with atrial pacing has to be adjunct to the left ventricular conductance catheter, to the left ventricular pressure catheter, to the vena cava balloon, and to afterload changes. Furthermore, a noninvasive index is needed. Noninvasive measurement of the pressure/volume ratio for increasing heart rates during stress in the echo lab could be the practical answer to this new clinical demand in the current years of a dramatic increase in the number of heart failure patients.

The feasibility of repeated left ventricular ejection fraction analysis with sequential single-dose radionuclide ventriculography

Repeated left ventricular ejection fraction (LVEF) analyses with sequential single-dose radionuclide ventriculography might be an interesting technique for monitoring the effect of positive inotropic interventions. The aim of the study was to assess the reproducibility of LVEF measurement with planar radionuclide ventriculography within 3 h, using a standard single dose of radioactive tracer. Sixteen patients underwent routine planar radionuclide ventriculography with a standard dose of 500 MBq of [Tc]pertechnetate and returned after 3 h for a repeat planar radionuclide ventriculography without administration of additional tracer. The average initial LVEF was 35.1+/-18.6%-point (range, 12%-point to 68%-point). The mean difference of the LVEF between the initial planar radionuclide ventriculography and the repeat planar radionuclide ventriculography was 2.8%+/-6.3% (range, -11.8% to 13.3%, P=NS). The correlation between both measurements was significant with a correlation coefficient of 0.995 (P<0.01). Bland-Altman analysis revealed a mean LVEF difference of 0.94%-point between the baseline planar radionuclide ventriculography and the repeat planar radionuclide ventriculography (95% confidence interval: -2.7%-point to 4.5%-point). The visual wall motion assessment showed excellent reproducibility, with a kappa-statistic of 0.98. Repeated radionuclide ventriculography with a 3 h interval using a single standard dose of 500 MBq of [Tc]pertechnetate is highly reproducible and will be useful for monitoring the effect of positive inotropic interventions.

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9–12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42–63%) compared to starch (ST)-fed controls. Cytosolic Ca 2+ removal attributable to Na +/Ca 2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca 2+ and cytosolic Ca 2+ clearing through NCX were normal, whereas Ca 2+ uptake by SERCA was significantly slower in SU myocytes (330 ± 29 ms) compared to ST cells (253 ± 16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca 2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca 2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).

Eucalyptol, an essential oil, reduces contractile activity in rat cardiac muscle

Eucalyptol is an essential oil that relaxes bronchial and vascular smooth muscle although its direct actions on isolated myocardium have not been reported. We investigated a putative negative inotropic effect of the oil on left ventricular papillary muscles from male Wistar rats weighing 250 to 300 g, as well as its effects on isometric force, rate of force development, time parameters, post-rest potentiation, positive inotropic interventions produced by Ca2+ and isoproterenol, and on tetanic tension. The effects of 0.3 mM eucalyptol on myosin ATPase activity were also investigated. Eucalyptol (0.003 to 0.3 mM) reduced isometric tension, the rate of force development and time parameters. The oil reduced the force developed by steady-state contractions (50% at 0.3 mM) but did not alter sarcoplasmic reticulum function or post-rest contractions and produced a progressive increase in relative potentiation. Increased extracellular Ca2+ concentration (0.62 to 5 mM) and isoproterenol (20 nM) administration counteracted the negative inotropic effects of the oil. The activity of the contractile machinery evaluated by tetanic force development was reduced by 30 to 50% but myosin ATPase activity was not affected by eucalyptol (0.3 mM), supporting the idea of a reduction of sarcolemmal Ca2+ influx. The present results suggest that eucalyptol depresses force development, probably acting as a calcium channel blocker.

Noninvasive assessment of left ventricular contractility by pacemaker stress echocardiography

Estimating contractility of the left ventricle with noninvasive techniques is an important yet elusive goal. Positive inotropic interventions are mirrored by smaller end-systolic volumes and higher end-systolic pressures. An increased heart rate progressively increases the force of ventricular contraction (Bowditch treppe or staircase phenomenon). To assess the feasibility of a noninvasive estimation of force-frequency relation (FFR) during pacing stress in the echo lab in patients with permanent pacemaker (PM). Transthoracic stress pacing echocardiography was performed in 26 patients with a permanent pacemaker (age 69+/-11 years; 21 men, 5 women). Seven patients had normal function at baseline and during stress ("normals"); eight had angiographically assessed coronary artery disease (three with and five without induced ischemia with stress echo); eleven patients had dilated cardiomyopathy (DC). To build the FFR, the force was determined at different steps as the ratio of the systolic pressure (SP, cuff sphygmomanometer)/end-systolic volume index (ESV, biplane Simpson rule/body surface area). Heart rate was determined from ECG. The absolute value of the FFR slope was highest in controls and lowest in DC patients. A flat-downsloping FFR was found in 12/19 patients but not for normals (p<0.01). Noninvasive pacemaker stress echocardiography (PASE) is a simple and efficient option to assess left ventricular (LV) contractility in patients with permanent pacemaker.

Nitroxyl gets to the heart of the matter

Heart failure (HF) is a progressively disabling and ultimately fatal disease, which is characterized by a decline in the heart's ability to pump blood efficiently enough to meet the body's metabolic demands. Despite substantial advances in our understanding of the underlying pathophysiology (1) and the therapeutic management of acute and chronic HF (2) in recent years, the outlook of patients with these conditions remains poor. Not only are mortality and morbidity discouragingly high, but also the patients' quality of life remains impaired because of a substantial symptom burden. In the United States alone, HF is responsible for almost 1 million hospital admissions (more than for all forms of cancer combined) and >50,000 deaths each year, with estimated annual costs exceeding $20 billion (3, 4). Despite improved patient information, beneficial changes in lifestyle and better treatment options, HF remains to be a major public health problem in industrialized nations and the leading cause of hospitalization in people older than 65 years. At a time when other cardiovascular diseases are on the decline, HF is rising and likely to further escalate over the coming decades because of an aging population and increased survival from the underlying causes such as coronary heart disease and hypertension. A broad spectrum of different drugs and various guidelines for the treatment of HF exist (5, 6). In the past, HF was mainly viewed as a problem of diminished cardiac output. Maximization of the latter with positive inotropic (contractility enhancing) agents led to therapies that initially improved functional capacity, but increased mortality. Today, the therapeutic focus is on reducing elevated filling pressures that lead to the symptoms of congestion (7). Although most recommendations agree on the major drug classes for the first-line and adjunct therapy of HF, there is considerable controversy about the role of positive inotropic agents (8). Despite a documented negative impact on survival, these agents are still widely used, often combined with vasodilators, to limit severe episodes of HF or as a bridge to transplantation. The rationale for combining vasodilatation with positive inotropic intervention lies in the possibility to “unload” the heart, i.e., to reduce its preload and afterload by venous and arterial dilatation, allowing to stimulate cardiac output without increasing oxygen consumption. Although conceptually ideal compounds, currently used inodilators (compounds with positive inotropic and vasodilatior properties) tend to increase myocardial oxygen demand at higher doses, precipitating ischemia in patients with coronary artery disease. Clearly, there is room for improvement in HF management, in particular with regard to quality of life and survival.

Compensatory changes in Ca(2+) and myocardial O(2) consumption in beta-tropomyosin transgenic hearts.

Transgenic mice overexpressing beta-tropomyosin have increased myofilament Ca(2+) sensitivity that we hypothesized would result in altered relationships among pressure and heart rates, intracellular Ca(2+), and myocardial O(2) consumption. In perfused hearts from transgenic mice there was a marked negative force-frequency response between 6 and 10 Hz with a 30 +/- 3% reduction in peak-positive first derivative of pressure development over time (dP/dt) compared with 14 +/- 2% in wild-type mice (P < 0.001). At 8 Hz systolic pressures were normal, though peak systolic intracellular Ca(2+) was significantly reduced in transgenic mice versus wild type (726 +/- 61 vs. 936 +/- 67 nM, P < 0.05) indicating an alteration in the pressure-Ca(2+) relationship. Over a wide range of positive and negative inotropic interventions there were normal developed pressures, though marked elevations in myocardial O(2) consumption (15-54%). Because pressures are normal and intracellular Ca(2+) decreased and myocardial O(2) consumption increased, this suggests that these abnormalities are at least in part compensatory mechanisms to the altered myofilament function.

Contractile adaptations preserving cardiac output predispose the hypertrophied canine heart to delayed afterdepolarization-dependent ventricular arrhythmias.

In dogs, chronic complete atrioventricular block (CAVB) results in structural (biventricular hypertrophy) and electrical (delayed repolarization) remodeling, which predisposes the heart to torsade de pointes arrhythmias. We assessed the contractile alterations in the CAVB dog and tested the hypothesis that these adaptations increase delayed afterdepolarization (DAD)-dependent triggered arrhythmias. Steady-state and dynamic (fast pacing: 1 to 68 stimuli) left and right ventricular systolic and diastolic parameters were determined by positive and negative inotropic interventions at acute AVB and CAVB. Concomitantly, left and right ventricular endocardial monophasic action potentials were registered. In CAVB, all systolic contractile parameters were markedly increased, resulting in preserved cardiac output. The increase was most pronounced at low heart rates, altering the force-frequency response. At both acute AVB and CAVB, the degree of potentiation of cardiac function with pacing was dependent on the number of stimuli and showed a maximum at 8 to 13 stimuli. With CAVB, this potentiation curve was shifted upward, and it was only then that pacing resulted in DADs (in 8 of 10 dogs) and ectopic beats (EBs, in 6 of 10 dogs). The incidence of EBs in relation to the number of stimuli also had a maximum at 8 to 13 stimuli. Ouabain increased the incidence of DADs and EBs, whereas the negative inotropic interventions prevented them completely. The alterations responsible for improvement in systolic contractile function in CAVB dogs predispose the hypertrophied heart to DAD-dependent triggered arrhythmias during positive inotropic interventions.

Mitochondria in heart ischaemia and aging.

We have examined the effect of dietary polyunsaturated fatty acids (PUFAs) upon mitochondrial Ca2+ content and dehydrogenase activation in the rat heart. Diets were either a conventional low-fat chow (Ref) or were rich in n-3 PUFAs from fish oils (n-3) or n-6 PUFAs from animal fat (n-6). We found that the n-3 diet minimized the rise in mitochondrial Ca2+ seen in response to positive inotropic intervention with noradrenaline, and also minimized the activation of pyruvate dehydrogenase, which is Ca2+ dependent. As the work output of all three groups of hearts was the same, this observation may explain the previous finding of increased thermodynamic efficiency of the n-3 heart relative to the n-6 heart. When hearts were subjected to low-flow ischaemia (15 min), followed by 5 min of reperfusion, increases of mitochondrial Ca2+ were less in the n-3 group than in the n-6 group. In more prolonged ischaemia and reperfusion, n-3 feeding may confer protection against mitochondrial Ca2+ overload, opening of the permeability transition pore and cell death. Notably, the effects of n-3 feeding on mitochondrial functioning were most apparent in hearts from senescent rats (23 months). This is consistent with our finding that the decrease in mitochondrial membrane cardiolipin content, and increase in phosphatidylcholine, which occurred with aging in the Ref and n-6 groups, was totally prevented by n-3 feeding. Thus there are a number of reasons to regard an n-3-rich diet as being protective of the heart in aging mammals.

Shortening versus isometric contractions in isolated human failing and non-failing left ventricular myocardium: dependency of external work and force on muscle length, heart rate and inotropic stimulation.

For reasons of simplicity, studies on isolated human myocardium have been conducted using exclusively isometric contractions, although positive inotropic interventions may differently influence force development, extent of shortening and myocardial work performance. We investigated human left ventricular failing and non-failing preparations comparing isometric versus isotonic, i.e., shortening contractions. (1) When muscle length is increased from 90% to 100% lMAX, peak developed force increases by 36% and 43% (p < 0.05) in non-failing and failing human left ventricular myocardium, respectively. Maximum performed work increases similarly in non-failing but decreases in failing myocardium. It can be shown that this discrepancy is due to significantly higher resting tension and does not present an insufficient intrinsic shortening capacity in failing myocardium. (2) When stimulation rate is increased from 0.5 to 2.0 Hz, isometric force increases significantly by 59% in non-failing and decreases by 27% in failing myocardium, whereas maximum performed work increases by 98% and decreases by 46%, respectively. (3) Pharmacological positive inotropic interventions by 7.2 mM calcium (n = 9), 3 x 10(-8) M isoproterenol (n = 7), 3 x 10(-8) M ouabain (n = 5), and 10(-5) M EMD 57033 (n = 3) equally increased force development and extent of shortening: When the fractional effect on shortening (y) was correlated to the fractional effect on force (x), the following linear regression equation was obtained: y = 0.91x + 0.26 (r = 0.86; p < 0.001). The data presented are of clinical and pharmacological importance: (1) The Frank-Starling mechanism is demonstrated to be existent in the failing human myocardium regarding both isometric force developed and maximum work performed. (2) Both force-frequency relations and--to a greater extent--work-frequency relations are reversed in failing human myocardium. (3) Independent of the pharmacological mode of action, positive inotropic compounds increase developed isometric force to the same extent as isotonic shortening and therefore potentiate maximum performed work.

Effect of milrinone and atrial pacing on stunned myocardium.

Most mammalian cardiac muscles show a positive force-frequency relation, which is turned into a negative relation in failing hearts. Stunned myocardium shows similar defects as failing myocardium, it has a functional reserve recruitable by positive inotropic interventions, and possibly shows a disturbed response to increased heart rate. The present experiments compare in vivo the response of stunned and intact myocardium to atrial pacing before and during inotropic stimulation by milrinone. In anaesthetised (piritramide) open chest pigs, heart rate, left ventricular and aortic pressure, left descending (LAD) and circumflex (LCX) coronary artery and aortic blood flow, myocardial systolic shortening in the LAD and LCX area were monitored, and myocardial power was calculated. The LAD region was subjected to ischaemia and reperfused. Heart rate was raised by right atrial pacing after 90 min reperfusion before and during i.v. milrinone (105 microg/kg bolus + 8 microg/kg per min infusion). The ischaemic/reperfused area was sliced post mortem and stained by triphenyl tetrazolium chloride to exclude myocardial infarction. Data from ten experiments are presented. After 90 min LAD reperfusion, LAD blood flow and power were 110 and 36% of preischaemic control, respectively, indicating myocardial stunning. The power of the intact area was not changed (102% of control). Pacing from 87 to 164 per min increased the power of the intact area (+96%), the power of the stunned myocardium decreased (-64%). Milrinone increased the power of the stunned region to 72% of the pre-stunning level and the power of the intact area by +51%. Pacing from 111 to 164 per min during milrinone increased the power of the intact myocardium to the same level as before milrinone, the power of the stunned region did not change. Stunned myocardium responds pathologically to atrial pacing with a negative staircase in contrast to the positive staircase of intact myocardium. Inotropic stimulation by the phosphodiesterase inhibitor milrinone recruited the functional reserve of stunned myocardium. Milrinone did not restore a positive staircase in stunned myocardium, but power was maintained during atrial pacing. The pathological staircase of stunned myocardium may arise from an impaired availability of cyclic AMP, but the data do not exclude defects in calcium handling, a dysfunction of the sarcoplasmic reticulum, or an impaired Ca-sensitivity of the myofilaments.

Force enhancement without changes in cross-bridge turnover kinetics: the effect of EMD 57033

The thiadiazinon derivative EMD 57033 has been found previously in cardiac muscle to increase isometric force generation without a proportional increase in fiber ATPase, thus causing a reduction in tension cost. To analyze the mechanism by which EMD 57033 affects the contractile system, we studied its effects on isometric force, isometric fiber ATPase, the rate constant of force redevelopment (k(redev)), active fiber stiffness, and its effect on Fo, which is the force contribution of a cross-bridge in the force-generating states. We used chemically skinned fibers of the rabbit psoas muscle. It was found that with 50 microM EMD 57033, isometric force increases by more than 50%, whereas Kredev, active stiffness, and isometric fiber ATPase increase by at most 10%. The results show that EMD 57033 causes no changes in cross-bridge turnover kinetics and no changes in active fiber stiffness that would result in a large enough increase in occupancy of the force-generating states to account for the increase in active force. However, plots of force versus length change recorded during stretches and releases (T plots) indicate that in the presence of EMD 57033 the y(o) value (x axis intercept) for the cross-bridges becomes more negative while its absolute value increases. This might suggest a larger cross-bridge strain as the basis for increased active force. Analysis of T plots with and without EMD 57033 shows that the increase in cross-bridge strain is not due to a redistribution of cross-bridges among different force-generating states favoring states of larger strain. Instead, it reflects an increased cross-bridge strain in the main force-generating state. The direct effect of EMD 57033 on the force contribution of cross-bridges in the force-generating states represents an alternative mechanism for a positive inotropic intervention.

Economy of contraction of cardiomyocytes as influenced by different positive inotropic interventions.

In the present study the effects of the novel cardiotonic agent EMD-57033 on contraction and energetic demand of isolated, electrically stimulated cardiomyocytes were investigated and compared with the effects of enhancement of extracellular calcium and of the beta-mimetic isoproterenol. In a specially designed setup [H. Rose, K.H. Strotmann, S. Pöpping, Y. Fischer, D. Kulsch, and H. Kammermeier. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1329-H1334, 1991] parameters of contractile behavior and metabolic demand (O2 consumption) of isolated cardiac myocytes were measured. For a given enhancement of contractile performance (cell shortening) the increase in energetic demand (VO2) after application of EMD-57033 were markedly lower than on treatment with elevated extracellular Ca2+ concentration or with isoproterenol. This economization of positive inotropic effects was proposed to be due to two factors. First, stimulation-related ion cycling was only slightly enhanced with marked increase in contraction amplitude after application of EMD-57033. Second, calcium sensitization reflected in a leftward shift of the calcium concentration needed for half-maximum force development could be interpreted to be mediated by modulation of the cross-bridge dynamics of the myofilaments, where reduction of the switch-off rate of the cross bridges and prolongation of their force-generating states were presumed to be involved. Lowered pH (7.0) decreased economy of contraction. EMD-57033 restored contraction amplitude and economy of contraction at lowered pH.