The isolated working mouse heart: methodological considerations

 Our aim was to develop a working isolated murine heart model, as the extensive use of genetically engineered mice in cardiovascular research requires development of new miniaturized technology. Left ventricular (LV) function was assessed in the isolated working mouse heart perfused with recirculated oxygenated Krebs-Henseleit bicarbonate buffer (37 °C pH 7.4) containing 11.1 mM glucose and 0.4 mM palmitate bound to 3% albumin. The hearts worked against an afterload reservoir at a height equivalent to 50 mmHg, and heart rate was controlled by electrical pacing of the right atrium. LV pressure was measured with a micromanometer connected to a small steel cannula inserted through the apex of the heart. The experimental protocol consisted of two interventions. First, following instrumentation and stabilization, the preload reservoir was raised from a pressure equivalent of 7 to 22.5 mmHg, while pacing at 390 beats·min^–1. Thereafter the height of the preload reservoir was set to 10 mmHg, and the pacing rate was varied from 260 to 600 beats·min^–1. Aortic and coronary flows were measured by timed collections of effluent from the afterload line and that dripping from the heart, respectively [aortic+coronary flow=cardiac output (CO)]. Elevation of LV end-diastolic pressure (LVEDP) from approximately 5 to 10 mmHg resulted in a twofold increase in average cardiac power [product of LV developed pressure (LVDevP) and CO], whereas myocardial contractility (first derivative of LV pressure, dP/dt) and LVDevP (LV systolic pressure–LVEDP) increased only minimally (5–10%). Measured LVEDP was lower than the equivalent height of the preload reservoir by an amount that was related to the heart rate. Cardiac power, LVDevP and dP/dt were stable at heart rates up to 400 beats·min^–1, but declined markedly with higher rates, consistent with the decrease in LVEDP. Thus, cardiac power was reduced to 50% of its maximum value when stimulated at approximately 500 beats·min^–1, and at even higher rates there was little ejection. By systematic manipulation of the height of the preload reservoir and heart rate, we conclude that LV afterload and preload can be assessed only by high-fidelity measurement of intraventricular pressures. The heights of the afterload column and the preload reservoir are unreliable and potentially misleading indicators of LV afterload and preload. Received: 28 September 1998 / Accepted: 25 January 1999     

In vivo heat shock preconditioning mitigates calcium overload during ischaemia/reperfusion in the isolated, perfused rat heart

Heat shock (HS) pretreatment of the heart is effective in mitigating the deleterious effects of ischaemia/reperfusion. The main objective of this study was to determine whether the beneficial effect of HS is associated with the preservation of intracellular Ca²⁺ handling in the ischaemic/reperfused, isolated rat heart. Twenty-four hours after raising body core temperature to 42 °C for 15 min, rat hearts were perfused according to Langendorff and subjected to 30 min ischaemia followed by 20 min reperfusion. Cyclic changes of cytoplasmic calcium ion [Ca²⁺_i] levels were measured by surface fluorometry using Indo-1 AM. Reperfused HS hearts showed improved recovery of contractile function compared with control hearts: end-diastolic pressure: 45±11 vs. 64±22 mm Hg; developed pressure: 72±12 vs. 41±20 mm Hg; maximum rate of pressure increase (+dP/dt_max): 1,513±305 vs. 938±500 mm Hg/s; maximum rate of pressure decrease (–dP/dt_max): –1,354±304 vs. –806±403 mm Hg/s. HS hearts displayed a significantly lower end-diastolic cytosolic [Ca²⁺] ([Ca²⁺]_i) after reinstallation of flow. The dynamic parameters of the Ca²⁺_i transients, i.e. the maximum rate of increase/decrease (±dCa²⁺_i/dt_max) and amplitude, did not differ between reperfused control and HS hearts. The novel finding of this study is that improved performance of the HS-preconditioned heart after an ischaemic insult is associated with a reduced end-diastolic Ca²⁺_i load, and most likely, preserved Ca²⁺ sensitivity of the myocardial contractile machinery.     

Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells

Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (K _d = 15.4 ± 4.6 μM; B _max = 1.7 ± 0.5 nmol mg⁻¹ protein); and a low affinity site (K _d = 170 ± 94.5 μM; B _max = 14.2 ± 3.9 nmol mg⁻¹ protein). In myotube membranes only a low affinity binding site for GTP (K _d = 169 ± 39 μM; B _max = 12.3 ± 1.4 nmol mg⁻¹ protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca²⁺]_i. The patterns of Ca²⁺ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca²⁺ responses: (1) a fast increase in [Ca²⁺]_i, followed by a sustained [Ca²⁺]_i elevation, and (2) a slow raising and steady prolonged increase in [Ca²⁺]_i. In myotubes, however only fast Ca²⁺ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca²⁺]_i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca²⁺ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Block of potassium outward currents in the crayfish stretch receptor neurons by 4-aminopyridine,tetraethylammonium chloride and some other chemical substances

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (K_d) of 2.3 ± 0.2 mm (mean ± SEM) in the slowly and 1.4 ± 0.2 mm in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1: 1 stoichiometry and the apparent K_d for TEA was 9.6 ± 3.4 mm with a cooperativity factor n= 0.43 ± 0.03 in the slowly adapting SRN and 34.5 ± 9.2 mm and 0.37 ± 0.03 respectively in the rapidly adapting SRN. Low Ca²⁺, apamin and charybdotoxin, which are known to block Ca²⁺-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (K_d= 0.8–1.6 mm) for TEA and the other a low affinity (K_d= 173–213 mm) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca²⁺-dependent K⁺ current in the SRNs.     

Minimal effects of nitric oxide on spatial blood flow heterogeneity of the dog heart

 Eleven Beagle dogs were studied to elucidate the possible role of L-arginine-derived nitric oxide on local blood flow distribution in left and right ventricular myocardium. Local blood flow was determined in 256 samples from the left and 64 samples from the right ventricle per heart using the tracer microsphere technique (mean sample mass 319 ± 131 mg). Nitric oxide production was effectively inhibited by intravenous infusion of 20 mg/kg nitro-L-arginine methylester (L-NAME) as evidenced by a shift of the dose/response curve for the effect of intracoronary administration of bradykinin (0.004–4.0 nmol/min) on coronary blood flow. L-NAME enhanced left and right ventricular systolic pressures from 132 ± 18 to 155 ± 15 mm Hg and from 26 ± 3 to 29 ± 3 mm Hg respectively (both P = 0.043). Mean left ventricular blood flow was 1.14 ± 0.38 before and 0.99 ± 0.28 ml min^–1 g^–1 after L-NAME (P = 0.068), while right ventricular blood flow fell from 0.72 ± 0.28 to 0.53 ± 0.20 ml min^–1 g^–1 (P = 0.043). Coronary conductance of left and right ventricular myocardium fell by 31 and 43% respectively (both P = 0.043). The coefficient of variation of left ventricular blood flow was 0.26 ± 0.07 before and 0.29 ± 0.07 after L-NAME (P = 0.068), that of right ventricular blood flow was 0.27 before and after L-NAME. Skewness (0.51) and kurtosis (4.23) of left ventricular blood flow distribution were unchanged after L-NAME, while in the right ventricle skewness decreased from 0.54 to 0.09 (P = 0.043) and kurtosis (3.68) tended to decrease after L-NAME (P = 0.080). The fractal dimension (D = 1.20–1.27) and the corresponding nearest-neighbor correlation coefficient (r _n = 0.37–0.53) of left and right ventricular myocardium remained unchanged after infusion of L-NAME. From these results it is concluded that firstly, local nitric oxide release does not explain the higher perfusion of physiological high flow samples and secondly, that spatial myocardial blood flow coordination is not dependent on nitric oxide. Received: 11 July 1996 / Received after revision: 29 October 1996 / Accepted: 17 December 1996     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

Na+/Ca2+ exchange during Ca2+ repletion is not a prerequisite for the Ca2+ paradox in isolated rat hearts

 Ca²⁺ paradox damage has been suggested to be determined by Na⁺ entry during Ca²⁺ depletion and exchange of Na⁺ for Ca²⁺ during Ca²⁺ repletion. Since previously a Ca²⁺ paradox without prior increase of total intracellular [Na⁺] ([Na⁺]_i) has been observed, we investigated whether local accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion plays a role in the Ca²⁺ paradox by replacing all extracellular Na⁺ by Li⁺ 5 min before and during 10 min Ca²⁺-free perfusion (37°C) in isolated rat hearts (group I). Subsequently, hearts were perfused with a standard, Na⁺- and Ca²⁺-containing solution. Verapamil was used to prevent contracture due to the absence of Na⁺/Ca²⁺ exchange during Na⁺-free perfusion in the presence of Ca²⁺. In group II, the Ca²⁺-free period was omitted, and in group III normal extracellular [Na⁺] was used throughout. ²³Na-NMR was used to monitor intra- and extracellular Na⁺ signals. Total creatine kinase release was 2,977±413, 36±24 and 3170±297 IU/g dry weight in groups I, II and III respectively, indicating a full Ca²⁺ paradox in groups I and III. [Na⁺]_i decreased from 11.3±0.6 mM during control perfusion to 1.2±0.4 mM after 10 min Ca²⁺ depletion in group I, whereas in group III [Na⁺]_i was 10.9±2.2 mM during control perfusion and did not change significantly after 10 min Ca²⁺-free perfusion. It is concluded that accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion is not a prerequisite for the Ca²⁺ paradox. Received: 2 February 1998 / Received after revision: 31 March 1998 / Accepted: 9 April 1998     

The inotropic effect of atrial natriuretic factor in the anesthetized rabbit

This study was designed to investigate whether atrial natriuretic factor (ANF) administered over the physiological, pathological and pharmacological range has a negative inotropic action on the heart. Anesthetized rabbits were infused with increasing doses of ANF (0.05, 0.25 and 0.5g kg^–1min^–1), while measuring hemodynamic variables including the maximum rate of change of left ventricular pressure (dP/dt _max) as an index of inotropic state. Plasma levels of immunoreactive ANF (iANF) were measured to relate the hemodynamic changes to actual plasma levels of the peptide. Administration of ANF was associated with decreases in blood pressure, left ventricular pressure and dP/dt _max so that after 0.5 g kg^–1 min^–1 infusion, these variables had decreased by 21±2 mmHg, 21±5.3 mmHg and 925±175 mmHg/s, respectively (P<0.01). There were no significant changes in right atrial pressure, left ventricular end-diastolic pressure or heart rate. Since dP/dt _max can be influenced by changing hemodynamic variables and baroreflex changes, a second group of rabbits was studied in which afterload and heart rate were held artificially constant. Again, in this group of rabbits, infusions of ANF led to decreasing inotropic state, so that at the highest infusion rate, a 14% decrease in dP/dt _max was observed (P<0.05). By comparison, hydralazine, a drug which causes active vasodilatation but no direct inotropic action, significantly (P<0.01) decreased blood pressure, left ventricular pressure and dP/dt _max when infused at a rate of 10 g kg^–1 min^–1. However, in animals in which afterload was controlled, hydralazine did not affect any of the variables measured. The results indicate that ANF does have a negative inotropic action in the anesthetized rabbit.     

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

 This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca²⁺]_i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca²⁺]_i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114±18 μmol l^–1 and K _d of 0.59±0.17 μM (n=8). During the systolic rise of [Ca²⁺]_i, the measured changes of [Ca²⁺]_i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca. Received: 14 October 1998 / Accepted: 6 November 1998     

A Na+-activated K+ current (I K,Na) is present in guinea-pig but not rat ventricular myocytes

 The effects of removing extracellular Ca²⁺ and Mg²⁺ on the membrane potential, membrane current and intracellular Na⁺ activity (a ⁱ _Na) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca²⁺ and Mg²⁺ are removed and the steady-state a ⁱ _Na increases rapidly from a resting value of 6.4± 0.6 mM to 33±3.8 mM in guinea-pig (n=9) and from 8.9±0.8 mM to 29.3±3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca²⁺ and Mg²⁺, K⁺ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at –20 mV, when the bathing divalent cations were removed. When the bathing K⁺ was removed after Ca²⁺ and Mg²⁺ depletion, a large outward K⁺ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of –80±0.7 mV and was not inhibited by high Mg²⁺ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li⁺ replaced the bathing Na⁺ and was blocked by R-56865, suggesting that it was due to the activation of K_Na channels. Received: 15 October 1998 / Received after revision: 22 January 1999 / Accepted: 5 February 1999     

Role of a Ca2+-activated K+ current in the maintenance of resting membrane potential of isolated, human, saphenous vein smooth muscle cells

 Calcium-activated potassium currents were studied in dissociated smooth muscle cells from human saphenous vein (HSV) using the patch-clamp technique in the whole-cell configuration. The average measured resting membrane potential (V _m) was –41±2 mV (n=39), when the cells were dialysed with an intracellular pipette solution (IPS) containing 0.1 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (IPS-0.1 mM EGTA). When the EGTA concentration was increased to 10 mM (IPS-10 mM EGTA) V _m became significantly less negative: –13±2 mV (n=23, P<0.05). These results suggest that 10 mM EGTA reduces a calcium-dependent current involved in the maintenance of V _m. Depolarizing voltage steps up to +60 mV from holding potentials of –60 mV resulted in large (1–10 nA) time- and voltage-dependent outward currents. The amplitudes of total whole-cell current densities measured at voltages above –20 mV were significantly greater in the cells dialysed with IPS-0.1 mM EGTA than in those dialysed with IPS-10 mM EGTA. In the cells dialysed with IPS-0.1 mM EGTA, 0.1 mM tetraethylammonium chloride (TEA) and 50 nM iberiotoxin (IBTX), which selectively block large conductance Ca²⁺-activated potassium channels (BK_Ca), diminished the total current recorded at +60 mV by 45±14% (P<0.05, n=5) and 50±6% (n=8, P<0.05), respectively. These blockers at the same concentrations did not affect the total current in cells dialysed with IPS-10 mM EGTA. When tested on intact HSV rings, both 0.1 mM TEA and 50 nM IBTX elicited vessel contraction. We conclude that BK_Ca channels present in HSV smooth muscle cells contribute to the maintenance of the V _m and sustain a significant portion of the total voltage-activated, outward current. Finally, BK_Ca channels appear to play a significant role in the regulation of HSV smooth muscle contractile activity. Received: 3 April 1998 / Received after revision: 23 September 1998 / Accepted: 13 October 1998     

Maximal lactate steady state concentration independent of pedal cadence in active individuals

The maximal lactate steady state (MLSS) is defined as the highest blood lactate concentration that can be maintained over time without a continual blood lactate accumulation. The objective of the present study was to analyze the effects of pedal cadence (50 vs. 100 rev min⁻¹) on MLSS and the exercise workload at MLSS (MLSS_workload) during cycling. Nine recreationally active males (20.9±2.9 years, 73.9±6.5 kg, 1.79±0.09 m) performed an incremental maximal load test (50 and 100 rev min⁻¹) to determine anaerobic threshold (AT) and peak workload (PW), and between two and four constant submaximal load tests (50 and 100 rev min⁻¹) on a mechanically braked cycle ergometer to determine MLSS_workload and MLSS. MLSS_workload was defined as the highest workload at which blood lactate concentration did not increase by more than 1 mM between minutes 10 and 30 of the constant workload. The maximal lactate steady state intensity (MLSS_intensity) was defined as the ratio between MLSS_workload and PW. MLSS_workload (186.1±21.2 W vs. 148.2±15.5 W) and MLSS_intensity (70.5±5.7% vs. 61.4±5.1%) were significantly higher during cycling at 50 rev min⁻¹ than at 100 rev min⁻¹, respectively. However, there was no significant difference in MLSS between 50 rev min⁻¹ (4.8±1.6 mM) and 100 rev min⁻¹ (4.7±0.8 mM). We conclude that MLSS_workload and MLSS_intensity are dependent on pedal cadence (50 vs. 100 rev min⁻¹) in recreationally active individuals. However, this study showed that MLSS is not influenced by the different pedal cadences analyzed.     

Stroke volume equation for impedance cardiography

The study's goal was to determine if cardiac output (CO), obtained by impedance cardiography (ICG), would be improved by a new equation N, implementing a square root transformation for dZ/dt_max/Z₀, and a variable magnitude, mass-based volume conductor V_c. Pulmonary artery catheterisation was performed on 106 cardiac surgery patients pre-operatively. Post-operatively, thermodilution cardiac output (TDCO) was simultaneously compared with ICG CO. dZ/dt_max/Z₀ and Z₀ were obtained from a proprietary bioimpedance device. The impedance variables, in addition to left ventricular ejection time T_LVE and patient height and weight, were input using four stroke volume (SV) equations: Kubicek (K), Sramek (S), Sramek-Bernstein (SB), and a new equation N. CO was calculated as SV × heart rate. Data are presented as mean ± SD. One way repeated measures of ANOVA followed by the Tukey test were used for inter-group comparisons. Bland-Altman methods were used to assess bias, precision and limits of agreement. P<0.05 was considered statistically significant. CO implementing N (6.06±1.48 l min⁻¹) was not different from TDCO (5.97±1.41 l min⁻¹). By contrast, CO calculated using K (3.70±1.53 l min⁻¹), S (4.16±1.83 l min⁻¹) and SB (4.37±1.82 l min⁻¹) was significantly less than TDCO. Bland-Altman analysis showed poor agreement between TDCO and K, S and SB, but not between TDCO and N. Compared with TDCO, equation N, using a square-root transformation for dZ/dt_max/Z₀, and a mass-based V_C was superior to existing transthoracic impedance techniques for SV and CO determination.     

Dissociation of force decline from calcium decline by preload in isolated rabbit myocardium

It is well known that the rate of intracellular calcium ([Ca²⁺]_i) decline is an important factor governing relaxation in unloaded myocardium. However, it remains unclear to what extent, under near physiological conditions, the intracellular calcium transient amplitude and kinetics contribute to the length-dependent increase in force and increase in duration of relaxation. We hypothesize that myofilament properties rather than calcium transient decline primarily determines the duration of relaxation in adult mammalian myocardium. To test this hypothesis, we simultaneously measured force of contraction and calibrated [Ca²⁺]_i transients in isolated, thin rabbit trabeculae at various lengths at 37°C. Time from peak tension to 50% relaxation (RT_50(tension)) increases significantly with length (from 49.8 ± 3.4 to 83.8 ± 7.4 ms at an [Ca²⁺]_o of 2.5 mM), whereas time from peak calcium to 50% decline (RT_50(calcium)) was not prolonged (from 124.8 ± 5.3 to 107.7 ± 11.4 ms at an [Ca²⁺]_o of 2.5 mM). Analysis of variance revealed that RT_50(tension) is significantly correlated with length (P < 0.0001). At optimal length, varying the extracellular calcium concentration increased both developed force and calcium transient amplitude, but RT_50(tension) remained unchanged (P = 0.90), whereas intracellular calcium decline actually accelerated (P < 0.05). Thus, an increase in muscle length will result in an increase in both force and duration of relaxation, whereas the latter is not primarily governed by the rate of [Ca²⁺]_i decline.     

Acute and adaptive responses in humans to exercise in a warm, humid environment

 Acute and repeated exposure for 8–13 consecutive days to exercise in humid heat was studied. Twelve fit subjects exercised at 150 W [45% of maximum O₂ uptake (V.O₂,_max)] in ambient conditions of 35°C and 87% relative humidity which resulted in exhaustion after 45 min. Average core temperature reached 39.9 ± 0.1°C, mean skin temperature (T– _sk) was 37.9 ± 0.1°C and heart rate (HR) 152 ± 6 beats min^–1 at this stage. No effect of the increasing core temperature was seen on cardiac output and leg blood flow (LBF) during acute heat stress. LBF was 5.2 ± 0.3 l min^–1 at 10 min and 5.3 ± 0.4 l min^–1 at exhaustion (n = 6). After acclimation the subjects reached exhaustion after 52 min with a core temperature of 39.9 ± 0.1°C, T– _sk 37.7 ± 0.2°C, HR 146 ± 4 beats min^–1. Acclimation induced physiological adaptations, as shown by an increased resting plasma volume (3918 ± 168 to 4256 ± 270 ml), the lower exercise heart rate at exhaustion, a 26% increase in sweating rate, lower sweat sodium concentration and a 6% reduction in exercise V.O₂. Neither in acute exposure nor after acclimation did the rise of core temperature to near 40°C affect metabolism and substrate utilization. The physiological adaptations were similar to those induced by dry heat acclimation. However, in humid heat the effect of acclimation on performance was small due to physical limitations for evaporative heat loss. Received: 3 July 1996 / Received after revision: 26 September 1996 / Accepted: 7 January 1997     

Cardiodepressant mediators are released after myocardial ischaemia: modulation by catecholamines and adenosine

The interaction of recently characterized cardiodepressant mediators with catecholamines and adenosine after myocardial ischaemia was investigated using a model of sequential perfusion of two isolated guinea-pig hearts. Sequential perfusion was initiated after 10, 20, and 30 min (group I, II, and III) of global ischaemia in the first heart. At the onset of sequential perfusion LVdP/dt_max and _min of Heart II decreased by 46 and 44% in group I, by 28 and 34% in group II, and increased by 60 and 24% in group III. Infusion of the β₁-receptor antagonist metoprolol (2.8 μmol L^–1) into Heart II did not modulate contractile changes after 10 min of ischaemia in Heart I, prevented the attenuation of the cardiodepressant effect after 20 min of ischaemia, and completely reversed the positive inotropic effect after 30 min of ischaemia. The A1- and A2-receptor antagonists DPCPX (2 μmol L^–1) and DMPX (20 μmol L^–1) enhanced the positive inotropic and lusitropic effects in Heart II (LVdP/dt_max +154%, LVdP/dt_min +71%) during sequential perfusion after 30 min of ischaemia in Heart I. It is concluded that the effects of cardiodepressant mediators released after myocardial ischaemia are counteracted by a time-dependent release of catecholamines. Endogenous cardiac adenosine, in turn, attenuates the modulatory effects of catecholamines.     

Noninvasive assessment of cardiac contractility by using (dP/dt)/P of carotid artery pulses during exercise

In earlier studies we have shown that both the pressure (P) of the carotid artery pulse (CAP) and its first derivative (CAP dP/dt) could be recorded during moderate exercise. To establish that the CAP (dP/dt)/P is a noninvasive substitute for the left ventricular (LV) value, LV (dP/dt)/P, an index of cardiac contractility, we studied CAP (dP/dt)/P under various states of activity in the autonomic nervous system in 12 healthy male subjects. Increased sympathetic nerve activities yielded by passive tilting, emotional load, or cold stress increased CAP (dP/dt)/P significantly (P< 0.05). Increased parasympathetic nerve activity by ocular compression, however, did not significantly affect the value. Moderate exercise at a heart rate of approximately 150 beats·min^–1 increased it significantly from 16.7 to 25.2·s^–1 in a supine position (P<0.001) and from 16.6 to 24.8·s^–1 in an upright position (P<0.001). It increased monotonically as heart rate increased, but the slope was steeper when the heart rate was greater than approximately 100 beats·min^–1 than it was when the rate was less than 100 beats·min^–1. In conclusion, the present study indicated that CAP (dP/dt)/P can be used as a noninvasive index of cardiac contractility even in moderate exercise.     

Differential actions of exogenous and intracellular spermine on contractile activity in smooth muscle of rat portal vein

Effects of the naturally occurring polyamine spermine on electrical and contractile properties of the rat portal vein were studied. 1 mM spermine nearly abolished spike activity and spontaneous contractions and decreased the intracellular Ca²⁺ concentration ([Ca²⁺],). The phasic force responses to 0.1 and 1 μM phenylephrine were partially inhibited, but not the sustain plateau contraction caused by 5 /IM phenylephrine. The Ca²⁺-force relation in high-K⁺ (128 mM)-depolarized veins was shifted to the right, EC₅₀ for Ca²⁺ increasing from 0.50 ± 0.03 mM (control, n= 8) to 0.65 ± 0.06 and to 0.94 ± 0.03 at 1 (n – 4) and 10 (n = 3) mM spermine, respectively. However, at a Ca²⁺ concentration of 2.5 mM, giving maximal force, there was no effect of spermine (1 mM) on either force or [Ca²⁺],. Whereas extracellular spermine thus reduced contractile activity at moderate levels of stimulation, increased intracellular concentration of spermine potentiated the force response to Ca²⁺. Intracellular loading of spermine by reversible permeabilization increased its concentration by 2–3 times. The spontaneous activity and response to phenylephrine were unchanged. However, the Ca²⁺-force relation of depolarized veins was shifted to the left, EC₅₀ decreasing from 0.51 ± 0.04 mM in controls (n= 7) to 0.36 ± 0.02 mM in the loaded veins (n= 9). Spermine increased Ca²⁺-activated force in portal veins permeabilized with β-escin. The degree of potentiation was consistent with observed effects in spermine-loaded intact veins. The results suggest that spermine at physiological intracellular concentration may contribute to the determination of Ca²⁺ sensitivity in vascular smooth muscle cells.