The dual effects of ouabain on45Ca2+ transport and contractility in adult rat ventricular myocytes

We used isolated ventricular myocytes to study⁴⁵Ca²⁺ transport in the presence of three concentrations of ouabain (10 nM, 1 M, and 100 M) in Tyrode solution containing 1 mM CaCl₂. The cells were quiescent and during⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux experiments 10 nM ouabain decreased Ca²⁺ content, 1 M, didn't change it appreciably, and 100 M increased it significantly. Qualitatively, the same results were obtained at 22°C and 35°C. Ouabain did not significantly affect the electrical activity of isolated, electrically stimulated myocytes, but it increased the amplitude of shortenings of these myocytes in a dose-dependent manner. Thus, the positive inotropic effect of ouabain at therapeutic doses (10 nM) occurs in spite of decreased Ca²⁺ content, while at high toxic doses the positive inotropic effect is accompanied by an increment in Ca²⁺ content. These data support the hypothesis that the mechanisms of positive inotropy of ouabain are different at therapeutic and toxic concentrations of this drug. Finally, our study demonstrates that the effects of low doses of ouabain are independent of the release of endogenous catecholamines.     

Osmolality- and Na+-dependent effects of hyperosmotic NaCl solution on contractile activity and Ca2+ cycling in rat ventricular myocytes

Hypertonic NaCl solutions have been used for small-volume resuscitation from hypovolemic shock. We sought to identify osmolality- and Na⁺-dependent components of the effects of the hyperosmotic NaCl solution (85 mOsm/kg increment) on contraction and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in isolated rat ventricular myocytes. The biphasic change in contraction and Ca²⁺ transient amplitude (decrease followed by recovery) was accompanied by qualitatively similar changes in sarcoplasmic reticulum (SR) Ca²⁺ content and fractional release and was mimicked by isosmotic, equimolar increase in extracellular [Na⁺] ([Na⁺]_o). Raising osmolality with sucrose, however, augmented systolic [Ca²⁺]_i monotonically without change in SR parameters and markedly decreased contraction amplitude and diastolic cell length. Functional SR inhibition with thapsigargin abolished hyperosmolality effects on [Ca²⁺]_i. After 15-min perfusion, both hyperosmotic solutions slowed mechanical relaxation during twitches and [Ca²⁺]_i decline during caffeine-evoked transients, raised diastolic and systolic [Ca²⁺]_i, and depressed systolic contractile activity. These effects were greater with sucrose solution, and were not observed after isosmotic [Na⁺]_o increase. We conclude that under the present experimental conditions, transmembrane Na⁺ redistribution apparently plays an important role in determining changes in SR Ca²⁺ mobilization, which markedly affect contractile response to hyperosmotic NaCl solutions and attenuate the osmotically induced depression of contractile activity.     

Effects of combined renovascular hypertension and diabetes mellitus on myocardial cells,non-vascular interstitium and capillaries: a stereological study on rat hearts

Summary The effects of combined renovascular hypertension and diabetes mellitus on the rat heart were investigated in order to detect possible synergistic effects of the two conditions. Hypertensive diabetic and hypertensive non-diabetic animals were compared to diabetic and non-diabetic controls. Hypertension was established for 12 weeks by a surgical stenosis of the left renal artery; diabetes mellitus was maintained for 8 weeks by a single intraperitoneal injection of 60 mg/kg streptozotocin. Light microscopic stereology did not reveal significant divergences between diabetic hypertensives and non-diabetic hypertensives. Hypertension induced a focal perivascular and interstitial fibrosis with increased volume densities of non-vascular interstitium and fibrosis (P<0.001). Capillary density (Q_A) was decreased in transverse sections (P<0.01) and increased in longitudinal sections (P<0.01). This indicates a three-dimensional remodelling of the capillary bed with an increased number of obliquely running capillaries. At least the length density (L_V) of capillaries (mm/mm³) tends to be normalized in long-term renovascular hypertension. At the ultrastructural level, a synergism of hypertension and diabetes mellitus was observed: the volume ratio of mitochondria to myofibrils was significantly decreased in hypertensive diabetics, but not in non-diabetic hypertensives or in diabetics. This may enhance the risk of cardiac deterioration. We conclude that the primary target of the synergistic damage in hypertensive diabetic heart muscle disease is the myocardial cell and not the cardiac interstitium.Preliminary results of this study have been published in: Mall G (1991) Morphometric study on the rat heart in combined renovascular hypertension and diabetes mellitus. In: Nagano N, Dhalla NS (eds) The diabetic heart. Raven Press, New York, pp 115–124Dedicated to Prof. Dr. med. G. Seifert on the occasion of his 70th birthday     

Regulation of Ca2+ current in frog ventricular myocytes by the holding potential, c-AMP and frequency

The whole-cell patch-clamp technique was used to study the effects of holding potential and frequency on the Ca²⁺ current in frog ventricular myocytes. I _Na was blocked by TTX, and i _ca was activated with depolarizing clamps from different holding potentials. Variation of the holding potential revealed three new effects on i _Ca: (1) At -40 mV i _Ca declined with a time constant of 15 min, while at-90 mV, this irreversible decline (run down) in i _Ca did not occur. (2) The decline of i _Ca at -40 mV was biphasic: run down was preceeded by a slow inactivation with a time constant of 40 s, which was reversible upon returning the holding potential to -90 mV. (3) Increasing the frequency of the clamp pulses from 0.1 to 1 Hz led to a rapid decline of i _Ca when the holding potential was positive to -60 mV, but at -90 mV had either no effect or increased i _Ca by 35%, if c-AMP was included in the dialyzing solution. On the other hand, c-AMP did not alter the time course of the run down and the slow inactivation. Replacement of extracellular Ca²⁺ by Ba²⁺ markedly slowed i _Ca kinetics, but did not change the very slow inactivation or the frequency-induced enhancement of i _Ca. Injection of c-AMP led to a transient increase of i _Ca. The phosphodiesterase inhibitor theophylline enhanced the amplitude of the transient and slowed its decay. This effect was mimicked by increased frequency. It is concluded that frequency-induced enhancement of i _Ca is highly dependent on the holding potential, independent of Ca²⁺, and may involve elevation of the intracellular level of c-AMP via inhibition of phosphodiesterase activity. The new type of very slow inactivation is probably under direct voltage control and independent of Ca²⁺ and c-AMP.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH,calcium and sodium in isolated rat ventricular myocytes

 The inhibitors of the Na⁺/H⁺-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Na_i) and calcium (Ca_i) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na⁺ and Ca²⁺ (pH_i, Na_i and Ca_i) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pH_i 7.2 to pH_i 6.8) and an increase in Ca_i from about 50 nmol/l to about 1 μmol/l. The decrease in pH_i began before the cells underwent hypoxic (rigor) contracture, whereas Ca_i only began to rise after rigor shortening had taken place. After reoxygenation, pH_i returned to its control value and Ca_i oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 μmol/l Hoe 642 was present from the beginning of the experiment, pH_i and Ca_i were not significantly different from control experiments. At reoxygenation, pH_i did not recover, but Ca_i oscillated and returned to its resting level. To monitor Na_i, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 μmol/l ouabain, Na_i increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 μmol/l) did not prevent the increase in Na_i. In contrast, the blocker of the persistent Na⁺ current R56865 (10 μmol/l) attenuated the CN^–-induced rise in Na_i. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell’s autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Ca_i overload, nor CN^–-induced Na_i and Ca_i overload. Hoe 642 prevented the recovery of pH_i from anoxic acidification. This low pH_i maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca²⁺ overload in mitochondria, cannot be ruled out. The increase in Na_i during anoxia is possibly due to an influx of Na⁺ via persistent Na⁺ channels. Received: 1 March 1996 / Received after revision: 30 April 1996 / Accepted: 12 July 1996