Diastolic dysfunction and abnormality of the Na+/Ca2+ exchanger in single uremic cardiac myocytes.

Cardiovascular disease is the most common cause of death in patients with end-stage renal disease, possibly due to a specific "uremic cardiomyopathy". This study investigated the function of the Na(+)/Ca(2+) exchanger in single cardiac myocytes from a model of early renal impairment. Mild uremia was induced by partial (5/6) nephrectomy in male Wistar rats. After 4 weeks, ventricular myocytes were isolated, loaded with the fluorescent Ca(2+) indicator indo-1, and contractile function and calcium transients recorded following electrical pacing at 0.2 Hz. Relaxation from rapid cooling contractures (RCCs) was also studied. Cells from uremic animals (U) were hypertrophied compared with controls (C), with a significant increase in width (14%; P<0.02) and cross-sectional area (13%; P<0.03). There was a significant increase in diastolic intracellular Ca(2+) ratio in the uremic cells (C, 0.33+/-0.00 vs U, 0.37+/-0.02; P<0.02), although the amount of calcium released per twitch was similar. Uremic cells were slower to relax following RCCs, however when Na(+)/Ca(2+) exchange was inhibited using a Na(+)-free/Ca(2+)-free solution, this difference was abolished. Under these conditions, there was little difference in the relaxation rate of control cells, indicating that the Na(+)/Ca(2+) exchanger plays only a minor role in relaxation in normal rat myocytes. However in uremia, the data indicate that the Na(+)/Ca(2+) exchanger actively interfered with relaxation, possibly by working in reverse rather than forward mode. These results indicate that myocyte relaxation and Ca(2+) handling are abnormal in early uremia and may provide further evidence for the existence of a specific "uremic cardiomyopathy".

Evidence for the action potential mediating the changes to contraction observed in cardiac hypertrophy in the rabbit.

BACKGROUND: We investigated the effects of cardiac hypertrophy on intracellular calcium (Ca(2+)) homeostasis, the amounts of proteins involved in calcium regulation and the influence of the action potential on such changes. METHODS: Cardiac hypertrophy was induced in rabbits by constriction of the ascending aorta. They were kept for 6 weeks then the heart was removed and left ventricular myocytes isolated. A portion of these myocytes was immediately frozen and stored for subsequent protein analyses using Western blotting. RESULTS: After aortic banding, cardiac myocyte two-dimensional area and membrane capacitance were increased by 53% and 23% respectively. Hypertrophy prolonged cell contraction and relaxation and the corresponding Indo-1 Ca(2+) transients. Hypertrophied cells displayed longer action potentials but Ca(2+) current densities were unchanged compared with myocytes from sham hearts. If Ca(2+) was released from the sarcoplasmic reticulum using rapid cooling, so bypassing the normal mechanisms involved in excitation-contraction coupling, then no functional differences between hypertrophied and control cells could be observed. Western blot analysis showed that the amounts of sarcoplasmic reticulum Ca(2+) ATPase, its regulatory protein phospholamban and the sodium/calcium exchanger were unchanged whereas the amount of calsequestrin was increased by 65% and the alpha(1) subunit of the sodium/potassium ATPase was reduced by 72%. These changes do not appear to evoke functional consequences under these conditions. CONCLUSION: In this model of cardiac hypertrophy, the increase in action potential duration is responsible for changes in contraction and relaxation.

Low-dose ramipril treatment improves relaxation and calcium cycling after established cardiac hypertrophy.

Rapid cooling contractures were used in this study to test whether low-dose ramipril improves sarcoplasmic reticulum (SR) Ca(2+) uptake and Na(+)/Ca(2+) exchanger function in isolated hypertrophied rat myocytes. Compensated cardiac hypertrophy was induced by abdominal aortic constriction for 5 wk followed by administration of ramipril (50 microg x kg(-1) x day(-1)) or vehicle for 4 wk. Myocyte cell length and cell width were significantly (P < 0.05) increased in both hypertrophied groups (+/-ramipril). Myocytes were loaded with indo 1, and relaxation was investigated after rapid cooling. Hypertrophied myocyte relaxation in Na(+)-free/Ca(2+)-free solution was 63% slower (P < 0.01) and the fall in intracellular Ca(2+) was 60% slower (P < 0.05) than the relaxation of control cells. After ramipril treatment both relaxation and the decline in intracellular Ca(2+) returned to control rates through improved SR Ca(2+)-ATPase function. Relaxation in caffeine showed no change after hypertrophy; however, after ramipril treatment the time to 50% relaxation in caffeine decreased by 30% (P < 0.05). The improvement in Ca(2+) extrusion across the sarcolemmal membrane occurred independently of changes in Na(+)/Ca(2+) exchanger mRNA and protein abundance. These data demonstrate that ramipril improves both SR-dependent and non-SR-dependent calcium cycling after established cardiac hypertrophy. However, the improvements in function are independent of transcriptional activation and likely to involve altered intracellular ion concentrations.

Voltage-dependent binding and calcium channel current inhibition by an anti-alpha 1D subunit antibody in rat dorsal root ganglion neurones and guinea-pig myocytes.

1. The presence of calcium channel alpha 1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea-pig cardiac myocytes was demonstrated using the reverse transcriptase-polymerase chain reaction. 2. An antipeptide antibody targeted at a region of the voltage-dependent calcium channel alpha 1D subunit C-terminal to the pore-forming SS1-SS2 loop in domain IV (amino acids 1417-1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+. 3. Incubation of cells under depolarizing conditions for 2-4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls. 4. S-(-)-Bay K 8644 (1 microM) enhanced calcium channel currents in DRGs by 75 +/- 19% (n = 5) in neurones incubated under depolarizing conditions with antibody that had been preabsorbed with its immunizing peptide (100 micrograms ml-1). This was significantly (P < 0.05) larger than the enhancement by S-(-)-Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 +/- 4% (n = 5). 5. These results demonstrate the presence of calcium channel alpha 1D subunits in rat DRG neurones and guinea-pig cardiac myocytes. They also show that amino acids 1417-1434 of the alpha 1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S-(-)-Bay K 8644 to enhance this current, indicating that it is an L-type current that is attenuated.

Effects of rest interval on the release of calcium from the sarcoplasmic reticulum in isolated guinea pig ventricular myocytes.

Guinea pig cardiac myocytes were loaded with the fluorescent dye indo 1, and cell contraction was measured by a video edge-detection system. Ca2+ was released from the sarcoplasmic reticulum (SR) by rapidly cooling the myocytes or by rapid application of 10 mmol/L caffeine. Estimates of the amount of Ca2+ released from the SR after different rest intervals (ie, under different loading conditions) were obtained by measuring the current evoked by rapid application of 10 mmol/L caffeine, which we call Na+/Ca2+ exchange current. This current is completely inhibited by removal of extracellular Na+ and Ca2+ or by application of 5 mmol/L Ni2+. SR Ca2+ release after rest intervals of 5 to 120 seconds (assuming cell volume to be 30 x 10(-12) L) was estimated to be 57.8 +/- 5.7 to 25.7 +/- 4.5 mumol/L accessible cell volume, respectively, equivalent to 23 to 10 mumol/kg wet wt, respectively. There was an exponential decline in Ca2+ release from the SR after rest intervals of 2 to 120 seconds (rate constant, 0.029 s-1; t1/2, 24 seconds); thereafter, there remained a portion (56%) of Ca2+ releasable to caffeine application. We found a similar exponential decay (rate constant, 0.020 s-1; t1/2, 35 seconds) of the size of rapid cooling contractures with increasing rest intervals. The time to peak of the Na+/Ca2+ exchange current in the presence of caffeine slowed at long rest intervals, ie, at smaller SR loads. A decrease in SR load of 50% increased the time to peak of the exchange current by 213 +/- 37% (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypertrophy on mechanisms of relaxation in isolated cardiac myocytes from guinea pig.

Modifications to cell relaxation and handling of intracellular Ca have been demonstrated in animals with cardiac cell hypertrophy leading to decompensated heart failure. A previously described model of renal hypertension leading to cardiac cell hypertrophy in the guinea pig, produced using the Goldblatt 2-kidney, 1-clip technique, was used to investigate which of the main mechanisms causing cell relaxation (the sarcoplasmic reticulum Ca-adenosinetriphosphatase and Na/Ca exchanger) are altered in hypertrophy. Relaxation upon rewarming from a rapid cooling contracture was slowed in hypertrophied (H) compared with control (C) cells. Relaxation was further slowed in H compared with C cells when Na/Ca exchange was inhibited by rewarming in a Na-free, Ca-free solution and slowed most markedly in H cells in the presence of 10 mM caffeine. Hypertrophy led to greater modification of cell length relaxation in comparison with the decline in the indo-1 transient, but the force-pCa relationship in skinned muscles showed that myofilament sensitivity was unchanged. Such results indicate that cell relaxation and Ca handling are affected in hypertrophy, possibly involving modifications of Na/Ca exchange activity.

Characteristics of myocytes isolated from hearts of renovascular hypertensive guinea pigs.

A model of renovascular hypertension has been developed in the guinea pig using the Goldblatt (2-kidney, 1-clip) operation. Systolic and diastolic blood pressures were significantly increased 3 and 7 wk after the operation, but levels fell to control values at 11 wk. The two-dimensional areas of myocytes isolated from the hearts of Goldblatt-operated (GB) animals were larger than those in control cells at 3 wk (cf. 3,397 +/- 87 and 2,208 +/- 125 microns 2, P < 0.01), and the difference was maintained at 7 and 11 wk. No change in cell contraction or relaxation characteristics were seen at either 3 or 7 wk after clipping. Myocytes from the 11-wk GB group showed a significantly reduced contraction amplitude and velocity at 32 degrees C in maximally activating Ca2+ or isoproterenol concentrations (%cell shortening in Ca2+, cf. 6.8 +/- 0.4 and 10.0 +/- 0.9, P < 0.01). Concentrations eliciting 50% of maximal response for Ca2+ or isoproterenol were unchanged, as was the ratio of isoproterenol to Ca2+ effect in the same cell. Increases in time to peak contraction (TTP) and time to 50% relaxation (R50) were observed in 11-wk GB myocytes, but only at room temperature. There was no lengthening of TTP or R50 of the Ca2+ transient, nor was there any change in Ca2+ current density or inactivation kinetics in these myocytes.