Expression of TASK-1, a pH-sensitive twin-pore domain K(+) channel,in rat myocytes

We have investigated the expression of TASK-1, a pH-sensitive, twin-pore domain K(+) channel in the rat heart. A mammalian cell line of Chinese hamster ovary cells (CHO), transfected with a plasmid containing mouse TASK-1, demonstrated the specificity of the anti-TASK-1 antibody. TASK-1 expression in cardiac tissue was initially demonstrated by Western blot and then localized by immunofluorescence. In single rat ventricular myocytes, strong staining of the TASK-1 protein was located at the intercalated disks and across the cell in a striated pattern, corresponding to the transverse axial tubular network (T tubules). In contrast, single rat atrial myocytes were stained at the intercalated disks with a weak punctate, striated pattern corresponding to underdeveloped T tubules. Also, formamide was used to induce the detubulation of ventricular myocytes, which enabled confirmation that TASK-1 protein expression occurs in T tubules. Consistent with this, RT-PCR revealed the expression of TASK-1 mRNA in total RNA from both the ventricles and atria. In this study, we conclusively demonstrated that TASK-1 protein and mRNA were expressed in rat atrial and ventricular tissue. The extensive distribution of TASK-1 shown to exist within myocyte membranes may provide a potential future target for antiarrhythmic drugs.     

Quantification of t-tubule area and protein distribution in rat cardiac ventricular myocytes

The transverse (t-) tubules of cardiac ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell. Many of the key proteins involved in excitation–contraction coupling appear to be located predominantly at the t-tubule membrane. Despite their importance, the fraction of cell membrane within the t-tubules remains unclear: measurement of cell capacitance following detubulation suggests 32%, whereas optical measurements suggest up to 65%. We have, therefore, investigated the factors that may account for this discrepancy. Calculation of the combinations of t-tubule radius, length and density that produce t-tubular membrane fractions of 32% or 56% suggest that the true fraction is at the upper end of this range. Assessment of detubulation using confocal and electron microscopy suggests that incomplete detubulation can account for some, but not all of the difference. High cholesterol, and a consequent decrease in specific capacitance, in the t-tubule membrane, may also cause the t-tubule fraction calculated from the loss of capacitance following detubulation to be underestimated. Correcting for both of these factors results in an estimate that is still lower than that obtained from optical measurements suggesting either that optical methods overestimate the fraction of membrane in the t-tubules, or that other, unknown, factors, reduce the apparent fraction obtained by detubulation. A biophysically realistic computer model of a rat ventricular myocyte, incorporating a t-tubule network, is used to assess the effect of the altered estimates of t-tubular membrane fraction on the calculated distribution of ion flux pathways.     

Transverse tubules are a common feature in large mammalian atrial myocytes including human

Transverse (t) tubules are surface membrane invaginations that are present in all mammalian cardiac ventricular cells. The apposition of L-type Ca(2+) channels on t tubules with the sarcoplasmic reticulum (SR) constitutes a "calcium release unit" and allows close coupling of excitation to the rise in systolic Ca(2+). T tubules are virtually absent in the atria of small mammals, and therefore Ca(2+) release from the SR occurs initially at the periphery of the cell and then propagates into the interior. Recent work has, however, shown the occurrence of t tubules in atrial myocytes from sheep. As in the ventricle, Ca(2+) release in these cells occurs simultaneously in central and peripheral regions. T tubules in both the atria and the ventricle are lost in disease, contributing to cellular dysfunction. The aim of this study was to determine if the occurrence of t tubules in the atrium is restricted to sheep or is a more general property of larger mammals including humans. In atrial tissue sections from human, horse, cow, and sheep, membranes were labeled using wheat germ agglutinin. As previously shown in sheep, extensive t-tubule networks were present in horse, cow, and human atrial myocytes. Analysis shows half the volume of the cell lies within 0.64 ± 0.03, 0.77 ± 0.03, 0.84 ± 0.03, and 1.56 ± 0.19 μm of t-tubule membrane in horse, cow, sheep, and human atrial myocytes, respectively. The presence of t tubules in the human atria may play an important role in determining the spatio-temporal properties of the systolic Ca(2+) transient and how this is perturbed in disease.     

MCI-154对大鼠心肌细胞的变力作用

钙增敏剂具有正性肌力作用,同时不增加细胞内钙浓度,因此可避免导致心律失常和最终心肌细胞死亡的钙超载。然而大部分钙增敏剂对心肌舒张功能有损害作用。MCI-154是一种钙增敏剂,但不损害舒张功能。为阐明其变力作用机制,我们应用离子成像技术研究了MCI-154对分离的单个大鼠心室肌细胞钙瞬变和收缩的影响,利用膜片钳技术观察了MCI-154对大鼠心室肌细胞L-型钙电流和Na^ ／Ca^2 交换电流的影响。结果表明：(1)MCI-154在1μmol／L至100μmol／L的浓度范围内对L-型钙电流（ICa-L)无直接影响：(2)MCI-154在轻微增加钙瞬变幅度和缩短心肌钙瞬变TR50和TR90的情况下,呈剂量依赖性地增加大鼠心室肌细胞的缩短;(3)MCI-154剂量依赖性地增加正常大鼠心室肌细胞的Na^ ／Ca^2 交换电流。这些结果提示：MCI-154不仅剂量依赖性地发挥了正性变力作用,对舒张功能也没有损害作用,明显不同于其它钙增敏剂,而且还轻微改善了大鼠心室肌细胞的舒张。其对内向Na^ ／Ca^2 交换电流的激动作用会加快钙内流,导致TR50和TR90的缩短,提示MCI-154是通过正向Na^ ／Ca^2 交换改善舒张功能的。     

Role of cyclooxygenase in ventricular effects of adrenomedullin: is adrenomedullin a double-edged sword in sepsis?

Adrenomedullin (ADM) is upregulated in cardiac tissue under various pathophysiological conditions. However, the direct inotropic effect of ADM on normal and compromised cardiomyocytes is not clear. In rat ventricular myocytes, ADM produced an initial (<30 min) increase in cell shortening and Ca(2+) transient and, on prolonged incubation (>1 h), a marked decrease in cell shortening and Ca(2+) transient. Both effects were sensitive to inhibition by the ADM antagonist ADM-(22-52). The increase and decrease in cell shortening and Ca(2+) transient were attenuated by pretreatment with indomethacin [a nonspecific cyclooxygenase (COX) inhibitor], nimesulide and SC-236 (specific COX-2 inhibitors), and tranylcypromine (a prostacyclin synthase inhibitor); SQ-29548 (a thromboxane receptor antagonist) was without effect. Cells isolated from LPS-treated rats that were in the late, hypodynamic phase of septic shock also showed a marked decrease in cell shortening and Ca(2+) transient. Because ADM is overexpressed in sepsis, we repeated the above protocol in cells isolated from LPS-treated rats. At 4 h after LPS injection, ADM levels markedly increased in plasma, ventricles, and freshly isolated ventricular myocytes. Decreases in cell shortening and Ca(2+) transient in LPS-treated cells were reversed by pretreatment with ADM-(22-52). Anti-ADM (rat) IgG also reversed the decrease in cell shortening and other parameters of cell kinetics. Indomethacin, SC-236, and tranylcypromine restored cell contractility and the decrease in Ca(2+) transient, whereas SQ-29548 had no effect, implying that prostacyclin played a role in both effects. However, with regard to cell-shortening kinetics, indomethacin and SQ-29548 decreased the amount of time taken by the cells to return to baseline, whereas SC-236 and tranylcypromine did not, implying that not only prostacyclin, but also thromboxane, is involved. The results indicate that ADM interacts with COX to yield prostanoids, which mediate its negative inotropic effect in LPS-treated rat ventricular myocytes.     

Effects of Na(+)/Ca(2+) exchanger downregulation on contractility and [Ca(2+)](i) transients in adult rat myocytes

Postmyocardial infarction (MI) rat myocytes demonstrated depressed Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. We investigated whether NCX1 downregulation in normal myocytes resulted in contractility changes observed in MI myocytes. Myocytes infected with adenovirus expressing antisense (AS) oligonucleotides to NCX1 had 30% less NCX1 at 3 days and 66% less NCX1 at 6 days. The half-time of relaxation from caffeine-induced contracture was twice as long in ASNCX1 myocytes. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase abundance, SR Ca(2+) uptake, resting membrane potential, action potential amplitude and duration, L-type Ca(2+) current density and cell size were not affected by ASNCX1 treatment. At extracellular Ca(2+) concentration ([Ca(2+)](o)) of 5 mM, ASNCX1 myocytes had significantly lower contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents than control myocytes. At 0.6 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents were significantly higher in ASNCX1 myocytes. At 1.8 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes were not different between control and ASNCX1 myocytes. This pattern of contractile and [Ca(2+)](i) transient abnormalities in ASNCX1 myocytes mimics that observed in rat MI myocytes. We conclude that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca(2+) influx and efflux during a twitch. We suggest that depressed NCX1 activity may partly account for the contractile abnormalities after MI.     

IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.     

Effects of phospholemman downregulation on contractility and [Ca(2+)]i transients in adult rat cardiac myocytes

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca(2+)](i) transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein (P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger (NCX1), Na(+)-K(+)-ATPase, and calsequestrin levels. SR Ca(2+) uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca(2+)](o), rASPLM myocytes had significantly (P < 0.003) lower contraction and [Ca(2+)](i) transient amplitudes than control GFP myocytes. At 5 mM [Ca(2+)](o), both contraction and [Ca(2+)](i) transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca(2+)](i) transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca(2+)](i) transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.     

Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes

In some forms of cardiac hypertrophy and failure, the gain of Ca(2+)-induced Ca(2+) release [CICR; i.e., the amount of Ca(2+) released from the sarcoplasmic reticulum normalized to Ca(2+) influx through L-type Ca(2+) channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca(2+) content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the "decreased CICR gain" may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca(2+)/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive β-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.     

Cellular redox state protects acetaldehyde-induced alteration in cardiomyocyte function by modifying Ca2+ release from sarcoplasmic reticulum

Recent studies indicate that low concentrations of acetaldehyde may function as the primary factor in alcoholic cardiomyopathy by disrupting Ca(2+) handling or disturbing cardiac excitation-contraction coupling. By producing reactive oxygen species, acetaldehyde shifts the intracellular redox potential from a reduced state to an oxidized state. We examined whether the redox state modulates acetaldehyde-induced Ca(2+) handling by measuring Ca(2+) transient using a confocal imaging system and single ryanodine receptor type 2 (RyR2) channel activity using the planar lipid bilayer method. Ca(2+) transient was recorded in isolated rat ventricular myocytes with incorporated fluo 3. Intracellular reduced glutathione level was estimated using the monochlorobimane fluorometric method. Acetaldehyde at 1 and 10 microM increased Ca(2+) transient amplitude and its relative area in intact myocytes, but acetaldehyde at 100 microM decreased Ca(2+) transient area significantly. Acetaldehyde showed a minor effect on Ca(2+) transient in myocytes in which intracellular reduced glutathione content had been decreased against challenge of diethylmaleate to a level comparable to that induced by exposure to approximately 50 microM acetaldehyde. Channel activity of the RyR2 with slightly reduced cytoplasmic redox potential from near resting state (-213 mV) or without redox fixation was augmented by all concentrations of acetaldehyde (1-100 microM) used here. However, acetaldehyde failed to activate the RyR2 channel, when the cytoplasmic redox potential was kept with a reduced (-230 mV) or markedly oxidized (-180 mV) state. This result was similar to effects of acetaldehyde on Ca(2+) transient in diethylmaleate-treated myocytes, probably being in oxidized redox potential. The present results suggest that acetaldehyde acts as an RyR2 activator to disturb cardiac muscle function, and redox potential protects the heart from acetaldehyde-induced alterations in myocytes.     

Calcium spark properties in ventricular myocytes are altered in aged mice

This study determined whether whole cell Ca(2+) transients and unitary sarcoplasmic reticulum (SR) Ca(2+) release events are constant throughout adult life or whether Ca(2+) release is altered in aging ventricular myocytes. Myocytes were isolated from young adult (approximately 5 mo old) and aged (approximately 24 mo old) mice. Spontaneous Ca(2+) sparks and Ca(2+) transients initiated by field stimulation were detected with fluo-4. All experiments were conducted at 37 degrees C. Ca(2+) transient amplitudes were reduced, and Ca(2+) transient rise times were abbreviated in aged cells stimulated at 8 Hz compared with young adult myocytes. Furthermore, the incidence and frequency of spontaneous Ca(2+) sparks were markedly higher in aged myocytes compared with young adult cells. Spark amplitudes and spatial widths were similar in young adult and aged myocytes. However, spark half-rise times and half-decay times were abbreviated in aged cells compared with younger cells. Resting cytosolic Ca(2+) levels and SR Ca(2+) stores were assessed by rapid application of caffeine in fura-2-loaded cells. Neither resting Ca(2+) levels nor SR Ca(2+) content differed between young adult and aged cells. Thus increased spark frequency in aging cells was not attributable to increased SR Ca(2+) stores. Furthermore, the decrease in Ca(2+) transient amplitude was not due to a decrease in SR Ca(2+) load. These results demonstrate that alterations in fundamental SR Ca(2+) release units occur in aging ventricular myocytes and raise the possibility that alterations in Ca(2+) release may reflect age-related changes in fundamental release events rather than changes in SR Ca(2+) stores and diastolic Ca(2+) levels.     

Altered beta-adrenergic signal transduction in nonfailing hypertrophied myocytes from Dahl salt-sensitive rats

Desensitization of the beta-adrenergic receptor (beta-AR) response is well documented in hypertrophied hearts. We investigated whether beta-AR desensitization is also present at the cellular level in hypertrophied myocardium, as well as the physiological role of inhibitory G (G(i)) proteins and the L-type Ca(2+) channel in mediating beta-AR desensitization. Left ventricular (LV) myocytes were isolated from hypertrophied hearts of hypertensive Dahl salt-sensitive (DS) rats and nonhypertrophied hearts of normotensive salt-resistant (DR) rats. Cells were paced at a rate of 300 beats/min at 37 degrees C, and myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) were simultaneously measured. In response to increasing concentrations of isoproterenol, DR myocytes displayed a dose-dependent augmentation of cell shortening and the [Ca(2+)](i) transient amplitude, whereas hypertrophied DS myocytes had a blunted response of both cell shortening and the [Ca(2+)](i) transient amplitude. Interestingly, inhibition of G(i) proteins did not restore beta-AR desensitization in DS myocytes. The responses to increases in extracellular Ca(2+) and an L-type Ca(2+) channel agonist were also similar in both DS and DR myocytes. Isoproterenol-stimulated adenylyl cyclase activity, however, was blunted in hypertrophied myocytes. We concluded that compensated ventricular hypertrophy results in a blunted contractile response to beta-AR stimulation, which is present at the cellular level and independent of alterations in inhibitory G proteins and the L-type Ca(2+) channel.     

Increases in diastolic [Ca2+] can contribute to positive inotropy in guinea pig ventricular myocytes in the absence of changes in amplitudes of Ca2+ transients

Increases in contraction amplitude following rest or in elevated extracellular Ca(2+) concentration ([Ca(2+)]) have been attributed to increased sarcoplasmic reticulum (SR) Ca(2+) stores and/or increased trigger Ca(2+). However, either manipulation also may elevate diastolic [Ca(2+)]. The objective of this study was to determine whether elevation of diastolic [Ca(2+)] could contribute to positive inotropy in isolated ventricular myocytes. Voltage-clamp experiments were conducted with high-resistance microelectrodes in isolated myocytes at 37 degrees C. Intracellular free [Ca(2+)] was measured with fura-2, and cell shortening was measured with an edge detector. SR Ca(2+) stores were assessed with 10 mM caffeine (0 mM Na(+), 0 mM Ca(2+)). Following a period of rest, cells were activated with trains of pulses, which generated contractions of increasing amplitude, called positive staircases. Positive staircases were accompanied by increasing diastolic [Ca(2+)] but no change in Ca(2+) transient amplitudes. When extracellular [Ca(2+)] was elevated from 2.0 to 5.0 mM, resting intracellular [Ca(2+)] increased and resting cell length decreased. Amplitudes of contractions and L-type Ca(2+) current increased in elevated extracellular [Ca(2+)], although SR Ca(2+) stores, assessed by rapid application of caffeine, did not increase. Although Ca(2+) transient amplitude did not increase in 5.0 mM extracellular [Ca(2+)], diastolic [Ca(2+)] continued to increase with increasing extracellular [Ca(2+)]. These data suggest that increased diastolic [Ca(2+)] contributes to positive inotropy following rest or with increasing extracellular [Ca(2+)] in guinea pig ventricular myocytes.     

Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange

Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.     

Mechanisms of impaired calcium handling underlying subclinical diastolic dysfunction in diabetes

Isolated diastolic dysfunction is found in almost half of asymptomatic patients with well-controlled diabetes and may precede diastolic heart failure. However, mechanisms that underlie diastolic dysfunction during diabetes are not well understood. We tested the hypothesis that isolated diastolic dysfunction is associated with impaired myocardial Ca(2+) handling during type 1 diabetes. Streptozotocin-induced diabetic rats were compared with age-matched placebo-treated rats. Global left ventricular myocardial performance and systolic function were preserved in diabetic animals. Diabetes-induced diastolic dysfunction was evident on Doppler flow imaging, based on the altered patterns of mitral inflow and pulmonary venous flows. In isolated ventricular myocytes, diabetes resulted in significant prolongation of action potential duration compared with controls, with afterdepolarizations occurring in diabetic myocytes (P < 0.05). Sustained outward K(+) current and peak outward component of the inward rectifier were reduced in diabetic myocytes, while transient outward current was increased. There was no significant change in L-type Ca(2+) current; however, Ca(2+) transient amplitude was reduced and transient decay was prolonged by 38% in diabetic compared with control myocytes (P < 0.05). Sarcoplasmic reticulum Ca(2+) load (estimated by measuring the integral of caffeine-evoked Na(+)-Ca(2+) exchanger current and Ca(2+) transient amplitudes) was reduced by approximately 50% in diabetic myocytes (P < 0.05). In permeabilized myocytes, Ca(2+) spark amplitude and frequency were reduced by 34 and 20%, respectively, in diabetic compared with control myocytes (P < 0.05). Sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a protein levels were decreased during diabetes. These data suggest that in vitro impairment of Ca(2+) reuptake during myocyte relaxation contributes to in vivo diastolic dysfunction, with preserved global systolic function, during diabetes.     

Quantification of calcium entry at the T-tubules and surface membrane in rat ventricular myocytes

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by approximately 60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 micromol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 micromol/L cytosol, respectively). However, more Ca entry occurs per microm(2) of junctional membrane at the cell surface than in the T-tubules (in nM/microm(2): 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions ( approximately 35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

Metabolic stress in isolated mouse ventricular myocytes leads to remodeling of t tubules

Cardiac ventricular myocytes possess an extensive t-tubular system that facilitates the propagation of membrane potential across the cell body. It is well established that ionic currents at the restricted t-tubular space may lead to significant changes in ion concentrations, which, in turn, may affect t-tubular membrane potential. In this study, we used the whole cell patch-clamp technique to study accumulation and depletion of t-tubular potassium by measuring inward rectifier potassium tail currents (I(K1,tail)), and inward rectifier potassium current (I(K1)) "inactivation". At room temperatures and in the absence of Mg(2+) ions in pipette solution, the amplitude of I(K1,tail) measured ~10 min after the establishment of whole cell configuration was reduced by ~18%, but declined nearly twofold in the presence of 1 mM cyanide. At ~35°C I(K1,tail) was essentially preserved in intact cells, but its amplitude declined by ~85% within 5 min of cell dialysis, even in the absence of cyanide. Intracellular Mg(2+) ions played protective role at all temperatures. Decline of I(K1,tail) was accompanied by characteristic changes in its kinetics, as well as by changes in the kinetics of I(K1) inactivation, a marker of depletion of t-tubular K(+). The data point to remodeling of t tubules as the primary reason for the observed effects. Consistent with this, detubulation of myocytes using formamide-induced osmotic stress significantly reduced I(K1,tail), as well as the inactivation of inward I(K1). Overall, the data provide strong evidence that changes in t tubule volume/structure may occur on a short time scale in response to various types of stress.     

Myofilament sensitivity to Ca2+ in ventricular myocytes from the Goto–Kakizaki diabetic rat

The chronic effects of type 2 diabetes mellitus on myofilament sensitivity to Ca(2+) in ventricular myocytes from the Goto-Kakizaki (GK) rat have been investigated. Experiments were performed in ventricular myocytes isolated from 17-month GK rats and age-matched Wistar controls. Myocytes were loaded with fura-2 (an indicator for intracellular Ca(2+) concentration) and the fura-2 ratio (340/380 nm), and shortening were measured simultaneously in electrically stimulated myocytes. Myofilament sensitivity to Ca(2+) was assessed from phase-plane diagrams of fura-2 versus cell length by measuring the gradient of the fura-2-cell length trajectory during late relaxation of the twitch contraction. Non-fasting and fasting blood glucose were elevated in GK rats compared to controls. Fasting blood glucose was 151.5 +/- 15.3 mg/dl (n = 8) in GK rats compared to 72.1 +/- 3.6 mg/dl (n = 9) in controls. At 120 min after intraperitoneal injection of glucose (2 g/kg body weight), blood glucose was 570.8 +/- 36.8 mg/dl in GK rats compared to 148 +/- 8.6 mg/dl in controls. Amplitude of shortening was significantly increased in myocytes from GK rats (6.56 +/- 0.54%, n = 31) compared to controls (5.05 +/- 0.43%, n = 36), and the amplitude of the Ca(2+) transient was decreased in myocytes from GK rats (0.23 +/- 0.02 RU, n = 31) compared to controls (0.30 +/- 0.02 RU, n = 36). The fura-2-cell length trajectory during the late stages of relaxation of the twitch contraction was steeper in myocytes from GK rats (89.2 +/- 16.6 microm/RU, n = 27) compared to controls (31.9 +/- 5.9 microm/RU, n = 35). Increased amplitude of shortening, accompanied by a decrease in amplitude of the Ca(2+) transient, might be explained by an increased myofilament sensitivity to Ca(2+).     

Numerical analysis of Ca2+ signaling in rat ventricular myocytes with realistic transverse-axial tubular geometry and inhibited sarcoplasmic reticulum

The t-tubules of mammalian ventricular myocytes are invaginations of the cell membrane that occur at each Z-line. These invaginations branch within the cell to form a complex network that allows rapid propagation of the electrical signal, and hence synchronous rise of intracellular calcium (Ca(2+)). To investigate how the t-tubule microanatomy and the distribution of membrane Ca(2+) flux affect cardiac excitation-contraction coupling we developed a 3-D continuum model of Ca(2+) signaling, buffering and diffusion in rat ventricular myocytes. The transverse-axial t-tubule geometry was derived from light microscopy structural data. To solve the nonlinear reaction-diffusion system we extended SMOL software tool (http://mccammon.ucsd.edu/smol/). The analysis suggests that the quantitative understanding of the Ca(2+) signaling requires more accurate knowledge of the t-tubule ultra-structure and Ca(2+) flux distribution along the sarcolemma. The results reveal the important role for mobile and stationary Ca(2+) buffers, including the Ca(2+) indicator dye. In agreement with experiment, in the presence of fluorescence dye and inhibited sarcoplasmic reticulum, the lack of detectible differences in the depolarization-evoked Ca(2+) transients was found when the Ca(2+) flux was heterogeneously distributed along the sarcolemma. In the absence of fluorescence dye, strongly non-uniform Ca(2+) signals are predicted. Even at modest elevation of Ca(2+), reached during Ca(2+) influx, large and steep Ca(2+) gradients are found in the narrow sub-sarcolemmal space. The model predicts that the branched t-tubule structure and changes in the normal Ca(2+) flux density along the cell membrane support initiation and propagation of Ca(2+) waves in rat myocytes.