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.     

Rescue of contractile abnormalities by Na+/Ca2+ exchanger overexpression in postinfarction rat myocytes.

Previous studies on myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated increased cell length, reduced Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. In the present study, we investigated whether NCX1 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. When myocytes were placed in culture under continued electrical-field stimulation conditions, differences in contraction amplitudes and cell lengths between sham and MI myocytes were preserved for at least 48 h. Infection of both sham and MI myocytes by adenovirus expressing green fluorescent protein resulted in >95% infection, as evidenced by green fluorescent protein fluorescence, but contraction amplitudes at 6-, 24-, and 48-h postinfection were not affected. NCX1 overexpression in MI myocytes resulted in lower diastolic [Ca(2+)](i) levels at all extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, suggesting enhanced forward NCX1 activity. At 5 mM [Ca(2+)](o), subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored toward normal levels by overexpressing NCX1. At 0.6 mM [Ca(2+)](o), supranormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were lowered by NCX1 overexpression. We conclude that overexpression of NCX1 in MI myocytes was effective in improving contractile dysfunction, most likely because of enhancement of both Ca(2+) efflux and influx during a cardiac cycle. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in postinfarction myocytes.     

Overexpression of phospholemman alters contractility and [Ca(2+)](i) transients in adult rat myocytes

Previous studies showed increased phospholemman (PLM) mRNA after myocardial infarction (MI) in rats (Sehl PD, Tai JTN, Hillan KJ, Brown LA, Goddard A, Yang R, Jin H, and Lowe DG. Circulation 101: 1990-1999, 2000). We tested the hypothesis that, in normal adult rat cardiac myocytes, PLM overexpression alters contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. Compared with myocytes infected by control adenovirus expressing green fluorescent protein (GFP) alone, Western blots indicated a 41% increase in PLM expression after 72 h (P < 0.001) but no changes in Na(+)/Ca(2+) exchanger, SERCA2, and calsequestrin levels in myocytes infected by adenovirus expressing GFP and PLM. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), maximal contraction amplitudes in PLM-overexpressed myocytes were 24% (P < 0.005) and [Ca(2+)](i) transient amplitudes were 18% (P < 0.05) lower than control myocytes. At 0.6 mM [Ca(2+)](o), however, contraction and [Ca(2+)](i) transient amplitudes were significantly (P < 0.05) higher in PLM-overexpressed than control myocytes (18% and 42%, respectively); at 1.8 mM [Ca(2+)](o), the differences in contraction and [Ca(2+)](i) transient amplitudes were narrowed. This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-overexpressed myocytes mimics that observed in post-MI rat myocytes. We suggest that PLM overexpression observed in post-MI myocytes may partly account for contractile abnormalities by perturbing Ca(2+) fluxes during excitation-contraction.     

Effects of sprint training on contractility and [Ca(2+)](i) transients in adult rat myocytes.

The effects of 6-8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant (P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain alpha-isoenzyme increased significantly (P < 0.02) from 0.566 +/- 0.077% in Sed rats to 0.871 +/- 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, maximal shortening amplitudes and maximal shortening velocities were significantly (P < 0.0001) lower and half-times of relaxation were significantly (P < 0.005) longer in HIST myocytes. HIST myocytes had significantly (P < 0.0001) higher diastolic [Ca(2+)](i) levels. Compared with Sed myocytes, systolic [Ca(2+)](i) levels in HIST myocytes were higher at 0.6 mM [Ca(2+)](o), similar at 1.8 mM [Ca(2+)](o), and lower at 5 mM [Ca(2+)](o). The amplitudes of [Ca(2+)](i) transients were significantly (P < 0.0001) lower in HIST myocytes. Half-times of [Ca(2+)](i) transient decline, an estimate of sarcoplasmic reticulum (SR) Ca(2+) uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant (P < 0.03) threefold decrease in Na(+)/Ca(2+) exchanger, but SR Ca(2+)-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca(2+)](i) transient amplitudes under the conditions studied. We speculate that downregulation of Na(+)/Ca(2+) exchanger may partly account for the decreased contractility in HIST myocytes.     

Sprint training improves contractility in postinfarction rat myocytes: role of Na+/Ca2+ exchange.

Previous studies in adult myocytes isolated from rat hearts 3-9 wk after myocardial infarction (MI) demonstrated abnormal contractility and decreased Na(+)/Ca(2+) exchanger (NCX1) activity. In addition, a program of high-intensity sprint training (HIST) instituted shortly after MI restored both contractility and NCX1 activity toward normal. The present study examined the hypotheses that reduced NCX1 activity caused abnormal contractility in myocytes isolated from sedentary (Sed) rat hearts 9-11 wk after coronary artery ligation and that HIST ameliorated contractile dysfunction in post-MI myocytes by increasing NCX1 activity. The approach was to upregulate NCX1 in MI-sedentary (MISed) myocytes and downregulate NCX1 in MI-exercised (MIHIST) myocytes by adenovirus-mediated gene transfer. Overexpression of NCX1 in MISed myocytes did not affect sarco(endo)plasmic reticulum Ca(2+)-ATPase and calsequestrin levels but rescued contractile abnormalities observed in MISed myocytes. That is, at 5 mM extracellular Ca(2+) concentration, the subnormal contraction amplitude in MISed myocytes (compared with Sham myocytes) was increased toward normal by NCX1 overexpression, whereas at 0.6 mM extracellular Ca(2+) concentration the supernormal contraction amplitude in MISed myocytes was lowered. Conversely, NCX1 downregulation by antisense in MIHIST myocytes abolished the beneficial effects of HIST on contraction amplitudes in MI myocytes. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in post-MI myocytes and that HIST ameliorated contractile dysfunction in post-MI myocytes partly by enhancing NCX1 activity.     

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.     

Phospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.     

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.     

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.     

Sprint training normalizes Ca(2+) transients and SR function in postinfarction rat myocytes.

Previous studies have shown that myocytes isolated from sedentary (Sed) rat hearts 3 wk after myocardial infarction (MI) undergo hypertrophy, exhibit altered intracellular Ca(2+) concentration ([Ca(2+)](i)) dynamics and abnormal contraction, and impaired sarcoplasmic reticulum (SR) function manifested as prolonged half-time of [Ca(2+)](i) decline. Because exercise training elicits positive adaptations in cardiac contractile function and myocardial Ca(2+) regulation, the present study examined whether 6-8 wk of high-intensity sprint training (HIST) would restore [Ca(2+)](i) dynamics and SR function in MI myocytes toward normal. In MI rats, HIST ameliorated myocyte hypertrophy as indicated by significant (P 相似文献   

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.     

In situ SR function in postinfarction myocytes.

Previous studies have shown lower systolic intracellular Ca(2+) concentrations ([Ca(2+)](i)) and reduced sarcoplasmic reticulum (SR)-releasable Ca(2+) contents in myocytes isolated from rat hearts 3 wk after moderate myocardial infarction (MI). Ca(2+) entry via L-type Ca(2+) channels was normal, but that via reverse Na(+)/Ca(2+) exchange was depressed in 3-wk MI myocytes. To elucidate mechanisms of reduced SR Ca(2+) contents in MI myocytes, we measured SR Ca(2+) uptake and SR Ca(2+) leak in situ, i.e., in intact cardiac myocytes. For sham and MI myocytes, we first demonstrated that caffeine application to release SR Ca(2+) and inhibit SR Ca(2+) uptake resulted in a 10-fold prolongation of half-time (t(1/2)) of [Ca(2+)](i) transient decline compared with that measured during a normal twitch. These observations indicate that early decline of the [Ca(2+)](i) transient during a twitch in rat myocytes was primarily mediated by SR Ca(2+)-ATPase and that the t(1/2) of [Ca(2+)](i) decline is a measure of SR Ca(2+) uptake in situ. At 5.0 mM extracellular Ca(2+), systolic [Ca(2+)](i) was significantly (P 相似文献   

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

Contribution of Ca(2+) transporters to relaxation in intact ventricular myocytes from developing rats

The relative contributions of Ca(2+) transporters to intracellular Ca(2+) concentration ([Ca(2+)](i)) decline associated with twitch relaxation were analyzed in intact ventricular myocytes from developing and adult rats. This was accomplished by estimation of individual integrated Ca(2+) fluxes with the use of kinetic parameters calculated from [Ca(2+)](i) measurements during twitches and caffeine-evoked contractures, and from myocardial passive Ca(2+) buffering data. Our main findings were the following: 1) twitch relaxation and [Ca(2+)](i) decline were significantly slower during the first postnatal week than in adults, 2) inhibition of sarcoplasmic reticulum (SR) Ca(2+) accumulation resulted in faster [Ca(2+)](i) decline in young cells than in adult cells, 3) the contributions of the SR Ca(2+) uptake and Na(+)/Ca(2+) exchange (NCX) to twitch relaxation increased from ~75 to 92%, and decreased from 24 to 5%, respectively, from birth to adulthood, and 4) Ca(2+) transport by the sarcolemmal Ca(2+)-ATPase was apparently increased in neonates. Our data indicate that despite a marked increase in NCX contribution to cell relaxation in immature rats, the SR Ca(2+)-ATPase appears to be the predominant transporter responsible for relaxation-associated [Ca(2+)](i) decline from birth to adulthood.     

Increased Ca(2+) sparks and sarcoplasmic reticulum Ca(2+) stores potentially determine the spontaneous activity of pulmonary vein cardiomyocytes

Pulmonary veins (PVs) contain cardiomyocytes with spontaneous activity that may be responsible for PV arrhythmia. Abnormal Ca(2+) regulation is known to contribute to PV arrhythmogenesis. The purpose of this study was to investigate whether PV cardiomyocytes with spontaneous activity have different intracellular Ca(2+) ([Ca(2+)](i)) transients, Ca(2+) sparks and responses to isoproterenol and ryanodine receptor modulators (magnesium and FK506) than do PV cardiomyocytes without spontaneous activity and left atrial (LA) cardiomyocytes. Through fluorescence and confocal microscopy, we evaluated the [Ca(2+)](i) transients and Ca(2+) sparks in isolated rabbit PV and LA cardiomyocytes. PV cardiomyocytes with spontaneous activity had larger [Ca(2+)](i) transients and sarcoplasmic reticulum (SR) Ca(2+) stores than PV cardiomyocytes without spontaneous activity or LA cardiomyocytes. PV cardiomyocytes with spontaneous activity also had a higher incidence and frequency of Ca(2+) sparks, and had Ca(2+) sparks with larger amplitudes than other cardiomyocytes. Magnesium (5.4 mM) reduced the [Ca(2+)](i) transient amplitude and beating rate in PV cardiomyocytes with spontaneous activity. However, in contrast with other cardiomyocytes, low doses (1.8 mM) of magnesium did not reduce the [Ca(2+)](i) transients amplitude in PV cardiomyocytes with spontaneous activity. FK506 (1 muM) diminished the SR Ca(2+) stores in PV cardiomyocytes with spontaneous activity to a lesser extent than that in other cardiomyocytes. Isoproterenol (10 nM) increased the [Ca(2+)](i) transient amplitude to a lesser extent in LA cardiomyocytes than in PV cardiomyocytes with or without spontaneous activity. In conclusion, our results suggest that enhanced [Ca(2+)](i) transients, increased Ca(2+) sparks and SR Ca(2+) stores may contribute to the spontaneous activity of PV cardiomyocytes.     

Na(+)/Ca(2+) exchanger 2 is neuroprotective by exporting Ca(2+) during a transient focal cerebral ischemia in the mouse

Na(+)/Ca(2+) exchanger (NCX), by mediating Na(+) and Ca(2+) fluxes bi-directionally, assumes a role in controlling the Ca(2+) homeostasis in the ischemic brain. It has been suggested that the three isoforms of NCX (NCX1, 2 and 3) may be differentially involved in permanent cerebral ischemia. However, the role of NCX2 has not been defined in ischemic reperfusion injury after a transient focal cerebral ischemia. Furthermore, it is not known whether NCX2 imports or exports intracellular Ca(2+) ([Ca(2+)](i)) following ischemia and reperfusion. To define the role of NCX2 in ischemia and reperfusion, we examined mice lacking NCX2, in vivo and in vitro. After an in vitro ischemia, a significantly slower recovery in population spike amplitudes, a sustained elevation of [Ca(2+)](i) and an increased membrane depolarization were developed in the NCX2-deficient hippocampus. Moreover, a transient focal cerebral ischemia in vivo produced a larger infarction and more cell death in the NCX2-deficient mouse brain. In particular, in the wild type brain, NCX2-expressing neurons were largely spared from cell death after ischemia. Our results suggest that NCX2 exports Ca(2+) in ischemia and thus protects neuronal cells from death by reducing [Ca(2+)](i) in the adult mouse brain.     

Mibefradil improves beta-adrenergic responsiveness and intracellular Ca(2+) handling in hypertrophied rat myocardium

The present study investigated the effects of mibefradil, a novel T-type channel blocker, on ventricular function and intracellular Ca(2+) handling in normal and hypertrophied rat myocardium. Ca(2+) transient was measured with the bioluminescent protein, aequorin. Mibefradil (2 microM) produced nonsignificant changes in isometric contraction and peak systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) in normal rat myocardium. Hypertrophied papillary muscles isolated from aortic-banded rats 10 weeks after operation demonstrated a prolonged duration of isometric contraction, as well as decreased amplitudes of developed tension and peak Ca(2+) transient compared with the sham-operated group. Additionally, diastolic [Ca(2+)](i) increased in hypertrophied rat myocardium. The positive inotropic effect of isoproterenol stimulation was blunted in hypertrophied muscles despite a large increase in Ca(2+) transient amplitude. Afterglimmers and corresponding aftercontractions were provoked with isoproterenol (10(-5) and 10(-4) M) stimulation in 4 out of 16 hypertrophied muscles, but were eliminated in the presence of mibefradil (2 microM). In addition, hypertrophied muscles in the presence of mibefradil had a significant improvement of contractile response to isoproterenol stimulation and a reduced diastolic [Ca(2+)](I), although a mild decrease of peak Ca(2+)-transient was also shown. However, verapamil (2 microM) did not restore the inotropic and Ca(2+) modulating effects of isoproterenol in hypertrophied myocardium. Mibefradil partly restores the positive inotropic response to beta-adrenergic stimulation in hypertrophied myocardium from aortic-banded rats, an effect that might be useful in hypertrophied myocardium with impaired [Ca(2+)](i) homeostasis.     

Hypoxia inhibits the Na(+)/Ca(2+) exchanger in pulmonary artery smooth muscle cells.

The cellular mechanisms underlying hypoxic pulmonary vasoconstriction are not fully understood. We examined the effect of hypoxia on Ca(2+) efflux from the cytosol in single Fura-2-loaded pulmonary artery myocytes. During mild hypoxia (pO(2)=50-60 Torr), peak [Ca(2+)](i) was increased and the rate of Ca(2+) removal from the cytosol was markedly slowed after stimuli that elevated [Ca(2+)](i). Removal of extracellular Na(+) potentiated the peak [Ca(2+)](i) rise and slowed the Ca(2+) decay rate in cells recorded under normoxic conditions; it did not further slow the Ca(2+) decay rate or potentiate the [Ca(2+)](i) increase in hypoxic cells. An Na(+)/Ca(2+) exchange current was recorded in isolated pulmonary artery myocytes. Switching from Li(+) to Na(+) (130 mM) revealed an inward current with reversal potential consistent with the Na(+)/Ca(2+) exchange current in cells in which [Ca(2+)](i) was clamped at 1 microM similar currents, although smaller, were observed with normal resting [Ca(2+)](i) using the perforated patch clamp technique. The Na(+)/Ca(2+) exchange current was markedly inhibited in myocytes exposed to mild hypoxia. RT-PCR revealed the expression of specific alternatively spliced RNAs of NCX1 in rat pulmonary arteries. These findings provide an enhanced understanding of the molecular mechanisms underlying hypoxic sensing in pulmonary arteries.     

Constitutive overexpression of phosphomimetic phospholemman S68E mutant results in arrhythmias, early mortality, and heart failure: potential involvement of Na+/Ca2+ exchanger

Expression and activity of cardiac Na(+)/Ca(2+) exchanger (NCX1) are altered in many disease states. We engineered mice in which the phosphomimetic phospholemman S68E mutant (inhibits NCX1 but not Na(+)-K(+)-ATPase) was constitutively overexpressed in a cardiac-specific manner (conS68E). At 4-6 wk, conS68E mice exhibited severe bradycardia, ventricular arrhythmias, increased left ventricular (LV) mass, decreased cardiac output (CO), and ～50% mortality compared with wild-type (WT) littermates. Protein levels of NCX1, calsequestrin, ryanodine receptor, and α(1)- and α(2)-subunits of Na(+)-K(+)-ATPase were similar, but sarco(endo)plasmic reticulum Ca(2+)-ATPase was lower, whereas L-type Ca(2+) channels were higher in conS68E hearts. Resting membrane potential and action potential amplitude were similar, but action potential duration was dramatically prolonged in conS68E myocytes. Diastolic intracellular Ca(2+) ([Ca(2+)](i)) was higher, [Ca(2+)](i) transient and maximal contraction amplitudes were lower, and half-time of [Ca(2+)](i) transient decline was longer in conS68E myocytes. Intracellular Na(+) reached maximum within 3 min after isoproterenol addition, followed by decline in WT but not in conS68E myocytes. Na(+)/Ca(2+) exchange, L-type Ca(2+), Na(+)-K(+)-ATPase, and depolarization-activated K(+) currents were decreased in conS68E myocytes. At 22 wk, bradycardia and increased LV mass persisted in conS68E survivors. Despite comparable baseline CO, conS68E survivors at 22 wk exhibited decreased chronotropic, inotropic, and lusitropic responses to isoproterenol. We conclude that constitutive overexpression of S68E mutant was detrimental, both in terms of depressed cardiac function and increased arrhythmogenesis.     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.