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D M Bers

Publications and source records attributed to D M Bers.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cyclic stretch down-regulates calcium transporter gene expression in neonatal rat ventricular myocytes.

Abnormal intracellular Ca2+ handling in hypertrophied and failing hearts is partly due to changes in Ca2+ transporter gene expression, but the mechanisms responsible for these alterations remain largely unknown. We previously showed that intrinsic mechanical load (i.e. spontaneous contractile activity) induced myocyte hypertrophy, and down-regulated SR Ca2+ ATPase (SERCA2) gene expression in cultured neonatal rat ventricular myocytes (NRVM). In the present study, we examined whether extrinsic mechanical load (i.e. cyclic stretch) also induced NRVM hypertrophy, and led to down-regulation of SERCA2 and other Ca2+ transporter genes which have been associated with cardiac hypertrophy and failure in vivo. NRVM were maintained in serum-free culture medium under control conditions, or subjected to cyclic mechanical deformation (1.0 Hz, 20% maximal strain, 48 h). Under these conditions, cyclic stretch induced NRVM hypertrophy, as evidenced by significant increases in total protein/DNA ratio, myosin heavy chain (MHC) content, and atrial natriuretic factor (ANF) secretion. Cyclic stretch also induced the MHC isoenzyme "switch" which is characteristic of hemodynamic overload of the rat heart in vivo. Cyclic stretch significantly down-regulated SERCA2 and ryanodine receptor (RyR) mRNA and protein levels, while simultaneously increasing ANF mRNA. In contrast, Na+-Ca2+ exchanger and phospholamban mRNA levels were unaffected. Load-dependent SERCA2 and RyR down-regulation was independent of Ca2+ influx via voltage-gated, L-type Ca2+ channels, as cyclic stretch down-regulated SERCA2 and RyR mRNA levels in both control and verapamil-treated NRVM. These results indicate that extrinsic mechanical load (in the absence of other exogenous stimuli) induces NRVM hypertrophy and causes down-regulation of Ca2+ transporter gene expression. This in vitro model system should prove useful to dissect the intracellular signaling pathways responsible for transducing this phenotype during cardiac hypertrophy and heart failure in vivo.

Animals↗
PubMed1998

Passive Ca2+ binding in ventricular myocardium of neonatal and adult rats.

In this study, passive Ca2+ binding was determined in ventricular homogenates (VH) from neonatal (4-6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca2+ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca2+ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca2+] ([Ca2+]F) was measured with Indo-1 and bound Ca2+ ([Ca2+]B) was the difference between [Ca2+]F and total Ca2+. Apparent Ca2+ dissociation constants (Kd) for BAPTA and Indo-1 were increased by 10-20 mg VH protein/ml (from 0.35 to 0.92 microM for Indo-1 and from 0.20 to 0.76 microM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl2 additions (2.5-10 nmoles) yielded delta[Ca2+]B/delta[Ca2+]F for the sum of [Ca2+]B at all three classes of binding sites. From this function, the apparent number of endogenous sites (Ben) and their Kd (Ken) were determined. Similar Ken values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 +/- 0.14 microM, 0.69 +/- 0.13 microM and 0.53 +/- 0.10 microM, respectively). However, Ben was significantly higher in adult myocytes than in adult VH (1.73 +/- 0.35 versus 0.70 +/- 0.12 nmol/mg protein, P < 0.01), which correspond to approximately 300 and 213 mumol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that Ben determined in VH underestimates cellular Ben by 29%. Although Ben values in nmol/mg protein were similar in adult and neonatal VH (0.69 +/- 0.12), protein content was much higher in adult ventricle (125 +/- 7 versus 80 +/- 1 mg protein/g wet weight, P < 0.01). Expressing Ben per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca2+ binding capacity at high-affinity sites is approximately 300 and 176 mmol/l cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca2+ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.

Aging↗
PubMed1998

Control of maximum sarcoplasmic reticulum Ca load in intact ferret ventricular myocytes. Effects Of thapsigargin and isoproterenol.

In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined by a balance among influx and efflux pathways. The SR Ca content may be limited mainly by the ATP-supplied chemical potential that is inherent in the gradient between SR and cytosol. That is, forward Ca pumping from cytosol to SR may be opposed by energetically conservative reverse pumping dependent on intra-SR free [Ca]. On the other hand, SR Ca loading may be limited by dissipative pathways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular myocytes cumulatively with known amounts of Ca via L-type Ca channels (ICa), using Na-free solutions to prevent Na/Ca exchange. We then measured the maximal resulting caffeine-released SR Ca content under control conditions, as well as when SR Ca pumping was accelerated by isoproterenol (1 micro M) or slowed by thapsigargin (0.2-0.4 micro M). Under control conditions, SR Ca content reached a limit of 137 micro mol.liter cytosol-1 (nonmitochondrial volume) when measured by integrating caffeine-induced Na/Ca exchange currents lintegraINaCaXdt) and of 119 micro mol.liter cytosol-1 when measured using fluorescence signals dependent on changes in cytosolic free Ca ([Ca]i). When Ca-ATPase pumping rate was slowed 39% by thapsigargin, the maximal SR Ca content decreased by 5 (integralINaCaXdt method) or 23% (fluorescence method); when pumping rate was increased 74% by isoproterenol, SR Ca content increased by 10% (fluorescence method) or 20% (integralINaCaXdt method). The relative stability of the SR Ca load suggests that dissipative losses have only a minor influence in setting the SR Ca content. Indeed, it appears that the SR Ca pump in intact cells can generate a [Ca] gradient approaching the thermodynamic limit.

Animals↗
PubMed1998

Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects.

Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H703-H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 microM) abolished the stimulation-dependent acceleration of twitch [Ca]i decline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]i decline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (tau) of [Ca]i decline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 +/- 6 vs. 188 +/- 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]i during rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 +/- 74 nM for WT, 1118 +/- 133 nM for PLB-KO, P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The tau for [Ca]i decline of the cafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 +/- 0.2 s in WT vs. 3.2 +/- 0.2 in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]i decline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.

Animals↗
PubMed1998

Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes.

Left ventricular hypertrophy (approximately 40%) was induced in rats by banding of the abdominal aorta. After 16 wk, ventricular homogenates were prepared for biochemical measurements and ventricular myocytes were isolated for functional studies. In myocytes, the effects of banding on intracellular Ca handling, contraction, and excitation-contraction (E-C) coupling were determined using indo 1 fluorescence and whole cell voltage clamp. After steady-state field or voltage-clamp stimulation to load the sarcoplasmic reticulum (SR), SR Ca content assessed by caffeine-induced Ca transients was the same in sham and banded groups. Despite this, cell shortening amplitudes were significantly depressed in the banded group, suggesting altered contractile properties. In banded rats, the SR Ca-adenosinetriphosphatase (Ca-ATPase) mRNA level was reduced, as was homogenate thapsigargin-sensitive SR Ca-ATPase, but cytosolic free Ca concentration ([Ca]i) decline attributed to SR Ca-ATPase activity in intact cells was not slowed. Banding also reduced Na/Ca exchange mRNA level but did not affect either Na-dependent sarcolemmal 45Ca transport in homogenate or the rate of [Ca]i decline in intact cells attributed to Na/Ca exchange (during caffeine-induced contractures). Banding also did not change the rate of [Ca]i decline mediated by the combined function of the mitochondrial Ca uptake and sarcolemmal Ca-ATPase in intact cells. Ca current (ICa) density and voltage dependence were the same in sham and banded groups. Ryanodine receptor mRNA, protein content, and ryanodine affinity were also unchanged in the banded group. At 1 mM extracellular Ca concentration ([Ca]o), banding did not affect E-C coupling efficacy in intact cells under voltage clamp (i.e., same contraction for given ICa and SR Ca load). However, when [Ca]o was reduced to 0.5 mM, the efficacy of E-C coupling was greatly depressed in the banded group (even though ICa and SR Ca content were matched). In summary, unloaded myocyte contraction was depressed in these hypertrophic hearts, but Ca transport was little altered, at 1 mM [Ca]o. However, reduction of [Ca]o to 0.5 mM appears to unmask a depressed fractional SR Ca release in response to a given ICa trigger and SR Ca load.

Animals↗
PubMed1998

Effects of left ventricular hypertrophy on force and Ca2+ handling in isolated rat myocardium.

To study the effect of left ventricular (LV) hypertrophy on force and Ca2+ handling in isolated rat myocardium, LV hypertrophy was induced in rats by banding of the abdominal aorta. After 16 wk, arterial pressure was assessed by catheterization. LV trabeculae were isolated and loaded with indo 1 salt by iontophoretic injection. Isometric force and intracellular free Ca2+ concentration ([Ca2+]i) were measured at stimulation frequencies between 0.25 and 3 Hz and rest intervals between 2 and 240 s. Sarcoplasmic reticulum (SR) Ca2+ content was also investigated using rapid cooling contractures (RCC). Systolic and diastolic pressure as well as heart weight-to-body weight ratios were significantly elevated in banded compared with control animals (167 vs. 117 mmHg, 108 vs. 83 mmHg, and 4.6 vs. 4.0 mg/g, respectively). At high frequencies, twitch relaxation and [Ca2+]i decline rates were significantly slower in banded compared with control rats, and diastolic [Ca2+]i was higher in the banded rat muscles (at 3 Hz, force half-time = 83 vs. 68 ms; time constant of [Ca2+]i decline = 208 vs. 118 ms; and diastolic [Ca2+]i = 505 vs. 353 nM). These differences could not be ascribed to altered Na+/Ca2+ exchange, since twitch relaxation and Ca2+ handling were not different between groups in the presence of caffeine (or cyclopiazonic acid plus ryanodine), where relaxation depends primarily on Na+/Ca2+ exchange. After long rest intervals (> or = 120 s), control rats showed a significant rest potentiation of force and Ca2+ transients, whereas banded rats did not. In addition, RCC amplitudes increased with rest in control but were unaltered in banded rats. In summary, pressure-overload hypertrophy was associated with slower twitch relaxation and [Ca2+]i decline but also with blunted rest potentiation of twitches and SR Ca2+ content of LV trabeculae. The decrease in SR Ca(2+)-ATPase function in banded rats may contribute to the observed diastolic dysfunction associated with pressure-overload hypertrophy.

Animals↗
PubMed1998

Subcellular properties of [Ca2+]i transients in phospholamban-deficient mouse ventricular cells.

The regulatory protein phospholamban exerts a physiological inhibitory effect on the sarcoplasmic reticulum (SR) Ca2+ pump that is relieved with phosphorylation. We have studied the subcellular properties of intracellular Ca2+ ([Ca2+]i) transients in ventricular myocytes isolated from wild-type (WT) and phospholamban-deficient (PLB-KO) mice. In PLB-KO myocytes, steady-state twitch [Ca2+]i transients revealed an accelerated relaxation and the occurrence of highly localized failures of Ca2+ release. The acceleration of SR Ca2+ uptake caused an increase in SR Ca2+ load with the frequent occurrence of spontaneous [Ca2+]i waves and Ca2+ sparks. [Ca2+]i waves in PLB-KO cells showed a marked decrease in spatial width and more frequently appeared to abort. Local Ca2+ release events (Ca2+ sparks) were larger and more variable in amplitude and [Ca2+]i declined faster in PLB-KO myocytes. Increased local buffering and reduction in the refractoriness of SR Ca2+ release caused by the increased SR pump rate led to an overall enhancement of local [Ca2+]i gradients and inhomogeneities in the [Ca2+]i distribution during spontaneous Ca2+ release, [Ca2+]i waves, and excitation-contraction coupling.

Animals↗
PubMed1997

The effect of Ca(2+)-calmodulin-dependent protein kinase II on cardiac excitation-contraction coupling in ferret ventricular myocytes.

1. The effect of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) on excitation-contraction coupling (E-C coupling) was studied in intact ferret cardiac myocytes using the selective inhibitor KN-93, KN-93 decreased steady-state (SS) twitch [Ca2+]i (by 51%), resting Ca2+ spark frequency (by 88%) and SS sarcoplasmic reticulum (SR) Ca2+ content evaluated by eaffeine application (by 37.5%). 2. Increasing extracellular Ca2+ concentration ([Ca2+]o) to 5 mM in KN-93 restored SR Ca2+ load and Ca2+ spark frequency towards that in control (2 mM Ca2+o), but SS twitch [Ca2+]i was still significantly depressed by KN-93. 3. KN-93 decreased Ca2+ transient amplitude of SS twitches much more strongly than the amplitude of post-rest (PR) twitches. In the control, the time constant (Tau) of [Ca2+]i decline of SS twitches was faster than that for PR twitches. This stimulation-dependent acceleration of [Ca2+]i decline was abolished by KN-93. 4. Voltage-clamp experiments demonstrated that KN-93 significantly inhibited sarcolemmal L-type Ca2+ current (ICa) during repetitive pulses by slowing recovery from inactivation. This may explain the preferential action of KN-93 to suppress SS vs. PR twitches. 5. In KN-93, even when both ICa and SR Ca2+ load were matched to the control levels by manipulation of conditioning voltage-clamp pulses, contraction and twitch Ca2+ transients were still both significantly depressed (to 39 and 49% of control, respectively). 6. Since KN-93 reduced SR Ca2+ release channel (RyR) activity during E-C coupling, even for matched SR Ca2+ load and trigger ICa, we infer that endogenous CaMKII is an important modulator of E-C coupling in intact cardiac myocytes. Effects of KN-93 on ICa and SS twitch [Ca2+]i decline also indicate that endogenous CaMKII may have stimulatory effects on ICa and SR Ca2+ uptake.

Animals↗
PubMed1997

Role of the sarcoplasmic reticulum in contraction and relaxation of immature rabbit ventricular myocytes.

Previous indirect studies of newborn hearts have suggested a diminished functional role of the SR and a greater dependency upon trans-sarcolemmal Ca2+ fluxes to directly elicit contraction and promote relaxation. We tested the hypothesis that the SR in newborn rabbit hearts is functionally incompetent by measuring contraction and relaxation in ventricular myocytes isolated from the hearts of 1-2-day-old (newborn), 10-12-day-old (juvenile) and >150-day-old (adult) rabbits. Electrically stimulated twitch characteristics were compared to those elicited by the rapid application of 10 mm caffeine in the presence and absence of functional sarcolemmal Na-Ca exchange (disabled using a Na+- and Ca2+-free extracellular solution). During steady state, electrically-induced contractions were lower in amplitude in newborn and juvenile compared to adult myocytes (2.9+/-0.5 and 3.4+/-0.3 v 8.5+/-0.9% of resting cell length, respectively; n=24-29) and relaxation was slower in immature myocytes (t0.75 values: newborn 250+/-20; juvenile 240+/-10; adult 130+/-20 ms, n=14-21). Contrary to our hypothesis, caffeine triggered sufficient SR Ca2+ release from immature myocytes to elicit contractions of similar magnitude to adults (newborn 12.8+/-1. 1; juvenile 14.0+/-0.9; adult 15.0+/-1.6% of resting cell length, n=25-29). The amplitude of indo-1 Ca2+ transients during steady-state twitch was 36+/-12% of the maximal caffeine-induced Ca2+ transient in newborns (n=6) and 59+/-4% in adults (n=6). Caffeine slightly prolonged relaxation in adult myocytes (t0. 75=200+/-30 ms), but accelerated relaxation in newborn and juvenile myocytes (t0.75=180+/-20 and 150+/-30 ms, respectively). When both the SR and Na-Ca exchanger were disabled, the rate of relaxation (attributable to the sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter) of newborn and juvenile myocytes was significantly faster than in the adults (1660+/-210 and 3030+/-180 v 4530+/-310 ms, respectively; n=14-21). We conclude that neonatal and adult rabbit ventricular myocytes have comparable SR Ca2+ load, but neonatal cells exhibit smaller fractional SR Ca2+ release during steady-state contractions and greater Ca2+ removal by sarcolemmal Na-Ca exchange during relaxation.

Animals↗
PubMed1997

Assessment of intra-SR free [Ca] and buffering in rat heart.

To measure the free intrasarcoplasmic reticulum [Ca] ([Ca]SR) in isolated rat cardiac microsomes, ventricular tissue was homogenized in the presence of the low-affinity Ca indicator furaptra. Stepwise increases in cuvette [Ca] ([Ca]c) in the presence of ATP caused progressive increases in steady-state intravesicular fluorescence ratio to a maximum (Rmax). Steady-state [Ca]SR/[Ca]c was approximately 7000. Therefore the resting [Ca]SR may approach 700 microM in the rat cardiac myocyte at [Ca]c = 100 nM. The sarcoplasmic reticulum (SR) Ca pump requires a free energy of deltaG approximately 44 kJ x mol(-1) to generate this [Ca] gradient (e.g., approximately 74% of deltaG(ATP)). Total SR 45Ca uptake was also measured in digitonin-permeabilized myocytes as a function of [Ca]c in the absence of precipitating ions. The steady-state SR Ca content at 100 nM [Ca]c was approximately 400 micromol/liter cytosolic volume. Used together, these data allowed evaluation of the in situ SR Ca-buffering properties. The SR Ca-binding site concentration was approximately 14 mM, and Kd(Ca) approximately 0.638 mM [Ca]SR.

Adenosine Triphosphate↗
PubMed1997

Myosin heavy chain gene expression in neonatal rat heart cells: effects of [Ca2+]i and contractile activity.

To determine if mechanical signals or alterations in intracellular Ca2+ concentration ([Ca2+]i) affect myosin heavy chain (MHC) gene expression in spontaneously beating, neonatal rat ventricular myocytes, contractile activity was inhibited with verapamil, KCl, or 2,3-butanedione monoxime (BDM), and their acute and chronic effects on myocyte shortening, [Ca2+]i, and MHC gene expression were examined. Despite their differing effects on [Ca2+]i, verapamil, KCl, and BDM all inhibited contractile activity and markedly downregulated beta-MHC mRNA levels to 24 +/- 5, 21 +/- 7, and 6 +/- 2% of contracting cells, respectively. In contrast, these inhibitors of contraction upregulated alpha-MHC mRNA levels to 163 +/- 19, 156 +/- 7, and 198 +/- 20% of contracting cells, respectively. Transient transfection with a rat beta-MHC promoter-luciferase expression plasmid demonstrated that all inhibitors of contraction significantly decreased beta-MHC promoter activity. Paradoxically, contractile arrest also inhibited alpha-MHC promoter activity, suggesting that increased alpha-MHC mRNA levels resulted from posttranscriptional mechanisms. Actinomycin D mRNA stability assays indicated that alpha-MHC mRNA half-life was prolonged in noncontracting cells (33 h) compared with contracting myocytes (14 h). Contraction-dependent alterations in MHC gene expression were not dependent on release of angiotensin II or other growth factors into the culture medium. Thus intrinsic mechanical signals rather than alterations in [Ca2+]i regulate alpha-MHC and beta-MHC gene expression by both transcriptional and posttranscriptional mechanisms.

Angiotensin II↗
PubMed1997

Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes.

In heart, spontaneous local increases in cytosolic Ca2+ concentration ([Ca2+]i) called "Ca2+ sparks" may be fundamental events underlying both excitation-contraction coupling and resting Ca2+ leak from the sarcoplasmic reticulum (SR). In this study, resting Ca2+ sparks were analyzed in rabbit and rat ventricular myocytes with laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo 3. During the first 20 s of rest after regular electrical stimulation, both the frequency of Ca2+ sparks and SR Ca2+ content gradually decreased in rabbit. When rabbit SR Ca2+ content was decreased by reduction of stimulation rate. the initial resting spark frequency was also decreased, even though resting [Ca2+]i was unchanged. The rest-dependent decrease in spark frequency in rabbit cells was prevented by inhibition of Na+/Ca2+ exchange (which also prevents SR Ca2+ depletion during rest). These results suggest that elevation of SR Ca2+ content can increase Ca2+ spark frequency. In contrast to rabbit cells, 20 s of rest produced a gradual increase in spark frequency in rat cells, although SR Ca2+ content was constant and Ca2+ influx was completely prevented. This indicates that there is a time-dependent increase in spark probability during rest that is independent of [Ca2+]i or SR Ca2+. This effect was also apparent in rabbit cells when SR Ca2+ depletion was prevented by blocking Na+/Ca2+ exchange. Stimulation of Ca2+ extrusion via Na+/Ca2+ exchange in the rat (by Ca2+-free superfusion, which slowly depletes SR Ca2+ content) converted the normal rest-dependent increase in spark frequency to a decrease. The amplitude of individual Ca2+ sparks increased with increasing SR Ca2+ content. In the Ca2+-overloaded state, fusion of sparks or long-lasting localized increases of [Ca2+]i were observed with increased spark frequency. We conclude that the resting frequency of Ca2+ sparks can be independently affected by changes in SR Ca2+ content, [Ca2+]i, or rest period. The latter may reflect recovery of the SR Ca2+ release channels from inactivation or adaptation.

Animals↗
PubMed1997

Downregulation of sarcoplasmic reticulum Ca(2+)-ATPase during progression of left ventricular hypertrophy.

To determine whether reduced sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) (SERCA2) activity contributes to delayed myocardial relaxation during chronic left ventricular hypertrophy (LVH) progression, LVH was produced in rats by abdominal aortic coarctation. Systolic and diastolic functions were assessed in vivo 8 and 16 wk after surgery, and compositional alterations in LV myocardium [SERCA2 concentration, myosin heavy chain (MHC) isoenzymes, and tissue collagen] were correlated with the development of prolonged isovolumic relaxation and impaired cardiac performance over time. Myocardial relaxation was prolonged in 8-wk banded rats, despite normal isovolumic systolic function and LV end-diastolic pressure (LVEDP). No significant alterations in SERCA2 protein, beta-MHC, or fibrillar collagen levels were observed at this early time point. In contrast, LV SERCA2, beta-MHC, and fibrillar collagen concentrations were all significantly altered in 16-wk banded rats. These late compositional changes were associated with reduced cardiac performance, as manifested by a significant elevation in LVEDP (14 +/- 2 mmHg). The 34% decrease in SERCA2 protein was associated with reduced SR Ca2+ uptake and an even greater reduction (76%) in SERCA2 mRNA. SERCA2 mRNA levels were also significantly reduced to 43 +/- 10% of sham-operated rats 8 wk after banding, despite unchanged SERCA2 protein levels and normal SR Ca2+ uptake. These results argue against a significant contribution of SERCA2 downregulation to the subtle alterations in myocardial relaxation observed in compensated LVH. However, the early reduction in SERCA2 mRNA levels may serve as a molecular marker for impaired cardiac performance during the transition from compensated LVH to heart failure.

Animals↗
PubMed1997

Cardiac myocyte volume, Ca2+ fluxes, and sarcoplasmic reticulum loading in pressure-overload hypertrophy.

Alterations in cellular Ca2+ transport and excitation-contraction coupling may contribute to dysfunction in cardiac hypertrophy. Left ventricular myocytes were isolated from rat hearts after 15-18 wk of suprarenal abdominal aortic banding to evaluate the hypothesis that hypertrophy alters the relationship between Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ load during steady-state voltage-clamp depolarization. Mean arterial pressure (MAP) and heart weight-to-body weight ratio of banded (B) animals were significantly higher than in control or sham-operated animals (C). Isolated myocyte dimensions and volume increased in parallel with whole heart hypertrophy and elevation in MAP. However, the relationship between membrane surface area (measured by capacitance) and cell volume (measured by laser scanning confocal microscopy) was unaltered (C: 8.9 +/- 0.3; B: 8.5 +/- 0.4 pF/pl). No differences in the voltage dependence of ICa activation, steady-state inactivation, or recovery from inactivation were detected between C and B myocytes. Maximal ICa density for the two groups was also not different either under basal conditions (C: 4.28 +/- 0.98; B: 4.57 +/- 0.60 pA/pF) or in the presence of 1 microM isoproterenol (C: 16.6 +/- 2.3; B: 16.5 +/- 2.3 pA/pF). The fraction of Ca2+ released from the SR by a single twitch was 55.4 +/- 9.4% in C and 37.1 +/- 6.9% in B (not significantly different). Steady-state Ca2+ influx during a twitch was calculated in units of micromoles per liter of nonmitochondrial volume from the integral of ICa (C: 13.4 +/- 0.7 microM; B: 13.3 +/- 0.8 microM). The SR Ca2+ load was similarly calculated by integration of Na+/Ca2+ exchange current induced by rapid caffeine application (C: 140 +/- 9 microM; B: 169 +/- 18 microM). We conclude that significant cellular hypertrophy is associated with proportional increases in sarcolemmal ICa influx, SR Ca2+ loading, and the amount of SR Ca2+ released in this model of pressure overload.

Animals↗
PubMed1997

Diastolic SR Ca efflux in atrial pacemaker cells and Ca-overloaded myocytes.

Evidence has shown that the sarcoplasmic reticulum (SR) of cardiac cells releases Ca not only during excitation-contraction coupling but also during diastole, albeit at a much lower rate. This diastolic SR Ca release (leak) has also been implicated in the generation of spontaneous depolarization in latent atrial pacemaker cells of the cat right atrium. In the present work, we sought to measure Ca transients in pacemaker and nonpacemaker cells of the cat using the fluorescent Ca indicator indo 1. Atrial latent pacemaker cells develop a slow Ca transient when rested in the presence of both Na- and Ca-free solution and thapsigargin [used to inhibit Na/Ca exchange and SR Ca adenosinetriphosphatase (Ca-ATPase), respectively]. This increase in cytosolic Ca concentration ([Ca]i) is probably caused by the rate of SR Ca leak exceeding the capacity of the remaining Ca transport systems (e.g., sarcolemmal Ca-ATPase and mitochondrial Ca uptake). However, neither cat sinoatrial (SA) node cells nor myocytes from cat atrium or ventricle exhibited a similar increase in [Ca]i during the same protocol. This indicates that SR Ca leak in these cells occurred at a rate low enough to be within the capacity of the slow Ca transporters, as observed previously in rabbit ventricular myocytes. When atrial and ventricular myocytes were stimulated at higher frequencies, sufficient to markedly increase diastolic and systolic [Ca]i and approach Ca overload (and spontaneous activity), they responded to inhibition of SR Ca-ATPase and Na/Ca exchange with a slow Ca transient similar to that normally observed in atrial latent pacemaker cells. Furthermore, the SR Ca depletion by thapsigargin did not affect spontaneous activity of SA node cells, but it prevented or slowed pacemaker activity in the atrial latent pacemaker cells. These findings suggest that enhanced diastolic SR Ca efflux contributes significantly to the generation of spontaneous activity in atrial subsidiary pacemakers under normal conditions and in Ca-overloaded myocytes but not in SA node cells.

Animals↗
PubMed1997

Intracellular Ca2+ increases the mitochondrial NADH concentration during elevated work in intact cardiac muscle.

It is not clear how mitochondrial energy production is regulated in intact tissue when energy consumption suddenly changes. Whereas mitochondrial [NADH] ([NADH]m) may regulate cellular respiration rate and energetic state, it is not clear how [NADH]m itself is controlled during increased work in vivo. We have varied work and [Ca2+] in intact cardiac muscle while assessing [NADH]m using fluorescence spectroscopy. When increased work was accompanied by increasing average [Ca2+]c (by increasing [Ca2+]c or pacing frequency), [NADH]m initially fell and subsequently recovered to a new steady state level. Upon reduction of work, [NADH]m overshot and then returned to control levels. In contrast, when work was increased without increasing average [Ca2+]o (by increasing sarcomere length), [NADH]m fell similarly, but no recovery or overshoot was observed. This Ca(2+)-dependent recovery and overshoot may be attributed to Ca(2+)-dependent stimulation of mitochondrial dehydrogenases. We conclude that the immediate initial increase in respiration rate upon elevation of work is not activated by increased [NADH]m (since [NADH]m rapidly fell) or by [Ca2+]o (since work could also be increased at constant [Ca2+]c). However, during sustained high work, a Ca(2+)-dependent mechanism causes slow recovery of [NADH]m toward control values. This demonstrates a Ca(2+)-dependent feed-forward control mechanism of cellular energetics in cardiac muscle during increased work.

Animals↗