点击显示 收起
The Department of Cell Biology and Molecular Medicine and the Cardiovascular Research Institute (Y.Q.P., S.-J.K., A.Y., R.K.K., Q.Z., L.H., B.G., D.E.V., S.F.V.), University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark
the Medical Biotechnology Center (L.-S.S., S.G., W.J.L.) University of Maryland Biotechnology Institute, Baltimore
the Department of Physiology and Biophysics (S.G.), Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
the Laboratory of Cardiovascular Science (H.C.), National Institute on Aging, National Institutes of Health, Baltimore, Md.
Abstract
In conscious dogs with severe left ventricular (LV) hypertrophy (H) (doubling of LV/body weight), which developed gradually over 1 to 2 years after aortic banding, baseline LV function was well compensated. The LV was able to generate twice the LV systolic pressure without an increase in LV end-diastolic pressure, or decrease in LV dP/dt or LV wall thickening. However, LV myocytes isolated from LVH dogs exhibited impaired contraction at baseline and in response to Ca2+. There was no change in L-type Ca2+ channel current (ICa) density but the ability of ICa to trigger Ca2+ release from the sarcoplasmic reticulum (SR) was reduced. Immunoblot analysis revealed a 68% decrease in SERCA2a, and a 35% decrease in the number of ryanodine receptors (RyR2), with no changes in protein level of calsequestrin, Na+/Ca2+ exchanger or phospholamban (PLB), but with both RyR2 and PLB hyperphosphorylated. Spontaneous Ca2+ sparks in LVH cells were found to have prolonged duration but similar intensities despite the reduced SR Ca2+ load. A higher Ca2+ spark rate was observed in LVH cells, but this is inconsistent with the reduced SR Ca2+ content. However, Ca2+ waves were found to be less frequent, slower and were more likely to be aborted in Ca2+-challenged LVH cells. These paradoxical observations could be accounted for by a nonuniform SR Ca2+ distribution, RyR2 hyperphosphorylation in the presence of decreased global SR Ca2+ load. We conclude that severe LVH with compensation masks cellular and subcellular Ca2+ defects that remain likely contributors to the limited contractile reserve of LVH.
Key Words: hypertrophy Ca2+ sparks E-C coupling myocyte contractility Ca2+ handling
Introduction
The extent to which left ventricular (LV) hypertrophy (H) is adaptive or maladaptive remains controversial.1 It is generally thought that during the stage of compensated LVH, cardiac function is maintained, but then decompensates with progression to heart failure (HF), resulting in a clear demarcation between compensated LVH and HF. However, several in vitro studies indicate that even in the stage of compensated LVH, isolated myocyte function and Ca2+ handling may be depressed;2eC6 whereas, other studies in isolated myocytes or tissues have demonstrated normal or even enhanced function and Ca2+ handling.7eC9 It is conceivable that, in part, the discrepancies in the literature can be ascribed to different experimental conditions, including species and model used. Indeed the great majority of prior studies on this topic have been conducted in rodent models.3,4,6eC9 With this in mind the current investigation was conducted in a large mammalian model of severe LVH, which mirrors the cardiac disease in human.10 This canine model is prepared by aortic banding in puppies, which induces a gradual development of LV/aortic pressure gradient and consequent LVH with growth and aging. The experiments are conducted in the fully-grown dogs 1 to 2 years later, at a time when LVH is severe, but still well compensated.10
The goal of this study is to improve our understanding of the cellular and molecular mechanisms underlying the functional consequences during the transition from cardiac hypertrophy to heart failure. To achieve this, we examined LV function at baseline and in response to stress in the same animals both in vivo, in chronically instrumented conscious dogs, and in vitro, using isolated myocytes. In the cellular studies, we examined excitation-contraction (E-C) coupling and sarcoplasmic reticulum (SR) function by using conventional patch-clamp techniques and confocal microscopy.11,12 In addition, we determined protein levels of SERCA2a, phospholamban (PLB), ryanodine receptor (RyR2) density and the phosphorylation state of RyR2 and PLB. All of these components have been examined in different models of HF4,9,13,14 but remain controversial in LVH in the absence of HF. Accordingly, the overall goal of the current investigation is to integrate these different methodologies into one study, based on the chronic large mammalian model of severe LVH, and broaden our molecular understanding of LVH.
Materials and Methods
Development of LVH Model, Instrumentation, and In Vivo Measurements
Forty-nine healthy canines (Marshall Farms; North Rose, NY) of either sex (8 to 10 weeks of age) were used in this study, 26 controls and 23 with LVH. LVH was created by placing a 1-cm wide polytetrafluoroethylene (Teflon) cuff around the ascending aorta above the coronary arteries. At 15 months after aortic banding, the animals were chronically instrumented to measure LV function as previously described,10 and noted further in the online data supplement available at http://circres.ahajournals.org.
Myocyte Isolation and Mechanical Function
After the in vivo experiments, single myocytes were isolated from LV free wall enzymatically.15 All functional measurements were performed at 35°C. Myocyte contraction was measured using a video motion edge detector as described previously.15
Simultaneous Recording of Ca2+ Currents (ICa) and Confocal Imaging of Ca2+ Release
Whole-cell currents were recorded under conditions that eliminated Na+ and K+ currents. Pipettes (1.2 to 2 M) were filled with (mmol/L) 10 NaCl, 120 CsCl, 20 TEA-Cl, 5 Mg-ATP, 10 HEPES (pH 7.2). Ca2+ indicator fluo-4 pentapotassium salt (100 eol/L) was also included in the pipette solution. Myocytes were perfused with external solution containing (mmol/L) 110 NaCl, 20 TEA-Cl, 5 CsCl, 0.5 MgCl2, 0.33 NaH2PO4, 10 glucose, 10 HEPES, 1.8 CaCl2, 5 4-aminopyridine, 0.03 niflumic acid (pH 7.4 adjusted with TEA-OH). Niflumic acid was used to block calcium-activated chloride currents. Tetrodotoxin (10 eol/L) was added to block Na+ currents. Cells were prestimulated with a train of 8 pulses from eC80 to 0 mV for 200 ms at 1.0 Hz. ICa was elicited by 10 steps of depolarization ranging from eC40 to 60 mV for 300 ms from a holding potential of eC45 mV. To simultaneously measure ICa-induced Ca2+ release from the SR, fluo-4 within myocytes was excited at 488 nm and the fluorescence was detected at 510 nm with laser scanning confocal microscope (LSM 510; Carl Zeiss International). Cell capacitances and ICa densities were calculated with Clampex 9.0 (Axon Instruments). Global Ca2+ release was analyzed by routines compiled with IDL 6.0 (Research System).4,16
Detection and Analysis of Ca2+ Sparks and Ca2+ Waves
Myocytes were incubated with fluo-4 AM (10 eol/L) and after a 20 minute period were superfused with an extracellular solution that contained 1.8 mmol/L Ca2+. Cells were field-stimulated at 1.0 Hz to produce steady-state conditions. Within the first 10 seconds after the last depolarization of a 15-pulse train, spontaneous nonpropagating Ca2+ releases (sparks) were recorded for the next 3 to 4 image frames, at high resolution (800 lines per frame, 1.92 ms per line) and identified as "diastolic Ca2+ sparks". The entire recording sequence was repeated 2 more times in each cell. The line-scan images were analyzed and Ca2+ sparks detected offline with a computer-based detection algorithm.12,16 Ca2+ waves were recorded under the same conditions as used for recording Ca2+ sparks except the Ca2+ challenge of 10 mmol/L extracellular Ca2+ was used.
Measurement of SR Ca2+ Load
SR Ca2+ content was assessed by a caffeine pulse protocol. In brief, 8 200-ms voltage-clamp steps from eC80 to 0 mV at 1 Hz were applied to achieve steady-state SR Ca2+ loading. After the prepulses, 10 mmol/L caffeine dissolved in a Na+- and Ca2+-free solution with 1 mmol/L EGTA was rapidly applied to the cell.
Western Blot Analysis
The antibodies used: a polyclonal antibody for SERCA2a (gift from Dr Frank Wuytack, Leuven, Belgium); a PLB monoclonal Ab (Affinity BioReagents; Golden, Colo), PLB PS-16 antibody (Fluorescience; Leeds, England); monoclonal anti-NCX (Research Diagnostics; Flanders, NJ); and Calsequestrin (CSQ; Research Diagnostics; Flanders, NJ). The intensity of target bands was evaluated by densitometric scanning using a Personal Densitometer SI with Image-Quant software (Amersham Biosciences) and normalized with loaded proteins.
Back-Phosphorylation
Tissue homogenates incubated with anti-RyR2 antibody (Affinity BioReagents, Golden, Co) overnight at 4°C. Back-phosphorylation was initiated with PKA (5 U) and Mg-ATP (containing 10% -32P ATP). After 8 minutes incubation, the reaction was stopped by adding 4x sample buffer. The samples were heated for 10 minutes at 37°C, and loaded on 6% SDS-PAGE.
Statistical Analysis
Statistical analysis was performed using Staview 5.0 statistical software (SAS Institute). The data are reported as mean±SEM. Multiple comparisons between groups were performed by 2-way ANOVA for repeated measures followed, if necessary, by a Student t test. One comparison between groups was analyzed using Student t test. Significance was recorded for P<0.05.
Results
LV Function
The extent of LVH and levels of baseline LV function in control and LVH dogs are shown in Table 1. The hemodynamics and extent of LVH were similar in other animals used for other aspects of this investigation. Although LV systolic pressure and LV/body weight ratio were doubled (P<0.001) in LVH, heart rate, mean arterial pressure, LVEDP, LV dP/dt, and LV wall thickening were similar between the 2 groups (Table 1). These results indicate that in this model, chronic aortic banding produced severe, but compensated LVH. Under normal physiological conditions, there is an increase in contraction as extracellular Ca2+ is acutely elevated. However, increases in LV wall thickening to intracoronary administration of Ca2+ (CaCl2 0.2 to 0.4 mg/kg/min) was blunted significantly in LVH dogs compared with controls (Figure 1A). These data suggest that baseline LV function is compensated in LVH but LV contractile reserve is reduced, because responses to stress are impaired.
Myocyte Contractile Dynamics in Single Cells
Myocyte shortening was decreased significantly and contraction and relaxation velocities were slower in LVH compared with control cells (Table 2). To confirm the stress response (by injecting Ca2+ through intracoronary in conscious animals), we further examined the contractile response to increased extracellular Ca2+ (3 and 4 mmol/L) in single myocytes from both groups (Figure 1B). Consistent with in vivo observations, LVH myocyte contractile response was significant but blunted compared with control cells; this suggests an impairment of Ca2+ handling at the cellular level in LVH myocytes, which is consistent with previous findings observed in a feline model.2
ICa Density and E-C Coupling
Cell capacitance in LVH myocytes was dramatically increased (control, 185.0±6.6 pF, n=34, LVH, 323.9±15.6 pF, n=19, P<0.001), indicating cellular hypertrophy. Because the increase in extracellular Ca2+ ("Ca2+ challenge") produced a blunted (ie, smaller than control) contractile response, we investigated the [Ca2+]i transient in LVH cells using fluo-4 pentapotassium salt and confocal microscopy (Figure 2). A clear reduction of the [Ca2+]i transient was observed at all membrane potentials in LVH cells. Figure 2A and 2B show examples of confocal line-scan images and spatial average of Ca2+ transients measured in control and LVH myocytes under voltage-clamp mode. LVH myocytes had smaller Ca2+ transients compared with control (shown as F/F0, Figure 2C) whereas ICa density (peak ICa normalized to cell capacitance) was not altered (Figure 2D) in LVH. Moreover, the ratios of peak Ca2+ transients to peak ICa densities (E-C coupling gain) were reduced in LVH compared with control myocytes (Figure 2E), suggesting that E-C coupling efficacy is reduced in LVH. Because of the important contribution of the SR Ca2+ content to E-C coupling process, the SR Ca2+ load was measured under steady-state conditions using rapid caffeine application. Maximal caffeine-induced Ca2+ transient (F/F0) was significantly reduced in LVH (by 16%) compared with control (Figure 2F), indicating that SR Ca2+ load was reduced in LVH. This may contribute to the decreased E-C coupling efficiency in LVH dogs.
Ca2+ Sparks and Ca2+ Waves
To further understand how Ca2+ signaling in LVH myocytes may change, the properties of Ca2+ sparks, the elementary SR Ca2+ release events, were examined (Figure 3A). We observed 829 diastolic Ca2+ sparks in 58 cells from 5 control dogs and 1421 diastolic Ca2+ sparks in 40 myocytes from 4 LVH dogs. The incidence of big (FWHM >2.0 e) and long-lasting (FDHM >40 ms) Ca2+ sparks was significantly higher in LVH than in control (Figure 3B and 3C). On average, the Ca2+ sparks occur at a higher frequency (LVH, 3.40±0.27; control, 1.5±0.15 events/100 e/s, P<0.01, Figure 3D) and have longer duration (LVH, 40.0±0.77, control, 32.1±0.69, P<0.01) in LVH cells, in spite of having similar Ca2+ spark amplitudes (LVH, 1.47±0.01; control, 1.48±0.01, Figure 3E). Overall, the distribution of Ca2+ sparks shifted to a larger and longer-lasting population in LVH myocytes. Experiments also showed that Ca2+ sparks observed between field-stimulated Ca2+ transients were similar to the diastolic Ca2+ sparks seen in LVH myocytes (data not shown). These results suggest that the characteristics of spontaneous Ca2+ release were significantly altered in LVH myocytes during diastole. Given the reduction of global SR Ca2+ content measured above (Figure 2F), these data suggest that there might be subcellular Ca2+ overload (see Discussion).
Exposure of normal myocytes to higher extracellular Ca2+ (ie, 10 mmol/L) should result in a SR Ca2+ overloaded state17 and induce cell-wide propagation of Ca2+ waves, which depends on normal SR function. Changes of Ca2+ spark properties are likely to reflect some important change in SR function including SERCA2a or RyR2 behavior. The effect of a steady state increase in extracellular Ca2+ (10 mmol/L) was examined within 10 seconds of the stopping of prolonged (steady-state) stimulation. We observed 675 Ca2+ sparks from 38 controls and 2268 sparks from 38 LVH myocytes. The Ca2+ spark frequency was increased by 43±5.5% and the Ca2+ spark amplitude was increased by 20±1.5% in LVH myocytes (Figure 3D and 3E). In contrast, both measures increased in control myocytes to a much smaller extent; Ca2+ spark frequency increased by 25±4.3% whereas Ca2+ spark amplitude increased by 12±1% (P<0.05; Figure 3D and 3E).
Despite the significant increase in number of Ca2+ sparks in LVH myocytes when extracellular Ca2+ was elevated (10 mmol/L), we observed many fewer propagating Ca2+ waves in the LVH cells (0 to 0.2 events/s; n=38) than in controls (1 to 1.5 events/s; n=38; P<0.01). Moreover, Ca2+ waves in LVH cells propagated at a slower rate (59±3.9 mm/s versus 78±4.0 mm/s in controls; P<0.05) than in control cells and tended to terminate the propagation spontaneously (Figure 3F and 3G). Given the observed increase in Ca2+ spark rate, these data suggest that there is spatial nonuniformity in the signaling mechanism that gives rise to both Ca2+ sparks and Ca2+ waves (see below and Discussion). We were concerned, however, that the SR Ca2+ transport proteins had somehow changed.
Ca2+ Regulatory Protein Expression
Quantitative immunoblotting showed that SERCA2a was reduced by 68% in LVH (Figure 4A) compared with control hearts but there was only a 16% reduction in SR Ca2+ content (Figure 2F). The full effect of the reduction of SERCA2a was mitigated by a constant amount of PLB (Figure 4B) but with increased PLB phosphorylation (measured using a phosphorylation specific antibody, see Figure 5C). PKA-dependent PLB phosphorylation was increased by 37% in LVH compared with controls. In addition, we did not detect any significant change in protein level of NCX and CSQ (Figure 4C and 4D).
Another protein that plays an important role for cellular Ca2+ handling is RyR2, the major SR Ca2+ release channel. Western blot analyses were performed to measure the abundance of RyR2 in LVH heart cells. The level of RyR2 phosphorylation was also measured by the back-phosphorylation method (Figure 5). The reduction of RyR2 expression of RyR2 (Figure 5A) will tend to decrease the Ca2+ efflux whereas the increased RyR2 phosphorylation level (Figure 5B) will tend to increase the efflux.18 The data on the SR Ca2+ regulatory proteins including RyR2 and SERCA2a are consistent with a decreased SR Ca2+ content but cannot readily account for the increased Ca2+ spark rate and the simultaneous decrease in Ca2+ wave propagation.
Discussion
We have investigated the nature of in vivo and cellular changes in Ca2+ handling in a dog model of LVH. An aortic band was applied in young animals so that aortic stenosis and the consequent LVH would develop gradually as the animal matured over 15 months. This leads to severe LVH (doubling of LV/body weight, normal systolic arterial pressure but LV systolic pressure is 228±7 mm Hg, and a gradient across the aortic valve of 121±10 mm Hg). By focusing on function at 2 levels in this study (in vivo and single cell), we identify and characterize one of the central "trade-offs" that develop late in LVH before frank heart failure is manifest. Contraction is maintained in vivo despite increased afterload but there is impaired Ca2+ response. At the single cell level, an important Ca2+ signaling paradox is identified. The SR is depleted of Ca2+, a feature consistent with the downregulation of SERCA2a,19,20 but there is an elevation of the diastolic Ca2+ spark rate, a finding that is normally attributable to elevated SR Ca2+ content. We discuss possible pathophysiological changes that may account for these observations.
Why Are Diastolic Ca2+ Sparks Increased in Frequency in LVH Cardiomyopathy
The observed increase in diastolic Ca2+ spark frequency in LVH is unexpected because of the measured decrease in cell-wide SR Ca2+ content. Others have clearly demonstrated that as SR Ca2+ content increases, Ca2+ spark frequency increases.17,21,22 The tendency of SR Ca2+ release increases as a power function of the SR Ca2+ content, (release loadn, where n >3).23,24 Mechanistically the increase in Ca2+ spark rate that is widely observed is thought to develop because as Ca2+ in the SR increases, the sensitivity of RyRs to be triggered by Ca2+ increases,21,25 a finding that is supported by Ca2+ spark modeling.26 However, we observe an increase in Ca2+ spark rate with a decrease in SR Ca2+ content. One hypothesis that may explain our findings is that the RyRs become more sensitive to [Ca2+]i for some other reason. In heart failure, several groups have reported that the RyR2 becomes more completely phosphorylated18 and such phosphorylation increases the sensitivity of the RyR2 in a complicated manner.18,27 In the present study, we observe such RyR2 hyperphosphorylation for the first time in late-stage LVH. Such an increased phosphorylation of RyR2 may contribute to an increase in Ca2+ spark rate. However, that alone may be inadequate to account for the changes that we see, because there is a degree of self-regulation produced under these conditions.19,23
Complex Ca2+ signaling may also arise from temporal-spatial heterogeneity of electrical behavior and E-C coupling. Sipido and coworkers have shown that, as transverse tubules are lost in ventricular myocytes in culture, dyssynchronous Ca2+ release develops.28 This, when combined with the development of dyssynchronous Ca2+ release in a model of heart failure29,30 and studies of T-tubules (TTs) and Ca2+ release by other groups31eC34 suggest that SR-TT changes may occur in LVH and HF, leading to Ca2+ signaling alterations. We hypothesize that such spatial heterogeneity may occur in the LVH dog ventricular myocytes. More specifically, we hypothesize that there are regions of "remnant" junctional SR (jSR) that are no longer controlled by nearby L-type Ca2+ channels. This would then lead to subcellular regions of the myocyte that were overloaded with Ca2+. These regions would behave like they were in a Ca2+ overloaded state and able to produce Ca2+ sparks at a high rate and support conducted waves of elevated [Ca2+]i. However, because these regions were in the minority, the overall SR Ca2+ content would be depressed. Furthermore the majority of the cell would act like "fire breaks" for the conducted Ca2+ waves; when Ca2+ waves were initiated, they would be stopped by the regions of low SR Ca2+ because they could not "jump" over them. This hypothesis can, in principle, account for all 3 of the observations. It would produce spatial heterogeneity of the RyR2 sensitivity to be triggered; in some regions, local SR Ca2+ content would be extremely high (eg, overloaded); whereas in other regions the SR Ca2+ content would be very low and produce few if any spontaneous Ca2+ sparks. On average, the SR Ca2+ content would be less than control if there were a preponderance of "under-loaded" regions. Combining the reports in the literature with our findings suggests the diagram in Figure 6.
Spatial heterogeneity can be tested directly using the methods described in the recent identification and characterization of "Ca2+ blinks."35 In these experiments, Brochet and colleagues measured local SR Ca2+ content (in individual jSR units) using the fluo-5N technique.35,36 Alternatively, release of Ca2+ from the jSR could be measured during the application of caffeine, using the "Ca2+ spike" method.37 After the application of caffeine, this technique should reveal the spatially resolved SR Ca2+ load. Each of these methods should work in principle but will be challenging because of the modest signal-to-noise ratios associated with each and the very high XY imaging that is required. New instrumentation has only recently been announced (Zeiss Live 5 confocal microscope) that has the specifications required to do these measurements.
The unusual changes in Ca2+ signaling seen in the LVH dog model that we have studied appear to be consistent with spatial remodeling at the level of the single ventricular myocytes similar to that observed by others. These findings are consistent with a combination of myocardial hypertrophy and single cell dysfunction. Testing the character of the Ca2+ distribution and signaling heterogeneity will require additional experiments.
Conclusions
We have found that a canine model of severe LVH caused by gradual afterload increase is adaptive, in vivo. The LVH permits a doubling of generated LV pressure and normal rate of LV pressure development without utilization of the Frank-Starling mechanism. One of the hidden costs of the compensated LVH is Ca2+ signaling dysfunction. Although hypertrophy appears to provide contractile compensation in vivo, the many other changes (eg, hyperphosphorylation of RyR2, and PLB, and downregulation of SERCA2a and RyR2) provide uncertain benefit. Interestingly, hyperphosphorylation of RyR2, previously described as a key mechanism in decompensated heart failure,18 was observed in the current model of well compensated hypertrophy. The paradox of elevated Ca2+ spark rate and aborted Ca2+ waves may be resolved by a putative heterogeneity of SR Ca2+ load. We conclude that severe LVH with compensation masks cellular and subcellular Ca2+ defects that remain likely contributors to the limited contractile reserve of LVH.
Acknowledgments
Supported by National Institutes of Health (NIH), National Heart, Lung, and Blood Institute, and the National Institutes of Health Aging Institute to S.F.V., D.E.V., S.J.K., A.Y., and W.J.L. (5R01HL62442, 5RO1HL65183, 5RO1HL65182, 1PO1HL69020, 2RO1AG14121, 2RO1HL33107, 2PO1HL59139, AG023137, HL65183, HL65182, and 2R01HL036974, 5R01HL025675, 5P01HL070709, 5P01HL067849) and H.C. (Intramural Research Program of NIH, National Institute on Aging).
References
Morisco C, Sadoshima J, Trimarco B, Arora R, Vatner DE, Vatner SF. Is treating cardiac hypertrophy salutary or detrimental: the two faces of Janus. Am J Physiol Heart Circ Physiol. 2003; 284: H1043eCH1047.
Bailey BA, Dipla K, Li S, Houser SR. Cellular basis of contractile derangements of hypertrophied feline ventricular myocytes. J Mol Cell Cardiol. 1997; 29: 1823eC1835.
Dorn GW II, Robbins J, Ball N, Walsh RA. Myosin heavy chain regulation and myocyte contractile depression after LV hypertrophy in aortic-banded mice. Am J Physiol. 1994; 267: H400eCH405.
Gomez AM, Valdivia HH, Cheng H, Lederer MR, Santana LF, Cannell MB, McCune SA, Altschuld RA, Lederer WJ. Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science. 1997; 276: 800eC806.
Gwathmey JK, Morgan JP. Altered calcium handling in experimental pressure-overload hypertrophy in the ferret. Circ Res. 1985; 57: 836eC843.
Siri FM, Krueger J, Nordin C, Ming Z, Aronson RS. Depressed intracellular calcium transients and contraction in myocytes from hypertrophied and failing guinea pig hearts. Am J Physiol. 1991; 261: H514eCH530.
Bing OH, Brooks WW, Conrad CH, Sen S, Perreault CL, Morgan JP. Intracellular calcium transients in myocardium from spontaneously hypertensive rats during the transition to heart failure. Circ Res. 1991; 68: 1390eC1400.
Brooksby P, Levi AJ, Jones JV. Investigation of the mechanisms underlying the increased contraction of hypertrophied ventricular myocytes isolated from the spontaneously hypertensive rat. Cardiovasc Res. 1993; 27: 1268eC1277.
Shorofsky SR, Aggarwal R, Corretti M, Baffa JM, Strum JM, Al-Seikhan BA, Kobayashi YM, Jones LR, Wier WG, Balke CW. Cellular mechanisms of altered contractility in the hypertrophied heart: big hearts, big sparks. Circ Res. 1999; 84: 424eC434.
Hittinger L, Shannon RP, Kohin S, Manders WT, Kelly P, Vatner SF. Exercise-induced subendocardial dysfunction in dogs with left ventricular hypertrophy. Circ Res. 1990; 66: 329eC343.
Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740eC744.
Cheng H, Song LS, Shirokova N, Gonzalez A, Lakatta EG, Rios E, Stern MD. Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method. Biophys J. 1999; 76: 606eC617.
Guatimosim S, Dilly K, Santana LF, Saleet Jafri M, Sobie EA, Lederer WJ. Local Ca(2+) signaling and EC coupling in heart: Ca(2+) sparks and the regulation of the [Ca(2+)](i) transient. J Mol Cell Cardiol. 2002; 34: 941eC950.
Lindner M, Bohle T, Beuckelmann DJ. Ca2+-handling in heart failureeCa review focusing on Ca2+ sparks. Basic Res Cardiol. 2002; 97 {suppl 1): I79eC82.
Kim SJ, Kudej RK, Yatani A, Kim YK, Takagi G, Honda R, Colantonio DA, Van Eyk JE, Vatner DE, Rasmusson RL, Vatner SF. A novel mechanism for myocardial stunning involving impaired Ca(2+) handling. Circ Res. 2001; 89: 831eC837.
Santana LF, Cheng H, Gomez AM, Cannell MB, Lederer WJ. Relation between the sarcolemmal Ca2+ current and Ca2+ sparks and local control theories for cardiac excitation-contraction coupling. Circ Res. 1996; 78: 166eC171.
Cheng H, Lederer MR, Lederer WJ, Cannell MB. Calcium sparks and [Ca2+]i waves in cardiac myocytes. Am J Physiol. 1996; 270: C148eC159.
Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365eC376.
Bers DM, Eisner DA, Valdivia HH. Sarcoplasmic reticulum Ca2+ and heart failure: roles of diastolic leak and Ca2+ transport. Circ Res. 2003; 93: 487eC490.
Hobai IA, O’Rourke B. Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure. Circulation. 2001; 103: 1577eC1584.
Lukyanenko V, Gyorke I, Gyorke S. Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes. Pflugers Arch. 1996; 432: 1047eC1054.
Satoh H, Blatter LA, Bers DM. Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes. Am J Physiol. 1997; 272: H657eC668.
Trafford AW, Diaz ME, Eisner DA. Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current. Circ Res. 2001; 88: 195eC201.
Diaz ME, O’Neill SC, Eisner DA. Sarcoplasmic reticulum calcium content fluctuation is the key to cardiac alternans. Circ Res. 2004; 94: 650eC656.
Gyorke I, Gyorke S. Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites. Biophys J. 1998; 75: 2801eC2810.
Sobie EA, Dilly KW, dos Santos Cruz J, Lederer WJ, Jafri MS. Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release. Biophys J. 2002; 83: 59eC78.
Valdivia HH, Kaplan JH, Ellis-Davies GC, Lederer WJ. Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation. Science. 1995; 267: 1997eC2000.
Louch WE, Bito V, Heinzel FR, Macianskiene R, Vanhaecke J, Flameng W, Mubagwa K, Sipido KR. Reduced synchrony of Ca2+ release with loss of T-tubules-a comparison to Ca2+ release in human failing cardiomyocytes. Cardiovasc Res. 2004; 62: 63eC73.
Litwin SE, Zhang D, Bridge JH. Dyssynchronous Ca(2+) sparks in myocytes from infarcted hearts. Circ Res. 2000; 87: 1040eC1047.
Sipido KR. Local Ca(2+) release in heart failure: timing is important. Circ Res. 2000; 87: 966eC968.
Balijepalli RC, Lokuta AJ, Maertz NA, Buck JM, Haworth RA, Valdivia HH, Kamp TJ. Depletion of T-tubules and specific subcellular changes in sarcolemmal proteins in tachycardia-induced heart failure. Cardiovasc Res. 2003; 59: 67eC77.
He J, Conklin MW, Foell JD, Wolff MR, Haworth RA, Coronado R, Kamp TJ. Reduction in density of transverse tubules and L-type Ca(2+) channels in canine tachycardia-induced heart failure. Cardiovasc Res. 2001; 49: 298eC307.
Heinzel FR, Bito V, Volders PG, Antoons G, Mubagwa K, Sipido KR. Spatial and temporal inhomogeneities during Ca2+ release from the sarcoplasmic reticulum in pig ventricular myocytes. Circ Res. 2002; 91: 1023eC1030.
Lipp P, Huser J, Pott L, Niggli E. Spatially non-uniform Ca2+ signals induced by the reduction of transverse tubules in citrate-loaded guinea-pig ventricular myocytes in culture. J Physiol. 1996; 497: 589eC597.
Brochet DX, Yang D, Maio AD, Lederer WJ, Franzini-Armstrong C, Cheng H. Ca2+ blinks: Rapid nanoscopic store calcium signaling. Proc Natl Acad Sci U S A. 2005; 102: 3099eC3104.
Shannon TR, Guo T, Bers DM. Ca2+ scraps: local depletions of free [Ca2+] in cardiac sarcoplasmic reticulum during contractions leave substantial Ca2+ reserve. Circ Res. 2003; 93: 40eC45.
Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. Direct measurement of SR release flux by tracking "Ca2+ spikes" in rat cardiac myocytes. J Physiol. 1998; 512 (Pt 3): 677eC691.
Drago GA, Colyer J. Discrimination between two sites of phosphorylation on adjacent amino acids by phosphorylation site-specific antibodies to phospholamban. J Biol Chem. 1994; 269: 25073eC25077.