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(Circulation Research. 2005;97:457.)
© 2005 American Heart Association, Inc.

Cellular Biology

Paradoxical Cellular Ca²⁺ Signaling in Severe but Compensated Canine Left Ventricular Hypertrophy

Long-Sheng Song, YeQing Pi, Song-Jung Kim, Atsuko Yatani, Silvia Guatimosim, Raymond K. Kudej, Qingxiu Zhang, Heping Cheng, Luc Hittinger, Bijan Ghaleh, Dorothy E. Vatner, W. Jonathan Lederer, Stephen F. Vatner

From 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; and the Laboratory of Cardiovascular Science (H.C.), National Institute on Aging, National Institutes of Health, Baltimore, Md.

Correspondence to Dr Stephen F. Vatner, Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 S Orange Ave, MSB G609, Newark, NJ 07103. E-mail vatnersf{at}umdnj.edu; or Dr W. Jonathan Lederer, Medical Biotechnology Center, University of Maryland Biotech Institute, 725 W Lombard St, Baltimore, MD 21201. E-mail: lederer@umbi.umd.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (I_Ca) density but the ability of I_Ca 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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;^2–6 whereas, other studies in isolated myocytes or tissues have demonstrated normal or even enhanced function and Ca²⁺ handling.^7–9 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.^3,4,6–9 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 HF^4,9,13,14 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (I_Ca) 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 µmol/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 µmol/L) was added to block Na⁺ currents. Cells were prestimulated with a train of 8 pulses from –80 to 0 mV for 200 ms at 1.0 Hz. I_Ca was elicited by 10 steps of depolarization ranging from –40 to 60 mV for 300 ms from a holding potential of –45 mV. To simultaneously measure I_Ca-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 I_Ca 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 µmol/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 –80 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

View this table: [in this window] [in a new window]   Table 1. Hemodynamics in Conscious Control and LVH Dogs

View larger version (15K): [in this window] [in a new window]   Figure 1. Blunted response of LV wall thickening and myocyte shortening to Ca2+ stress in LVH model. A, Changes in LV wall thickening in control and LVH dogs in response to intracoronary infusion of CaCl2 (control n=7, LVH n=12). At baseline, there was no difference in LV wall thickening between groups (see Table 1). However, responses to Ca2+ were depressed in LVH (*P<0.01 vs control). Because the agent was infused intracoronary, heart rate and mean arterial pressure remained unchanged in both groups of animals. B, Increases in myocyte shortening in response to Ca2+ (control n=6, LVH n=8). Response to Ca2+ was evaluated in 50 cells of control and 60 of LVH. The response was decreased in LVH cells (*P<0.05 vs control).

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

View this table: [in this window] [in a new window]   Table 2. Single Myocyte Dynamics

I_Ca 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/F_0, Figure 2C) whereas I_Ca density (peak I_Ca normalized to cell capacitance) was not altered (Figure 2D) in LVH. Moreover, the ratios of peak Ca²⁺ transients to peak I_Ca 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.

View larger version (41K): [in this window] [in a new window]   Figure 2. Suppression of Ca2+ release triggered by Ca2+ influx through ICa in single myocytes under whole-cell voltage clamp mode. A and B, Representative recordings of confocal Ca2+ images and spatial average of Ca2+ transients at indicated voltages. The top panel shows voltage steps. Underneath are line-scan images and traces of averaged Ca2+ transients from control (A) and LVH (B) myocytes. C and D, Average of Ca2+ transients and ICa as a function of membrane potentials (*P<0.05; cells from 7 control n=35 and LVH n=20). The peak Ca2+ transients were significantly reduced in LVH in comparison with control, although ICa current density was not different between the 2 groups. E, The gain of E-C coupling, defined as the ratio of peak Ca2+ release to ICa, was decreased in LVH myocytes. The inset shows reduced CICR gain at even higher voltages. F, SR Ca2+ content, assessed with caffeine-induced Ca2+ release.

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 µm) 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 µm/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).

View larger version (47K): [in this window] [in a new window]   Figure 3. Characteristics of Ca2+ sparks and waves in control and LVH myocytes. Ca2+ sparks were acquired after steady-state field stimulation, in myocytes from 5 control dogs and 4 LVH dogs. A, Typical Ca2+ sparks recorded from control and LVH myocytes in line-scan mode in 1.8 mmol/L Ca2+. B and C, Comparison of Ca2+ spark properties: The LVH myocytes displayed a tendency with larger (spatial width, FWHM) and longer-lasting (duration, FDHM) sparks. (*P<0.01 vs control). D, Comparison of Ca2+ spark frequency between control and LVH myocytes exposed to 1.8 mmol/L and 10.0 mmol/L Ca2+. At both concentrations, Ca2+ sparks occur more frequently (*P<0.01 vs control) in LVH than in control myocytes. E, Ca2+ spark amplitude was greater at 10 mmol/L Ca2+ than those measured at 1.8 mmol/L Ca2+ in either control or LVH myocytes (*P<0.05 vs control). The amplitude was not different at 1.8 mmol/L between the 2 groups. F, Typical examples of Ca2+ waves recorded in 10 mmol/L Ca2+. Note: Arrows show the direction of wave propagation. The Ca2+ wave was started and then aborted after a short distance of propagation in LVH cell. Black dashed circles indicate almost distinguishable Ca2+ sparks in close junctions. G, The velocity of Ca2+ wave propagation was significantly slower in LVH compared with those from control myocytes (*P<0.05 vs control).

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).

View larger version (28K): [in this window] [in a new window]   Figure 4. Expression level of Ca2+ handling proteins in control and LVH heart tissues. Immunoblotting of SERCA2a (A), phospholamban (B), Na+/Ca2+ exchanger (NCX; C), and Calsequestrin (CSQ; D) from control and LVH dogs. The number of animals per experiment appears in each graph. SERCA2a expression was markedly reduced in LVH compared with control (*P<0.01 vs control). In contrast, there was no significant difference in the density of any of the other proteins between the 2 groups.

View larger version (20K): [in this window] [in a new window]   Figure 5. RyR2 density and phosphorylation state of RyR2 and PLB in canine hearts. RyR2 density (A) and PKA phosphorylation state (B) of RyR2 in LV homogenates. RyR2 density and back phosphorylation level of RyR2 were both reduced in LVH dogs (*P<0.01 vs control), indicating that RyR2 was hyperphosphorylated in LVH. C, PLB phosphorylation state was also significantly increased in LVH hearts (n=5) using a phosphorylation specific antibody (phospholamban-PS-16 antibody38) compared with controls (P<0.05, n=3).

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 failure^29,30 and studies of T-tubules (TTs) and Ca²⁺ release by other groups^31–34 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.

View larger version (29K): [in this window] [in a new window]   Figure 6. Model of spatial heterogeneity of SR Ca2+ load in LVH. A, Schematic diagram of a control ventricular myocyte with homogeneous SR Ca2+ loading. The SR Ca2+ load is represented as a filled circle, where the color corresponds to the load. Normal (blue), overload (red), and underload (gray) conditions are considered. Control myocytes produce Ca2+ sparks at a low rate, and a Ca2+ spark does not activate neighboring SR regions. Additionally, there are no Ca2+ waves initiated or conducted (right). B, Ca2+ overloaded myocytes. When many of the SR regions are "overloaded," the sensitivity of the release sites to cytosolic Ca2+ is much higher. Ca2+ sparks occur at a much higher rate, and a Ca2+ spark tends to trigger neighboring Ca2+ spark regions if they too are overloaded. Under these conditions, Ca2+ sparks tend to trigger Ca2+ waves (right). C, LVH and HF myocytes (see Figure 3F). The cell-wide average SR Ca2+ load is decreased. Additionally, there may be a high degree of heterogeneity of SR Ca2+ loading, where some regions are overloaded. Such regions have a higher than normal Ca2+ spark rate. In addition, Ca2+ sparks that occur will initiate Ca2+ wave propagation if a neighboring region is overloaded. However, these propagating waves of elevated Ca2+ tend to be aborted by regions of underload. These regions of underload act like firebreaks to the propagation of a brush fire and they stop the conducted Ca2+ wave (right).

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).

	Footnotes

 
Original received November 24, 2004; revision received March 1, 2005;resubmission received June 15, 2005; revised resubmission received July 20, 2005; accepted July 20, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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