Cellular Mechanisms of Contractile Dysfunction in Hibernating Myocardium
Ischemic heart disease is a leading cause of chronic heart failure. Hibernation (ie, a chronic reduction of myocardial contractility distal to a severe coronary stenosis and reversible on revascularization) is an important contributing factor. The underlying cellular mechanisms remain however poorly understood. In young pigs (n=13, ISCH), an acquired coronary stenosis >90% (4 to 6 weeks) resulted in the development of hibernating myocardium. Single cardiac myocytes from the ISCH area were compared with cells from the same area obtained from matched normal pigs (n=12, CTRL). Myocytes from ISCH were larger than from CTRL. In field stimulation, unloaded cell shortening was reduced and slower in ISCH; relaxation was not significantly different. The amplitude of the [Ca2+]i transient was not significantly reduced, but reducing [Ca2+]o for CTRL cells could mimic the properties of ISCH, inducing a significant reduction of contraction, but not of [Ca2+]i. Action potentials were longer in ISCH. With square voltage-clamp pulses of equal duration in ISCH and CTRL, the amplitude of the [Ca2+]i transient was significantly smaller in ISCH, as was the Ca2+ current. Near-maximal activation of the myofilaments resulted in smaller contractions of ISCH than of CTRL cells. There was no evidence for increased degradation of Troponin I. In conclusion, cellular remodeling is a major factor in the contractile dysfunction of the hibernating myocardium. Myocytes are hypertrophied, action potentials are prolonged, and L-type Ca2+ currents and Ca2+ release are decreased. The steep [Ca2+]i dependence of contraction and possibly a reduction of maximal myofilament responsiveness further enhance the contractile deficit.
Cardiac failure is one of the leading causes of morbidity and mortality with death related to pump failure as well as to arrhythmias.1 Coronary artery disease due to atherosclerosis is the most common underlying etiology. In recent years, the mortality with acute myocardial infarction has decreased as efficient thrombolytic therapy and acute revascularization (angioplasty, stenting) have become available. However, preventing or reversing ventricular remodeling and the evolution toward end-stage heart failure presents a new challenge.2 Novel strategies are being explored, and one of the targets is the so-called “hibernating” myocardium.3 The term hibernation describes a condition where myocardial contractility is chronically reduced in the presence of a flow-limiting severe coronary stenosis (but without infarction), and which is reversible after revascularization.4 Hallmarks of hibernating myocardium are its partially preserved contractile reserve5,6 and its maintained metabolic activity, despite reduced perfusion.7
The reduced contraction of the hibernating myocardium is considered to be an adaptive response with contraction-perfusion matching,6,8 but the mechanisms underlying the reduced contractility are still unclear. Myocardial biopsies during revascularization surgery have shown variable numbers of cells that are dedifferentiated with loss of myofilaments and glycogen accumulation.7 Signs of degeneration, rather than adaptation have also been reported.9 Animal models of semiacute to chronic coronary stenosis recapitulate many of the characteristics of the hibernating human myocardium,10–15 and have been used to study mechanisms of contractile dysfunction. Loss of myocytes through apoptosis, and the presence of dedifferentiated cells, lead to a reduced number of functional myocytes, with hypertrophy of the remaining myocytes.14 In the model of Canty, Fallavollita, and coworkers,16 expression of SERCA and phospholamban were reduced. However, until now there have been no functional studies of myocytes isolated from hibernating myocardium. It is unknown whether the reduced contractility in vivo is the result of intrinsic remodeling or of in situ factors, and whether the reported reduction of Ca2+ handling proteins leads to altered Ca2+ handling. Recently, a pig model of severe nonocclusive coronary stenosis and contractile dysfunction with the hallmarks of hibernation has been developed in our institution.17 The closed-chest, nonsurgical approach, uncomplicated by pericardial opening, allows for superior echocardiographic analysis to identify ischemic but viable myocardium.18 After excision of the heart the coronary stenosis can easily be identified, and intact single myocytes can successfully be isolated from the area of interest. Functional studies of the isolated myocytes then allow distinguishing between in situ factors and intrinsic remodeling. We used this approach to identify the cellular mechanisms underlying the reduced contractility of the hibernating myocardium.
Materials and Methods
A copper stent that would induce intima proliferation was inserted in the proximal circumflex artery of young domestic pigs (27 to 32 kg; Seghers GenTech, Lebbeke, Belgium). This resulted in a stenosis >90% within 10 to 15 days.17 The procedure and protocols were approved by the local ethics committee. Animals were housed and treated according to the Guide for the Care and Use of Laboratory Animals (NIH). Severity of stenosis was evaluated by angiography. There was no evidence of collateral circulation development after the 6 weeks of follow-up. The contraction of the posterior wall was quantified by ultrasonic strain and strain rate analysis. This allowed identification of transmural or nontransmural infarction, or chronic ischemic myocardium.18 For the cellular studies, pigs with regional deformation characteristic of hibernation were selected (ISCH, n=13, 35 to 45 kg at the time of euthanasia). This was based on the presence of moderately decreased systolic thickening in the “at risk” segment (systolic strain 42±7% versus 58±5% in matched control animals, CTRL; P<0.01), which was associated with postsystolic thickening at rest (15±4% versus 2±2% in CTRL) and which showed a biphasic response during dobutamine challenge.19 None of the animals had signs of heart failure, and the LV end-diastolic pressure was not increased. However, the +dP/dt was reduced in ISCH (1638±80 versus 1952±83 mm Hg · s−1 in CTRL). The LV end-diastolic diameter was not significantly increased (38±6 mm in ISCH, versus 42±4 mm in CTRL) and wall thickness of the septum or of the posterior wall was not different between ISCH and CTRL either. In a separate group of animals with similar contractile characteristics, but not included in this study, PET studies had shown a reduction of baseline flow and preserved metabolic activity in the “at risk” segment. Histology confirmed the absence of myocardial infarction, although small subendocardial foci of patchy necrosis could sometimes be observed. The subendocardial and midmyocardial layers contained cells with increased glycogen content, as described for hibernating myocardium.7
Animals were euthanized 4 to 5 weeks after the stenosis had developed. The procedure for cell isolation was as described before.20 ISCH cells were obtained from the area distal to the stenosis. CTRL cells were isolated from the same area of matched normal pigs (n=12). Only cells from the midmyocardial layer were used, cells were stored at room temperature (23°C) and used within 14 hours.
All experiments were performed in normal Tyrode solution (in mmol/L, NaCl 137, KCl 5.4, MgCl2 0.5, CaCl2 1.8, Na-HEPES 11.8, and glucose 10; pH 7.40) at 37°C. The pipette solution for whole-cell patch clamp contained (in mmol/L, K-aspartate 120, NaCl 10, KCl 20, K-HEPES 10, MgATP 5, and K5fluo-3 0.05; pH 7.2). For measuring [Ca2+]i during field stimulation, cells were loaded with fluo-3-AM (5 μmol/L for 10 minutes).
Measurement of Unloaded Cell Shortening, Membrane Currents, and [Ca2+]i
Cells were placed in a perfusion chamber on the stage of an inverted microscope (Nikon Diaphot). Unloaded cell shortening was measured with a video edge-detector (Crescent Electronics, USA). The setup for fluorescence recording, procedures for calibration to [Ca2+]i, and recording of membrane currents were as described before.21
L-type Ca2+ current was measured as the nifedipine-sensitive current (20 μmol/L). The Ca2+ content of the sarcoplasmic reticulum (SR) was measured as the integrated inward Na+-Ca2+ exchange current during caffeine-induced Ca2+ release (10 mmol/L, fast application for 10 seconds), in the presence of 200 μmol/L DIDS to block Ca2+-activated Cl− currents.
Protein Expression Studies
Transmural needle biopsies from the ischemic area were taken in situ at the time of euthanasia. Control tissue was obtained from the same area in hearts of CTRL pigs. The tissue was immediately frozen at −80°C. Protein levels were determined in tissue homogenates by immunoblot analysis, essentially as described previously,22 using commercially available antibodies (TnI, Mab clone 8I-7, Spectral Diagnostics;23 SERCA2, Mab clone 2A7-A1, Affinity Bioreagents; NCX Mab clone C2C12, Affinity Bioreagents). The fluorescent signals were quantified on the Storm840 FluorImager with ImageQuant Software (Molecular Dynamics).
All data are shown as mean±SEM. Paired or unpaired Student t test, or 2-way ANOVA, were used as appropriate. A value of P<0.05 was considered significant.
Morphology of Isolated Myocytes
The yield of quiescent, regularly striated, viable myocytes was comparable for CTRL and ISCH pigs (40% to 60%), and there were no obvious differences in appearance of the myocytes in transmitted light (Figure 1A). There was a significant increase in mean cell length and width of ISCH (179±3 μm versus 139±2 μm in CTRL, and 27±0.6 μm versus 24±0.5 μm in CTRL; P<0.05); the distribution of cell size in ISCH was broader indicating a larger variability (Figure 1B).
Sarcomere length, measured by analysis of the striation pattern as in Figure 1A, was not different (1.92±0.01 μm for CTRL, versus 1.93±0.01 μm for ISCH).
Contraction and [Ca2+]i During Field Stimulation
Figure 2A shows a representative example of unloaded cell shortening in a ISCH and CTRL myocyte; pooled data are shown in Figure 2B. In ISCH cells, ΔL/L0 is significantly reduced, and the time to peak of the contraction is significantly increased. Consequently, maximum rate of contraction (−dL/dt) was significantly decreased in ISCH. Relaxation tended to be slower in ISCH. Adrenergic stimulation increased contraction to a larger extent in CTRL cells than in ISCH, increasing the contractile deficit of the ischemic cells (shortening in the presence of 300 nmol/L isoproterenol was 12±4% in CTRL, n=5, versus 6±1% in ISCH, n=7).
In a subset of cells, [Ca2+]i was measured simultaneously with shortening; there were no significant differences in amplitude nor time course of [Ca2+]i (Figure 3), despite significantly smaller contractions (Figure 4A). However, reducing extracellular [Ca2+] in CTRL cells revealed that the resultant, nonstatistically significant, reduction of [Ca2+]i transient amplitude led to significant changes in cell shortening, mimicking the properties of ISCH (Figure 4B). To some extent the lack of significance for the reduction in [Ca2+]i can be related to the properties of Fluo-3, which may not be sensitive enough in terms of Kd and signal-to-noise to report these small differences. The data obtained in different [Ca2+]o in CTRL cells (Figure 4B) illustrate that the [Ca2+]i dependence of contraction is very steep, with large changes in contraction for small changes in [Ca2+]i, small changes that we cannot detect with Fluo-3. As these observations strongly suggest that alterations in [Ca2+]i can explain the reduced contraction, we studied Ca2+ handling in more detail during whole-cell recording.
Cell Shortening and [Ca2+]i During Whole-Cell Recording
In current-clamp mode, after the cytosol had equilibrated with the pipette solution, shortening was still significantly less in ISCH. The amplitude of [Ca2+]i transients was not significantly different, but action potentials were longer (Figure 5A). Resting [Ca2+]i was comparable for ISCH and CTRL (129±12 nmol/L, n=24 cells from 5 hearts, versus 134±16 nmol/L, n=23 cells from 9 hearts).
To exclude the influence of altered action potentials, myocytes were studied in voltage-clamp mode with pulses of similar duration. The amplitude of [Ca2+]i transients tended to be smaller in ISCH (P=0.051, Figure 5C); the SR content was not significantly different (Figure 5D).
The voltage dependence of [Ca2+]i was similar in ISCH and CTRL; [Ca2+]i transients were significantly smaller in ISCH (Figure 6A, protocol as in Figure 5B but with a prepulse to −50 mV). The L-type Ca2+ current was decreased in ISCH (Figures 6B and 6C with intracellular [Ca2+]i buffering).
Protein levels of SERCA, Na+-Ca2+ exchanger, and phospholamban were not significantly different (Figure 7). Fitting of the decline of the caffeine-induced [Ca2+]i transients, which depends on Ca2+ removal by the Na+-Ca2+ exchanger, did not show differences between CTRL and ISCH (data not shown).
Myofilament Ca2+ Response and TnI
The data above suggest that the reduction in [Ca2+]i could explain the reduced shortening of ISCH, and predict that the contraction would normalize for a larger [Ca2+]i transient. We measured the amplitude of shortening during caffeine application. Despite the larger [Ca2+]i transients, shortening was still smaller in ISCH (Figure 8A).
We examined whether there was evidence for TnI degradation in tissue homogenates. The degraded fraction of the total TnI was very small, and not significantly different between ISCH and CTRL (Figure 8B).
In this pig model of myocardial hibernation, we found that ISCH myocytes had a reduced and slowed contraction. The global [Ca2+]i transient was reduced, in particular after eliminating the influence of the prolonged action potential of ISCH. Analysis of Ca2+ fluxes revealed that ICaL was reduced, but SR Ca2+ content was preserved. Expression of SERCA, Na+-Ca2+ exchange, and phospholamban were unchanged. The maximal myofilament response to Ca2+ in the intact cell was apparently reduced, but this was not associated with a significantly increased degradation of TnI.
Correlation of Cellular Findings With In Vivo Contractility
The reduced contraction of myocytes isolated from hibernating myocardium indicates that the reduced contraction in vivo is at least to a large extent due to intrinsic cellular remodeling. To our knowledge these are the first data on cellular function in a model of severe coronary stenosis of more than 1-week duration. They support the concept that downregulation of cellular function is an adaptive process that contributes to protection of the compromised myocardium.6 The reduced cellular contraction accounts for the reduced wall motion per se, but may also contribute to the specific contraction pattern observed in vivo, ie, the presence of postsystolic thickening.24
Characteristics of the Isolated Myocytes
One of the hallmarks of the human hibernating myocardium is the presence of glycogen-containing, dedifferentiated cells.9,25 Such changes have also been described in animal models (see review6). In the present pig model, such cells were clearly abundant in the histology studies (data not shown). However, in the freshly isolated myocytes used for functional studies, such cells cannot be identified as there are no live stains for glycogen available. In the light-transmitted image, there was no obvious reduction of the myofilaments, previously described during electron microscopy in human cells,7 and calculation of intracellular Ca2+ buffering did not reveal significant differences between CTRL and ISCH (data not shown). However, it is important to remember that reduction of myofilaments in human samples was observed only in a fraction of cells (on average 27%),26 and the reduction in a given cell could be as little as 10%. The similar yield of viable cells in the ISCH and CTRL hearts indicates that the cells we have studied are a representative sample.
Myocytes were significantly larger and this is consistent with previous reports.14 Similar signaling pathways as in mechanical overload and/or specific pathways related to ischemia/stunning could be involved.27 The loss of surrounding myocytes through apoptosis could indeed induce a local stretch. During episodes of increased demand, local ischemia could prevail, as supported by the reduced contraction at high doses of dobutamine. Although there were no signs of heart failure, the reduced +dP/dt indicates that overall cardiac function is compromised. Therefore, systemic factors could also be involved. However, we did not observe hypertrophy in the remote myocardium (measurements of septal thickness with echocardiography), which would plead against systemic activation of hypertrophic factors.
Evidence for Electrical Remodeling
We found a significant increase in action potential duration, although preliminary data do not show a significant increase in the overall QT, probably due to the relatively small mass of cells with APD prolongation and the posterior wall location. These data suggest that the presence of hibernating myocardium increases the dispersion of repolarization between the normal LV and the chronically ischemic area. This can provide an arrhythmogenic substrate, contributing to sudden cardiac death in patients with hibernating myocardium.28 The underlying alterations in ionic currents are presently under investigation.
Mechanisms Underlying the Reduced Cellular Contraction: Alterations in [Ca2+]i Handling
In our study, the decrease in the global [Ca2+]i transient during field stimulation was small. However, during voltage clamp, a clear and significant decrease of [Ca2+]i was observed; the Ca2+ current, ICaL, was reduced, whereas the SR Ca2+ content was not. The latter would minimize the effect of a reduction of ICaL on the global [Ca2+]i transient and could result from autoregulation of the SR content in the presence of a reduced trigger.29 These observations indicate that there are important changes in excitation-contraction coupling underlying the reduced contraction of cells from hibernating myocardium. Alterations in action potential duration and/or INa are partially compensatory mechanisms in the intact cells. The reduced Ca2+ release could by itself be partially responsible for the action potential prolongation by reducing the rate of inactivation of the L-type Ca2+ current.30 It is possible that modulating factors in vivo, such as an increase in heart rate and acute ischemia with stunning, may lead to further reductions in Ca2+ release.23 Indeed, under adrenergic stimulation the contractile differences between ISCH and CTRL were exacerbated.
Are There Additional Alterations in Myofilament Properties?
As the [Ca2+]i-contraction relation is very steep (Figure 4B), the small changes in [Ca2+]i transient amplitude can explain the reduced contraction. Yet the persistently reduced contraction during high activating [Ca2+]i (Figure 8A), suggests that the myofilament response is reduced as well. Such a reduced Ca2+ response is responsible for the depressed contraction in stunned rat myocardium, and is due to Ca2+-dependent degradation of TnI.31,32 In our pig model, we could not detect a significant increase in TnI degradation, although the immunoblot technique may lack sensitivity to detect small changes. A 1D-gel analysis indicated no changes in isoform composition of MHC and TnT (6 samples each of CTRL and ISCH; data not shown), but further detailed study of other proteins and phosphorylation status is needed. It is also quite possible that the intracellular milieu, rather than intrinsic changes of myofibrillar proteins, reduces the myofilament response to Ca2+.
Another potential factor reducing the contraction amplitude and rate is an increased internal load related to increased glycogen content, or to alterations in the cytoskeleton.33 If the internal load is increased, the rate of relengthening for a similar degree of shortening should be slower.34 A first analysis does not indicate that such is the case as the relation between shortening amplitude, and dL/dt is not different for ISCH and CTRL (data not shown).
Hibernation or/and Stunning: Differences and Similarities in Phenotype
In many but not all human studies, the resting blood flow of hibernating myocardium has been observed to be reduced (see reviews6,35–37) and coronary reserve diminished. Therefore, repeated episodes of acute ischemia and reperfusion, leading to stunning, will contribute to the development of the hibernating phenotype.10,11 The phenotype of our animal model should therefore be compared with previously described models of hibernation and of stunning.
The absence of a reduction in SERCA and PLB is consistent with the findings in short-term hibernation, induced by 24 hours of low perfusion.12 In this model, the increase in contractility in response to an increase in [Ca2+] of the perfusate was reduced, which may correspond to the apparent reduction of the myofilament Ca2+ responsiveness of the single cells in the present study. On the other hand, in their well-characterized model of a single LAD occluder in juvenile pigs (around 8.8 kg at the time of surgery14), Fallavollita, Canty, and collaborators16 reported that the SR Ca2+ handling proteins were downregulated after 3 months but not at 1 month. In this model, the stenosis is not very pronounced at 1 month, around 75%, but develops as the pigs grow,38 and it was proposed that initially repeated stunning is the dominant problem.39 Our model is different in that the critical stenosis develops fast and is more severe, and our animals are older and grow less during the study period. It is possible that at a later stage downregulation of SERCA will occur, but our data indicate that contractile dysfunction in the setting of a critical stenosis with in vivo contractility consistent with hibernation, can occur in the absence of these changes.
Important features of the myocytes in our model are distinct from the stunning phenotype induced by acute occlusion and reperfusion.23 The hypertrophy of the cells in our study indicates the presence of long-term remodeling. The action potential prolongation, as opposed to shortening with stunning,23 indicates that alterations in membrane fluxes other than a reduction in ICaL are present.
It is quite possible that episodes of stunning occurred in our animal model, during, eg, feeding or the dobutamine stress echo. We carefully observed a rest period after any intervention, before euthanasia and cell isolation, to avoid potential acute stunning. We should however consider that in vivo, the phenotype of the myocardium distal to a critical coronary stenosis is determined by the combination of continuous low flow leading to hibernation and repeated stunning. The overlap between stunning and hibernation was recently underscored by the study of Kim et al,40 who showed that repeated occlusions followed by full restoration of flow could lead to a hibernation phenotype.
Alterations in Ca2+ handling and action potential prolongation are common features of remodeling in hypertrophy and heart failure, but depending on the stimulus and trigger, different patterns result (eg, see Sipido et al41). Considering that the primary event in hibernation is a reduction in perfusion, but that secondary triggers such as increased mechanical loading and intermittent stunning may also contribute, the specific pattern of altered Ca2+ handling in the present study is not entirely unexpected.
Limitations of the Study
In our study, we limited ourselves to study myocytes from the midmyocardial layer and did not compare them to myocytes from the remote zone. This was a deliberate choice. In the pig, as in other species, regional differences in excitation-contraction coupling exist.20 In addition, the reduced contractility of the hypoperfused zone may by itself increase loading of remote myocardium. These factors would have complicated the interpretation of the changes induced in the region of interest.
We have limited our analysis to a single time point. Further longitudinal studies are needed to look at the time dependence of the phenotype.39,42
Cellular remodeling is a major factor in the contractile dysfunction of the hibernating myocardium. Myocytes are hypertrophied, action potentials are prolonged, and L-type Ca2+ currents and Ca2+ release are decreased. The steep Ca2+ dependence of contraction and possibly a reduction of maximal myofilament responsiveness further enhance the contractile deficit. In addition to this contractile remodeling, there is evidence for electrical remodeling.
This study was supported by the FWO, the Flanders Fund for Scientific Research (K.R.S., B.B., G.E.S.), and the “Herz- und Kreizlaufzentrum, Essen” (F.R.H.).
Original received October 7, 2003; revision received February 18, 2004; accepted February 20, 2004.
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