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Transmural stretch-dependent regulation of contractile properties in rat heart and its alteration after myocardial infarction

INSERM U-637, CHU Arnaud de Villeneuve, Montpellier, France

¹Correspondence: INSERM U-637, CHU Arnaud de Villeneuve, Montpellier 34295, France. E-mail: vassort{at}montp.inserm.fr

SPECIFIC AIMS

The Frank-Starling law describes a major aspect of cardiac modulation: an increase in end-diastolic volume improves systolic contraction. Although this phenomenon has long been known, the molecular mechanisms of this "stretch-sensitization" modulation remain unclear and controversial. This modulation of contraction is dramatically altered at the organ level during heart failure.

The aim of this study was to provide new insight into the physiological and pathological stretch-dependent regulation of cardiac contractility using rat isolated skinned cardiomyocytes from normal and diseased hearts. We hypothesized that 1) myofibrillar Ca²⁺ sensitivity was not uniformly regulated across the ventricular wall and 2) acute stretch modulates phosphorylation of contractile and/or regulatory proteins.

PRINCIPAL FINDINGS

1. Stretch-dependent contractile properties across the left ventricular wall
Single cells were isolated from subepicardial and subendocardial layers of sham and PMI hearts; they were skinned, attached to a force transducer, and maintained in isometric conditions. Myofilaments were activated with various Ca²⁺-containing solutions. At 1.9 µm sarcomere length (SL), Ca²⁺ sensitivity of myofilament activation (pCa₅₀) did not significantly differ between cell layers and between sham and PMI animals. Stretch sensitized sham cells by shifting the tension-pCa curve (pCa₅₀) more efficiently in ENDO cells (0.24±0.02 pCa unit, n=10) than in EPI cells (0.11±0.01 pCa unit, n=10). In PMI hearts, EPI and ENDO pCa₅₀ were identical (0.14±0.01 pCa unit, n=8 and 11, respectively). Thus, myocardial infarction predominantly affects the stretch-dependent regulation of the contractile machinery in ENDO cells.

2. Passive properties of control and PMI cardiomyocytes
Passive tension was measured by stretching the cell in relaxing solution from slack length to 2.3 µm SL. In sham and PMI hearts, ENDO cells were significantly stiffer than EPI cells (Fig. 1 ). Cell stiffness is essentially due to titin in the physiological SL range. Titin content and isoforms were investigated by 2.5–7% SDS-PAGE analysis. This analysis revealed a gradient in the number of titin molecules per half-sarcomere that correlates with the stiffness gradient without change in isoform. The amount of titin relative to MHC was lower in EPI (0.22±0.02) than in ENDO (0.31±0.02) sham cells. A similar observation was made in PMI rats in which the titin to MHC ratios were 0.26 ± 0.02 in EPI and 0.34 ± 0.03 in ENDO.

View larger version (19K): [in this window] [in a new window]   Figure 1. Correlation between passive tension and stretch-induced Ca2+ sensitization across the wall. Variations in pCa50 induced by stretching cells from 1.9 to 2.3 µm SL (pCa50) were plotted against mean passive tension for each tissue area (ENDO: , EPI: •; MID: midwall: *) in sham (filled) and PMI (open) rat hearts. Linear regressions were calculated on a scatter plot including all individual data in sham (slope 3.2±0.5, r=0.85) and PMI (slope 0.1±0.3, r=0.07).

3. Transmural gradient of stretch-dependent contractile properties
It has been proposed that pCa₅₀ is closely correlated to titin-based passive tension rather than to SL. We studied the relationship between pCa₅₀ and the passive tension developed at 2.3 µm SL. Figure 1 represents the average values of pCa₅₀ established in cells isolated from sham and PMI rats, taking into account their location in the left ventricular free wall. Passive tension was positively correlated with Ca²⁺ sensitivity of myofilament activation. EPI cells were more compliant and the stretch-dependent sensitization of myofilament was less effective than in ENDO cells. Midmyocardial (MID) cells exhibited intermediate properties. This representation demonstrates a gradient of passive and active properties across the left ventricular free wall. After myocardial infarction, this positive correlation disappears due to a reduced pCa₅₀ in ENDO cells.

4. Phosphorylation of thin and thick filament regulatory proteins
To explain the gradient in stretch sensitization across the left ventricular free wall and its alteration in PMI, we explored phosphorylation of various sarcomeric proteins before (1.9 µm SL) and after 1 min stretch (2.3 µm SL). Phosphorylation of the thin filament regulatory protein troponin I (TnI) by PKA is known to affect Ca²⁺ sensitivity and was proposed to enhance length-dependent activation. Using a specific antibody that recognized the PKA-phosphorylated form of TnI, we observed no modification of TnI phosphorylation between regions of sham and PMI rats before or after 1 min stretch (Fig. 2 A). Using urea gel separation, four bands of MLC2 were detected corresponding to VLC2a and VLC2b and their phosphorylated forms (P-VLC2a and P-VLC2b). Phosphorylation of VLC2a was significantly lower in ENDO-PMI than in ENDO-sham tissues. In ENDO-sham tissues, VLC2b phosphorylation was hardly detectable at rest but was increased 6-fold by stretch. This stretch-dependent phosphorylation of VLC2b was lost in ENDO-PMI cells (Fig. 2B ).

View larger version (59K): [in this window] [in a new window]   Figure 2. Localized stretch-dependent effect on TnI and MLC2 phosphorylations in control and diseased hearts. Immunoblots were performed on permeabilized strips of sham (clear column) and PMI (hatched column) myocardium dissected from EPI and ENDO layers before (white) and after 1-min stretch (gray). A) Immunoblots with an anticardiac TnI and anti-cTnI phosphorylated by PKA showed that TnI-phosphorylation was unaffected by stretch (n=6 animals/group). The specificity of the antiphospho TnI was tested on control cells before and after PKA treatment (blots, right panel). B) Immunoblots (upper panels) show 4 bands of MLC2 corresponding to the 2 ventricular isoforms (VLC2a and VLC2b) and their phosphorylated forms (P-VLC2a and P-VLC2b). For phosphorylation quantification each isoform was separated and expressed as a percent of its total amount (phosphorylated+nonphosphorylated forms) for VLC2a and VLC2b. VLC2b is 30% of the total amount of MLC2. This form did not vary with localization in sham hearts (34±3% in EPI vs. 29±2% in ENDO) whereas it decreased significantly in ENDO (23±1%) compared with EPI PMI strips (31±1%).(n=7 animals/group).

CONCLUSIONS AND SIGNIFICANCE

This study characterizes the stretch-dependent contractile properties of cardiomyocytes isolated from sham and failing animals, taking into account localization within the left ventricular wall. This work extends an earlier observation that Ca²⁺ sensitivity of the contractile machinery is correlated more to stretch-induced modification in passive tension than to sarcomere length. It demonstrates for the first time the presence of a gradient of stretch-dependent Ca²⁺sensitivities of the contractile machinery within the left ventricular wall accompanied with a marked stretch-dependent increase in VLC2 phosphorylation in subendocardial cells. Following myocardial infarction this transmural gradient of passive tension-dependent Ca²⁺ sensitivity is lost together with the passive tension-induced MLC2 phosphorylation. The remaining component of length-dependent Ca²⁺ sensitization is similar to that seen in control or diseased epicardium.

Passive and active contractile properties follow a positive gradient from epicardium to endocardium. ENDO cells are stiffer and demonstrate a larger increase in Ca²⁺sensitization following stretch. The SL range across the wall and its changes during cardiac cycle are still not known precisely. It has been proposed that during filling, the nonuniform transmural SL distribution observed in the unloaded state becomes more uniform with the inner sarcomeres elongating more than the outer ones. This is in accordance with higher circumferential strains and ejection fraction observed in vivo in the endocardium. In line with these observations, our study shows that heterogeneous cellular mechanical properties may contribute to the nonuniform deformation of the wall. It may also be anticipated that higher stiffness in the endocardium allows for faster relaxation of this layer during the early diastolic refilling due to titin-based restoring force.

pCa₅₀ is more closely correlated to passive tension than to sarcomere length as previously demonstrated. Because titin isoform switch was not detected across the left ventricular wall, it cannot explain the stiffness gradient. Instead, we noticed a differential titin amount with stiffer ENDO cells expressing more titin per half-sarcomere than EPI cells. The difference in stiffness across the free wall seems to be essentially a titin-based mechanism since we observed that degradation of titin with mild trypsin treatment homogenized passive tension across the wall to around 2 µm/mm². From this study we cannot exclude that other mechanisms may participate in the passive tension modulation across the wall (e.g., PKA phosphorylation, titin-actin interaction, Ca²⁺ binding for titin-based modulation, or a reduced myofibrillar area in the EPI cells).

Titin has been proposed to be involved in the Frank-Starling mechanism mostly by reducing the interfilament lattice spacing. Stretch-dependent structural changes in the thick filaments in response to passive stretch has been proposed for cardiac muscle. We postulated that, in ENDO cells, a passive tension-induced signaling pathway is tuned on top of the stretch-induced effect that could be explained by a change in interfilament lattice spacing. It has recently been suggested that the phosphorylation level of cTnI regulates length dependence of activation. This hypothesis implies a dephosphorylation of TnI with stretch to account for increased activation. In our conditions, cTnI phosphorylation is similar in both EPI and ENDO cells and is unaffected by stretch.

Phosphorylating MLC2 represents an important and ubiquitous regulatory mechanism to modulate force generation of smooth, skeletal, and cardiac muscles by increasing myofilament Ca²⁺sensitivity. We proposed that stretch could induce MLC2 phosphorylation. We observed that stretch mostly activates phosphorylation of VLC-2b in ENDO cells that have a very low basal phosphorylation level compared with EPI cells.

The mechanisms underlying the decline in cardiac pump function during cardiac insufficiency are poorly understood. Isolated whole heart from the PMI rat model develops a reduced Frank-Starling relationship. At the cellular level, epicardium layer seems unaffected. Subendocardial cells exhibit a markedly reduced stretch-dependent activation. This occurs without any effect on the Ca²⁺ sensitivity at resting length (1.9 µm) and without changes in titin content or isoform expression consistently with unchanged cell passive tension. The normal, unaffected Ca²⁺ sensitivity of ENDO- and EPI-PMI cells at resting length is in accordance with previous work in which it was observed up to 6 wk postinfarction, remodeling of myocytes but normal contractile function and intracellular Ca²⁺ transient in unloaded intact myocytes.

In the ENDO-PMI cells, reduction of stretch-dependent myofilament activation is associated with a lack of VLC 2b phosphorylation. Decreased phosphorylation has been associated with depressed responsiveness of the contractile apparatus to systolic rise of activating Ca²⁺, accounting in part for severe heart failure seen in patients in whom both VLC2 isoforms are dephosphorylated. It was mentioned that incubation in cardioplegic solution used to keep human tissues might have dephosphorylated MLC2. Reduced stretch-dependent contractile properties and VLC-2 phosphorylation seen in the ENDO-PMI cells supports the hypothesis that MLC2 is actively involved in the stretch-sensing regulation of contractility.

As summarized in Fig. 3 , on top of the transmural gradient of stretch-dependent component seen in all cells, control or diseased, that could be in part attributable to interfilament spacing, there is a stretch-induced component of active tension that correlates with MLC2 phosphorylation and is observed only in healthy ENDO cells. Nonuniformity is a major characteristic of the normal adult left ventricle that might be caused not only by the morphological heterogeneity of the tissue in the LV wall, but also by the nonuniform contractile properties of the myocytes across the wall. The loss of a contractile transmural gradient after myocardial infarction should contribute to the impaired LV function.

View larger version (18K): [in this window] [in a new window]   Figure 3. 3-Dimensional schematic representation of the stretch-induced Ca2+sensitization of contractile proteins in sham and postmyocardial infarcted (PMI), subepicardial (EPI) and subendocardial (ENDO) cells. Note that the EPI to ENDO gradient of Ca2+sensitization associated with phosphorylation occurs only in sham cells, on top of the ubiquitous component of stretch-dependent Ca2+ sensitization seen in sham and PMI cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2066fje;

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