The present study demonstrates that T-tubule structure in murine cardiomyocytes becomes progressively disorganized during early CHF following MI. This structural rearrangement caused irregular gaps to appear between adjacent T-tubules, especially in 3-week CHF cells. Regions of delayed Ca2+ release observed in line-scan images occurred at these gaps between T-tubules. An increased occurrence of delayed Ca2+ release sites in 3-week CHF contributed to a more dyssynchronous pattern of Ca2+ release. This pattern was consistent between beats and not altered by forskolin treatment. Increased dyssynchrony contributed to slowing of overall Ca2+ release in 3-week CHF cells, although a uniform slowing of Ca2+ release was also observed beginning at 1-week CHF.
T-tubule alterations in heart failure
Few studies have examined the structure of the T-tubule network in heart failure. Kamp and collaborators observed a marked loss of T-tubules in canine ventricular myocytes following tachycardia-induced heart failure (He et al. 2001; Balijepalli et al. 2003). However, only a single preliminary study has reported decreased T-tubule density in human failing cardiomyocytes (Wong et al. 2001). An unchanged (Ohler et al. 2001) and even an increased T-tubule density (Kaprielian et al. 2000) have also been reported. Louch et al. (2004) observed that a prominent T-tubule network was in place in failing human ventricular myocytes, although that study was conducted without human controls. Thus, based on the small amount of literature to date it seems unlikely that there is marked T-tubule loss in human heart failure. However, several reports agree that the T-tubule network may be re-structured in this condition. These changes may include dilation of the T-tubules (Kostin et al. 1998; Kaprielian et al. 2000; Louch et al. 2004) or more complex structural disorganization. Kaprielian et al. (2000) observed more twisted T-tubules in failing human cells, with an increased proportion of tubules running in the longitudinal direction. Similar observations were made in a preliminary report of failing human myocytes (Wong et al. 2001) and in cells from spontaneously hypertensive rats which had developed heart failure (Song et al. 2006). In the present study, we also observed T-tubule disorganization that involved an increased number of longitudinal tubules. At present the trigger for T-tubule disorganization in heart failure is unknown, but possible mechanisms include activation of fetal genes and/or loss of structural proteins.
Local alterations in Ca2+ homeostasis: implications for heart failure
This study directly links alterations in T-tubule structure to alterations in Ca2+ handling during heart failure. However, previous work has shown that similar mechanisms can contribute to dyssynchronous Ca2+ release in healthy cells. Heinzel et al. (2002) observed large inhomogeneities during Ca2+ release in pig ventricular myocytes, and showed that these regions of delayed Ca2+ release occurred at gaps between T-tubules; they observed much more synchronous release of Ca2+ in mouse ventricular myocytes and attributed this difference to a higher density of T-tubules in these cells. In the present study, we have also observed quite uniform release of Ca2+ in SHAM mouse cells. The regions of delayed Ca2+ release and the corresponding gaps between adjacent T-tubules that we observed in these cells were indeed smaller and fewer than those reported in healthy pig myocytes. In the present study, and in the study by Heinzel et al. (2002), regions of delayed Ca2+ release occurred in the same part of the cell during consecutive beats and Ca2+ transients were not synchronized by stimulation of the β-adrenergic signalling pathway.
We observed that T-tubules were disorganized in nearly all 3-week CHF cells examined. However, in some cells the degree of disorganization was not sufficient to cause the formation of irregular gaps between T-tubules, and few delayed Ca2+ release regions were observed. Even in those 3-week CHF cells with the most pronounced T-tubule disorganization delayed release regions were usually not large. In both 3-week SHAM and 3-week CHF, most delayed release regions had a width of less than 4 μm, which is likely to correspond to only one or two missing transverse segments of tubule since the intertubule distance is normally ∼2 μm. If T-tubule disorganization continues with progression of CHF beyond 3 weeks, the size of delayed regions might increase as neighbouring gaps fuse. This effect has been observed previously during progressive loss of T-tubules in cell culture (Louch et al. 2004). This had not happened to any great extent in the current study because of the relatively low number of delayed regions per cell, but might explain the occurrence of a few large delayed release regions (>10 μm) in 3-week CHF.
It seems possible that in 3-week SHAM and 3-week CHF, Ca2+ influx is triggered in early release regions where T-tubules and L-type Ca2+ channels are present, and Ca2+ then diffuses more slowly into delayed release regions where T-tubules are absent (Lipp et al. 1996; Yang et al. 2002; Heinzel et al. 2002; Louch et al. 2004; Song et al. 2006). We believe that this diffusing Ca2+ then triggers SR Ca2+ release in delayed release regions. In support of this view, local transients in delayed release regions were not smaller in magnitude than those in early release regions. Although we have not examined the uniformity of ryanodine receptor distribution and function in CHF, previous studies have also suggested that SR function might remain intact despite the local absence of T-tubules and Ca2+ channels (Lipp et al. 1996; Yang et al. 2002; Heinzel et al. 2002; Louch et al. 2004; Song et al. 2006). Similarly, atrial and Purkinje cells which completely lack T-tubules exhibit SR Ca2+ release throughout the cell which is triggered solely by propagation of CICR (Berlin, 1995; Cordeiro et al. 2001). An important point for our study is that the local Ca2+ transients in delayed release regions had a similar temporal profile in 3-week CHF and 3-week SHAM, suggesting that the mechanisms underlying delayed Ca2+ release are analogous in the two cell types.
Our findings are consistent with the proposal that the ability of Ca2+ current to trigger SR Ca2+ release is compromised in heart failure (Gomez et al. 2001; Sjaastad et al. 2002; Sjaastad et al. 2005; Song et al. 2006). A decrease in CICR gain could be expected if T-tubule remodelling leads to ‘orphan’ ryanodine receptors that are cut off from the triggering Ca2+ signal (Gomez et al. 2001; Song et al. 2006). However, in this situation the effect of ‘autoregulation’ should also be considered (Trafford et al. 2002). Local absence of Ca2+ channels in areas devoid of T-tubules could lead to a compensatory increase in SR content in these regions. This would increase ryanodine receptor conductance and open probability (Sitsapesan & Williams, 1994; Lukyanenko et al. 1996) which could partially offset a decrease in gain of CICR. An increased gap between the T-tubules and SR could also occur so that all ryanodine receptors were located further from Ca2+ channels (Gomez et al. 2001). Such changes could explain the increase in latency of Ca2+ release we have observed in 3-week CHF, as there would be a longer delay between Ca2+ influx and CICR throughout the cell. Perhaps T-tubule disorganization leads to both types of structural changes in CHF: the appearance of ‘orphan’ ryanodine receptors and an increased distance between Ca2+ channels and ryanodine receptors.
Our statistical analyses of F50 profiles over consecutive beats showed that a common signal could be extracted to describe the shape of Ca2+ release along cells. Importantly, this common signal was more dyssynchronous in 3-week CHF than 3-week SHAM, supporting a causative role of structural alterations. Beat-to-beat variability in F50 profiles was also larger in 3-week CHF than 3-week SHAM, suggesting that non-structural mechanisms may also promote dyssynchrony of Ca2+ release. One such mechanism could be alteration of the stochastic activity of ryanodine receptors (Cannell et al. 1994). Beat-to-beat variability in Ca2+ release synchrony can also result from alterations in action potential configuration, as shown previously in normal (Sah et al. 2002) and failing (Harris et al. 2005) ventricular myocytes. Finally, decreased phosphorylation of Ca2+ handling proteins can trigger randomly dyssynchronous Ca2+ release, as reported in MI border zone myocytes (Litwin et al. 2000). However, this latter mechanism for dyssynchrony may be restricted to the myocardium neighbouring the infarction and is an unlikely explanation for our findings since forskolin treatment did not alter the synchrony of Ca2+ release in 3-week CHF.
Our findings are in agreement with previous studies on freshly isolated and cultured cells which have observed that cell regions where T-tubules are absent exhibit delayed Ca2+ release that is not synchronized by isoproterenol (Louch et al. 2004; Heinzel et al. 2002). However, isoproterenol treatment has been reported to synchronize Ca2+ release in de-tubulated myocytes (Brette et al. 2004). In this latter study, synchronization of the Ca2+ transient was attributed to enhanced coupling of clusters of ryanodine receptors and more rapid propagation of Ca2+-induced Ca2+ release due to increased SR Ca2+ content and ryanodine receptor phosphorylation. The apparent discrepancy between de-tubulated and intact myocyte studies could be explained by different regulation of ryanodine receptors in the two situations. Althought the de-tubulation procedure seals off T-tubules from the surface sarcolemma, T-tubules remain inside the cell and dyads may remain intact. Observations from the present study and other studies (Heinzel et al. 2002; Louch et al. 2004; Song et al. 2006) suggest that some ryanodine receptors are ‘orphaned’ in intact cells. Altered regulation of these ryanodine receptors not found in dyads may make them less susceptible to the effects of β-adrenergic stimulation.
In failing human heart, Ca2+ transients are prolonged and exhibit a characteristic broadening of the peak (Gwathmey et al. 1987; Beuckelmann & Erdmann, 1992; Piacentino et al. 2003). In our model of CHF, we observed that peak broadening partly resulted from an increased incidence of delayed release regions. We calculated that increased dyssynchrony of Ca2+ release in 3-week CHF increased time-to-peak values by approximately 3 ms. This is not unexpected based on the relatively small size and incidence of delayed release regions we have observed. Experimentally promoting large decreases in T-tubule density has been observed to slow time-to-peak values by only about 7 ms (Louch et al. 2004). However, Ca2+ transients were actually about 16 ms slower to peak in 3-week CHF than in 3-week SHAM (Fig. 7), and marked slowing of Ca2+ release was observed in 1-week CHF without increased dyssynchrony (Figs 2 and 7). Therefore, factors other than dyssynchrony must also contribute to the slowing of Ca2+ release in CHF. In support of this view, Ca2+ transients in 3-week CHF cells exhibited increased latency (Fig. 7C), the first time-point to reach F50 occurred later in 3-week CHF (Fig. 3A), and local transients in early release regions were slower (Fig. 8B and C). Thus, CHF in our model appears to involve a uniform slowing of Ca2+ release throughout the cell which begins early in CHF. With progression of CHF, a non-uniform dyssynchronous slowing of Ca2+ release also occurs as a result of T-tubule disorganization.
The mechanisms underlying the uniform slowing of Ca2+ release we have observed in CHF are unclear. Hyper-phosphorylation and uncoupling of neighbouring ryanodine receptors (Marx et al. 2000, 2001) could theoretically produce such effects in heart failure. As well, less coordinated Ca2+ channel openings might result in an overall slowing of Ca2+ release. We have previously postulated that this mechanism might result in loss of ‘high-gain’ CICR at relatively negative voltages (Sjaastad et al. 2005). Such alterations might also contribute to increased beat-to-beat variability in the timing of Ca2+ release. Finally, changes in SR content should be considered. Reduced SR content in chronically failing myocytes (for review see Houser et al. 2000) could contribute to slowing of Ca2+ release by reducing Ca2+ conductance and open probability of ryanodine receptors (Sitsapesan & Williams, 1994; Lukyanenko et al. 1996). Reduced SR content could also promote dyssynchronous Ca2+ release if non-uniform alterations in Ca2+ stores occur throughout the cell. However, in the present study we have employed a model of early CHF in which SR function is not reduced. Thus, it seems that reduced SR content is not a prerequisite for either slower or dyssynchronous Ca2+ release in CHF.
In conclusion, murine cardiomyocytes exhibit progressive disorganization of T-tubules during CHF following MI. This structural rearrangement causes irregular gaps to form between adjacent T-tubules which promotes more dyssynchronous Ca2+ release. Our results show that this mechanism can contribute to slowing of the Ca2+ transient in CHF.