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Ca²⁺ channel remodeling in perfused heart: effects of mechanical work and interventions affecting Ca²⁺ cycling on sarcolemmal and sarcoplasmic reticulum Ca²⁺ channels ¹

Dipartimento di Scienze dell’Uomo e dell’Ambiente, University of Pisa, Pisa, Italy

²Correspondence: Dip. Scienze dell’Uomo e dell’Ambiente, Sezione di Biochimica, via Roma 55, I-56100 Pisa, Italy. E-mail: r.zucchi{at}med.unipi.it

SPECIFIC AIMS

By "Ca²⁺ channel remodeling" we refer to intermediate- or long-term modulation of the expression of sarcolemmal L-type Ca²⁺ channels (dihydropyridine receptors, DHPRs) and sarcoplasmic reticulum (SR) Ca²⁺ release channels (ryanodine receptors, RyRs), i.e., to modulatory processes occurring over a time scale exceeding a few minutes. In this work, we investigated whether Ca²⁺ channel remodeling can be induced by changes in mechanical work or in Ca²⁺ fluxes in an isolated rat heart model over a period of about an hour.

PRINCIPAL FINDINGS

1. Perfusion at low Ca²⁺ concentration determines DHPR and RyR down-regulation
Isolated working rat hearts were perfused with hypocalcemic or normocalcemic buffer (0.8 mM and 1.5 mM Ca²⁺, respectively). After 60 min, the expression of DHPR and RyR proteins was assayed by determining [³H]-PN 200–110 and [³H]-ryanodine binding in ventricular homogenate. Hypocalcemic perfusion reduced [³H]-PN 200–110 and [³H]-ryanodine binding. Changes in binding consisted in 30% reduction of the B_max whereas the K_d was unaffected (Fig. 1 ). Experiments aimed at determining the time course of the phenomenon showed that 10 or 30 min of hypocalcemic perfusion was not sufficient to induce significant changes in [³H]-PN 200–110 and [³H]-ryanodine binding. On the other hand, DHPR and RyR down-regulation was reversible with restoration of normocalcemic perfusion: after 60 min of hypocalcemic perfusion followed by 60 min of normocalcemic perfusion, B_max values reverted to the baseline.

View larger version (16K): [in this window] [in a new window]   Figure 1. Saturation binding curves for [3H]-PN 200–110 (above) and [3H]-ryanodine (below), obtained in hearts subjected to 60 min of control perfusion (•, n=14) or 60 min of hypocalcemic perfusion (, n=7). Data points represent mean ± SE. Scatchard analysis yielded significant reduction in Bmax (57±7 vs. 85±4 fmol/mg for [3H]-PN 200–110; 327±29 vs. 459±15 fmol/mg for [3H]-ryanodine; P<0.01 in both cases), with unchanged Kd.

2. The effect is related to changes in Ca²⁺ fluxes rather than to mechanical work per se
Hypocalcemic perfusion reduces Ca²⁺ entry through the sarcolemma, depleting intracellular Ca²⁺ pools and decreasing contractile performance. Perfusions at a low preload (height of the atrial chamber set at 5 cm instead of 20 cm) and in the presence of nifedipine (100 nM) were carried out to discriminate between the role of mechanical work and the role of Ca²⁺ homeostasis. Low preload perfusion decreases mechanical work in the absence of major effects on Ca²⁺ homeostasis; nifedipine antagonizes Ca²⁺ entry through DHPRs and therefore causes SR Ca²⁺ depletion and eventually reduces SR Ca²⁺ release. In our model, contractile performance was similar in the hypocalcemia, low preload, and nifedipine groups (e.g., aortic flow decreased by 30–50%). As shown in Fig. 2 , low preload perfusion determined no significant variation of either [³H]-PN 200–110 or [³H]-ryanodine binding whereas nifedipine produced similar effects as hypocalcemic perfusion.

View larger version (35K): [in this window] [in a new window]   Figure 2. Bmax values for [3H]-PN 200–110 (above) and [3H]-ryanodine (below) obtained in homogenates derived from perfused hearts. Bars represent mean ± SE of 6–14 experiments in each group. *P < 0.05, **P < 0.01 vs. control, by ANOVA and Dunnett’s test. Kd values were not significantly different in any case.

In other experiments, we investigated whether the effects were proportional to the triggering stimulus, using a cardioplegia model. Cardioplegia produced DHPR and RyR down-regulation, but the changes were similar in extent to those observed in the hypocalcemia and nifedipine groups.

3. Gene expression is not affected but the phenomenon is abolished by inhibitors of protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)
To unravel the molecular mechanisms underlying channel remodeling, we determined DHPR and RyR gene expression by RT-PCR technique in hearts subjected to normocalcemic perfusion, hypocalcemic perfusion, and cardioplegia: no significant change in DHPR and RyR mRNA was observed in any group. We also investigated whether remodeling was affected by protein kinase inhibitors by performing hypocalcemic experiments in the presence of: 10 nM H-89 (protein kinase A inhibitor), 2 µM chelerythrine (PKC inhibitor), or 1 µM lavendustin C (CaMKII inhibitor). Perfusion with chelerythrine or lavendustin C abolished the remodeling process, since no change in [³H]-PN 200–110 and [³H]-ryanodine binding occurred after 60 min of hypocalcemic perfusion. In the presence of H-89, however, hypocalcemic perfusion still induced significant decrease in [³H]-PN 200–110 and [³H]-ryanodine B_max.

CONCLUSIONS AND SIGNIFICANCE

Sarcolemmal L-type Ca²⁺ channels (dihydropyridine receptors) and sarcoplasmic reticulum Ca²⁺ release channels (RyRs) are subjected to short-term regulation in cardiomyocytes, e.g., allosteric modulation by Ca²⁺ and other ions, or changes in channel gating produced by covalent modifications, such as phosphorylation of specific sites or nitrosylation of reactive thiols. On a longer time scale, modifications of RyR and/or DHPRs have been described under pathological conditions such as ischemia, hypertrophy, heart failure, and chronic atrial fibrillation, suggesting the existence of intermediate- or long-term modulatory mechanisms (here designated as Ca²⁺ channel remodeling), which have not been characterized so far.

In this work, evidence of Ca²⁺ channel remodeling was obtained in isolated rat heart after perfusion at a low Ca²⁺ concentration that determined a significant decrease in [³H]-PN 200–110 and [³H]-ryanodine binding. The time scale of the process was on the order of an hour and changes were reversible. Hypocalcemic perfusion reduces mechanical performance by decreasing Ca²⁺ availability. To assess the role of Ca²⁺ homeostasis vs. contractile function, we evaluated the response to preload reduction and nifedipine. Although mechanical performance was similar in all groups, low preload perfusion determined only minor changes in [³H]-PN 200–110 and [³H]-ryanodine binding, whereas nifedipine reproduced the effects of hypocalcemic perfusion. We conclude that remodeling is not related to cardiac work per se, but to some variable associated with Ca²⁺ handling. We speculate that crucial factor may be represented by Ca²⁺ transients or by time-averaged Ca²⁺ concentration, in relevant subcellular compartments (e.g., in the cleft of the triadic junction).

We also investigated the molecular mechanisms underlying the remodeling process. Gene expression was not modified, suggesting that we were dealing with post-translational events. Proteolysis cannot be excluded, but it appears unlikely because of the quick reversibility of the process. Variations in radioligand binding may reflect changes in protein conformation, as observed in several experimental settings with regard to DHPR or RyR. The effect of hypocalcemic perfusion was abolished by the protein kinase inhibitors chelerythrine and lavendustin C. CaMKII has been proposed as a sensor of Ca²⁺ oscillations and as a mediator of long-term potentiation in neurons. Both PKC and CaMKII have been reported to phosphorylate either DHPR or RyR, although the functional consequences of phosphorylation are unclear. In our experimental model > 30 min was necessary to observe significant effects. This time course suggests that remodeling is not due to direct channel phosphorylation by PKC and/or CaMKII, but rather is the outcome of a more complex process. There is evidence that DHPR and RyR are closely associated with kinases, phosphatases, and scaffolding proteins, and CaMKII targeting to the SR has been observed in skeletal muscle. Therefore, we hypothesize that the remodeling process we have observed may be mediated by signaling complexes including PKC and CaMKII, possibly affecting the interaction of channel proteins with associated scaffolding proteins (Fig. 3 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. Hypothetical interpretation of our results. Ca2+ concentration in the triadic cleft activates a signaling module, possibly including PKC and CaMKII, that is responsible for the assemblage of the triadic junction. Reduction of triadic Ca2+ leads to remodeling of the triadic complexes.

In neurons, ion channels show a high degree of plasticity and their expression is modulated by synaptic activity. Similar mechanisms have not been described in muscle, but our findings can be interpreted as a form of plasticity involving myocardial sarcolemma and SR Ca²⁺ channels, since reduction of Ca²⁺ cycling caused channel down-regulation. This phenomenon might have important physiological, pathophysiological, and pharmacological implications.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0402fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0402fje

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This Article Full Text (PDF) All Versions of this Article: 16/14/1976 02-0402fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZUCCHI, R. Articles by RONCA-TESTONI, S. Search for Related Content PubMed PubMed Citation Articles by ZUCCHI, R. Articles by RONCA-TESTONI, S.