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The voltage-independent B-type Ca²⁺ channel modulates apoptosis of cardiac myocytes ¹

INSERM Unité 460, Faculté de Médecine Xavier Bichat, Paris, France; and
^* CNRS ESA 8078, Hôpital Marie-Lannelongue, Le Plessis Robinson, France

²Correspondence: INSERM U460, 16 rue Henri Huchard, 75018 Paris, France. E-mail: hatem{at}bichat.inserm.fr

SPECIFIC AIMS

Calcium is involved in various aspects of the physiology and growth of cardiac myocytes including apoptosis, an important factor underlying the progression of a number of cardiovascular diseases. The aim of this study was to determine how Ca²⁺ regulates cardiac myocyte apoptosis induced by ceramide.

PRINCIPAL FINDINGS

1. Ceramide-induced apoptosis of adult rat ventricular myocytes is sensitive to calcium
After 6 h of ceramide (Cer) treatment, 38.5 ± 1.3% of myocytes exhibited an apoptotic phenotype. The permeant Ca²⁺ chelator BAPTA-AM (50 µM) reduced Cer-treated myocyte apoptosis whereas pretreatment with the Ca²⁺ ionophore ionomycin (1 µM) markedly increased it.

2. Calcium homeostasis is altered in the late stages of Cer-induced myocyte apoptosis
In control conditions (control), few local Ca²⁺ events were observed and rarely propagated Ca²⁺ events. In contrast, after more than 3 h both local and propagated Ca²⁺ events were increased, indicating that altered Ca²⁺ homeostasis. Ni²⁺, which blocks L- and T-type Ca²⁺ channels and the Na⁺/Ca²⁺ exchanger, or ryanodine, which inhibits the SR Ca²⁺ release channel, did not modify Cer-induced apoptosis. The overexpression of SERCA_1a, which lowers [Ca²⁺]_i but increases SR Ca²⁺ load, inhibited Cer-induced myocyte apoptosis. These results indicated that myocytes maintained normal Ca²⁺ homeostasis during the first 2 h of incubation with Cer.

3. Abnormal B-type Ca²⁺ channel activity in Cer-treated myocytes
In cell-attached patch-clamp experiments, B-type Ca²⁺ channel activity was scarce and appeared as irregular bursts, separated by long quiescent periods in 20% of patches recorded on a given myocyte. In myocytes preincubated with Cer for 2 h, channel activity still appeared in 20% of the patches, but the burst duration was much longer than in control conditions (Fig. 1 a, c).

View larger version (45K): [in this window] [in a new window]   Figure 1. Cer activated B-type Ca2+ channels of ventricular myocytes. a) Sample of channel current recording showing a long burst of activity in a cell-attached membrane patch from a myocyte incubated with Cer. b) In an inside-out patch, representative channel activity induced by cytosolic superfusion of Cer and its inhibition by eosin. c) Bar graphs summarizing significant changes in the relative mean patch-current observed in the above experiments. Data are means of n experiments ± SD. *P < 0.01 and **P < 0.001 vs. inside-out control. d) Dihydrocer failed to induce channel activity in inside-out patches. Lower traces ( and ß in a, b, and d) are expanded time scale extracts illustrating the same complex gating behavior usually observed for B-type Ca2+ channels.

Application of Cer to the cytosolic face during the quiescent period of inside-out membrane patches (Fig. 1b ) induced single channel activity after a few seconds. This activity exhibited a complex gating behavior with at least three current amplitudes (Fig. 1b, c ), as described for chlorpromazine (CPZ) -activated B-type Ca²⁺ channels. Eosin, a potent inhibitor of B-type Ca²⁺ channels, completely blocked Cer-activated channels (Fig. 1b , c ). The inactive analog dihydro N-acetyl-D-erythro-sphingosine (dihydroCer) had no effect on channel activity (Fig. 1d ). Direct application of Cer in the inside-out configuration caused a sevenfold increase in mean patch current (Fig. 1 c , ß).

4. Pharmacological modulation of B-type channels regulates myocyte apoptosis
Eosin reduced Cer-induced apoptosis whereas CPZ markedly enhanced it. CPZ had no effect on the survival of control myocytes. The proapoptotic effect of CPZ was suppressed by both eosin and BAPTA-AM. These results indicated that the modulation of B-type Ca²⁺ channel activity regulated Cer-induced cardiomyocyte apoptosis.

5. B-type calcium channels modulate mitochondrial functions of apoptotic myocytes
In control conditions, MitoFluor® Red and Mitotracker® Green accumulated in mitochondria, indicating an intact mitochondrial membrane potential (m) (Fig. 2 b, j). After the first hour of Cer treatment (Fig. 2e-j ), a majority of mitochondria had lost their m. Eosin prevented this phenomenon. Caspase-9 activity, which increased in the first hour after Cer application, was reduced by eosin (Fig. 2k ). Cer-induced myocyte apoptosis was reduced by incubating myocytes with caspase-9 inhibitor or cyclosporine A, showing the involvement of mitochondria in this death process.

View larger version (29K): [in this window] [in a new window]   Figure 2. a, d, g) Double labeling of myocytes with MitoTracker® Green and b, e, h) MitoFluor® Red to assess changes in m in control (a, b, c), ceramide (d, e, f), and eosin-pretreatment + ceramide (g, h, i) conditions. c, f, i) The green and red images were superimposed to assess the proportion of mitochondria that had lost their m. j) Mitochondrial membrane potential changes assessed by the red/green fluorescence ratio in various conditions. ***P < 0.001 vs. control. k) Caspase-9 activity vs. control in various experimental conditions. *P < 0.05 and **P < 0.01 vs. ceramide.

6. Evidences for alteration of the mitochondrial calcium content in Cer-treated myocytes
Mitochondrial Ca²⁺ content was evaluated by recording [Ca²⁺]_i changes after myocyte exposure to FCCP. Application of FCCP for 10 s caused a slow and transient rise in [Ca²⁺]_i whose magnitude and duration were greater in Cer-treated than in control cells.

CONCLUSIONS

Cardiomyocyte apoptosis induced by the lipid second messenger C₂-ceramide was highly sensitive to intracellular Ca²⁺ concentration changes. In the present study, myocytes maintained their functional integrity and Ca²⁺ homeostasis during the first hours of ceramide accumulation. Likewise, involvement of the SR Ca²⁺ pool in the modulation of ceramide-induced apoptosis is very unlikely. Abnormal sarcolemmal Ca²⁺ influx through L- or T-type channels or the Na⁺-Ca²⁺ exchanger is unlikely, as Ni²⁺ did not modify ceramide-induced apoptosis. Thus, the Ca²⁺ sensitivity of apoptotic myocytes does not result from a massive loss of cell integrity leading to nonspecific Ca²⁺-dependent necrosis. Rather, Ca²⁺ might act as a second messenger in some of the signaling pathways activated by ceramide.

B-type Ca²⁺ channels are good candidates for the Ni²⁺-insensitive Ca²⁺ entry because their activity was increased by chronic and acute applications of ceramide, an effect blocked by eosin. Moreover, pharmacological modulations of B-type Ca²⁺ channels regulate the apoptosis of myocytes induced by C₂-ceramide. There is a specific coupling between B-type Ca²⁺ channels and mitochondria. Mitochondria appear to be involved in ceramide-induced apoptosis of cardiomyocytes, as indicated by the 1) early loss of m in the majority of mitochondria of ceramide-treated myocytes, 2) activation of caspase-9, which depends on the activation of mitochondria, and 3) protective effect against apoptosis of caspase-9 inhibitor and cyclosporine A. It is conceivable that the increased B-type Ca²⁺ channel activity in ceramide-treated myocytes causes a sustained increase in mitochondrial Ca²⁺ content that contributes, together with other factors, to extensive mitochondrial structural impairment and the release of proapoptotic components.

In control myocytes, activation of B-type Ca²⁺ channels by chlorpromazine failed to trigger apoptosis whereas in Cer-treated myocytes, channel blockade attenuated but not suppressed cell death. Altogether, these findings indicate that the Ca²⁺ entry transported by these channels is a regulatory but not a triggering event of ceramide-induced myocyte apoptosis. Indeed, ceramide can induce apoptosis by various mechanisms.

View larger version (50K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized B-type Ca2+ channel involvement in ceramide-induced apoptosis of cardiac myocytes. B-type Ca2+ channels are functionally coupled to mitochondria through Ca2+. During ceramide-induced apoptosis, the activation of B-type Ca2+ channels causes a Ca2+ accumulation in mitochondria that modulates the mitochondrial apoptotic pathway. PMCA: plasma membrane calcium-ATPase.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0389fje; to cite this article, use FASEB J. (November 29, 2001) 10.1096/fj.01-0389fje

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This Article Full Text (PDF) All Versions of this Article: 16/1/99 01-0389fjev1    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 HÉNAFF, M. Articles by HATEM, S. N. Search for Related Content PubMed PubMed Citation Articles by HÉNAFF, M. Articles by HATEM, S. N.