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. 2014 Jul 15;111(28):E2905-14.
doi: 10.1073/pnas.1402544111. Epub 2014 Jun 26.

Impaired functional communication between the L-type calcium channel and mitochondria contributes to metabolic inhibition in the mdx heart

Helena M Viola  1 , Abbie M Adams  2 , Stefan M K Davies  3 , Susan Fletcher  4 , Aleksandra Filipovska  3 , Livia C Hool  5
Affiliations

Impaired functional communication between the L-type calcium channel and mitochondria contributes to metabolic inhibition in the mdx heart

Helena M Viola et al. Proc Natl Acad Sci U S A. .

Abstract

Duchenne muscular dystrophy is a fatal X-linked disease characterized by the absence of dystrophin. Approximately 20% of boys will die of dilated cardiomyopathy that is associated with cytoskeletal protein disarray, contractile dysfunction, and reduced energy production. However, the mechanisms for altered energy metabolism are not yet fully clarified. Calcium influx through the L-type Ca(2+) channel is critical for maintaining cardiac excitation and contraction. The L-type Ca(2+) channel also regulates mitochondrial function and metabolic activity via transmission of movement of the auxiliary beta subunit through intermediate filament proteins. Here, we find that activation of the L-type Ca(2+) channel is unable to induce increases in mitochondrial membrane potential and metabolic activity in intact cardiac myocytes from the murine model of Duchenne muscular dystrophy (mdx) despite robust increases recorded in wt myocytes. Treatment of mdx mice with morpholino oligomers to induce exon skipping of dystrophin exon 23 (that results in functional dystrophin accumulation) or application of a peptide that resulted in block of voltage-dependent anion channel (VDAC) "rescued" mitochondrial membrane potential and metabolic activity in mdx myocytes. The mitochondrial VDAC coimmunoprecipitated with the L-type Ca(2+) channel. We conclude that the absence of dystrophin in the mdx ventricular myocyte leads to impaired functional communication between the L-type Ca(2+) channel and mitochondrial VDAC. This appears to contribute to metabolic inhibition. These findings provide new mechanistic and functional insight into cardiomyopathy associated with Duchenne muscular dystrophy.

Keywords: cytoskeleton; ion channels; structure.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Absence of dystrophin alters ICa-L current and Ψm in mdx myocytes. (A) ICa-L current traces recorded from a myocyte from a mdx heart and a myocyte from a wt heart as indicated. (A, Inset) ICa-L current traces from a wt myocyte (250 pF) exposed to a peptide derived against the alpha-interacting domain of ICa-L (AID-TAT, 1 μM) or a wt myocyte (220 pF) exposed to a scrambled control peptide [AID(S)-TAT, 1 μM] as indicated. Microelectrodes contained the following: 115 mM CsCl, 10 mM Hepes, 10 mM EGTA, 20 mM tetraethylammonium chloride, 5 mM MgATP, 0.1 mM Tris-GTP, 10 mM phosphocreatine, and 1 mM CaCl2 (pH adjusted to 7.05 at 37 °C with CsOH). Currents were measured in extracellular modified Tyrode’s solution containing the following: 140 mM NaCl, 5.4 mM CsCl, 2.5 mM CaCl2, 0.5 mM MgCl2, 5.5 mM Hepes, and 11 mM glucose (pH adjusted to 7.4 with NaOH). (B) Mean ± SEM of rate of inactivation (tau) for mdx myocytes and wt myocytes. (C–F) Direct activation of ICa-L results in an increase in Ψm in wt but not mdx myocytes. Representative ratiometric JC-1 fluorescence recorded from wt myocytes (C), and mdx myocytes (D), before and after exposure to 10 μM BayK(-) or 10 μM BayK(+). Vertical arrow indicates when drug was added. Four millimolar KCN was added to collapse Ψm as indicated. Mean ± SEM of increases in JC-1 fluorescence for all wt (E) and mdx (F) myocytes exposed to treatments as indicated. Nisol, 10 µM nisoldipine; Oligo, 20 µM oligomycin. Oligo alone induced a robust increase in JC-1 signal in mdx myocytes.
Fig. 2.
Fig. 2.
Direct activation of ICa-L results in an increase in Ψm in wt but not mdx myocytes under calcium-free conditions. (A) Representative ratiometric JC-1 fluorescence recorded from wt myocytes before and after exposure to 10 μM BayK(+) or 10 μM BayK(-) under calcium-free conditions (0 mM Ca2+) as indicated. Vertical arrow indicates when drug was added. Four micromolar KCN was added to collapse Ψm as indicated. ES, external solution; IS, internal solution. (B) Representative traces of JC-1 fluorescence recorded from mdx myocytes and myocytes from mdx mice treated with PMO (“mdx rescue”; see text for detail) before and after exposure to 10 μM BayK(+) or 10 μM BayK(-) under calcium-free conditions (0 mM Ca2+). Vertical arrow indicates when drug was added. (C) Representative traces of JC-1 fluorescence recorded from wt myocytes before and after exposure to 2 μM caffeine (Caff) or 2 μM Caff and 10 μM BayK(-) under calcium-free conditions (0 mM Ca2+) as indicated. Vertical arrow indicates when drug was added. (D and E) Mean ± SEM of increases in JC-1 fluorescence for all wt myocytes (D), and all mdx myocytes including myocytes from mdx mice treated with PMO (“mdx rescue”) (E), exposed to treatments as indicated. Nisol: 10 μM nisoldipine; Oligo: 20 µM oligomycin. Oligo alone induced a robust increase in JC-1 signal in mdx myocytes. (F, i) RT-PCR performed on cardiac muscle RNA from mdx mice treated with PMO demonstrating exon 23 skipping (Δ23), as indicated by arrow. (F, ii) Immunoblot performed on cardiac muscle from C57BL/10 control mice, untreated mdx mice (mdx control), and mdx mice treated with PMO demonstrating presence of dystrophin (mdx treated), as indicated by arrow (2% and 1% dilution shown). (G) Immunostaining of heart and diaphragm cryosections from a C57BL/10 control mouse, untreated mdx mouse (mdx control), and mdx mouse treated with PMO (mouse 12-08-50) demonstrating presence of dystrophin (mdx treated). (Scale bars: 100 µm.)
Fig. 3.
Fig. 3.
Direct activation of ICa-L results in an increase in metabolic activity and flavoprotein oxidation in wt but not mdx myocytes. (A) Formation of formazan measured as change in absorbance in 8-wk-old wt and 8-wk-old mdx myocytes after addition of 10 µM BayK(+) or 10 µM BayK(-). (B and C) Mean ± SEM of increases in absorbance for wt myocytes (B) and mdx myocytes (C), exposed to treatments as indicated. Dant,10 µM dantrolene; Nisol, 10 µM nisoldipine; Oligo: 20 µM oligomycin; Ru360, 10 µM Ru360. (D and E) Representative traces of flavoprotein fluorescence recorded from wt myocytes (D), and mdx myocytes and myocytes from mdx mice treated with PMO (“mdx rescue”) (E), before and after exposure to 10 μM BayK(+) or 10 μM BayK(-). Vertical arrow indicates when drug was added. 10 µM FCCP was added to increase flavoprotein signal, confirming the signal was mitochondrial in origin. (F and G) Mean ± SEM of increases in flavoprotein fluorescence for all wt myocytes (F), and all mdx myocytes including myocytes from mdx mice treated with PMO (“mdx rescue”) (G), exposed to treatments as indicated. FCCP, 10 μM FCCP; Nisol, 10 μM nisoldipine.
Fig. 4.
Fig. 4.
VDAC coimmunoprecipitates with ICa-L, and direct block of VDAC restores the increase in Ψm in mdx myocytes. (A) Regulation of Ψm by ICa-L requires an intact cytoskeleton. Representative ratiometric JC-1 fluorescence is recorded from wt myocytes before and after addition of 10 µM BayK(-) in the presence or absence of 5 µM Latrunculin A (Latrunc) (i), or 1 μM AID(S)-TAT or 1 μM active AID-TAT peptide (ii), under calcium-free conditions (0 mM Ca2+). Vertical arrow indicates when drug was added. Four micromolar KCN was added to collapse Ψm as indicated. ES, external solution; IS, internal solution. (B) ICa-L associates with mitochondrial VDAC via cytoskeletal proteins. (i) Immunoblot of 10 μg of immunoprecipitated VDAC protein from wt (Left) and mdx (Right) heart probed with anti-ICa-L antibody (Upper) and 10 μg immunoprecipitated ICa-L protein probed with anti-VDAC antibody (Lower). Negative control immunoblots were performed on 10 μg of immunoprecipitated VDAC protein from wt (Left) and mdx (Right) heart by probing with MS antibody (Upper), and on 10 μg of immunoprecipitated ICa-L protein from wt (Left) and mdx (Right) heart by probing with α-GOLD antibody (Lower). (ii) Mass spectrometry analysis of immunoprecipitated ICa-L protein. AKAP, A-kinase anchor protein; ANT, adenine nucleotide translocator; CaMKII, Ca2+/calmodulin-dependent protein kinase II; LTCC, L-type Ca2+ channel; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; TK, tyrosine kinase. (C) Application of a peptide that blocks VDAC mimics the increase in Ψm in wt myocytes. Representative ratiometric JC-1 fluorescence is recorded from wt myocytes before and after exposure to 10 μM BayK(-), 10 µM VDAC peptide (VDAC), or 10 µM VDAC scrambled peptide [VDAC(S)] under calcium-free (0 mM Ca2+) conditions (i), or 2.5 mM calcium (ii). Vertical arrow indicates when drug was added. Four millimolar KCN was added to collapse Ψm as indicated. ES, external solution; IS, internal solution. (D) Application of a peptide that blocks VDAC restores the increase in Ψm in mdx myocytes. Representative ratiometric JC-1 fluorescence recorded from mdx myocytes before and after exposure to 10 μM BayK(-), 10 µM VDAC peptide (VDAC), or 10 µM VDAC scrambled peptide [VDAC(S)] under calcium-free (0 mM Ca2+) conditions (i) or 2.5 mM calcium (ii). Vertical arrow indicates when drug was added. ES, external solution; IS, internal solution. (E and F) Mean ± SEM of increases in JC-1 fluorescence for all wt myocytes (E), and all mdx myocytes (F), exposed to treatments as indicated.
Fig. 5.
Fig. 5.
Direct activation of ICa-L results in an increase in Ψm and metabolic activity in wt but not mdx myocytes isolated from 43-wk-old mice. (A and B) Representative ratiometric JC-1 fluorescence recorded from wt myocytes (A), and mdx myocytes (B), before and after exposure to 10 μM BayK(+) or 10 μM BayK(-). Vertical arrow indicates when drug was added. Four micromolar KCN was added to collapse Ψm as indicated. (C and D) Mean ± SEM of increases in JC-1 fluorescence for all wt (C) and mdx (D) myocytes exposed to treatments as indicated. Nisol, 10 µM nisoldipine; Oligo, 20 µM oligomycin. Oligo alone induced a robust increase in JC-1 signal in mdx myocytes. (E) Formation of formazan measured as change in absorbance in wt and mdx myocytes after addition of 10 μM BayK(+) or 10 μM BayK(-). (F and G) Mean ± SEM of increases in absorbance for wt myocytes (F) and mdx myocytes (G), exposed to treatments as indicated. Dant, 10 µM dantrolene; Nisol, 10 µM nisoldipine; Oligo, 20 µM oligomycin; Ru360, 10 µM Ru360. (H) Respiration and complex activity in mitochondria isolated from 43-wk-old wt hearts and 43-wk-old mdx hearts (see Materials and Methods for details).
Fig. 6.
Fig. 6.
Schematic representation of communication of ICa-L with the outer membrane mitochondrial protein VDAC via cytoskeletal proteins (A), and altered cytoskeletal network in hearts lacking dystrophin (B) (see text for details).
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