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

Significance

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease that results in cardiomyopathy and heart failure. The cardiomyopathy is characterized by cytoskeletal protein disarray, contractile dysfunction, and reduced energy production. The mechanisms for altered energy metabolism are not yet fully clarified. The L-type Ca²⁺ channel regulates excitation and contraction in the heart, and can regulate mitochondrial function via the movement of cytoskeletal proteins. Here, we find that myocytes from the murine model of DMD (mdx) exhibit impaired communication between the L-type Ca²⁺ channel and the mitochondria that results in poor energy production. Morpholino oligomer therapy targeting dystrophin or block of the mitochondrial voltage-dependent anion channel (VDAC) “rescues” metabolic function, indicating that impaired communication between the L-type Ca²⁺ channel and VDAC contributes to the cardiomyopathy.

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 Ca²⁺ channel is critical for maintaining cardiac excitation and contraction. The L-type Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ channel. We conclude that the absence of dystrophin in the mdx ventricular myocyte leads to impaired functional communication between the L-type Ca²⁺ 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.

Duchenne muscular dystrophy affects 1:3,500 males, and in addition to skeletal muscle damage and atrophy, boys also develop a dilated cardiomyopathy due to the absence of expression of dystrophin. This pathology involves myocyte remodelling, disorganization of cytoskeletal proteins, and contractile dysfunction (1, 2). Early metabolic and signaling alterations have been reported in 10- to 12-wk-old murine model of Duchenne muscular dystrophy (mdx) hearts before the development of overt contractile dysfunction (2). However, the mechanisms by which absence of dystrophin leads to mitochondrial dysfunction and compromised cardiac function in mdx hearts are not fully clarified yet.

Cytoskeletal proteins stabilize cell structure. In mature muscle, intermediate filaments form a 3D scaffold that extend from the Z disks to the plasma membrane and traverse cellular organelles such as t-tubules, sarcoplasmic reticulum, and mitochondria (3). Intermediate filaments and microtubules interact directly with mitochondria by binding to outer mitochondrial membrane proteins. In addition to a physical association, cytoskeletal proteins also regulate the function of proteins in the plasma membrane and within the cell (4). The L-type Ca²⁺ channel (I_Ca-L) or dihydropyridine receptor (DHPR) is anchored to F-actin networks by subsarcolemmal stabilizing proteins that also tightly regulate the function of the channel (5–7). Disruption of actin filaments significantly alters I_Ca-L current (5, 7, 8).

Calcium influx through I_Ca-L is a requirement for contraction. I_Ca-L can also regulate mitochondrial function. Activation of I_Ca-L with application of the DHPR agonist BayK(-) or voltage clamp of the plasma membrane can influence mitochondrial superoxide production, NADH production, and metabolic activity in a calcium-dependent manner (9, 10). Activation of I_Ca-L can also increase mitochondrial membrane potential (Ψ_m) in a calcium-independent manner (9). The response is reversible upon inactivation of I_Ca-L. The response also depends on actin filaments because depolymerization of actin prevents the increase in Ψ_m (9). Similarly preventing movement of the beta auxiliary subunit of I_Ca-L with application of a peptide derived against the alpha-interacting domain of the channel attenuates the increase in Ψ_m (9). Therefore, we have proposed that I_Ca-L influences metabolic activity through transmission of movement of the channel via cytoskeletal proteins. Here, we sought to identify whether cytoskeletal disruption due to the absence of dystrophin leads to mitochondrial dysfunction and compromised cardiac function in mdx hearts. Specifically, we investigated whether the absence of dystrophin in ventricular myocytes from mdx mice results in impaired communication between I_Ca-L and mitochondria and, subsequently, metabolic inhibition.

Results

I_Ca-L Measured in mdx Myocytes Exhibit Altered Inactivation Kinetics.

We measured I_Ca-L currents in myocytes isolated from hearts of mdx mice and compared them with currents recorded from wt myocytes. Consistent with previous reports (11, 12) we find I_Ca-L current density in myocytes from 8-wk-old wt mice is not different from current density recorded in myocytes from 8-wk-old mdx mice (6.2 ± 0.8 pA/pF, n = 10 vs. 7.9 ± 1.6 pA/pF, n = 9; P = not significant). In addition, there was no difference in cell size between mdx and wt myocytes (12). Through immunoblotting, we confirm that channel expression is not altered in mdx myocytes (Fig. S1). However, in mdx myocytes, the inactivation of the current was significantly slower (τ = 26.15 ± 1.75 vs. 21.06 ± 1.29 ms; Fig. 1 A and B). Consistent with a delayed inactivation rate, we have demonstrated that the inactivation integral of current and total integral of current are significantly greater in myocytes from mdx hearts compared with myocytes from wt hearts (12). Additionally, the activation integral of current in mdx and wt myocytes does not differ (12). The delayed inactivation of the current persists in the mdx myocyte when barium is used as a carrier (13). We cannot definitively state that alterations in intracellular calcium are not responsible for the response; however, these findings suggest that the delay in inactivation may occur as a result of alterations in cytoskeletal structure. Consistent with this argument and with previous reports, we find that immobilizing the beta subunit of I_Ca-L by exposing wt myocytes to a peptide derived against the alpha-interacting domain (AID) of I_Ca-L slows inactivation of the current (AID-TAT; Fig. 1 A, Inset) (6, 9, 10). Because the auxiliary beta subunit regulates inactivation of I_Ca-L current (14, 15), we propose that the alterations in I_Ca-L inactivation in the mdx myocyte result from disruption of the cytoskeleton due to the absence of dystrophin.

Fig. 1.

Absence of dystrophin alters I_Ca-L current and Ψ_m in mdx myocytes. (A) I_Ca-L current traces recorded from a myocyte from a mdx heart and a myocyte from a wt heart as indicated. (A, Inset) I_Ca-L current traces from a wt myocyte (250 pF) exposed to a peptide derived against the alpha-interacting domain of I_Ca-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 I_Ca-L results in an increase in Ψ_m 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 Ψ_m 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.

Direct Activation of I_Ca-L Increases Ψ_m in wt but Not mdx Myocytes.

We have demonstrated that activation of I_Ca-L with application of DHPR agonist BayK(-) or voltage clamp of the plasma membrane results in an increase in Ψ_m in guinea-pig ventricular myocytes (9). BayK(-) increases I_Ca-L in the mdx myocyte (12). We examined whether the absence of dystrophin altered the increase in Ψ_m after application of Bay K(-) in myocytes from mdx mice. We performed experiments in quiescent ventricular myocytes with consistent ATP utilization because this approach allowed us to more readily explore the effects of I_Ca-L activation on mitochondrial function. Exposure of wild-type (wt) myocytes to BayK(-) increased Ψ_m measured as changes in JC-1 signal by 7.61 ± 1.04% compared with wt myocytes exposed to inactive enatiomer BayK(+) (Fig. 1 C and E). The response could be attenuated with application of I_Ca-L antagonist nisoldipine. However, in mdx myocytes, BayK(-) could not elicit an increase in JC-1 signal (Fig. 1 D and F). Myocytes from mdx hearts exhibited functional electron transport because the ATP synthase blocker oligomycin induced a significant increase in JC-1 signal (Fig. 1 D and F). To further confirm that the signal was measuring changes in Ψ_m, we induced a collapse of Ψ_m with application of potassium cyanide (KCN) (Fig. 1 C and D).

Alterations in Ψ_m After Activation of I_Ca-L Do Not Require Calcium.

Mitochondrial membrane potential can function independent of changes in intracellular calcium in the range of 0–400 nM (16). We tested the effect of activation of I_Ca-L on Ψ_m in wt and mdx myocytes in calcium-free and EGTA-containing Hepes-buffered solution (HBS). Exposure of wt myocytes to BayK(-) increased Ψ_m measured as changes in JC-1 signal by 12.1 ± 1.4% compared with wt myocytes exposed to inactive enatiomer BayK(+) (Fig. 2 A, i and D). The response could be attenuated with application of I_Ca-L antagonist nisoldipine. In some experiments, wt myocytes were incubated in calcium-free EGTA containing HBS for at least 3 h and perfused intracellularly with 3 mM EGTA and 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) to deplete intracellular calcium stores. The wt myocytes exhibited similar increases in Ψ_m after activation of I_Ca-L (Fig. 2 A, ii). To further confirm whether calcium was involved in the response, wt myocytes that were incubated in calcium-free EGTA containing HBS for at least 3 h as described above were also exposed to caffeine (2 μM) that stimulates release of calcium from ryanodine receptors in the sarcoplasmic reticulum. There was no significant alteration in JC-1 signal after the addition of caffeine (Fig. 2 C and D). Exposure of wt myocytes to caffeine and BayK(-) resulted in an increase in JC-1 signal similar to that of BayK(-) alone (Fig. 2 C and D).

Fig. 2.

Direct activation of I_Ca-L results in an increase in Ψ_m 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 Ψ_m 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.)

However, in mdx myocytes, BayK(-) could not elicit an increase in JC-1 signal (Fig. 2 B and E). The mitochondria in myocytes from mdx hearts exhibited functional electron transport because the ATP synthase blocker oligomycin induced a significant increase in JC-1 signal (Fig. 2E). These data confirm that the alteration in Ψ_m in response to BayK(-) does not depend on changes in intracellular calcium (9, 17). We propose that the loss of regulation of Ψ_m by I_Ca-L in the mdx myocyte results from disruption of the cytoskeleton due to the absence of dystrophin.

To determine whether the response depended on the presence of dystrophin, we injected mdx mice with 10 mg/kg per wk phosphorodiamidate morpholino oligomer (PMO) targeting mouse dystrophin exon 23 for 24 wk. This treatment induces removal of exon 23 during processing of the primary transcript and results in expression of a slightly shorter dystrophin protein (18). Similar therapy is being administered to Duchenne muscular dystrophy patients in clinical trials with excellent results demonstrated as maintaining ambulation (19, 20). Uptake of the oligomers in mouse cardiac muscle can be detected as evidence of exon skipping on RT-PCR (Fig. 2 F, i), by the presence of dystrophin on immunoblot (Fig. 2 F, ii) and immunohistochemistry staining in cryosections (Fig. 2G). We find that BayK(-) can elicit a significant increase in JC-1 signal in myocytes isolated from hearts of mdx mice treated with PMOs, indicating that treatment with oligomers “rescued” the JC-1 response (Fig. 2 B and E). These data confirm that the alteration in Ψ_m in response to BayK(-) requires dystrophin.

Activation of I_Ca-L Increases Metabolic Activity in wt but Not mdx Myocytes.

We examined whether activation of I_Ca-L altered metabolic activity in myocytes isolated from mdx hearts. Consistent with previous results (9), BayK(-) elicited a significant increase in metabolic activity (assessed as formation of formazan from tetrazolium salt) in myocytes from wt mice (Fig. 3 A and B). The response could be attenuated with application of nisoldipine or block of calcium uptake into the mitochondria (Ru360), but not with an inhibitor of the ryanodine receptor (dantrolene; Fig. 3B). However, in myocytes from mdx hearts, BayK(-) could not elicit an increase in metabolic activity (Fig. 3 A and C). Application of oligomycin caused a significant decrease in metabolic activity, indicating the cells were metabolically active (Fig. 3C).

Fig. 3.

Direct activation of I_Ca-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.

We also examined changes in mitochondrial electron transport by measuring alterations in flavoprotein oxidation (as autofluorescence) in the myocytes in response to activation of I_Ca-L. In wt myocytes, BayK(-) caused a small but significant increase in flavoprotein oxidation that could be attenuated with application of nisoldipine (Fig. 3 D and F). However, BayK(-) could not elicit an increase in flavoprotein oxidation in mdx myocytes despite an increase recorded in response to application of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (Fig. 3 E and G). To determine whether the response depended on the presence of dystrophin, we examined the effect of BayK(-) on flavoprotein oxidation in myocytes isolated from hearts of PMO-treated mdx mice. We find that BayK(-) can elicit a significant increase in flavoprotein oxidation in myocytes isolated from hearts of PMO-treated mdx mice, indicating that treatment with oligomers partially rescued the response (Fig. 3 E and G). These data provide evidence that loss of regulation of metabolic activity by I_Ca-L in the mdx myocyte results from disruption of the cytoskeleton due to the absence of dystrophin.

I_Ca-L Associates with the Mitochondrial VDAC via Cytoskeletal Proteins.

We have demonstrated that activation of I_Ca-L results in an increase in Ψ_m in guinea-pig ventricular myocytes, and the response depends on an intact cytoskeleton because disruption of F-actin filaments attenuated the increase in Ψ_m (9). We assessed alterations in Ψ_m in myocytes from wt mice treated with either latrunculin A to depolymerize F-actin filaments, or a peptide directed toward the AID of I_Ca-L (AID-TAT) to immobilize the auxiliary beta subunit of I_Ca-L. Myocytes treated with either latrunculin A or AID-TAT demonstrated attenuation of increased Ψ_m following exposure to BayK(-) (Fig. 4 A and E). These data suggest that the response depends on an intact cytoskeletal architecture.

Fig. 4.

VDAC coimmunoprecipitates with I_Ca-L, and direct block of VDAC restores the increase in Ψ_m in mdx myocytes. (A) Regulation of Ψ_m by I_Ca-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 Ψ_m as indicated. ES, external solution; IS, internal solution. (B) I_Ca-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-I_Ca-L antibody (Upper) and 10 μg immunoprecipitated I_Ca-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 I_Ca-L protein from wt (Left) and mdx (Right) heart by probing with α-GOLD antibody (Lower). (ii) Mass spectrometry analysis of immunoprecipitated I_Ca-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 Ψ_m 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 Ψ_m as indicated. ES, external solution; IS, internal solution. (D) Application of a peptide that blocks VDAC restores the increase in Ψ_m 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.

Voltage-dependent anion channel (VDAC) resides in the outer mitochondrial membrane and associates with the adenine nucleotide translocator (ANT) that is located in the inner mitochondrial membrane (21). The VDAC/ANT complex is responsible for trafficking of ATP/ADP in and out of the mitochondria (22). Because Ψ_m depends on the function of the VDAC/ANT complex, we investigated whether I_Ca-L was associated with the outer mitochondrial membrane protein VDAC. We immunoprecipitated VDAC protein from hearts of 8-wk-old wt and mdx mice and immunoblotted the protein against an I_Ca-L antibody. We identified a band at ∼220 kDa on immunoblot consistent with I_Ca-L protein (Fig. 4 B, i). Conversely, when we immunoprecipitated I_Ca-L protein and probed the protein with anti-VDAC antibody, we identified a protein at 37 kDa, consistent with VDAC (Fig. 4 B, i). The immunoprecipitated I_Ca-L proteins from wt heart and mdx heart were analyzed by mass spectrometry on a Velos Orbitrap (Tables S1–S4). A number of similar proteins were identified in immunoprecipitated protein from wt heart and mdx heart, including the α_1C pore forming and auxiliary beta subunits of I_Ca-L. Known regulators of I_Ca-L function were identified, including β-tubulin, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and A-kinase anchor protein (AKAP) (23–25) (Fig. 4 B, ii). Proteins that were identified in wt hearts and not mdx hearts, and proteins identified in mdx hearts but not wt hearts, are listed in Table S4. Consistent with our in vitro data (Fig. 4 A and E), F-actin was identified as a cytoskeletal partner with I_Ca-L. In addition, VDAC and ANT were identified in the immunoprecipitated I_Ca-L protein. These data suggest I_Ca-L associates with the mitochondrial VDAC via cytoskeletal proteins, one of which is F-actin. The disrupted cytoskeletal architecture due to the absence of dystrophin leads to altered function between I_Ca-L and mitochondria.

Preventing Anion Flux Through the VDAC “Restores” the Increase in Ψ_m in mdx Myocytes.

We directly blocked anion transport from the outer mitochondrial membrane with application of a peptide derived against VDAC. The VDAC peptide blocks VDAC at the mitochondrial outer membrane. In wt myocytes, application of the VDAC peptide increased JC-1 signal in a similar manner to BayK(-) (Fig. 4 C, ii and E). A scrambled VDAC peptide had no effect. When the VDAC peptide was applied to mdx myocytes, the JC-1 signal increased significantly (Fig. 4 D, ii and F) in a similar manner to the response recorded after application of BayK(-) alone in wt myocytes (Fig. 4 C, ii and E). This response was also observed in myocytes incubated in calcium-free EGTA containing HBS for at least 3 h and perfused intracellularly with EGTA and BAPTA to deplete intracellular calcium stores (wt, Fig. 4 C, i; mdx, Fig. 4 D, i). These data confirm that mitochondria exhibit functional electron transport in mdx myocytes because a block of VDAC can increase Ψ_m. Application of VDAC restored the increase in Ψ_m in mdx myocytes. Taking into account that I_Ca-L immunoprecipitates with VDAC (Fig. 4 B, i and ii), we suggest that activation of I_Ca-L may increase Ψ_m in wt myocytes via transmission of movement of cytoskeletal proteins to VDAC.

Activation of I_Ca-L Increases Ψ_m in wt but Not mdx Myocytes with Established Cardiomyopathy.

We isolated myocytes from 43-wk-old mdx hearts that had developed cardiomyopathy as assessed on echocardiography (26) (Table S5) and examined the effect of activation of I_Ca-L on Ψ_m. Similar to results recorded in myocytes from 8-wk-old mdx mice, exposure of 43-wk-old wt myocytes to BayK(-) increased Ψ_m measured as changes in JC-1 signal by 9.74 ± 2.76% compared with wt myocytes exposed to inactive enantiomer BayK(+) (Fig. 5 A and C). The response could be attenuated with application of I_Ca-L antagonist nisoldipine. However, in 43-wk-old mdx myocytes BayK(-) could not elicit an increase in JC-1 signal (Fig. 5 B and D). Myocytes from mdx hearts exhibited functional electron transport because the ATP synthase blocker oligomycin induced a significant increase in JC-1 signal (Fig. 5D). To further confirm that the signal was measuring changes in Ψ_m, we induced a collapse of Ψ_m with application of KCN.

Fig. 5.

Direct activation of I_Ca-L results in an increase in Ψ_m 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 Ψ_m 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).

We also examined the effect of activation of I_Ca-L on metabolic activity in myocytes isolated from 43-wk-old mdx hearts. Similar to results recorded in myocytes from 8-wk-old wt mice, BayK(-) elicited a significant increase in metabolic activity in myocytes from wt mice (Fig. 5 E and F). The response could be attenuated with application of nisoldipine or block of calcium uptake into the mitochondria (Ru360) but not with an inhibitor of the ryanodine receptor (dantrolene; Fig. 5F). However, in myocytes from 43-wk-old mdx hearts BayK(-) could not elicit a further increase in metabolic activity (Fig. 5 E and G). Application of oligomycin caused a significant decrease in metabolic activity, indicating the cells were metabolically active (Fig. 5G).

To determine whether the alterations in metabolic activity in the mdx myocyte were specific to the mitochondria, we isolated mitochondria from hearts of 43-wk-old mdx mice. Respiratory complex activity and oxygen consumption were similar to that recorded in mitochondria isolated from 43-wk-old wt hearts (Fig. 5H). These data confirm that alterations in mitochondrial function occur early and persist up to the development of cardiomyopathy.

Taken together, our data suggest that the alterations in mitochondrial function depend on activation and inactivation of I_Ca-L in the intact cell and occur because of altered transmission of movement from I_Ca-L through cytoskeletal proteins to the mitochondria.

Discussion

Duchenne muscular dystrophy is a fatal X-linked disease characterized by the absence of dystrophin that is associated with cytoskeletal protein disarray, contractile dysfunction, and reduced energy production (1, 2). However, the mechanisms for altered energy metabolism are not fully understood. We have shown that activation of I_Ca-L alters mitochondrial function via the movement of cytoskeletal proteins (9, 12). The response depends on an intact cytoskeleton (9). In this study, we examined whether the lack of dystrophin (and associated cytoskeletal disarray; refs. 27–29) in ventricular myocytes isolated from mdx mice results in impaired communication between I_Ca-L and the mitochondria.

First, we characterized the kinetics of I_Ca-L current inactivation. We and others have demonstrated that disruption of the cytoskeleton alters I_Ca-L kinetics (11–13). We find that myocytes from mdx hearts exhibit a delayed inactivation of I_Ca-L (Fig. 1A). This finding is consistent with an alteration in movement of the auxiliary beta subunit of I_Ca-L because immobilization of the beta subunit with a peptide derived against the alpha-interacting domain also slows inactivation of the current (Fig. 1A, Inset). This finding is also consistent with previous studies that have demonstrated that dissociation of microtubules or depolymerization of actin alters the inactivation rate of I_Ca-L (5, 23, 29).

Disruption of the cytoskeleton is associated with alterations in mitochondrial function. Unlike wt myocytes, Ψ_m did not increase after activation of I_Ca-L in mdx myocytes (Figs. 1 and 2). In addition, metabolic activity and oxidation of flavoprotein did not increase after activation of I_Ca-L in mdx myocytes (Fig. 3). Consistent with previous studies (2), these data suggest that metabolic activity in the mdx myocyte is significantly different from the wt myocyte. Similar responses were observed in mdx myocytes isolated from hearts of 43-wk-old mice that had developed cardiomyopathy (Fig. 5). However, respiration was normal in mitochondria isolated from 43-wk-old mice, indicating that the responses depend on an association between I_Ca-L and the cytoskeleton in the intact cell (Fig. 5). Consistent with this finding, previous studies in desmin-null mice have demonstrated abnormal mitochondrial respiration in vivo, whereas the rates of maximal respiration in isolated mitochondria from desmin-null mice were similar to mitochondria isolated from wt mice (30). In another study, although mitochondrial distribution was normal in the hearts of 10- to 11-mo-old mdx mice, a lack of dystrophin was associated with impaired coupling of mitochondrial-creatine kinase to oxidative phosphorylation and altered ADP uptake by the mitochondria only in intact skinned fibers (31). Our data demonstrate that cytoskeletal disruption due to the absence of dystrophin results in the loss of regulation of mitochondrial function by I_Ca-L in intact mdx myocytes. Because I_Ca-L initiates contraction in the beating heart, we propose that altered communication between I_Ca-L and the mitochondria contributes to poor metabolic activity in the mdx contracting heart demonstrated as a decrease in fractional shortening on echocardiography (Table S5). Although cytosolic and mitochondrial calcium are increased in the mdx myocyte (12, 32, 33), we propose that altered regulation of VDAC by I_Ca-L may contribute to the poor metabolic activity.

In this study, we demonstrate that treatment of mice with a PMO targeting dystrophin restores regulation of mitochondrial function by I_Ca-L in the mdx myocyte (Figs. 2 and 3). These data confirm that the absence of dystrophin is responsible for the lack of response of the mitochondria to activation of I_Ca-L. The data also demonstrate that treatment with PMOs offers a promising therapy for Duchenne muscular dystrophy cardiomyopathy.

VDAC resides in the outer mitochondrial membrane and associates with ANT that is located in the inner mitochondrial membrane (21). The VDAC/ANT complex is responsible for trafficking of ATP/ADP in and out of the mitochondria (22). VDAC and the cytoskeletal protein β-tubulin are closely associated and may play a role in regulation of mitochondrial respiration (34, 35). We examined whether I_Ca-L is associated with VDAC by immunoprecipitating the I_Ca-L protein. Immunoblot and pull-down analysis confirmed that VDAC associates with I_Ca-L (Fig. 4B). In addition, we provide functional evidence that the I_Ca-L communicates with VDAC. We demonstrate that preventing the opening of VDAC with application of a peptide directed toward VDAC restores the increase in JC-1 in mdx hearts (Fig. 4 C and D). These data to our knowledge are the first to demonstrate that I_Ca-L communicates with the mitochondria through an association with outer membrane mitochondrial protein VDAC via cytoskeletal proteins. We propose that communication between I_Ca-L and the mitochondria occurs via transmission of movement from I_Ca-L (during activation and inactivation of I_Ca-L) to mitochondrial VDAC through F-actin (Fig. 6A). We find that this structural and functional communication between I_Ca-L and the mitochondria is altered in hearts lacking dystrophin (Fig. 6B). Abnormalities occur only in the intact cell.

Fig. 6.

Schematic representation of communication of I_Ca-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).

The VDAC/ANT complex is responsible for shuttling ATP out of the mitochondria (36). This shuttling is critical to meeting energy demands of the heart. Here, we find that the absence of dystrophin and disrupted cytoskeletal architecture in the mdx ventricular myocyte leads to impaired communication between I_Ca-L and mitochondrial VDAC. This alteration results in reduced metabolic activity in the mdx ventricular myocyte. These findings provide new mechanistic and functional insight into cardiomyopathy associated with Duchenne muscular dystrophy.

Materials and Methods

Isolation of Ventricular Myocytes.

Myocytes were isolated from 8-wk-old and 43-wk-old male C57BL/10ScSn-Dmdmdx/Arc (mdx) and C57BL/10ScSnArc wt mice. A total number of 110 wt and 67 mdx mice were used. Four mdx mice were treated with PMO at 10 mg/kg per week by i.p. injection for 24 wk from 7 d of age. Cells were isolated as described (37). For full details, see SI Materials and Methods.

Data Acquisition for Patch-Clamp Studies.

The whole-cell configuration of the patch-clamp technique was used to measure changes in I_Ca-L currents in intact ventricular myocytes (37, 38). Microelectrodes with tip diameters of 3–5 μm and resistances of 0.5–1.5 MΩ 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). Barium currents were measured by using internal solution containing the following: 125 mM CsCl, 10 mM Hepes, 0.1 mM EGTA, 20 mM tetraethylammonium chloride, 5 mM MgATP, 0.1 mM Tris-GTP, and 10 mM phosphocreatine (pH adjusted to 7.05 at 37 °C with CsOH), and external solution containing the following: 152.5 mM tetraethylammonium chloride, 10 mM Hepes, and 10 mM BaCl₂ [pH adjusted to 7.4 with Ba(OH)₂]. Macroscopic currents were recorded by using an Axopatch 200B voltage-clamp amplifier (Molecular Devices) and an IBM compatible computer with a Digidata 1322A interface and pClamp9 software (Molecular Devices). A Ag/AgCl electrode was used to ground the bath. Once the whole-cell configuration was achieved, the holding potential was set at –80 mV. Na⁺ channels and T-type Ca²⁺ channels were inactivated by applying a 50-ms prepulse to –30 mV immediately before each test pulse. The time course of changes in Ca²⁺ conductance were monitored by applying a 100-ms test pulse to 10 mV once every 10 s. All experiments were performed at 37 °C.

Fluorescent Studies.

The fluorescent indicator 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide was used to measure Ψ_m at 37 °C as described (38). For full details, see SI Materials and Methods. To confirm that the JC-1 signal was indicative of Ψ_m, 4 mM KCN was added at the end of each experiment to collapse Ψ_m. For calcium-free experiments, cells were exposed to calcium-free HBS (containing 3 mM EGTA) for at least 3 h, before measuring changes in Ψ_m. In some experiments, cells were exposed to calcium-free HBS for at least 3 h and then patch-clamped with pipette solution containing the following: 115 mM CsCl, 10 mM Hepes, 5 mM BAPTA, 3 mM EGTA, 20 mM tetraethylammonium chloride, 5 mM MgATP, 0.1 mM Tris-GTP, 10 mM phosphocreatine (pH adjusted to 7.05 at 37 °C with CsOH), and 200 nM JC-1. Changes in Ψ_m were then assessed.

Flavoprotein autofluorescence was used to measure flavoprotein oxidation in intact mouse cardiac myocytes based on described methods (39). For full details, see SI Materials and Methods. Ten micromolar FCCP was added at the end of each experiment to achieve a maximum fluorescence value indicative of maximum flavoprotein oxidation.

RNA Preparation and RT-PCR Analysis, Immunostaining, and Immunoblot.

Total RNA was extracted from the hearts, and RT-PCR was performed for analysis of exons 22–24 (Table S6). Dystrophin protein was stained on diaphragm and heart cryosections by using NCL-Dys2 and Zenon Alexafluor 488. Sections from untreated mdx and wt mice are included for comparison. All images were captured and processed by using identical parameters (18, 40). Dystrophin protein was assessed on immunoblot as described (40, 41). For full details, see SI Materials and Methods.

Yellow Tetrazolium Salt Assay.

The rate of cleavage of yellow tetrazolium salt to purple formazan crystals by the mitochondrial electron transport chain was measured spectrophotometrically at 570 nm with a reference wavelength of 620 nm at 37 °C as described (9). Each n represents number of animals used in each treatment group for the assay. For full details, see SI Materials and Methods.

Immunoprecipitation of I_Ca-L and VDAC Protein and Immunoblot.

Membrane fractions were prepared from crude heart homogenates as described (42). Immunoprecipitation and immunoblot were performed as described (25, 42), using either anti-I_Ca-L antibody (ACC-004; Alomone Labs) or anti-porin antibody (MSA03; MitoSciences). Immunoprecipitation was performed by using PureProteome Protein G Magnetic Beads (Millipore). Rabbit polyclonal antibody raised in house against a gold N-heterocyclic carbene compound (α-GOLD) or serum from pooled BALB/c and C57BL/6 mice (MS) were used as negative controls. For full details, see SI Materials and Methods.

Mass Spectrometry Analysis of Immunoprecipitated I_Ca-L Protein.

Digest peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex) at The Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney. Samples were analyzed by using an Orbitrap Velos (Thermo Electron) mass spectrometer. For full details, see SI Materials and Methods.

Mitochondrial Respiration Studies.

Mitochondrial respiration was measured in mitochondria isolated from three pooled wt mouse hearts and from three pooled mdx mouse hearts by using a OROBOROS high resolution respirometer thermostatically maintained at 37 °C as described (43). For full details, see SI Materials and Methods.

Statistical Analysis.

Results are reported as mean ± SEM. Statistical comparisons of responses between unpaired data were made by using the Student t test or between groups of cells by using one-way ANOVA and the Tukey’s post hoc test (GraphPad Prism version 5.04).