The Role of the L-Type Ca2+ Channel in Altered Metabolic Activity in a Murine Model of Hypertrophic Cardiomyopathy.

Heterozygous mice (αMHC403/+ ) expressing the human disease-causing mutation Arg403Gln exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+ mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+ mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+ cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+ . These increases were attenuated with ICa-L antagonists and following F-actin or β-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+ mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the Arg403Gln mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.

Myosin heavy chain (MHC) consists of a myosin carboxyl terminal rod and an amino terminal globular head that interacts with actin 1, 2. During contraction, force is transduced via a hinge region between these 2 domains, allowing attachment–detachment of the myosin head with actin filaments. There are 2 cardiac-specific isoforms of MHC: α-cardiac MHC and β-cardiac MHC. In humans, β-MHC predominates in adult life, accounting for >90% of ventricular myosin (3). Genetic mutations in contractile protein β-MHC account for approximately 40% of genotyped families with hypertrophic cardiomyopathy (HCM) 4, 5, 6.

The human β-MHC mutation MYH7 Arg403Gln causes a severe form of HCM characterized by early-onset and progressive myocardial dysfunction with a high incidence of sudden cardiac death (7). However, the relationship between the gene mutation and phenotype is poorly understood.

Heterozygous mice expressing the human disease–causing mutation Arg403Gln (αMHC) exhibit hallmark features of the cardiomyopathy, including hypertrophy, myocyte disarray, and increased myocardial fibrosis (8). We have previously demonstrated that treatment of αMHC mice with the L-type calcium channel (ICa-L) antagonist diltiazem decreases left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis 9, 10. In addition, administration of diltiazem to patients with HCM improves left ventricular end-diastolic diameter and left ventricular wall thickness-to-dimension ratio (10). However, the role of ICa-L in development of the cardiomyopathy is currently unknown.

The cytoskeleton consists of microtubules composed of tubulin, microfilaments composed of actin, and intermediate filaments, and is recognized as a modulator of cell morphology, motility, intracytoplasmic transport, and mitosis 11, 12. Cytoskeletal proteins also regulate the function of proteins in the plasma membrane 13, 14. ICa-L is anchored to F-actin and β-tubulin that regulate ICa-L activation and inactivation kinetics 15, 16, 17, 18. In addition, the mitochondrial outer membrane contains docking sites for cytoskeletal proteins that can regulate mitochondrial function 12, 19, 20.

Calcium influx through the ICa-L or dihydropyridine channel is critical to cardiac excitation and contraction. ICa-L can also regulate mitochondrial function. Activation of ICa-L with voltage clamp of the plasma membrane or with application of the dihydropyridine receptor agonist Bay K8644 (BayK(-)) is sufficient to increase cytosolic and mitochondrial calcium, in addition to NADH production, superoxide generation, and metabolic activity in a calcium-dependent manner 21, 22. Activation of ICa-L can also increase mitochondrial membrane potential (Ψm) in a calcium-independent manner (21). The response is reversible upon inactivation of ICa-L and is in part dependent on F-actin filaments because depolymerization of F-actin prevents the response (21). The beta subunit (β2) of ICa-L is tethered to cytoskeletal proteins. Preventing movement of the β2 subunit with application of a peptide derived against the alpha-interacting domain of ICa-L attenuates the increase in Ψm (21). Therefore, ICa-L influences metabolic activity through transmission of movement of ICa-L via cytoskeletal proteins.

We and others have demonstrated that αMHC mice exhibit increased actin–myosin sliding velocity, force generation, increased ATPase activity, and ADP concentration 23, 24. Here, we sought to identify whether the Arg403Gln mutation leads to mitochondrial dysfunction in cardiac myocytes isolated from 30- to 50-week-old αMHC mice with established cardiomyopathy 8, 25. Specifically, we investigated whether the mutation resulted in altered communication between the ICa-L and mitochondria, and subsequently, altered metabolic activity.

Methods

Mouse models

Male 30- to 50-week-old and 10- to 15-week-old mice expressing the human disease–causing mutation Arg403Gln (αMHC) were generated (8) and studied. The mice develop cardiomyopathy by 30 to 50 weeks as evidenced by echocardiography and heart weight to body weight measurements (Supplemental Table 1). Genotype-negative littermate age-matched male mice were used as wild-type (wt) controls. Hearts were extracted as approved by The Animal Ethics Committee of The University of Western Australia in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NH&MRC, 8th Edition, 2013). Cardiac myocytes were isolated as previously described 26, 27. Detailed methods are provided in the Supplemental Methods.

Data acquisition for patch clamp studies

Whole-cell configuration of the patch clamp technique was used to measure changes in ICa-L currents in intact cardiac myocytes as described previously 28, 29. Detailed methods are provided in the Supplemental Methods.

Fluorescent studies

All studies were performed in intact mouse cardiac myocytes at 37°C as previously described. Intracellular calcium ([Ca2+]i) was monitored using Fura-2 (29). Superoxide generation was assessed using dihydroethidium (29). Fluorescent indicator JC-1 was used to measure Ψm (29). Flavoprotein autofluorescence was used to measure flavoprotein oxidation (30). Detailed methods are provided in the Supplemental Methods.

MTT assay

The rate of cleavage of the tetrazolium salt MTT to formazan by the mitochondrial electron transport chain was measured spectrophotometrically as previously described 21, 26. Detailed methods are provided in the Supplemental Methods.

Mitochondrial respiration studies and DNA copy number

Mitochondrial respiration was measured in mitochondria isolated from 3 pooled wt and 3 pooled αMHC mouse hearts at 37°C as previously described (31). Detailed methods are provided in the Supplemental Methods. Mitochondrial DNA copy number was determined by quantitative reverse-transcription polymerase chain reaction as previously described (32).

Quantitative reverse-transcription polymerase chain reaction

Transcript abundance of mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma (PPARγ) and peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 was measured as previously described (33). Detailed methods are provided in the Supplemental Methods.

Sample preparation for transmission electron microscopy and confocal imaging

For transmission electron microscopy, cardiac tissue samples were imaged on a JEOL JEM-2100 electron microscope (JEOL, Akishima, Japan). For confocal imaging, cardiac myocytes tripled stained with MitoTracker (mitochondria), phalloidin (F-actin) and DAPI (nuclei) (Thermo Fisher Scientific, Massachusetts) were imaged on an Olympus IX71 inverted fluorescent microscope (Olympus, Tokyo, Japan).

Detailed methods are provided in the Supplemental Methods.

Statistical analysis

Results are reported as mean ± SEM or SD where indicated. Statistical comparisons of parametric data were made using the unpaired Student t test (GraphPad Prism version 5.04, GraphPad Software, La Jolla, California). Statistical comparisons of non-parametric data were made using the Mann-Whitney U test, or Kruskal-Wallis test (GraphPad Prism version 5.04).

Results

αMHC cardiac myocytes exhibit altered ICa-L inactivation kinetics

Using the patch clamp technique, we measured ICa-L currents in αMHC myocytes (Figure 1A). We found no difference in ICa-L current density recorded in αMHC versus wt myocytes (αMHC 3.86 ± 0.26 pA/pF vs. wt 3.91 ± 0.30, p = NS) (Figures 1C and 1F). These data suggest that ICa-L expression is not altered in αMHC myocytes. To further confirm this, we probed immunoblots of ICa-L protein isolated from αMHC hearts with an antibody directed against the pore-forming α1C subunit. Densitometry analysis indicated a slight increase (8.2 ± 0.6%) in α1C subunit expression in αMHC hearts (Supplemental Figures 1A and 1B), but this did not appear to be sufficient to increase peak inward current and current density (Figures 1A, 1C, and 1F). However, inactivation of the current was significantly faster in αMHC versus wt myocytes (αMHC: τ1 = 32.76 ± 1.96 versus wt: τ1 = 40.68 ± 2.49, p < 0.05) (Figure 1B). Similar results were obtained when barium was used as the charge carrier indicating that changes in calcium were not mediating alterations in current inactivation (Figures 1H to 1J). The total integral of current in αMHC myocytes were significantly less compared with wt myocytes, whereas no difference in activation integral was observed (Figures 1D and 1E). No difference in steady-state inactivation was observed in αMHC versus wt myocytes (Figure 1G). Consistent with our results indicating no difference in ICa-L current density or peak inward current in αMHC myocytes, we found no difference in intracellular calcium ([Ca2+]i) in αMHC versus wt myocytes (Supplemental Figures 2A to 2C).

Figure 1

Myocytes Isolated From αMHC Hearts Exhibit Altered Inactivation Kinetics

(A) Representative ICa-L current traces from αMHC (130 pF) and wild-type (wt) (120 pF) myocytes. (Inset) Pulse protocol. (B) Mean ± SEM rate of inactivation (tau) of current for αMHC and wt myocytes fitted with 2 exponential functions (i, τ 1; and ii, τ 2). Mean ± SEM of (C) current density, and (D) activation integral and (E) total integral of current for αMHC and wt myocytes. (F) Current/voltage (I–V) relationship and (G) voltage dependency of steady-state inactivation measured in αMHC and wt myocytes. (Insets) Pulse protocols. (H) Representative ICa-L current traces recorded from αMHC (100 pF) and wt (100 pF) with barium as charge carrier. (Inset) Pulse protocol. (I) Mean ± SEM of inactivation (tau) of current for αMHC and wt myocytes fitted with 2 exponential functions with barium as charge carrier (i, τ 1; and ii, τ 2). (J) Mean ± SEM of current density for all myocytes with barium as charge carrier. The unpaired Student t test was used for comparisons in B and C; Mann-Whitney test was used for comparisons in D to G and I to J.

The β2 subunit of ICa-L is bound to the α1C subunit of ICa-L and plays an important role in ICa-L kinetics (34). We probed immunoblots of ICa-L protein with an antibody directed against the β2 subunit. No significant alteration in β2 subunit expression was observed in αMHC versus wt hearts (Supplemental Figures 1C and 1D). Because the β2 subunit of ICa-L is tethered to F-actin filaments that also tightly regulate the function of ICa-L 15, 16, 17, these data suggest that cytoskeletal architecture rather than altered α1C subunit or β2 subunit expression may be responsible for altered inactivation of ICa-L current in αMHC myocytes.

αMHC cardiac myocytes exhibit a significantly larger increase in Ψm following activation of ICa-L

Increased mitochondrial Ca2+ uptake is associated with an increase in Ψm. However, Ψm can function independently of changes in [Ca2+]i in the range of 0 to 400 nmol/l (35). We have previously shown that adult guinea pig cardiac myocytes exhibit increased Ψm following activation of ICa-L under calcium-free conditions (21). The response is dependent upon an intact cytoskeletal architecture (21).

Here, we find that application of BayK(-) elicits a significant increase in Ψm in αMHC and wt myocytes pre-incubated in calcium-free and EGTA containing HEPES-Buffered Solution for at least 3 hours (assessed as changes in JC-1 fluorescence) (Figures 2A to 2C). The responses were similar to those recorded in 2.5 mmol/l calcium containing Hepes-Buffered Solution (Supplemental Figure 3A). However the ratio of the response was significantly larger in αMHC versus wt myocytes (Figure 2D). The responses could be prevented with application of ICa-L antagonists nisoldipine or diltiazem (Figures 2A to 2C). Application of BayK(+) did not significantly alter Ψm in αMHC or wt myocytes (Figures 2A to 2C). Sodium cyanide was added to collapse Ψm demonstrating that the signal was mitochondrial and indicative of Ψm (Figures 2A and 2B). No difference was observed in basal Ψm in αMHC versus wt myocytes (Supplemental Figures 3B and 3C). These data demonstrate that activation of ICa-L causes a significantly greater increase in Ψm in myocytes isolated from αMHC hearts compared with wt myocytes, and the response does not require calcium.

Figure 2

αMHC Cardiac Myocytes Exhibit a Significantly Larger Increase in Ψm Following Activation of ICa-L

Representative ratiometric JC-1 fluorescence recorded in (A)wt myocytes and (B)αMHC myocytes before and after exposure to 10 μmol/l BayK(+) or BayK(-) ± 15 μM nisoldipine (Nisol) or diltiazem (Dilt) under calcium-free conditions (0 mmol/l Ca2+). Arrow indicates addition of drugs. NaCN: 40 mmol/l sodium cyanide. (C) Mean ± SEM of JC-1 fluorescence for all myocytes exposed to BayK(+), BayK(-), Nisol, Dilt, 5 μmol/l latrunculin A (Latrunc), or 1 μmol/l colchicine (Colch) as indicated. Latrunc and Colch were added 20 min and 3.5 h before commencing basal Ψm recording, respectively. (D) Ratio of increase in JC-1 fluorescence after addition of BayK(-). The Kruskal-Wallis test was used for all comparisons. ICa-L = L-type Ca2+ channel.

The β2 subunit of ICa-L is tethered to F-actin via subsarcolemmal stabilizing protein AHNAK (15). Mitochondria also associate with F-actin via mitochondrial docking proteins 36, 37, 38. We have previously demonstrated that ICa-L regulates mitochondrial function due to an association between ICa-L and the mitochondria via cytoskeletal protein F-actin 21, 26. We exposed αMHC myocytes to F-actin depolymerizing agent latrunculin A. Under calcium-free conditions, the increase in Ψm in response to BayK(-) was attenuated in αMHC and wt myocytes (Figure 2C). These data indicate that elevated Ψm in response to activation of ICa-L is dependent on cytoskeletal protein F-actin.

Regulation of Ψm is in part dependent on the mitochondrial voltage-dependent anion channel (VDAC) 39, 40. We have previously demonstrated that directly blocking VDAC (and anion transport from the outer mitochondrial membrane) mimics the effect of BayK(-) on Ψm in wt mouse myocytes 26, 41. In addition, it is known that the cytoskeletal protein β-tubulin associates with and regulates the function of VDAC (42). Therefore, we examined whether BayK(-)–induced alterations in Ψm were dependent upon β-tubulin in αMHC myocytes by incubating myocytes in the β-tubulin depolymerizing agent colchicine. Under calcium-free conditions, the increase in Ψm in response to BayK(-) was attenuated in αMHC and wt myocytes (Figure 2C). These data indicate that the increase in Ψm in response to activation of ICa-L is dependent on cytoskeletal protein β-tubulin.

αMHC cardiac myocytes exhibit a significantly larger increase in metabolic activity in response to activation of ICa-L

Metabolic activity is dependent upon oxygen consumption and electron flow down the inner mitochondrial membrane. Application of BayK(-) elicited a significant increase in metabolic activity in both αMHC and wt myocytes (Figures 3A and 3B). However, the ratio of the response was significantly larger in αMHC versus wt myocytes (Figure 3C). Both responses could be prevented with application of nisoldipine or the mitochondrial Ca2+ uniporter inhibitor Ru360, but not ryanodine (Figure 3B). Application of BayK(+) did not significantly alter metabolic activity in αMHC or wt myocytes (Figures 3A and 3B). Application of ATP synthase blocker oligomycin significantly decreased metabolic activity in αMHC and wt myocytes confirming the cells were metabolically active (Figure 3B).

Figure 3

αMHC Cardiac Myocytes Exhibit a Significantly Larger Increase in Metabolic Activity in Response to Activation of ICa-L

(A) Formation of formazan measured as change in absorbance in wt and αMHC myocytes after addition of 10 μmol/l BayK(+) or BayK(-). (B) Mean ± SEM of increases in absorbance for all myocytes exposed to BayK(+), BayK(-), 10 μmol/l nisoldipine (Nisol), 5 μmol/l Ru360, 5 μmol/l ryanodine (RyR), or 20 μmol/l oligomycin (Oligo) as indicated. (C) Ratio of increase in absorbance after addition of BayK(-). The Kruskal-Wallis test was used for all comparisons. Abbreviations as in Figure 1, Figure 2.

We examined changes in mitochondrial electron transport by measuring alterations in flavoprotein oxidation in myocytes isolated from αMHC hearts in response to activation of ICa-L. Application of BayK(-) caused a significant increase in flavoprotein oxidation in αMHC and wt myocytes (Figures 4A to 4C). The ratio of the increase in flavoprotein oxidation was significantly larger in αMHC versus wt myocytes (Figure 4D). Both responses could be prevented with application of nisoldipine (Figure 4C). Application of BayK(+) did not significantly alter flavoprotein oxidation in αMHC or wt myocytes (Figures 4A to 4C). FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) was added at the end of each experiment to increase flavoprotein signal confirming the signal was mitochondrial in origin (Figures 4A to 4C). These data indicate that activation of ICa-L causes a significantly greater increase in metabolic activity in myocytes isolated from αMHC compared to wt hearts.

Figure 4

αMHC Cardiac Myocytes Exhibit a Significantly Larger Increase In Flavoprotein Oxidation in Response to Activation of ICa-L

Representative traces of flavoprotein fluorescence recorded in (A)wt and (B)αMHC myocytes before and after exposure to 10 μmol/l BayK(-) or BayK(+). Arrow indicates addition of drugs. (C) Mean ± SEM of flavoprotein fluorescence for all myocytes exposed to BayK(+), BayK(-), 15 μmol/l nisoldipine (Nisol), or 50 μmol/l FCCP as indicated. (D) Ratio of increase in flavoprotein fluorescence after addition of BayK(-). The Kruskal-Wallis test was used for all comparisons. Abbreviations as in Figure 1, Figure 2.

Respiratory complex activity is similar in mitochondria isolated from hearts of αMHC and wt hearts

We performed respiratory electron transport chain complex activity and oxygen consumption measurements on mitochondria isolated from αMHC and wt hearts. No differences were observed in mitochondria isolated from αMHC versus wt hearts (Figure 5A). These data suggest that alterations in mitochondrial function observed in αMHC myocytes were cell intrinsic, and not secondary to hypertrophic remodeling of the ventricle.

Figure 5

Respiration Is Normal, but Gene Expression Is Altered in Mitochondria Isolated From αMHC Hearts

(A) Respiration and mitochondrial electron transport chain complex activity in mitochondria isolated from 3 pooled wt and 3 pooled αMHC hearts. (B) Mean ± SD of MtDNA copy number normalized to 18S rDNA. (C) Mean ± SD of gene expression of αMHC MtDNA copy number regulators TFAM, PPARγ and PGC1 relative to wt. (D) Mean ± SD of FGF21. (E and F) Representative TEM images demonstrating disordered distribution of mitochondria in αMHC(F) versus wt(E) heart sections. M = mitochondria; S = sarcomere. Scale = 1 μm. (G and H) Confocal imaging demonstrating disordered mitochondrial distribution and disorganization of F-actin in representative αMHC(H) versus wt(G) myocytes. Mitochondria shown in red (MitoTracker), F-actin shown in green (phalloidin), nuclei shown in blue (DAPI). Scale = 10 μm. The Mann-Whitney test was used for all comparisons. MtDNA = mitochondrial DNA; TEM = transmission electron microscopy; wt = wild type.

Mitochondrial DNA copy number and gene expression are altered in αMHC hearts

We found that mitochondrial DNA copy number was increased in αMHC versus wt hearts (Figure 5B). This correlated with increased expression of nuclear encoded regulators of the mitochondrial genome TFAM, PPARγ, and PGC-1 (Figure 5C). Because fibroblast growth factor 21 (FGF21) is a marker for mitochondrial dysfunction in myocytes 43, 44, we measured FGF21 in circulating blood from the mice. We measured a significant increase in FGF21 levels in αMHC versus wt mice, correlating with the onset of cardiomyopathy (Figure 5D). Transmission electron microscopy imaging revealed disordered mitochondrial distribution in αMHC versus wt hearts (Figures 5E and 5F). Additionally, confocal imaging revealed disordered mitochondrial distribution and disorganization of F-actin in αMHC versus wt myocytes (Figures 5G and 5H).

Pre-cardiomyopathic αMHC cardiac myocytes exhibit altered ICa-L inactivation kinetics and larger increases in Ψm and metabolic activity following activation of ICa-L

We assessed alterations in ICa-L inactivation kinetics and mitochondrial responses in cardiac myocytes from 10- to 15-week-old αMHC hearts that had not yet developed cardiomyopathy (Supplemental Table 1) 8, 25. Using the patch clamp technique, we measured ICa-L currents in the myocytes (Supplemental Figure 4A). Similar to 30- to 50-week-old myocytes, we found no difference in ICa-L current density in 10- to 15-week-old MHC versus age-matched wt myocytes (Supplemental Figure 4C). However, inactivation of the current was significantly faster in αMHC myocytes (Supplemental Figure 4B). Additionally, application of BayK(-) elicited a significantly larger increase in Ψm and flavoprotein oxidation in 10- to 15-week-old MHC versus age-matched wt myocytes (Supplemental Figure 5). The responses could be prevented with ICa-L antagonist nisoldipine. These data suggest that altered communication between ICa-L and the mitochondria precedes the development of αMHC cardiomyopathy.

Discussion

The L-type Ca2+ channel plays an important role in cardiac excitation and contraction. It can also influence metabolic activity through transmission of movement of the β subunit via cytoskeletal proteins 21, 26. We investigated whether the Arg403Gln mutation in contractile protein β-MHC results in impaired communication between ICa-L and the mitochondria, and subsequently, altered metabolic function. We find that ICa-L current inactivates more rapidly in myocytes from αMHC hearts (Figure 1). This appears to occur as a result of tethering of ICa-L to cytoskeletal proteins, and is consistent with findings that dissociation of microtubules or depolymerization of actin alters ICa-L inactivation rate 16, 17, 45, 46. Peak inward current, current density, and ICa-L expression were not significantly altered in αMHC myocytes. Consistent with this, basal and BayK(-)–stimulated increases in [Ca2+]i and superoxide production were also no different from wt myocytes (Supplemental Figure 2). Previous studies have demonstrated that the relaxation rate of αMHC cardiac myocytes is slowed and calcium transients are smaller due to reduced expression of ryanodine receptors and calsequestrin, leading to diminished sarcoplasmic reticulum stores 9, 47. Taken together, the findings demonstrate that the Arg403Gln mutation is associated with altered sarcoplasmic reticulum calcium cycling but does not appear to be associated with significant changes in diastolic Ca2+ or superoxide production in aged αMHC hearts.

One factor that influences metabolic activity and mitochondrial ATP production is electron flow down the inner mitochondrial membrane. We demonstrate that αMHC cardiac myocytes exhibit a significantly larger increase in Ψm, oxygen consumption and flavoprotein oxidation in response to activation of ICa-L that can be attenuated by ICa-L antagonist nisoldipine (Figure 2, Figure 3, Figure 4). The increase in Ψm can also be attenuated by diltiazem. These data indicate that metabolic activity in αMHC myocytes is higher versus wt myocytes. We demonstrate that this is dependent upon the intact cellular environment because respiration was normal in mitochondria isolated from αMHC hearts (Figure 5A). In support of this, desmin-null mice exhibit normal rates of maximal respiration in isolated mitochondria, but in vivo mitochondrial respiration is abnormal (20). Because alterations in mitochondrial function are observed only in the intact myocyte, we conclude that alterations to the cell’s intrinsic environment (as evidenced in Figures 5G and 5H) result in altered communication between ICa-L and mitochondria, contributing to a hypermetabolic state in the αMHC cardiac myocyte.

We have demonstrated previously that ICa-L co-immunoprecipitates with many cytoskeletal proteins 21, 26. We investigated how the mutation in the MHC gene leads to alterations in protein- protein interactions through the cytoskeletal network. The β2 subunit of the L-type Ca2+ channel is tightly bound to the α1C subunit via the alpha-interacting domain 48, 49. The β2 subunit of the channel is also tethered to F-actin via subsarcolemmal stabilizing protein AHNAK (15). Mitochondria also associate with actin via mitochondrial docking proteins 36, 37, 38, and with β-tubulin via VDAC (42). Here, we demonstrate that elevated Ψm in response to activation of ICa-L is dependent on cytoskeletal proteins F-actin and β-tubulin in the αMHC cardiac myocyte because exposure of myocytes to either F-actin depolymerizing agent latrunculin A or β-tubulin depolymerizing agent colchicine attenuates elevated Ψm in response to activation of ICa-L (Figure 2C). These data indicate that the Arg403Gln mutation is associated with altered functional communication between ICa-L and mitochondria via the cytoskeletal network, and increased cardiac metabolic activity.

To determine whether alterations in mitochondrial responses occurred before the onset of cardiomyopathy, we examined ICa-L kinetics and the effect of activation of ICa-L on Ψm and flavoprotein oxidation in myocytes isolated from pre-cardiomyopathic 10- to 15-week-old αMHC hearts. Similar responses were recorded to those observed in myocytes from 30- to 50-week-old αMHC hearts that had developed cardiomyopathy (Supplemental Figures 4 and 5). Because the responses were observed before the development of the cardiomyopathy, we conclude that altered communication between the ICa-L and mitochondria may contribute to the histology and pathophysiology, specifically altered energy reserve and hypercontractility, which has been identified in patients with HCM (50). The ICa-L antagonist diltiazem is effective in preventing the development of cardiomyopathy in αMHC mice and in some patients with identified MYH7 gene mutations. Our findings indicate that targeting ICa-L may be effective in the treatment of cardiomyopathy by modulating the activity of the ICa-L and decreasing/restoring metabolic activity. We speculate that early intervention involving treatment with ICa-L antagonist diltiazem may prove beneficial in regulating metabolic activity and subsequently, preventing the development of cardiomyopathy in “at-risk” patients with identified MYH7 gene mutations.

COMPETENCY IN MEDICAL KNOWLEDGE: Mutations in contractile protein β-myosin heavy chain account for approximately 40% of genotyped families with HCM. Altered energy reserve has been identified in patients with HCM, however the relationship between the gene mutation and phenotype is poorly understood. L-type Ca2+ channel antagonists are used clinically to treat patients but the role of the L-type Ca2+ channel in the development of the cardiomyopathy is unknown. Here we find that the β-myosin heavy chain mutation Arg403Gln leads to altered functional communication between the L-type Ca2+ channel and mitochondria that is associated with increased cardiac metabolic activity. This may contribute to the development of the cardiomyopathy because the response is present prior to the development of cardiomyopathy.

TRANSLATIONAL OUTLOOK: Further studies are needed to determine cardiac metabolic activity in “at risk” patients with identified MYH7 gene mutations before the development of HCM. On the basis of our findings, we speculate that utilizing L-type Ca2+ channel antagonists as a means of modulating cardiac metabolic activity may prove beneficial in early intervention and subsequent prevention of HCM in at-risk patients with identified MYH7 gene mutations.