The L-type Ca(2+) channel facilitates abnormal metabolic activity in the cTnI-G203S mouse model of hypertrophic cardiomyopathy

Abstract

Key points: Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterized by myocyte remodelling, disorganization of cytoskeletal proteins and altered energy metabolism. The L-type Ca(2+) channel is the main route for calcium influx and is crucial to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network. We find that L-type Ca(2+) channel kinetics are altered in cTnI-G203S cardiac myocytes and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI-G203S cardiac myocytes. These responses occur as a result of impaired communication between the L-type Ca(2+) channel and cytoskeletal protein F-actin, involving decreased movement of actin-myosin and block of the mitochondrial voltage-dependent anion channel, resulting in a 'hypermetabolic' mitochondrial state. We propose that L-type Ca(2+) channel antagonists, such as diltiazem, might be effective in reducing the cardiomyopathy by normalizing mitochondrial metabolic activity.

Abstract: Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is associated with disorganization of cytoskeletal proteins and altered energy metabolism. The L-type Ca(2+) channel (ICa-L ) plays an important role in regulating mitochondrial function. This involves a functional communication between the channel and mitochondria via the cytoskeletal network. We investigate the role of ICa-L in regulating mitochondrial function in 25- to 30-week-old cardiomyopathic mice expressing the human disease-causing mutation Gly203Ser in cTnI (cTnI-G203S). The inactivation rate of ICa-L is significantly faster in cTnI-G203S myocytes [cTnI-G203S: τ1 = 40.68 ± 3.22, n = 10 vs. wild-type (wt): τ1 = 59.05 ± 6.40, n = 6, P < 0.05]. Activation of ICa-L caused a greater increase in mitochondrial membrane potential (Ψm , 29.19 ± 1.85%, n = 15 vs. wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 vs. wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred because of impaired communication between ICa-L and F-actin, involving lack of dynamic movement of actin-myosin and block of the mitochondrial voltage-dependent anion channel. Similar responses were observed in precardiomyopathic mice. ICa-L antagonists nisoldipine and diltiazem decreased Ψm to basal levels. We conclude that the Gly203Ser mutation leads to impaired functional communication between ICa-L and mitochondria, resulting in a 'hypermetabolic' state. This might contribute to development of cTnI-G203S cardiomyopathy because the response is present in young precardiomyopathic mice. ICa-L antagonists might be effective in reducing the cardiomyopathy by altering mitochondrial function.

Figure 1. Myocytes isolated from cTnI‐G203S hearts exhibit altered inactivation kinetics

A, representative L‐type Ca²⁺ (I_Ca‐L) current traces from cTnI‐G203S (120 pF) and wt myocytes (120 pF). Inset: pulse protocol. Mean ± SEM of rate of inactivation (τ, calculated bi‐exponentially; B), current density (C), 50 ms inactivation integral of current (D), total integral of current (E) and activation integral of current (F) for cTnI‐G203S myocytes and wt myocytes. G, current–voltage (I–V) relationship measured in cTnI‐G203S (n = 10) and wt myocytes (n = 7; P = n.s.). Inset above: pulse protocol. H, voltage dependency of steady‐state inactivation measured in cTnI‐G203S (n = 10) and wt myocytes (n = 7; P = n.s.). Inset above: pulse protocol. I, representative I_Ca‐L current traces recorded from cTnI‐G203S (95 pF) and wt myocytes (90 pF) with barium as the charge carrier. Inset above: pulse protocol. Mean ± SEM of rate of inactivation (τ; J) and current density (K) for cTnI‐G203S myocytes and wt myocytes with barium as the charge carrier. n, number of myocytes.

Figure 2. Immunoblot analysis of L‐type Ca²⁺ channel α_1C and β₂ subunit protein expression

L‐type Ca²⁺ channel (I_Ca‐L) protein was extracted and pooled from three 25‐ to 30‐week‐old wt hearts and and three 25‐ to 30‐week‐old cTnI‐G203S hearts. A and B, representative immunoblots of I_Ca‐L protein extracted from wt and cTnI‐G203S hearts probed with I_Ca‐L α_1C subunit antibody (α_1C) and porin monoclonal antibody (A) or β₂ subunit antibody (β₂) and porin monoclonal antibody (B). Four experimental repeats were run against α_1C and porin, or β₂ and porin for both wt and cTnI‐G203S. C and D, densitometry analysis of α_1C (C) and β₂ (D) protein expression for wt and cTnI‐G203S presented as a ratio of wt α_1C and β₂ protein expression, respectively, each normalized to associated porin expression.

Figure 3. Wild‐type and cTnI‐G203S cardiac myocytes exhibit similar alterations in intracellular Ca²⁺ concentrations and mitochondrial superoxide production following activation of the L‐type Ca²⁺ channel

A, representative traces of intracellular calcium recorded in wt and cTnI‐G203S myocytes before and after exposure to 10 μm BayK(−) or BayK(+). Vertical arrow indicates when drug was added. B, mean ± SEM of intracellular Ca²⁺ for all myocytes exposed to treatments as indicated. Abbreviations: Nisol, 15 μm nisoldipine; RyR, 5 μm ryanodine. C, representative traces of DHE fluorescence recorded in wt and cTnI‐G203S myocytes before and after exposure to 10 μm BayK(−) or BayK(+). Addition of drugs is indicated by the vertical arrow. Slopes of signals are as indicated. D, mean ± SEM of changes in DHE fluorescence for all myocytes exposed to treatments as indicated. Abbreviations: Nisol, 15 μm nisoldipine; 2 μm Ru360; Myx, 7 nm myxothiazol; n, number of myocytes.

Figure 4. cTnI‐G203S cardiac myocytes exhibit a significantly larger increase in Ψ_m in response to activation of the L‐type Ca²⁺ channel compared with wt myocytes

Representative traces of ratiometric JC‐1 fluorescence recorded in wt (A) and cTnI‐G203S (B) myocytes before and after exposure to 10 μm BayK(+) or BayK(−) with or without 15 μm nisoldipine (Nisol) or 15 μM diltiazem (Dilt) in Ca²⁺‐free conditions (0 mm Ca²⁺). Vertical arrow indicates addition of drugs. Abbreviation: NaCN, 40 mm sodium cyanide. Insets show representative ratiometric JC‐1 fluorescence recorded in wt (A) and cTnI‐G203S (B) myocytes exposed to BayK(+) and BayK(−) in the presence of 2.5 mm calcium. C, mean ± SEM of JC‐1 fluorescence for all myocytes exposed to treatments as indicated. Inset shows mean ± SEM of JC‐1 fluorescence for all myocytes exposed to treatments in the presence of 2.5 mm calcium. D, ratio of increase in JC‐1 fluorescence after addition of BayK(−) for wt and cTnI‐G203S myocytes compared with wt BayK(+) response. n, number of myocytes.

Figure 5. cTnI‐G203S cardiac myocytes exhibit a significantly larger increase in metabolic activity in response to activation of the L‐type Ca²⁺ channel compared with wt myocytes

A and B, representative traces of flavoprotein fluorescence recorded in wt (A) and cTnI‐G203S (B) myocytes before and after exposure to 10 μm BayK(−) or BayK(+). Addition of drugs is indicated by the arrow. Abbreviation: FCCP, 50 μm carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone. C, mean ± SEM of flavoprotein fluorescence for all myocytes exposed to treatments as indicated. Abbreviation: Nisol, 15 μm nisoldipine. D, ratio of increase in flavoprotein fluorescence after addition of BayK(−) for wt and cTnI‐G203S myocytes compared with wt BayK(+) response. n, number of myocytes. E, formation of formazan measured as the change in absorbance in wt and cTnI‐G203S myocytes after addition of 10 μm BayK(+) or BayK(−). F, mean ± SEM of increases in absorbance for all myocytes exposed to treatments as indicated. Abbreviations: Nisol, 10 μm nisoldipine; 5 μm Ru360; 5 μm ryanodine; Oligo, 20 μm oligomycin. G, ratio of increase in formation of formazan after addition of BayK(−) for wt and cTnI‐G203S myocytes compared with wt BayK(+) response. n represents the number of replicates for each treatment group.

Figure 6. Regulation of mitochondrial membrane potential (Ψ_m) by the L‐type Ca²⁺ channel involves F‐actin and mitochondrial voltage‐dependent anion channel (VDAC)

A, representative traces of ratiometric JC‐1 fluorescence recorded from wt and cTnI‐G203S myocytes before and after addition of 10 μm BayK(−) in the presence or absence of 5 μm latrunculin A (Latrunc) in calcium‐free conditions (0 mm Ca²⁺). Vertical arrow indicates when drug was added. Abbreviation: NaCN, 40 mm sodium cyanide. B, mean ± SEM of JC‐1 fluorescence for all myocytes exposed to treatments as indicated. n, number of myocytes. C, exposure of wt myocytes to an inhibitor of actin–myosin interaction mimics the increase in Ψ_m observed in cTnI‐G203S myocytes. Representative traces of ratiometric JC‐1 fluorescence recorded from wt and cTnI‐G203S myocytes before and after exposure to BayK(−) with or without 10 μm blebbistatin (Blebb) in 0 mm Ca²⁺ conditions. D, mean ± SEM of JC‐1 fluorescence for all myocytes exposed to treatments as indicated. E, exposure of wt myocytes to a peptide that blocks VDAC mimics the increase in Ψ_m observed in cTnI‐G203S myocytes. Representative traces of ratiometric JC‐1 fluorescence recorded from wt and cTnI‐G203S myocytes before and after exposure to BayK(−), 10 μm VDAC peptide (VDAC) or 10 μm VDAC scrambled peptide [VDAC (S)] in 0 mm Ca²⁺ conditions. Inset shows ratiometric JC‐1 fluorescence recorded from cTnI‐G203S myocytes before and after exposure to BayK(−) plus VDAC. F, mean ± SEM of JC‐1 fluorescence for all myocytes exposed to treatments as indicated. G and H, I_Ca‐L associates with mitochondrial VDAC via cytoskeletal proteins. Mass spectrometry analysis of immunoprecipitated I_Ca‐L protein from wt (n = 4) and cTnI‐G203S (n = 4) hearts. Abbreviations: AKAP, A‐kinase anchor protein; CaMKII, Ca²⁺/calmodulin‐dependent protein kinase II; LTCC, L‐type Ca²⁺ channel; MAPK, mitogen‐activated protein kinase; PKC, protein kinase C; TK, tyrosine kinase; VDAC, voltage‐dependent anion channel.

Figure 7. Respiration is normal but mitochondrial characteristics are altered in mitochondria isolated from cTnI‐G203S hearts

A, respiration and mitochondrial electron transport chain complex activity in mitochondria isolated from 25‐ to 30‐week‐old wt and cTnI‐G203S hearts. Measurements were performed in triplicate. B, mitochondrial (Mt) DNA copy number in wt and cTnI‐G203S hearts, normalized to 18S rDNA. Data are represented as means ± SD. C and D, representative transmission electron microscopy sections from ventricles of wt (C) and cTnI‐G203S hearts (D). Abbreviations: M, mitochondria; S, sarcomere. Scale bars represent 0.5 μm. E, mitochondrial (Mt) surface area in wt and cTnI‐G203S hearts measured from 11 sections from each of four wt and four cTnI‐G203S hearts. F, number of mitochondria (Mt) per square micrometre determined from 11 sections from each of four wt and four cTnI‐G203S hearts.

Figure 8. cTnI‐G203S cardiac myocytes from young (10‐ to 15‐week‐old) precardiomyopathic mice exhibit altered inactivation kinetics and a significantly larger increase in Ψ_m and metabolic activity in response to activation of the L‐type Ca²⁺ channel compared with wt myocytes

A, representative I_Ca‐L traces recorded from cTnI‐G203S (350 pF) and wt myocytes (400 pF). Inset: pulse protocol. B and C, mean ± SEM of rate of inactivation (τ, calculated bi‐exponentially; B) and current density (C) for cTnI‐G203S myocytes and cTnI‐G203S myocytes. D, representative traces of ratiometric JC‐1 fluorescence recorded in wt and cTnI‐G203S myocytes before and after exposure to 10 μm BayK(−) with or without 15 μm nisoldipine (Nisol) in Ca²⁺‐free conditions (0 mm Ca²⁺). Vertical arrow indicates when the drug was added. Abbreviation: NaCN, 40 mm sodium cyanide. E, mean ± SEM of JC‐1 fluorescence for all myocytes exposed to treatments as indicated. F, ratio of increase in JC‐1 fluorescence after addition of BayK(−) for wt and cTnI‐G203S myocytes compared with wt BayK(+) response. n, number of myocytes. G, representative traces of flavoprotein fluorescence recorded in wt and cTnI‐G203S myocytes before and after exposure to 10 μm BayK(−) or BayK(+). Addition of drugs is indicated by the arrow. FCCP: 50 μm. H, mean ± SEM of flavoprotein fluorescence for all myocytes exposed to treatments as indicated. Abbreviation: Nisol, 15 μm nisoldipine. I, ratio of increase in flavoprotein fluorescence after addition of BayK(−) for wt and cTnl‐G203S myocytes compared with wt BayK(+) response. wt, number of myocytes.