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Cardiovascular Research 2006 69(2):370-380; doi:10.1016/j.cardiores.2005.11.009
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Copyright © 2005, European Society of Cardiology

Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice

Olivier Cazorlaa,b,*,1, Szabolcs Szilagyia,b,1, Nicolas Vignierc,d, Guillermo Salazara,b, Elisabeth Krämere, Guy Vassorta,b, Lucie Carrierc,d,e and Alain Lacampagnea,b

aINSERM, U 637, Montpellier, F-34295, France
bUniversité MONTPELLIER1, UFR de Médecine, Montpellier, F-34295, France
cINSERM, U 582, Paris, F-75013, France
dUniversité Pierre et Marie Curie, UFR de Médecine, Paris, F-75013, France
eInstitute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

* Corresponding author. INSERM U-637, CHU Arnaud de Villeneuve, 34295 Montpellier, France. Tel.: +33 467 41 52 44; fax: +33 467 41 52 42. Email address: cazorla{at}montp.inserm.fr

Received 13 September 2005; revised 17 October 2005; accepted 4 November 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: β-Adrenergic stimulation modulates cardiac contractility through protein kinase A (PKA), which phosphorylates proteins such as troponin I (cTnI) and C-protein (cMyBP-C). The relative contributions of cTnI and cMyBP-C to the regulation of myofilament Ca2+ sensitivity are still controversial because of difficulty in targeting specific protein phosphorylation. Recently, impaired relaxation was found in cMyBP-C-deficient mice (KO) in vivo under basal conditions and after β-adrenergic stimulation. The goal of this study was to analyse the length-dependent and PKA-dependent modulations of the cardiac contractile machinery in a mouse model lacking cMyBP-C.

Methods: In the present work, we studied the PKA effect on myofilament Ca2+ sensitivity of left ventricular skinned myocytes isolated from 5-week- and 55-week-old wild-type (WT) and cMyBP-C knockout (KO) mice at 1.9 and 2.3 µm sarcomere lengths (SL). The cTnI content and phosphorylation status were examined by Western blot analysis.

Results: Without PKA stimulation and at the shorter SL, Ca2+ sensitivity was higher in KO compared to WT. The difference disappeared at the longer SL. No difference in passive tension or maximal active tension was observed. PKA stimulation induced a desensitization of WT myofilaments at both SL but had almost no effect in KO myofilaments despite similar levels of cTnI phosphorylation. We also observed expression of slow skeletal TnI in KO animals that was not correlated with the PKA effects.

Conclusion: The results suggest that cMyBP-C contributes to the regulation of cardiac contraction at short sarcomere length and that myofilament desensitization induced by PKA requires the presence of cMyBP-C and does not depend only upon TnI phosphorylation.

KEYWORDS Ca2+ sensitivity; β-Adrenergic; Frank–Starling law; TnI

See Editorial by H. Kögler (pages 304–306) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Cardiac myosin binding protein C (cMyBP-C) is a component of the thick filament of the sarcomere, which is expressed exclusively in the heart during development, and differs in three specific regions from the two skeletal isoforms (reviewed in Winegrad [1]). Particularly, it contains phosphorylation sites in the MyBP-C motif, which connects the C1 and C2 domains. MyBP-C has sites for binding, respectively, to myosin, titin, and actin (reviewed in Flashman et al. [2]). There are two myosin-binding sites, one in the C-terminal C10 domain that binds to the light meromyosin and another in the C1C2 region that binds to the S2 region of myosin. The interest in cMyBP-C intensified with the discovery that mutations in the human gene, as well as in other genes encoding sarcomeric proteins, cause familial hypertrophic cardiomyopathy [3,4]. The regulatory role of cMyBP-C on contractility is still controversial since studies showing that extraction, truncation or KO of cMyBP-C resulted in different effects on Ca2+ sensitivity being increased [5], unchanged [6] or decreased [7]. Another unknown parameter is the role of cMyBP-C in the length-dependent activation. cMyBP-C is of particular interest in the heart because it can be phosphorylated by different kinases 3 phosphorylation sites leading to a total of 4 potential phosphorylation states. One of the sites is regulated by a Ca2+-calmodulin kinase bound to the thick filament and three sites by a cAMP-regulated protein kinase (PKA) [8]. The dephosphorylated and PKA-dependent phosphorylated forms of cMyBP-C interact differently with actin and myosin and have different effects on actin-activated myosin ATPase activities [9,10]. However, the precise role of cMyBP-C in cardiac contraction remains unclear, especially because PKA also phosphorylates cardiac troponin I (cTnI).

β-Adrenergic stimulation is a well-known mechanism for modulating cardiac contractility and hemodynamics through activation of PKA, which predominantly targets various proteins involved in Ca2+ handling (reviewed in Solaro and Van Eyk [11]). In addition, PKA phosphorylates regulatory proteins of the myofilaments like cTnI, titin, and cMyBP-C, which leads to a decrease in myofilaments Ca2+ sensitivity [12]. Konhilas et al. showed that replacement of native cTnI by the slow skeletal isoform (ssTnI), which is not phosphorylated by PKA, eliminates the effect of PKA on Ca2+ sensitivity [13]. These observations led to suggest that cTnI modulates Ca2+ sensitivity of activation and is necessary for the PKA effect on myofilament activation.

Recently, Carrier and colleagues have developed a mouse model lacking cMyBP-C [14]. Hemodynamic exploration of this model revealed a 2-fold increase in relaxation time and a blunted relaxation phase after β-adrenergic stimulation. This strongly suggests an alteration in the Ca2+ sensitization of the myofilament in this model. The goal of this study was therefore to determine the effects of sarcomere length (SL) and PKA-mediated phosphorylation on the myofibrillar Ca2+ sensitivity in skinned single cardiomyocytes obtained from these cMyBP-C null mice (KO) compared to wild type mice (WT). Animals were studied at 5 and 55 weeks of age at a mild and severe degree of hypertrophy, respectively. We found that Ca2+ sensitivity of myofilaments was higher in KO than in WT cells at short SL, but the difference disappeared at long SL, in both young and older animals. PKA treatment induced a desensitization of WT myofilaments at both SL but had significantly less effect on KO myofilaments. These data suggest that cMyBP-C is involved in cardiac contraction modulation and contributes to the PKA effects on Ca2+ sensitivity of myofilaments.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Animal model
Homozygous cardiac cMyBP-C-null mice (KO) were generated as previously described [14]. Briefly, the mouse model was based upon the targeted deletion of exons 1–2 of the mouse cMyBP-C gene, which also includes the transcription initiation site. In the present study, female WT and KO mice were studied at 5 weeks old (WT-5 n=7, and KO-5 n=5, body weight (BW) was ~18 g) and 55 weeks old (WT-55 n=12, and KO-55 n=10, BW ~35 g). Electrophoretic and Western blot analysis confirmed the absence of cMyBP-C at both ages (Fig. 1). Morphological analysis of hearts estimated by the ventricular weight/body weight ratio (VW/BW) indicated an overall hypertrophy of the KO animals that was already observed at birth and then dramatically increased between the age of 6 and 9 weeks (Fig. 1C). The two groups of animals belong to the two different stable levels of hypertrophy (~20% from birth up to 6 weeks, and more than 44% after 9 weeks). The investigation conforms to the guide for the care and use of laboratory animals published by the NIH. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital.


Figure 1
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  		Fig. 1 cMyBP-C content and cTnI isoforms. (A) The myofibrillar protein expression in WT-55 and cMyBP-C KO-55 cardiomyocytes was analyzed by 15% SDS–PAGE. TnI, troponin I; TnT, troponin T; TnC, troponin C; MLC-2, myosin light chain 2. *Appearance of a darker band (B) cMyBP-C content was analyzed in WT and KO ventricular myocardium using Coomassie-stained 2.5–7% SDS–PAGE and Western blot analysis with antibodies directed against the C0C1 domains of cMyBP-C. Soleus muscle was used as a control of the skeletal isoforms. MHC is myosin heavy chain. Antibody directed against MLC-2 was used as a loading control. (C) Increase in ventricular weight/body weight ratio (VW/BW) in KO mice relative to WT animals at the same age.

 
2.2 Myocyte preparation
Ventricular myocytes were dissociated mechanically as previously described [15]. The heart was perfused retrogradely (1.6 ml/min) with a Ca2+-free Hanks–HEPES buffered solution for 5 min at room temperature then pre-skinned by perfusing with relaxing solution containing 1% Triton X-100 and protease inhibitors for 7 to 10 min (see below). Left ventricular strips (8-mm long, 2-mm width, 1 mm thick) were dissected from the same heart, frozen in liquid nitrogen for biochemistry or further skinned for 10 min at 4 °C in relaxing solution containing 1% Triton X-100 for mechanical experiments. The strips were then blended in fresh ice-cold relaxing solution at 11,000 rpm for 2–3 s. The resulting suspension was further skinned in 0.3% Triton X-100 solution for 6 min at 22 °C. Cells were kept in ice and used within the day.

2.3 Solutions
Ca2+ activating solutions were prepared daily by mixing relaxing (pCa 9.0) and maximal activating (pCa 4.5) stock solutions. The relaxing and activating solutions contained (in mM): phosphocreatine 12, imidazole 30, free Mg2+ 1, EGTA 10, Na2ATP 3.3, and dithiothreitol 0.3 with pCa 9.0 (relaxing solution) and pCa 4.5 (maximal activating solution), pH 7.1 adjusted with KOH. Ionic strength was adjusted to 180 mM with K-acetate. For PKA stimulation, cells were preincubated for 50 min at room temperature (22 °C) with 200 UI of catalytic subunit of PKA (Sigma Chemicals) per ml of relaxing solution. Some myocytes were used for micromechanical experiments while others were directly solubilized in Laemmli buffer to evaluate the level of TnI phosphorylation (see below). All solutions contained protease inhibitors (in mM: PMSF 0.5; leupeptin 0.04 and E64 0.01).

2.4 Force measurements
The procedure of cell attachment has been previously described [15]. Skinned myocyte was attached to a piezoresistive strain gauge (AE801 sensor, Memscap, Crolle, France; 500 Hz unloaded resonant frequency, compliance of the strain gauge 0.03 µm/µN) and to a stepper motor driven micromanipulator (MP-285, Sutter instrument company, Novato CA, USA) with thin needles and optical glue (NOA 63, Norland products Inc., North Brunswick, NJ, USA) that polymerized by 2 min UV illumination. SL was determined online throughout the experiment at 50 Hz by using a fast Fourier transform algorithm on the video images of the cell. Force was normalized by the cross-sectional area measured from the imaged cross-section as previously described [16]. Slack SL was measured before attachment to serve as origin and did not differ significantly between WT (1.80 ± 0.01 µm SL, n=48) and KO (1.81 ± 0.01 µm SL, n=42) myocytes. After a test-activation at pCa 4.5, the cell was stretched to various SL in relaxing solution using a stepper motor driven micromanipulator at a speed of 0.1 length/s to evaluate passive tension. Steady state passive tension (after the rapid phase of stress relaxation) was sequentially measured at 1.9, 2.1, and 2.3 µm SL. Then pCa–force relationships were established at two SL, 1.9 and 2.3 µm at 22 °C. The cell was kept 5 min at slack length in relaxing solution between each phase of the protocol for complete refolding of titin. Active tension at each pCa was the difference between total tension and relaxed tension. Cells that did not maintain 80% of the first maximal tension or a visible striation pattern were discarded. Although cells were kept isometric during contraction, sarcomeres typically changed length, especially when activation was maximal [13,17]. The SL change varied somewhat from cell to cell, and we continued with cells that were well attached with minimal SL changes (<0.1 µm). When required, cell length was varied during contraction in order to keep SL constant. Active tensions at submaximal activations were normalized to maximal isometric tension (classically obtained at pCa 5) at the same SL. The maximal active force at pCa 4.5 decreased slightly with the number of imposed contractions; the mean reduction during the measurement of two force–pCa curves was 7 ± 3% (n=22) and 6 ± 2% (n=27) for WT and KO without PKA stimulation, respectively.

2.5 Western blot analysis
Total protein homogenates (20 µg) were separated on 15% SDS–PAGE (1 h, 200 V) for cTnI analysis and blotted onto nitrocellulose membrane (1 h, 0.08 A, Protran®, Schleicher & Schuele). Total cTnI content was determined with an antibody that recognizes the skeletal and cardiac TnI (clone 6F9, Hytest, Finland). PKA treatment was controlled with an antibody directed against the Ser23/24-phosphorylated form of cTnI (clone 5E6, Hytest, Finland). Both antibodies were diluted at 1/10000. Immunodetection was revealed with ECL system (ECL-Plus kit, Amersham Pharmacia).

Quantification of signals was performed by densitometry using an imaging system (Kodak Image Station 2000R). Results were expressed relative to the total amount of the studied protein.

2.6 Real-time quantitative RT–PCR
Real-time quantitative RT–PCR was performed as previously described [14]. Total RNA was isolated from 5 mg of mouse ventricles using SV Total RNA Isolation kit (Promega #Z3100). Reverse transcription was performed on 50 ng of total RNA with superscript III reverse transcriptase (Invitrogen #18080-051). cDNA was purified using Microcon MY50 column (Millipore #42416) and the concentration was determined with Nanodrop technology. RT–PCR was performed using the LightCycler system (Roche) with specific primers for ssTnI, cTnI, and Emerin (EMD) for internal control (Fig. 5). Absolute mRNA levels were calculated from standard curves generated by RT–PCR on serials dilutions of cloned EMD, ssTnI and cTnI. EMD was used as endogenous control to normalize the quantification of the mRNA target for differences in the amount of total RNA added to each reaction. Genomic DNA contamination was tested with Agilent bioanalyser and a sterile water sample subjected to RT–PCR in every experiment.


Figure 5
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  		Fig. 5 Troponin I expression in cardiomyocytes lacking cMyBP-C. (A) Immunoblots of myocardium isolated from 5-week- and 55-week-old WT and KO mice with an anti-TnI antibody, which recognizes both the skeletal and cardiac TnI isoforms, and an anti-MLC2 antibody. Degraded myocardium (degradation) and soleus muscle were co-electrophoresed to provide standards for cTnI degradation products and for the slow skeletal and fetal TnI isoforms, respectively. (B) Expression of ssTnI relative to the total amount of TnI (4–6 animals). *P<0.05 between WT and KO. (C) ssTnI and cTnI mRNA levels were determined in young and adult WT and KO mice (n=7 animals in each group). Amplification curves generated during quantitative RT–PCR (left). Amount of messengers in KO relative to WT (right).

 
2.7 Data analysis
Individual pCa–force relationships were fitted to the following equation: normalized force=[Ca2+]nH/(K+[Ca2+]nH), where nH is the Hill coefficient and pCa50, pCa for half-maximal activation, equals –(log K)/nH. The {Delta}pCa50 was calculated as the difference in pCa50 at 1.9 and 2.3 µm SL for each experiment. One-way or two-way ANOVA was applied for comparison between groups. When significant interactions were found, a Holm–Sidak t-test was applied (Sigmastat3 software) with P<0.05. The average Hill fit parameters of the pCa–tension relationships obtained from WT and KO myocytes with or without PKA treatment at the two SL are presented as mean ± S.E.M (n=number of cells).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Passive properties of cardiomyocytes
First, we analyzed the passive mechanical properties of myocytes isolated from transgenic mice lacking cMyBP-C in young (~5 weeks, KO-5) and adult (~55 weeks, KO-55) mice exhibiting a mild and severe hypertrophy, respectively. Passive tension did not differ between myocytes isolated from WT and KO mice at the same age (Fig. 2A–B). Passive properties were also analyzed in myocytes preincubated with PKA. In both WT and KO at both ages, a decrease in passive tension was noticed after PKA treatment. This is in agreement with the report that PKA-dependent phosphorylation of titin in the I-band decreases passive tension [18]. The level of PKA-induced phosphorylation was evaluated in 55-week-old myocytes by Western blot with specific anti-total TnI and anti-Ser23/24 phospho-cTnI antibodies. Basal phosphorylation levels of cTnI were similar in WT and KO cells, while PKA phosphorylated cTnI to the same extent in both WT and KO (27% and 30%, respectively; Fig. 2C).


Figure 2
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  		Fig. 2 Sarcomere length–passive tension relationships of myocytes isolated from 5-week- (A) and 55-week-old (B) WT (left) and KO (right) animals without (open symbols, line) and after a PKA treatment (close symbols, dash line). (C) Immunoblots with an anti-cTnI and anti-phospho-Ser23–24cTnI showed that TnI phosphorylation in 55-week-old mice with or without cMyBP-C was similar (n=4 animals/group). Phosphorylation levels of cTnI were significantly increased by PKA incubation in both WT and KO myocytes. *P<0.05, from values obtained from WT myocytes with or without pre-treatment.

 
3.2 Relation between sarcomere length and Ca2+ sensitivity
The length-dependent properties of WT and KO myocytes were then compared after activation of the myofilaments with various Ca2+ concentrations. Myocytes isolated from the left ventricle of KO mice had a higher Ca2+-sensitivity at 1.9 µm SL than WT, at both ages (Fig. 3). The values of pCa50 in WT and KO myocytes were 5.79 ± 0.03 and 5.87 ± 0.02 in 5-week-old mice and 5.77 ± 0.01 and 5.85 ± 0.01 in 55-week-old mice, respectively (P<0.01; Table 1). In WT, increasing SL to 2.3 µm produced a leftward shift of the Ca2+–tension relationship ({Delta}pCa50). At 2.3 µm SL, there was no significant difference in the pCa50 between WT and KO at both ages. The length-dependent activation, represented by the {Delta}pCa50, significantly decreased in KO-5 and KO-55 myofilaments compared with WT (Table 1). However, this effect was more prominent in adult animals (in pCa unit, WT-55=0.23 ± 0.01, KO-55=0.16 ± 0.01) compared with young mice (WT-5=0.18 ± 0.02, KO-5=0.14 ± 0.01). Deficiency of cMyBP-C was also associated with reduced slopes of the Ca2+–force relationships at both SL as indicated by the lower Hill coefficients in KO myofilaments although significance in KO-5 mice at short SL was not reached (P=0.17; Table 1). Tmax was not significantly different between the WT and KO groups at either SL and ages (Table 1).


Figure 3
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  		Fig. 3 Length-dependent Ca2+ activation in skinned cardiomyocytes. (A) Representative measurement of isometric tension elicited on attached cardiomyocyte at 1.9 µm and 2.3 µm SL. Contractions were induced by application of solutions containing various pCa (from 6.2 to 4.5). Tension (normalized to maximal tension)–pCa curves of myocytes isolated from 5-week- (A) and 55-week-old (B) WT (open symbols, line) or KO (closed symbols, dash line) mice, were established at two SL: 1.9 µm (circle) and 2.3 µm (square), respectively. Contractile parameters are given in Table 1.

 

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  		Table 1 Characteristics of stretch-dependent Ca2+ sensitivity of skinned cardiomyocytes isolated from WT and KO mice

 
3.3 Effect of PKA-dependent phosphorylation on Ca2+ sensitivity
The myofilament Ca2+ sensitivity of cells preincubated with PKA was determined at 1.9 and 2.3 µm SL and was compared with previously obtained values in control cells. PKA treatment of WT myocytes induced a rightward shift of the tension–pCa curves at both SL and ages (Fig. 4A, B) resulting in an overall desensitization of the myofilaments to Ca2+ and a shift in pCa50 of ~0.13 pCa unit (Table 1). The sensitivity of Ca2+ activation to length after PKA treatment remained unchanged (P<0.01; Table 1) even when expressed as Ca2+ concentration ({Delta}EC50; Fig. 4C). In KO-5 and KO-55 myocytes, PKA-induced phosphorylation reduced the pCa50 by ~0.03–0.04 pCa unit and had a significant effect only at short SL in young animal where pCa50 was reduced by 0.07 pCa unit (P=0.012; Fig. 4A, B and Table 1). In both KO, the response of Ca2+ sensitivity to change in SL remain unchanged after PKA treatment (Fig. 4C). Maximum tension was also unaffected (Table 1). The Hill coefficient in KO myofilaments was increased to the same extent as in WT (Table 1). Thus, the difference in the response of Ca2+ sensitivity to PKA-induced phosphorylation depending on whether cMyBP-C was present (WT) or not (KO) suggests that the presence of cMyBP-C or its phosphorylation status participates in producing the change in contractility.


Figure 4
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  		Fig. 4 Effect of PKA on length-dependent Ca2+ activation. Tension–pCa curves of myocytes isolated from WT (top panel) or KO (bottom panel) mice at 5-week-old (A) and 55-week-old (B), without (open symbol) or after PKA treatment (closed symbol) at 1.9 (circle) and 2.3 µm (square) SL, respectively. Contractile parameters are given in Table 1. (C) Length-dependent effect on Ca2+ sensitivity expressed in Ca2+ concentration ({Delta}EC50) in WT and KO myocytes treated with or without PKA in 5-week- (left) and 55-week-old (right) animals. *P<0.05, difference between WT and KO with or without PKA pre-treatment.

 
3.4 Troponin I expression in the absence of cMyBP-C
TnI expression in WT and KO myocytes was analyzed by Western blot with an antibody that recognizes both skeletal and cardiac TnI (Fig. 5A). In KO, myocardium appeared a lower band of TnI preferentially in young animals, and with a molecular weight similar to that of the ssTnI isoform present in soleus muscle. This band had a different electrophoretic motility to that of the degradation products of cTnI obtained from either WT or KO myocardium kept on the bench for several hours without protease inhibitors. Expression of ssTnI in the heart has been previously reported during development [19]. In our conditions, ssTnI represented only 1% and 4% of total TnI in WT-5 and WT-55 mice, and 24% and 7% in KO-5 and KO-55 mice, respectively (Fig. 5B). The amount of mRNA for ssTnI and cTnI, determined by quantitative RT–PCR did not vary between WT and KO animals and was independent of the age (Fig. 5C). The crossing point cycle number (CP), used as an index of mRNA expression, did not vary in KO mice when expressed relative to WT at both ages (Fig. 5C, right panel). Taking WT and KO data altogether, we observed that the CP were 24.5 ± 1.2 and 30.4 ± 0.8 for cTnI and ssTnI, respectively, indicating that cTnI mRNA amount was approximately 125-fold higher than that of ssTnI mRNA.

Since ssTnI does not contain the PKA phosphorylation sites [20], its expression could be in part responsible for the altered PKA effect observed in KO mice. To test this hypothesis, Ca2+ sensitivity expressed in Ca2+ concentration (EC50 i.e. the Ca2+ concentration at which half-maximal force is generated) and the PKA-induced Ca2+ desensitization of myofilaments relative to the basal level were expressed in a bar graph as a function of the ssTnI amount (Fig. 6). PKA treatment significantly reduced Ca2+ sensitivity in WT myocytes as indicated by the increase in EC50. The PKA induced shift in EC50 was more pronounced in WT-55 mice that also expressed slightly more ssTnI and at 2.3 µm SL (34% and 55% shift of EC50 at 1.9 and 2.3 µm SL after PKA treatment in WT-55 mice). KO-55 mice lost most of that desensitization (10% and 5% shift of EC50). Desensitization was slightly higher only in KO-5 mice that expressed also the highest amount of ssTnI (14% and 9% shift of EC50). Thus no correlation was found between contractile parameters after PKA treatment and the amount of ssTnI, rather the Ca2+ sensitivity of myofilaments at both SL was dependent upon the presence of cMyBP-C (Fig. 6).


Figure 6
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  		Fig. 6 Correlation between Ca2+ activation of myocytes lacking cMyBP-C and their ssTnI content. Myofibrillar Ca2+ activation, expressed in Ca2+ concentration (EC50), measured at 1.9 (left) and 2.3 (right) µm SL in 5-week- (open bars) and 55-week-old (dash bars) WT (white bars) or KO (gray bars) myocytes without (A) or after PKA treatment (B). The PKA-induced shift of EC50 was preferentially observed in WT animals and was not correlated with the amount of ssTnI (C).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the present study, we examined the contractile properties of cardiomyocytes isolated from cMyBP-C KO and WT mice, at two different ages and degree of hypertrophy. We used two well-established, physiologically relevant methods for changing the Ca2+ sensitivity of the cardiac contractile system: change in SL and PKA-induced phosphorylation. We demonstrate that the lack of cMyBP-C (1) reduced the SL-dependent change in Ca2+ sensitivity and (2) almost abolished the PKA-induced decrease in Ca2+ sensitivity that occurred in control. This implies that the presence of cMyBP-C is required for a normal regulation of cardiac contractility. This is in agreement with the impaired relaxation found in the KO mice in vivo under basal and after β-adrenergic stimulation [14].

4.1 cMyBP-C and Ca2+ sensitization of myofilaments
The sensitization of Ca2+ myofilaments in cMyBP-C KO mice observed in the present study at short SL is in agreement with previous data in which cMyBP-C was partially removed [21]. Although we cannot totally rule out that the effects are due to hypertrophy rather than to cMyBP-C deficiency, it is to note that the Ca2+ sensitization observed in this study is similar at both ages and, therefore, independent of the degree of cardiac hypertrophy. We have recently reported in cardiomyocytes isolated from hypertrophied failing rat hearts that Ca2+ sensitivity, at variance with the present results, was unchanged at 1.9 µm SL but reduced at 2.3 µm SL [15]. Similar observations were also made in a rat model of compensated hypertrophy obtained by abdominal aortic banding (unpublished observations). This is also consistent with another transgenic model lacking the light chain binding domains on MHC, which develops hypertrophy without alteration of myofilament Ca2+ sensitivity [22]. Altogether, these observations reinforce the view that the reduced length-dependent activation is attributable to the absence of cMyBP-C.

As initially shown with phosphorylation of myosin light chain 2 in cardiac muscle, increase in Ca2+ sensitivity and decrease in cooperativity occur together with increased flexibility and decreased order of myosin heads [23]. The increase in Ca2+ sensitivity at 1.9 µm SL in cMyBP-C KO is accompanied by a significant decrease in the slope of the tension–pCa curve reflecting a decrease in cooperativity in myosin head attachment. This is in agreement with data showing that the presence of cMyBP-C appears to be necessary for order, and disorder of myosin heads [9]. The cMyBP-C dependence of Ca2+ sensitivity is consistent with studies showing that disruption of normal cMyBP-C by the addition of S2 [24] or C1C2 [25,26], replacement by an truncated form [5], and partial MyBP-C extraction [21]. In another cMyBP-C KO mouse model, myofilament Ca2+ sensitivity was reduced in single skinned mouse cardiomyocytes when compared to WT [7,27]. More recently, using the same model, a slight increase in Ca2+ sensitivity, although not significant, was found in multicellular preparations at 2.3 µm SL in KO vs. WT [26]. Our results suggest that, for the most part, differences in SL used in the studies may be responsible for the differences in Ca2+ sensitivity that have been reported. Slight molecular differences between the two cMyBP-C KO models, one accumulating mutant mRNA [7] and the other one without accumulation of any mutant mRNA (present model) could also be responsible for these discrepancies.

4.2 cMyBP-C and stretch-dependent regulation of Ca2+-sensitization
cMyBP-C appears to modulate Ca2+ sensitivity through a mechanism activated by cell lengthening. In the absence of normal cMyBP-C, Ca2+ sensitivity is already enhanced, and therefore further increase in SL has substantially less effect. Among published papers on cMyBP-C, a wide range of SLs was used although none used more than one SL. Sarcomere lengths varied from 1.8 µm [24], to 2.1 µm [21,28], 2.2 µm [29], and finally to 2.25–2.3 µm [7,27]. In line with our results, these studies at the shortest SL report large effects of cMyBP-C deficiency on Ca2+ sensitization. We would predict that the effects of cMyBP-C manipulation is reduced or abolished with stretch and even become the opposite with longer stretch if there is an optimal SL for Ca2+ sensitivity.

The mechanisms of how changes in SL affect Ca2+ sensitivity are not clear. Interfilament distance is an obvious consideration especially in view of the facts that both cMyBP-C phosphorylation (by itself) and sarcomere lengthening decrease interfilament distance [13], and that both influence the consequences of different degrees of order of myosin heads. It was also proposed that length dependence of activation could be a variable function of the state of phosphorylation of cTnI [13], and MLC-2 [15]. From the present study, it is suggested that cMyBP-C should be also considered as a myofilament regulatory protein. Another consequence of SL increase is to increase titin-based passive tension. Titin has been shown to affect cardiac contractility by altering the likelihood of actomyosin interaction via either modulating interfilament spacing or straining the thick filament [30,31]. Since cMyBP-C can interact with both titin and myosin, it could transmit part of the tension to the myosin head. Such a mechanism was observed in skeletal muscle [32].

4.3 Effect of PKA on length-dependent activation
Konhilas et al. reported a PKA effect on length-dependent activation, unobserved in the present study [13]. This may be explained by methodological differences. For example, in their study intact hearts were perfused with a physiological solution containing β-blockers and carbamyl choline to decrease the overall level of phosphorylation [13]. The PKA effect we observed is in the range of amplitude described in the literature that varies from 0.12 pCa unit [33] to 0.28 pCa units [13] at 2.3 µm SL. Various basal levels of phosphorylation before the PKA treatment could also explain the different effects of PKA on length-dependence: reduced [34], unchanged [35] or increased [13].

PKA-induced phosphorylation had almost no effect on Ca2+ sensitivity in KO myocytes at long or short SL, despite equal increases in cTnI phosphorylation of WT and KO. This is in line with the work by Yang et al. showing an impaired response during isoproterenol infusion of mice expressing a mutated cMyBP-C lacking both the titin and myosin binding sites at the C-terminus [36]. The lack of effect by β-adrenergic stimulation when cMyBP-C is absent is similar to the observations obtained when cTnI was replaced with ssTnI [13]. Combining the two sets of results supports the idea that PKA effect on myofilament activation requires both cMyBP-C and the phosphorylation of cTnI. From the present work, we cannot say if it is the protein itself that is required or its phosphorylation and this requires further experiments.

4.4 Implication of ssTnI
ssTnI expression induces a sensitization of myofilaments in normal conditions [13,37] and makes the myofilaments insensitive to β-adrenergic stimulation because ssTnI does not contain the PKA phosphorylation sites [37]. ssTnI expression in the cMyBP-C-deficient mice could in part influence the results observed. This isoform is transiently expressed in the heart during development. Particularly, ssTnI mRNA is present in rat neonatal cardiomyocytes [38]. In mouse ssTnI, the major form of TnI at early embryonic stage, decreases to 40% of total TnI at 7 days post-natal and almost disappears at 6–8 weeks of age [19]. Interestingly, it has been recently shown that ssTnI is re-expressed in various pathological models such as respiratory hypercapnic acidosis [39], and endotoxemia [40]. The authors suggested that replacement of cTnI with ssTnI in the heart provides significant protection against endotoxemia- or hypercapania-induced cardiac contractile dysfunction, most probably by preserving myofilament desensitization induced by β-adrenergic stimulation. This concept has been reinforced using a transgenic model in which cTnI was totally replaced by ssTnI [13,37]. In the present study, a maintained expression or a re-expression of ssTnI was observed in young KO animal that disappeared with age, although RT–PCR analysis shows that mRNA levels are similar in KO and WT at both ages. Differential expression of ssTnI between WT and KO animals might result from a post-transcriptional regulation. However, we cannot completely exclude that the lower band in Fig 5A is a specific degradation product in the KO model. In contrast with data showing expression of ssTnI in normal and pathological models, the PKA-induced desensitization of myofilaments did not correlate with the level of ssTnI expression. In addition, replacement of cTnI by ssTnI has been shown to be also associated with an increase in maximal active tension [41], which is not the case in the present study. It seems thus unlikely that ssTnI is responsible for the observed effects but rather linked to the lack of cMyBP-C. Various hypotheses can be proposed: (1) the small level of ssTnI is not enough to play a functional role; and (2) ssTnI is only partially incorporated in sarcomeres and its effects remain negligible.

In summary, alterations of the regulation of Ca2+ sensitivity by either length- or PKA-mediated phosphorylation of cardiomyocytes isolated from KO mice suggest that cMyBP-C is necessary for the regulation of cardiac contractility. The dobutamine effect on heart relaxation was unaffected in the heterozygote cMyBP-C mice in vivo and almost completely blunted in the KO mice [14]. The leftover of β-adrenergic responsiveness in vivo may be due to proteins involved in Ca2+ homeostasis or the other sarcomeric regulatory proteins unaltered in the KO model (cTnI and titin). Our results further suggest that at least some of the effects of PKA-induced cTnI phosphorylation on Ca2+-sensitivity require cMyBP-C, although one cannot distinguish from the present work whether it is the protein itself or its phosphorylated form that is involved.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by INSERM (PNRMC), Association Française contre les Myopathies, Fondation pour la Recherche Médicale, Fondation de France and Conseil Régional du Languedoc-Roussillon. S.S. was a Eurogendis Marie-Curie fellow. Thanks are due to Patrice Bideaux for technical assistance. We are thankful to Saul Winegrad (Philadelphia) for helpful discussion on this study.


   Notes
 
1 These authors contributed equally to this work. Back

Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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