Olivier Cazorla

F.P Alexandre Lucas

Florence Poirier

Alain Lacampagne
email: alain.lacampagne@inserm.fr

Frank Lezoualc'h
email: frank.lezoualch@u-psud.fr.

F L Lacampagne

The cAMP binding protein Epac regulates cardiac myofilament function

In the heart, cAMP is a key regulator of excitation-contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8-pCPT) reduced Ca 2؉ transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca 2؉ sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca 2؉ sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca 2؉ sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca 2؉ calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function. calmodulin kinase II ͉ contraction ͉ exchange protein activated by cyclic AMP ͉ sarcomeric proteins ͉ protein kinase C

T he second messenger cAMP is a key mediator of the sympathetic system and is involved in the control of cardiac function. Besides the cyclic nucleotide pacemaker channel, cAMP acts through the serine/threonine-specific protein kinase A (PKA) to modulate cardiac contractility via intracellular Ca 2ϩ movements [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF]. Ca 2ϩ is essential for cardiac electrical activity and directly activates myofilaments, thus inducing their contraction. In cardiac myocytes, PKA targets various Ca 2ϩ handling proteins involved in excitation-contraction (EC) coupling, such as the sarcolemmal L-type Ca 2ϩ channel and the sarcoplasmic reticulum (SR) ryanodine receptor (RYR) [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF]. The effect of PKA on myofilament protein phosphorylation is also critical for cardiac dynamics and contractility [START_REF] Solaro | Modulation of cardiac myofilament activity by protein phosphorylation[END_REF]. For instance, under ␤-adrenergic stimulation, PKA-dependent phosphorylation of the thin filament protein cardiac Troponin I (cTnI) results in reduction of myofilament Ca 2ϩ sensitivity and increase of crossbridge cycling rate, leading to acceleration of relaxation [START_REF] Layland | Regulation of cardiac contractile function by troponin I phosphorylation[END_REF]. Phosphorylation of the thick filament protein cardiac Myosin Binding Protein-C (cMyBP-C) by PKA appears to affect actin and myosin interactions [START_REF] Kunst | Myosin binding protein C, a phosphorylation-dependent force regulator in muscle that controls the attachment of myosin heads by its interaction with myosin S2[END_REF] and contributes to PKA effects on Ca 2ϩ sensitivity [START_REF] Cazorla | Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice[END_REF]. In addition, PKA-dependent phosphorylation of Titin has been shown to reduce cardiomyocyte stiffness and consequently heart diastolic force [START_REF] Fukuda | Phosphorylation of Titin modulates passive stiffness of cardiac muscle in a Titin isoform-dependent manner[END_REF].

A decade ago, a family of proteins directly activated by cAMP was discovered, adding another layer of complexity to the cAMP-mediated signaling cascade [START_REF] Kawasaki | A family of cAMP-binding proteins that directly activate Rap1[END_REF][START_REF] De Rooij | Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP[END_REF]. These proteins, named Epac (Exchange proteins directly activated by cAMP), are guanine nucleotide exchange factors (GEFs) for Rap1 and Rap2 small GTPases [START_REF] Bos | Epac proteins: Multi-purpose cAMP targets[END_REF]. Two variants of Epac exist (Epac1 and Epac2), both of which are activated by physiologically relevant concentrations of cAMP [START_REF] Bos | Epac proteins: Multi-purpose cAMP targets[END_REF]. Epac1 is highly expressed in the heart and displays comparable affinity for cAMP as a PKA holoenzyme [START_REF] De Rooij | Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP[END_REF][START_REF] Dao | Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition[END_REF]. With the recent availability of a selective Epac activator, the cAMP analog 8-(4-chlorophenylthio)-2Ј-Omethyl-cAMP (8-pCPT) [START_REF] Enserink | A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK[END_REF], several studies have revealed the critical role of Epac in various cellular processes such as cell permeability and cardiomyocyte hypertrophy [START_REF] Morel | cAMP-binding protein Epac induces cardiomyocyte hypertrophy[END_REF][START_REF] Somekawa | Enhanced functional gap junction neoformation by protein kinase A-dependent and Epac-dependent signals downstream of cAMP in cardiac myocytes[END_REF][START_REF] Lezoualc'h F | Small GTP-binding proteins and their regulators in cardiac hypertrophy[END_REF]. Interestingly, recent evidence indicates that Epac activation alters Ca 2ϩ signaling in the SR [START_REF] Pereira | The cAMP binding protein Epac modulates Ca2ϩ sparks by a Ca2ϩ/calmodulin kinase signalling pathway in rat cardiac myocytes[END_REF][START_REF] Oestreich | Epac-mediated activation of phospholipase C{epsilon} plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2ϩ mobilization in cardiac myocytes[END_REF]. However, the role of Epac in the regulation of cardiomyocyte contractility is still unknown.

Here we report that Epac potentiates cardiac contraction despite a decrease in the amplitude of Ca 2ϩ transient. We show that specific activation of Epac or overexpression of a constitutively active form of Epac increases myofilament Ca 2ϩ sensitivity in permeabilized ventricular cardiac myocytes in a PKAindependent manner. This is correlated with an increase in phosphorylation of cMyBP-C and cTnI. In addition, we report that Epac-dependent effects on myofilament proteins involve both protein kinase C (PKC) and Ca 2ϩ Calmodulin-Kinase II (CaMKII). Taken together our data show that independently of its effect on SR function, Epac has a direct effect on the contractile machinery and is a new piece of the regulatory cascade of cardiac contractile function.

Results

Epac Regulates Myofilament Ca 2؉ Sensitivity in a PKA-Independent Manner. To test the effect of Epac activation on cell contraction, we recorded simultaneously changes in sarcomere length (SL) and intracellular Ca 2ϩ in indo-1-loaded intact cardiomyocytes stimulated at 1 Hz prior to and during treatment with the Epac selective activator 8-pCPT (1 M) (Fig. 1A). SL shortening started to increase progressively Ϸ1 min after addition of 8-pCPT to reach a plateau within 5 min. 8-pCPT increased SL shortening and decreased calcium transient in a concentrationdependent manner (Fig. 1 B andC). The steady-state maximal inhibition of Ca 2ϩ transient could not be determined because arrhythmic events occurred at high concentrations (starting at 1 M), probably because of the increase in the diastolic Ca 2ϩ level (Fig. 1D). The gain of function (SL shortening-Ca 2ϩ transient ratio) that is a good indicator of the myofilament Ca 2ϩ sensitivity increased from 0.1 to 1 M 8-pCPT (Fig. 1E). The speed of SL shortening increased [supporting information (SI) Fig. S1 A] and diastolic SL decreased (Fig. S1C), both in a concentration-dependent manner. The speed of SL relaxation tended to increase with 8-pCPT but the differences did not reach significance (Fig. S1B). Altogether, these data suggest a dual effect of Epac activation on SR Ca 2ϩ signaling (i.e., Ca 2ϩ basal level and transient amplitude) and a direct effect on the contractile machinery (SL shortening).

To determine the effect of Epac activation on the contractile machinery properties, we measured the relationship between Ca 2ϩ -activated tension and internal concentration of Ca 2ϩ expressed as pCa (ϭ Ϫlog[Ca 2ϩ ]) in permeabilized cardiomyocytes (Fig. 2A). Permeabilized cardiomyocytes allow us to study the properties of the contractile machinery and its relation to calcium independently of the amount released by SR [START_REF] Kunst | Myosin binding protein C, a phosphorylation-dependent force regulator in muscle that controls the attachment of myosin heads by its interaction with myosin S2[END_REF][START_REF] Cazorla | Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice[END_REF][START_REF] Cazorla | Transmural stretch-dependent regulation of contractile properties in rat heart and its alteration after myocardial infarction[END_REF][START_REF] Noland | Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2ϩ)-stimulated MgATPase of reconstituted actomyosin S-1[END_REF]. After incubation with 8-pCPT (1 M), the curve representing the tension-pCa relationship was significantly shifted to the left (i.e., increase in pCa 50 ), indicating that for a given amount of Ca 2ϩ , more force was produced by the myofilaments upon Epac activation. The other contractile parameters such as maximal active tension, passive tension, and the Hill coefficient were not affected by Epac activation (Table S1). Conversely, permeabilized cells incubated with either a recombinant catalytic subunit of PKA (200 UI) or 6-BnZ-cAMP (200 M), a selective and membrane-permeant activator of PKA, showed a significant decrease in pCa 50 (Fig. 2 A, Fig. S2) . These data demonstrate that Epac and PKA have opposite effects on Ca 2ϩ -activated force and myofilament Ca 2ϩ sensitivity (Fig. 2 A, Fig. S2). Similarly, we found that ventricular myocytes isolated from myocardial tissues infected with a bicistronic adenovirus bearing a constitutively active form of Epac1 and green fluorescent protein (GFP) (Ad-Epac ⌬cAMP ) showed an increase in pCa 50 (Fig. 2B). The effect of Epac ⌬cAMP on pCa 50 was similar to the effect obtained with 8-pCPT (1 M) and was not further increased by addition of this drug (Fig. 2B). Finally, we found that Epac effect on myofilament Ca 2ϩ sensitivity was independent of PKA because a PKA inhibitor, PKI (5 M), failed to inhibit the effect of 8-pCPT on pCa 50 (Fig. 2C). Altogether, our data show that Epac activation increases myofilament Ca 2ϩ sensitivity in a PKAindependent manner in adult ventricular cardiac myocytes.

Epac Regulates Phosphorylation of cMyBP-C and cTnI. Myofilament Ca 2ϩ sensitivity is regulated through phosphorylation of sarcomeric proteins, such as cTnI and cMyBP-C, which are known to mediate myocardial responses to cAMP via PKA (3). Therefore we checked whether activated Epac could also regulate the phosphorylation of such proteins. Indeed, activation of endogenous Epac by 8-pCPT (1 M) increased phosphorylation of cMyBP-C at Ser 282 (P-cMyBP-C) in freshly isolated adult cardiomyocytes (Fig. 3A). The level of P-cMyBP-C following Epac activation was comparable to that observed in cells treated with isoproterenol (ISO) (100 nM), the standard ␤-adrenergic receptor agonist (Fig. 3A). Similarly, infection of cardiomyocytes with adenoviruses encoding the wild-type form of human Epac1 (Ad-Epac WT ) induced a partial increase in P-cMyBP-C that was further enhanced by treatment with 8-pCPT (1 M) (Fig. 3B). In myocytes expressing Ad-Epac ⌬cAMP , the P-cMyBP-C level was comparable to that obtained in myocytes infected with Ad-Epac WT and treated with 8-pCPT (Fig. 3B). In addition, Epacinduced P-cMyBP-C was independent of PKA, because PKI failed to block the effect of 8-pCPT on P-cMyBP-C phosphorylation (Fig. 3C). As previously reported, cMyBP-C was phosphorylated by PKA after ISO stimulation (Fig. 3D); however, ISO-induced cMyBP-C phosphorylation was only partially re- duced by treatment with PKI, suggesting that Epac contributes to the regulation of cMyBP-C phosphorylation induced by ␤-adrenergic receptors (␤-AR) (Fig. 3D). These results may explain the remaining increase in intact cell shortening after ISO stimulation in the presence of a PKA inhibitor, KT5720 (Fig. S1D). Accordingly, myocytes treated with ISO (100 nM) and infected with a short hairpin RNA targeting Epac1 (Ad-shEpac) to knock down its expression showed a decreased level of P-cMyBP-C compared to cells infected with shRNA sequence control (Ad-shCT) and stimulated with ISO (Fig. S3 A andB). Knockdown of Epac1 slightly decreased pCa 50 in basal condition but this was not statistically significant as compared to Ad-shCT control cells (Fig. S3C). ISO decreased myofilament Ca 2ϩ sensitivity in a similar fashion in Ad-shCT and Ad-shEpac cells (Fig. S3C). Importantly, 8-pCPT failed to increase myofilament Ca 2ϩ sensitivity in cells infected with Ad-shEpac, indicating that Epac specifically regulates the contractile properties of cardiomyocytes (Fig. S3C).

We next investigated whether Epac activation could also regulate phosphorylation of cTnI. Differently from ISO, 8-pCPT failed to induce cTnI phosphorylation at Ser 22/23 even in cells infected with Ad-Epac WT or Ad-Epac ⌬cAMP (Fig. 4A). To determine whether Epac activation could influence cTnI phosphorylation at other sites, myofilament proteins were extracted from myocytes infected with either Ad-GFP or Ad-Epac WT and treated or not with 1 M 8-pCPT or infected with Ad-Epac ⌬cAMP . In extracts from cells treated with 1 M 8-pCPT or infected with Ad-Epac ⌬cAMP , 1 band of Ϸ23 kDa showed a significant increase in phosphorylation, as determined with the ProQ phospho-protein stain (Fig. 4B). Tandem mass spectrometry revealed that this 23-kDa band corresponded to cTnI (MASCOT score: 196; number of identified peptides: 19). The effect of Epac activation on cTnI phosphorylation was not abolished by PKI (Fig. 4C), which, on the other hand, completely prevented PKA-dependent phosphorylation of cTnI (Fig. S4). To confirm a change in the phosphorylation status of cTnI upon Epac activation, myocytes treated or not with 8-pCPT were processed for 2D electrophoresis and subjected to ProQ Diamond staining to reveal phosphoproteins. In an area of interest corresponding to a theoretical spot of cTnI (pI 9.57; M r 25 kDa), we identified 5 phosphorylated spots corresponding to cTnI by tandem mass spectrometry of peptides separated by reverse phase liquid chromatography (LC/MS/MS). Fig. 4D shows that cTnI was resolved as a train of 5 spots differing in their phosphorylation level in basal condition. The intensity of the train of phosphorylation was modified in the presence of 8-pCPT with spot 5 being the most phosphorylated. Altogether these data show that Epac induces a change in the phosphorylation status of cTnI and cMyBP-C in a PKA-independent manner.

Signaling Pathways Involved in Myofilament Phosphorylation Induced by Epac. Because the permeabilization process could induce a loss of Epac expression and its potential effectors, we first analyzed their expression in permeabilized cardiomyocytes. We found that all these proteins are present in permeabilized cardiomyocytes although some of them showed a decreased expression level as compared to intact cardiac myocytes (Fig. 5A). We next investigated the signaling pathways involved in Epac-induced myofilament phosphorylation. The primary function of Epac is to act as GEFs for Rap GTPases [START_REF] Bos | Epac proteins: Multi-purpose cAMP targets[END_REF]. Thus, we examined whether Rap1 was involved in the effect of Epac on myofilament phosphorylation. A Rap1 GTPase activating protein (RapGAP), which has been previously shown to abolish 8-pCPT-induced Rap1 activation in adult cardiac myocytes [START_REF] Metrich | Epac mediates {beta}-adrenergic receptor-induced cardiomyocyte hypertrophy[END_REF], failed to inhibit an Epac effect on P-cMyBP-C (Fig. S5). This finding demonstrates that Epac acts on myofilaments via another effector than Rap1.

Previous studies have suggested that phospholipase C (PLC) may be involved in Epac functional effects [START_REF] Oestreich | Epac-mediated activation of phospholipase C{epsilon} plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2ϩ mobilization in cardiac myocytes[END_REF][START_REF] Schmidt | A new phospholipase-C-calcium signalling pathway mediated by cyclic AMP and a Rap GTPase[END_REF]. PLC hydrolyzes phosphatidylinositol bisphosphate (PIP2) to produce diacylglycerol (DAG) and inositol triphosphate (IP3), leading to protein PKC activation or IP3 receptor-dependent Ca 2ϩ release. The stimulating effect of 8-pCPT on myofilament Ca 2ϩ sensitivity was blocked by treatment with U73122, a PLC inhibitor, or calphostin-C, a PKC inhibitor (Fig. 5B). Calphostin-C also decreased cTnI and cMyBP-C phosphorylation induced by 8-pCPT (Fig. 5 C andE). Because we previously identified CaMKII as an effector of Epac in cardiac myocytes [START_REF] Pereira | The cAMP binding protein Epac modulates Ca2ϩ sparks by a Ca2ϩ/calmodulin kinase signalling pathway in rat cardiac myocytes[END_REF][START_REF] Metrich | Epac mediates {beta}-adrenergic receptor-induced cardiomyocyte hypertrophy[END_REF], we also evaluated whether CaMKII played a role in Epac effect on Ca 2ϩ sensitivity. We found that KN-93, a pharmacological inhibitor of CaMKII, significantly inhibited the 8-pCPT effect on Ca 2ϩ sensitivity in myofilaments (Fig. 5B). Consistent with these data, 8-pCPT-induced cTnI and cMyBP-C phosphorylation was blocked when myocytes were preincubated with KN-93 or infected with adenoviruses coding for a CaMKII peptide inhibitor (Ad-CaMKIIN) (Fig. 5 D and F, Fig. S6). Taken together, these data show that Epac regulates myofilament Ca 2ϩ sensitivity and sarcomeric protein phosphorylation through PLC-, PKC-, and CaMKII-dependent pathways.

Discussion

In this study we demonstrate that Epac regulates the contractile properties of cardiomyocytes by modulating Ca 2ϩ signaling and Ca 2ϩ sensitivity of sarcomeric proteins. We report that both PKA and Epac, 2 major effectors of cAMP, increase cardiac myocyte contraction but through opposite mechanisms. Indeed, Epac decreases the amount of Ca 2ϩ released by the SR and sensitizes myofilaments to Ca 2ϩ , whereas PKA does the opposite.

Epac activation increased rapidly the rate of SL shortening and reduced significantly Ca 2ϩ transient amplitude, both in a concentration-dependent manner, thus suggesting that Epac affects both myofilament properties and SR Ca 2ϩ function independently. Similarly, we previously observed a decrease in SR Ca 2ϩ load upon Epac activation in adult rat cardiac myocytes subsequent to CaMKII-dependent RyR phosphorylation and an increase in SR Ca 2ϩ leak during diastole [START_REF] Pereira | The cAMP binding protein Epac modulates Ca2ϩ sparks by a Ca2ϩ/calmodulin kinase signalling pathway in rat cardiac myocytes[END_REF]. These data could explain the reduced Ca 2ϩ transient amplitude in the presence of 8-pCPT and the increase in diastolic Ca 2ϩ level (Fig. 1C). Consistent with this finding, Curran and colleagues (21) also found that ␤-AR stimulation enhanced SR Ca 2ϩ leak in ventricular myocytes in a CaMKII-dependent (and PKAindependent) manner. Conversely, Oestreich and colleagues [START_REF] Oestreich | Epac-mediated activation of phospholipase C{epsilon} plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2ϩ mobilization in cardiac myocytes[END_REF] showed that acute treatment of single mouse ventricular cardiac myocytes with 8-pCPT increased Ca 2ϩ transient amplitude in field-stimulated cells. This process was dependent on Rap1 and PLC- [START_REF] Oestreich | Epac-mediated activation of phospholipase C{epsilon} plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2ϩ mobilization in cardiac myocytes[END_REF]. The reasons for these discrepancies are unclear and may involve species differences and/or methodological approaches such as the frequency of cardiac myocyte electrical stimulation.

PKA-dependent phosphorylation of cTnI is known to reduce myofilament Ca 2ϩ sensitivity and to shift the tension-pCa curve toward the right [START_REF] Solaro | Multiplex kinase signaling modifies cardiac function at the level of sarcomeric proteins[END_REF]. This effect involves phosphorylation of cTnI at Ser 22 /Ser 23 (3) and of cMyBP-C [START_REF] Cazorla | Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice[END_REF]. Here, we report that activated Epac increases myofilament Ca 2ϩ sensitivity and modulates phosphorylation of cTnI and cMyBP-C, both in a PKAindependent manner. We also show that a PLC inhibitor abolishes the stimulating effect of Epac on myofilament Ca 2ϩ sensitivity, suggesting that a downstream effector of PLC is involved in Epac signaling leading to sarcomeric protein phosphorylation. Such a candidate could be PKC because inhibition of PKC decreased 8-pCPT-induced myofilament Ca 2ϩ sensitization and cTnI and cMyBP-C phosphorylation. PKC activation has been reported to potentially induce phosphorylation of cTnI at Ser 22/23 , Ser 43/45 , and Thr 144 (3). However, PKC-dependent phosphorylation of Ser 22/23 is unlikely to be involved in an Epac effect because we did not detect increased cTnI phosphorylation at this site after Epac activation (Fig. 4A). Moreover, phosphorylation at this site would induce the same functional effects of PKA activation (i.e., decrease in myofilament Ca 2ϩ sensitivity) [START_REF] Noland | Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2ϩ)-stimulated MgATPase of reconstituted actomyosin S-1[END_REF][START_REF] Noland | Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants[END_REF].

PKC phosphorylates also cMyBP-C but the functional significance of these phosphorylations in the control of cardiac function is unknown [START_REF] Solaro | Multiplex kinase signaling modifies cardiac function at the level of sarcomeric proteins[END_REF][START_REF] Flashman | Cardiac myosin binding protein C: Its role in physiology and disease[END_REF]. Interestingly, PKC leads to CaMKII activation upon Epac activation, suggesting that a linear cascade involving PLC, PKC, and CaMKII could be involved in Epac-dependent myofilament regulation (25) (Fig. S7). Alternatively, CaMKII could directly phosphorylate cTnI and cMyBP-C. Indeed, we found that inhibition of CaMKII activity blocked the effect of Epac on myofilament Ca 2ϩ sensitivity and phosphorylation of sarcomeric proteins. The residual phosphorylation of cTnI and cMyBP-C could be because of PKC activation by Epac. Consistent with our study, cMyBP-C has also been reported to be phosphorylated by CaMKII (26 -28), resulting in modulation of the rates of force development and relaxation [START_REF] Tong | Roles of phosphorylation of myosin binding protein-C and troponin I in mouse cardiac muscle twitch dynamics[END_REF]. It is now known that cardiac contraction can be differentially regulated by a restricted number of sarcomeric proteins [START_REF] Solaro | Multiplex kinase signaling modifies cardiac function at the level of sarcomeric proteins[END_REF]. Indeed, phosphorylation of cTnI and cMyBP-C can exert opposite regulatory effects depending on the type of kinase involved and on the site of phosphorylation. Our data suggest that the functional effect of Epac on myofilaments results from phosphorylations of both cTnI and cMyBP-C following dual activation of PKC and CaMKII (Fig. 5). This particular pathway may explain the discrepancies between the myofilament Ca 2ϩ sensitization observed in the present study and the myofilament Ca 2ϩ desensitization observed after specific PKC stimulation in other studies [START_REF] Noland | Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2ϩ)-stimulated MgATPase of reconstituted actomyosin S-1[END_REF][START_REF] Noland | Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants[END_REF]. Interestingly, PKC isoforms differentially regulate sarcomeric protein phosphorylation such as cTnI, MLC-2, and TnT [START_REF] Wu | Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins[END_REF][START_REF] Kanaya | Propofol increases phosphorylation of troponin I and myosin light chain 2 via protein kinase C activation in cardiomyocytes[END_REF]. In our study, we did not find any effect of Epac activation on the phosphorylation status of myosin light chain 2 (Fig. S8). Consistent with our data on PKC-dependent myofilament sensitization upon Epac activation, the PKC-dependent pathway involving the phosphoryla-tion of cTnI has been previously shown to increase myofilament Ca 2ϩ sensitivity [START_REF] Kanaya | Propofol increases phosphorylation of troponin I and myosin light chain 2 via protein kinase C activation in cardiomyocytes[END_REF][START_REF] Wang | PKC-␤II sensitizes cardiac myofilaments to Ca2ϩ by phosphorylating troponin I on threonine-144[END_REF]. At present, we still do not know which PKC isoform(s) is (are) specifically involved in the regulation of myofilament protein phosphorylation induced by Epac. Further studies will be needed to identify the specific Epac-dependent phosphorylation sites in cTnI and cMyBP-C.

An important question concerns the physiological or pathophysiological relevance of Epac effects on the phosphorylation of myofilament protein. We show here that ISO increases cMyBP-C phosphorylation in a PKA-independent fashion, suggesting that ␤-AR may influence sarcomeric phosphorylation via Epac. It is therefore crucial to understand the context in which Epac is regulated by ␤-AR and the associated functional effects. Epac activity depends on the level of cAMP, which is in turn regulated by adenylate cyclases and phosphodiesterases (PDE). Some PDE inhibitors, such as adibendan and saterinone referenced as Ca 2ϩ sensitizers [START_REF] Blinks | Modification of myofibrillar responsiveness to Caϩϩ as an inotropic mechanism[END_REF], behave like Epac. Because PDEs are key enzymes for the regulation of cAMP concentration and diffusion in cardiac cells [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: The role of cyclic nucleotide phosphodiesterases[END_REF], one can speculate that PDE inhibitors may influence the Epac signaling pathway to account for their Ca 2ϩ sensitizing effects.

Chronic stimulation of ␤-AR causes hypertrophy in cardiac myocytes [START_REF] Lohse | What is the role of beta-adrenergic signaling in heart failure?[END_REF]. Our previous work indicated that Epac contributes to the hypertrophic effect of ␤-AR in a CaMKII-dependent, but PKA-independent, fashion [START_REF] Metrich | Epac mediates {beta}-adrenergic receptor-induced cardiomyocyte hypertrophy[END_REF]. Consistent with its role in cardiac remodeling, Epac is increased in different animal models of myocardial hypertrophy and upregulates markers of cardiac hypertrophy [START_REF] Morel | cAMP-binding protein Epac induces cardiomyocyte hypertrophy[END_REF][START_REF] Metrich | Epac mediates {beta}-adrenergic receptor-induced cardiomyocyte hypertrophy[END_REF][START_REF] Ulucan | Developmental changes in gene expression of Epac and its upregulation in myocardial hypertrophy[END_REF]. Altogether these findings, combined with the observation that myofilament properties are altered in cardiac hypertrophy and heart failure, suggest that Epac may play a role in the changes of sarcomeric proteins observed in these pathologies [START_REF] Layland | Regulation of cardiac contractile function by troponin I phosphorylation[END_REF].

In conclusion, our data show that the cAMP-binding protein Epac has an opposite effect from PKA on myofilament Ca 2ϩ sensitivity. We demonstrate that Epac is a mediator of sarcomeric proteins' phosphorylation and may contribute to the regulation of myofilament function. Our data suggest that altered Epac activity in disease may impact on contractile function.

Materials and Methods

Myocyte Preparation. All experiments were carried out according to the ethical principles laid down by the French (Ministry of Agriculture) and European Union Council Directives for the care of laboratory animals. Male Wistar rats (250 -300 g) were anesthetized by sodium pentobarbital i.p. injection (2 g/kg). Cardiac ventricular myocytes were isolated by standard enzymatic methods as previously described [START_REF] Cazorla | SR33805, a Ca2ϩ antagonist with length-dependent Ca2ϩ-sensitizing properties in cardiac myocytes[END_REF]. Briefly, the heart was excised and perfused retrogradely for 5 min at 37 °C with a Ca 2ϩ -free Hepes-buffered solution (117 mM NaCl, 5.7 mM KCl, 4.4 mM NaHCO3, 1.5 mM KH2PO4, 1.7 mM MgCl2, 21 mM Hepes, 11 mM glucose, 20 mM taurine) adjusted to pH 7.2 with NaOH and bubbled with 100% O2. The heart was then perfused with an enzyme-containing solution for 20 -30 min (1.3 mg⅐mL Ϫ1 collagenase type IV (Worthington). Cells were then filtered and washed several times in the Hepes-buffered solution containing 0.3 mM Ca 2ϩ . Finally, myocytes were kept in Hepes-buffered solution containing 1 mM Ca 2ϩ and 0.5% BSA. The specific relationship between the amount of Ca 2ϩ and the force developed by myofilaments was studied in myocytes permeabilized with 0.3% Triton X-100 in relaxing solution, resulting in a full permeabilization of sarcolemmal, SR, nuclear, and mitochondrial membranes. Myofilaments are activated by perfusing the cell with an internal solution containing increasing amounts of Ca 2ϩ . With permeabilized myocytes it is thus possible to measure precisely the relationship between force developed by the myofilaments and the exact amount of Ca 2ϩ . Permeabilized cells were incubated with either 8-pCPT (1 M, 10 min) or 6-BnZ-cAMP (200 M, 60 min) in relaxing solution at room temperature (22 °C) to activate Epac and PKA, respectively. We also activated the PKA pathway, using the recombinant catalytic subunit of PKA. For that, permeabilized cells were preincubated for 50 min at room temperature with 200 UI of recombinant catalytic subunits of PKA per milliliter of relaxing solution.

For a description of other methods, see SI Materials and Methods.

ACKNOWLEDGMENTS. We are grateful to H. Lum and L. Carrier for providing Ad-PKI and the antibody against cMy-BPC, respectively. We are grateful to Aurore Germain, Florence Lefebvre, Guillermo Salazar, Patrice Bideaux,

Fig. 1 .

 1 Fig. 1. Epac regulates the contractile machinery. (A) Effect of 8-pCPT (1 M) perfusion on sarcomere shortening (Left, Upper) and intracellular calcium transient amplitude (Left, Lower) in intact ventricular cardiomyocytes stimulated at 1 Hz. Activation of Epac increased progressively SL shortening and reduced the amplitude of Ca 2ϩ transient. The maximal effect was observed within 5 min. Right, a time control of SL shortening and calcium transient. (B-E) Concentration-dependent effect of 8-pCPT on SL shortening (B), Ca 2ϩ transient amplitude (C), diastolic calcium level (D), and the gain of function (E), which corresponds to the ratio between SL shortening and calcium transient amplitudes (n ϭ 11 cells). * , P Ͻ 0.05 versus control.

Fig. 2 .

 2 Fig. 2. Epac regulates myofilament Ca 2ϩ sensitivity in a PKA-independent manner. (A) The relationship between Ca 2ϩ -activated tension and intracellular Ca 2ϩ content was measured in isolated, permeabilized cardiomyocytes at 2.3 m SL. The relationship was fitted with a modified Hill equation and the pCa at which half of the maximal tension is developed (pCa 50) was determined as an index of myofilament Ca 2ϩ sensitivity (see SI Materials and Methods for more details). Preincubation of cells with 8-pCPT (1 M for 10 min) increased myofilament Ca 2ϩ sensitivity as indicated by the shift toward the left of the curve and the increase in pCa 50 (n ϭ 14 cells). In similar conditions, recombinant PKA catalytic subunit induced an opposite effect that reflects desensitization of the myofilaments (n ϭ 10 cells). (B) Constitutively active Epac (Epac ⌬cAMP ) increased myofilament Ca 2ϩ sensitivity as indexed by pCa50. The left ventricular free wall of rats was infected with adenoviruses encoding GFP (Ad-GFP) (control) or bicistronic adenoviruses coexpressing GFP and Epac ⌬cAMP (Ad-Epac ⌬cAMP ). Three days later, cells were isolated and myofilament Ca 2ϩ sensitivity of permeabilized ventricular cardiomyocytes expressing GFP was measured after incubation, or not, with 8-pCPT (1 M) for 10 min. Ad-GFP, n ϭ 10; Ad-Epac ⌬cAMP , n ϭ 16 cells (3 rats per condition). (C) Effect of PKI, a PKA inhibitor, on 8-pCPT-induced myofilament Ca 2ϩ sensitization. Myofilament Ca 2ϩ sensitivity (pCa50) was determined in permeabilized adult ventricular cardiomyocytes treated, or not, with 8-pCPT (1 M) for 10 min and in the presence or absence of PKI (5 M); n ϭ 16 cells (3 rats). Results are expressed as means Ϯ SEM. ** , P Ͻ 0.01 compared with nontreated cells.

Fig. 3 .

 3 Fig. 3. Epac regulates phosphorylation of the thick filament protein cMyBP-C. (A) Effect of 8-pCPT (1 M) or ISO (100 nM) on cMyBP-C phosphorylation (P-cMyBP-C) in freshly isolated adult cardiomyocytes. Cells were incubated with the drugs for 10 min and P-cMyBP-C was determined by Western blotting using an anti-P-cMyBP-C (P-Ser 282 ) antibody as described in SI Materials and Methods. Membranes were then stripped and probed for total cMyBP-C expression to confirm equivalent protein loading. Lower, immunoblots were quantified and data were normalized to total cMyBP-C expression. (B and C) Effect of 8-pCPT (1 M) and (D) effect of ISO (100 nM) on P-cMyBP-C in adult cardiomyocytes infected with Ad-GFP, Ad-Epac WT , Ad-Epac ⌬cAMP , or Ad-PKI (PKA inhibitor). Immunoblots against P-cMyBP-C and total cMyBP-C were performed as in A. (B) An anti-HA antibody was used to monitor the expression levels of Epac WT and Epac ⌬cAMP . (C and D) Immunoblots were quantified and data normalized to total cMyBP-C. Results are means Ϯ SEM from 6 (A) or 4 (B-D) independent experiments. * , P Ͻ 0.05; ** , P Ͻ 0.01; *** , P Ͻ 0.001 compared with control or indicated values. CT, control.

Fig. 4 .

 4 Fig. 4. Epac regulates phosphorylation of cTnI in a PKA-independent manner. (A) Representative Western blot showing cTnI phosphorylation (P-cTnI) at PKA sites (P-Ser 22 /P-Ser 23 ) (Top). Isolated adult cardiac myocytes were infected with control Ad-GFP (CT), Ad-Epac WT , or Ad-Epac ⌬cAMP for 36 h. Cells were then treated, or not, with 1 M 8-pCPT or 100 nM ISO for 10 min and P-Ser 22 /P-Ser 23 cTnI was determined by Western blotting as described in SI Materials and Methods. Total cTnI expression is shown (Middle), and anti-HA antibody was used to monitor the expression levels of Epac WT and Epac ⌬cAMP (Bottom). (B) Adult cardiac myocytes were infected for 36 h with control Ad-GFP (CT, lane 1), Ad-GFP treated with 1 M 8-pCPT (lane 2), Ad-Epac WT treated with 1 M 8-pCPT (lane 3), or Ad-Epac ⌬cAMP (lane 4). (C) Myocytes were infected with Ad-PKI and were then treated or not with 1 M 8-pCPT for 10 min as in A and B. Protein phosphorylation was visualized with ProQ Diamond phospho-protein gel stain, followed by total protein staining with Sypro Ruby to confirm equal loading. Right, protein phosphorylation was normalized to the total protein content revealed by SYPRO Ruby staining. Results are means Ϯ SEM of 8 (B) or 4 (C) independent experiments. * , P Ͻ 0.05; ** , P Ͻ 0.01 compared with control values. Tandem mass spectrometry revealed that the phosphorylated 23-kDa band corresponded to cTnI. (D) ProQ Diamond and EZ Coomassie Blue staining of a representative 2D gel. Isolated cardiomyocytes were incubated in the absence or the presence of 8-pCPT (1 M, 10 min). Protein extracts were separated by 2D electrophoresis using isoelectric focusing strips (18 cm, pH 3-11, nonlinear). A representative enlargement of the gel showing the marked region is illustrated. Five spots were excised and analyzed by LC/MS/MS to show the presence of cTnI. Densitometric analysis of intensities of protein spots was performed and ratios of volume values are indicated in the table. Changes in intensities of protein spots are indicated as increased (up) or decreased (down) in the stimulated 8-pCPT vs. control (CT). The 5 protein spots were identified using the MASCOT Search Engine. The score refers to the degree of similarity between a sample and a searched database match. A score of Ն65 is considered a properly identified match. ID, identity.

Fig. 5 .

 5 Fig. 5. Epac signaling pathways involved in myofilament protein regulation. (A) Expression analysis of Epac and its potential downstream effectors in nonpermeabilized (intact) and permeabilized cardiac myocytes. The indicated proteins were revealed by Western blotting as described in SI Materials and Methods. (B) Shift of myofilament Ca 2ϩ sensitivity of activation (⌬pCa50) induced by 8-pCPT in permeabilized cardiomyocytes preincubated with a PLC inhibitor (U73122, 5 M), a PKC inhibitor (Calphostin C, 0.5 M) or a CaMKII inhibitor (KN-93, 2 M). Average values were expressed as the difference in pCa 50 between nonstimulated (no inhibitor) and 8-pCPT-treated cells (n ϭ 8 -13 cells per condition). (C and E) Effect of a PKC inhibitor, calphostin-C (0.5 M), and (D and F) effect of a CaMKII inhibitor, KN-93 (0.5 M), on P-cTnI and P-cMyBP-C in isolated cardiomyocytes. In C and D phosphorylation of the 23-kDa band corresponding to P-cTnI was revealed by ProQ Diamond phosphoprotein gel staining, followed by total protein staining with Sypro Ruby to confirm equal loading. Representative gels are shown of 4 independent experiments. (E and F) Western blots were performed to probe for P-cMyBP-C and total cMyBP-C expression. Lower, immunoblots were quantified and data normalized to total cMyBP-C expression. Results are means Ϯ SEM from 5 (E) and 4 (F) independent experiments. * , P Ͻ 0.05; *** , P Ͻ 0.001 compared with control and indicated values.

͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812536106 Cazorla et al.

͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812536106 Cazorla et al.

Chloe ´Godard, and Ce ´line Boursier for their technical assistance. We acknowledge Bertrand Crozatier for the critical reading of the manuscript. This work was supported by grants from the Agence Nationale de la Recherche (Epac-06 to F.L.), the Association Franc ¸aise Contre les Myopathies (AFM-11590 to O.C.), the Leducq Foundation (Caerus Network) (A.L.), and the Fondation pour la Recherche Me ´dicale (e ´quipe FRM) (F.L.).


Olivier Cazorla

A Freiburg

M Helmes

T Centner

M Mcnabb

Yiming Wu

K Trombitás

S Labeit

Henk Granzier
email: granzier@wsunix.wsu.edu

Differential Expression of Cardiac Titin Isoforms and Modulation of Cellular Stiffness

Keywords: Compliance, passive tension, diastolic force, mechanical properties, myocyte, connectin

Introduction

During diastole, the myocardium stretches and passive force is generated. A major contributor to this force is the giant protein titin, spanning the half sarcomere from Z-band to M-line. When sarcomere length (SL) increases during diastole, titin's I-band region extends and passive force (F) develops. The shape of titin's F-SL relation is expected to influence ventricular filling during diastole and ventricular emptying during systole. In addition to influencing ventricular filling, titin also helps to maintain the structural integrity of the contracting sarcomere (for reviews see 1-5 ).

Titin's force arises from its extensible I-band region which consists of two main segment types:

1) a segment rich in proline (P), glutamate (E), valine (V) and lysine (K) residues (the so-called PEVK segment) and 2) serially linked immunoglobulin-like domains (tandem Ig segments) flanking this PEVK segment. 6 Several titin isoforms are now known, all of which contain PEVK and tandem Ig segments. In addition to these common segments the extensible region of the N2B isoform contains the N2B element (3 Ig domains and a 572-residue unique sequence) while the N2A class of isoforms contains the N2A element (4 Ig domains and a 106-residue unique sequence). N2B titin is found exclusively in cardiac muscle and contains within its central I-band region a 163 residue PEVK segment and tandem Ig segments with 37 Ig domains. N2A titins are found in skeletal muscles with PEVK and tandem Ig segments that differ in length in different muscles. [START_REF] Labeit | Titins: giant proteins in charge of muscle ultrastructure and elasticity[END_REF] Heart muscle expresses not only N2B transcripts but also N2A transcripts; the latter are associated with a larger PEVK segment than the N2B transcripts. [START_REF] Labeit | Titins: giant proteins in charge of muscle ultrastructure and elasticity[END_REF] Here we investigated cardiac titin isoform expression by high resolution SDS-PAGE and by various immuno-labeling techniques (western blotting, immunoelectron microscopy and immunofluorescence) with isoform-specific antibodies.

In slack sarcomeres titin's extensible segment has a short end-to-end length as a result of thermally-induced bending motions that lead to a state of maximal entropy. 7, 8 Straightening of the extensible region by extending the sarcomere lowers the conformational entropy and results in a force, known as entropic force. The extensible region of titin does not straighten uniformly, rather tandem Ig and PEVK segments extend sequentially. 9-11 In addition, it was recently found that the 572 residue unique sequence of the N2B element is extensible as well, explaining why the extensible region of N2B titin can be stretched to lengths much longer than the combined contour length (end-to-end-length when completely straight) of the tandem Ig and PEVK segments. [START_REF] Helmes | Mechanically driven contour-length adjustment in rat cardiac titin`s unique N2B sequence: titin is an adjustable spring[END_REF][START_REF] Linke | I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure[END_REF] Variation in the length of the extensible region is predicted to influence the developed force upon extension. Cardiac myocytes that express a larger isoform (referred to below as N2BA titin) have a longer extensible region and are predicted to develop less force than those that express N2B-titin, as for a given SL the fractional extension (end-to-end length of extensible region divided by its contour length) of N2BA titin will be less than that of the N2B isoform (for details see Discussion).

Here we investigated titin expression at the protein level. A number of mammalian species were surveyed for the expression of different cardiac titin isoforms. SDS-PAGE revealed that in most species both small and large isoforms are co-expressed, but in widely varying ratios. Western blot studies with N2B and N2A antibodies suggest that the smaller isoform contains the N2B element (N2B titin), whereas the larger isoform contains both the N2B and N2A elements (N2BA titin). To test the hypothesis that the expression of the N2BA isoform leads to a decrease in cell stiffness, mechanical experiments were performed on single cardiac myocytes isolated from hearts showing a high or low N2BA expression level. The results indicate that expressing small and large titin isoforms at different ratios is a means to modulate cardiac myocyte stiffness.

Material and Methods

Cardiac myocyte isolation. Ventricular and atrial myocytes were isolated from rat, mouse, and pig by digesting their extracellular matrix with collagenase and hyaluronidase. The protocol to isolate cells from rat has been explained earlier 14 , the protocol for mouse and pig was adapted from Wolska 15 . For isolating pig atrial cells we used the blending method developed by Moss 16 . The myocytes were chemically skinned as previously described. [START_REF] Granzier | Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments[END_REF] To prevent degradation, all solutions contained protease inhibitors (see [START_REF] Granzier | Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments[END_REF]. See Material and Methods online.

Gel electrophoresis. Myocardial and skeletal muscle samples were quick-frozen in liquid nitrogen, pulverized and then rapidly solubilized. 14 The samples were analyzed with SDS-PAGE (2-9.5% acrylamide gradient gels) and Coomassie Blue stained. Titin and myosin heavy chain (MHC) bands were analyzed as previously described 14 with some adaptations (see Material and Methods online).

Western blotting.

Western blotting was performed with affinity purified anti-titin antibodies (raised in rabbit) specific to the N-terminal end of titin (Z1/Z2 and Zr), the C-terminal end of titin (T51), the C-terminus of the PEVK segment (bk283/4), N2A (X105-X106) and N2B (X150-X151, X214-X215 and X216-X217). 8 (For details see Fig. 3A Electron microscopy. Electron microcopy was used to obtain cross-sections of skinned cells to determine the myofibrillar fractional area. Immunoelectron microscopy was used to study rat and pig left ventricular cells labeled with N2A (X105-106) and N2B (X150-151) antibodies 8,18 .

Results

SDS-PAGE analysis.

Comparative gel electrophoresis of human skeletal muscle (soleus) and myocardium of rat, rabbit, bovine and human confirmed earlier findings and provided novel results as well. Confirming earlier findings, skeletal muscle and rat myocardium contain a single major titin band at the top of the gel (T1 in Fig. 1, left two lanes), representing the full-length titin molecule 8 . T1 mobility greatly varies, reflecting the difference in molecular mass of the 3.7 MDa soleus titin and the 2.97 MDa rat cardiac titin. [START_REF] Labeit | Titins: giant proteins in charge of muscle ultrastructure and elasticity[END_REF] The samples also reveal a minor band with higher mobility (T2) derived from a large titin degradation product that contains the A-band segment of titin. 8 The T2 band is barely detectable in samples carefully prepared to minimize degradation (Fig. 1). Novel findings were obtained when using myocardium of rabbit, bovine and human.

These species contain two T1 bands, a bottom band which co-migrates with the T1 band of rat and a top band with clearly reduced mobility (Fig. 1). The top T1 is just visible in rabbit (Fig. 1 inset) but easily detectable in bovine and human. The fact that two T1 bands in cardiac muscle have gone unnoticed in our earlier works, is because our previous studies focussed on rat and rabbit cardiac titin and in these species the top T1 band is minor or absent. Limited gel resolution is another explanation why two T1 bands may have been missed in our previous studies. Only large pore gels with high spatial resolution allow the two T1 bands in bovine and human to be clearly separated.

To follow up on these findings we surveyed additional species, examined ventricular and atrial myocardium in some of them, and determined the ratio of the top to bottom T1 bands using quantitative densitometry. Two T1 bands were consistently found in many species, but the density of the bands varied greatly. Rat, rabbit and mouse myocardium contain barely detectable levels of the top T1 band, in dog and human the two bands are present at similar levels, and in pig and cow the top T1 band dominates (Figs. 2A andB). Furthermore, in the two species in which we examined both left ventricular and left atrial myocardium, the expression ratio of top to bottom band is much higher in the atrium than in the ventricle (Figs. 2A andB). For example, in the left ventricle of the cow the top T1 band is 1.8 times more abundant than the bottom band, while in the left atrium the top band is 21 times more abundant. Finally, we also studied titin expression in the sub-endocardial, mid-wall and sub-epicardial regions of the ventricular free wall of the pig.

Significantly higher levels of the large isoform were found in the sub-endocardium (Fig. 2C).

Immuno-labeling experiments. Using the mobility of human soleus T1 (Mr 3.7 MDa) and rat cardiac titin T1 (Mr 2.97 MDa) as standards suggests that the two T1 bands of pig differ by ~0.35 MDa in mass. To test whether both T1 bands represent full-length titin molecules (and not degradation products), western blot experiments were performed with antibodies against the terminal ends of the molecule (Fig. 3A). The presence of the N-terminal end of titin was investigated with Z1/Z2 and Zr antibodies (Z1/Z2 labels the 200 first N-terminal amino acid residues and Zr labels epitopes ~ 500 residues from the N-terminus, [START_REF] Gregorio | Muscle assembly: a titanic achievement? Current opinion in[END_REF] ) and the presence of titin's C-terminal end with T51 antibody (labels ~200 residues from the C-terminus, 20 ). Western blot results show that all antibodies reacted with the two T1 bands (Fig. 3B). Thus it is likely that the two T1 bands represent distinct full-length titin molecules.

Cardiac muscle differentially expresses transcripts, which code for the N2B and N2A elements, respectively. 6 To determine the identity of the titin bands seen on SDS gels, western blot studies were performed using a library of antibodies raised against sequences from the N2B and N2A elements (see Fig. 3A). These antibodies were first tested on skeletal muscle titin (human soleus) which is known to contain only N2A titin 6 and, as expected, the N2A-element specific antibody reacted with skeletal muscle titin, while the N2B-element specific antibodies did not (Fig. 3C).

Western blot experiments were also performed using pig myocardium, because it contains high levels of both T1 bands (Fig. 2). The top T1 band reacted with both N2A and N2B specific antibodies while the bottom T1 band reacted only with the N2B antibody (Fig. 3D). These results are consistent with the top T1 band representing a titin isoform containing both the N2B and N2A elements (referred as N2BA cardiac titin) and the bottom band an isoform that contains only the N2B element (N2B cardiac titin).

To investigate titin in the sarcomere, immunoelectron microscopy was performed on pig and rat ventricular cells using N2A-and N2B-specific antibodies. Pig cells were positive for both N2B and N2A antibodies and rat cells only for the N2B-specific antibodies (Fig. 3E). These results are consistent with the conclusion that pig myocardium expresses the N2BA isoform and rat myocardium predominately the N2B isoform. Furthermore, in the pig cells the N2B epitope was on average closer to the Z-line than the N2A epitope. For example at a SL of 2.45 µm the distance between the epitope and the middle of the Z-line was 175 ± 21 nm (n=9) for the N2B epitope and 279 ± 15 nm (n=10) for the N2A epitope. Thus, in the N2BA isoform the N2B element is N-terminal of the N2A element. This result is consistent with recent sequencing work on cardiac titins (Freiburg A. and Labeit S., unpublished results, 1999).

Immuno-fluorescence (IF).

The SDS-PAGE and westernblot results were performed at the tissue level and the results may be explained either by co-expression of isoforms in cells, or by discrete expression in cells (i.e., cells either express N2B or N2BA titin, but not both) with species variation in the ratio of cells that are N2B or N2BA pure. To distinguish between co-expression and discrete expression, IF studies were carried out on single cardiac myocytes. Considering that only N2BA titin reacts with the N2A-element specific antibodies, discrete expression predicts that a certain fraction of cells (N2B cells) will not stain with this antibody. This fraction can be estimated from the expression ratio of N2B and N2BA titins determined by solubilizing a large number of cells from the cell suspension used for IF, followed by electrophoresis and quantitative densitometry. Results indicate that 60 ± 5% of total titin in pig left ventricle is N2BA titin and 40 ± 7% is N2B titin (Table 1). Thus, the discrete expression hypothesis predicts that only 60% of the pig cells stain with the N2A antibodies. Antibodies specific for the N2B element are predicted to stain all pig cells. For a comparison mouse myocytes were studied as well. Considering that mouse myocardium contains a barely detectable N2BA band (Fig. 2), discrete expression predicts that only few mouse cells are positive for the N2A antibodies while all cells are predicted to be positive for the N2B-element specific antibodies.

Experiments with the N2B antibodies revealed that all pig and mouse cells were positive (Table 1; Figs. 4A andB), consistent with the results of the above discussed western blot studies. When using the N2A antibodies, we obtained positive cells (Figs. 4C,E), and cells that contained a faint background-fluorescence level without a clear banding pattern (Figs. 4D,F) similar to that obtained in control experiments in which the primary antibodies were omitted from the labeling protocol (no-primary antibody control). To test whether absence of labeling with the N2A antibodies may have resulted from titin degradation or inability of the N2A antibody to penetrate some of the cells, experiments with the anti-titin antibody 9D10 were performed. 9D10 labels the PEVK region of titin 11 , a site which is sensitive to degradation. 21 Furthermore, 9D10 is an IgM type antibody 17 , which is the largest of all antibody types and, therefore, 9D10 is expected to diffuse slowly into the cells. Results revealed that all cells were 9D10 positive (Figs. 4G,I; Table 1), suggesting that antibodies can fully penetrate the cells and that all cells contain intact titin. Therefore, cells that gave rise to a fluorescence similar to that of 'no-primary antibody control cells' were scored as N2A negative cells.

It was found that 92% of the pig cells are positive for the N2A antibody (Table 1), a value much higher value than the 60 ± 5 % positive cells predicted by the discrete expression hypothesis (cells express only one of the isoforms). On the other hand, the co-expression model (all cells co-express isoforms at the same ratio) predicts that all pig cells would be N2A positive while we found that 8% of pig cells are N2A-negative. These findings may be explained by co-expression that varies from cell-to-cell with a small fraction of the cells expressing predominantly one of the isoforms. indicate that the average stiffness of the mouse myocytes is much higher than that of the pig (inset Fig. 5). To test for statistical differences between mouse and pig cells, the tension developed at a SL of 2.4 µm was compared using a Mann-Whitney test (this test was used because results were not normally distributed). This revealed that mouse ventricular cells are significantly stiffer than pig myocytes (p<0.05). To determine whether the differences in stiffness may have resulted from differences in the fractional area of myofibrils, cells were prepared for electron microscopy and their cross-sections were analyzed. Results revealed that this explanation is unlikely because mouse and pig cells contain similar fractional areas of myofibrils (Table 2). We also studied whether differences in the cellular content of titin could explain the mechanical differences between pig and mouse cells. The ratio of titin relative to myosin heavy chain (MHC) was determined using quantitative densitometry (for details see Methods). It was found that the titin/MHC ratios of mouse and pig cells are not significantly different (Table 2). The titin/MHC ratio was also used to determine the number of titin molecules per half-thick filament (for details see Methods). Results indicate 6.0 ± 0.3 and 6.5 ± 0.4 titin molecules per half-thick filament in mouse and pig cells, respectively (Table 2). Thus a difference in the titin content of mouse and pig cells is an unlikely explanation for their different passive properties.

Diastolic properties of cardiac

It may be noted from Fig. 5 that a few of the pig cells are as stiff as most of the mouse cells while one of the mouse cells is as compliant as most pig cells. Although experimental error may underlie part of this variation, differences in cell behavior may also result from cell to cell variation in the expression ratio of N2B and N2BA titins. For example, the compliant mouse cells may express high levels of N2BA titin. To further explore cell-to-cell variation in cell stiffness, cells isolated from pig atrium and rat ventricle were also studied. Results indicate that most of the pig atrial cells are compliant (i.e. their T-SL curve are shallow) and that a few cells are rigid and behaved like the average mouse cell (Fig. 6 red curves). In contrast, rat cardiac cells were all stiff (Fig. 6). Considering that the rat expresses predominantly N2B titin, cell-to-cell variation appears more prominent in cells isolated from tissue that co-expresses both titin isoforms.

Discussion

We found that the myocardium co-expresses two titin isoforms. One of the isoforms contains the N2B element and the other both the N2B and N2A elements. The expression ratio of these isoforms varies in different species and in different locations within the heart. Furthermore, results suggest that co-expression of isoforms takes place at the level of the single cell. The mechanical properties of single cardiac myocytes were measured and this revealed that on average cells isolated from a species that contains high levels of N2BA titin (pig) are significantly less stiff than those from species that contain high levels of N2B titin (rat and mouse). Below we discuss these findings and their functional significance.

Cardiac titin isoforms.

Previous RT-PCR studies of human cardiac titin revealed the existence of N2A and N2B isoform transcripts. 6 Here we studied isoform expression at the protein level. SDS-PAGE indicates that myocardium of many species contains two T1 bands, a top band which reacts with N2A-and N2B-element specific antibodies and a bottom band with only N2Bspecific antibodies. That the two T1 bands represent full-length titin molecules is supported by western blot experiments with antibodies against the N-terminal end (Z1/Z2 and Zr) and the Cterminal end (T51) of titin. This showed that both T1 bands of pig are full-length molecules and not degradation products. Absence of titin degradation is also supported by the IEM data.

Degradation results in retraction of titin towards the Z-line and the A-band 22 , and no such retraction was found in the cells that we investigated. Hence we conclude that the two T1 bands represent distinct isoforms. This conclusion is in accordance with recent RT-PCR studies using rabbit cardiac mRNA which revealed that differential splicing gives rise to two main classes of cardiac titin isoforms: the N2B and N2BA titins; so named because they contain either the N2B element or both the N2B and N2A elements (Freiburg, A. and Labeit S., unpublished results, 1999). Cardiac N2B titin is the smallest isoform known to date and the small size results from skipping the I20-I75 segment and splicing together the I19 to I76 encoding exons. The splicing of the exon coding for I19 to exons in the I47-I60 segment leads to a family of isoforms, all including the N2A segment, and thus these isoforms are referred to as N2BA titins.

Due to the sequence differences, the molecular mass of the cardiac titin isoforms varies from ~3.3 MDa for the N2BA titin isoform to ~3.0 MDa for N2B titin ( 6 ; Freiburg, A. and Labeit S., unpublished results, 1999). Our current work indicates that this mass difference is large enough to allow separation of N2B and N2BA titins on high resolution gels (Figs. 123). This enables studies of the expression levels of the isoforms in different species and in different locations within the heart. Results establish that co-expression of isoforms takes places in a wide range of species, but at widely varying ratios, and that atria express higher N2BA levels than the ventricles. The quantitative densitometric study of titin in mouse and pig (expressing high levels of N2B and N2BA titin, respectively) reveals that while the expression ratio of titin isoforms varies greatly, the number of titin molecules per half-thick filament appears constant (Table 2). Thus, titin's stoichiometry is well controlled while the isoform expression ratio is variable.

Co-expression of titin isoforms at the single cardiac myocyte level.

The titin isoform coexpression results were obtained with SDS-PAGE and western blot techniques using myocardial tissue as starting material. Co-expression of isoforms at the tissue level can be explained by assuming (1) that individual cells express only one of the isoforms (discrete expression hypothesis) and ( 2) that the ratio of N2B cells to N2BA cells determines the isoform expression ratio of the myocardial tissue. However, we found that the ratio of positive and negative cells is inconsistent with a discrete expression of isoforms (Table 1, see Results). Instead results are consistent with a co-expression of titin isoforms within individual cells. The small fraction of negative cells can be explained by cell-to-cell variation in the co-expression ratios resulting in some cells with predominantly one isoform. This 'variable co-expression' hypothesis is also consistent with recent immunoelectron microscopical studies that revealed co-expression of isoforms within the same Iband and cell-to-cell variation in the co-expression level (Trombitás, K., Labeit, S., Granzier, H., 1999. Unpublished data). In summary, our findings indicate that the titin isoform co-expression seen at the myocardial level results from co-expression at the level of the single cardiac myocyte.

Diastolic properties of cardiac myocytes.

Whether diastolic properties of N2B cells are predicted to be different from those of N2BA cells can be ascertained from the molecular mechanism of titin-based passive force development. The molecular mechanism that underlies titin's force has been investigated in dynamic light scattering studies on titin in solution 23 , mechanical studies on single titin molecules 24;-27 , and immunoelectron microscopic studies on skeletal and cardiac muscle titin 11; 28; 29 . From these, a model has emerged in which the tandem-Ig segments (containing folded Ig domains) and the PEVK segment (acting largely as an unfolded polypeptide) behave as serially-linked entropic springs. In short sarcomeres, these springs are in a contracted state with high entropy and upon sarcomere extension the springs straighten, lowering their conformational entropy and resulting in a force, known as entropic force. This force increases with the fractional extension of the segment (end-to-end length divided by the contour length). (see 8 ).

The serially-linked entropic springs model of passive force development may be applied to size variants of titin's elastic segment by adapting the entropic forces to the fractional extensions multiplied by the contour lengths of the size variant's tandem Ig and PEVK segments. The contour lengths of tandem Ig and PEVK segments are ~100 and ~250 nm longer, respectively, in N2BA than in N2B titin (assuming a 5 nm repeat per Ig domain and 3.8Å per PEVK residue, see [START_REF] Trombitas | Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments[END_REF][START_REF] Helmes | Mechanically driven contour-length adjustment in rat cardiac titin`s unique N2B sequence: titin is an adjustable spring[END_REF].

It follows that at a given SL the fractional extension of tandem Ig and PEVK segments is considerably less for N2BA titin than for N2B titin and, therefore, passive force will be lower. This prediction is qualitatively unaffected by the extensibility of the unique N2B sequence 12 because both cardiac titin isoforms contain this sequence. In conclusion, the serially-linked entropic springs model of passive force development predicts that the passive force-SL relation increases less steeply (i.e., the compliance is higher) for N2BA containing cells than for N2B cells.

We studied the passive tension -SL relation of single cardiac myocytes isolated from mouse, rat and pig myocardium. The mouse and rat were chosen because their cardiac myocytes are predicted to be stiff (they express high levels of N2B titin), and the pig because its cells are predicted to be compliant (high levels of N2BA titin). Consistent with the predicted tension differences, pig myocytes are on average significantly more compliant than rat and mouse cells (Figs. 5 and6). Considering the similar fractional cell areas of myofibrils and the similar number of titin molecules per half thick filament (Table 2), it is unlikely that these mechanical results can be explained by differences in the amount of titin per unit cross-sectional cell area. Rather it is likely that these mechanical differences result from within titin itself. It is also worthwhile to point out that studies performed more than 20 years ago by Fabiato and Fabiato 30 are consistent with our findings. In this earlier work, it was shown that dog myocytes are more compliant than rat myocytes, consistent with the high levels of N2BA titin that we found in dog myocardium and high levels of N2B titin in rat (Fig. 2). Finally, Brady 31 has reported the passive stiffness modulus (normalized to the cross-sectional area) of myocytes from several species and this revealed lower stiffness in rabbit than in rat. This finding is in agreement with the higher N2BA levels of rabbit (Fig. 3).

Mechanical results of cells isolated from the same ventricle often showed considerable variation (Figs. 5 and6). Although some of this variation may result from experimental error (such as errors in the cross-sectional area measurement), it seems more likely that additional sources of variation exist as well. It is also worth noting that variation in mechanical properties has been reported in intact guinea-pig ventricular cells by Gannier et al 32 and Cazorla et al 33 with a tendency of the cells to separate into stiff and compliant sub-populations. Variation in cellular stiffness is consistent with the variable co-expression hypothesis that was derived from the immunofluorescence study (see above). Variation in the titin isoform co-expression ratio of cardiac myocytes is expected to give rise to variation in the level of passive tension for a given SL with an increase in cell compliance as the N2BA expression level increases. In summary, our mechanical studies reveal cell-to-cell variation in cell stiffness, but on average cells isolated from myocardium that expresses high levels of N2B titin are stiff and those from myocardium that expresses N2BA titin are more compliant. Thus, the diastolic properties of cardiac myocytes isolated from different species are not the same, but instead they vary with the expression ratio of the titin isoforms.

Functional significance. Work on rat cardiac muscle has shown that both titin and collagen are major contributors to passive stiffness of the myocardium, with titin's contribution dominating at the short to mid-range of the physiological SL range and collagen contributing more at longer lengths 14 . Although comparative muscle studies in different species remain to be done, it is likely that variation in titin-based stiffness of the myocytes will translate into variation in stiffness at the level of the myocardium. Thus, we speculate that as the expression level of N2BA titin increases and cell stiffness decreases, myocardial stiffness will decrease as well. Variation in myocardial stiffness is expected to influence filling of the heart. For example, lower myocardial stiffness will allow for faster filling and larger end-diastolic volumes for a given filling pressure. The variation in expression levels of titin isoforms in different species may thus be related to variation in filling rate and/or filling volume of the heart. We speculate that the cell-to-cell stiffness variation within species is related to strain equalization of muscle fibers in different layers of the wall. Consistent with this explanation is the variation in the N2BA/N2B isoform expression ratio that was found in different layers of the ventricular wall (Fig. 2C).

In conclusion, this work revealed that the myocardium co-expresses titin isoforms and that the expression ratio of the isoforms varies between species and within a species in different locations of the heart. Co-expressing titin isoforms at different levels modulates cellular stiffness and we hypothesize that this influences filling of the heart. Research at the multicellular and organ levels will be required to test this hypothesis and to fully elucidate the functional significance of the present findings.

Captions

Fig. 1: Comparative SDS-PAGE analysis of left ventricular myocardium (rat, rabbit, bovine, and human) and skeletal muscle (human soleus). Myocardium contains a prominent T1 band that has a similar mobility in all species. In addition a lower mobility T1 band is clearly present in bovine and human, just detectable in rabbit, but absent from rat. The inset shows an enlargement of the titin bands of rat and rabbit. (Human soleus was included as a titin molecular mass marker (Mr 3.7 Mda.)) Fig. 2. A) SDS-PAGE based analysis of titin expression in left ventricle and right atrium of various species. Dog, human, pig, and cow contain two T1 bands, while mouse, rat, and rabbit contain a strong bottom T1 band and a barely detectable top band. The atrium contains predominately the top T1 band. B) Densitometric quantification of the T1 bands. The ratio of top to bottom T1 bands varies greatly in the different species. In mouse a faint top band was sometimes detectable by eye but the band was at the detection limit of our gel scanner and the obtained result (asterisk) has a limited accuracy. For the rat, no top band was detectable. C) Analysis of titin expression across the left ventricular wall of pig. The ratio of top to bottom bands in the sub-endocardium (endo) is significantly higher than the ratio in the mid-wall and the sub-epicardium (epi) (P<0.05, ANOVA, n= 3 pigs). Values are the mean ± SD. Titin / MHC ratio (1) 0.29 ± 0.02 (n=5) 0.34 ± 0.02 (n=5)

# titin molecules / half-thick filament (2) 6.0 ± 0.3 (n=5) 6.5 ± 0.4 (n=5)

Values are expressed as mean ± SEM. (1) To determine the amount of titin relative to that of MHC, gels were scanned and the total OD (optical density) of the MHC and of all titin peaks was determined for a range of loadings. The slope of the linear range of the OD vs. loading relation of titin and MHC was measured and the slope ratio was taken as a measure of relative amount of titin in the samples. (2) This relative amount was converted to the number of titin molecules per half thick filament assuming 150 myosin molecules per half-thick filament, a molecular mass of 0.205 MDa for MHC 34 , 2.97 MDa for N2B titin and 3.35 MDa for N2BA 6 . For myocardium that co-expresses titin isoforms, the average molecular mass was calculated from the isoform expression ratio.

-5 -

  and Material and Methods online.) Immunofluorescence. Single or double labeling experiments with PEVK (9D10) 17 and N2B (X151-X151) or N2A (X105-X106) antibodies were performed on cardiac myocytes. (For details see Material and Methods online.) Passive tension -SL measurement of cardiac myocytes. For technical details of force and online sarcomere length measurement see 12,14 . The passive force -SL relationship of the cells was measured by imposing a slow ramp stretch-release (0.1 length/s) on cells in relaxing solution.

  myocytes. To investigate the functional significance of coexpressing titin isoforms, mechanical experiments were performed on single cardiac myocytes and their passive force -SL relation was measured. The force generated by the intermediate filament network was determined by extracting the thin and thick filaments from the myocytes using KCl and KI containing relaxing solutions (see 14 ). Extraction removes titin as a passive force generator as the extraction-induced depolymerization of actin and myosin removes titin's anchors in the sarcomere. The force after extraction was subtracted from the force before extraction, resulting in data that represent the force produced by titin alone. This force was converted to tension (T) by dividing it by the cross-sectional area of the cell. The cells studied had been isolated by either blending myocardial tissue (cf. 16 ) or by enzymatic digestion of the heart (see captions of Figures). When ventricular cells isolated by blending or digestion were compared (data not shown), no statistically significant differences were found in either the titin content or the titin-based stiffness of the cells. Titin-based T-SL curves of 15 mouse and 10 pig ventricular cells are shown in figure 5. Results

Fig. 3 :

 3 Fig. 3: Immuno-labeling of pig cardiac titin. A) Layout of the titin molecule in the half-sarcomere with localization of the epitopes of the antibodies used. B) Western blot analysis of the pig myocardial proteins with antibodies raised against the N-terminal end of titin (Z1/Z2 and Zr) and the C-terminal end of titin (T51). All antibodies react with the two T1 bands, indicating that both bands represent full-length titin molecules. C) and D) Western blot analysis of human soleus muscle titin (C) and pig cardiac titin (D) with antibodies specific to the N2-sequences. Skeletal muscle titin reacts with N2A antibodies only. The top T1 band of pig cardiac titin reacts with all N2A and N2B antibodies and the bottom band with the N2B antibodies only. E) Immunoelectron micrographs of rat myocardium (left) and pig myocardium (right) double-labeled with the N2B antibody (X150-X151), and the N2A antibody (X105-X106). The rat contains a single epitope derived from the N2B antibody (as concluded from single antibody labeling experiments; results

Fig. 4 :

 4 Fig. 4: Immunofluorescence of cardiac myocytes from mouse and pig left ventricles. A) Typical example of pig cell labeled with the N2B (X150-X151) antibody that reveals strong labeling in narrow repeating bands. B) Comparison of phase contrast image (top) and immuno-fluorescence (bottom) of pig myofibril labeled with N2A (X105-X106) antibody. Note that labeling is restricted to the I-band region of the sarcomere. C)-F): Cells labeled with N2A antibodies. Examples are shown of positive cells (C, E) and negative cells that only contain a faint background fluorescence (D, F). G) and H) Examples of cells labeled with the 9D10 antibody (labels the PEVK segment). Scale bar denotes 10 µm.

Fig. 5 :Fig. 6 :

 56 Fig. 5: Passive tension (T) -sarcomere length (SL) relations of mouse and pig left ventricular myocytes. T-SL curves of 15 mouse (black) and 10 pig ventricular cells (red) are shown superimposed. Contribution of intermediate filaments (IFs) to tension was subtracted from total measured tension (see Results) and only the titin-based tension is shown. Inset shows the mean ± SEM of the cells (black symbols: mouse; red symbols: pig). Mean IF-based tension is shown in the inset as well. IF results from mouse and pig superimpose. (Cells obtained by enzymatic digestion of the ventricles.)

Table 1 : Immunofluoresence of cardiac myocytes.

 1

		Mouse			Pig
		# positive cells # negative cells	# positive cells # negative cells
	N2B (X150-X151)	126 (100%)	0	(0%)	134 (99%)	1 (1%)
	N2A (X105-X106)	20 (12%)	149 (88%)	154 (92%)	14 (8%)
	PEVK (9D10)	51 (100%)	0	(0%)	43 (100%)	0 (0%)
	Predicted N2A results by "discrete hypothesis"	5%		95%	60%	40%

Table 2 : Cross-sectional analysis and titin content of cardiac myocytes.

 2

		Mouse	Pig
	Cross section (µm 2 )	271 ± 18 (n=16)	329 ± 10 (n=10)
	% myofibrillar area	68 ± 3 (n=7)	73 ± 7 (n=3)
	N2BA:N2B ratio	< 0.05 (n=6)	1.48 ± 0.11 (n=8)

Acknowledgments.

This work was supported by grants from NIHLBI (HL61497 and HL62881) to HG, the Human Frontier Science Program (SL) the Deutsche Forschungsgemeinschaft (La668/5-1 to SL) and a postdoctoral fellowship awarded to OC from the American Heart Association (Washington State Affiliate, #98-WA-115). HG is an Established investigator of the American Heart Association.