Abstract
RATIONALE In addition to its typical rod-shape, the mammalian adult cardiomyocyte (CM) harbors a unique lateral membrane surface architecture with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) the role of which is still unknown.
OBJECTIVE To investigate the development and functional role of CM crests during the postnatal period.
METHODS AND RESULTS Electron/confocal microscopy and western-blot of left ventricular tissues from rat hearts indicated a late CM surface crest maturation, between postnatal day 20 (P20) and P60, as shown by substantial SSM swelling and increased claudin-5 cell surface expression. The P20-P60 postnatal stage also correlates with an ultimate maturation of the T-Tubules and the intercalated disk. At the cellular level, we identified an atypical CM hypertrophy characterized by an increase in long- and short-axes without myofibril addition and with sarcomere lateral stretching, indicative of lateral stretch-based CM hypertrophy. We confirmed the P20-P60 hypertrophy at the organ level by echocardiography but also demonstrated a transcriptomic program after P20 targeting all the cardiac cell populations. At the functional level, using Doppler echocardiography, we found that the P20-P60 period is specifically dedicated to the improvement of relaxation. Mechanistically, using CM-specific knock-out mice, we identified ephrin-B1 as a determinant of CM crest maturation after P20 controlling lateral CM stretch-hypertrophy and relaxation. Interestingly, while young adult Efnb1CMspe−/− mice essentially show a relaxation impairment with exercise intolerance, they progressively switch toward heart failure with 100% KO mice dying after 13 months.
CONCLUSIONS This study highlights a new late P20-P60 postnatal developmental stage of the heart in rodents during which the CM surface crests mature through an ephrin-B1-dependant mechanism and regulate the diastolic function. Moreover, we demonstrate for the first time that the CM crest architecture is cardioprotective.
Introduction
The mammalian adult cardiomyocyte (CM) harbors a typical rod shape specifically dedicated to the function of the adult heart. Understanding the maturation of the adult rod-shaped CM can provide important insights for regenerative medicine, especially for the differentiation of hiPSC cells into fully mature rod-shaped adult CMs which still remains an obstacle. To date, the molecular events leading to the setting of the CM rod-shape during the post-natal stage are still unknown.
Indeed, despite fetal and adult CMs were extensively studied, the postnatal stage has only recently garnered an unprecedented interest only recently with the discovery of the potential of adult CMs to proliferate and regenerate the heart1. Thus, strategies in this field have focused on boosting this regenerative process by exploiting factors specifically involved in the early CM proliferation arrest that occurs during the postnatal period2. However, despite the postnatal maturation period characterizing the exit of the CM from the cell cycle, it also coincides with a morphogenesis step during which the CM switches from a proliferative rounded shape to a non-proliferative mature rod shape3. In situ, mammalian CMs stop dividing and start their morphogenesis maturation around postnatal day 7 (P7)4,5.
The postnatal morphogenesis stage of the CM relies on a polarization process that results in the asymmetric organization of the plasma membrane components underlying specific functions with an atypical basolateral polarity. It begins with a longitudinal elongation process of the CM to progressively evolve into the rod-shaped characteristic of the mature adult state. Contrary to epithelial cells harboring a basolateral and apical side, the adult CM lacks the apical side and looks like a barrel surrounded by a unique basal side with a basement membrane connecting the fibrillar extracellular matrix (ECM) and flanked by two intercalated disks (ID) involved in the CM-CM tight interactions, shaping the longitudinal alignment of myofibers within the tissue6,7. The ID phenocopies the lateral side of polarized epithelial cells with the presence of tight, adherent, gap junctions and desmosomes that play a specific role in the anchorage of the contractile myofilaments but also in the synchronization of the contraction. One architectural feature of the basal side of the adult CM, also called the lateral membrane, also relies on its important intracellular invaginations into transverse T-tubules, which play a key role in both action potential propagation and Ca2+ handling/excitation-contraction coupling8. Likewise, the extracellular side of the LM seems more complex than initially suspected, since, besides the presence of a costamere structure expressing classical transmembrane receptors (integrin and DGC dystroglycan-glycoprotein complex) that connect the ECM components to the intracellular myofibrils, we and others have reported the atypical presence of transmembrane proteins, claudin-5 and ephrin-B19,10, more likely involved in cell-cell communication. This finding was unexpected since, contrary to the ID, the lateral membrane was so far viewed as a CM side lacking physical interactions with neighboring CMs. However, in recent years, using high-resolution nanoscale imaging, i.e. SCIM, AFM and MET, we and Gorelik’s lab have described a highly organized architecture of the lateral membrane on adult CMs with periodic crests11–13 filled with subsarcolemmal mitochondria (SSM) whose role is unknown. More recently, we provided evidence for the existence in the 3D cardiac tissue of intermittent lateral crest-crest contacts all along the lateral membrane through claudin5/claudin-5 tight junction interactions12, thus reconciling the presence of such proteins on the lateral face of the adult CM. Exactly when and how the lateral membrane crests of the CM maturate is completely unknown.
In this study, we investigated the maturation of the CM crests and their role during the postnatal period. We provide evidence for a late postnatal development stage, following the set-up of the rod shape, during which crests fully maturate through SSM swelling. We also show that this maturation step of the crests is ephrin-B1-dependent and specifically regulates the diastolic function of the adult heart.
METHODS
The methods are described in detail in the Supplemental Material.
RESULTS
CM surface crests maturate late after postnatal day 20
We investigated crest maturation during the postnatal period on the left ventricle tissue of male rat hearts from different postnatal days (P0/birth, P5, P20, P60/young adult) using transmission electron microscopy (TEM) as previously described12. Surprisingly, at P0, while the contractile apparatus is disorganized in the neonatal CM, myofibrils are already orientated on the longitudinal axis of the cell with the first layer already anchored to the plasma membrane through Z-lines, and thus outlining a periodic crest-like architecture, more likely plasma membrane protrusions, which can yet be visualized all along the CM surface (Figure 1A; Figure I in the Supplemental Material). These immature and disordered crests already attempt to interact with crests from a neighboring CM. At this stage, no SSM could be observed. Then, crest maturation throughout the postnatal period occurs in two steps. A first early step, already completed by P5, relies on the maturation of the Z-lines, which delimits each sarcomere from the outer myofibril, allowing better visualization of the surface crest structure, concomitant to the myofilament alignment/organization that follows the morphological elongation of the CM (Figure 1A, P5). However, crests still display an unstructured morphology and their heights, directly correlated with the SSM number as previously described in adult CMs12, are small (“flat”-appearance) due to the lack or the presence of very small SSM, likely immature SSM (Figure 1A). By comparison, much larger but disorganized interfibrillar mitochondria (IFM) can be visualized in CMs at birth (P0) predominantly around CM nuclei (Figure I in the Supplemental Material), while they mature until P60 through both swelling (completed at P20) and alignment along the myofilaments (Figure II in the Supplemental Material). The crest immaturity persists until P20, a late stage of the postnatal period, while the CM has already implemented its rod shape (Figure III in the Supplemental Material) and completed its whole cytoarchitecture14, as indicated by the perfect alignment of sarcomere Z-lines between myofibril layers inferred from the α-actinin staining (Figure IV in the Supplemental Material). It is worth noting, that at P20, CMs display different morphologies with both rod-shaped and spindle-shaped CMs when compared with only rod-shaped CMs at P60 (Figure III lower panels in the Supplemental Material). Interestingly, a second but delayed maturation step of the surface crests occurs between P20 and the adult stage (P60) through substantial SSM swelling (Figure 1A), during which crest heights significantly expand up, correlating with an increase in the SSM number and area (Figure 1B). We further confirmed this maturation of the CM surface crests after P20 by analyzing the expression and localization of claudin-5, a tight-junctional protein that we previously described as a determinant of the lateral crest-crest interactions between the LM of neighbouring CMs12. While claudin-5 protein expression is progressively induced in the cardiac tissue at birth (P0), reaching its maximal expression at P10 (Figure 1C, left panel), complete localization of the protein at the lateral membrane of the CM occurs only between P20 and P60 (Figure 1C, right panel), most likely in agreement with the implementation of claudin-5/claudin-5-dependent interactions necessary to clip crests from neighboring CMs that we previously described at the adult stage 12. In line with this mechanism, atypical tight junctions connecting crests from the lateral face of neighbor CMs could be observed only in the P60 adult stage (Figure V in the Supplemental Material). Remarkably, the maturation of the CM lateral surface occurring between P20 and P60, at least in part through the setting of crest-crest interactions, occurs concomitantly with an ultimate maturation of both the ID and the T-tubules, relying on a spatial reorganization of their specific components. Thus, while connexin 43 (gap junctions), desmoplakin 1/2 (desmosomes) and N-cadherin (Adherens junctions) are located on both the lateral membrane and the ID at P20, they fully relocalize to the ID at P60 (Figure VI in the Supplemental Material). Likewise, RyR and caveolin-3, T-tubule markers (see Methods), are still misaligned at P20 while a perfect alignment along the sarcomere Z-lines on the short CM axis can be observed at P60 (Figure VII A, B in the Supplemental Material). In agreement, the TT power (TT regularity) tends to increase after P20 while the TT periodicity (frequency) is already established at P20 (Figure VII C and Methods in the Supplemental Material).
Overall, these results indicate that the entire plasma membrane architecture of the CM maturates late, after P20, following the establishment of the rod shape.
Evidence for a new late postnatal maturation stage of the CM and the mammalian heart dedicated to the development of the diastolic function
We next performed a more detailed analysis of the late P20/P60 maturation stage of the rodent heart.
At the cellular level, CMs from the left ventricle of rat hearts undergo significant hypertrophy between P20 and P60 as indicated by the substantial increase in their cross-sectional area (Figure 2A), which peaks at P45 (Figure VIII in the Supplemental Material), and in both their long and short axes (Figure 2B and Figure IX in the Supplemental Material). Similar late CM hypertrophy was observed in male mice (Figure X in the Supplemental Material). This P20-P60 CM hypertrophy was confirmed by echocardiography with a significant increase in the left ventricle posterior wall thickening (LVPWd) and cavity size (LVEDV, LVEDD) (Figure 2C), all indicative of an overall heart growth. Surprisingly, this physiological CM hypertrophy is atypical since it is not correlated with an expected increase in the myofilament compartment as indicated by the constant number of CM myofibrils between P20 and P60 (Figure 2D). This is corroborated by a marked decrease in the heart weight to body weight ratio during this period (Figure 2E), as previously reported14. Interestingly, we also noticed a significant and specific increase in the sarcomere heights with no variation of the sarcomere lengths (Figure 2F) which was inversely correlated with a decrease in the inter-lateral space between two CMs (Figure 2G), likely indicative of a lateral stretch of the CMs and a cardiac tissue compaction. Further supporting the CM lateral stretch that should distend the myofibrils, we observed larger distances between the thick myosin filaments at P60 than at P20 (Figure XI in the Supplemental Material). Taken together, these results highlight a new type of physiological cardiac hypertrophy that occurs during late postnatal development and that presumably relies, at least in part from the lateral vantage point, on the stretching of the CM lateral membrane.
The existence of a new late postnatal maturation stage between P20 and P60 of the mammalian heart was confirmed by transcriptional analysis of left ventricular tissue from male mouse hearts. Volcano-plot analysis of the transcriptome and the heat map clearly show a significant difference between P20 and P60 heart gene expression (Figure 3A, B) with 1000 protein-coding genes that are up- or downregulated between P20 and P60 (p < 0.05, fold change > 1.5). The gene ontology (GO) enrichment analysis revealed significantly affected biological pathways (p < 0.05) (Figure 3C and Figure XII in the Supplemental Material) that are upregulated and mainly related to processes of the immune defence system, muscle cell differentiation, angiogenesis, positive regulation of cell death, different metabolisms including antioxidant defense, plasma membrane-related transport/signaling together with many pathways related to the nervous system development. By contrast, many ECM and developmental processes are downregulated. It is worth noting that some upregulated metabolic pathways relate to heart fuels other than fatty acids and glucose (tryptophan, glutamate, glycine/serine/threonine metabolisms), thus suggesting that the heart increases its metabolic flexibility between P20 and P60. We next performed gene clustering according to the cardiac cell populations established by Tucker et al15 and assuming similar cardiac cell populations at P20 and P60 (Methods in the Supplemental Material). In line with the GO analysis, we found that the main downregulation of the transcriptome between P20 and P60 occurs primarily in the fibroblast populations known to be involved in the synthesis of the fibrillar ECM but also to a lesser extent in the CM populations (Figure 3D), consistent with the skeletal system and heart development pathways. By contrast, although all cardiac cells seem to be involved in the transcriptional maturation, the P20-P60 upregulated transcriptome largely referred to the ventricular CM populations when compared to the downregulated one (Figure 3D), in agreement with the evidence of a prominent muscle cell differentiation pathway depicted through the GO analysis. The existence of an important transcriptional maturation step of the CM during the P20-P60 postnatal window is further reinforced by the modulation of key CM-specific protein-encoding genes, especially several proteins from the contractile apparatus or regulating the contractile machinery at the lateral membrane (Figure XIIIA in the Supplemental Material). Another CM maturation also occurs at the metabolic level while the metabolic switch of the heart from glycolysis to fatty acid (FA) oxidation was already established during the early postnatal period16, with the remarkable upregulation of the BDH1 gene that encodes the β-hydroxybutyrate dehydrogenase, the limiting mitochondrial enzyme for ketone body (KB) uptake during FA catabolism, but also to a lesser extent the upregulation of key actors in the glycolytic metabolism (PFKFB2, SLC2A4) (Figure XIIIB in the Supplemental Material).
To better understand the functional impact of the P20-P60 maturation of the CM, we evaluated both the systolic and diastolic functions of rat hearts during this stage. Longitudinal echocardiographic evaluation reveals a similar left ventricular ejection fraction (LVEF) in both P20 and P60 rats (Figure 4A), indicating earlier maturation of the systolic function during the postnatal period. By contrast, we observed specific changes in the diastolic function between P20 and P60 as measured by non invasive Doppler imaging and showing a significant increase in passive filling (E/A) and an improvement in relaxation (decrease in the isovolumic relaxation time (IVRT), increase in the e’/a’ and the early diastolic mitral annular tissue velocity e’, without change in the LV filling pressures E/e’) (Figure 4A), most likely indicating that the diastolic function of the rat heart maturates during the late postnatal period, between P20 and P60. Further confirming the P20-P60 set-up of the adult diastolic function, while P20 and P60 rats display similar heart rates, invasive left ventricle catheter analysis shows increased systolic and diastolic blood pressure (SBP/DBP) as well as end-diastolic pressure (EDP) and an improvement in diastolic relaxation reflected by the dP/dtmin and the decreased time constant of isovolumic relaxation (Tau) (Figure 4B).
Efnb1-specific knockdown in the CM impairs the late maturation of surface crests and the diastolic function
We have previously shown that the CM hypertrophy between P20 and P60 occurs concomitantly with the maturation of the crests that coat the whole CM lateral surface and, more specifically, with the implementation of the crest-crest interactions between neighbouring CMs. This observation raises questions about a specific contribution of the crest-crest interactions and the setting of intermittent tight junctions in the lateral stretch of the CM and the control of the diastole. Thus, we next sought to evaluate whether the maturation of the CM surface crests occurring after P20 could directly control the CM hypertrophy and the diastolic function of the heart.
For that purpose, we examined the role of ephrin-B1, a transmembrane protein that we previously identified as a new protein of the LM of the adult CM independent from the integrin or the dystroglycan complex, both connecting the ECM to the intracellular contractile machinery9. We showed that ephrin-B1 directly interacts with claudin-5 at the lateral membrane of the adult CM and controls its expression. Also, given the importance of claudin-5 in the setting of the crest-crest lateral interactions within the adult cardiac tissue12, we questioned about a potential role for ephrin-B1 as a crest determinant at the CM surface.
We first studied ephrin-B1 expression / localization in the cardiac tissue from rat hearts during the early postnatal development. As shown in Figure 5A and similarly to what we observed for claudin-5, ephrin-B1 reaches maximal expression very early during the postnatal period (P5). However, the complete trafficking of ephrin-B1 from the cytosol to the CM surface is only achieved at P60. To further explore the role of ephrin-B1, we took advantage of a knock-out (KO) mouse model harboring a CM-specific deletion of Efnb1 that we previously described9. At the cellular level, the lack of ephrin-B1 in the CM partially impairs the P20-P60 physiological hypertrophy of the CM (Figure 5B), without modification of the myofibril number (Figure 5C). Consistent with ephrin-B1-specific expression at the lateral membrane, Efnb1 deletion more specifically impedes the lateral stretch (short axis) of the CM but not the longitudinal one (Figure 5D) and accordingly leads to a decrease in both the sarcomere heights (Figure XIVA in the Supplemental Material) and the tissue compaction (increased lateral interspace) (Figure XIVB in the Supplemental Material). Of note, Efnb1 deletion only partially prevents CM hypertrophy (partial CM short-axis elongation), most likely due to the other molecular events taking place at the LM during the P20-P60 stage, i.e. the interactions with the ECM (integrin, dystroglycan complex) that we previously showed to be independent of ephrin-B19. At the lateral membrane level, P20 Efnb1CM−/−KO and WT mice harbor similar unstructured surface crests with immature SSM but only WT mice underwent crest maturation at P60 (Figure 5E), thus demonstrating a key role for ephrin-B1 in the postnatal maturation of the surface crest/SSM.
We next assessed the role of ephrin-B1 in the cardiac function of young adult male mice (P60/2-months). We did not notice differences in heart rate and LVEF between 2-month-old WT and Efnb1CM-spe KO mice (Figure 6A). However, and consistent with the role of ephrin-B1 in the lateral stretch-based hypertrophy of the CM, Efnb1CM−/− KO mice display a significant decrease in LVPWd conversely to an increase in left ventricular internal diameter (Left ventricular internal diameter end diastole, LVIDd) and volume (LV end-diastolic volume, LVEDV) (Figure 6A), in agreement with our previous study9. Moreover, compared with WT mice, Efnb1CM−/− KO mice display significantly elongated IVRT, increased left atrial volume (left atrial to aortic root ratio, LA/Ao) and decreased E/A as well as heterogeneous E/E’(Figure 6A), all indicative of impaired LV relaxation. Despite preservation of the LVEF, we also measured a significant decrease in ventricular global longitudinal strain (LV-GLS) in KO mice (Figure 6A) reflecting abnormal longitudinal systolic function. Diastolic dysfunction in KO mice was further confirmed by cardiac catheterization since 2-month-old Efnb1CM−/− KO mice exhibit increased EDP and heterogeneous dP/dt but with similar relaxation time constants (tau) compared with age-matched WT mice (Figure 6B). The diastolic defects of Efnb1CM−/− KO mice rely on a specific impairment of the CM relaxation since both the diastolic basal sarcomere length (SL) shortening and relaxation velocities are significantly reduced in isolated intact CMs from Efnb1CM−/− KO compared to WT mice (Figure 6C). Furthermore, consistent with heart failure with preserved ejection fraction (HFpEF) phenotype in which CM contraction defects co-exist with the relaxation impairment 17–19, the auxotonic contraction indexed by the SL shortening as well as the SL shortening velocities are also significantly decreased in CMs from Efnb1CM−/− KO compared to WT CMs (Figure 6C). Finally, Efnb1CM−/− KO mice also display significant exercise intolerance compared with WT mice, as indicated by the significant decrease in maximum oxygen uptake (Figure 6D). Together, these results indicate that young adult male Efnb1CM−/− KO mice recapitulate some features of clinical HFpEF, thus demonstrating a pivotal role for ephrin-B1 in the control of the diastolic function. In line with this assumption, a recent RNAseq study performed on ventricles from HFpEF, HFrEF (heart failure with reduced ejection fraction) and control patients reported a specific and more significant downregulation of the Efnb1 gene in HFpEF than HFrEF patients (p = 9.10−14 HFpEF vs control, p = 2.10−5 HFrEF versus control, p = 0.005 HFpEF versus HFrEF)20.
More globally, these results demonstrate that ephrin-B1-dependent maturation of the CM surface crests during the P20-P60 postnatal period allows maturing the adult diastolic function.
Efnb1CM−/− mice switched progressively from HFpeF to HFrEF and all died at 14 months of age due to T-tubule disorganization
Finally, we monitored changes in the cardiac function of Efnb1CM−/− KO and WT mice in medium and long term. Interestingly, while in young adulthood (2 months) Efnb1CM−/− KO mice display LV diastolic dysfunction with preserved ejection fraction compared to WT mice, LVEF decreased progressively over time in these mice, which start developing moderate HFrEF at 9 months, evolving toward severe HFrEF by 13 months (Figure 7A) and 100% mortality after 15 months (Figure 7B). Accordingly, LVIDd and LVEDV significantly increase over time only in KO mice (Figure 7A) concurrently with the development of compensatory cardiac hypertrophy (IVSd, Figure 7A, C, D) and fibrosis around 12 months of age (Figure 7E). Corroborating the HF progression in Efnb1CM−/− KO mice, while T-tubule (HFrEF marker) misalignment from Z-lines is already detectable but limited to discrete local regions in the cardiac tissue of some KO CMs at 2 months of age, this disorganization progressively spreads over time to all CMs after 12 months compared with WT mice (Figure 7F).
Collectively, these data indicate that the lack of ephrin-B1 in CM primarily impairs the diastolic function of young adult mice, which progressively switch toward a systolic defect with ageing.
DISCUSSION
In this study, we describe a new developmental stage occurring in the late postnatal period between P20 and P60, during which LM crests maturate through SSM swelling and crest-crest interactions within the tissue, thus allowing CM lateral stretching and a global compaction of the cardiac tissue (Figure 8). We demonstrate that this mechanism is ephrin-B1-dependent and regulates the adult diastolic function. Taken together, our findings identify crest subdomains of the adult CM lateral surface as novel specific determinants of cardiac diastole.
Postnatal maturation of the heart in mammals has been paid much less attention than embryonic and fetal development. This would be a prerequisite for future pediatric clinical trials, which are seriously lacking. It also contributes to an significant knowledge gap that impedes research progression in regenerative medicine, since this postnatal maturation coincides with the proliferation blockage of the CM. Up to now, postnatal cardiac maturation has been essentially described until P20 in rodents, most likely related to the implementation of the typical adult rod shape of the CM. Very few studies have examined the transition from P20 to the adult stage, assuming that the genetic maturation of the heart is completed by P20 and that the heart merely undergoes a substantial growth beyond P20 until the young adult stage14. However, this period coincides with the weaning time in rodents (~P21) and thus with a critical nutritional/metabolic switch in the organism likely to influence heart function. So far, research has focused on the P0 (birth) to P20 postnatal window, during which the heart switches from a hyperplasic growth (CM proliferation) (~ P3-P5) to an hypertrophic growth (increase in CM size) (P20-21)21–23. Recent omics studies have detailed the molecular changes associated with the postnatal development of the mouse heart but only until P2024,25. Thus, the P0-P20 postnatal development period essentially relies on the ECM remodelling of the cardiac tissue and, at the CM level, myofibril and Ca2+ handling maturation, metabolic switch from glycolysis to major fatty acid oxidation and the transition from CM proliferation to CM hypertrophy26,27. This metabolic change most likely occurs as an adaptation of the postnatal heart to cardiac tissue oxygenation and the newborn diet, which essentially consists of mother’s milk and thus on high fatty acid availability. Our transcriptome analysis of mouse hearts reveals an additional developmental genetic program between P20 and P60 originating from all the cardiac cells of the left ventricle, thus indicating that the heart and the CM had not completed maturation by P20. Of interest, while the primary metabolic reliance of the heart on fatty acids and then glucose is already programmed between birth and P20, we found that the P20-P60 late postnatal stage programs the metabolic diversification of the heart. Such a new final metabolic adaptation of the heart coincides with the weaning process occurring around P20 in rodents, during which the newborn switches from milk to solid food28. This step is also in line with the capacity of the heart to metabolize a large panel of substrates to meet the energy demands of the adult stage29. Remarkably, we found that the Bdh1 gene was increased by more than 37-fold between P20 and P60, most likely indicating that the capacity of the heart to oxidize KB (coming essentially from the liver) as a fuel is not only restricted to the adaptation of the failing heart, as previously shown30, but is also necessary for the adult physiological state. These results also agree with recent findings demonstrating that the human adult heart can use a large panel of substrates as a fuel, including a large proportion of KB29. Horton et al did not report a major cardiac phenotype under basal conditions in Bdh1CM−/− mice30. However, more in-depth cardiac phenotyping of these mice would undoubtedly be necessary to depict the role of KB in the adult cardiac physiology, such as diastolic function.
An intriguing finding from our study is the physiological cardiac hypertrophy of the heart between P20 and P60, which, from a CM lateral standpoint, does not rely on a classical myofibril addition in the CM but, at least in part, on a ephrin-B1-dependent-i/ crest maturation through SSM swelling at the CM surface and -ii/ lateral stretching of the CM. During postnatal development, heart growth occurs primarily through CM proliferation (hyperplasia) but transitions rapidly after birth (~P5-7) to CM growth (hypertrophy)22,23 through new myofibril biogenesis. Although physiological CM hypertrophy has been described beyond P20 and is related to increased cardiac mass14,31, the underlying molecular mechanisms have never been explored. Piquerau et al14 already reported and questioned such atypical hypertrophy of the rat heart after P21 without fiber addition, which was not classically related to the increase in heart-weight to body-weight index but conversely to a marked decrease. Here, we show that the P20-P60 hypertrophy relies on both an enlargement of the short and long axes of the CM. Interestingly, we demonstrated that the short axis elongation is dependent on the architectural maturation of the CM surface trough an ephrin-B1 mechanism. Thus, the ephrin-B1 lateral membrane protein, a partner and regulator of claudin-59, participates in P20-P60 lateral CM hypertrophy, likely by bringing claudin-5 into the vicinity of neighboring CM, allowing intermittent but direct lateral crest-crest interactions (claudin-5 / claudin-5 interactions), crests/SSM maturation and the ensuing stretching of the CM lateral membrane (Figure 8). In agreement with this model, we previously showed that CMs from Efnb1CM−/− KO mice exhibit substantially decreased levels of claudin-5 expression9, likely accounting for the lack of crest maturation and lateral crest-crest interactions in these adult KO mice. However, other mechanisms also likely contribute to the short-axis elongation, since it was only partially prevented by ephrin-B1 deletion. More specifically, lateral membrane interactions with the ECM, i.e. through integrin or the dystroglycan complex, that we previously showed to be independent of ephrin-B19, could also play a role. In line with this assumption, our transcriptomic analysis identified the regulation of genes from the ECM remodelling pathway during the P20-P60 maturation period and the dystrobrevin-encoding gene (DTNA, Figure X in the Supplemental Material) from the dystroglycan complex. Apart from the short-axis, P20-P60 CM hypertrophy also depends on the elongation of the CM long-axis, which we show here to be independent from ephrin-B1 and which probably depends on the classical assembly of new sarcomeres at the myofibril extremities.
A new finding of our study is the specific maturation of SSM during the late P20-P60 postnatal period of the CM. Coinciding with this late cardiac mitochondria maturation in the CM, Piquerau et al14 previously reported a substantial increase in maximum respiratory capacity occurring after P21 and the adult stage in cardiac tissues from rats while the postnatal maturation of IFM occurred earlier. Biogenesis/maturation of cardiac mitochondria occur early during the embryogenesis, concurrently with the energy demand of the heart during the development period16,32. Thus, they can adapt their morphology and function according to the energy need and metabolic conditions of the cell33,34. In this field, most of the works on the postnatal period have focused on the characterization of the IFM (the most abundant in the adult CM) or the global cardiac mitochondria activity dedicated to the energy supply to the CMs for the contractile machinery, without distinguishing the different mitochondria subpopulations of the adult CM35. IFM maturation during the P0-P7 stage occurs both through swelling and through architectural reorganization along the myofibrils14, concomitantly with the well-known metabolic shift of the heart from glycolysis to a central oxidative metabolism more efficient for adult CM contraction16. It follows that IFM function is primarily dedicated to supplying the CM with the energy necessary for adult contraction. The delayed maturation of SSM at the CM surface compared to intracellular IFM supports the concept that these mitochondria regulate different CM functions. In agreement with this notion, we demonstrated in this study that crests/SSM specifically regulate heart diastole. Several results support this conclusion: i/ young adult Efnb1CM−/− KO mice with immature SSM display diastolic defects with no impairment of systolic function, ii/ systolic function is already mature by P20 in rat hearts while the diastolic function is highly variable. Moreover, supporting our results about the late maturation of diastole, Zhou et al previously reported that the diastolic function in the left ventricle of mice matures around the weaning period36. In the future, it would be interesting to investigate whether cardiac SSM defects are a specific feature of diastolic dysfunction pathologies, such as HFpEF frequently associated with a metabolic syndrome37. In support of this concept, a recent study of myocardial gene expression signatures in human HFpEF reported that the ephrin-B1-encoding gene, that we demonstrated here as a specific determinant of the CM crests/SSM, is specifically downregulated in HFpEF heart patients compared with HFrEF20. Unfortunately, while several works have reported the influence of different cardiac pathologies on specific subpopulations of cardiac mitochondria35, these results require some caution, given the technical difficulty of specifically purifying and distinguishing the populations in the absence of reliable markers and due to the fact that these mitochondria are morphologically highly similar12. Today, electron microscopy still remains the gold standard for accurately examining the cardiac mitochondria subpopulations.
An essential and new finding of our study is the identification of the ephrin-B1/crest/SSM module at the lateral membrane of the CM as a specific determinant of the physiological diastolic function of the adult heart. So far, the diastolic determinants have been proposed only in the context of diastolic dysfunction in cardiac pathologies such as HFpEF38. However, until rather recently, the lack of specific and compelling therapeutics in HFpEF underlines our limited knowledge of the control of diastole. This is overcomplicated in the context of HFpEF due to the contributions of several comorbidities delineating highly heterogeneous clinical features38. These last months, the discovery of empaglifozine, a iSGLT2 antidiabetic drug, as the first effective therapeutics for HFpEF patients39, questioned the molecular mechanisms underlying the cardiac benefit, given the lack of SGTL2 expression in the heart. However, a beneficial and indirect role through metabolism regulation has been postulated40. It follows that the control of the diastole could arise, in part, from a systemic metabolic regulation. Thus, the extent to which iSGTL2 could regulate the crest/SSM module at the CM surface in a pathological context should be consider in the future. Here, our study demonstrates that Efnb1 deletion specifically in the CM recapitulates at the young adult stage (2 months) some features of the diastolic dysfunction depicted in HFpEF, i.e. an elevated EDP and altered filling patterns combined with exercise intolerance. One important concern is understanding how ephrin-B1 at the lateral membrane of the adult CM can impact diastolic function. We previously demonstrated that ephrin-B1 controls the architecture of the lateral membrane and the overall adult rod shape of the CM9. Here, we now further demonstrated that ephrin-B1 controls the maturity of the crests/SSM architectural motif at the lateral membrane, thus playing a key role in the adult crest-crest interactions between neighboring CMs and in overall tissue cohesion. These lateral CM interactions likely dictate a mechanical lateral stretch of the CM, allowing perfect stacking between myofibril layers. Consequently, crest-crest interactions might contribute to controlling the relaxation length of the sarcomere, a mechanism that we previously suggested depends on crest height12. This ephrin-B1-dependent lateral stretch of the CM is also necessary for the spatial arrangement of the T-tubules and most likely its ensuing function during the P20-P60 development, as supported by the T-tubule disorganization in the Efnb1CM−/− KO mice. Thus, by altering the T-tubule structure, lack of ephrin-B1 could influence the Ca2+ entry / Ca2+ exit. It is worth noting that T-tubule disorganization in the CMs of Efnb1CM−/− KO mice is progressive with mild disorganization correlating with diastolic dysfunction only, while extensive disorganization is observed in HFrEF. Although defects in T-tubule architecture/function are a hallmark of HFrEF41, it has been poorly examined in HFpEF, only one recent study reported that this mechanism is etiology-dependent42. An exciting feature of the Efnb1CM−/− KO mice is the progressive cardiac phenotype starting from a primarily diastolic defect that progressively switches toward a mild and then severe HFrEF and finally to death, thus highlighting the substantial cardioprotective role of the surface crests/SSM of the lateral membrane. Although this switch toward HFrEF is not a common feature of HFpEF patients43, definitive conclusions cannot be reached on the natural evolution of HFpEF, given that HFpEF patients received medication for their different comorbidities, which could protect them from HFrEF. In the future, an accurate characterization of the CM surface crests/SSM in different HFpEF models with diastolic dysfunction will undoubtedly shed light on whether crest disruption is a hallmark of HFpEF and could thus contribute to the setting of the pathology.
Sources of funding
This work was in part supported by the “Fondation Bettencourt Schueller” (to C.G.), the “Fondation de France” grant n°75807 (to C.G.), the “Fondation pour la Recherche Médicale” grant DEQ20170336733 (to C.G.) and grant FDM201906008682 (to B.T.) and the “Société Francophone du Diabète” (to B.T.).
Acknowledgements
We thank Claire Naylies and Yannick Lippi (Toxalim, Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, Toulouse, France) for their contribution to microarray fingerprints acquisition and microarray data analysis carried out at GeT Genopole Toulouse Midi-Pyrénées facility (https://doi.org/10.15454/1.5572370921303193E12).
We are also grateful to TRI Genotoul network facilities, specifically ANEXPLO platform (Toulouse) for help with echocardiography, the “Centre de Microscopie Electronique Appliquée à la biologie-CMEAB» (Faculté de Médecine Rangueil-Toulouse) and the Cellular Imaging Facility-I2MC (Toulouse).
Footnotes
The authors declared that no conflict of interest exists.
NONSTANDARD ABBREVIATIONS AND ACRONYMS
- AFM
- Atomic force microscopy
- CM
- Cardiomyocyte
- DBP
- Diastolic blood pressure
- ECM
- Extracellular matrix
- EDP
- End diastolic pressure
- FA
- Fatty acid
- GO
- Gene ontology
- HF
- Heart Failure
- HFrEF
- Heart Failure with reduced ejection fraction
- HFpEF
- Heart Failure with preserved ejection fraction
- ID
- Intercalated disk
- IFM
- Interfibrillar mitochondria
- IVRT
- Isovolumic relaxation time
- KB
- Keytone bodies
- KO
- Knock-out
- LVPWd
- left ventricular posterior wall in diastole
- LVEDV
- Left ventricular end-diastolic volume
- LVEDD
- Left ventricular end-diastolic diameter
- LVIDd
- left ventricular internal diameter in diastole
- LV-GLS
- left ventricular global longitudinal strain
- P5
- Postnatal day 5
- SBP
- Systolic blood pressure
- SSM
- Subsarcolemmal mitochondria
- SCIM
- Scanning ion-conductance microscopy
- TEM
- Transmission electron microscopy
- WT
- wild-type