Protein geranylgeranylation: a possible new player in congenital heart defects

This editorial refers to ‘Geranylgeranyl pyrophosphate synthase facilitates the organization of cardiomyocytes during mid-gestation through modulating protein geranylgeranylation in mouse heart’ by Z. Chen et al., pp. 965–978. The morphogenesis of the myocardial wall is a complex process and disruption can lead to congenital heart disease (CHD). Thus, identifying genes and intracellular processes that are essential for heart development will provide pivotal genetic information for the management of CHD. The exact details of how the cardiomyocytes assemble and interact to form the correct myocardial mass and dimensions are still part of ongoing research in many laboratories. In this issue of Cardiovascular Research, Chen et al.1 introduces the novel idea that the process of protein geranylgeranylation is required for the arrangement of early cardiomyocytes during ventricular chamber maturation. Protein geranylgeranylation is a form of prenylation and is an essential type of post-translational modification of proteins. It involves the attachment of lipophilic geranylgeranyl isoprene residues to a cysteine located at the C-terminus of specific proteins. This subsequently acts as a membrane anchor for the modified target protein, triggering the translocation of the protein from the cytosol to the membrane, hence activating the protein. The mevalonate pathway regulates the process of geranylgeranylation. It leads to the production of farnesyl pyrophosphate (FPP), which is synthesized into geranylgeranyl pyrophosphate (GGPP) by the enzyme, geranylgeranyl pyrophosphate synthase (Ggpps). Downstream, the enzyme, geranylgeranyl transferase I (GGTase I), then catalyses the addition of one geranylgeranyl group from GGPP onto the target protein. The heart initially consists of a single layer of cardiomyocytes, which mature and proliferate under the control of many different genetic pathways, to form thick compact myocardium and trabeculae layers. Early embryonic cardiomyocytes are polygonal in shape with the cell–cell junctions arranged uniformly around the cell membrane.2 The adherens junctions are the major anchorage sites for actin cytoskeleton, the myofibrils, which initially run in random orientations during embryonic development. Disruption of cellular junctions early in development leads to heart defects and embryonic lethality. During trabeculae formation, trabecular cardiomyocytes undergo directional migration or orientation cell division, forming new cell–cell junctions. Adherens junctions are required to establish both of these processes as loss of N-cadherin led to trabeculation defects.3 Hence, the integrity of cell-cell junctions are essential to maintain the rigidity of the cytoarchitecture required for development of the ventricular wall. This is the first publication to study specifically the role of Ggpps in the developing mammalian heart. Previous publications showed that enzymes involved in the mevalonate pathway, including GGPPS, in Drosophila and zebrafish, control the early stages of heart formation.4,5 Chen et al. have previously published the deletion of Ggpps in postnatal cardiomyocytes. These mutant mice developed heart failure and died at 2 months, highlighting the role of Ggpps in controlling postnatal heart growth by regulating cardiomyocyte cell size.6 These studies did not identify the target substrate that required geranylgeranylation, but alluded to the possibility that the Rho family members are involved. Links between decreased GGPP levels and reduced RhoA activation have been reported in adult rat hearts.7 The authors used two Cre lines, Nkx2.5-Cre and α-SMA-Cre, to delete Ggpps, leading to disruption of protein geranylgeranylation at E9.5 and E13.5, respectively. Disruption of protein geranylgeranylation, in the former, affected ventricular chamber maturation and caused embryonic lethality between E12.0 and E13.0, whereas later disruption of the process using the α-SMA-Cre line had no effect on the phenotype. The organization of the cytoskeleton was affected, as abnormal sarcomeres were present and proteins associated with the adherens and gap junctions in the ventricular wall were disrupted at E11.5; they attributed this to specific reduction of geranylgeranylation of Rho GTPases, RhoA, and Rac1. The abnormal cytoarchitecture of the cardiomyocytes coincided with reduced proliferation, hence accounting for the thin myocardium. However, the direct link between geranylgeranylation of Rho GTPases, cell junction maintenance and proliferation is still unclear. As Nkx2.5-Cre is expressed in cardiomyocytes, smooth muscle, and endothelial lineages of the developing heart8 the cell specificity of protein geranylgeranylation could be further addressed by using alternative transgenic mouse Cre lines to target individual cell populations, for example TnT-Cre (cardiomyocytes only). The authors, focused on RhoA and Rac1 as downstream targets of geranylgeranylation, and proved by western blot analysis that there was reduced membrane bound and prenylated forms of each of these proteins, without a reduction in total protein. Further dual staining, could establish if the altered cellular distribution of RhoA and Rac1 is located in the endocardial, cardiomyocytes and/or epicardial cell lineages. They confirmed that this reduction in geranylgeranylation of RhoA and Rac1 led to reduced levels of active GTP-bound form of each protein. Chemical administration of GGPP in vitro and in vivo (via GGOH) rescued the phenotype, but the authors did not show that this specifically increased geranylgeranylation of RhoA and Rac1. Another enzyme in the mevalonate pathway is farnesyl pyrophosphate synthase (FPPS), which is required for the production of FPP. As described, FFP can be converted into GGPP or directly leads to another type of protein prenylation, farnesylation. In their previous publication, the deletion of Ggpps in postnatal hearts, led to increased farnesylation as a consequence of decreased geranylgeranylation6 and overexpression of FPPS is a known regulator of myocardial remodeling.9 However, the level of farnesylation was not addressed in the current publication and may be involved in heart development. This study is the first to implicate that the geranylgeranylation of RhoA and Rac1 is essential for cardiomyocyte integrity and hence ventricular development, and opens up a number of questions. Both of these Rho GTPases have a number of downstream targets and establishing, which are affected by the reduced geranylgeranylation, would generate a mechanism of the exact cause of the heart defects described. Furthermore, as there are over 22 members of the Rho GTPase family, the deletion of Ggpps may impact on these through altered geranylgeranylation or farnesylation.10 For example, the activity of Cdc42 is dependent upon prenylation11 and is essential for the establishment of adherens junctions. Embryonic deletion of Cdc42 in cardiomyocytes, caused embryonic lethality after E12.5 with heart defects including thin ventricle walls, which the authors attributed to reduced cardiomyocyte proliferation and impaired formation of adherens junction cell–cell contacts,12 thus presenting a similar phenotype as described in this article by Chen et al. Therefore, this opens up the possibility that the geranylgeranylation of Cdc42 may be altered in the Nkx2.5Cre/+; Ggppsfl/fl mutants as well. How exactly these RhoGTPases control adhesion between cardiomyocytes remains elusive, but the level of protein geranylgeranylation of other members of this family is worth pursuing. In conclusion, Chen et al., have introduced the idea that protein geranylgeranylation is an important step during cardiomyocyte maturation and is required for the maintenance of cellular junctions. This raises the possibility that other components upstream and downstream in the mevalonate pathway and other members of the Rho GTPase family may be potential disease causing genes in CHD (Figure 1) . Potential role of protein geranylgeranylation in the maturation of the ventricular wall. The mevalonate pathway leads to protein geranylgeranylation of downstream targets. In normal heart development the enzyme, Ggpps, converts FPP into GGPP, from which a geranylgeranyl group is transferred by GGTase I, to activate members of the RhoGTPase family. The membrane bound RhoGTPases are required for proliferation, and the maintenance of the cell–cell junctions and the actin cytoskeleton, hence establishing the polygonal shape of embryonic cardiomyocytes, and subsequent maturation of the ventricular wall (A). Deletion of Ggpps reduces the activity of RhoA and Rac1, and potentially further members of the RhoGTPase family, through decreased geranylgeranylation at E9.5; possibly leading to an increase in cytoplasmic localisation of RhoGTPases. Loss of cell junction and actin cytoskeleton integrity alters the cell shape of the cardiomyocytes, leading to a visually abnormal cytoarchitecture of the ventricular wall at E11.5, and subsequent embryonic death a day later. Deletion of Cdc42 was reported to lead to a very similar phenotype12 highlighting the importance of active RhoGTPases in early heart development (B). FPPS, farnesyl pyrophosphate synthase; FPP, farnesyl pyrophosphate; Ggpps, geranylgeranyl pyrophosphate synthase; GGPP, geranylgeranyl pyrophosphate; GGTase I, geranylgeranyl transferase I. Potential role of protein geranylgeranylation in the maturation of the ventricular wall. The mevalonate pathway leads to protein geranylgeranylation of downstream targets. In normal heart development the enzyme, Ggpps, converts FPP into GGPP, from which a geranylgeranyl group is transferred by GGTase I, to activate members of the RhoGTPase family. The membrane bound RhoGTPases are required for proliferation, and the maintenance of the cell–cell junctions and the actin cytoskeleton, hence establishing the polygonal shape of embryonic cardiomyocytes, and subsequent maturation of the ventricular wall (A). Deletion of Ggpps reduces the activity of RhoA and Rac1, and potentially further members of the RhoGTPase family, through decreased geranylgeranylation at E9.5; possibly leading to an increase in cytoplasmic localisation of RhoGTPases. Loss of cell junction and actin cytoskeleton integrity alters the cell shape of the cardiomyocytes, leading to a visually abnormal cytoarchitecture of the ventricular wall at E11.5, and subsequent embryonic death a day later. Deletion of Cdc42 was reported to lead to a very similar phenotype12 highlighting the importance of active RhoGTPases in early heart development (B). FPPS, farnesyl pyrophosphate synthase; FPP, farnesyl pyrophosphate; Ggpps, geranylgeranyl pyrophosphate synthase; GGPP, geranylgeranyl pyrophosphate; GGTase I, geranylgeranyl transferase I. Conflict of interest: none declared.