RhoA: a dubious molecule in cardiac pathophysiology.

The Ras homolog gene family member A (RhoA) is the founding member of Rho GTPase superfamily originally studied in cancer cells where it was found to stimulate cell cycle progression and migration. RhoA acts as a master switch control of actin dynamics essential for maintaining cytoarchitecture of a cell. In the last two decades, however, RhoA has been coined and increasingly investigated as an essential molecule involved in signal transduction and regulation of gene transcription thereby affecting physiological functions such as cell division, survival, proliferation and migration. RhoA has been shown to play an important role in cardiac remodeling and cardiomyopathies; underlying mechanisms are however still poorly understood since the results derived from in vitro and in vivo experiments are still inconclusive. Interestingly its role in the development of cardiomyopathies or heart failure remains largely unclear due to anomalies in the current data available that indicate both cardioprotective and deleterious effects. In this review, we aimed to outline the molecular mechanisms of RhoA activation, to give an overview of its regulators, and the probable mechanisms of signal transduction leading to RhoA activation and induction of downstream effector pathways and corresponding cellular responses in cardiac (patho)physiology. Furthermore, we discuss the existing studies assessing the presented results and shedding light on the often-ambiguous data. Overall, we provide an update of the molecular, physiological and pathological functions of RhoA in the heart and its potential in cardiac therapeutics.

Rho-family GTPase 1 (Rnd1) is a biomechanical stress-sensitive activator of cardiomyocyte hypertrophy.

Cardiac remodeling is induced by mechanical or humoral stress causing pathological changes to the heart. Here, we aimed at identifying the role of differentially regulated genes upon dynamic mechanical stretch. Microarray of dynamic stretch induced neonatal rat ventricular cardiomyocytes (NRVCMs) discovered Rho family GTPase 1 (Rnd1) as one of the significantly upregulated genes, a cardiac role of which is not known yet. Rnd1 was consistently upregulated in NRVCMs after dynamic stretch or phenylephrine (PE) stimulation, and in a mouse model of pressure overload. Overexpression of Rnd1 in NRVCMs activated the fetal gene program (including nppa and nppb) effected into a significant increase in cell surface area in untreated, stretched or PE-treated cells. Furthermore, Rnd1 overexpression showed a positive effect on cell proliferation as detected by significant increase in Ki67, Phosphohistone H3, and EdU positive NRVCMs. Through a Yeast two-hybrid screen and immunoprecipitation analysis, we identified Myozap, an intercalated disc protein, as novel interaction partner of Rnd1. Importantly, functional analysis of this interaction revealed the importance of RND1 in the RhoA and Myozap protein network that activates serum-response factor (SRF) signaling. In summary, we identified Rnd1 as a novel stretch-sensitive gene which influences cell proliferation and cellular hypertrophy via activation of RhoA-mediated SRF dependent and independent signaling pathways.

A cardiac α-actin (ACTC1) p. Gly247Asp mutation inhibits SRF-signaling in vitro in neonatal rat cardiomyocytes.

We recently identified a novel, heterozygous, and non-synonymous ACTC1 mutation (p.Gly247Asp or G247D) in a large, multi-generational family, causing atrial-septal defect followed by late-onset dilated cardiomyopathy (DCM). Molecular dynamics studies revealed possible actin polymerization defects as G247D mutation resides at the juncture of side-chain interaction, which was indeed confirmed by in vitro actin polymerization assays. Since polymerization/de-polymerization is important for the activation of Rho-GTPase-mediated serum response factor (SRF)-signaling, we studied the effect of G247D mutation using luciferase assay. Overexpression of native human ACTC1 in neonatal rat cardiomyocytes (NRVCMs) strongly activated SRF-signaling both in C2C12 cells and NRVCMs, whereas, G247D mutation abolished this activation. Mechanistically, we found reduced GTP-bound Rho-GTPase and increased nuclear localization of globular actin in NRVCMs overexpressing mutant ACTC1 possibly causing inhibition of SRF-signaling activation. In conclusion, our data suggests that human G247D ACTC1 mutation negatively regulates SRF-signaling likely contributing to the late-onset DCM observed in mutation carrier patients.

Increased Density of Growth Differentiation Factor-15+ Immunoreactive M1/M2 Macrophages in Prostate Cancer of Different Gleason Scores Compared with Benign Prostate Hyperplasia.

Although growth differentiation factor-15 (GDF-15) is highly expressed in PCa, its role in the development and progression of PCa is unclear. The present study aims to determine the density of GDF-15+ cells and immune cells (M1-/M2 macrophages [MΦ], lymphocytes) in PCa of different Gleason scores (GS) compared to BPH. Immunohistochemistry and double immunofluorescence were performed on paraffin-embedded human PCa and BPH biopsies with antibodies directed against GDF-15, CD68 (M1 MΦ), CD163 (M2 MΦ), CD4, CD8, CD19 (T /B lymphocytes), or PD-L1. PGP9.5 served as a marker for innervation and neuroendocrine cells. GDF-15+ cell density was higher in all GS than in BPH. CD68+ MΦ density in GS9 and CD163+ MΦ exceeded that in BPH. GDF-15+ cell density correlated significantly positively with CD68+ or CD163+ MΦ density in extratumoral areas. Double immunoreactive GDF-15+/CD68+ cells were found as transepithelial migrating MΦ. Stromal CD68+ MΦ lacked GDF-15+. The area of PGP9.5+ innervation was higher in GS9 than in BPH. PGP9.5+ cells, occasionally copositive for GDF-15+, also occurred in the glandular epithelium. In GS6, but not in BPH, GDF-15+, PD-L1+, and CD68+ cells were found in epithelium within luminal excrescences. The degree of extra-/intra-tumoral GDF-15 increases in M1/M2Φ is proposed to be useful to stratify progredient malignancy of PCa. GDF-15 is a potential target for anti-tumor therapy.

RhoA Signaling in Immune Cell Response and Cardiac Disease.

Chronic inflammation, the activation of immune cells and their cross-talk with cardiomyocytes in the pathogenesis and progression of heart diseases has long been overlooked. However, with the latest research developments, it is increasingly accepted that a vicious cycle exists where cardiomyocytes release cardiocrine signaling molecules that spiral down to immune cell activation and chronic state of low-level inflammation. For example, cardiocrine molecules released from injured or stressed cardiomyocytes can stimulate macrophages, dendritic cells, neutrophils and even T-cells, which then subsequently increase cardiac inflammation by co-stimulation and positive feedback loops. One of the key proteins involved in stress-mediated cardiomyocyte signal transduction is a small GTPase RhoA. Importantly, the regulation of RhoA activation is critical for effective immune cell response and is being considered as one of the potential therapeutic targets in many immune-cell-mediated inflammatory diseases. In this review we provide an update on the role of RhoA at the juncture of immune cell activation, inflammation and cardiac disease.

Data on the role of cardiac α-actin (ACTC1) gene mutations on SRF-signaling.

We recently reported a novel, heterozygous, and non-synonymous ACTC1 mutation (p.Gly247Asp or G247D) in a large, multi-generational family, causing atrial-septal defect followed by late-onset dilated cardiomyopathy (DCM). We also found that the G247D ACTC1 mutation negatively regulated serum response (SRF)-signaling thereby contributing to the late-onset DCM observed in human patients carrying this mutation ("A cardiac α-actin (ACTC1) p. Gly247Asp mutation inhibits SRF-signaling in vitro in neonatal rat cardiomyocytes" [1]). There are some ACTC1 mutations known to date, majority of which, though, have not been investigated for their functional consequence. We thus aimed at determining the functional impact of various ACTC1 gene mutations on SRF-signaling using SM22-response element driven firefly luciferase activity assays in C2C12 cells.