MBNL1 Regulates Programmed Postnatal Switching Between Regenerative and Differentiated Cardiac States.

BACKGROUND: Discovering determinants of cardiomyocyte maturity is critical for deeply understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration, but elucidation of endogenous developmental regulators of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. MBNL1 (muscleblind-like 1) regulates fibroblast, thymocyte, and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. METHODS: MBNL1 gain- and loss-of-function mouse models were studied at several developmental time points and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro assays to determine the mechanisms through which MBNL1 exerts its effects. RESULTS: MBNL1 is coexpressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deficiency was insufficient to promote regeneration in the adult heart because of cell cycle checkpoint activation. CONCLUSIONS: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Targeting loss of cardiomyocyte maturity and downregulation of cell cycle inhibitors through MBNL1 deletion was not sufficient to promote adult regeneration.

MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states.

Discovering determinants of cardiomyocyte maturity and the maintenance of differentiated states is critical to both understanding development and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Here, the RNA binding protein Muscleblind-like 1 (MBNL1) was identified as a critical regulator of cardiomyocyte differentiated states and their regenerative potential through transcriptome-wide control of RNA stability. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of the estrogen-related receptor signaling axis was essential for maintaining cardiomyocyte maturity. In accordance with these data, modulating MBNL1 dose tuned the temporal window of cardiac regeneration, where enhanced MBNL1 activity arrested myocyte proliferation, and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. Collectively these data suggest MBNL1 acts as a transcriptome-wide switch between regenerative and mature myocyte states postnatally and throughout adulthood.

MBNL1 drives dynamic transitions between fibroblasts and myofibroblasts in cardiac wound healing.

Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response. Because transcriptome maturation by muscleblind-like 1 (MBNL1) promotes differentiated cell states, this study investigated whether tactical control of MBNL1 activity could alter myofibroblast activity and fibrotic outcomes. In healthy mice, cardiac fibroblast-specific overexpression of MBNL1 transitioned the fibroblast transcriptome to that of a myofibroblast and after injury promoted myocyte remodeling and scar maturation. Both fibroblast- and myofibroblast-specific loss of MBNL1 limited scar production and stabilization, which was ascribed to negligible myofibroblast activity. The combination of MBNL1 deletion and injury caused quiescent fibroblasts to expand and adopt features of cardiac mesenchymal stem cells, whereas transgenic MBNL1 expression blocked fibroblast proliferation and drove the population into a mature myofibroblast state. These data suggest MBNL1 is a post-transcriptional switch, controlling fibroblast state plasticity during cardiac wound healing.

Infarct Collagen Topography Regulates Fibroblast Fate via p38-Yes-Associated Protein Transcriptional Enhanced Associate Domain Signals.

RATIONALE: Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart's ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart. OBJECTIVE: To investigate the role of collagen organization in cardiac fibroblast fate determination. METHODS AND RESULTS: Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFß (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here. CONCLUSIONS: Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.

Sonic Hedgehog upregulation does not enhance the survival and engraftment of stem cell-derived cardiomyocytes in infarcted hearts.

The engraftment of human stem cell-derived cardiomyocytes (hSC-CMs) is a promising treatment for remuscularizing the heart wall post-infarction, but it is plagued by low survival of transplanted cells. We hypothesize that this low survival rate is due to continued ischemia within the infarct, and that increasing the vascularization of the scar will ameliorate the ischemia and improve hSC-CM survival and engraftment. An adenovirus expressing the vascular growth factor Sonic Hedgehog (Shh) was injected into the infarcted myocardium of rats immediately after ischemia/reperfusion, four days prior to hSC-CM injection. By two weeks post-cell injection, Shh treatment had successfully increased capillary density outside the scar, but not within the scar. In addition, there was no change in vessel size or percent vascular volume when compared to cell injection alone. Micro-computed tomography revealed that Shh failed to increase the number and size of larger vessels. It also had no effect on graft size or heart function when compared to cell engraftment alone. Our data suggests that, when combined with the engraftment of hSC-CMs, expression of Shh within the infarct scar and surrounding myocardium is unable to increase vascularization of the infarct scar, and it does not improve survival or function of hSC-CM grafts.

Fibroblast-Specific Genetic Manipulation of p38 Mitogen-Activated Protein Kinase In Vivo Reveals Its Central Regulatory Role in Fibrosis.

BACKGROUND: In the heart, acute injury induces a fibrotic healing response that generates collagen-rich scarring that is at first protective but if inappropriately sustained can worsen heart disease. The fibrotic process is initiated by cytokines, neuroendocrine effectors, and mechanical strain that promote resident fibroblast differentiation into contractile and extracellular matrix-producing myofibroblasts. The mitogen-activated protein kinase p38α (Mapk14 gene) is known to influence the cardiac injury response, but its direct role in orchestrating programmed fibroblast differentiation and fibrosis in vivo is unknown. METHODS: A conditional Mapk14 allele was used to delete the p38α encoding gene specifically in cardiac fibroblasts or myofibroblasts with 2 different tamoxifen-inducible Cre recombinase-expressing gene-targeted mouse lines. Mice were subjected to ischemic injury or chronic neurohumoral stimulation and monitored for survival, cardiac function, and fibrotic remodeling. Antithetically, mice with fibroblast-specific transgenic overexpression of activated mitogen-activated protein kinase kinase 6, a direct inducer of p38, were generated to investigate whether this pathway can directly drive myofibroblast formation and the cardiac fibrotic response. RESULTS: In mice, loss of Mapk14 blocked cardiac fibroblast differentiation into myofibroblasts and ensuing fibrosis in response to ischemic injury or chronic neurohumoral stimulation. A similar inhibition of myofibroblast formation and healing was also observed in a dermal wounding model with deletion of Mapk14. Transgenic mice with fibroblast-specific activation of mitogen-activated protein kinase kinase 6-p38 developed interstitial and perivascular fibrosis in the heart, lung, and kidney as a result of enhanced myofibroblast numbers. Mechanistic experiments show that p38 transduces cytokine and mechanical signals into myofibroblast differentiation through the transcription factor serum response factor and the signaling effector calcineurin. CONCLUSIONS: These findings suggest that signals from diverse modes of injury converge on p38α mitogen-activated protein kinase within the fibroblast to program the fibrotic response and myofibroblast formation in vivo, suggesting a novel therapeutic approach with p38 inhibitors for future clinical application.

Regulator of G-protein signaling-5 is a marker of hepatic stellate cells and expression mediates response to liver injury.

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4)-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.

Regulator of G-protein signaling - 5 (RGS5) is a novel repressor of hedgehog signaling.

Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.

PDGF-dependent regulation of regulator of G protein signaling-5 expression and vascular smooth muscle cell functionality.

Regulator of G protein signaling (RGS) proteins, and notably members of the RGS-R4 subfamily, control vasocontractility by accelerating the inactivation of Gα-dependent signaling. RGS5 is the most highly and differently expressed RGS-R4 subfamily member in arterial smooth muscle. Expression of RGS5 first appears in pericytes during development of the afferent vascular tree, suggesting that RGS5 is a good candidate for a regulator of arterial contractility and, perhaps, for determining the mass of the smooth muscle coats required to regulate blood flow in the branches of the arterial tree. Consistent with this hypothesis, using cultured vascular smooth muscle cells (VSMCs), we demonstrate RGS5 overexpression inhibits G protein-coupled receptor (GPCR)-mediated hypertrophic responses. The next objective was to determine which physiological agonists directly control RGS5 expression in VSMCs. GPCR agonists failed to directly regulate RGS5 mRNA expression; however, platelet-derived growth factor (PDGF) acutely represses expression. Downregulation of RGS5 results in the induction of migration and the activation of the GPCR-mediated signaling pathways. This stimulation leads to the activation of mitogen-activated protein kinases directly downstream of receptor stimulation, and ultimately VSMC hypertrophy. These results demonstrate that RGS5 expression is a critical mediator of both VSMC contraction and potentially, arterial remodeling.

RGS5, RGS4, and RGS2 expression and aortic contractibility are dynamically co-regulated during aortic banding-induced hypertrophy.

Overexpression of regulator of G protein signaling 5 (RGS5) in arteries over veins is the most striking difference observed using microarray analysis. The obvious question is what arterial function might require RGS5. Based on functions of homologous proteins in regulating cardiac mass and G-protein-coupled receptor (GPCR) signaling, we proposed that RGS5 and vascular expressed RGS2 and RGS4 could participate in regulating arterial hypertrophy. We used the suprarenal abdominal aorta banding model to induce hypertension and hypertrophy. All 3 RGS messages were expressed in unmanipulated aorta with RGS5 predominating. After 2 days, thoracic aorta lost expression of RGS5, 4, and 2. At 1 week, all three returned to normal, and at 28 days, they increased many fold above normal. Valsartan blockade of angiotensin II (angII)/angII type 1 receptor signaling prevented upregulation of RGS messages but only delayed mass increases, implying wall mass regulation involves both angII-dependent and angII-independent pathways. The abdominal aorta showed less dramatic expression changes in RGS5 and 4, but not 2. Again, those changes were delayed by valsartan treatment with no mass changes. Thoracic aorta contraction to GPCR agonists was examined in aortic explant rings to identify vessel wall physiological changes. In 2-day aorta, the response to Galphaq/i agonists increased above normal, while 28-day aorta had attenuated induced contraction via Galphaq/i agonist, implicating a connection between RGS message levels and changes in GPCR-induced contraction. In vitro overexpression studies showed RGS5 inhibits angII-induced signaling in smooth muscle cells. This study is the first experimental evidence that changes in RGS expression and function correlate with vascular remodeling.