Multi-Omics Approach to Dissect the Mechanisms of Rexinoid Signaling in Myoblast Differentiation

Stem cells represent a key resource in regenerative medicine, however, there is a critical need for pharmacological modulators to promote efficient conversion of stem cells into a myogenic lineage. We have previously shown that bexarotene, an agonist of retinoid X receptor (RXR) approved for cancer therapy, promotes the specification and differentiation of skeletal muscle progenitors. To decipher the molecular regulation of rexinoid signaling in myogenic differentiation, we have integrated RNA-seq transcription profiles with ChIP-seq of H4K8, H3K9, H3K18, H3K27 acetylation, and H3K27 methylation in addition to that of histone acetyl-transferase p300 in rexinoid-promoted myoblast differentiation. Here, we provide details regarding data collection, validation and omics integration analyses to offer strategies for future data application and replication. Our analyses also reveal molecular pathways underlying different patterns of gene expression and p300-associated histone acetylation at distinct chromatin states in rexinoid-enhanced myoblast differentiation. These datasets can be repurposed for future studies to examine the relationship between signaling molecules, chromatin modifiers and histone acetylation in myogenic regulation, providing a framework for discovery and functional characterization of muscle-specific loci.

Abnormal myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization contribute to impaired myogenic regulation of cerebral arterial diameter in the type 2 diabetic Goto-Kakizaki rat

The myogenic response of cerebral resistance arterial smooth muscle to intraluminal pressure elevation is a key physiological mechanism regulating blood flow to the brain. Rho-associated kinase plays a critical role in the myogenic response by activating Ca2+ sensitization mechanisms: (i) Rho-associated kinase inhibits myosin light chain phosphatase by phosphorylating its targeting subunit myosin phosphatase targeting subunit 1 (at T855), augmenting 20 kDa myosin regulatory light chain (LC20) phosphorylation and force generation; and (ii) Rho-associated kinase stimulates cytoskeletal actin polymerization, enhancing force transmission to the cell membrane. Here, we tested the hypothesis that abnormal Rho-associated kinase-mediated myosin light chain phosphatase regulation underlies the dysfunctional cerebral myogenic response of the Goto-Kakizaki rat model of type 2 diabetes. Basal levels of myogenic tone, LC20, and MYPT1-T855 phosphorylation were elevated and G-actin content was reduced in arteries of pre-diabetic 8–10 weeks Goto-Kakizaki rats with normal serum insulin and glucose levels. Pressure-dependent myogenic constriction, LC20, and myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization were suppressed in both pre-diabetic Goto-Kakizaki and diabetic (18–20 weeks) Goto-Kakizaki rats, whereas RhoA, ROK2, and MYPT1 expression were unaffected. We conclude that abnormal Rho-associated kinase-mediated Ca2+ sensitization contributes to the dysfunctional cerebral myogenic response in the Goto-Kakizaki model of type 2 diabetes.

Abnormal Rho-associated kinase activity contributes to the dysfunctional myogenic response of cerebral arteries in type 2 diabetes

The structural and functional integrity of the brain, and therefore, cognition, are critically dependent on the appropriate control of blood flow within the cerebral circulation. Inadequate flow leads to ischemia, whereas excessive flow causes small vessel rupture and (or) blood–brain-barrier disruption. Cerebral blood flow is controlled through the interplay of several physiological mechanisms that regulate the contractile state of vascular smooth muscle cells (VSMCs) within the walls of cerebral resistance arteries and arterioles. The myogenic response of cerebral VSMCs is a key mechanism that is responsible for maintaining constant blood flow during variations in systemic pressure, i.e., flow autoregulation. Inappropriate myogenic control of cerebral blood flow is associated with, and prognostic of, neurological deterioration and poor outcome in patients with several conditions, including type 2 diabetes. Here, we review recent advances in our understanding of the role of inappropriate Rho-associated kinase activity as a cause of impaired myogenic regulation of cerebral arterial diameter in type 2 diabetes.

Altered myogenic vasoconstriction and regulation of whole kidney blood flow in the ASIC2 knockout mouse

Previous studies from our laboratory have suggested that degenerin proteins contribute to myogenic constriction, a mechanism of blood flow regulation and protection against pressure-dependent organ injury, in renal vessels. The goal of the present study was to determine the importance of one family member, acid-sensing ion channel 2 (ASIC2), in myogenic constriction of renal interlobar arteries, myogenic regulation of whole kidney blood flow, renal injury, and blood pressure using ASIC2+/+, ASIC2+/−, and ASIC2−/− mice. Myogenic constriction in renal interlobar arteries was impaired in ASIC2+/− and ASIC2−/− mice, whereas constriction to KCl/phenylephrine was unchanged. Correction of whole kidney renal vascular resistance (RVR) during the first 5 s after a 10- to 20-mmHg step increase in perfusion pressure, a timeframe associated with myogenic-mediated correction of RVR, was slowed (4.2 ± 0.9, 0.3 ± 0.7, and 2.4 ± 0.3 resistance units/s in ASIC2+/+, ASIC2+/−, and ASIC2−/− mice). Although modest reductions in function were observed in ASIC2−/− mice, greater reductions were observed in ASIC2+/− mice, which may be explained by protein-protein interactions of ASIC2 with other degenerins. Isolated glomeruli from ASIC2+/− and ASIC2−/− mice had modest alterations in the expression of inflammation and injury markers (transforming growth factor-β, mouse anti-target of antiproliferative antibody-1, and nephrin), whereas ASIC2+/− mice had an increase in the remodeling marker collagen type III. Consistent with a more severe loss of function, mean arterial pressure was increased in ASIC2+/− mice (131 ± 3 mmHg) but not in ASIC2−/− mice (122 ± 3 vs. 117 ± 2 mmHg in ASIC2+/+ mice). These results suggest that ASIC2 contributes to transduction of the renal myogenic response and are consistent with the protective role of myogenic constriction against renal injury and hypertension.

TRPM4 Channels Couple Purinergic Receptor Mechanoactivation and Myogenic Tone Development in Cerebral Parenchymal Arterioles

Cerebral parenchymal arterioles (PAs) have a critical role in assuring appropriate blood flow and perfusion pressure within the brain. They are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to chemical stimulants, and enhanced myogenic tone development. The objective of the present study was to reveal some of the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. Here, we report that in vivo suppression of TRPM4 (transient receptor potential) channel expression, or inhibition of TRPM4 channels with 9-phenanthrol substantially reduced myogenic tone of isolated PAs, supporting a key role of TRPM4 channels in PA myogenic tone development. Further, downregulation of TRPM4 channels inhibited vasoconstriction induced by the specific P2Y4 and P2Y6 receptor ligands (UTP γS and UDP) by 37% and 42%, respectively. In addition, 9-phenanthrol substantially attenuated purinergic ligand-induced membrane depolarization and constriction of PAs, and inhibited ligand-evoked TRPM4 channel activation in isolated PA myocytes. In concert with our previous work showing the essential contributions of P2Y4 and P2Y6 receptors to myogenic regulation of PAs, the current results point to TRPM4 channels as an important link between mechanosensitive P2Y receptor activation and myogenic constriction of cerebral PAs.