G Protein-Biased Agonists for Intracellular Angiotensin Receptors Promote Collagen Secretion in Myofibroblasts.

Photoactivatable ligands remain valuable tools to study the spatiotemporal aspects of cellular signaling. However, the synthesis, handling, and biological validation of such compounds remain challenging, especially when dealing with peptides. We report an optimized synthetic strategy, where laborious preparation of dimethoxy-nitrobenzyl-tyrosine building blocks was replaced by direct functionalization of amino acid side chains while peptides remained coupled to resin, reducing both preparation time and cost. Our caged peptides were designed to investigate cellular responses mediated by intracellular angiotensin II receptors (iATR) upon interaction with known biased and unbiased ligands. The pathophysiological roles of iATRs remain poorly understood, and we sought to develop ligands to explore this. Initial validation showed that our caged ligands undergo rapid photolysis and produced functionally active peptides upon UV exposure. We also show, for the first time, that different biased ligands (ß-arrestin- vs G protein-biased analogues) evoked distinct responses when uncaged in adult rat myofibroblasts. Intracellularly targeted versions of Ang II (unbiased) or G protein-biased analogues (TRV055, TRV056) were more effective than ß-arrestin-biased Ang II analogues (SI, TRV026, and TRV27) in inducing collagen secretion, suggesting a divergent role in regulating the fibrotic response.

The complicated lives of GPCRs in cardiac fibroblasts.

The role of different G protein-coupled receptors (GPCRs) in the cardiovascular system is well understood in cardiomyocytes and vascular smooth muscle cells (VSMCs). In the former, stimulation of Gs-coupled receptors leads to increases in contractility, whereas stimulation of Gq-coupled receptors modulates cellular survival and hypertrophic responses. In VSMCs, stimulation of GPCRs also modulates contractile and cell growth phenotypes. Here, we will focus on the relatively less well-studied effects of GPCRs in cardiac fibroblasts, focusing on key signaling events involved in the activation and differentiation of these cells. We also review the hierarchy of signaling events driving the fibrotic response and the communications between fibroblasts and other cells in the heart. We discuss how such events may be distinct depending on where the GPCRs and their associated signaling machinery are localized in these cells with an emphasis on nuclear membrane-localized receptors. Finally, we explore what such connections between the cell surface and nuclear GPCR signaling might mean for cardiac fibrosis.

Implications of enigmatic transglutaminase 2 (TG2) in cardiac diseases and therapeutic developments.

Cardiac diseases are the leading cause of mortality and morbidity worldwide. Mounting evidence suggests that transglutaminases (TGs), tissue TG (TG2) in particular, are involved in numerous molecular responses underlying the pathogenesis of cardiac diseases. The TG family has several intra- and extracellular functions in the human body, including collagen cross-linking, angiogenesis, cell growth, differentiation, migration, adhesion as well as survival. TGs are thiol- and calcium-dependent acyl transferases that catalyze the formation of a covalent bond between the γ-carboxamide group of a glutamine residue and an amine group, thus increasing the stability, rigidity, and stiffness of the myocardial extracellular matrix (ECM). Excessive accumulation of cross-linked collagen leads to increase myocardial stiffness and fibrosis. Beyond TG2 extracellular protein cross-linking action, increasing evidence suggests that this pleiotropic TG isozyme may also promote fibrotic diseases through cell survival and profibrotic pathway activation at the signaling, transcriptional and translational levels. Due to its multiple functions and localizations, TG2 fulfils critical yet incompletely understood roles in myocardial fibrosis and associated heart diseases, such as cardiac hypertrophy, heart failure, and age-related myocardial stiffness under several conditions. This review summarizes current knowledge and existing gaps regarding the ECM-dependent and ECM-independent roles of TG2 and highlights the therapeutic prospects of targeting TG2 to treat cardiac diseases.

Protein tyrosine phosphatase 1B regulates miR-208b-argonaute 2 association and thyroid hormone responsiveness in cardiac hypertrophy.

Increased production of reactive oxygen species plays an essential role in the pathogenesis of several diseases, including cardiac hypertrophy. In our search to identify redox-sensitive targets that contribute to redox signaling, we found that protein tyrosine phosphatase 1B (PTP1B) was reversibly oxidized and inactivated in hearts undergoing hypertrophy. Cardiomyocyte-specific deletion of PTP1B in mice (PTP1B cKO mice) caused a hypertrophic phenotype that was exacerbated by pressure overload. Furthermore, we showed that argonaute 2 (AGO2), a key component of the RNA-induced silencing complex, was a substrate of PTP1B in cardiomyocytes and in the heart. Our results revealed that phosphorylation at Tyr393 and inactivation of AGO2 in PTP1B cKO mice prevented miR-208b-mediated repression of thyroid hormone receptor-associated protein 1 (THRAP1; also known as MED13) and contributed to thyroid hormone-mediated cardiac hypertrophy. In support of this conclusion, inhibiting the synthesis of triiodothyronine (T3) with propylthiouracil rescued pressure overload-induced hypertrophy and improved myocardial contractility and systolic function in PTP1B cKO mice. Together, our data illustrate that PTP1B activity is cardioprotective and that redox signaling is linked to thyroid hormone responsiveness and microRNA-mediated gene silencing in pathological hypertrophy.

Isolation and culture of adult murine cardiac atrial and ventricular fibroblasts and myofibroblasts.

Cardiac fibroblasts play a critical role in extracellular matrix homeostasis, wound healing, and cardiac interstitial fibrosis: the latter being a pathophysiological response to a chronic increase in afterload. Using a standard protocol to isolate cardiac fibroblasts and maintain them in their quiescent phenotype in vitro will enable a better understanding of cardiac fibroblast biology and their role in the response to profibrotic stimuli. Here, we describe an enzymatic method for isolating cardiac fibroblasts. The resulting cells are maintained on either a collagen-coated hydrogel-bound polystyrene (compliant) substrate or standard polystyrene culture dishes (non-compliant) to obtain quiescent fibroblasts and activated fibroblasts (myofibroblasts), respectively. Fibroblasts maintained on a non-compliant substrate developed a myofibroblast phenotype, in which the αSMA immunoreactivity was markedly elevated and incorporated into the stress fibers. In contrast, ventricular and atrial fibroblasts retain their quiescent phenotype for up to 3 passages when maintained on a compliant substrate. Hence, the methodology described herein provides a simple and reproducible way to isolate adult murine atrial and ventricular cardiac fibroblasts from a single animal and, by selecting a substrate with the appropriate compliance, examine the mediators of fibroblast activation or inactivation.

The brain-derived neurotrophic factor prompts platelet aggregation and secretion.

Brain-derived neurotrophic factor (BDNF) has both autocrine and paracrine roles in neurons, and its release and signaling mechanisms have been extensively studied in the central nervous system. Large quantities of BDNF have been reported in circulation, essentially stored in platelets with concentrations reaching 100- to 1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. At low concentrations, BDNF primed platelets, acting synergistically with classical agonists. At high concentrations, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. Neurotrophin-4, but not nerve growth factor, and an activating antibody against the canonical BDNF receptor tropomyosin-related kinase B (TrkB) induced similar platelet responses to BDNF, suggesting TrkB could be the mediator. Platelets expressed, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking its tyrosine kinase domain. BDNF-induced platelet aggregation was prevented by inhibitors of Ras-related C3 botulinum toxin substrate 1 (Rac1), protein kinase C, and phosphoinositide 3-kinase. BDNF-stimulated platelets secreted a panel of angiogenic and inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Two families with autism spectrum disorder were found to carry rare missense variants in the BDNF gene. Platelet studies revealed defects in platelet aggregation to low concentrations of collagen, as well as reduced adenosine triphosphate secretion in response to adenosine diphosphate. In summary, circulating BDNF levels appear to regulate platelet activation, aggregation, and secretion through activation of a truncated TrkB receptor and downstream kinase-dependent signaling.

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.

MK2-Deficient Mice Are Bradycardic and Display Delayed Hypertrophic Remodeling in Response to a Chronic Increase in Afterload.

Background Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is a protein serine/threonine kinase activated by p38α/ß. Herein, we examine the cardiac phenotype of pan MK2-null (MK2-/-) mice. Methods and Results Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P=0.580). At 12 weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, aging was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3, were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+ sensitivity to mitochondrial permeability transition pore opening. Eight weeks of pressure overload increased left ventricular mass in MK2+/+ and MK2-/- mice; however, after 2 weeks the increase was significant in MK2+/+ but not MK2-/- mice. Finally, the pressure overload-induced decrease in systolic function was attenuated in MK2-/- mice 2 weeks, but not 8 weeks, after constriction of the transverse aorta. Conclusions Collectively, these results implicate MK2 in (1) autonomic regulation of heart rate, (2) cardiac mitochondrial function, and (3) the early stages of myocardial remodeling in response to chronic pressure overload.

Filamentous nestin and nonmuscle myosin IIB are associated with a migratory phenotype in neonatal rat cardiomyocytes.

Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13-dibutyrate (PDBu) and the p38α/ß mitogen-activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild-type and heterozygous NMIIB embryonic hearts at E11.5-E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes.

Current knowledge into the role of the peptidylarginine deiminase (PAD) enzyme family in cardiovascular disease.

Peptidylarginine deiminase (PAD) family members have a vital role in maintaining the stability of the extracellular matrix (ECM) during remodelling in several heart diseases. PAD-mediated deamination, or citrullination, has been studied in different physiological and pathological conditions in the body. However, the role of PAD isoforms has not been fully studied in cardiovascular system. Citrullination is a post-translational modification that involves conversion of peptidyl-based arginine to peptidyl-based citrulline by PAD family members in a calcium-dependent manner. Upregulation of PADs have been observed in various cardiovascular diseases, including venous thrombosis, cardiac fibrosis, heart failure, atherosclerosis, coronary heart disease and acute inflammation. In this review, experimental aspects of in vivo and in vitro studies related to the roles PAD isoforms in cardiovascular diseases including mechanisms, pathophysiological and therapeutic properties are discussed. Pharmacological strategies for targeting PAD family proteins in cardiac diseases have not yet been studied. Furthermore, the role played by PAD family members in the remodelling process during the progression of cardiovascular diseases is not fully understood.

A putative protein-RNA complex regulates posttranscriptional processing of cytochrome P450 aromatase (CYP19A1) in bovine granulosa cells.

Follicle growth and granulosa cell health are dependent on the secretion of estradiol from granulosa cells. Estradiol is synthesized from androgen precursor by cytochrome P450 aromatase (CYP19A1), and in cattle CYP19A1 messenger RNA has a short half-life but a long (3.5 kb) 3'-untranslated region (3'UTR), suggesting that posttranscriptional regulation may be important for control of enzyme activity. We tested this hypothesis by inserting the CYP19A1 3'UTR and fragments thereof into a reporter vector between the end of the luciferase coding region and the polyadenylation signal. The full-length aromatase 3'UTR suppressed luciferase activity to 10% of control levels, and smaller fragments showed that this inhibitory activity lies between +926 and +1134 of the 3'UTR. Protein-RNA cross-linking experiments revealed that these 3'UTR fragments formed an RNA-protein complex of approximately 70 kDa that was present in granulosa cells but not in corpus luteum, lung, liver, kidney, pancreas, or bladder extracts. The RNA-binding activity was specific to the 3'UTR, as shown by competition experiments with unlabeled RNA, and was present only in 3'UTR constructs that inhibited luciferase activity. These data suggest that posttranscriptional regulation is an important component of the control of CYP19A1 expression and involves protein binding to a specific sequence in the 3'UTR.

Role of the lysyl oxidase enzyme family in cardiac function and disease.

Heart diseases are a major cause of morbidity and mortality world-wide. Lysyl oxidase (LOX) and related LOX-like (LOXL) isoforms play a vital role in remodelling the extracellular matrix (ECM). The LOX family controls ECM formation by cross-linking collagen and elastin chains. LOX/LOXL proteins are copper-dependent amine oxidases that catalyse the oxidation of lysine, causing cross-linking between the lysine moieties of lysine-rich proteins. Dynamic changes in LOX and LOXL protein-expression occur in a variety of cardiac pathologies; these changes are believed to be central to the associated tissue-fibrosis. An awareness of the potential pathophysiological importance of LOX has led to the evaluation of interventions that target LOX/LOXL proteins for heart-disease therapy. The purposes of this review article are: (i) to summarize the basic biochemistry and enzyme function of LOX and LOXL proteins; (ii) to consider their tissue and species distribution; and (iii) to review the results of experimental studies of the roles of LOX and LOXL proteins in heart disease, addressing involvement in the mechanisms, pathophysiology and therapeutic responses based on observations in patient samples and relevant animal models. Therapeutic targeting of LOX family enzymes has shown promising results in animal models, but small-molecule approaches have been limited by non-specificity and off-target effects. Biological approaches show potential promise but are in their infancy. While there is strong evidence for LOX-family protein participation in heart failure, myocardial infarction, cardiac hypertrophy, dilated cardiomyopathy, atrial fibrillation and hypertension, as well as potential interest as therapeutic targets, the precise involvement of LOX-family proteins in heart disease requires further investigation.

Transcript levels for extracellular matrix proteins are altered in MK5-deficient cardiac ventricular fibroblasts.

MK5 is a protein serine/threonine kinase activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. Although little is known of the physiological role of MK5 in the heart, both hypertrophic growth and the increase in collagen 1-α1 mRNA induced by increased afterload are attenuated in hearts of MK5 haploinsufficient (MK5+/-) mice. MK5 transcripts are detected at high levels in the left ventricular myocardium; however, MK5 immunoreactivity is detected in adult cardiac fibroblasts, but not myocytes. The present study was to determine if MK5 has a potential role in remodeling of the extracellular matrix. Ventricular fibroblasts were isolated from MK5+/+, MK5+/-, or MK5-/- mice and maintained in culture on either compliant (8 kPa) or rigid substrates to obtain quiescent fibroblasts or activated myofibroblasts, respectively. In quiescent fibroblasts, reduced MK5 had little effect: BMP7 and TGF-ß1 mRNA was increased in MK5+/- and MK5-/-.cells, respectively. Ang-II altered the abundance of numerous transcripts in an MK5-sensitive manner. Both collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity were increased by Ang-II in wild type but not MK5-deficient fibroblasts. The effects of deleting MK5 were quite different in myofibroblasts: both the abundance of collagen 1-α1 mRNA and secreted type 1 collagen immunoreactivity elevated in the absence of added Ang-II and addition of Ang-II failed to evoke a further increase in either. In addition, whereas type I collagen immunoreactivity was distributed throughout the cytosol of wild-type myofibroblasts, it was perinuclear in MK5-/- myofibroblasts. Furthermore, in MK5-deficient myofibroblasts the abundance of collagen 3-α2, Timp3, Smad 6, Smad 7, TGF-ß3, and snail homolog 1 transcripts was increased whereas integrin ß3, latent TGF-ß binding protein 1, thrombospondin 1, hepatocyte growth factor, and interleukin 13 were decreased. Finally, fibroblast contraction was decreased upon knocking down MK5. These results indicate that MK5 may be involved in fibroblast-mediated regulation of extracellular matrix homeostasis.

MK5 haplodeficiency decreases collagen deposition and scar size during post-myocardial infarction wound repair.

MK5 is a protein serine/threonine kinase activated by p38, ERK3, and ERK4 MAPKs. MK5 mRNA and immunoreactivity are detected in mouse cardiac fibroblasts, and MK5 haplodeficiency attenuates the increase in collagen 1-α1 mRNA evoked by pressure overload. The present study examined the effect of MK5 haplodeficiency on reparative fibrosis following myocardial infarction (MI). Twelve-week-old MK5+/- and wild-type littermate (MK5+/+) mice underwent ligation of the left anterior descending coronary artery (LADL). Surviving mice were euthanized 8 or 21 days post-MI. Survival rates did not differ significantly between MK5+/+ and MK5+/- mice, with rupture of the LV wall being the primary cause of death. Echocardiographic imaging revealed similar increases in LV end-diastolic diameter, myocardial performance index, and wall motion score index in LADL-MK5+/+ and LADL-MK5+/- mice. Area at risk did not differ between LADL-MK5+/+ and LADL-MK5+/- hearts. In contrast, infarct size, scar area, and scar collagen content were reduced in LADL-MK5+/- hearts. Immunohistochemical analysis of mice experiencing heart rupture revealed increased MMP-9 immunoreactivity in the infarct border zone of LADL-MK5+/- hearts compared with LADL-MK5+/+. Although inflammatory cell infiltration was similar in LADL-MK5+/+ and LADL-MK5+/- hearts, angiogenesis was more pronounced in the infarct border zone of LADL-MK5+/- mice. Characterization of ventricular fibroblasts revealed reduced motility and proliferation in fibroblasts isolated from MK5-/- mice compared with those from both wild-type and haplodeficient mice. siRNA-mediated knockdown of MK5 in fibroblasts from wild-type mice also impaired motility. Hence, reduced MK5 expression alters fibroblast function and scar morphology but not mortality post-MI. NEW & NOTEWORTHY MK5/PRAK is a protein serine/threonine kinase activated by p38 MAPK and/or atypical MAPKs ERK3/4. MK5 haplodeficiency reduced infarct size, scar area, and scar collagen content post-myocardial infarction. Motility and proliferation were reduced in cultured MK5-null cardiac myofibroblasts.

Subclinical Inflammation in Heart Failure: A Neutrophil Perspective.

Although it is widely recognized that inflammation plays a critical role in the development and pathology of heart failure (HF), very little is known about the involvement of one of the most abundant immune cells in the blood, a primary immune response cell: the neutrophil. This review summarizes the current literature on the role of subclinical inflammation, with a focus on the neutrophil in the pathophysiology of the HF syndrome. Some emerging therapeutic strategies are also discussed.

Nucleoligands-repurposing G Protein-coupled Receptor Ligands to Modulate Nuclear-localized G Protein-coupled Receptors in the Cardiovascular System.

There is significant evidence that internal pools of G protein-coupled receptors (GPCRs) exist and may be affected by both endogenous signaling molecules and hydrophobic pharmaceutical ligands, once assumed to only affect cell surface versions of these receptors. Here, we discuss evidence that the biology of nuclear GPCRs in particular is complex, rich, and highly interactive with GPCR signaling from the cell surface. Caging existing GPCR ligands may be an excellent means of further stratifying the phenotypic effects of known pharmacophores such as ß-adrenergic, angiotensin II, and type B endothelin receptor ligands in the cardiovascular system. We describe some synthetic strategies we have used to design ligands to go from in cellulo to in vivo experiments. We also consider how surface and intracellular GPCR signaling might be integrated and ways to dissect this. If they could be selectively targeted, nuclear GPCRs and their associated nucleoligands would represent a completely novel area for exploration by Pharma.

5-Azacytidine engages an IRE1α-EGFR-ERK1/2 signaling pathway that stabilizes the LDL receptor mRNA.

Hepatic low-density lipoprotein receptor (LDLR) is the primary conduit for the clearance of plasma LDL-cholesterol and increasing its expression represents a central goal for treating cardiovascular disease. However, LDLR mRNA is unstable and undergoes rapid turnover mainly due to the three AU-rich elements (ARE) in its proximal 3'-untranslated region (3'-UTR). Herein, our data revealed that 5-azacytidine (5-AzaC), an antimetabolite used in the treatment of myelodysplastic syndrome, stabilizes the LDLR mRNA through a previously unrecognized signaling pathway resulting in a strong increase of its protein level in human hepatocytes in culture. 5-AzaC caused a sustained activation of the inositol-requiring enzyme 1α (IRE1α) kinase domain and c-Jun N-terminal kinase (JNK) independently of endoplasmic reticulum stress. This resulted in activation of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase1/2 (ERK1/2) that, in turn, stabilized LDLR mRNA. Systematic mutation of the AREs (ARE1-3) in the LDLR 3'UTR and expression of each mutant coupled to a luciferase reporter in Huh7 cells demonstrated that ARE1 is required for rapid LDLR mRNA decay and 5-AzaC-induced mRNA stabilization via the IRE1α-EGFR-ERK1/2 signaling cascade. The characterization of this pathway will help to reveal potential targets to enhance plasma LDL clearance and novel cholesterol-lowering therapeutic strategies.

MK5: A novel regulator of cardiac fibroblast function?

MAP kinase-activated protein kinases (MKs), protein serine/threonine kinases downstream of the MAPKs, regulate a number of biological functions. MK5 was initially identified as a substrate for p38 MAPK but subsequent studies revealed that MK5 activity is regulated by atypical MAPKs ERK3 and ERK4. However, the roles of these MAPKs in activating MK5 remain controversial. The interactome and physiological function of MK5 are just beginning to be understood. Here, we provide an overview of the structure-function of MK5 including recent progress in determining its role in cardiac structure and function. The cardiac phenotype of MK5 haplodeficient mice, and the effect of reduced MK5 expression on cardiac remodeling, is also discussed. © 2017 IUBMB Life, 69(10):785-794, 2017.

Proteolytic cleavage of the hydrophobic domain in the CaVα2δ1 subunit improves assembly and activity of cardiac CaV1.2 channels.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVß, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.