Johanna Magga

Increasing knowledge of the components involved in left ventricle (LV) remodelling and fibrotic processes after a myocardial infarction is crucial to understanding heart pathology. We have here analysed collagen XV (ColXV) expression in human myocardial infarct samples and assessed how its deficiency affects cardiac responses, such as fibrogenesis and tissue stiffness, after acute myocardial infarction (AMI) in mice. We first observed high ColXV expression in human infarction scars. After ligating the left anterior descending artery in mice, cardiac function and remodelling were monitored by echocardiography, elasticity assessment, immunohistochemical analysis and ultrastructural assessments. After AMI, Col15a1 ^−/− mice showed significantly increased tissue stiffness and upregulation of fibrosis‐related genes in the remote myocardium. Striking differences were observed between the genotypes in the scar ultrastructure, protein compositions, cardiomyocyte morphology and intracellular architecture. Furthermore, the proportion of immature collagen fibres in the infarct border zone increased in Col15a1 ^−/− mice, suggesting fragility and poor scar resistance to mechanical stress. Structural parameters indicated more substantial LV remodelling in the knockout mice, leading to a more dilated ventricle. Functionally, the ejection fraction and fractional shortening decreased significantly in Col15a1 ^−/− mice, indicating impaired heart contractile capacity. The results show that in the event of an AMI, ColXV plays an essential role in sustaining cardiac structure and function. In the absence of ColXV, dysregulated remodelling results in disrupted scar and infarct border zone, and stiffer left ventricle. These changes lead to a more severe cardiac phenotype and may affect long‐term survival after AMI.

The mRNA export factor GLE1 protein plays critical yet enigmatic functions in RNA processing and has been linked with multiple developmental disorders, including lethal congenital contracture syndrome 1 (LCCS1). Using in vivo genetic engineering to study disturbed GLE1 functions under physiological conditions, we demonstrate that total inactivation of GLE1 results in disorganization of the blastocyst inner cell mass and early embryonic lethality due to defects in lineage specification. In contrast, the knock‐in mice genocopying the LCCS1‐associated GLE1 _FinMajor variant ( Gle1 ^PFQ/PFQ ) survive the prenatal period but die suddenly at midadulthood. Gle1 ^PFQ/PFQ mice present an irregular count and distribution of spinal motor neurons as well as impaired development of neural crest‐derived tissues, as demonstrated by defects in the sympathetic innervation of heart ventricles, irregularities in the paravertebral sympathetic ganglia volume, and decreased adrenal chromaffin cell counts. Unlike previously reported for yeast and HeLa cells, analysis of the molecular consequences of the GLE1 _FinMajor variant identified normal poly(A) + RNA distribution in Gle1 ^PFQ/PFQ cells; however, cells were impaired in RNA and protein synthesis and simultaneously showed severely disturbed formation of G3BP stress granule assembly factor 1 (G3BP1)‐positive stress granules. Intriguingly, stressed Gle1 ^PFQ/PFQ cells show microRNA profiles indicative of impaired transcription, protein metabolism, nervous system development, and axon guidance, further corroborating our functional findings. Our results show the necessity of functional GLE1 for life and indicate that LCCS1 etiology is a result of the pathogenic GLE1 _FinMajor variant impinging differentiation of neural crest derivatives and leading to complex multiorgan defects.

N‐terminal nonsynonymous single‐nucleotide polymorphisms (SNPs) of G protein‐coupled receptors (GPCRs) are common and often affect receptor post‐translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human β₁‐adrenergic receptor (β₁AR) is O‐glycosylated in the N‐terminal extracellular domain by polypeptide GalNAc transferase‐2 that co‐regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O‐glycosylation assays, analysis of native receptor N‐terminal O‐glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O‐glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O‐glycosites that enhanced or inhibited cleavage at the adjacent sites (P⁵²↓L⁵³ and R³¹↓L³²), respectively, or abolished the major site at R³¹↓L³² (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full‐length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol‐mediated signaling in an enhanced bystander bioluminescence energy transfer β‐arrestin2 recruitment assay in a coordinated manner with the common C‐terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a β₁AR gain‐of‐function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.

Prior studies show that glycogen synthase kinase 3β (GSK3β) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3β is constitutionally active and phosphorylation of GSK3β at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3β is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3β S389 phosphorylation in diseased hearts and utilized overexpression of GSK3β carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3β in primary cardiomyocytes. We found that phosphorylation of GSK3β at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3β S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia–reoxygenation. Overexpression of double GSK3β mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3β S389A or GSK3β S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3β S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3β at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy.

Signaling through activin receptors regulates skeletal muscle mass and activin receptor 2B (ACVR2B) ligands are also suggested to participate in myocardial infarction (MI) pathology in the heart. In this study, we determined the effect of systemic blockade of ACVR2B ligands on cardiac function in experimental MI, and defined its efficacy to revert muscle wasting in ischemic heart failure (HF). Mice were treated with soluble ACVR2B decoy receptor (ACVR2B‐Fc) to study its effect on post‐MI cardiac remodeling and on later HF. Cardiac function was determined with echocardiography, and myocardium analyzed with histological and biochemical methods for hypertrophy and fibrosis. Pharmacological blockade of ACVR2B ligands did not rescue the heart from ischemic injury or alleviate post‐MI remodeling and ischemic HF. Collectively, ACVR2B‐Fc did not affect cardiomyocyte hypertrophy, fibrosis, angiogenesis, nor factors associated with cardiac regeneration except modification of certain genes involved in metabolism or cell growth/survival. ACVR2B‐Fc, however, was able to reduce skeletal muscle wasting in chronic ischemic HF, accompanied by reduced LC3II as a marker of autophagy and increased mTOR signaling and Cited4 expression as markers of physiological hypertrophy in quadriceps muscle. Our results ascertain pharmacological blockade of ACVR2B ligands as a possible therapy for skeletal muscle wasting in ischemic HF. Pharmacological blockade of ACVR2B ligands preserved myofiber size in ischemic HF, but did not compromise cardiac function nor exacerbate cardiac remodeling after ischemic injury.

Sprouty1 (Spry1) is a negative modulator of receptor tyrosine kinase signaling, but its role in cardiomyocyte survival has not been elucidated. The aim of this study was to investigate the potential role of cardiomyocyte Spry1 in cardiac ischemia–reperfusion (I/R) injury. Infarct areas of mouse hearts showed an increase in Spry1 protein expression, which localized to cardiomyocytes. To investigate if cardiomyocyte Spry1 regulates I/R injury, 8-week-old inducible cardiomyocyte Spry1 knockout (Spry1 cKO) mice and control mice were subjected to cardiac I/R injury. Spry1 cKO mice showed reduction in release of cardiac troponin I and reduced infarct size after I/R injury compared to control mice. Similar to Spry1 knockdown in cardiomyocytes in vivo, RNAi-mediated Spry1 silencing in isolated cardiomyocytes improved cardiomyocyte survival following simulated ischemia injury. Mechanistically, Spry1 knockdown induced cardiomyocyte extracellular signal-regulated kinase (ERK) phosphorylation in healthy hearts and isolated cardiomyocytes, and enhanced ERK phosphorylation after I/R injury. Spry1-deficient cardiomyocytes showed better preserved mitochondrial membrane potential following ischemic injury and an increase in levels of phosphorylated ERK and phosphorylated glycogen synthase kinase-3β (GSK-3β) in mitochondria of hypoxic cardiomyocytes. Overexpression of constitutively active GSK-3β abrogated the protective effect of Spry1 knockdown. Moreover, pharmacological inhibition of GSK-3β protected wild-type cardiomyocytes from cell death, but did not further protect Spry1-silenced cardiomyocytes from hypoxia-induced injury. Cardiomyocyte Spry1 knockdown promotes ERK phosphorylation and offers protection from I/R injury. Our findings indicate that Spry1 is an important regulator of cardiomyocyte viability during ischemia–reperfusion injury.