Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Functional amyloid signaling via the inflammasome, necrosome, and signalosome: New therapeutic targets in heart failure

As the most common cause of death and disability globally, heart disease remains an incompletely understood enigma. A growing number of cardiac diseases are being characterized by the presence of misfolded proteins underlying their pathophysiology, including cardiac amyloidosis and dilated cardiomyopathy (DCM). At least nine precursor proteins have been implicated in the development of cardiac amyloidosis, most commonly caused by multiple myeloma (MM) light chain disease and disease-causing mutant or wildtype transthyretin (TTR). Similarly aggregates with PSEN1 and COFILIN-2 have been identified in up to 1/3 of idiopathic DCM cases studied indicating the potential predominance of misfolded proteins in heart failure. In this review, we present recent evidence linking misfolded proteins mechanistically with heart failure and present multiple lines of new therapeutic approaches that target the prevention of misfolded proteins in cardiac TTR amyloid disease. These include multiple small molecule pharmacological chaperones now in clinical trials designed specifically to support TTR folding by rational design, such as tafamidis, and chaperones previously developed for other purposes, such as doxycycline and tauroursodeoxycholic acid. Lastly, we present newly discovered non-pathological functional amyloid structures, such as the inflammasome and necrosome signaling complexes, which can be activated directly by amyloid. These may represent future targets to successfully attenuate amyloid-induced proteotoxicity in heart failure as the inflammasome, for example, is being therapeutically inhibited experimentally in autoimmune disease. Together, these studies demonstrate multiple novel points in which new therapies may be used to primarily prevent misfolded proteins or to inhibit their downstream amyloid-mediated effectors, such as the inflammasome, to prevent proteotoxicity in heart failure

Transient receptor potential canonical type 6 (TRPC6) O-GlcNAcylation at Threonine-221 plays potent role in channel regulation

Summary: Transient receptor potential canonical type 6 (TRPC6) is a non-voltage-gated channel that principally conducts calcium. Elevated channel activation contributes to fibrosis, hypertrophy, and proteinuria, often coupled to stimulation of nuclear factor of activated T-cells (NFAT). TRPC6 is post-translationally regulated, but a role for O-linked β-N-acetyl glucosamine (O-GlcNAcylation) as elevated by diabetes, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated at Ser14, Thr70, and Thr221 in the N-terminus ankryn-4 (AR4) and linker (LH1) domains. Mutagenesis to alanine reveals T221 as a critical controller of resting TRPC6 conductance, and associated NFAT activity and pro-hypertrophic signaling. T→A mutations at sites homologous in closely related TRPC3 and TRPC7 also increases their activity. Molecular modeling predicts interactions between Thr221-O-GlcNAc and Ser199, Glu200, and Glu246, and combined alanine substitutions of the latter similarly elevates resting NFAT activity. Thus, O-GlcNAcylated T221 and interactions with coordinating residues is required for normal TRPC6 channel conductance and NFAT activation

Abstract 15709: Antagonist of Growth Hormone-Releasing Hormone Receptor Protects Against Cardiopulmonary Injury Induced by rVSV-SARS-CoV-2-S Virus Through Rag2 Regulation

Abstract only Background: SARS-CoV-2 (COVID-19) transmits a multi-systemic disease that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to regulate inflammation in animal models and immune cell responses to bleomycin lung injury. Using a BSL2-compatible recombinant VSV-eGFP-SARS-CoV-2-S virus (rVSV-SARS-CoV-2-S) which mimics native SARS-CoV-2 infection in K18 hACE2tg mice, we tested our hypothesis that MIA-602 attenuates COVID-19-induced cardiopulmonary injury by reducing inflammation. Methods: Male and female K18-hACE2tg mice were infected with SARS-CoV-2/USA-WA1/2020, rVSV-SARS-CoV-2-S, or PBS and lung viral load, weight-loss and histopathology were compared (N=8). Mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle (control) starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed, and blood and tissues collected for histopathological analyses, H&E staining, RNA and protein work. Heart and lung tissues were used for RNASeq (n=3 per group). T-test or One-way ANOVA-test was used for statistical analysis. Results: SARS-CoV-2 and rVSV-SARS-CoV-2-S presented similar pathology for weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung inflammation, pneumonia and pulmonary dysfunction evidenced by rescued respiratory rate, expiratory parameters, and dysregulated airway parameters (p<.05). MIA-602 normalized the high expression of the inflammatory protein ICAM-1 in heart and lung (p<0.01), and master immune modulator Rag2 in lung (RNA:10-FC; Protein: 2-FC; p<.001). Conclusions: The results indicate a possible role for pulmonary Rag2 in protecting against pulmonary dysfunction and heart/lung inflammation by peptide GHRH-R antagonist MIA-602 in a novel animal model of COVID-19 pneumonia

Growth hormone-releasing hormone receptor antagonist MIA-602 attenuates cardiopulmonary injury induced by BSL-2 rVSV-SARS-CoV-2 in hACE2 mice

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia