Metabolic Communication by SGLT2 Inhibition.

BACKGROUND: SGLT2 (sodium-glucose cotransporter 2) inhibitors (SGLT2i) can protect the kidneys and heart, but the underlying mechanism remains poorly understood. METHODS: To gain insights on primary effects of SGLT2i that are not confounded by pathophysiologic processes or are secondary to improvement by SGLT2i, we performed an in-depth proteomics, phosphoproteomics, and metabolomics analysis by integrating signatures from multiple metabolic organs and body fluids after 1 week of SGLT2i treatment of nondiabetic as well as diabetic mice with early and uncomplicated hyperglycemia. RESULTS: Kidneys of nondiabetic mice reacted most strongly to SGLT2i in terms of proteomic reconfiguration, including evidence for less early proximal tubule glucotoxicity and a broad downregulation of the apical uptake transport machinery (including sodium, glucose, urate, purine bases, and amino acids), supported by mouse and human SGLT2 interactome studies. SGLT2i affected heart and liver signaling, but more reactive organs included the white adipose tissue, showing more lipolysis, and, particularly, the gut microbiome, with a lower relative abundance of bacteria taxa capable of fermenting phenylalanine and tryptophan to cardiovascular uremic toxins, resulting in lower plasma levels of these compounds (including p-cresol sulfate). SGLT2i was detectable in murine stool samples and its addition to human stool microbiota fermentation recapitulated some murine microbiome findings, suggesting direct inhibition of fermentation of aromatic amino acids and tryptophan. In mice lacking SGLT2 and in patients with decompensated heart failure or diabetes, the SGLT2i likewise reduced circulating p-cresol sulfate, and p-cresol impaired contractility and rhythm in human induced pluripotent stem cell-derived engineered heart tissue. CONCLUSIONS: SGLT2i reduced microbiome formation of uremic toxins such as p-cresol sulfate and thereby their body exposure and need for renal detoxification, which, combined with direct kidney effects of SGLT2i, including less proximal tubule glucotoxicity and a broad downregulation of apical transporters (including sodium, amino acid, and urate uptake), provides a metabolic foundation for kidney and cardiovascular protection.

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents.

The contraction and relaxation of the heart is controlled by stimulation of the ß1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of ß1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Dicoumarol Inhibits Multidrug Resistance Protein 1-Mediated Export Processes in Cultured Primary Rat Astrocytes.

Dicoumarol is frequently used as inhibitor of the detoxifying enzyme NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1). In order to test whether dicoumarol may also affect the cellular glutathione (GSH) metabolism, we have exposed cultured primary astrocytes to dicoumarol and investigated potential effects of this compound on the cell viability as well as on the cellular and extracellular contents of GSH and its metabolites. Incubation of astrocytes with dicoumarol in concentrations of up to 100 µM did not acutely compromise cell viability nor was any GSH consumption or GSH oxidation to glutathione disulfide (GSSG) observed. However, unexpectedly dicoumarol inhibited the cellular multidrug resistance protein (Mrp) 1-dependent export of GSH in a time- and concentration-dependent manner with half-maximal effects observed at low micromolar concentrations of dicoumarol. Inhibition of GSH export by dicoumarol was not additive to that observed for the known Mrp1 inhibitor MK571. In addition, dicoumarol inhibited also the Mrp1-mediated export of GSSG during menadione-induced oxidative stress and the export of the GSH-bimane-conjugate (GS-B) that had been generated in the cells after exposure to monochlorobimane. Half-maximal inhibition of the export of Mrp1 substrates was observed at dicoumarol concentrations of around 4 µM (GSH and GSSG) and 30 µM (GS-B). These data demonstrate that dicoumarol strongly affects the GSH metabolism of viable cultured astrocytes by inhibiting Mrp1-mediated export processes and identifies for the first time Mrp1 as additional cellular target of dicoumarol.

CMYA5 is a novel interaction partner of FHL2 in cardiac myocytes.

Four-and-a-half LIM domains protein 2 (FHL2) is an anti-hypertrophic adaptor protein that regulates cardiac myocyte signalling and function. Herein, we identified cardiomyopathy-associated 5 (CMYA5) as a novel FHL2 interaction partner in cardiac myocytes. In vitro pull-down assays demonstrated interaction between FHL2 and the N- and C-terminal regions of CMYA5. The interaction was verified in adult cardiac myocytes by proximity ligation assays. Immunofluorescence and confocal microscopy demonstrated co-localisation in the same subcellular compartment. The binding interface between FHL2 and CMYA5 was mapped by peptide arrays. Exposure of neonatal rat ventricular myocytes to a CMYA5 peptide covering one of the FHL2 interaction sites led to an increase in cell area at baseline, but a blunted response to chronic phenylephrine treatment. In contrast to wild-type hearts, loss or reduced FHL2 expression in Fhl2-targeted knockout mouse hearts or in a humanised mouse model of hypertrophic cardiomyopathy led to redistribution of CMYA5 into the perinuclear and intercalated disc region. Taken together, our results indicate a direct interaction of the two adaptor proteins FHL2 and CMYA5 in cardiac myocytes, which might impact subcellular compartmentation of CMYA5.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue.

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation.

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+ -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow-up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non-failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+ -binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+ -scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.