Kyung Chan Park

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca²⁺, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with β-alanine buildup. Raising β-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Background: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Methods: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. Results: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. Conclusions: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.

With the ever-mounting evidence for a profound and direct effect of SGLT2 inhibitors (SGLT2is) on the heart, understanding their mechanism of action becomes increasingly important. So, we are pleased that our paper ¹ published in this edition of Cardiovascular Research has generated a lively debate. ^{2, 3} In our work, we use a variety of methods to show that, at least in our hands, empagliflozin (EMPA) from two independent suppliers, as well as two other chemically related SGLT2is, are not potent inhibitors of the cardiac Na⁺/H⁺ exchanger-1 (NHE1) and, related to this, have no effect on intracellular Na⁺ concentration ([Na⁺]_i) in the healthy heart. This is contrary to several previous reports (see references 1–4 in Zuurbier et al. ²).

Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na⁺/H⁺ exchanger 1 (NHE1), causing a reduction in intracellular [Na⁺] ([Na⁺]_i). However, at resting intracellular pH (pH_i), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na⁺]_i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pH_i recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na⁺]_i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na⁺]_i rise upon superfusion of Na⁺-depleted cells with Na⁺-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na⁺]_i at baseline nor pH_i recovery following acute acidosis, as measured by ²³Na triple quantum filtered NMR and ³¹P NMR, respectively. Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na⁺]_i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na⁺].

SARS‐CoV‐2 coronavirus infection is characterised by a marked inflammatory state and viral pneumonitis. A striking clinical feature is severe hypoxaemia, often in the presence of near‐normal lung mechanics.

A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid–base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO₂/HCO₃⁻ and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.

Iron deficiency is present in ~50% of heart failure (HF) patients. Large multicenter trials have shown that treatment of iron deficiency with i.v. iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet, and some received i.v. ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca ²⁺ signaling, a pathway linked to the contractile deficit in failing hearts, was also significantly affected. Ventricular myocytes isolated from iron-deficient animals produced smaller Ca ²⁺ transients from an elevated diastolic baseline but had unchanged sarcoplasmic reticulum (SR) Ca ²⁺ load, trigger L-type Ca ²⁺ current, or cytoplasmic Ca ²⁺ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message levels. The constancy of diastolic SR Ca ²⁺ load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due to dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca ²⁺ signaling and ejection fraction. Thus, 2 Ca ²⁺ -handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by i.v. iron supplementation.

Propionic acidaemia (PA) is an autosomal recessive disorder characterised by malfunctional mitochondrial propionyl-CoA carboxylase. Consequences of this inborn error of metabolism (IEM) is defective catabolism of propiogenic substrates (branched-chain amino acids, odd-number fatty acids, etc.) leading to mitochondrial accumulation of propionyl CoA and its derivatives. Accumulation of propionate derivatives in the cytoplasm and plasma leads to intra- and extracellular metabolic acidosis. The clinical manifestation is severe; arrhythmia and cardiomyopathy (hypertrophic and dilated) are cited as major causes of death. Little is known of the underlying mechanisms. Since butyrate is in the same homologous series as propionate, and is itself a well-established inhibitor of histone deacetylases (HDACs), it is plausible pro-hypertrophic signalling in PA is epigenetically regulated. Therefore, we investigated the effects of propionate on cardiomyocytes. In preliminary work, culturing neonatal rat ventricular myocytes (NRVMs) in a chemical milieu mimicking PA (6 mM propionate; 200 M ammonium; 18 mM bicarbonate; pH 7.15) increased significantly cardiomyocyte area. Treating adult rat VMs with PA-medium increased significantly histone-3 lysine-9 acetylation, phenocopying the effects of butyrate. To determine the gene expression profile associated with PA, RNA-sequencing was performed on polyadenylated RNA extracted from NRVMs, treated with control-, butyrate-, or PA-medium. In PA-medium, Differentially Expressed Gene analysis demonstrated 3.0% of genes were upregulated and 3.0% downregulated (FDR<0.0001). Gene Ontology Enrichment Analysis using DAVID revealed ‘cardiac muscle contraction’ and ‘metabolic pathways’ as being significantly enriched. 1.9% of genes were upregulated in both PA and butyrate, representing candidates that could be regulated by HDACs. We are currently validating these hits.

Elucidation of the physiologically distinct subunits of troponin in 1973 greatly facilitated our understanding of cardiac contraction. Although troponins are expressed in both skeletal and cardiac muscle, there are isoforms of troponin I/T expressed selectively in the heart. By exploiting cardiac-restricted epitopes within these proteins, one of the most successful diagnostic tests to date has been developed: cardiac troponin (cTn) assays. For the past decade, cTn has been regarded as the gold-standard marker for acute myocardial necrosis: the pathological hallmark of acute myocardial infarction (AMI). Whilst cTn is the cornerstone for ruling-out AMI in patients presenting with a suspected acute coronary syndrome (ACS), elevated cTn is frequently observed in those without clinical signs indicative of AMI, often reflecting myocardial injury of ‘unknown origin’. cTn is commonly elevated in acute non-ACS conditions, as well as in chronic diseases. It is unclear why these elevations occur; yet they cannot be ignored as cTn levels in chronically unwell patients are directly correlated to prognosis. Paradoxically, improvements in assay sensitivity have meant more differential diagnoses have to be considered due to decreased specificity, since cTn is now more easily detected in these non-ACS conditions. It is important to be aware cTn is highly specific for myocardial injury, which could be attributable to a myriad of underlying causes, emphasizing the notion that cTn is an organ-specific, not disease-specific biomarker. Furthermore, the ability to detect increased cTn using high-sensitivity assays following extreme exercise is disconcerting. It has been suggested troponin release can occur without cardiomyocyte necrosis, contradicting conventional dogma, emphasizing a need to understand the mechanisms of such release. This review discusses basic troponin biology, the physiology behind its detection in serum, its use in the diagnosis of AMI, and some key concepts and experimental evidence as to why cTn can be elevated in chronic diseases.