Bjorn C. Knollmann

BACKGROUND The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel–dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. METHODS Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine …

A868T variant cardiac tissue displayed increased myofilament Ca2þ sensitivity of isometric force, lower sinusoidal stiffness (SS), and faster tension redevelopment (kTR) rates at all Ca2þ levels. Small-angle X-ray diffraction indicated larger lattice spacing (LS) and increased I1, 1/I1, 0. As previously reported by our group, larger LS can explain faster kTR. Despite more myosin heads positioned away from the thick filament backbone and closer to thin filaments, as indicated by higher I1, 1/I1, 0, the heads either weakly interact with actin or generate less force, as suggested by lower SS. Molecular dynamics simulations showed that the variant likely alters a-actinin 2 binding to titin. Our results suggest evidence for two communications pathways (CPs) with a-actinin 2 at the nexus: the CP1 pathway from the Z-disc directly to the thin filaments and the CP2 pathway from the Z-disc directly to titin and indirectly to the thick …

Introduction The voltage gated potassium ion channel KV11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which can be rescued by pharmacological chaperones like E-4031. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery, comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants, and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. Methods We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes in human embryonic kidney (HEK293) cells expressing wild-type (WT) KV11.1 or trafficking-deficient channel variants in the presence or absence of E 4031. Results We identified 573 core KV11.1 protein interactors. Both variants KV11.1-G601S and KV11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We found that proteasomal degradation is a key component for KV11.1 degradation and that the KV11.1-G601S-G965* variant was more responsive to E-4031 treatment. This suggests a role in the C-terminal domain and the ER retention motif of KV11.1 in regulating trafficking. Conclusion Our report …

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and high rate of sudden cardiac arrest at juvenile age. cTnT-I79N is located in the cTnT N-terminal (TNT1) loop region and is well-known for its pathological and prognostic relevance. Recent structural study revealed that I79 is part of hydrophobic interface between TNT1 loop and actin which stabilizes the OFF state of the cardiac thin filament. Given the importance of understanding: i) the role of TNT1 loop region on the regulation of the cardiac thin filament, and ii) the underlying mechanisms of cTnT-I79N-linked pathogenesis; we sought to investigate the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased passive force and passive sinusoidal stiffness, which could be due in part to an increase in force-generating cross-bridges …

The ACTN2 gene encodes α-actinin 2, located in the Z-disc of the sarcomeres in striated muscle. In this study, we sought to investigate the effects of an ACTN2 missense variant of unknown significance (p.A868T) on cardiac muscle structure and function. Left ventricular free wall samples were obtained at the time of cardiac transplantation from a heart failure patient with the ACTN2 A868T heterozygous variant. This variant is in the EF 3–4 domain known to interact with titin and α-actinin. At the ultrastructural level, ACTN2 A868T cardiac samples presented small structural changes in cardiomyocytes when compared to healthy donor samples. However, contractile mechanics of permeabilized ACTN2 A868T variant cardiac tissue displayed higher myofilament Ca2+ sensitivity of isometric force, reduced sinusoidal stiffness, and faster rates of tension redevelopment at all Ca2+ levels. Small-angle X-ray diffraction indicated increased separation between thick and thin filaments, possibly contributing to changes in muscle kinetics. Molecular dynamics simulations indicated that while the mutation does not significantly impact the structure of α-actinin on its own, it likely alters the conformation associated with titin binding. Our results can be explained by two Z-disc mediated communication pathways: one pathway that involves α-actinin’s interaction with actin, affecting thin filament regulation, and the other pathway that involves α-actinin’s interaction with titin, affecting thick filament activation. This work establishes the role of α-actinin 2 in modulating cross-bridge kinetics and force development in the human myocardium as well as how it can be involved in …

INTRODUCTIONSudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) are a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)-mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD.OBJECTIVETo test the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function.METHODSWe used a nonischemic HF guinea pig model that replicates key aspects of human clinical HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF and intention-to-treat analysis was done. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation.RESULTSDS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient β-adrenergic challenge, improved heart rate …

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/−) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/−) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca …

The brief opening mode of the mitochondrial permeability transition pore (mPTP) serves as a calcium (Ca2+) release valve to prevent mitochondrial Ca2+ (mCa2+) overload. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to mutations in the Ca2+ release channel complex of ryanodine receptor 2 (RyR2). We hypothesize that inhibiting the mPTP opening in CPVT exacerbates the disease phenotype. By crossbreeding a CPVT model of CASQ2 knockout (KO) with a mouse missing CypD, an activator of mPTP, a double KO model (DKO) was generated. Echocardiography, cardiac histology, and live-cell imaging were employed to assess the severity of cardiac pathology. Western blot and RNAseq were performed to evaluate the contribution of various signaling pathways. Although exacerbated arrhythmias were reported, the DKO model did not exhibit pathological remodeling. Myocyte Ca2+ handling was similar to that of the CASQ2 KO mouse at a low pacing frequency. However, increased ROS production, activation of the CaMKII pathway, and hyperphosphorylation of RyR2 were detected in DKO. Transcriptome analysis identified altered gene expression profiles associated with electrical instability in DKO. Our study provides evidence that genetic inhibition of mPTP exacerbates RyR2 dysfunction in CPVT by increasing activation of the CaMKII pathway and subsequent hyperphosphorylation of RyR2.