Atrial-paced, exercise-similar heart rate envelope induces myocardial protection from ischaemic injury.

AIMS: The study investigates the role and mechanisms of clinically translatable exercise heart rate (HR) envelope effects, without dyssynchrony, on myocardial ischaemia tolerance compared to standard preconditioning methods. Since the magnitude and duration of exercise HR acceleration are tightly correlated with beneficial cardiac outcomes, it is hypothesized that a paced exercise-similar HR envelope, delivered in a maximally physiologic way that avoids the toxic effects of chamber dyssynchrony, may be more than simply a readout, but rather also a significant trigger of myocardial conditioning and stress resistance. METHODS AND RESULTS: For 8 days over 2 weeks, sedated mice were atrial-paced once daily via an oesophageal electrode to deliver an exercise-similar HR pattern with preserved atrioventricular and interventricular synchrony. Effects on cardiac calcium handling, protein expression/modification, and tolerance to ischaemia-reperfusion (IR) injury were assessed and compared to those in sham-paced mice and to the effects of exercise and ischaemic preconditioning (IPC). The paced cohort displayed improved myocardial IR injury tolerance vs. sham controls with an effect size similar to that afforded by treadmill exercise or IPC. Hearts from paced mice displayed changes in Ca2+ handling, coupled with changes in phosphorylation of calcium/calmodulin protein kinase II, phospholamban and ryanodine receptor channel, and transcriptional remodelling associated with a cardioprotective paradigm. CONCLUSIONS: The HR pattern of exercise, delivered by atrial pacing that preserves intracardiac synchrony, induces cardiac conditioning and enhances ischaemic stress resistance. This identifies the HR pattern as a signal for conditioning and suggests the potential to repurpose atrial pacing for cardioprotection.

Functional reclassification of variants of uncertain significance in the HCN4 gene identified in sudden unexpected death.

The HCN4 gene encodes a subunit of the hyperpolarization-activated cyclic nucleotide-gated channel, type 4 that is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are all missense alterations, leading to single amino acid changes: p.E66Q in the N-terminus, p.D546N in the C-linker domain, and both p.S935Y and p.R1044Q in the C-terminus distal to the CNBD. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild-type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C-linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild-type. With functional assays, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time-consuming further study. Our studies also provide a new lead to investigate how a VUS located in the C-linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response.

Musclin is an activity-stimulated myokine that enhances physical endurance.

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this study we identify musclin-a peptide with high homology to natriuretic peptides (NP)-as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca(2+)-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin.

Sarcolemmal ATP-sensitive potassium (KATP) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle KATP channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle KATP channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of KATP channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) - an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish KATP channel-dependent musclin production as a potential mechanistic link coupling "local" skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities.

Disruption of KATP channel expression in skeletal muscle by targeted oligonucleotide delivery promotes activity-linked thermogenesis.

Despite the medical, social, and economic impact of obesity, only a few therapeutic options, focused largely on reducing caloric intake, are currently available and these have limited success rates. A major impediment is that any challenge by caloric restriction is counterbalanced by activation of systems that conserve energy to prevent body weight loss. Therefore, targeting energy-conserving mechanisms to promote energy expenditure is an attractive strategy for obesity treatment. Here, in order to suppress muscle energy efficiency, we target sarcolemmal ATP-sensitive potassium (KATP) channels which have previously been shown to be important in maintaining muscle energy economy. Specifically, we employ intramuscular injections of cell-penetrating vivo-morpholinos to prevent translation of the channel pore-forming subunit. This intervention results in significant reduction of KATP channel expression and function in treated areas, without affecting the channel expression in nontargeted tissues. Furthermore, suppression of KATP channel function in a group of hind limb muscles causes a substantial increase in activity-related energy consumption, with little effect on exercise tolerance. These findings establish a proof-of-principle that selective skeletal muscle targeting of sarcolemmal KATP channel function is possible and that this intervention can alter overall bodily energetics without a disabling impact on muscle mechanical function.

Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II.

Cardiac ATP-sensitive potassium (K(ATP)) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac K(ATP) channels. We used real-time monitoring of K(ATP) channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant K(ATP) channel subunits to track the dynamics of K(ATP) channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of K(ATP) channels. This process required phosphorylation of threonine at 180 and 224 and an intact (330)YSKF(333) endocytosis motif of the K(ATP) channel Kir6.2 pore-forming subunit. A molecular model of the µ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that µ2 docks by interaction with (330)YSKF(333) and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on µ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac K(ATP) channel subunits. This mechanism couples the surface expression of cardiac K(ATP) channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance.

Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia.

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K(ATP)) channels. K(ATP) channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K(ATP) channel subunit were compared with littermate controls. Evaluation of K(ATP) channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80-85% reduction in cardiac K(ATP) channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K(ATP) channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.

Epidemiology of Viliuisk encephalomyelitis in Eastern Siberia.

BACKGROUND: Viliuisk encephalomyelitis is a disorder that starts, in most cases, as an acute meningoencephalitis. Survivors of the acute phase develop a slowly progressing neurologic syndrome characterized by dementia, dysarthria, and spasticity. An epidemic of this disease has been spreading throughout the Yakut Republic of the Russian Federation. Although clinical, neuropathologic, and epidemiologic data suggest infectious etiology, multiple attempts at pathogen isolation have been unsuccessful. METHODS: Detailed clinical, pathologic, laboratory, and epidemiologic studies have identified 414 patients with definite Viliuisk encephalomyelitis in 15 of 33 administrative regions of the Yakut Republic between 1940 and 1999. All data are documented in a Registry. RESULTS: The average annual Viliuisk encephalomyelitis incidence rate at the height of the epidemic reached 8.8 per 100,000 population and affected predominantly young adults. The initial outbreak occurred in a remote isolated area of the middle reaches of Viliui River; the disease spread to adjacent areas and further in the direction of more densely populated regions. The results suggest that intensified human migration from endemic villages led to the emergence of this disease in new communities. Recent social and demographic changes have presumably contributed to a subsequent decline in disease incidence. CONCLUSIONS: Based on the largest known set of diagnostically verified Viliuisk encephalomyelitis cases, we demonstrate how a previously little-known disease that was endemic in a small indigenous population subsequently reached densely populated areas and produced an epidemic involving hundreds of persons.

Functional characterization of ABCC9 variants identified in sudden unexpected natural death.

BACKGROUND: Genetic variation in ion channel genes ('channelopathies') are often associated with inherited arrhythmias and sudden death. Genetic testing ('molecular autopsies') of channelopathy genes can be used to assist in determining the likely causes of sudden unexpected death. However, different in silico approaches can yield conflicting pathogenicity predictions and assessing their impact on ion channel function can assist in this regard. METHODS AND RESULTS: We performed genetic testing of cases of sudden expected death in the New York City metropolitan area and found four rare or novel variants in ABCC9, which codes for the regulatory SUR2 subunit of KATP channels. All were missense variants, causing amino acid changes in the protein. Three of the variants (A355S, M941V, and K1379Q) were in cases of infants less than six-months old and one (H1305Y) was in an adult. The predicted pathogenicities of the variants were conflicting. We have introduced these variants into a human SUR2A cDNA, which we coexpressed with the Kir6.2 pore-forming subunit in HEK-293 cells and subjected to patch clamp and biochemical assays. Each of the four variants led to gain-of-function phenotypes. The A355S and M941V variants increased in the overall patch current. The sensitivity of the KATP channels to inhibitory 'cytosolic' ATP was repressed for the M941V, H1305Y and K1379Q variants. None of the variants had any effect on the unitary KATP channel current or the surface expression of KATP channels, as determined with biotinylation assays, suggesting that all of the variants led to an enhanced open state. CONCLUSIONS: All four variants caused a gain-of-function phenotype. Given the expression of SUR2-containing KATP channels in the heart and specialized cardiac conduction, vascular smooth muscle and respiratory neurons, it is conceivable that electrical silencing of these cells may contribute to the vulnerability element, which is a component of the triple risk model of sudden explained death in infants. The gain-of-function phenotype of these ABCC9 variants should be considered when assessing their potential pathogenicity.

Functional characterization of TRPM4 variants identified in sudden unexpected natural death.

BACKGROUND: The TRPM4 gene encodes the subunit of the Ca2+-activated nonselective cation channel, which is enriched in the specialized cardiac conduction system and Purkinje fibers. To date, several putative disease-causing variants in TRPM4 have been reported to be associated with cardiac arrhythmia and progressive conduction disease. Here, we report the functional effects of previously uncharacterized variants of uncertain significance (VUS) that we have found while performing a "genetic autopsy" in individuals who have suffered sudden unexpected death (SUD) in the New York City area. METHODS AND RESULTS: We have identified thirteen uncommon missense VUS in TRPM4 by testing 95 targeted genes implicated in channelopathy and cardiomyopathy in 330 cases of SUD. In several cases there were co-existing VUS in one or more other genes that were tested. We selected four TRPM4 VUS (C20S, A380V, L595V and I1082S) for functional characterization, since these cases lacked detectable variants in other genes of our testing panel. Two of the cases were infants, one was a child and one an adult. RNA-seq data analysis showed that the longer TRPM4b splice variant is predominantly expressed in adult and fetal human heart. We therefore used site-directed mutagenesis to introduce these variants in a TRPM4b cDNA. HEK293 cells were transfected with the cDNAs and patch clamping was performed to assess the functional consequences of the TRPM4 mutants. The TRPM4 current was recorded in excised patches and was significantly reduced by each of the mutants. The total protein level of TRPM4-C20S was markedly decreased, whereas the A380V and L595V mutants exhibited decreased surface expression. The TRPM4-A380V current rapidly desensitized following patch excision. CONCLUSIONS: Each of the VUS tested caused a defect in TRPM4 channel function via distinctly different mechanisms, hence, it lays the foundation for further co-segregation family studies and animal studies of the TRPM4 variants.

Molecular autopsy: using the discovery of a novel de novo pathogenic variant in the KCNH2 gene to inform healthcare of surviving family.

BACKGROUND: Molecular testing of the deceased (Molecular Autopsy) is an overlooked area in the United States healthcare system and is not covered by medical insurance, leading to ineffective care for surviving families of thousands of sudden unexpected natural deaths each year. We demonstrated the precision management of surviving family members through the discovery of a novel de novo pathogenic variant in a decedent. METHODS: Forensic investigation and molecular autopsy were performed on an 18-year-old female who died suddenly and unexpectedly. Co-segregation family study of the first-degree relatives and functional characterization of the variant were conducted. FINDINGS: We identified a novel nonsense variant, NP_000229.1:p.Gln1068Ter, in the long QT syndrome type II gene KCNH2 in the decedent. This finding correlated with her ante-mortem electrocardiograms. Patch clamp functional studies using transfected COS-7 cells show that hERG-ΔQ1068 has a mixed phenotype, with both gain- (negative voltage shift of steady-state activation curve, the positive shift of the steady-state inactivation curve, and accelerated activation) and loss-of function (reduced current density, reduced surface expression and accelerated deactivation) hallmarks. Loss of cumulative activation during rapid pacing demonstrates that the loss-of-function phenotype predominates. The wild-type channel did not rescue the hERG-ΔQ1068 defects, demonstrating haploinsufficiency of the heterozygous state. Targeted variant testing in the family showed that the variant in KCNH2 arose de novo, which eliminated the need for exhaustive genome testing and annual cardiac follow-up for the parents and four siblings. INTERPRETATION: Molecular testing enables accurate determination of natural causes of death and precision care of the surviving family members in a time and cost-saving manner. We advocate for molecular autopsy being included under the healthcare coverage in US.

Cardiovascular KATP channels and advanced aging.

With advanced aging, there is a decline in innate cardiovascular function. This decline is not general in nature. Instead, specific changes occur that impact the basic cardiovascular function, which include alterations in biochemical pathways and ion channel function. This review focuses on a particular ion channel that couple the latter two processes, namely the KATP channel, which opening is promoted by alterations in intracellular energy metabolism. We show that the intrinsic properties of the KATP channel changes with advanced aging and argue that the channel can be further modulated by biochemical changes. The importance is widespread, given the ubiquitous nature of the KATP channel in the cardiovascular system where it can regulate processes as diverse as cardiac function, blood flow and protection mechanisms against superimposed stress, such as cardiac ischemia. We highlight questions that remain to be answered before the KATP channel can be considered as a viable target for therapeutic intervention.

Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury.

The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (KATP) channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane KATP channels ensures a rapid energy-sparing reduction in action potential duration (APD) in response to metabolic challenges, while cellular signaling that reduces surface KATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kir6.2 KATP channel subunit initiating a cascade responsible for KATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB) model of heart failure is coupled with a 35-40% reduction in surface expression of KATP channels compared to hearts from sham-operated mice. Linkage between KATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of KATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial KATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by KATP channels. Recognition of CaMKII-dependent downregulation of KATP channel expression as a mechanism for vulnerability to injury in failing hearts points to strategies targeting this interaction for potential preventives or treatments.

Sarcolemmal ATP-sensitive potassium channels modulate skeletal muscle function under low-intensity workloads.

ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle-specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.

Genetic factors of Ebola virus virulence in guinea pigs.

Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in primates, whereas in guinea pigs it induces a nonlethal infection with a mild fever and subsequent recovery. We performed 7 selective passages in guinea pigs resulted in obtaining of guinea pig-adapted strain (GPA-P7) strain. By the 7th passage, the infection with EBOV induced a lethal disease in animals accompanied by the characteristic hematological changes: leukocytosis (primarily due to neutrophilia) as well as pronounced deficiencies in platelets, lymphocytes, monocytes and significant decrease of blood neutrophils phagocytic capacity. Increasing of virulence correlated with appearance of several nucleotide substitutions: in the genes NP, A2166G (N566S), VP24, U10784C (L147P), G10557A (M71I), G10805U (R154L), and L, G12286A (V236I). It has been theoretically calculated that the mutations associated with an increase in EBOV virulence can confer characteristic secondary structure on the proteins NP (C-terminal region) and full-sized VP24.