Innate Immune Activation and Mitochondrial ROS Invoke Persistent Cardiac Conduction System Dysfunction after COVID-19.

Background: Cardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome in humans, but the mechanisms behind COVID-19-linked arrhythmias are unknown. This study explores the acute and long term effects of SARS-CoV-2 on the cardiac conduction system (CCS) in a hamster model of COVID-19. Methods: Radiotelemetry in conscious animals was used to non-invasively record electrocardiograms and subpleural pressures after intranasal SARS-CoV-2 infection. Cardiac cytokines, interferon-stimulated gene expression, and macrophage infiltration of the CCS, were assessed at 4 days and 4 weeks post-infection. A double-stranded RNA mimetic, polyinosinic:polycytidylic acid (PIC), was used in vivo and in vitro to activate viral pattern recognition receptors in the absence of SARS-CoV-2 infection. Results: COVID-19 induced pronounced tachypnea and severe cardiac conduction system (CCS) dysfunction, spanning from bradycardia to persistent atrioventricular block, although no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped after the pulmonary infection was resolved, indicating persistent CCS injury. Increased cardiac cytokines, interferon-stimulated gene expression, and macrophage remodeling in the CCS accompanied the electrophysiological abnormalities. Interestingly, the arrhythmia phenotype was reproduced by cardiac injection of PIC in the absence of virus, indicating that innate immune activation was sufficient to drive the response. PIC also strongly induced cytokine secretion and robust interferon signaling in hearts, human iPSC-derived cardiomyocytes (hiPSC-CMs), and engineered heart tissues, accompanied by alterations in electrical and Ca 2+ handling properties. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by in vivo inhibition of JAK/STAT signaling or by a mitochondrially-targeted antioxidant. Conclusions: The findings indicate that long term dysfunction and immune cell remodeling of the CCS is induced by COVID-19, arising indirectly from oxidative stress and excessive activation of cardiac innate immune responses during infection, with implications for long COVID Syndrome.

Mitochondrial membrane potential instability on reperfusion after ischemia does not depend on mitochondrial Ca2+ uptake.

Physiologic Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload but may also contribute to cell death during ischemia/reperfusion (I/R) injury. The MCU has been identified as the primary mode of Ca2+ import into mitochondria. Several groups have tested the hypothesis that Ca2+ import via MCU is detrimental during I/R injury using genetically-engineered mouse models, yet the results from these studies are inconclusive. Furthermore, mitochondria exhibit unstable or oscillatory membrane potentials (ΔΨm) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is an excess influx of mitochondrial Ca2+ (mCa2+), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca2+ uptake during I/R is involved in ΔΨm instability, or sustained mitochondrial depolarization, during reperfusion by acutely knocking out MCU in neonatal mouse ventricular myocyte (NMVM) monolayers subjected to simulated I/R. Unexpectedly, we find that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect ΔΨm recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa2+ increase during Ischemia but did not affect ΔΨm recovery or the frequency of ΔΨm oscillations during reperfusion, indicating that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨm oscillations. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCE activity and supporting ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury.

Oxidative stress in the mitochondrial matrix underlies ischemia/reperfusion-induced mitochondrial instability.

Ischemia and reperfusion affect multiple elements of cardiomyocyte electrophysiology, especially within the mitochondria. We previously showed that in cardiac monolayers, upon reperfusion after coverslip-induced ischemia, mitochondrial inner membrane potential (ΔΨ) unstably oscillates between polarized and depolarized states, and ΔΨ instability corresponds with arrhythmias. Here, through confocal microscopy of compartment-specific molecular probes, we investigate the mechanisms underlying the postischemic ΔΨ oscillations, focusing on the role of Ca2+ and oxidative stress. During reperfusion, transient ΔΨ depolarizations occurred concurrently with periods of increased mitochondrial oxidative stress (5.07 ± 1.71 oscillations/15 min, N = 100). Supplementing the antioxidant system with GSH monoethyl ester suppressed ΔΨ oscillations (1.84 ± 1.07 oscillations/15 min, N = 119, t test p = 0.027) with 37% of mitochondrial clusters showing no ΔΨ oscillations (versus 4% in control, odds ratio = 14.08, Fisher's exact test p < 0.001). We found that limiting the production of reactive oxygen species using cyanide inhibited postischemic ΔΨ oscillations (N = 15, t test p < 10-5). Furthermore, ΔΨ oscillations were not associated with any discernable pattern in cell-wide oxidative stress or with the changes in cytosolic or mitochondrial Ca2+. Sustained ΔΨ depolarization followed cytosolic and mitochondrial Ca2+ increase and was associated with increased cell-wide oxidative stress. Collectively, these findings suggest that transient bouts of increased mitochondrial oxidative stress underlie postischemic ΔΨ oscillations, regardless of Ca2+ dynamics.

MCU Overexpression Rescues Inotropy and Reverses Heart Failure by Reducing SR Ca2+ Leak.

[Figure: see text].

Mitochondrial ROS Drive Sudden Cardiac Death and Chronic Proteome Remodeling in Heart Failure.

RATIONALE: Despite increasing prevalence and incidence of heart failure (HF), therapeutic options remain limited. In early stages of HF, sudden cardiac death (SCD) from ventricular arrhythmias claims many lives. Reactive oxygen species (ROS) have been implicated in both arrhythmias and contractile dysfunction. However, little is known about how ROS in specific subcellular compartments contribute to HF or SCD pathophysiology. The role of ROS in chronic proteome remodeling has not been explored. OBJECTIVE: We will test the hypothesis that elevated mitochondrial ROS (mROS) is a principal source of oxidative stress in HF and in vivo reduction of mROS mitigates SCD. METHODS AND RESULTS: Using a unique guinea pig model of nonischemic HF that recapitulates important features of human HF, including prolonged QT interval and high incidence of spontaneous arrhythmic SCD, compartment-specific ROS sensors revealed increased mROS in resting and contracting left ventricular myocytes in failing hearts. Importantly, the mitochondrially targeted antioxidant (MitoTEMPO) normalized global cellular ROS. Further, in vivo MitoTEMPO treatment of HF animals prevented and reversed HF, eliminated SCD by decreasing dispersion of repolarization and ventricular arrhythmias, suppressed chronic HF-induced remodeling of the expression proteome, and prevented specific phosphoproteome alterations. Pathway analysis of mROS-sensitive networks indicated that increased mROS in HF disrupts the normal coupling between cytosolic signals and nuclear gene programs driving mitochondrial function, antioxidant enzymes, Ca2+ handling, and action potential repolarization, suggesting new targets for therapeutic intervention. CONCLUSIONS: mROS drive both acute emergent events, such as electrical instability responsible for SCD, and those that mediate chronic HF remodeling, characterized by suppression or altered phosphorylation of metabolic, antioxidant, and ion transport protein networks. In vivo reduction of mROS prevents and reverses electrical instability, SCD, and HF. Our findings support the feasibility of targeting the mitochondria as a potential new therapy for HF and SCD while identifying new mROS-sensitive protein modifications.

Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes.

Oxidative stress arises from an imbalance in the production and scavenging rates of reactive oxygen species (ROS) and is a key factor in the pathophysiology of cardiovascular disease and aging. The presence of parallel pathways and multiple intracellular compartments, each having its own ROS sources and antioxidant enzymes, complicates the determination of the most important regulatory nodes of the redox network. Here we quantified ROS dynamics within specific intracellular compartments in the cytosol and mitochondria and determined which scavenging enzymes exert the most control over antioxidant fluxes in H9c2 cardiac myoblasts. We used novel targeted viral gene transfer vectors expressing redox-sensitive GFP fused to sensor domains to measure H2O2 or oxidized glutathione. Using genetic manipulation in heart-derived H9c2 cells, we explored the contribution of specific antioxidant enzymes to ROS scavenging and glutathione redox potential within each intracellular compartment. Our findings reveal that antioxidant flux is strongly dependent on mitochondrial substrate catabolism, with availability of NADPH as a major rate-controlling step. Moreover, ROS scavenging by mitochondria significantly contributes to cytoplasmic ROS handling. The findings provide fundamental information about the control of ROS scavenging by the redox network and suggest novel interventions for circumventing oxidative stress in cardiac cells.

Inhibiting mitochondrial Na+/Ca2+ exchange prevents sudden death in a Guinea pig model of heart failure.

RATIONALE: In cardiomyocytes from failing hearts, insufficient mitochondrial Ca(2+) accumulation secondary to cytoplasmic Na(+) overload decreases NAD(P)H/NAD(P)(+) redox potential and increases oxidative stress when workload increases. These effects are abolished by enhancing mitochondrial Ca(2+) with acute treatment with CGP-37157 (CGP), an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger. OBJECTIVE: Our aim was to determine whether chronic CGP treatment mitigates contractile dysfunction and arrhythmias in an animal model of heart failure (HF) and sudden cardiac death (SCD). METHODS AND RESULTS: Here, we describe a novel guinea pig HF/SCD model using aortic constriction combined with daily ß-adrenergic receptor stimulation (ACi) and show that chronic CGP treatment (ACi plus CGP) attenuates cardiac hypertrophic remodeling, pulmonary edema, and interstitial fibrosis and prevents cardiac dysfunction and SCD. In the ACi group 4 weeks after pressure overload, fractional shortening and the rate of left ventricular pressure development decreased by 36% and 32%, respectively, compared with sham-operated controls; in contrast, cardiac function was completely preserved in the ACi plus CGP group. CGP treatment also significantly reduced the incidence of premature ventricular beats and prevented fatal episodes of ventricular fibrillation, but did not prevent QT prolongation. Without CGP treatment, mortality was 61% in the ACi group <4 weeks of aortic constriction, whereas the death rate in the ACi plus CGP group was not different from sham-operated animals. CONCLUSIONS: The findings demonstrate the critical role played by altered mitochondrial Ca(2+) dynamics in the development of HF and HF-associated SCD; moreover, they reveal a novel strategy for treating SCD and cardiac decompensation in HF.

Mitochondrial ROMK channel is a molecular component of mitoK(ATP).

RATIONALE: Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. OBJECTIVE: To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP). METHODS AND RESULTS: Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase ß. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death. CONCLUSIONS: The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.

Insulin effects on cardiac Na+/Ca2+ exchanger activity: role of the cytoplasmic regulatory loop.

Insulin can alter myocardial contractility, in part through an effect on the cardiac sarcolemmal Na(+)/Ca(2+) exchanger (NCX), but little is known about its mechanism of action. The large cytoplasmic domain (f-loop) of NCX is required for regulation by various intracellular factors, and we have shown previously that residues 562-679 are determinants of NCX inhibition by exchanger inhibitory peptide (XIP). Here we show that the same f-loop deletion eliminates the enhancement of NCX current by insulin, and we examine the signal pathways involved in the insulin response. NCX current (I(NCX)) was measured in freshly isolated or cultured (up to 48 h) adult guinea pig myocytes and in myocytes expressing canine NCX1.1 with the 562-679 f-loop deletion (NCX-(Delta562-679)) via adenoviral gene transfer. I(NCX) was recorded by whole-cell patch clamp as the Ni(2+)-sensitive current at 37 degrees C with intracellular Ca(2+) buffered. Insulin (1 microm) increased I(NCX) (at +80 mV) by 110 and 83% in fresh and cultured myocytes, respectively, whereas in myocytes expressing NCX-(Delta562-679) the response was eliminated (with 100 microm XIP included to suppress any native guinea pig I(NCX)). The insulin effect on I(NCX) was not inhibited by wortmannin, a nitric-oxide synthase inhibitor, or disruption of caveolae but was blocked by chelerythrine, implicating protein kinase C, but not phosphatidylinositol-3-kinase, in the mechanism. The insulin effect was also not additive with phosphatidylinositol-4,5-bisphosphate-induced activation of I(NCX). The finding that the 562-670 f-loop domain is implicated in both XIP and receptor-mediated modulation of NCX highlights its important role in acute physiological or pathophysiological regulation of Ca(2+) balance in the heart.

Beta-adrenergic stimulation of L-type Ca2+ channels in cardiac myocytes requires the distal carboxyl terminus of alpha1C but not serine 1928.

Beta-adrenoceptor stimulation robustly increases cardiac L-type Ca2+ current (ICaL); yet the molecular mechanism of this effect is still not well understood. Previous reports have shown in vitro phosphorylation of a consensus protein kinase A site at serine 1928 on the carboxyl terminus of the alpha1C subunit; however, the functional role of this site has not been investigated in cardiac myocytes. Here, we examine the effects of truncating the distal carboxyl terminus of the alpha1C subunit at amino acid residue 1905 or mutating the putative protein kinase A site at serine 1928 to alanine in adult guinea pig myocytes, using novel dihydropyridine-insensitive alpha1C adenoviruses, coexpressed with beta2 subunits. Expression of alpha1C truncated at 1905 dramatically attenuated the increase of peak ICaL induced by isoproterenol. However, the point mutation S1928A did not significantly attenuate the beta-adrenergic response. The findings indicate that the distal carboxyl-terminus of alpha1C plays an important role in beta-adrenergic upregulation of cardiac L-type Ca2+ channels, but that phosphorylation of serine 1928 is not required for this effect.

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors.

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.

Cardiac sodium-calcium exchanger is regulated by allosteric calcium and exchanger inhibitory peptide at distinct sites.

The sarcolemmal Na+-Ca2+ exchanger (NCX) is the main Ca2+ extrusion mechanism in cardiac myocytes and is thus essential for the regulation of Ca2+ homeostasis and contractile function. A cytosolic region (f-loop) of the protein mediates regulation of NCX function by intracellular factors including inhibition by exchanger inhibitory peptide (XIP), a 20 amino acid peptide matching the sequence of an autoinhibitory region involved in allosteric regulation of NCX by intracellular Na+, Ca2+, and phosphatidylinositol-4,5-biphosphate (PIP2). Previous evidence indicates that the XIP interaction domain can be eliminated by large deletions of the f-loop that also remove activation of NCX by intracellular Ca2+. By whole-cell voltage clamping experiments, we demonstrate that deletion of residues 562-679, but not 440- 456, 498-510, or 680-685 of the f-loop selectively eliminates XIP-mediated inhibition of NCX expressed either heterologously (HEK293 and A549 cells) or in guinea pig cardiac myocytes. In contrast, by plotting I(NCX) against reverse-mode NCX-mediated Ca2+ transients in myocytes, we demonstrate that Ca2+-dependent regulation of NCX is preserved in Delta562-679, but significantly reduced in the other three deletion mutants. The findings indicate that f-loop residues 562-679 may contain the regulatory site for endogenous XIP, but this site is distinct from the Ca2+-regulatory domains of the NCX. Because regulation of the NCX by Na+ and PIP2 involves the endogenous XIP region, the Delta562-679 mutant NCX may be a useful tool to investigate this regulation in the context of the whole cardiac myocyte.

Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane.

Ion channels on the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to cell survival. At least one type of potassium (K+) channel, the mitochondrial adenosine triphosphate-sensitive K+ channel (mitoKATP), is an important effector of protection against necrotic and apoptotic cell injury after ischemia. Here another channel with properties similar to the surface membrane calcium-activated K+ channel was found on the mitochondrial inner membrane (mitoKCa) of guinea pig ventricular cells. MitoKCa significantly contributed to mitochondrial K+ uptake of the myocyte, and an opener of mitoKCa protected hearts against infarction.