BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes.

Carnicer R, Duglan D, Ziberna K, Recalde A, ... Channon KM, Casadei B
Rationale
In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype.
Objective
Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes.
Methods and results
In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca²⁺]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post-diabetes induction (streptozotocin, 42-45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by ³¹phosphorous magnetic resonance spectroscopy), and myocardial function.
Conclusions
We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.



Circ Res: 04 Mar 2021; 128:585-601

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca] Leak-Load Relationship.

Yan J, Bare DJ, DeSantiago J, Zhao W, ... Chen SRW, Ai X
Rationale
We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca²⁺) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca²⁺-ATPase), essential in maintaining SR Ca²⁺ content cycling during each heartbeat, is completely unknown.
Objective
To test the hypothesis that JNK2 increases SERCA2 activity SR Ca²⁺ content and exacerbates an arrhythmic SR Ca²⁺ content leak-load relationship.
Methods and results
We used confocal Ca²⁺ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca²⁺/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca²⁺ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca²⁺ affinity. Unlike the CaMKII (Ca²⁺/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca²⁺ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca²⁺ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca²⁺ leak present, the JNK2-enhanced SR Ca²⁺ uptake limited leak-induced reduction in SR Ca²⁺, normalizing Ca²⁺ transient amplitude, but at a higher arrhythmogenic SR Ca²⁺ leak. JNK2-specific inhibition completely normalized SR Ca²⁺ handling, attenuated arrhythmic Ca²⁺ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts.
Conclusions
We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca²⁺ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca²⁺ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.



Circ Res: 18 Feb 2021; 128:455-470

Klotho Deficiency Causes Heart Aging via Impairing the Nrf2-GR Pathway.

Chen K, Wang S, Sun QW, Zhang B, Ullah M, Sun Z
Rationale
Cardiac aging is an important contributing factor for heart failure, which affects a large population but remains poorly understood.
Objective
The purpose of this study is to investigate whether Klotho plays a role in cardiac aging.
Methods and results
Heart function declined in old mice (24 months), as evidenced by decreases in fractional shortening, ejection fraction, and cardiac output. Heart size and weight, cardiomyocyte size, and cardiac fibrosis were increased in old mice, indicating that aging causes cardiac hypertrophy and remodeling. Circulating Klotho levels were dramatically decreased in old mice, which prompted us to investigate whether the Klotho decline may cause heart aging. We found that Klotho gene mutation (KL-/-) largely decreased serum klotho levels and impaired heart function. Interestingly, supplement of exogenous secreted Klotho prevented heart failure, hypertrophy, and remodeling in both old mice and KL (-/-) mice. Secreted Klotho treatment inhibited excessive cardiac oxidative stress, senescence and apoptosis in old mice and KL (-/-) mice. Serum phosphate levels in KL (-/-) mice were kept in the normal range, suggesting that Klotho deficiency-induced heart aging is independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed GR (glutathione reductase) expression and activity in the heart via inhibition of transcription factor Nrf2 (nuclear factor-erythroid 2 p45-related factor 2). Furthermore, cardiac-specific overexpression of GR prevented excessive oxidative stress, apoptosis, and heart failure in both old and KL (-/-) mice.
Conclusions
Klotho deficiency causes cardiac aging via impairing the Nrf2-GR pathway. Supplement of exogenous secreted Klotho represents a promising therapeutic strategy for aging-associated cardiomyopathy and heart failure.



Circ Res: 18 Feb 2021; 128:492-507

Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently.

Gadi I, Fatima S, Elwakiel A, Nazir S, ... Isermann B, Shahzad K
Rationale
While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa (factor Xa) or aPC (activated protein C), independently modulate intracellular signaling via partially distinct receptors.
Objectives
To study the differential effects of fXa or fIIa (factor IIa) inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury.
Methods and results
Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail-bleeding assay and FeCl₃-induced thrombosis). Myocardial ischemia-reperfusion injury was induced via left anterior descending ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNA sequencing, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL [interleukin]-1β, IL-6, and TNFα [tumor necrosis factor alpha]), as well as NF-κB (nuclear factor kappa B) and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post-myocardial ischemia-reperfusion injury. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect.
Conclusions
We showed that specific inhibition of coagulation via direct oral anticoagulants had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.



Circ Res: 18 Feb 2021; 128:513-529

SIRT6 Protects Smooth Muscle Cells From Senescence and Reduces Atherosclerosis.

Grootaert MOJ, Finigan A, Figg NL, Uryga AK, Bennett MR
Rationale
Vascular smooth muscle cell (VSMC) senescence promotes atherosclerosis and features of plaque instability, in part, through lipid-mediated oxidative DNA damage and telomere dysfunction. SIRT6 (Sirtuin 6) is a nuclear deacetylase involved in DNA damage response signaling, inflammation, and metabolism; however, its role in regulating VSMC senescence and atherosclerosis is unclear.
Objective
We examined SIRT6 expression in human VSMCs, the role, regulation, and downstream pathways activated by SIRT6, and how VSMC SIRT6 regulates atherogenesis.
Methods and results
SIRT6 protein, but not mRNA, expression was markedly reduced in VSMCs in human and mouse atherosclerotic plaques, and in human VSMCs derived from plaques or undergoing replicative or palmitate-induced senescence versus healthy aortic VSMCs. The ubiquitin ligase CHIP (C terminus of HSC70-interacting protein) promoted SIRT6 stability, but CHIP expression was reduced in human and mouse plaque VSMCs and by palmitate in a p38- and c-Jun N-terminal kinase-dependent manner. SIRT6 bound to telomeres, while SIRT6 inhibition using shRNA or a deacetylase-inactive mutant (SIRT6^H133Y) shortened human VSMC lifespan and induced senescence, associated with telomeric H3K9 (histone H3 lysine 9) hyperacetylation and 53BP1 (p53 binding protein 1) binding, indicative of telomere damage. In contrast, SIRT6 overexpression preserved telomere integrity, delayed cellular senescence, and reduced inflammatory cytokine expression and changes in VSMC metabolism associated with senescence. SIRT6, but not SIRT6^H133Y, promoted proliferation and lifespan of mouse VSMCs, and prevented senescence-associated metabolic changes. ApoE^-/- (apolipoprotein E) mice were generated that overexpress SIRT6 or SIRT6^H133Y in VSMCs only. SM22α-hSIRT6/ApoE^-/- mice had reduced atherosclerosis, markers of senescence and inflammation compared with littermate controls, while plaques of SM22α-hSIRT6^H133Y/ApoE^-/- mice showed increased features of plaque instability.
Conclusions
SIRT6 protein expression is reduced in human and mouse plaque VSMCs and is positively regulated by CHIP. SIRT6 regulates telomere maintenance and VSMC lifespan and inhibits atherogenesis, all dependent on its deacetylase activity. Our data show that endogenous SIRT6 deacetylase is an important and unrecognized inhibitor of VSMC senescence and atherosclerosis.



Circ Res: 18 Feb 2021; 128:474-491

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss.

Mäe MA, He L, Nordling S, Vazquez-Liebanas E, ... Keller A, Betsholtz C
Rationale
Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive.
Objective
To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level and to correlate them with regional heterogeneities in BBB function and vascular phenotype.
Methods and results
We reveal transcriptional, morphological, and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses, and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfb^ret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfb^ret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB.
Conclusions
By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence, and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 (angiopoietin 2) is paradoxical given its wider role as TIE2 (TEK receptor tyrosine kinase) receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.



Circ Res: 18 Feb 2021; 128:e46-e62

Ca-CaM Dependent Inactivation of RyR2 Underlies Ca Alternans in Intact Heart.

Wei J, Yao J, Belke D, Guo W, ... Echebarria B, Chen SRW
Rationale
Ca²⁺ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca²⁺ alternans remains undefined. Increasing evidence suggests that Ca²⁺ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca²⁺ alternans are unknown.
Objective
To determine the role of CaM (calmodulin) on Ca²⁺ alternans in intact working mouse hearts.
Methods and results
We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored Ca²⁺ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca²⁺ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca²⁺ alternans and prolonged Ca²⁺ transient recovery in intact RyR2 wild type and mutant hearts, whereas CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca²⁺ current but had no significant impact on sarcoplasmic reticulum Ca²⁺ content. Furthermore, we developed a novel numerical myocyte model of Ca²⁺ alternans that incorporates Ca²⁺-CaM-dependent regulation of RyR2 and the L-type Ca²⁺ channel. Remarkably, the new model recapitulates the impact on Ca²⁺ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca²⁺ elevation as a result of rapid pacing triggers Ca²⁺-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca²⁺ release, which, in turn, reduces diastolic cytosolic Ca²⁺, leading to alternations in diastolic cytosolic Ca²⁺, RyR2 inactivation, and sarcoplasmic reticulum Ca²⁺ release (ie, Ca²⁺ alternans).
Conclusions
Our results demonstrate that inactivation of RyR2 by Ca²⁺-CaM is a major determinant of Ca²⁺ alternans, making Ca²⁺-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.



Circ Res: 18 Feb 2021; 128:e63-e83

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation.

Ikeda J, Scipione CA, Hyduk SJ, Althagafi MG, ... MacParland SA, Cybulsky MI
Rationale
Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT.
Objective
To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms.
Methods and results
Ldlr^-/- mice underwent total body γ-irradiation, bone marrow reconstitution, and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared with mice without BMT. In BMT mice, neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet for 3, 6, and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation, and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL (low-density lipoprotein) accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70 kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr^-/- relative to Ldlr^+/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single-cell resolution, was performed to give insights into pathways modulated by BMT.
Conclusions
Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single-cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.



Circ Res: 18 Feb 2021; 128:530-543

Structural and Functional Characterization of a Na1.5-Mitochondrial Couplon.

Pérez-Hernández M, Leo-Macias A, Keegan S, Jouni M, ... Rothenberg E, Delmar M
Rationale
The cardiac sodium channel Na_V1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined.
Objective
To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria.
Methods and results
Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a Na_V1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na⁺/Ca²⁺ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near Na_V1.5 channels accumulated more Ca²⁺ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between Na_V1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding Na_V1.5) and SLC8B1 (which encode Na_V1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A.
Conclusions
We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca²⁺ from the mitochondria is powered, at least in part, by the entry of sodium through Na_V1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.



Circ Res: 04 Feb 2021; 128:419-432

Cell-Extracellular Matrix Interactions Play Multiple Essential Roles in Aortic Arch Development.

Warkala M, Chen D, Ramirez A, Jubran A, ... Zhao H, Astrof S
Rationale
Defects in the morphogenesis of the fourth pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease.
Objective
Cell-ECM (extracellular matrix) interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5β1 and Fn1 (fibronectin) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5β1 and Fn1 regulate aortic arch artery morphogenesis.
Methods and results
Using temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and contribute to pharyngeal arch endothelium between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches form a plexus of small blood vessels, which remodels into the PAAs by 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on the pharyngeal ECM microenvironment, extrinsic to the endothelium. Conditional ablation of integrin α5β1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates aortic arch artery morphogenesis at multiple steps: (1) accumulation of SHF-derived ECs in the pharyngeal arches, (2) remodeling of the EC plexus in the fourth arches into the PAAs, and (3) differentiation of neural crest-derived cells adjacent to the PAA endothelium into vascular smooth muscle cells.
Conclusions
PAA formation is a multistep process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the aortic arch artery and its major branches. Cell-ECM interactions regulated by integrin α5β1 and Fn1 play essential roles at each of these developmental stages.



Circ Res: 04 Feb 2021; 128:e27-e44

Lipid Profiles and Heart Failure Risk: Results From Two Prospective Studies.

Wittenbecher C, Eichelmann F, Toledo E, Guasch-Ferré M, ... Martínez-González MÁ, Hu FB
Rationale
Altered lipid metabolism has been implicated in heart failure (HF) development, but no prospective studies have examined comprehensive lipidomics data and subsequent risk of HF.
Objective
We aimed to link single lipid metabolites and lipidomics networks to the risk of developing HF.
Methods and results
Discovery analyses were based on 216 targeted lipids in a case-control study (331 incident HF cases and 507 controls, matched by age, sex, and study center), nested within the PREDIMED (Prevención con Dieta Mediterránea) study. Associations of single lipids were examined in conditional logistic regression models. Furthermore, lipidomics networks were linked to HF risk in a multistep workflow, including machine learning-based identification of the HF-related network clusters, and regression-based discovery of the HF-related lipid patterns within these clusters. If available, significant findings were externally validated in a subsample of the EPIC-Potsdam cohort (2414 at-risk participants, including 87 incident HF cases). After confounder-adjustments, 2 lipids were significantly associated with HF risk in both cohorts: CER (ceramide) 16:0 (relative risk [RR] per SD in PREDIMED, 1.28 [95% CI, 1.13-1.47]) and phosphatidylcholine 32_0 (RR per SD in PREDIMED, 1.23 [95% CI, 1.08-1.41]). Additionally, lipid patterns in several network clusters were associated with HF risk in PREDIMED. Adjusted for standard risk factors, an internally cross-validated score based on the significant HF-related lipids that were identified in the network analysis in PREDIMED was associated with a higher HF risk (20 lipids, RR per SD, 2.33 [95% CI, 1.93%-2.81%). Moreover, a lipid score restricted to the externally available lipids was significantly associated with HF incidence in both cohorts (6 lipids, RRs per SD, 1.30 [95% CI, 1.14-1.47] in PREDIMED, and 1.46 [95% CI, 1.17-1.82] in EPIC-Potsdam).
Conclusions
Our study identified and validated 2 lipid metabolites and several lipidomics patterns as potential novel biomarkers of HF risk. Lipid profiling may capture preclinical molecular alterations that predispose for incident HF. Registration: URL: https://www.isrctn.com/ISRCTN35739639; Unique identifier: ISRCTN35739639.



Circ Res: 04 Feb 2021; 128:309-320

Endothelial S1P Signaling Counteracts Infarct Expansion in Ischemic Stroke.

Nitzsche A, Poittevin M, Benarab A, Bonnin P, ... Hla T, Camerer E
Rationale
Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P₁ (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P₁ also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P₁ modulation in stroke.
Objective
To address roles and mechanisms of engagement of endothelial cell S1P₁ in the naive and ischemic brain and its potential as a target for cerebrovascular therapy.
Methods and results
Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P₁ in the mouse brain. With an S1P₁ signaling reporter, we reveal that abluminal polarization shields S1P₁ from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P₁ signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P₁ receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P₁ provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P₁ supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P₁-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion.
Conclusions
This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P₁ agonists.



Circ Res: 04 Feb 2021; 128:363-382

RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT.

Kryshtal DO, Blackwell DJ, Egly CL, Smith AN, ... Laver DR, Knollmann BC
Rationale
The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide\'s efficacy in CPVT.
Objective
To determine whether RyR2 block independently contributes to flecainide\'s efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo.
Methods and results
We synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide\'s inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide\'s inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block.
Conclusions
Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.



Circ Res: 04 Feb 2021; 128:321-331

PPARγ-p53-Mediated Vasculoregenerative Program to Reverse Pulmonary Hypertension.

Hennigs JK, Cao A, Li CG, Shi M, ... Snyder MP, Rabinovitch M
Rationale
In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53.
Objective
We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium.
Methods and results
In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 (ephrin type-A receptor 2), FHL2 (four and a half LIM domains protein 2), JAG1 (jagged 1), SULF2 (extracellular sulfatase Sulf-2), and TIGAR (TP53-inducible glycolysis and apoptosis regulator). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2-knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN (apelin) and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis.
Conclusions
We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.



Circ Res: 04 Feb 2021; 128:401-418

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

Kyriazis ID, Hoffman M, Gaignebet L, Lucchese AM, ... Kararigas G, Drosatos K
Rationale
Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism.
Objective
In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation.
Methods and results
KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice.
Conclusions
Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.



Circ Res: 04 Feb 2021; 128:335-357

Loss of Endogenously Cycling Adult Cardiomyocytes Worsens Myocardial Function.

Bradley LA, Young A, Li H, Billcheck HO, Wolf MJ
Rationale
Endogenously cycling adult cardiomyocytes increase after myocardial infarction (MI) but remain scarce and are generally thought not to contribute to myocardial function. However, this broadly held assumption has not been tested, mainly because of the lack of transgenic reporters that restrict Cre expression to adult cardiomyocytes that reenter the cell cycle.
Objective
We created and validated a new transgenic mouse, αMHC (alpha myosin heavy chain)-MerDreMer-Ki67p-RoxedCre (denoted αDKRC [cardiomyocyte-specific αMHC-MerDreMer-Ki67p-RoxedCre]) that restricts Cre expression to cycling adult cardiomyocytes and uniquely integrates spatial and temporal adult cardiomyocyte cycling events based on the DNA specificities of orthologous Dre and Cre recombinases. We then created αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes and examined the effects of ablating these endogenously cycling cardiomyocytes on myocardial function after ischemic-reperfusion (I/R) MI.
Methods and results
A tandem αDKRC transgene was designed, validated in cultured cells, and used to make transgenic mice. The αDKRC transgene integrated between MYH6 and MYH7 and did not disrupt expression of the surrounding genes. Compared with controls, αDKRC::RLTG (Rox-Lox-tdTomato-eGFP) mice treated with Tamoxifen expressed tdTomato+ in cardiomyocytes with rare Bromodeoxyuridine+, eGFP+ cardiomyocytes, consistent with reentry of the cell cycle. We then pretreated αDKRC::RLTG mice with Tamoxifen to activate the reporter before sham or reperfusion (I/R) MI surgeries. Compared with Sham surgery, the I/R MI group had increased single and paired eGFP+ (enhanced green fluorescent protein)+ cardiomyocytes predominantly in the border zones (5.8±0.5 versus 3.3±0.3 cardiomyocytes per 10-micron section, N=8-9 mice per group, n=16-24 sections per mouse), indicative of cycled cardiomyocytes. The single to paired eGFP+ cardiomyocyte ratio was ≈9 to 1 (5.2±0.4 single versus 0.6±0.2 paired cardiomyocytes) in the I/R MI group after MI, suggesting that cycling cardiomyocytes were more likely to undergo polyploidy than replication. The ablation of endogenously cycling adult cardiomyocytes in αDKRC::DTA (diphtheria) mice caused progressive worsening left ventricular chamber size and function after I/R MI, compared with controls.
Conclusions
Although scarce, endogenously cycling adult cardiomyocytes contribute to myocardial function after injury, suggesting that these cells may be physiologically relevant.



Circ Res: 21 Jan 2021; 128:155-168

Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure.

Abplanalp WT, Cremer S, John D, Hoffmann J, ... Zeiher AM, Dimmeler S
Rationale
Clonal hematopoiesis driven by mutations of DNMT3A (DNA methyltransferase 3a) is associated with increased incidence of cardiovascular disease and poor prognosis of patients with chronic heart failure (HF) and aortic stenosis. Although experimental studies suggest that DNMT3A clonal hematopoiesis-driver mutations may enhance inflammation, specific signatures of inflammatory cells in humans are missing.
Objective
To define subsets of immune cells mediating inflammation in humans using single-cell RNA sequencing.
Methods and results
Transcriptomic profiles of peripheral blood mononuclear cells were analyzed in n=6 patients with HF harboring DNMT3A clonal hematopoiesis-driver mutations and n=4 patients with HF and no DNMT3A mutations by single-cell RNA sequencing. Monocytes of patients with HF carrying DNMT3A mutations demonstrated a significantly increased expression of inflammatory genes compared with monocytes derived from patients with HF without DNMT3A mutations. Among the specific upregulated genes were the prototypic inflammatory IL (interleukin) IL1B (interleukin 1B), IL6, IL8, the inflammasome NLRP3, and the macrophage inflammatory proteins CCL3 and CCL4 as well as resistin, which augments monocyte-endothelial adhesion. Silencing of DNMT3A in monocytes induced a paracrine proinflammatory activation and increased adhesion to endothelial cells. Furthermore, the classical monocyte subset of DNMT3A mutation carriers showed increased expression of T-cell stimulating immunoglobulin superfamily members CD300LB, CD83, SIGLEC12, as well as the CD2 ligand and cell adhesion molecule CD58, all of which may be involved in monocyte-T-cell interactions. DNMT3A mutation carriers were further characterized by increased expression of the T-cell alpha receptor constant chain and changes in T helper cell 1, T helper cell 2, T helper cell 17, CD8+ effector, CD4+ memory, and regulatory T-cell-specific signatures.
Conclusions
This study demonstrates that circulating monocytes and T cells of patients with HF harboring clonal hematopoiesis-driver mutations in DNMT3A exhibit a highly inflamed transcriptome, which may contribute to the aggravation of chronic HF.



Circ Res: 21 Jan 2021; 128:216-228

Eosinophils Protect Mice From Angiotensin-II Perfusion-Induced Abdominal Aortic Aneurysm.

Liu CL, Liu X, Zhang Y, Liu J, ... Zhang J, Shi GP
Rationale
Blood eosinophil count and ECP (eosinophil cationic protein) associate with human cardiovascular diseases. Yet, whether eosinophils play a role in cardiovascular disease remains untested. The current study detected eosinophil accumulation in human and murine abdominal aortic aneurysm (AAA) lesions, suggesting eosinophil participation in this aortic disease.
Objective
To test whether and how eosinophils affect AAA growth.
Methods and results
Population-based randomized clinically controlled screening trials revealed higher blood eosinophil count in 579 male patients with AAA than in 5063 non-AAA control (0.236±0.182 versus 0.211±0.154, 10⁹/L, P<0.001). Univariate (odds ratio, 1.381, P<0.001) and multivariate (odds ratio, 1.237, P=0.031) logistic regression analyses indicated that increased blood eosinophil count in patients with AAA served as an independent risk factor of human AAA. Immunostaining and immunoblot analyses detected eosinophil accumulation and eosinophil cationic protein expression in human and murine AAA lesions. Results showed that eosinophil deficiency exacerbated AAA growth with increased lesion inflammatory cell contents, matrix-degrading protease activity, angiogenesis, cell proliferation and apoptosis, and smooth muscle cell loss using angiotensin-II perfusion-induced AAA in Apoe^-/- and eosinophil-deficient Apoe^-/-ΔdblGATA mice. Eosinophil deficiency increased lesion chemokine expression, muted lesion expression of IL (interleukin) 4 and eosinophil-associated-ribonuclease-1 (mEar1 [mouse EOS-associated-ribonuclease-1], human ECP homolog), and slanted M1 macrophage polarization. In cultured macrophages and monocytes, eosinophil-derived IL4 and mEar1 polarized M2 macrophages, suppressed CD11b⁺Ly6C^hi monocytes, and increased CD11b⁺Ly6C^lo monocytes. mEar1 treatment or adoptive transfer of eosinophil from wild-type and Il13^-/- mice, but not eosinophil from Il4^-/- mice, blocked AAA growth in Apoe^-/-ΔdblGATA mice. Immunofluorescent staining and immunoblot analyses demonstrated a role for eosinophil IL4 and mEar1 in blocking NF-κB (nuclear factor-κB) activation in macrophages, smooth muscle cells, and endothelial cells.
Conclusions
Eosinophils play a protective role in AAA by releasing IL4 and cationic proteins such as mEar1 to regulate macrophage and monocyte polarization and to block NF-κB activation in aortic inflammatory and vascular cells.



Circ Res: 21 Jan 2021; 128:188-202

Machine Learned Cellular Phenotypes in Cardiomyopathy Predict Sudden Death.

Rogers AJ, Selvalingam A, Alhusseini MI, Krummen DE, ... Zaharia M, Narayan SM
Rationale
Susceptibility to VT/VF (ventricular tachycardia/fibrillation) is difficult to predict in patients with ischemic cardiomyopathy either by clinical tools or by attempting to translate cellular mechanisms to the bedside.
Objective
To develop computational phenotypes of patients with ischemic cardiomyopathy, by training then interpreting machine learning of ventricular monophasic action potentials (MAPs) to reveal phenotypes that predict long-term outcomes.
Methods and results
We recorded 5706 ventricular MAPs in 42 patients with coronary artery disease and left ventricular ejection fraction ≤40% during steady-state pacing. Patients were randomly allocated to independent training and testing cohorts in a 70:30 ratio, repeated K=10-fold. Support vector machines and convolutional neural networks were trained to 2 end points: (1) sustained VT/VF or (2) mortality at 3 years. Support vector machines provided superior classification. For patient-level predictions, we computed personalized MAP scores as the proportion of MAP beats predicting each end point. Patient-level predictions in independent test cohorts yielded c-statistics of 0.90 for sustained VT/VF (95% CI, 0.76-1.00) and 0.91 for mortality (95% CI, 0.83-1.00) and were the most significant multivariate predictors. Interpreting trained support vector machine revealed MAP morphologies that, using in silico modeling, revealed higher L-type calcium current or sodium-calcium exchanger as predominant phenotypes for VT/VF.
Conclusions
Machine learning of action potential recordings in patients revealed novel phenotypes for long-term outcomes in ischemic cardiomyopathy. Such computational phenotypes provide an approach which may reveal cellular mechanisms for clinical outcomes and could be applied to other conditions.



Circ Res: 21 Jan 2021; 128:172-184

Targeting Mitochondria-Inflammation Circuit by β-Hydroxybutyrate Mitigates HFpEF.

Deng Y, Xie M, Li Q, Xu X, ... Tian R, Li T
Rationale
Over 50% of patients with heart failure have preserved ejection fraction (HFpEF), rather than reduced ejection fraction. Complexity of its pathophysiology and the lack of animal models hamper the development of effective therapy for HFpEF.
Objective
This study was designed to investigate the metabolic mechanisms of HFpEF and test therapeutic interventions using a novel animal model.
Methods and results
By combining the age, long-term high-fat diet, and desoxycorticosterone pivalate challenge in a mouse model, we were able to recapture the myriad features of HFpEF. In these mice, mitochondrial hyperacetylation exacerbated while increasing ketone body availability rescued the phenotypes. The HFpEF mice exhibited overproduction of IL (interleukin)-1β/IL-18 and tissue fibrosis due to increased assembly of NLPR3 inflammasome on hyperacetylated mitochondria. Increasing β-hydroxybutyrate level attenuated NLPR3 inflammasome formation and antagonized proinflammatory cytokine-triggered mitochondrial dysfunction and fibrosis. Moreover, β-hydroxybutyrate downregulated the acetyl-CoA pool and mitochondrial acetylation, partially via activation of CS (citrate synthase) and inhibition of fatty acid uptake.
Conclusions
Therefore, we identify the interplay of mitochondrial hyperacetylation and inflammation as a key driver in HFpEF pathogenesis, which can be ameliorated by promoting β-hydroxybutyrate abundance.



Circ Res: 21 Jan 2021; 128:232-245

Intracellular β-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

Wang Y, Shi Q, Li M, Zhao M, ... Bers DM, Xiang YK
Rationale
β₁ARs (β₁-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β₁AR in cardiac contractility remains to be elucidated.
Objective
Test localization and function of intracellular β₁AR on cardiac contractility.
Methods and results
Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β₁ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca²⁺-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility.
Conclusions
Functional β₁ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β₁ARs requires catecholamine transport via OCT3.



Circ Res: 21 Jan 2021; 128:246-261

Heterodimerization With 5-HTR Is Indispensable for βAR-Mediated Cardioprotection.

Song Y, Xu C, Liu J, Li Y, ... Woo AY, Xiao RP
Rationale
The β₂-adrenoceptor (β₂-AR), a prototypical GPCR (G protein-coupled receptor), couples to both G_s and G_i proteins. Stimulation of the β₂-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream G_i-PI3K-Akt cell survival pathway. Cardiac β₂-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated.
Objective
Here, we aim to investigate the potential cardioprotective effect of β₂-adrenergic stimulation with a subtype-selective agonist, (R,R\')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of β₂-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT_2BRs).
Methods and results
Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the β₂-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H₂O₂) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H₂O₂ or hypoxia/reoxygenation. Mechanistically, we found that MNF or another β₂-agonist zinterol markedly promoted heterodimerization of β₂-ARs with 5-HT_2BRs. Upregulation of the heterodimerized 5-HT_2BRs and β₂-ARs enhanced β₂-AR-stimulated G_i-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT_2BR attenuated β₂-AR-stimulated G_i signaling and cardioprotection.
Conclusions
These data demonstrate that the β₂-AR-stimulated cardioprotective G_i signaling depends on the heterodimerization of β₂-ARs and 5-HT_2BRs.



Circ Res: 21 Jan 2021; 128:262-277

Beat-by-Beat Cardiomyocyte T-Tubule Deformation Drives Tubular Content Exchange.

Rog-Zielinska EA, Scardigli M, Peyronnet R, Zgierski-Johnston CM, ... Sacconi L, Kohl P
Rationale
The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates.
Objective
We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid.
Methods and results
Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics.
Conclusions
Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.



Circ Res: 21 Jan 2021; 128:203-215