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The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca(2+) challenge and led to a greater Ca(2+) uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca(2+) overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy.

Antagonists of L-type Ca²⁺ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C⁻/⁺ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C⁻/⁺ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C⁻/⁺ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C⁺/⁺ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca²⁺ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease. [ABSTRACT FROM AUTHOR]

The most common mutation in surfactant protein C gene (SFTPC), SFTPCI73T, causes interstitial lung disease with few therapeutic options. We previously demonstrated that EMC3, an important component of the multiprotein endoplasmic reticulum membrane complex (EMC), is required for surfactant homeostasis in alveolar type 2 epithelial (AT2) cells at birth. In the present study, we investigated the role of EMC3 in the control of SFTPCI73T metabolism and its associated alveolar dysfunction. Using a knock-in mouse model phenocopying the I73T mutation, we demonstrated that conditional deletion of Emc3 in AT2 cells rescued alveolar remodeling/simplification defects in neonatal and adult mice. Proteomic analysis revealed that Emc3 depletion reversed the disruption of vesicle trafficking pathways and rescued the mitochondrial dysfunction associated with I73T mutation. Affinity purification-mass spectrometry analysis identified potential EMC3 interacting proteins in lung AT2 cells, including Valosin Containing Protein (VCP) and its interactors. Treatment of SftpcI73T knock-in mice and SFTPCI73T expressing iAT2 cells derived from SFTPCI73T patient-specific iPSCs with the specific VCP inhibitor CB5083 restored alveolar structure and SFTPCI73T trafficking respectively. Taken together, the present work identifies the EMC complex and VCP in the metabolism of the disease-associated SFTPCI73T mutant, providing novel therapeutical targets for SFTPCI73T-associated interstitial lung disease.

Fluctuations in mitochondrial matrix Ca 2+ plays a critical role in matching energy production to cellular demand through direct effects on oxidative phosphorylation and ATP production. Disruption in mitochondrial Ca 2+ homeostasis, particularly under pathological conditions such as ischemia or heart failure, can lead to mitochondrial dysfunction, energy deficit, and eventually death of cardiomyocytes. The primary channel regulating acute mitochondrial Ca 2+ influx is the mitochondrial Ca 2+ uniporter (mtCU), which is regulated by the mitochondrial Ca 2+ uptake (MICU) proteins that were examined here., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Skeletal muscle has a major impact on total body metabolism and obesity, and is characterized by dynamic regulation of substrate utilization. While it is accepted that acute increases in mitochondrial matrix Ca 2+ increase carbohydrate usage to augment ATP production, recent studies in mice with deleted genes for components of the mitochondrial Ca 2+ uniporter (MCU) complex have suggested a more complicated regulatory scenario. Indeed, mice with a deleted Mcu gene in muscle, which lack acute mitochondrial Ca 2+ uptake, have greater fatty acid oxidation (FAO) and less adiposity. By contrast, mice deleted for the inhibitory Mcub gene in skeletal muscle, which have greater acute mitochondrial Ca 2+ uptake, antithetically display reduced FAO and progressive obesity. In this review we discuss the emerging concept that dynamic fluxing of mitochondrial matrix Ca 2+ regulates metabolism., Competing Interests: Declaration of interests The authors have declared that no conflicts of interest exist., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor β (TGFβ)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFβ-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1 -/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFβ-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFβ-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondrial Ca 2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca 2+ influx across the sarcolemma that causes mitochondrial Ca 2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca 2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca 2+ uptake. One strategy for preventing mitochondrial Ca 2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca 2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca 2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca 2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca 2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo., (© 2024. The Author(s).)

During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic β- and γ-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the non-sarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width. NEW & NOTEWORTHY Here, we performed an unbiased analysis of the total phosphoproteome downstream of MEK1-ERK1/2 kinase signaling in cardiomyocytes. Pathway analysis suggested that proteins of the non-sarcomeric cytoskeleton were the most differentially affected. We showed that cytoplasmic β-actin and γ-actin isoforms, regulated by MEK1-ERK1/2, are localized to the subcortical space at both lateral membranes and intercalated discs of adult cardiomyocytes suggesting how MEK1-ERK1/2 signaling might underlie directional growth of adult cardiomyocytes.

S-palmitoylation is a reversible lipid modification catalyzed by 23 S-acyltransferases with a conserved zinc finger aspartate-histidine-histidine-cysteine (zDHHC) domain that facilitates targeting of proteins to specific intracellular membranes. Here we performed a gain-of-function screen in the mouse and identified the Golgi-localized enzymes zDHHC3 and zDHHC7 as regulators of cardiac hypertrophy. Cardiomyocyte-specific transgenic mice overexpressing zDHHC3 show cardiac disease, and S-acyl proteomics identified the small GTPase Rac1 as a novel substrate of zDHHC3. Notably, cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice is preceded by enhanced Rac1 S-palmitoylation, membrane localization, activity, downstream hypertrophic signaling, and concomitant induction of all Rho family small GTPases whereas mice overexpressing an enzymatically dead zDHHC3 mutant show no discernible effect. However, loss of Rac1 or other identified zDHHC3 targets Gαq/11 or galectin-1 does not diminish zDHHC3-induced cardiomyopathy, suggesting multiple effectors and pathways promoting decompensation with sustained zDHHC3 activity. Genetic deletion of Zdhhc3 in combination with Zdhhc7 reduces cardiac hypertrophy during the early response to pressure overload stimulation but not over longer time periods. Indeed, cardiac hypertrophy in response to 2 weeks of angiotensin-II infusion is not diminished by Zdhhc3/7 deletion, again suggesting other S-acyltransferases or signaling mechanisms compensate to promote hypertrophic signaling. Taken together, these data indicate that the activity of zDHHC3 and zDHHC7 at the cardiomyocyte Golgi promote Rac1 signaling and maladaptive cardiac remodeling, but redundant signaling effectors compensate to maintain cardiac hypertrophy with sustained pathological stimulation in the absence of zDHHC3/7., Competing Interests: Conflict of interest The authors declare that there are no conflicts of interests with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria use the electron transport chain to generate high-energy phosphate from oxidative phosphorylation, a process also regulated by the mitochondrial Ca 2+ uniporter (MCU) and Ca 2+ levels. Here, we show that MCUb, an inhibitor of MCU-mediated Ca 2+ influx, is induced by caloric restriction, where it increases mitochondrial fatty acid utilization. To mimic the fasted state with reduced mitochondrial Ca 2+ influx, we generated genetically altered mice with skeletal muscle-specific MCUb expression that showed greater fatty acid usage, less fat accumulation, and lower body weight. In contrast, mice lacking Mcub in skeletal muscle showed increased pyruvate dehydrogenase activity, increased muscle malonyl coenzyme A (CoA), reduced fatty acid utilization, glucose intolerance, and increased adiposity. Mechanistically, pyruvate dehydrogenase kinase 4 (PDK4) overexpression in muscle of Mcub-deleted mice abolished altered substrate preference. Thus, MCUb is an inducible control point in regulating skeletal muscle mitochondrial Ca 2+ levels and substrate utilization that impacts total metabolic balance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rationale: The adult cardiac extracellular matrix (ECM) is largely comprised of type I collagen. In addition to serving as the primary structural support component of the cardiac ECM, type I collagen also provides an organizational platform for other ECM proteins, matricellular proteins, and signaling components that impact cellular stress sensing in vivo., Objective: Here we investigated how the content and integrity of type I collagen affect cardiac structure function and response to injury., Methods and Results: We generated and characterized Col1a2 -/- mice using standard gene targeting. Col1a2 -/- mice were viable, although by young adulthood their hearts showed alterations in ECM mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included augmented TGFβ activity, increases in fibroblast number, and progressive cardiac hypertrophy, with reduced functional performance by 9 months of age. Col1a2-loxP -targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer mice to delete the Col1a2 gene in myofibroblasts with pressure overload injury. Interestingly, while germline Col1a2 -/- mice showed gradual pathologic hypertrophy and fibrosis with aging, the acute deletion of Col1a2 from activated adult myofibroblasts showed a loss of total collagen deposition with acute cardiac injury and an acute reduction in pressure overload-induce cardiac hypertrophy. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was lost after 2 and 6 weeks of pressure overload, as fibrotic deposition accumulated., Conclusions: Defective type I collagen in the heart alters the structural integrity of the ECM and leads to cardiomyopathy in adulthood, with fibroblast expansion, activation, and alternate fibrotic ECM deposition. However, acute inhibition of type I collagen production can have an anti-fibrotic and anti-hypertrophic effect.

Mitochondrial permeability transition pore (MPTP) formation contributes to ischemia-reperfusion injury in the heart and several degenerative diseases, including muscular dystrophy (MD). MD is a family of genetic disorders characterized by progressive muscle necrosis and premature death. It has been proposed that the MPTP has two molecular components, the adenine nucleotide translocase (ANT) family of proteins and an unknown component that requires the chaperone cyclophilin D (CypD) to activate. This model was examined in vivo by deleting the gene encoding ANT1 ( Slc25a4 ) or CypD ( Ppif ) in a δ-sarcoglycan ( Sgcd ) gene-deleted mouse model of MD, revealing that dystrophic mice lacking Slc25a4 were partially protected from cell death and MD pathology. Dystrophic mice lacking both Slc25a4 and Ppif together were almost completely protected from necrotic cell death and MD disease. This study provides direct evidence that ANT1 and CypD are required MPTP components governing in vivo cell death, suggesting a previously unrecognized therapeutic approach in MD and other necrotic diseases.

To better understand the genetic basis of heart disease, we identified a variant in the Flightless-I homolog ( FLII ) gene that generates a R1243H missense change and predisposes to cardiac remodeling across multiple previous human genome-wide association studies (GWAS). Since this gene is of unknown function in the mammalian heart we generated gain- and loss-of-function genetically altered mice, as well as knock-in mice with the syntenic R1245H amino acid substitution, which showed that Flii protein binds the sarcomeric actin thin filament and influences its length. Deletion of Flii from the heart, or mice with the R1245H amino acid substitution, show cardiomyopathy due to shortening of the actin thin filaments. Mechanistically, Flii is a known actin binding protein that we show associates with tropomodulin-1 (TMOD1) to regulate sarcomere thin filament length. Indeed, overexpression of leiomodin-2 in the heart, which lengthens the actin-containing thin filaments, partially rescued disease due to heart-specific deletion of Flii . Collectively, the identified FLII human variant likely increases cardiomyopathy risk through an alteration in sarcomere structure and associated contractile dynamics, like other sarcomere gene-based familial cardiomyopathies.

Background: Acute kidney injury (AKI) is a short-term life-threatening condition that, if survived, can lead to renal insufficiency and development of chronic kidney disease. The pathogenesis of AKI and chronic kidney disease involves direct effects on the heart and the development of hypertrophy and cardiomyopathy., Methods: We used mouse models of ischemia/reperfusion AKI and unilateral ureteral obstruction to investigate the role of IL-33 (interleukin-33) and its receptor-encoding gene Il1rl1 (also called ST2L [suppression of tumorigenicity 2]) in cardiac remodeling after AKI. Mice with cell type-specific genetic disruption of the IL-33/ST2L axis were used, and IL-33 monoclonal antibody, adeno-associated virus encoding IL-33 or ST2L, and recombinant IL-33, as well., Results: Mice deficient in Il33 were refractory to cardiomyopathy associated with 2 models of kidney injury. Treatment of mice with monoclonal IL-33 antibody also protected the heart after AKI. Moreover, overexpression of IL-33 or injection of recombinant IL-33 induced cardiac hypertrophy or cardiomyopathy, but not in mice lacking Il1rl1 . AKI-induced cardiomyopathy was also reduced in mice with cardiac myocyte-specific deletion of Il1rl1 but not in endothelial cell- or fibroblast-specific deletion of Il1rl1 . Last, overexpression of the ST2L receptor in cardiac myocytes recapitulated induction of cardiac hypertrophy., Conclusions: These results indicate that IL-33 released from the kidney during AKI underlies cardiorenal syndrome by directly signaling to cardiac myocytes, suggesting that antagonism of IL-33/ST2 axis would be cardioprotective in patients with kidney disease.

Introduction: The ribosomal protein L3-like (RPL3L) is a heart and skeletal muscle-specific ribosomal protein and paralogue of the more ubiquitously expressed RPL3 protein. Mutations in the human RPL3L gene are linked to childhood cardiomyopathy and age-related atrial fibrillation, yet the function of RPL3L in the mammalian heart remains unknown. Methods and Results: Here, we observed that mouse cardiac ventricles express RPL3 at birth, where it is gradually replaced by RPL3L in adulthood but re-expressed with induction of hypertrophy in adults. Rpl3l gene-deleted mice were generated to examine the role of this gene in the heart, although Rpl3l -/- mice showed no overt changes in cardiac structure or function at baseline or after pressure overload hypertrophy, likely because RPL3 expression was upregulated and maintained in adulthood. mRNA expression analysis and ribosome profiling failed to show differences between the hearts of Rpl3l null and wild type mice in adulthood. Moreover, ribosomes lacking RPL3L showed no differences in localization within cardiomyocytes compared to wild type controls, nor was there an alteration in cardiac tissue ultrastructure or mitochondrial function in adult Rpl3l -/- mice. Similarly, overexpression of either RPL3 or RPL3L with adeno-associated virus -9 in the hearts of mice did not cause discernable pathology. However, by 18 months of age Rpl3l -/- null mice had significantly smaller hearts compared to wild type littermates. Conclusion: Thus, deletion of Rpl3l forces maintenance of RPL3 expression within the heart that appears to fully compensate for the loss of RPL3L, although older Rpl3l -/- mice showed a mild but significant reduction in heart weight., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Grimes, Prasad, Huo, Kuwabara, Vanhoutte, Baldwin, Bowers, Johansen, Sargent, Lin and Molkentin.)

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals., Competing Interests: Declaration of interests R.Y., J.K., J.A.G., V.C., and K.D.P. are listed as inventors on a patent application filed by Duke University on methods for enhancing tissue regeneration. C.A.G. is an inventor on patents and patent applications related to epigenome editing, is a co-founder/advisor of Tune Therapeutics and Locus Biosciences, and is an advisor to Sarepta Therapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In vivo testing of glucocorticoid steroids in dystrophic mice offers important insights in benefits and risks of those drugs in the pathological context of muscular dystrophy. Frequency of dosing changes the spectrum of glucocorticoid effects on muscle and metabolic homeostasis. Here, we describe a combination of non-invasive and invasive methods to quantitatively discriminate the specific effects of intermittent (once-weekly) versus mainstay (once-daily) regimens on muscle fibrosis, muscle function, and metabolic homeostasis in murine models of Duchenne and limb-girdle muscular dystrophies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2/SMαA in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair remained unknown. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal post-MI survival rate. Moreover, Acta2 deletion did not affect the function or histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT and Acta2-null cardiac myofibroblasts. Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a significant compensatory increase in the transcription level of non-Acta2 actin isoforms, especially Actg2 and Acta1. Moreover, in myofibroblasts, the transcription levels of cytoplasmic actin isoforms were significantly higher than those of muscle actin isoforms. In addition, we found that myocardin-related transcription factor-A is critical for myofibroblast differentiation but is not required for the compensatory effects of non-Acta2 isoforms. In conclusion, the Acta2 deletion does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increased expression and stress fiber formation of non-SMαA actin isoforms and the functional redundancy between actin isoforms are able to compensate for the loss of Acta2 in cardiac myofibroblasts., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca 2+ ) entry through Ca v 1 channels triggered Ca 2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca 2+ -dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Objective: Mitochondrial capacity is critical to adapt the high energy demand of the heart to circadian oscillations and diseased states. Glucocorticoids regulate the circadian cycle of energy metabolism, but little is known about how circadian timing of exogenous glucocorticoid dosing directly regulates heart metabolism through cardiomyocyte-autonomous mechanisms. While chronic once-daily intake of glucocorticoids promotes metabolic stress and heart failure, we recently discovered that intermittent once-weekly dosing of exogenous glucocorticoids promoted muscle metabolism in normal and obese skeletal muscle. However, the effects of glucocorticoid intermittence on heart metabolism and heart failure remain unknown. Here we investigated the extent to which circadian time of dosing regulates the effects of the glucocorticoid prednisone in heart metabolism and function in conditions of single pulse or chronic intermittent dosing., Methods and Results: In WT mice, we found that prednisone improved cardiac content of NAD + and ATP with light-phase dosing (ZT0), while the effects were blocked by dark-phase dosing (ZT12). The drug effects on mitochondrial function were cardiomyocyte-autonomous, as shown by inducible cardiomyocyte-restricted glucocorticoid receptor (GR) ablation, and depended on an intact cardiomyocyte clock, as shown by inducible cardiomyocyte-restricted ablation of Brain and Muscle ARNT-like 1 (BMAL1). Conjugating time-of-dosing with chronic intermittence, we found that once-weekly prednisone improved metabolism and function in heart after myocardial injury dependent on circadian time of intake, i.e. with light-phase but not dark-phase dosing., Conclusions: Our study identifies cardiac-autonomous mechanisms through which circadian-specific intermittent dosing reconverts glucocorticoid drugs to metabolic boosters for the heart., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Skeletal muscle can repair and regenerate due to resident stem cells known as satellite cells. The muscular dystrophies are progressive muscle wasting diseases underscored by chronic muscle damage that is continually repaired by satellite cell-driven regeneration. Here we generate a genetic strategy to mediate satellite cell ablation in dystrophic mouse models to investigate how satellite cells impact disease trajectory. Unexpectedly, we observe that depletion of satellite cells reduces dystrophic disease features, with improved histopathology, enhanced sarcolemmal stability and augmented muscle performance. Mechanistically, we demonstrate that satellite cells initiate expression of the myogenic transcription factor MyoD, which then induces re-expression of fetal genes in the myofibers that destabilize the sarcolemma. Indeed, MyoD re-expression in wildtype adult skeletal muscle reduces membrane stability and promotes histopathology, while MyoD inhibition in a mouse model of muscular dystrophy improved membrane stability. Taken together these observations suggest that satellite cell activation and the fetal gene program is maladaptive in chronic dystrophic skeletal muscle., (© 2022. The Author(s).)

Cell communication is needed for organ function and stress responses, especially in the heart. Cardiac fibroblasts, cardiomyocytes, immune cells, and endothelial cells comprise the major cell types in ventricular myocardium that together coordinate all functional processes. Critical to this cellular network is the non-cellular extracellular matrix (ECM) that provides structure and harbors growth factors and other signaling proteins that affect cell behavior. The ECM is not only produced and modified by cells within the myocardium, largely cardiac fibroblasts, it also acts as an avenue for communication among all myocardial cells. In this Review, we discuss how the development of therapeutics to combat cardiac diseases, specifically fibrosis, relies on a deeper understanding of how the cardiac ECM is intertwined with signaling processes that underlie cellular activation and behavior., Competing Interests: Competing Interests All authors declare no conflicts of interest.

The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both the UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (Ub G76V -mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both Ub G76V -mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with Zfp418 overexpression in HEK293 cells, we evaluated Zfp418 knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from Zfp418 knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activated the promoters of Dapk2 and Fyco1 , which are involved in autophagy. RNA-seq from Zfp418 knockdown revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function. Abbreviations: ACTN2, actinin alpha 2; ALP, autophagy-lysosomal pathway; COPB1, COPI coat complex subunit beta 1; DAPK2, death associated protein kinase 2; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; HEK293, human embryonic kidney cells 293; HTS, high-throughput screen; LC3, microtubule associated protein 1 light chain 3; NRVMs, neonatal rat ventricular myocytes; RNA-seq, RNA sequencing; RPS6, ribosomal protein S6; TNNI3, troponin I, cardiac 3; UPS, ubiquitin-proteasome system; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; VPS28, VPS28 subunit of ESCRT-I; ZNF418/ZFP418, zinc finger protein 418.

Background: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy., Methods: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1 WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1 WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1 WT/S52F mice and determine its effects on PH and RV hypertrophy., Results: Foxf1 WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1 WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1 WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1 WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling., Conclusions: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

The world is currently in a pandemic of COVID-19 (Coronavirus disease-2019) caused by a novel positive-sense, single-stranded RNA β-coronavirus referred to as SARS-CoV-2. Here we investigated rates of SARS-CoV-2 infection in the greater Cincinnati, Ohio, USA metropolitan area from August 13 to December 8, 2020, just prior to initiation of the national vaccination program. Examination of 9,550 adult blood donor volunteers for serum IgG antibody positivity against the SARS-CoV-2 Spike protein showed an overall prevalence of 8.40%, measured as 7.56% in the first 58 days and 9.24% in the last 58 days, and 12.86% in December 2020, which we extrapolated to ~20% as of March, 2021. Males and females showed similar rates of past infection, and rates among Hispanic or Latinos, African Americans and Whites were also investigated. Donors under 30 years of age had the highest rates of past infection, while those over 60 had the lowest. Geographic analysis showed higher rates of infectivity on the West side of Cincinnati compared with the East side (split by I-75) and the lowest rates in the adjoining region of Kentucky (across the Ohio river). These results in regional seroprevalence will help inform efforts to best achieve herd immunity in conjunction with the national vaccination campaign., Competing Interests: he authors have declared that no competing interests exist.

The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1 -/- mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart.

Aims: One unaddressed aspect of healing after myocardial infarction (MI) is how non-myocyte cells that survived the ischemic injury, keep withstanding additional cellular damage by stress forms typically arising during the post-infarction inflammation. Here we aimed to determine if cell survival is conferred by expression of a mitochondrial protein novel to the cardiac proteome, known as steroidogenic acute regulatory protein, (StAR/STARD1). Further studies aimed to unravel the regulation and role of the non-steroidogenic cardiac StAR after MI., Methods and Results: Following permanent ligation of the left anterior descending coronary artery in mouse heart, timeline western blot analyses showed that StAR expression corresponds to the inflammatory response to MI. Following the identification of StAR in mitochondria of cardiac fibroblasts in culture, confocal microscopy immunohistochemistry (IHC) identified StAR expression in left ventricular (LV) activated interstitial fibroblasts, adventitial fibroblasts and endothelial cells. Further work with the primary fibroblasts model revealed that interleukin-1α (IL-1α) signaling via NF-κB and p38 MAPK pathways efficiently upregulates the expression of the Star gene products. At the functional level, IL-1α primed fibroblasts were protected against apoptosis when exposed to cisplatin mimicry of in vivo apoptotic stress; yet, the protective impact of IL-1α was lost upon siRNA mediated StAR downregulation. At the physiological level, StAR expression was nullified during post-MI inflammation in a mouse model with global IL-1α deficiency, concomitantly resulting in a 4-fold elevation of apoptotic fibroblasts. Serial echocardiography and IHC studies of mice examined 24 days after MI revealed aggravation of LV dysfunction, LV dilatation, anterior wall thinning and adverse tissue remodeling when compared with loxP control hearts., Conclusions: This study calls attention to overlooked aspects of cellular responses evolved under the stress conditions associated with the default inflammatory response to MI. Our observations suggest that LV IL-1α is cardioprotective, and at least one mechanism of this action is mediated by induction of StAR expression in border zone fibroblasts, which renders them apoptosis resistant. This acquired survival feature also has long-term ramifications on the heart recovery by diminishing adverse remodeling and improving the heart function after MI., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

Aims: Cyclophilin-D is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischaemia/reperfusion injury. However, the binding of CypD to the PTP is poorly understood. Cysteine 202 (C202) of CypD is highly conserved among species and can undergo redox-sensitive post-translational modifications. We investigated whether C202 regulates the opening of PTP., Methods and Results: We developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). Infarct size is reduced in CypD-C202S Langendorff perfused hearts compared to wild type (WT). Cardiac mitochondria from CypD-C202S mice also have higher calcium retention capacity compared to WT. Therefore, we hypothesized that oxidation of C202 might target CypD to the PTP. Indeed, isolated cardiac mitochondria subjected to oxidative stress exhibit less binding of CypD-C202S to the proposed PTP component F1F0-ATP-synthase. We previously found C202 to be S-nitrosylated in ischaemic preconditioning. Cysteine residues can also undergo S-acylation, and C202 matched an S-acylation motif. S-acylation of CypD-C202 was assessed using a resin-assisted capture (Acyl-RAC). WT hearts are abundantly S-acylated on CypD C202 under baseline conditions indicating that S-acylation on C202 per se does not lead to PTP opening. CypD C202S knock-in hearts are protected from ischaemia/reperfusion injury suggesting further that lack of CypD S-acylation at C202 is not detrimental (when C is mutated to S) and does not induce PTP opening. However, we find that ischaemia leads to de-acylation of C202 and that calcium overload in isolated mitochondria promotes de-acylation of CypD. Furthermore, a high bolus of calcium in WT cardiac mitochondria displaces CypD from its physiological binding partners and possibly renders it available for interaction with the PTP., Conclusions: Taken together the data suggest that with ischaemia CypD is de-acylated at C202 allowing the free cysteine residue to undergo oxidation during the first minutes of reperfusion which in turn targets it to the PTP., (Published by Oxford University Press on behalf of the European Society of Cardiology 2020. This work is written by US Government employees and is in the public domain in the US.)

Recent genome-wide association studies (GWAS) identified DUSP8, encoding a dual-specificity phosphatase targeting mitogen-activated protein kinases, as a type 2 diabetes (T2D) risk gene. Here, we reveal that Dusp8 is a gatekeeper in the hypothalamic control of glucose homeostasis in mice and humans. Male, but not female, Dusp8 loss-of-function mice, either with global or corticotropin-releasing hormone neuron-specific deletion, had impaired systemic glucose tolerance and insulin sensitivity when exposed to high-fat diet (HFD). Mechanistically, we found impaired hypothalamic-pituitary-adrenal axis feedback, blunted sympathetic responsiveness, and chronically elevated corticosterone levels driven by hypothalamic hyperactivation of Jnk signaling. Accordingly, global Jnk1 ablation, AAV-mediated Dusp8 overexpression in the mediobasal hypothalamus, or metyrapone-induced chemical adrenalectomy rescued the impaired glucose homeostasis of obese male Dusp8-KO mice, respectively. The sex-specific role of murine Dusp8 in governing hypothalamic Jnk signaling, insulin sensitivity, and systemic glucose tolerance was consistent with functional MRI data in human volunteers that revealed an association of the DUSP8 rs2334499 risk variant with hypothalamic insulin resistance in men. Further, expression of DUSP8 was increased in the infundibular nucleus of T2D humans. In summary, our findings suggest the GWAS-identified gene Dusp8 as a novel hypothalamic factor that plays a functional role in the etiology of T2D.

Arterial macrophages have different developmental origins, but the association of macrophage ontogeny with their phenotypes and functions in adulthood is still unclear. Here, we combine macrophage fate-mapping analysis with single-cell RNA sequencing to establish their cellular identity during homeostasis, and in response to angiotensin-II (AngII)-induced arterial inflammation. Yolk sac erythro-myeloid progenitors (EMP) contribute substantially to adventitial macrophages and give rise to a defined cluster of resident immune cells with homeostatic functions that is stable in adult mice, but declines in numbers during ageing and is not replenished by bone marrow (BM)-derived macrophages. In response to AngII inflammation, increase in adventitial macrophages is driven by recruitment of BM monocytes, while EMP-derived macrophages proliferate locally and provide a distinct transcriptional response that is linked to tissue regeneration. Our findings thus contribute to the understanding of macrophage heterogeneity, and associate macrophage ontogeny with distinct functions in health and disease.

During the postnatal period in mammals, the cardiac muscle transitions from hyperplasic to hypertrophic growth, the extracellular matrix (ECM) undergoes remodeling, and the heart loses regenerative capacity. While ECM maturation and crosstalk between cardiac fibroblasts (CFs) and cardiomyocytes (CMs) have been implicated in neonatal heart development, not much is known about specialized fibroblast heterogeneity and function in the early postnatal period. In order to better understand CF functions in heart maturation and postnatal cardiomyocyte cell-cycle arrest, we have performed gene expression profiling and ablation of postnatal CF populations. Fibroblast lineages expressing Tcf21 or Periostin were traced in transgenic GFP reporter mice, and their biological functions and transitions during the postnatal period were examined in sorted cells using RNA sequencing. Highly proliferative Periostin (Postn)+ lineage CFs were found from postnatal day 1 (P1) to P11 but were not detected at P30, due to a repression of Postn gene expression. This population was less abundant and transcriptionally different from Tcf21+ resident CFs. The specialized Postn+ population preferentially expresses genes related to cell proliferation and neuronal development, while Tcf21+ CFs differentially express genes related to ECM maturation at P7 and immune crosstalk at P30. Ablation of the Postn+ CFs from P0 to P6 led to altered cardiac sympathetic nerve patterning and a reduction in binucleation and hypertrophic growth with increased fetal troponin (TroponinI1) expression in CM. Thus, postnatal CFs are heterogeneous and include a transient proliferative Postn+ population required for cardiac nerve development and cardiomyocyte maturation soon after birth., Competing Interests: The authors declare no competing interest.

Rationale: Mitochondrial Ca 2+ loading augments oxidative metabolism to match functional demands during times of increased work or injury. However, mitochondrial Ca 2+ overload also directly causes mitochondrial rupture and cardiomyocyte death during ischemia-reperfusion injury by inducing mitochondrial permeability transition pore opening. The MCU (mitochondrial Ca 2+ uniporter) mediates mitochondrial Ca 2+ influx, and its activity is modulated by partner proteins in its molecular complex, including the MCUb subunit., Objective: Here, we sought to examine the function of the MCUb subunit of the MCU-complex in regulating mitochondria Ca 2+ influx dynamics, acute cardiac injury, and long-term adaptation after ischemic injury., Methods and Results: Cardiomyocyte-specific MCUb overexpressing transgenic mice and Mcub gene-deleted ( Mcub - /- ) mice were generated to dissect the molecular function of this protein in the heart. We observed that MCUb protein is undetectable in the adult mouse heart at baseline, but mRNA and protein are induced after ischemia-reperfusion injury. MCUb overexpressing mice demonstrated inhibited mitochondrial Ca 2+ uptake in cardiomyocytes and partial protection from ischemia-reperfusion injury by reducing mitochondrial permeability transition pore opening. Antithetically, deletion of the Mcub gene exacerbated pathological cardiac remodeling and infarct expansion after ischemic injury in association with greater mitochondrial Ca 2+ uptake. Furthermore, hindlimb remote ischemic preconditioning induced MCUb expression in the heart, which was associated with decreased mitochondrial Ca 2+ uptake, collectively suggesting that induction of MCUb protein in the heart is protective. Similarly, mouse embryonic fibroblasts from Mcub -/- mice were more sensitive to Ca 2+ overload., Conclusions: Our studies suggest that Mcub is a protective cardiac inducible gene that reduces mitochondrial Ca 2+ influx and permeability transition pore opening after ischemic injury to reduce ongoing pathological remodeling.

The adenosine nucleotide translocase (ANT) family of proteins are inner mitochondrial membrane proteins involved in energy homeostasis and cell death. The primary function of ANT proteins is to exchange cytosolic ADP with matrix ATP, facilitating the export of newly synthesized ATP to the cell while providing new ADP substrate to the mitochondria. As such, the ANT proteins are central to maintaining energy homeostasis in all eukaryotic cells. Evidence also suggests that the ANTs constitute a pore-forming component of the mitochondrial permeability transition pore (MPTP), a structure that forms in the inner mitochondrial membrane that is thought to underlie regulated necrotic cell death. Additionally, emerging studies suggest that ANT proteins are also critical for mitochondrial uncoupling and for promoting mitophagy. Thus, the ANTs are multifunctional proteins that are poised to participate in several aspects of mitochondrial biology and the greater regulation of cell death, which will be discussed here., Competing Interests: Declaration of Competing Interest All authors confirm no conflict of interest with the current study., (Copyright © 2020 Elsevier Ltd. All rights reserved.)