A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Protein Kinase A Is a Master Regulator of Physiological and Pathological Cardiac Hypertrophy.

BACKGROUND: The sympathoadrenergic system and its major effector PKA (protein kinase A) are activated to maintain cardiac output coping with physiological or pathological stressors. If and how PKA plays a role in physiological cardiac hypertrophy (PhCH) and pathological CH (PaCH) are not clear. METHODS: Transgenic mouse models expressing the PKA inhibition domain (PKAi) of PKA inhibition peptide alpha (PKIalpha)-green fluorescence protein (GFP) fusion protein (PKAi-GFP) in a cardiac-specific and inducible manner (cPKAi) were used to determine the roles of PKA in physiological CH during postnatal growth or induced by swimming, and in PaCH induced by transaortic constriction (TAC) or augmented Ca2+ influx. Kinase profiling was used to determine cPKAi specificity. Echocardiography was used to determine cardiac morphology and function. Western blotting and immunostaining were used to measure protein abundance and phosphorylation. Protein synthesis was assessed by puromycin incorporation and protein degradation by measuring protein ubiquitination and proteasome activity. Neonatal rat cardiomyocytes (NRCMs) infected with AdGFP (GFP adenovirus) or AdPKAi-GFP (PKAi-GFP adenovirus) were used to determine the effects and mechanisms of cPKAi on myocyte hypertrophy. rAAV9.PKAi-GFP was used to treat TAC mice. RESULTS: (1) cPKAi delayed postnatal cardiac growth and blunted exercise-induced PhCH; (2) PKA was activated in hearts after TAC due to activated sympathoadrenergic system, the loss of endogenous PKIα (PKA inhibition peptide α), and the stimulation by noncanonical PKA activators; (3) cPKAi ameliorated PaCH induced by TAC and increased Ca2+ influxes and blunted neonatal rat cardiomyocyte hypertrophy by isoproterenol and phenylephrine; (4) cPKAi prevented TAC-induced protein synthesis by inhibiting mTOR (mammalian target of rapamycin) signaling through reducing Akt (protein kinase B) activity, but enhancing inhibitory GSK-3α (glycogen synthase kinase-3α) and GSK-3ß signals; (5) cPKAi reduced protein degradation by the ubiquitin-proteasome system via decreasing RPN6 phosphorylation; (6) cPKAi increased the expression of antihypertrophic atrial natriuretic peptide (ANP); (7) cPKAi ameliorated established PaCH and improved animal survival. CONCLUSIONS: Cardiomyocyte PKA is a master regulator of PhCH and PaCH through regulating protein synthesis and degradation. cPKAi can be a novel approach to treat PaCH.

Metabolic Reprogramming: A Byproduct or a Driver of Cardiomyocyte Proliferation?

Defining mechanisms of cardiomyocyte proliferation should guide the understanding of endogenous cardiac regeneration and could lead to novel treatments for diseases such as myocardial infarction. In the neonatal heart, energy metabolic reprogramming (phenotypic alteration of glucose, fatty acid, and amino acid metabolism) parallels cell cycle arrest of cardiomyocytes. The metabolic reprogramming occurring shortly after birth is associated with alterations in blood oxygen levels, metabolic substrate availability, hemodynamic stress, and hormone release. In the adult heart, myocardial infarction causes metabolic reprogramming but these changes cannot stimulate sufficient cardiomyocyte proliferation to replace those lost by the ischemic injury. Some putative pro-proliferative interventions can induce the metabolic reprogramming. Recent data show that altering the metabolic enzymes PKM2 [pyruvate kinase 2], LDHA [lactate dehydrogenase A], PDK4 [pyruvate dehydrogenase kinase 4], SDH [succinate dehydrogenase], CPT1b [carnitine palmitoyl transferase 1b], or HMGCS2 [3-hydroxy-3-methylglutaryl-CoA synthase 2] is sufficient to partially reverse metabolic reprogramming and promotes adult cardiomyocyte proliferation. How metabolic reprogramming regulates cardiomyocyte proliferation is not clearly defined. The possible mechanisms involve biosynthetic pathways from the glycolysis shunts and the epigenetic regulation induced by metabolic intermediates. Metabolic manipulation could represent a new approach to stimulate cardiac regeneration; however, the efficacy of these manipulations requires optimization, and novel molecular targets need to be defined. In this review, we summarize the features, triggers, and molecular regulatory networks responsible for metabolic reprogramming and discuss the current understanding of metabolic reprogramming as a critical determinant of cardiomyocyte proliferation.

The American Heart Association at 100: A Century of Scientific Progress and the Future of Cardiovascular Science: A Presidential Advisory From the American Heart Association.

In 1924, the founders of the American Heart Association (AHA) envisioned an international society focused on the heart and aimed at facilitating research, disseminating information, increasing public awareness, and developing public health policy related to heart disease. This presidential advisory provides a comprehensive review of the past century of cardiovascular and stroke science, with a focus on the AHA's contributions, as well as informed speculation about the future of cardiovascular science into the next century of the organization's history. The AHA is a leader in fundamental, translational, clinical, and population science, and it promotes the concept of the "learning health system," in which a continuous cycle of evidence-based practice leads to practice-based evidence, permitting an iterative refinement in clinical evidence and care. This advisory presents the AHA's journey over the past century from instituting professional membership to establishing extraordinary research funding programs; translating evidence to practice through clinical practice guidelines; affecting systems of care through quality programs, certification, and implementation; leading important advocacy efforts at the federal, state and local levels; and building global coalitions around cardiovascular and stroke science and public health. Recognizing an exciting potential future for science and medicine, the advisory offers a vision for even greater impact for the AHA's second century in its continued mission to be a relentless force for longer, healthier lives.

Functional LTCC-ß2AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure.

BACKGROUND: Beta-2 adrenergic receptors (ß2ARs) but not beta-2 adrenergic receptors (ß1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and ß adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. METHODS: Global signaling between LTCCs and ß adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and ß1AR or ß2AR in different membrane microdomains in control and failing cardiomyocytes. RESULTS: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when ß2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and ß2AR was lost. Interestingly, local stimulation of ß1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via ß1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the ß2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream ß1AR and results in an increase in LTCC current. CONCLUSIONS: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via ß2AR, but not ß1AR. This may explain how ß2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

Systemic Hypoxemia Induces Cardiomyocyte Hypertrophy and Right Ventricular Specific Induction of Proliferation.

BACKGROUND: A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce adult cardiomyocyte cell cycle reentry, and to determine if hypoxemia also induces cardiomyocyte proliferation in female mice. METHODS: EdU-containing mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were placed in a hypoxia chamber, and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% oxygen for 2 weeks before terminal studies. Myocyte proliferation was also studied with a mosaic analysis with double markers mouse model. RESULTS: Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced an increase (0.01±0.01% in normoxia to 0.11±0.09% in hypoxia) in the number of EdU+ RV cardiomyocytes, with no effect on LV myocytes in male C57BL/6 mice. Similar results were observed in female mice. Furthermore, in mosaic analysis with double markers mice, hypoxia induced a significant increase in RV myocyte proliferation (0.03±0.03% in normoxia to 0.32±0.15% in hypoxia of RFP+ myocytes), with no significant change in LV myocyte proliferation. RNA sequencing showed upregulation of mitotic cell cycle genes and a downregulation of Cullin genes, which promote the G1 to S phase transition in hypoxic mice. There was significant proliferation of nonmyocytes and mild cardiac fibrosis in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression following hypoxia. CONCLUSIONS: Systemic hypoxia induces a global hypertrophic stress response that was associated with increased RV proliferation, and while LV myocytes did not show increased proliferation, our results minimally confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in vivo.

RNA-Binding Protein LIN28a Regulates New Myocyte Formation in the Heart Through Long Noncoding RNA-H19.

BACKGROUND: Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear diploid cardiomyocytes. On maturation, mononuclear diploid cardiomyocytes become binucleated or polyploid and exit the cell cycle. Cardiomyocyte metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative mononuclear diploid cardiomyocytes enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism in cardiac repair after injury. METHODS: LIN28a overexpression was tested using mouse transgenesis on postnatal cardiomyocyte numbers, cell cycle, and response to apical resection injury. With the use of neonatal and adult cell culture systems and adult and Mosaic Analysis with Double Markers myocardial injury models in mice, the effect of LIN28a overexpression on cardiomyocyte cell cycle and metabolism was tested. Last, isolated adult cardiomyocytes from LIN28a and wild-type mice 4 days after myocardial injury were used for RNA-immunoprecipitation sequencing. RESULTS: LIN28a was found to be active primarily during cardiac development and rapidly decreases after birth. LIN28a reintroduction at postnatal day (P) 1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing cardiomyocyte cell cycle activity in LIN28a transgenic pups compared with wild-type littermates. Moreover, LIN28a overexpression extended cardiomyocyte cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated cardiomyocyte apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared with wild-type littermate controls. Instead, LIN28a small molecule inhibitor attenuated the proreparative effects of LIN28a on the heart. Neonatal rat ventricular myocytes overexpressing LIN28a mechanistically showed increased glycolysis, ATP production, and levels of metabolic enzymes compared with control. LIN28a immunoprecipitation followed by RNA-immunoprecipitation sequencing in cardiomyocytes isolated from LIN28a-overexpressing hearts after injury identified long noncoding RNA-H19 as its most significantly altered target. Ablation of long noncoding RNA-H19 blunted LIN28a-induced enhancement on cardiomyocyte metabolism and cell cycle activity. CONCLUSIONS: Collectively, LIN28a reprograms cardiomyocyte metabolism and promotes persistence of mononuclear diploid cardiomyocytes in the injured heart, enhancing proreparative processes, thereby linking cardiomyocyte metabolism to regulation of ploidy/nucleation and repair in the heart.

Assessing the Impact of the American Heart Association's Research Portfolio: A Scientific Statement From the American Heart Association.

A task force composed of American Heart Association (AHA) Research Committee members established processes to measure the performance of the AHA's research portfolio and evaluated key outcomes that are fundamental to the overall success of the program. This report reviews progress that the AHA research program has had in achieving its goals relevant to the research programs in the AHA's research portfolio from 2008 to 2017. Comprehensive performance metrics were identified to assess the impact of AHA funding on researchers' career progress and research outcomes. Metrics included bibliometric analysis (ie, tracking of publications and their impact) and career development measures (ie, subsequent grant funding, intellectual property, faculty appointment/promotion, or industry position). Publication rates ranged from ≈0.5 to 4 publications per year, with a strong correlation between number of publications per year and later career stage. The Field-Weighted Citation Index, a metric of bibliometric impact, was between 1.5 and 3.0 for all programs, indicating that AHA awardee publications had a higher citation impact compared with similar publications. To gain insight into the career progression of AHA awardees, a 2-year postaward survey was distributed. Of the Postdoctoral Fellowship recipient respondents, 72% obtained academic research positions, with the remaining working in industry or government research settings; 72% of those in academic positions obtained additional funding. Among respondents who were Beginning Grant-in-Aid and Scientist Development Grant awardees, 45% received academic promotions and 83% obtained additional funding. Measuring performance of the AHA's research portfolio is critical to ensure that its strategic goals are met and to show the AHA's commitment to high-quality, impactful research.

Effects of maternal hypothyroidism on postnatal cardiomyocyte proliferation and cardiac disease responses of the progeny.

Maternal hypothyroidism (MH) could adversely affect the cardiac disease responses of the progeny. This study tested the hypothesis that MH reduces early postnatal cardiomyocyte (CM) proliferation so that the adult heart of MH progeny has a smaller number of larger cardiac myocytes, which imparts adverse cardiac disease responses following injury. Thyroidectomy (TX) was used to establish MH. The progeny from mice that underwent sham or TX surgery were termed Ctrl (control) or MH (maternal hypothyroidism) progeny, respectively. MH progeny had similar heart weight (HW) to body weight (BW) ratios and larger CM size consistent with fewer CMs at postnatal day 60 (P60) compared with Ctrl (control) progeny. MH progeny had lower numbers of EdU+, Ki67+, and phosphorylated histone H3 (PH3)+ CMs, which suggests they had a decreased CM proliferation in the postnatal timeframe. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH hearts, including bone morphogenetic protein 10 (Bmp10). Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation. After transverse aortic constriction (TAC), the MH progeny had more severe cardiac pathological remodeling compared with the Ctrl progeny. Thyroid hormone (T4) treatment for MH mothers preserved their progeny's postnatal CM proliferation capacity and prevented excessive pathological remodeling after TAC. Our results suggest that CM proliferation during early postnatal development was significantly reduced in MH progeny, resulting in fewer CMs with hypertrophy in adulthood. These changes were associated with more severe cardiac disease responses after pressure overload.NEW & NOTEWORTHY Our study shows that compared with Ctrl (control) progeny, the adult progeny of mothers who have MH (MH progeny) had fewer CMs. This reduction of CM numbers was associated with decreased postnatal CM proliferation. Gene expression studies showed a reduced expression of Bmp10 in MH progeny. Bmp10 has been linked to myocyte proliferation. In vivo and in vitro studies showed that Bmp10 treatment of MH progeny and their myocytes could increase CM proliferation. Differences in CM number and size in adult hearts of MH progeny were linked to more severe cardiac structural and functional remodeling after pressure overload. T4 (synthetic thyroxine) treatment of MH mothers during their pregnancy, prevented the reduction in CM number in their progeny and the adverse response to disease stress.

Combining three independent pathological stressors induces a heart failure with preserved ejection fraction phenotype.

Heart failure (HF) with preserved ejection fraction (HFpEF) is defined as HF with an ejection fraction (EF) ≥ 50% and elevated cardiac diastolic filling pressures. The underlying causes of HFpEF are multifactorial and not well-defined. A transgenic mouse with low levels of cardiomyocyte (CM)-specific inducible Cavß2a expression (ß2a-Tg mice) showed increased cytosolic CM Ca2+, and modest levels of CM hypertrophy, and fibrosis. This study aimed to determine if ß2a-Tg mice develop an HFpEF phenotype when challenged with two additional stressors, high-fat diet (HFD) and Nω-nitro-l-arginine methyl ester (l-NAME, LN). Four-month-old wild-type (WT) and ß2a-Tg mice were given either normal chow (WT-N, ß2a-N) or HFD and/or l-NAME (WT-HFD, WT-LN, WT-HFD-LN, ß2a-HFD, ß2a-LN, and ß2a-HFD-LN). Some animals were treated with the histone deacetylase (HDAC) (hypertrophy regulators) inhibitor suberoylanilide hydroxamic acid (SAHA) (ß2a-HFD-LN-SAHA). Echocardiography was performed monthly. After 4 mo of treatment, terminal studies were performed including invasive hemodynamics and organs weight measurements. Cardiac tissue was collected. Four months of HFD plus l-NAME treatment did not induce a profound HFpEF phenotype in FVB WT mice. ß2a-HFD-LN (3-Hit) mice developed features of HFpEF, including increased atrial natriuretic peptide (ANP) levels, preserved EF, diastolic dysfunction, robust CM hypertrophy, increased M2-macrophage population, and myocardial fibrosis. SAHA reduced the HFpEF phenotype in the 3-Hit mouse model, by attenuating these effects. The 3-Hit mouse model induced a reliable HFpEF phenotype with CM hypertrophy, cardiac fibrosis, and increased M2-macrophage population. This model could be used for identifying and preclinical testing of novel therapeutic strategies.NEW & NOTEWORTHY Our study shows that three independent pathological stressors (increased Ca2+ influx, high-fat diet, and l-NAME) together produce a profound HFpEF phenotype. The primary mechanisms include HDAC-dependent-CM hypertrophy, necrosis, increased M2-macrophage population, fibroblast activation, and myocardial fibrosis. A role for HDAC activation in the HFpEF phenotype was shown in studies with SAHA treatment, which prevented the severe HFpEF phenotype. This "3-Hit" mouse model could be helpful in identifying novel therapeutic strategies to treat HFpEF.

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

RATIONALE: Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. OBJECTIVE: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. METHODS AND RESULTS: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after ß-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. CONCLUSIONS: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability.

Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.

SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore.

Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca(2+)- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca(2+) retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca(2+) and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca(2+)- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.

HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling.

BACKGROUND: Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction. METHODS: Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. RESULTS: HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements. CONCLUSIONS: These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.

Cardiac Remodeling During Pregnancy With Metabolic Syndrome: Prologue of Pathological Remodeling.

BACKGROUND: The heart undergoes physiological hypertrophy during pregnancy in healthy individuals. Metabolic syndrome (MetS) is now prevalent in women of child-bearing age and might add risks of adverse cardiovascular events during pregnancy. The present study asks if cardiac remodeling during pregnancy in obese individuals with MetS is abnormal and whether this predisposes them to a higher risk for cardiovascular disorders. METHODS: The idea that MetS induces pathological cardiac remodeling during pregnancy was studied in a long-term (15 weeks) Western diet-feeding animal model that recapitulated features of human MetS. Pregnant female mice with Western diet (45% kcal fat)-induced MetS were compared with pregnant and nonpregnant females fed a control diet (10% kcal fat). RESULTS: Pregnant mice fed a Western diet had increased heart mass and exhibited key features of pathological hypertrophy, including fibrosis and upregulation of fetal genes associated with pathological hypertrophy. Hearts from pregnant animals with WD-induced MetS had a distinct gene expression profile that could underlie their pathological remodeling. Concurrently, pregnant female mice with MetS showed more severe cardiac hypertrophy and exacerbated cardiac dysfunction when challenged with angiotensin II/phenylephrine infusion after delivery. CONCLUSIONS: These results suggest that preexisting MetS could disrupt physiological hypertrophy during pregnancy to produce pathological cardiac remodeling that could predispose the heart to chronic disorders.

Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Loss of Protease-Activated Receptor 4 Prevents Inflammation Resolution and Predisposes the Heart to Cardiac Rupture After Myocardial Infarction.

BACKGROUND: Cardiac rupture is a major lethal complication of acute myocardial infarction (MI). Despite significant advances in reperfusion strategies, mortality from cardiac rupture remains high. Studies suggest that cardiac rupture can be accelerated by thrombolytic therapy, but the relevance of this risk factor remains controversial. METHODS: We analyzed protease-activated receptor 4 (Par4) expression in mouse hearts with MI and investigated the effects of Par4 deletion on cardiac remodeling and function after MI by echocardiography, quantitative immunohistochemistry, and flow cytometry. RESULTS: Par4 mRNA and protein levels were increased in mouse hearts after MI and in isolated cardiomyocytes in response to hypertrophic and inflammatory stimuli. Par4-deficient mice showed less myocyte apoptosis, reduced infarct size, and improved functional recovery after acute MI relative to wild-type (WT). Conversely, Par4-/- mice showed impaired cardiac function, greater rates of myocardial rupture, and increased mortality after chronic MI relative to WT. Pathological evaluation of hearts from Par4-/- mice demonstrated a greater infarct expansion, increased cardiac hemorrhage, and delayed neutrophil accumulation, which resulted in impaired post-MI healing compared with WT. Par4 deficiency also attenuated neutrophil apoptosis in vitro and after MI in vivo and impaired inflammation resolution in infarcted myocardium. Transfer of Par4-/- neutrophils, but not of Par4-/- platelets, in WT recipient mice delayed inflammation resolution, increased cardiac hemorrhage, and enhanced cardiac dysfunction. In parallel, adoptive transfer of WT neutrophils into Par4-/- mice restored inflammation resolution, reduced cardiac rupture incidence, and improved cardiac function after MI. CONCLUSIONS: These findings reveal essential roles of Par4 in neutrophil apoptosis and inflammation resolution during myocardial healing and point to Par4 inhibition as a potential therapy that should be limited to the acute phases of ischemic insult and avoided for long-term treatment after MI.

Postsurgery echocardiography can predict the amount of ischemia-reperfusion injury and the resultant scar size.

Despite advances in the diagnosis and treatment of ischemic heart disease (IHD), it remains the leading cause of death globally. Thus, there is a need to investigate the underlying pathophysiology and develop new therapies for the prevention and treatment of IHD. Murine models are widely used in IHD research because they are readily available, relatively inexpensive, and can be genetically modified to explore mechanistic questions. Ischemia-reperfusion (I/R)-induced myocardial infarction in mice is produced by the blockage followed by reperfusion of the left anterior descending branch (LAD) to imitate human IHD disease and its treatment. This I/R model can be widely used to investigate the potential reparative effect of putative treatments in the setting of reperfusion. However, the surgical technique is demanding and can produce an inconsistent amount of damage, which can make identification of treatment effects challenging. Therefore, determining which hearts have been significantly damaged by I/R is an important consideration in studies designed to either explore the mechanisms of disrupted function or test possible therapies. Noninvasive echocardiography (ECHO) is often used to determine structural and functional changes in the mouse heart following injury. In the present study, we determined that ECHO performed 3 days post I/R surgery could predict the permanent injury produced by the ischemic insult.NEW & NOTEWORTHY We believe our work is noteworthy due to its creation of standards for early evaluation of the level of myocardial injury in mouse models of ischemia-reperfusion. This improvement to study design could reduce the sample sizes used in evaluating therapeutics and lead to increased confidence in conclusions drawn regarding the therapeutic efficacy of treatments tested in these translational mouse models.

Cortical bone stem cell-derived exosomes' therapeutic effect on myocardial ischemia-reperfusion and cardiac remodeling.

Heart failure is the one of the leading causes of death in the United States. Heart failure is a complex syndrome caused by numerous diseases, including severe myocardial infarction (MI). MI occurs after an occlusion of a cardiac artery causing downstream ischemia. MI is followed by cardiac remodeling involving extensive remodeling and fibrosis, which, if the original insult is severe or prolonged, can ultimately progress into heart failure. There is no "cure" for heart failure because therapies to regenerate dead tissue are not yet available. Previous studies have shown that in both post-MI and post-ischemia-reperfusion (I/R) models of heart failure, administration of cortical bone stem cell (CBSC) treatment leads to a reduction in scar size and improved cardiac function. Our first study investigated the ability of mouse CBSC-derived exosomes (mCBSC-dEXO) to recapitulate mouse CBSCs (mCBSC) therapeutic effects in a 24-h post-I/R model. This study showed that injection of mCBSCs and mCBSC-dEXOs into the ischemic region of an infarct had a protective effect against I/R injury. mCBSC-dEXOs recapitulated the effects of CBSC treatment post-I/R, indicating exosomes are partly responsible for CBSC's beneficial effects. To examine if exosomes decrease fibrotic activation, adult rat ventricular fibroblasts (ARVFs) and adult human cardiac fibroblasts (NHCFs) were treated with transforming growth factor ß (TGFß) to activate fibrotic signaling before treatment with mCBSC- and human CBSC (hCBSC)-dEXOs. hCBSC-dEXOs caused a 100-fold decrease in human fibroblast activation. To further understand the signaling mechanisms regulating the protective decrease in fibrosis, we performed RNA sequencing on the NHCFs after hCBSC-dEXO treatment. The group treated with both TGFß and exosomes showed a decrease in small nucleolar RNA (snoRNA), known to be involved with ribosome stability.NEW & NOTEWORTHY Our work is noteworthy due to the identification of factors within stem cell-derived exosomes (dEXOs) that alter fibroblast activation through the hereto-unknown mechanism of decreasing small nucleolar RNA (snoRNA) signaling within cardiac fibroblasts. The study also shows that the injection of stem cells or a stem-cell-derived exosome therapy at the onset of reperfusion elicits cardioprotection, emphasizing the importance of early treatment in the post-ischemia-reperfusion (I/R) wounded heart.

Cortical bone stem cells modify cardiac inflammation after myocardial infarction by inducing a novel macrophage phenotype.

Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury that is part of a complex and highly organized wound-healing process. Cortical bone stem cell (CBSC) therapy after MI has been shown to reduce adverse structural and functional remodeling of the heart after MI in both mouse and swine models. The basis for these CBSC treatment effects on wound healing are unknown. The present experiments show that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably macrophage colony-stimulating factor (M-CSF) and transforming growth factor-ß, but not IL-4. CBSC therapy increased the number of galectin-3+ macrophages, CD4+ T cells, and fibroblasts in the heart while decreasing apoptosis in an in vivo swine model of MI. Macrophages treated with CBSC medium in vitro polarized to a proreparative phenotype are characterized by increased CD206 expression, increased efferocytic ability, increased IL-10, TGF-ß, and IL-1RA secretion, and increased mitochondrial respiration. Next generation sequencing revealed a transcriptome significantly different from M2a or M2c macrophage phenotypes. Paracrine factors from CBSC-treated macrophages increased proliferation, decreased α-smooth muscle actin expression, and decreased contraction by fibroblasts in vitro. These data support the idea that CBSCs are modulating the immune response to MI to favor cardiac repair through a unique macrophage polarization that ultimately reduces cell death and alters fibroblast populations that may result in smaller scar size and preserved cardiac geometry and function.NEW & NOTEWORTHY Cortical bone stem cell (CBSC) therapy after myocardial infarction alters the inflammatory response to cardiac injury. We found that cortical bone stem cell therapy induces a unique macrophage phenotype in vitro and can modulate macrophage/fibroblast cross talk.