Future Impact of mRNA Therapy on Cardiovascular Diseases.

The silver lining of the recent pandemic was that it accelerated the emergence of messenger ribonucleic acid (mRNA) therapeutics. The great promise of mRNA therapeutics was highlighted by the speed at which the vaccines were created, tested, and proven to be relatively safe and highly effective. There are a wide variety of mRNA therapeutics now under development, and dozens of these are in clinical trials. These therapeutics are generating a major paradigm shift in medical therapy, including the treatment of cardiovascular disease. Most of the cardiovascular mRNA therapies are still in preclinical development, although a phase 2a trial of mRNA therapy for myocardial ischemia has been completed with promising results.1 The application of mRNA therapies to cardiovascular diseases is virtually limitless, and ongoing work includes mRNA therapies for myocardial ischemia, heart failure, arrhythmias, hypercholesterolemia, and arterial occlusive diseases. In addition, mRNA may be used to enhance cell therapies. In the future, mRNA therapies for cardiovascular disease are destined to supplant some of our current biologics and pharmacotherapies and will be used to treat previously untreatable cardiovascular diseases. Furthermore, mRNA therapies can be personalized, and they can be rapidly generated in current Good Manufacturing Practice facilities with a modest footprint, facilitating the rise of hospital-based regional centers of RNA therapeutics.

An In Vitro Platform to Study Reversible Endothelial-to-Mesenchymal Transition.

Endothelial cells can acquire a mesenchymal phenotype in response to external stimuli through both mechanical and biological factors, using a process known as endothelial-to-mesenchymal (EndoMT) transition. EndoMT is characterized by the decrease in endothelial characteristics, increase in mesenchymal markers, and morphological changes. It has been recognized not only during development but also in different pathological conditions including organ/tissue fibrosis in adults. The ability to modulate the EndoMT process could have a therapeutic potential in many fibrotic diseases. An in vitro method is presented here to induce EndoMT with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) and angiotensin II (Ang II) followed by a protocol to study the reversibility of EndoMT. Using this method, we furnish evidence that the combination of L-NAME and Ang II can stimulate EndoMT in Human umbilical vascular endothelial cells (HUVECs) and this process can be reversed as observed using endothelial functionality assays. This method may serve as a model to screen and identify potential pharmacological molecules to target and regulate the EndoMT process, with applications in drug discovery for human diseases.

STK35 Gene Therapy Attenuates Endothelial Dysfunction and Improves Cardiac Function in Diabetes.

Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis that leads to progressive heart failure. The mechanisms underlying DCM pathogenesis remain obscure, and no effective treatments for the disease have been available. In the present study, we observed that STK35, a novel kinase, is decreased in the diabetic human heart. High glucose treatment, mimicking hyperglycemia in diabetes, downregulated STK35 expression in mouse cardiac endothelial cells (MCEC). Knockdown of STK35 attenuated MCEC proliferation, migration, and tube formation, whereas STK35 overexpression restored the high glucose-suppressed MCEC migration and tube formation. Angiogenesis gene PCR array analysis revealed that HG downregulated the expression of several angiogenic genes, and this suppression was fully restored by STK35 overexpression. Intravenous injection of AAV9-STK35 viral particles successfully overexpressed STK35 in diabetic mouse hearts, leading to increased vascular density, suppression of fibrosis in the heart, and amelioration of left ventricular function. Altogether, our results suggest that hyperglycemia downregulates endothelial STK35 expression, leading to microvascular dysfunction in diabetic hearts, representing a novel mechanism underlying DCM pathogenesis. Our study also emerges STK35 is a novel gene therapeutic target for preventing and treating DCM.

Endothelial Dysfunction-related Neurological Bleeds with Continuous Flow-Left Ventricular Assist Devices Measured by Digital Thermal Monitor.

Endothelial dysfunction has been demonstrated in patients with Continuous Flow-Left Ventricular Assist Devices (CF-LVADs) but association with adverse events has not been shown. We used a noninvasive, operator-independent device called VENDYS® to assess vasodilatory function based on digital thermal measurements postrelease of a brachial artery occlusion in ambulatory patients with CF-LVAD (n = 56). Aortic valve opening and pulse perception were also documented before the test. Median duration of CF-LVAD support was 438 days. The VENDYS® test generates a vascular reactivity index (VRI). Outcomes for the CF-LVAD patients were compared between VRI < 1 and VRI ≥ 1. The bleeding events were driven primarily by a difference in neurologic bleeds. Multivariate analysis showed that VRI < 1 correlated with future bleeding events (HR: 5.56; P = 0.01). The C-statistic with the VRI dichotomized as above was 0.82. There was a trend toward a worse survival in patients with poor endothelial function. Endothelial vasodilatory dysfunction measured by a simple test utilizing digital thermal monitoring can predict adverse bleeding events in patients with CF-LVADs.

Small molecule disruption of G protein ßγ subunit signaling reprograms human macrophage phenotype and prevents autoimmune myocarditis in rats.

The purpose of this study was to determine whether blocking of G protein ßγ (Gßγ) signaling halts heart failure (HF) progression by macrophage phenotype manipulation. Cardiac Gßγ signaling plays a crucial role in HF pathogenesis. Previous data suggested that inhibiting Gßγ signaling reprograms T helper cell 1 (Th1) and Th2 cytokines, suggesting that Gßγ might be a useful drug target for treating HF. We investigated the efficacy of a small molecule Gßγ inhibitor, gallein, in a clinically relevant, experimental autoimmune myocarditis (EAM) model of HF as well as in human macrophage phenotypes in vitro. In the myocardium of HF patients, we observed that G protein coupled receptor kinase (GRK)2 levels were down-regulated compared with healthy controls. In rat EAM, treatment with gallein effectively improved survival and cardiac function, suppressed cardiac remodeling, and further attenuated myocardial protein expression of GRK2 as well as high mobility group box (HMGB)1 and its cascade signaling proteins. Furthermore, gallein effectively inhibited M1 polarization and promoted M2 polarization in vivo in the EAM heart and in vitro in human monocyte-derived macrophages. Taken together, these data suggest that the small molecule Gßγ inhibitor, gallein, could be an important pharmacologic therapy for HF as it can switch the phenotypic reprogramming from M1 to M2 phenotype in a rat model of EAM heart and in human macrophages.

Efficacy of sustained delivery of GC-1 from a Nanofluidic system in a spontaneously obese non-human primate: a case study.

With nearly 40% of U.S. adults obese, and childhood and adolescent rates rising, obesity and associated comorbidities are serious public health concerns with massive societal costs. Often, lifestyle interventions do not offer sufficient weight loss to improve health, requiring surgery and medications as adjunct management strategies. Here, we present a 4-month case study in which the sustained, low-dose, and constant administration of the thyroid receptor ß selective agonist GC-1 (sobetirome) from a novel nanochannel membrane implant was assessed in an obese, pre-diabetic rhesus macaque. Dramatic loss of white adipose tissue in the abdomen from 36 to 18% was observed via magnetic resonance imaging in conjunction with normalized serum insulin and glycemia, with no signs of cardiotoxicity shown. The non-human primate study highlights sustained low-dose delivery of GC-1 from our minimally invasive subcutaneous implant as a valuable approach to induce weight loss and manage obesity and comorbidities, including type 2 diabetes.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.IMPORTANCE Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.

Functionally redundant control of cardiac hypertrophic signaling by inositol 1,4,5-trisphosphate receptors.

Calcium plays an integral role to many cellular processes including contraction, energy metabolism, gene expression, and cell death. The inositol 1, 4, 5-trisphosphate receptor (IP3R) is a calcium channel expressed in cardiac tissue. There are three IP3R isoforms encoded by separate genes. In the heart, the IP3R-2 isoform is reported to being most predominant with regards to expression levels and functional significance. The functional roles of IP3R-1 and IP3R-3 in the heart are essentially unexplored despite measureable expression levels. Here we show that all three IP3Rs isoforms are expressed in both neonatal and adult rat ventricular cardiomyocytes, and in human heart tissue. The three IP3R proteins are expressed throughout the cardiomyocyte sarcoplasmic reticulum. Using isoform specific siRNA, we found that expression of all three IP3R isoforms are required for hypertrophic signaling downstream of endothelin-1 stimulation. Mechanistically, IP3Rs specifically contribute to activation of the hypertrophic program by mediating the positive inotropic effects of endothelin-1 and leading to downstream activation of nuclear factor of activated T-cells. Our findings highlight previously unidentified functions for IP3R isoforms in the heart with specific implications for hypertrophic signaling in animal models and in human disease.

Rapamycin nanoparticles localize in diseased lung vasculature and prevent pulmonary arterial hypertension.

Vascular remodeling resulting from pulmonary arterial hypertension (PAH) leads to endothelial fenestrations. This feature can be exploited by nanoparticles (NP), allowing them to extravasate from circulation and accumulate in remodeled pulmonary vessels. Hyperactivation of the mTOR pathway in PAH drives pulmonary arterial smooth muscle cell proliferation. We hypothesized that rapamycin (RAP)-loaded NPs, an mTOR inhibitor, would accumulate in diseased lungs, selectively targeting vascular mTOR and preventing PAH progression. RAP poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) NPs were fabricated. NP accumulation and efficacy were examined in a rat monocrotaline model of PAH. Following intravenous (IV) administration, NP accumulation in diseased lungs was verified via LC/MS analysis and confocal imaging. Pulmonary arteriole thickness, right ventricular systolic pressures, and ventricular remodeling were determined to assess the therapeutic potential of RAP NPs. Monocrotaline-exposed rats showed increased NP accumulation within lungs compared to healthy controls, with NPs present to a high extent within pulmonary perivascular regions. RAP, in both free and NP form, attenuated PAH development, with histological analysis revealing minimal changes in pulmonary arteriole thickness and no ventricular remodeling. Importantly, NP-treated rats showed reduced systemic side effects compared to free RAP. This study demonstrates the potential for nanoparticles to significantly impact PAH through site-specific delivery of therapeutics.

AIBP Limits Angiogenesis Through γ-Secretase-Mediated Upregulation of Notch Signaling.

RATIONALE: Angiogenesis improves perfusion to the ischemic tissue after acute vascular obstruction. Angiogenesis in pathophysiological settings reactivates signaling pathways involved in developmental angiogenesis. We showed previously that AIBP (apolipoprotein A-I [apoA-I]-binding protein)-regulated cholesterol efflux in endothelial cells controls zebra fish embryonic angiogenesis. OBJECTIVE: This study is to determine whether loss of AIBP affects angiogenesis in mice during development and under pathological conditions and to explore the underlying molecular mechanism. METHODS AND RESULTS: In this article, we report the generation of AIBP knockout (Apoa1bp-/-) mice, which are characterized of accelerated postnatal retinal angiogenesis. Mechanistically, AIBP triggered relocalization of γ-secretase from lipid rafts to nonlipid rafts where it cleaved Notch. Consistently, AIBP treatment enhanced DLL4 (delta-like ligand 4)-stimulated Notch activation in human retinal endothelial cells. Increasing high-density lipoprotein levels in Apoa1bp-/- mice by crossing them with apoA-I transgenic mice rescued Notch activation and corrected dysregulated retinal angiogenesis. Notably, the retinal vessels in Apoa1bp-/- mice manifested normal pericyte coverage and vascular integrity. Similarly, in the subcutaneous Matrigel plug assay, which mimics ischemic/inflammatory neovascularization, angiogenesis was dramatically upregulated in Apoa1bp-/- mice and associated with a profound inhibition of Notch activation and reduced expression of downstream targets. Furthermore, loss of AIBP increased vascular density and facilitated the recovery of blood vessel perfusion function in a murine hindlimb ischemia model. In addition, AIBP expression was significantly increased in human patients with ischemic cardiomyopathy. CONCLUSIONS: Our data reveal a novel mechanistic connection between AIBP-mediated cholesterol metabolism and Notch signaling, implicating AIBP as a possible druggable target to modulate angiogenesis under pathological conditions.

MicroRNA-126 overexpression rescues diabetes-induced impairment in efferocytosis of apoptotic cardiomyocytes.

Efferocytosis, a process of clearance of apoptotic cells by phagocytes, is essential for successful resolution of inflammation and maintenance of tissue homeostasis. Diabetes compromises the function of macrophages leading to adverse inflammatory response during wound healing, myocardial injury, atherosclerosis and autoimmune disorders. However, the effect of diabetes on macrophage-mediated efferocytosis of apoptotic cardiomyocytes (ACM) and the molecular mechanisms involved are not understood so far. In the present study we found that invitro efferocytosis of ACM was impaired in macrophages from db/db (diabetic) mice. Macrophages exposed to high glucose (HG) decreases microRNA-126 (miR-126) expression with a corresponding increase in ADAM9 expression. Dual-luciferase reporter assay confirms that ADAM9 3'UTR contains miR-126 target site. ADAM9 inhibition reduces HG-induced proteolytic cleavage of Mer tyrosine receptor kinase (MerTK, a proto-oncogene that plays a critical role in phagocytosis), resulting in shedding of soluble-Mer (sMER) and loss of MERTK function. Over-expression of miR-126 attenuates HG-induced impairment of efferocytosis. Furthermore, human diabetic hearts show lower miR-126 expression with a corresponding increase in ADAM9 expression vs. normal counterparts. These data suggests that diabetes impairs efferocytosis of ACM and that strategies to enhance efferocytosis might attenuate diabetes-induced impairment in inflammation resolution and cardiac repair after injury.

MicroRNA-9 inhibits hyperglycemia-induced pyroptosis in human ventricular cardiomyocytes by targeting ELAVL1.

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associated with underlying chronic inflammation and cardiac cell death, subsequently leading to heart failure (HF). ELAV-like protein 1 (ELAVL1) plays a critical role in the progression of inflammation and HF. However the role of ELAVL-1 in inflammation induced cardiac cell death (pyroptosis) under hyperglycemic condition remains elusive. Our data demonstrates that ELAVL1 expression augmented with a concomitant increase in caspase-1 and IL-1 beta expression in human hearts and human ventricular cardiomyocytes under hyperglycemic condition. Furthermore, ELAVL1 knockdown abrogates TNF-α induced canonical pyroptosis via NLRP3, caspase-1 and IL-1beta suppression. Bioinformatics analysis and target validation assays showed that miR-9 directly targets ELAVL1. Interestingly, miRNA-9 expression significantly reduced in high glucose treated cardiomyocytes and in human diabetic hearts. Inhibition of miR-9 upregulates ELAVL1 expression and activates caspase-1. Alternatively, treatment with miR-9 mimics attenuates hyperglycemia-induced ELAVL1 and inhibits cardiomyocyte pyroptosis. Taken together our study highlights the potential therapeutic implications of targeting miR-9/ELAVL1 in preventing cardiomyocyte cell loss during HF in diabetics.

Full Expression of Cardiomyopathy Is Partly Dependent on B-Cells: A Pathway That Involves Cytokine Activation, Immunoglobulin Deposition, and Activation of Apoptosis.

BACKGROUND: Limited information exists on the role of B-cell-dependent mechanisms in the progression of heart failure (HF). However, in failing human myocardium, there is evidence of deposition of activated complement components as well as anticardiac antibodies. We aimed to determine the contribution of B-cells in HF progression using a nonsurgical mouse model of nonischemic cardiomyopathy (CMP). METHODS AND RESULTS: CMP protocol involved the use of l-NAME and NaCl in the drinking water and angiotensin-II infusion for 35 days. At day 35, mice were analyzed by cardiac magnetic resonance imaging, gene expression, and histology. Mice (12 weeks old) were divided into 4 groups, all in C57BL/6 background: wild-type (WT) CMP; severe combined immunodeficiency (SCID) CMP (T- and B-cell deficient); CD22(-) CMP (B-cell depleted); and Nude CMP (T-cell deficient), with their respective controls. We performed B-cell depletion and reconstitution protocols. The protective effect of B-cell depletion was demonstrated by a significant reduction of cell hypertrophy and collagen deposition and a preserved ejection fraction in the CD22(-) CMP group compared to WT CMP. Once SCID mice underwent B-cell reconstitution with isolated CMP B-cells, the CMP phenotype was restored. Furthermore, deposition of IgG3 and apoptosis in the myocardium follows the development of CMP; in addition, in vitro studies demonstrated that activated B-cells stimulate collagen production by cardiac fibroblasts. CONCLUSIONS: The absence of B-cells in this model of HF resulted in less hypertrophy and collagen deposition, preservation of left ventricular function, and, in association with these changes, a reduction in expression of proinflammatory cytokines, immunoglobulin G deposition, and apoptosis in the myocardium. Taken together, these data suggest that B-cells play a contributory role in an angiotensin-II-induced HF model.

Rnd3/RhoE Modulates Hypoxia-Inducible Factor 1α/Vascular Endothelial Growth Factor Signaling by Stabilizing Hypoxia-Inducible Factor 1α and Regulates Responsive Cardiac Angiogenesis.

The insufficiency of compensatory angiogenesis in the heart of patients with hypertension contributes to heart failure transition. The hypoxia-inducible factor 1α-vascular endothelial growth factor (HIF1α-VEGF) signaling cascade controls responsive angiogenesis. One of the challenges in reprograming the insufficient angiogenesis is to achieve a sustainable tissue exposure to the proangiogenic factors, such as HIF1α stabilization. In this study, we identified Rnd3, a small Rho GTPase, as a proangiogenic factor participating in the regulation of the HIF1α-VEGF signaling cascade. Rnd3 physically interacted with and stabilized HIF1α, and consequently promoted VEGFA expression and endothelial cell tube formation. To demonstrate this proangiogenic role of Rnd3 in vivo, we generated Rnd3 knockout mice. Rnd3 haploinsufficient (Rnd3(+/-)) mice were viable, yet developed dilated cardiomyopathy with heart failure after transverse aortic constriction stress. The poststress Rnd3(+/-) hearts showed significantly impaired angiogenesis and decreased HIF1α and VEGFA expression. The angiogenesis defect and heart failure phenotype were partially rescued by cobalt chloride treatment, a HIF1α stabilizer, confirming a critical role of Rnd3 in stress-responsive angiogenesis. Furthermore, we generated Rnd3 transgenic mice and demonstrated that Rnd3 overexpression in heart had a cardioprotective effect through reserved cardiac function and preserved responsive angiogenesis after pressure overload. Finally, we assessed the expression levels of Rnd3 in the human heart and detected significant downregulation of Rnd3 in patients with end-stage heart failure. We concluded that Rnd3 acted as a novel proangiogenic factor involved in cardiac responsive angiogenesis through HIF1α-VEGFA signaling promotion. Rnd3 downregulation observed in patients with heart failure may explain the insufficient compensatory angiogenesis involved in the transition to heart failure.

Combination of angiotensin II and l-NG-nitroarginine methyl ester exacerbates mitochondrial dysfunction and oxidative stress to cause heart failure.

Mitochondrial dysfunction has been implicated as a cause of energy deprivation in heart failure (HF). Herein, we tested individual and combined effects of two pathogenic factors of nonischemic HF, inhibition of nitric oxide synthesis [with l-N(G)-nitroarginine methyl ester (l-NAME)] and hypertension [with angiotensin II (AngII)], on myocardial mitochondrial function, oxidative stress, and metabolic gene expression. l-NAME and AngII were administered individually and in combination to mice for 5 wk. Although all treatments increased blood pressure and reduced cardiac contractile function, the l-NAME + AngII group was associated with the most severe HF, as characterized by edema, hypertrophy, oxidative stress, increased expression of Nppa and Nppb, and decreased expression of Atp2a2 and Camk2b. l-NAME + AngII-treated mice exhibited robust deterioration of cardiac mitochondrial function, as observed by reduced respiratory control ratios in subsarcolemmal mitochondria and reduced state 3 levels in interfibrillar mitochondria for complex I but not for complex II substrates. Cardiac myofibrils showed reduced ADP-supported and oligomycin-inhibited oxygen consumption. Mitochondrial functional impairment was accompanied by reduced mitochondrial DNA content and activities of pyruvate dehydrogenase and complex I but increased H2O2 production and tissue protein carbonyls in hearts from AngII and l-NAME + AngII groups. Microarray analyses revealed the majority of the gene changes attributed to the l-NAME + AngII group. Pathway analyses indicated significant changes in metabolic pathways, such as oxidative phosphorylation, mitochondrial function, cardiac hypertrophy, and fatty acid metabolism in l-NAME + AngII hearts. We conclude that l-NAME + AngII is associated with impaired mitochondrial respiratory function and increased oxidative stress compared with either l-NAME or AngII alone, resulting in nonischemic HF.

A specifically designed nanoconstruct associates, internalizes, traffics in cardiovascular cells, and accumulates in failing myocardium: a new strategy for heart failure diagnostics and therapeutics.

AIMS: Ongoing inflammation and endothelial dysfunction occurs within the local microenvironment of heart failure, creating an appropriate scenario for successful use and delivery of nanovectors. This study sought to investigate whether cardiovascular cells associate, internalize, and traffic a nanoplatform called mesoporous silicon vector (MSV), and determine its intravenous accumulation in cardiac tissue in a murine model of heart failure. METHODS AND RESULTS: In vitro cellular uptake and intracellular trafficking of MSVs was examined by scanning electron microscopy, confocal microscopy, time-lapse microscopy, and flow cytometry in cardiac myocytes, fibroblasts, smooth muscle cells, and endothelial cells. The MSVs were internalized within the first hours, and trafficked to perinuclear regions in all the cell lines. Cytotoxicity was investigated by annexin V and cell cycle assays. No significant evidence of toxicity was found. In vivo intravenous cardiac accumulation of MSVs was examined by high content fluorescence and confocal microscopy, with results showing increased accumulation of particles in failing hearts compared with normal hearts. Similar to observations in vitro, MSVs were able to associate, internalize, and traffic to the perinuclear region of cardiomyocytes in vivo. CONCLUSIONS: Results show that MSVs associate, internalize, and traffic in cardiovascular cells without any significant toxicity. Furthermore, MSVs accumulate in failing myocardium after intravenous administration, reaching intracellular regions of the cardiomyocytes. These findings represent a novel avenue to develop nanotechnology-based therapeutics and diagnostics in heart failure.

Differential Mitochondrial Function in Remodeled Right and Nonremodeled Left Ventricles in Pulmonary Hypertension.

OBJECTIVES: Right ventricular failure is the primary reason for mortality in pulmonary hypertension (PH), but little is understood about the energetics of the failing right myocardium. Our aim was to examine mitochondrial function and proteomic signatures in paired remodeled right (RM-RV) and non-remodeled left (NRM-LV) ventricular tissue samples procured during heart-lung transplantation. METHODS AND RESULTS: Contractile dysfunction in RM-RV and preserved contractile function in NRM-LV were determined clinically and by echocardiography. Mitochondria were isolated from fresh paired RV and LV wall specimens of explanted hearts. Respiratory states in response to 4 substrates and an uncoupler were analyzed. Proteomic analysis on the mitochondrial isolates was performed with the use of liquid chromatography-mass spectrometry. The RM-RV mitochondria exhibited higher succinate state 4 levels with lower respiratory control ratio (RCR) compared with state 4 levels for pyruvate-malate (PM) and glutamate-malate (GM). RM-RV mitochondria also exhibited lower state 3 for palmitoyl-carnitine (PC) and state 4 for all complex I substrates compared with NRM-LV. The mean RCR were greater in RM-RVs than in NRM-LVs for PM and GM, which is consistent with tight coupling (low state 4 rates, higher RCRs); however, low RM-RV state 3 rates suggest concurrent substrate-dependent impairment in respiratory capacity. Mitochondrial proteomics revealed greater levels of mitochondrial ADP-ATP translocase and proteins of ATP synthesis, mitochondrial pyruvate and short branched chain acyl-CoA metabolism in RM-RV. CONCLUSIONS: The mitochondrial respiration and proteomics in RM-RV are different from NRM-LV. These results have important implications in expanding our understanding of RV metabolism and future management of RV failure.

Enhanced Cardiac Regenerative Ability of Stem Cells After Ischemia-Reperfusion Injury: Role of Human CD34+ Cells Deficient in MicroRNA-377.

BACKGROUND: MicroRNA (miR) dysregulation in the myocardium has been implicated in cardiac remodeling after injury or stress. OBJECTIVES: The aim of this study was to explore the role of miR in human CD34(+) cell (hCD34(+)) dysfunction in vivo after transplantation into the myocardium under ischemia-reperfusion (I-R) conditions. METHODS: In response to inflammatory stimuli, the miR array profile of endothelial progenitor cells was analyzed using a polymerase chain reaction-based miR microarray. miR-377 expression was assessed in myocardial tissue from human patients with heart failure (HF). We investigated the effect of miR-377 inhibition on an hCD34(+) cell angiogenic proteome profile in vitro and on cardiac repair and function after I-R injury in immunodeficient mice. RESULTS: The miR array data from endothelial progenitor cells in response to inflammatory stimuli indicated changes in numerous miR, with a robust decrease in the levels of miR-377. Human cardiac biopsies from patients with HF showed significant increases in miR-377 expression compared with nonfailing control hearts. The proteome profile of hCD34(+) cells transfected with miR-377 mimics showed significant decrease in the levels of proangiogenic proteins versus nonspecific control-transfected cells. We also validated that serine/threonine kinase 35 is a target of miR-377 using a dual luciferase reporter assay. In a mouse model of myocardial I-R, intramyocardial transplantation of miR-377 silenced hCD34(+) cells in immunodeficient mice, promoting neovascularization (at 28 days, post-I-R) and lower interstitial fibrosis, leading to improved left ventricular function. CONCLUSIONS: These findings indicate that HF increased miR-377 expression in the myocardium, which is detrimental to stem cell function, and transplantation of miR-377 knockdown hCD34(+) cells into ischemic myocardium promoted their angiogenic ability, attenuating left ventricular remodeling and cardiac fibrosis.

Mechanical unloading promotes myocardial energy recovery in human heart failure.

BACKGROUND: Impaired bioenergetics is a prominent feature of the failing heart, but the underlying metabolic perturbations are poorly understood. METHODS AND RESULTS: We compared metabolomic, gene transcript, and protein data from 6 paired samples of failing human left ventricular tissue obtained during left ventricular assist device insertion (heart failure samples) and at heart transplant (post-left ventricular assist device samples). Nonfailing left ventricular wall samples procured from explanted hearts of patients with right heart failure served as novel comparison samples. Metabolomic analyses uncovered a distinct pattern in heart failure tissue: 2.6-fold increased pyruvate concentrations coupled with reduced Krebs cycle intermediates and short-chain acylcarnitines, suggesting a global reduction in substrate oxidation. These findings were associated with decreased transcript levels for enzymes that catalyze fatty acid oxidation and pyruvate metabolism and for key transcriptional regulators of mitochondrial metabolism and biogenesis, peroxisome proliferator-activated receptor γ coactivator 1α (PGC1A, 1.3-fold) and estrogen-related receptor α (ERRA, 1.2-fold) and γ (ERRG, 2.2-fold). Thus, parallel decreases in key transcription factors and their target metabolic enzyme genes can explain the decreases in associated metabolic intermediates. Mechanical support with left ventricular assist device improved all of these metabolic and transcriptional defects. CONCLUSIONS: These observations underscore an important pathophysiologic role for severely defective metabolism in heart failure, while the reversibility of these defects by left ventricular assist device suggests metabolic resilience of the human heart.