Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure.

During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure. Significance Statement: Recently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.

Unlocking the Role of sMyBP-C: A Key Player in Skeletal Muscle Development and Growth.

Skeletal muscle is the largest organ in the body, responsible for gross movement and metabolic regulation. Recently, variants in the MYBPC1 gene have been implicated in a variety of developmental muscle diseases, such as distal arthrogryposis. How MYBPC1 variants cause disease is not well understood. Here, through a collection of novel gene-edited mouse models, we define a critical role for slow myosin binding protein-C (sMyBP-C), encoded by MYBPC1, across muscle development, growth, and maintenance during prenatal, perinatal, postnatal and adult stages. Specifically, Mybpc1 knockout mice exhibited early postnatal lethality and impaired skeletal muscle formation and structure, skeletal deformity, and respiratory failure. Moreover, a conditional knockout of Mybpc1 in perinatal, postnatal and adult stages demonstrates impaired postnatal muscle growth and function secondary to disrupted actomyosin interaction and sarcomere structural integrity. These findings confirm the essential role of sMyBP-C in skeletal muscle and reveal specific functions in both prenatal embryonic musculoskeletal development and postnatal muscle growth and function.

HuR inhibition reduces post-ischemic cardiac remodeling by dampening acute inflammatory gene expression and the innate immune response.

Myocardial ischemia/reperfusion (I/R) injury and the resulting cardiac remodeling is a common cause of heart failure. The RNA binding protein Human Antigen R (HuR) has been previously shown to reduce cardiac remodeling following both I/R and cardiac pressure overload, but the full extent of the HuR-dependent mechanisms within cells of the myocardium have yet to be elucidated. In this study, we applied a novel small molecule inhibitor of HuR to define the functional role of HuR in the acute response to I/R injury and gain a better understanding of the HuR-dependent mechanisms during post-ischemic myocardial remodeling. Our results show an early (two hours post-I/R) increase in HuR activity that is necessary for early inflammatory gene expression by cardiomyocytes in response to I/R. Surprisingly, despite the reductions in early inflammatory gene expression at two hours post-I/R, HuR inhibition has no effect on initial infarct size at 24-hours post-I/R. However, in agreement with previously published work, we do see a reduction in pathological remodeling and preserved cardiac function at two weeks post-I/R upon HuR inhibition. RNA-sequencing analysis of neonatal rat ventricular myocytes (NRVMs) at two hours post-LPS treatment to model damage associated molecular pattern (DAMP)-mediated activation of toll like receptors (TLRs) demonstrates a broad HuR-dependent regulation of pro-inflammatory chemokine and cytokine gene expression in cardiomyocytes. We show that conditioned media from NRVMs pre-treated with HuR inhibitor loses the ability to induce inflammatory gene expression in bone marrow derived macrophages (BMDMs) compared to NRVMs treated with LPS alone. Functionally, HuR inhibition in NRVMs also reduces their ability to induce endocrine migration of peripheral blood monocytes in vitro and reduces post-ischemic macrophage infiltration to the heart in vivo. In summary, these results suggest a HuR-dependent expression of pro-inflammatory gene expression by cardiomyocytes that leads to subsequent monocyte recruitment and macrophage activation in the post-ischemic myocardium.

Pharmacologic Inhibition of Pain Response to Incomplete Vascular Occlusion Blunts Cardiovascular Preconditioning Response.

Complete vascular occlusion to distant tissue prior to an ischemic cardiac event can provide significant cardioprotection via remote ischemic preconditioning (RIPC). Despite understanding its mechanistic basis, its translation to clinical practice has been unsuccessful, likely secondary to the inherent impossibility of predicting (and therefore preconditioning) an ischemic event, as well as the discomfort that is associated with traditional, fully occlusive RIPC stimuli. Our laboratory has previously shown that non-occlusive banding (NOB) via wrapping of a leather band (similar to a traditional Jewish ritual) can elicit an RIPC response in healthy human subjects. This study sought to further the pain-mediated aspect of this observation in a mouse model of NOB with healthy mice that were exposed to treatment with and without lidocaine to inhibit pain sensation prior to ischemia/reperfusion injury. We demonstrated that NOB downregulates key inflammatory markers resulting in a preconditioning response that is partially mediated via pain sensation.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Autonomic dysfunction following traumatic brain injury: translational insights.

Although there is a substantial amount of research on the neurological consequences of traumatic brain injury (TBI), there is a knowledge gap regarding the relationship between TBI and the pathophysiology of organ system dysfunction and autonomic dysregulation. In particular, the mechanisms or incidences of renal or cardiac complications after TBI are mostly unknown. Autonomic dysfunction following TBI exacerbates secondary injury and may contribute to nonneurologial complications that prolong hospital length of stay. Gaining insights into the mechanisms of autonomic dysfunction can guide advancements in monitoring and treatment paradigms to improve acute survival and long-term prognosis of TBI patients. In this paper, the authors will review the literature on autonomic dysfunction after TBI and possible mechanisms of paroxysmal sympathetic hyperactivity. Specifically, they will discuss the link among the brain, heart, and kidneys and review data to direct future research on and interventions for TBI-induced autonomic dysfunction.

V2a Neurons Constrain Extradiaphragmatic Respiratory Muscle Activity at Rest.

Breathing requires precise control of respiratory muscles to ensure adequate ventilation. Neurons within discrete regions of the brainstem produce oscillatory activity to control the frequency of breathing. Less is understood about how spinal and pontomedullary networks modulate the activity of respiratory motor neurons to produce different patterns of activity during different behaviors (i.e., during exercise, coughing, swallowing, vocalizing, or at rest) or following disease or injury. Here, we use a chemogenetic approach to inhibit the activity of glutamatergic V2a neurons in the brainstem and spinal cord of neonatal and adult mice to assess their potential roles in respiratory rhythm generation and patterning respiratory muscle activity. Using whole-body plethysmography (WBP), we show that V2a neuron function is required in neonatal mice to maintain the frequency and regularity of respiratory rhythm. However, silencing V2a neurons in adult mice increases respiratory frequency and ventilation, without affecting regularity. Thus, the excitatory drive provided by V2a neurons is less critical for respiratory rhythm generation in adult compared to neonatal mice. In addition, we used simultaneous EMG recordings of the diaphragm and extradiaphragmatic respiratory muscles in conscious adult mice to examine the role of V2a neurons in patterning respiratory muscle activity. We find that silencing V2a neurons activates extradiaphragmatic respiratory muscles at rest, when they are normally inactive, with little impact on diaphragm activity. Thus, our results indicate that V2a neurons participate in a circuit that serves to constrain the activity of extradiaphragmatic respiratory muscles so that they are active only when needed.

Acoustic droplet vaporization-mediated dissolved oxygen scavenging in blood-mimicking fluids, plasma, and blood.

Acoustic droplet vaporization (ADV) has been shown to reduce the partial pressure of oxygen (PO2) in a fluid. The goals of this study were three-fold: 1) to determine the ADV pressure amplitude threshold in fluids that had physiologically relevant values for surface tension, protein concentration, and viscosity; 2) to assess whether these parameters and fluid mixing affect ADV-mediated PO2 reduction; and 3) to assess the feasibility of ADV-mediated PO2 reduction in plasma and whole blood. In vitro ADV experiments were conducted using perfluoropentane droplets (number density: 5 × 106 ±â€¯0.2 × 106/mL) dispersed in fluids (saline, polyvinylpyrrolidone solutions, porcine plasma, or porcine whole blood) that had a physiological range of surface tensions (62-68 mN/m), protein concentrations (0 and 68.7 mg/mL), and viscosities (0.7-4 cP). Droplets were exposed to pulsed ultrasound (5 MHz, 4.25 MPa peak negative pressure) while passing through a 37 °C flow system with inline PO2 sensors. In select experiments, the fluid also passed through mixing channels after ultrasound exposure. Our results revealed that the ADV pressure thresholds were the same for all fluids. Surface tension and protein concentration had no effect on PO2 reduction. Increasing viscosity attenuated PO2 reduction. However, the attenuated effect was absent after fluid mixing. Furthermore, ADV-mediated PO2 reduction in whole blood (30.8 ±â€¯3.2 mmHg) was less than that in a polyvinylpyrrolidone solution (40.2 ±â€¯2.1 mmHg) with equal viscosity. These findings should be considered when planning clinical studies of ADV-mediated PO2 reduction and other biomedical applications of ADV.

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Human antigen R as a therapeutic target in pathological cardiac hypertrophy.

RNA binding proteins represent an emerging class of proteins with a role in cardiac dysfunction. We show that activation of the RNA binding protein human antigen R (HuR) is increased in the failing human heart. To determine the functional role of HuR in pathological cardiac hypertrophy, we created an inducible cardiomyocyte-specific HuR-deletion mouse and showed that HuR deletion reduces left ventricular hypertrophy, dilation, and fibrosis while preserving cardiac function in a transverse aortic constriction (TAC) model of pressure overload-induced hypertrophy. Assessment of HuR-dependent changes in global gene expression suggests that the mechanistic basis for this protection occurs through a reduction in fibrotic signaling, specifically through a reduction in TGF-ß (Tgfb) expression. Finally, pharmacological inhibition of HuR at a clinically relevant time point following the initial development of pathological hypertrophy after TAC also yielded a significant reduction in pathological progression, as marked by a reduction in hypertrophy, dilation, and fibrosis and preserved function. In summary, this study demonstrates a functional role for HuR in the progression of pressure overload-induced cardiac hypertrophy and establishes HuR inhibition as a viable therapeutic approach for pathological cardiac hypertrophy and heart failure.

NBCe1 Na+-HCO3- cotransporter ablation causes reduced apoptosis following cardiac ischemia-reperfusion injury in vivo.

AIM: To investigate the hypothesis that cardiomyocyte-specific loss of the electrogenic NBCe1 Na+-HCO3 - cotransporter is cardioprotective during in vivo ischemia-reperfusion (IR) injury. METHODS: An NBCe1 (Slc4a4 gene) conditional knockout mouse (KO) model was prepared by gene targeting. Cardiovascular performance of wildtype (WT) and cardiac-specific NBCe1 KO mice was analyzed by intraventricular pressure measurements, and changes in cardiac gene expression were determined by RNA Seq analysis. Response to in vivo IR injury was analyzed after 30 min occlusion of the left anterior descending artery followed by 3 h of reperfusion. RESULTS: Loss of NBCe1 in cardiac myocytes did not impair cardiac contractility or relaxation under basal conditions or in response to ß-adrenergic stimulation, and caused only limited changes in gene expression patterns, such as those for electrical excitability. However, following ischemia and reperfusion, KO heart sections exhibited significantly fewer apoptotic nuclei than WT sections. CONCLUSION: These studies indicate that cardiac-specific loss of NBCe1 does not impair cardiovascular performance, causes only minimal changes in gene expression patterns, and protects against IR injury in vivo .

Cardiac Dysfunction in the Sigma 1 Receptor Knockout Mouse Associated With Impaired Mitochondrial Dynamics and Bioenergetics.

Background The Sigma 1 receptor (Sigmar1) functions as an interorganelle signaling molecule and elicits cytoprotective functions. The presence of Sigmar1 in the heart was first reported on the basis of a ligand-binding assay, and all studies to date have been limited to pharmacological approaches using less-selective ligands for Sigmar1. However, the physiological function of cardiac Sigmar1 remains unknown. We investigated the physiological function of Sigmar1 in regulating cardiac hemodynamics using the Sigmar1 knockout mouse (Sigmar1-/-). Methods and Results Sigmar1-/- hearts at 3 to 4 months of age showed significantly increased contractility as assessed by left ventricular catheterization with stimulation by increasing doses of a ß1-adrenoceptor agonist. Noninvasive echocardiographic measurements were also used to measure cardiac function over time, and the data showed the development of cardiac contractile dysfunction in Sigmar1 -/- hearts as the animals aged. Histochemistry demonstrated significant cardiac fibrosis, collagen deposition, and increased periostin in the Sigmar1 -/- hearts compared with wild-type hearts. Ultrastructural analysis of Sigmar1-/- cardiomyocytes revealed an irregularly shaped, highly fused mitochondrial network with abnormal cristae. Mitochondrial size was larger in Sigmar1-/- hearts, resulting in decreased numbers of mitochondria per microscopic field. In addition, Sigmar1-/- hearts showed altered expression of mitochondrial dynamics regulatory proteins. Real-time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1-/- hearts compared with wild-type hearts. Conclusions We demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Sigmar1 loss of function led to mitochondrial dysfunction, abnormal mitochondrial architecture, and adverse cardiac remodeling, culminating in cardiac contractile dysfunction.

Tranilast Blunts the Hypertrophic and Fibrotic Response to Increased Afterload Independent of Cardiomyocyte Transient Receptor Potential Vanilloid 2 Channels.

Tranilast is clinically indicated for the treatment of allergic disorders and is also a nonselective blocker of the transient receptor potential vanilloid 2 (TRPV2) channel. Previous studies have found that it has protective effects in various animal models of cardiac disease. Our laboratory has found that genetic deletion of TRPV2 results in a blunted hypertrophic response to increased afterload; thus, this study tested the hypothesis that tranilast through cardiomyocyte TRPV2 blockade can inhibit the hypertrophic response to pressure overload in vivo through transverse aortic constriction and ex vivo through isolated myocyte studies. The in vivo studies demonstrated that tranilast blunted the fibrotic response to increased afterload and, to a lesser extent, the hypertrophic response. After 4 weeks, this blunting was associated with improved cardiac function, although at 8 weeks, the cardiac function deteriorated similarly to the control group. Finally, the in vitro studies demonstrated that tranilast was not inhibiting these responses at the cardiomyocyte level. In conclusion, we demonstrated that tranilast blunting of the fibrotic and hypertrophic response occurs independently of cardiac TRPV2 channels and may be cardioprotective in the short term but not after prolonged administration.

Inhibiting Fibronectin Attenuates Fibrosis and Improves Cardiac Function in a Model of Heart Failure.

BACKGROUND: Fibronectin (FN) polymerization is necessary for collagen matrix deposition and is a key contributor to increased abundance of cardiac myofibroblasts (MFs) after cardiac injury. We hypothesized that interfering with FN polymerization or its genetic ablation in fibroblasts would attenuate MF and fibrosis and improve cardiac function after ischemia/reperfusion (I/R) injury. METHODS: Mouse and human MFs were used to assess the impact of the FN polymerization inhibitor (pUR4) in attenuating pathological cellular features such as proliferation, migration, extracellular matrix deposition, and associated mechanisms. To evaluate the therapeutic potential of inhibiting FN polymerization in vivo, wild-type mice received daily intraperitoneal injections of either pUR4 or control peptide (III-11C) immediately after cardiac surgery for 7 consecutive days. Mice were analyzed 7 days after I/R to assess MF markers and inflammatory cell infiltration or 4 weeks after I/R to evaluate long-term effects of FN inhibition on cardiac function and fibrosis. Furthermore, inducible, fibroblast-restricted, FN gene-ablated (Tcf21MerCreMer; Fnflox) mice were used to evaluate cell specificity of FN expression and polymerization in the heart. RESULTS: pUR4 administration on activated MFs reduced FN and collagen deposition into the extracellular matrix and attenuated cell proliferation, likely mediated through decreased c-myc signaling. pUR4 also ameliorated fibroblast migration accompanied by increased ß1 integrin internalization and reduced levels of phosphorylated focal adhesion kinase protein. In vivo, daily administration of pUR4 for 7 days after I/R significantly reduced MF markers and neutrophil infiltration. This treatment regimen also significantly attenuated myocardial dysfunction, pathological cardiac remodeling, and fibrosis up to 4 weeks after I/R. Last, inducible ablation of FN in fibroblasts after I/R resulted in significant functional cardioprotection with reduced hypertrophy and fibrosis. The addition of pUR4 to the FN-ablated mice did not confer further cardioprotection, suggesting that the salutary effects of inhibiting FN polymerization may be mediated largely through effects on FN secreted from the cardiac fibroblast lineage. CONCLUSIONS: Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

RNA SEQ Analysis Indicates that the AE3 Cl-/HCO3- Exchanger Contributes to Active Transport-Mediated CO2 Disposal in Heart.

Loss of the AE3 Cl-/HCO3- exchanger (Slc4a3) in mice causes an impaired cardiac force-frequency response and heart failure under some conditions but the mechanisms are not known. To better understand the functions of AE3, we performed RNA Seq analysis of AE3-null and wild-type mouse hearts and evaluated the data with respect to three hypotheses (CO2 disposal, facilitation of Na+-loading, and recovery from an alkaline load) that have been proposed for its physiological functions. Gene Ontology and PubMatrix analyses of differentially expressed genes revealed a hypoxia response and changes in vasodilation and angiogenesis genes that strongly support the CO2 disposal hypothesis. Differential expression of energy metabolism genes, which indicated increased glucose utilization and decreased fatty acid utilization, were consistent with adaptive responses to perturbations of O2/CO2 balance in AE3-null myocytes. Given that the myocardium is an obligate aerobic tissue and consumes large amounts of O2, the data suggest that loss of AE3, which has the potential to extrude CO2 in the form of HCO3-, impairs O2/CO2 balance in cardiac myocytes. These results support a model in which the AE3 Cl-/HCO3- exchanger, coupled with parallel Cl- and H+-extrusion mechanisms and extracellular carbonic anhydrase, is responsible for active transport-mediated disposal of CO2.

Pharmacological and Activated Fibroblast Targeting of Gßγ-GRK2 After Myocardial Ischemia Attenuates Heart Failure Progression.

BACKGROUND: Cardiac fibroblasts are a critical cell population responsible for myocardial extracellular matrix homeostasis. Upon injury or pathological stimulation, these cells transform to an activated myofibroblast state and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation, a hallmark of heart failure (HF), induces pathological signaling through G protein ßγ (Gßγ) subunits and their interaction with G protein-coupled receptor kinase 2 (GRK2). OBJECTIVES: This study investigated the hypothesis that Gßγ-GRK2 inhibition and/or ablation after myocardial injury would attenuate pathological myofibroblast activation and cardiac remodeling. METHODS: The therapeutic potential of small molecule Gßγ-GRK2 inhibition, alone or in combination with activated fibroblast- or myocyte-specific GRK2 ablation-each initiated after myocardial ischemia-reperfusion (I/R) injury-was investigated to evaluate the possible salutary effects on post-I/R fibroblast activation, pathological remodeling, and cardiac dysfunction. RESULTS: Small molecule Gßγ-GRK2 inhibition initiated 1 week post-injury was cardioprotective in the I/R model of chronic HF, including preservation of cardiac contractility and a reduction in cardiac fibrotic remodeling. Systemic small molecule Gßγ-GRK2 inhibition initiated 1 week post-I/R in cardiomyocyte-restricted GRK2 ablated mice (also post-I/R) still demonstrated significant cardioprotection, which suggested a potential protective role beyond the cardiomyocyte. Inducible ablation of GRK2 in activated fibroblasts (i.e., myofibroblasts) post-I/R injury demonstrated significant functional cardioprotection with reduced myofibroblast transformation and fibrosis. Systemic small molecule Gßγ-GRK2 inhibition initiated 1 week post-I/R provided little to no further protection in mice with ablation of GRK2 in activated fibroblasts alone. Finally, Gßγ-GRK2 inhibition significantly attenuated activation characteristics of failing human cardiac fibroblasts isolated from end-stage HF patients. CONCLUSIONS: These findings suggested consideration of a paradigm shift in the understanding of the therapeutic role of Gßγ-GRK2 inhibition in treating HF and the potential therapeutic role for Gßγ-GRK2 inhibition in limiting pathological myofibroblast activation, interstitial fibrosis, and HF progression.

The non-diuretic hypotensive effects of thiazides are enhanced during volume depletion states.

Thiazide derivatives including Hydrochlorothiazide (HCTZ) represent the most common treatment of mild to moderate hypertension. Thiazides initially enhance diuresis via inhibition of the kidney Na+-Cl- Cotransporter (NCC). However, chronic volume depletion and diuresis are minimal while lowered blood pressure (BP) is maintained on thiazides. Thus, a vasodilator action of thiazides is proposed, likely via Ca2+-activated K+ (BK) channels in vascular smooth muscles. This study ascertains the role of volume depletion induced by salt restriction or salt wasting in NCC KO mice on the non-diuretic hypotensive action of HCTZ. HCTZ (20mg/kg s.c.) lowered BP in 1) NCC KO on a salt restricted diet but not with normal diet; 2) in volume depleted but not in volume resuscitated pendrin/NCC dKO mice; the BP reduction occurs without any enhancement in salt excretion or reduction in cardiac output. HCTZ still lowered BP following treatment of NCC KO on salt restricted diet with paxilline (8 mg/kg, i.p.), a BK channel blocker, and in BK KO and BK/NCC dKO mice on salt restricted diet. In aortic rings from NCC KO mice on normal and low salt diet, HCTZ did not alter and minimally decreased maximal phenylephrine contraction, respectively, while contractile sensitivity remained unchanged. These results demonstrate 1) the non-diuretic hypotensive effects of thiazides are augmented with volume depletion and 2) that the BP reduction is likely the result of HCTZ inhibition of vasoconstriction through a pathway dependent on factors present in vivo, is unrelated to BK channel activation, and involves processes associated with intravascular volume depletion.

Transient receptor potential vanilloid 2 function regulates cardiac hypertrophy via stretch-induced activation.

OBJECTIVE: Hypertension (increased afterload) results in cardiomyocyte hypertrophy leading to left ventricular hypertrophy and subsequently, heart failure with preserved ejection fraction. This study was performed to test the hypothesis that transient receptor potential vanilloid 2 subtype (TRPV2) function regulates hypertrophy under increased afterload conditions. METHODS: We used functional (pore specific) TRPV2 knockout mice to evaluate the effects of increased afterload-induced stretch on cardiac size and function via transverse aortic constriction (TAC) as well as hypertrophic stimuli including adrenergic and angiotensin stimulation via subcutaneous pumps. Wild-type animals served as control for all experiments. Expression and localization of TRPV2 was investigated in wild-type cardiac samples. Changes in cardiac function were measured in vivo via echocardiography and invasive catheterization. Molecular changes, including protein and real-time PCR markers of hypertrophy, were measured in addition to myocyte size. RESULTS: TRPV2 is significantly upregulated in wild-type mice after TAC, though not in response to beta-adrenergic or angiotensin stimulation. TAC-induced stretch stimulus caused an upregulation of TRPV2 in the sarcolemmal membrane. The absence of functional TRPV2 resulted in significantly reduced left ventricular hypertrophy after TAC, though not in response to beta-adrenergic or angiotensin stimulation. The decreased development of hypertrophy was not associated with significant deterioration of cardiac function. CONCLUSION: We conclude that TRPV2 function, as a stretch-activated channel, regulates the development of cardiomyocyte hypertrophy in response to increased afterload.

In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin.

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.