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.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8, while the surviving proximal tubules (PTs) showed restored transcriptional signature. Furthermore, we found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

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.

HuR-dependent expression of Wisp1 is necessary for TGFß-induced cardiac myofibroblast activity.

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFß-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFß-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFß treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFß. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFß, in part through post-transcriptional regulation of Wisp1.

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.

Adipocyte-specific deletion of HuR induces spontaneous cardiac hypertrophy and fibrosis.

Adipose tissue homeostasis plays a central role in cardiovascular physiology, and the presence of thermogenically active brown adipose tissue (BAT) has recently been associated with cardiometabolic health. We have previously shown that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) reduces BAT-mediated adaptive thermogenesis, and the goal of this work was to identify the cardiovascular impacts of Adipo-HuR-/-. We found that Adipo-HuR-/- mice exhibit a hypercontractile phenotype that is accompanied by increased left ventricle wall thickness and hypertrophic gene expression. Furthermore, hearts from Adipo-HuR-/- mice display increased fibrosis via picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene coexpression network analysis (WGCNA) across both cardiac and adipose tissue to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and cardiac fibrosis. RNA-seq results demonstrated a significant increase in proinflammatory gene expression in both cardiac and subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF-α and IL-6. In addition to inflammation-related genes, WGCNA identified a significant enrichment in extracellular vesicle-mediated transport and exosome-associated genes in scWAT, whose expression most significantly associated with the degree of cardiac fibrosis observed in Adipo-HuR-/- mice, implicating these processes as a likely adipose-to-cardiac paracrine mechanism. These results are significant in that they demonstrate the spontaneous onset of cardiovascular pathology in an adipose tissue-specific gene deletion model and contribute to our understanding of how disruptions in adipose tissue homeostasis may mediate cardiovascular disease.NEW & NOTEWORTHY The presence of functional brown adipose tissue in humans is known to be associated with cardiovascular health. Here, we show that adipocyte-specific deletion of the RNA binding protein HuR, which we have previously shown to reduce BAT-mediated thermogenesis, is sufficient to mediate a spontaneous development of cardiac hypertrophy and fibrosis. These results may have implications on the mechanisms by which BAT function and adipose tissue homeostasis directly mediate cardiovascular disease.

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.

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.

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.

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.

SERCA2 Haploinsufficiency in a Mouse Model of Darier Disease Causes a Selective Predisposition to Heart Failure.

Null mutations in one copy of ATP2A2, the gene encoding sarco/endoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), cause Darier disease in humans, a skin condition involving keratinocytes. Cardiac function appears to be unimpaired in Darier disease patients, with no evidence that SERCA2 haploinsufficiency itself causes heart disease. However, SERCA2 deficiency is widely considered a contributing factor in heart failure. We therefore analyzed Atp2a2 heterozygous mice to determine whether SERCA2 haploinsufficiency can exacerbate specific heart disease conditions. Despite reduced SERCA2a levels in heart, Atp2a2 heterozygous mice resembled humans in exhibiting normal cardiac physiology. When subjected to hypothyroidism or crossed with a transgenic model of reduced myofibrillar Ca(2+)-sensitivity, SERCA2 deficiency caused no enhancement of the disease state. However, when combined with a transgenic model of increased myofibrillar Ca(2+)-sensitivity, SERCA2 haploinsufficiency caused rapid onset of hypertrophy, decompensation, and death. These effects were associated with reduced expression of the antiapoptotic Hax1, increased levels of the proapoptotic genes Chop and Casp12, and evidence of perturbations in energy metabolism. These data reveal myofibrillar Ca(2+)-sensitivity to be an important determinant of the cardiac effects of SERCA2 haploinsufficiency and raise the possibility that Darier disease patients are more susceptible to heart failure under certain conditions.

Ablation of plasma membrane Ca(2+)-ATPase isoform 4 prevents development of hypertrophy in a model of hypertrophic cardiomyopathy.

The mechanisms linking the expression of sarcomeric mutant proteins to the development of pathological hypertrophy in hypertrophic cardiomyopathy (HCM) remain poorly understood. We investigated the role of the plasma membrane Ca(2+)-ATPase PMCA4 in the HCM phenotype using a transgenic model that expresses mutant (Glu180Gly) α-tropomyosin (Tm180) in heart. Immunoblot analysis revealed that cardiac PMCA4 expression was upregulated early in Tm180 disease pathogenesis. This was accompanied by an increase in levels of the L-type Ca(2+)-channel, which is implicated in pathological hypertrophy. When Tm180 mice were crossed with a PMCA4-null line, loss of PMCA4 caused the abrogation of hypertrophy in Tm180/PMCA4-null double mutant mice. RT-PCR analysis of Tm180/PMCA4-null hearts revealed blunting of the fetal program and reversion of pro-fibrotic Col1a1 and Col3a1 gene expression to wild-type levels. This was accompanied by evidence of reduced L-type Ca(2+)-channel expression, and diminished calcineurin activity. Expression of the metabolic substrate transporters glucose transporter 4 and carnitine palmitoyltransferase 1b was preserved and Tm180-related changes in mRNA levels of various contractile stress-related proteins including the cardiac ankyrin protein CARP and the N2B isoform of titin were reversed in Tm180/PMCA4-null hearts. cGMP levels were increased and phosphorylation of vasodilator-stimulated phosphoprotein was elevated in Tm180/PMCA4-null hearts. These changes were associated with a sharp reduction in left ventricular end-diastolic pressure in Tm180/PMCA4-null hearts, which occurred despite persistence of Tm180-related impairment of relaxation dynamics. These results reveal a novel and specific role for PMCA4 in the Tm180 hypertrophic phenotype, with the "protective" effects of PMCA4 deficiency encompassing multiple determinants of HCM-related hypertrophy.

Loss of NHE1 activity leads to reduced oxidative stress in heart and mitigates high-fat diet-induced myocardial stress.

Acute inhibition of the NHE1 Na(+)/H(+) exchanger protects against ischemia-reperfusion injury and chronic inhibition attenuates development of cardiac hypertrophy and failure. To determine the cardiac effects of chronic inhibition of NHE1 under non-pathological conditions we used NHE1-null mice as a model of long-term NHE1 inhibition. Cardiovascular performance was relatively normal in Nhe1(-/-) mice although cardiac contractility and relaxation were slightly improved in mutant mice of the FVB/N background. GSH levels and GSH:GSSG ratios were elevated in Nhe1(-/-) hearts indicating an enhanced redox potential. Consistent with a reduced need for antioxidant protection, expression of heat shock proteins Hsp60 and Hsp25 was lower in Nhe1(-/-) hearts. Similarly, expression of mitochondrial superoxide dismutase 2 was reduced, with no increase in expression of other ROS scavenging enzymes. GLUT1 levels were increased in Nhe1(-/-) hearts, the number of lipid droplets in myocytes was reduced, and PDK4 expression was refractory to high-fat diet-induced upregulation observed in wild-type hearts. High-fat diet-induced stress was attenuated in Nhe1(-/-) hearts, as indicated by smaller increases in phosphorylation of Hsp25 and α-B crystallin, and there was better preservation of insulin sensitivity, as evidenced by PKB/Akt phosphorylation. Plasma glucose and insulin levels were lower and high-fat diet-induced hepatic lipid accumulation was reduced in Nhe1(-/-) mice, demonstrating extracardiac effects of NHE1 ablation. These data indicate that long-term ablation of NHE1 activity increases the redox potential, mitigates high-fat diet-induced myocardial stress and fatty liver disease, leads to better preservation of insulin sensitivity, and may alter both cardiac and systemic metabolic substrate handling in mice.

Knockout of the Na,K-ATPase α2-isoform in cardiac myocytes delays pressure overload-induced cardiac dysfunction.

The α2-isoform of the Na,K-ATPase (α2) is the minor isoform of the Na,K-ATPase expressed in the cardiovascular system and is thought to play a critical role in the regulation of cardiovascular hemodynamics. However, the organ system/cell type expressing α2 that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of α2 to further define the tissue-specific role of α2 in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model using the Cre/loxP system to generate a tissue-specific knockout of α2 in the heart using ß-myosin heavy chain Cre. We have achieved a 90% knockout of α2 expression in the heart of the knockout mice. Interestingly, the heart-specific knockout mice exhibit normal basal cardiac function and systolic blood pressure, and in addition, these mice develop ACTH-induced hypertension in response to ACTH treatment similar to control mice. Surprisingly, the heart-specific knockout mice display delayed onset of cardiac dysfunction compared with control mice in response to pressure overload induced by transverse aortic constriction; however, the heart-specific knockout mice deteriorated to control levels by 9 wk post-transverse aortic constriction. These results suggest that heart expression of α2 does not play a role in the regulation of basal cardiovascular function or blood pressure; however, heart expression of α2 plays a role in the hypertrophic response to pressure overload. This study further emphasizes that the tissue localization of α2 determines its unique roles in the regulation of cardiovascular function.

Loss of the AE3 Cl(-)/HCO(-) 3 exchanger in mice affects rate-dependent inotropy and stress-related AKT signaling in heart.

Cl(-)/HCO(-) 3 exchangers are expressed abundantly in cardiac muscle, suggesting that HCO(-) 3 extrusion serves an important function in heart. Mice lacking Anion Exchanger Isoform 3 (AE3), a major cardiac Cl(-)/HCO(-) 3 exchanger, appear healthy, but loss of AE3 causes decompensation in a hypertrophic cardiomyopathy (HCM) model. Using intra-ventricular pressure analysis, in vivo pacing, and molecular studies we identified physiological and biochemical changes caused by loss of AE3 that may contribute to decompensation in HCM. AE3-null mice had normal cardiac contractility under basal conditions and after ß-adrenergic stimulation, but pacing of hearts revealed that frequency-dependent inotropy was blunted, suggesting that AE3-mediated HCO(-) 3 extrusion is required for a robust force-frequency response (FFR) during acute biomechanical stress in vivo. Modest changes in expression of proteins that affect Ca(2+)-handling were observed, but Ca(2+)-transient analysis of AE3-null myocytes showed normal twitch-amplitude and Ca(2+)-clearance. Phosphorylation and expression of several proteins implicated in HCM and FFR, including phospholamban (PLN), myosin binding protein C, and troponin I were not altered in hearts of paced AE3-null mice; however, phosphorylation of Akt, which plays a central role in mechanosensory signaling, was significantly higher in paced AE3-null hearts than in wild-type controls and phosphorylation of AMPK, which is affected by Akt and is involved in energy metabolism and some cases of HCM, was reduced. These data show loss of AE3 leads to impaired rate-dependent inotropy, appears to affect mechanical stress-responsive signaling, and reduces activation of AMPK, which may contribute to decompensation in heart failure.

Knockout of the Na,K-ATPase α2-isoform in the cardiovascular system does not alter basal blood pressure but prevents ACTH-induced hypertension.

The α(2)-isoform of Na,K-ATPase (α(2)) is thought to play a role in blood pressure regulation, but the specific cell type(s) involved have not been identified. Therefore, it is important to study the role of the α(2) in individual cell types in the cardiovascular system. The present study demonstrates the role of vascular smooth muscle α(2) in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model utilizing the Cre/LoxP system to generate a cell type-specific knockout of the α(2) in vascular smooth muscle cells using the SM22α Cre. We achieved a 90% reduction in the α(2)-expression in heart and vascular smooth muscle in the knockout mice. Interestingly, tail-cuff blood pressure analysis reveals that basal systolic blood pressure is unaffected by the knockout of α(2) in the knockout mice. However, knockout mice do fail to develop ACTH-induced hypertension, as seen in wild-type mice, following 5 days of treatment with ACTH (Cortrosyn; wild type = 119.0 ± 6.8 mmHg; knockout = 103.0 ± 2.0 mmHg). These results demonstrate that α(2)-expression in heart and vascular smooth muscle is not essential for regulation of basal systolic blood pressure, but α(2) is critical for blood pressure regulation under chronic stress such as ACTH-induced hypertension.

Renovascular hypertension using a modified two-kidney, one-clip approach in mice is not dependent on the α1 or α2 Na-K-ATPase ouabain-binding site.

Endogenous cardiotonic steroids, through their interaction with the ouabain-binding site of the Na-K-ATPase α-subunit, have been implicated in a variety of cardiovascular disease states including hypertension. We have previously shown that ACTH-induced hypertension is abolished in mutant mice expressing ouabain-resistant α1- and α2-subunits. To further evaluate hypertension resistance in these mutant mice, we examined blood pressure changes in a modified model of 2-kidney, 1-clip (2K1C) renovascular hypertension. To reliably generate 2K1C hypertension, we used polyvinyl tubing (inner diameter: ∼0.27 mm) to accurately gauge the degree of renal artery stenosis. Using this method, virtually all of the clipped mice became hypertensive and there was no incidence of apparent renal ischemia. By telemetry, in response to renal artery clipping, blood pressure in wild-type mice (α1 ouabain-resistant, α2 ouabain-sensitive) increased from 97 ± 3 to 136 ± 7 mmHg. In α1-resistant, α2-resistant mice, pressure increased from 93 ± 2 to 123 ± 4 mmHg, and in α1-sensitive, α2-resistant mice, blood pressure increased from 95 ± 2 to 139 ± 5 mmHg. Blood pressure changes were equivalent in all three groups. In sham mice, blood pressure did not change (96 ± 1 to 95 ± 2 mmHg). Renin mRNA expression was dramatically elevated in the left vs. the right kidney, and plasma renin concentration was elevated similarly in all genotypes. These data indicate that sensitivity of the α1- or α2-Na-K-ATPase binding site to cardiotonic steroids is not a prerequisite for the development of 2K1C renovascular hypertension. In addition, use of a polyurethane cuff to constrict the renal artery provides a reliable method for producing 2K1C hypertension in mice.