Treatment with αvß3-integrin-specific 29P attenuates pressure-overload induced cardiac remodelling after transverse aortic constriction in mice.

Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvß3-specific RGD-mimetic, 29P , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 µg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvß3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.

Cardiomyocyte-specific loss of plasma membrane calcium ATPase 1 impacts cardiac rhythm and is associated with ventricular repolarisation dysfunction.

Plasma membrane calcium ATPase 1 (PMCA1, Atp2b1) is emerging as a key contributor to cardiac physiology, involved in calcium handling and myocardial signalling. In addition, genome wide association studies have associated PMCA1 in several areas of cardiovascular disease including hypertension and myocardial infarction. Here, we investigated the role of PMCA1 in basal cardiac function and heart rhythm stability. Cardiac structure, heart rhythm and arrhythmia susceptibility were assessed in a cardiomyocyte-specific PMCA1 deletion (PMCA1CKO) mouse model. PMCA1CKO mice developed abnormal heart rhythms related to ventricular repolarisation dysfunction and displayed an increased susceptibility to ventricular arrhythmias. We further assessed the levels of cardiac ion channels using qPCR and found a downregulation of the voltage-dependent potassium channels, Kv4.2, with a corresponding reduction in the transient outward potassium current which underlies ventricular repolarisation in the murine heart. The changes in heart rhythm were found to occur in the absence of any structural cardiomyopathy. To further assess the molecular changes occurring in PMCA1CKO hearts, we performed proteomic analysis. Functional characterisation of differentially expressed proteins suggested changes in pathways related to metabolism, protein-binding, and pathways associated cardiac function including ß-adrenergic signalling. Together, these data suggest an important role for PMCA1 in basal cardiac function in relation to heart rhythm control, with reduced cardiac PMCA1 expression resulting in an increased risk of arrhythmia development.

PMCA4 inhibition does not affect cardiac remodelling following myocardial infarction, but may reduce susceptibility to arrhythmia.

Ischaemic heart disease is the world's leading cause of mortality. Survival rates from acute myocardial infarction (MI) have improved in recent years; however, this has led to an increase in the prevalence of heart failure (HF) due to chronic remodelling of the infarcted myocardium, for which treatment options remain poor. We have previously shown that inhibition of isoform 4 of the plasma membrane calcium ATPase (PMCA4) prevents chronic remodelling and HF development during pressure overload, through fibroblast mediated Wnt signalling modulation. Given that Wnt signalling also plays a prominent role during remodelling of the infarcted heart, this study investigated the effect of genetic and functional loss of PMCA4 on cardiac outcomes following MI. Neither genetic deletion nor pharmacological inhibition of PMCA4 affected chronic remodelling of the post-MI myocardium. This was the case when PMCA4 was deleted globally, or specifically from cardiomyocytes or fibroblasts. PMCA4-ablated hearts were however less prone to acute arrhythmic events, which may offer a slight survival benefit. Overall, this study demonstrates that PMCA4 inhibition does not affect chronic outcomes following MI.

Acute inhibition of PMCA4, but not global ablation, reduces blood pressure and arterial contractility via a nNOS-dependent mechanism.

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild-type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild-type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl-l-NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries.

Hypertension is a well-established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht ) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age-matched wild-type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility.

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

The oxoglutarate receptor 1 (OXGR1) modulates pressure overload-induced cardiac hypertrophy in mice.

The G-protein-coupled receptors (GPCRs) family of proteins play essential roles in the heart, including in the regulation of cardiac hypertrophy. One member of this family, the oxoglutarate receptor 1 (OXGR1), may have a crucial role in the heart because it acts as a receptor for α-ketoglutarate, a metabolite that is elevated in heart failure patients. OXGR1 is expressed in the heart but its precise function during cardiac pathophysiological process is unknown. Here we used both in vivo and in vitro approaches to investigate the role of OXGR1 in cardiac hypertrophy. Genetic ablation of Oxgr1 in mice (OXGR1-/-) resulted in a significant increase in hypertrophy following transverse aortic constriction (TAC). This was accompanied by reduction in contractile function as indicated by cardiac fractional shortening and ejection fraction. Conversely, adenoviral mediated overexpression of OXGR1 in neonatal rat cardiomyocytes significantly reduced phenylephrine-induced cardiomyocyte hypertrophy, a result that was consistent with the in vivo data. Using a combination of yeast two hybrid screening and phospho-antibody array analysis we identified novel interacting partner and downstream signalling pathway that might be regulated by the OXGR1. First, we found that OXGR1 forms a molecular complex with the COP9 signalosome complex subunit 5 (CSN5). Secondly, we observed that the STAT3 signalling pathway was upregulated in OXGR1-/- hearts. Since CSN5 interacts with TYK2, a major upstream regulator of STAT3, OXGR1 might regulate the pro-hypertrophic STAT3 pathway via interaction with the CSN5-TYK2 complex. In conclusion, our study has identified OXGR1 as a novel regulator of pathological hypertrophy via the regulation of the STAT3. Identification of molecules that can specifically activate or inhibit this receptor may be very useful in the development of novel therapeutic approach for pathological cardiac hypertrophy.

Calcium Extrusion Pump PMCA4: A New Player in Renal Calcium Handling?

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy.

The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition.

The mammalian Ste20-like kinase 2 (Mst2) modulates stress-induced cardiac hypertrophy.

The Hippo signaling pathway has recently moved to center stage in cardiac research because of its key role in cardiomyocyte proliferation and regeneration of the embryonic and newborn heart. However, its role in the adult heart is incompletely understood. We investigate here the role of mammalian Ste20-like kinase 2 (Mst2), one of the central regulators of this pathway. Mst2(-/-) mice showed no alteration in cardiomyocyte proliferation. However, Mst2(-/-) mice exhibited a significant reduction of hypertrophy and fibrosis in response to pressure overload. Consistently, overexpression of MST2 in neonatal rat cardiomyocytes significantly enhanced phenylephrine-induced cellular hypertrophy. Mechanistically, Mst2 positively modulated the prohypertrophic Raf1-ERK1/2 pathway. However, activation of the downstream effectors of the Hippo pathway (Yes-associated protein) was not affected by Mst2 ablation. An initial genetic study in mitral valve prolapse patients revealed an association between a polymorphism in the human MST2 gene and adverse cardiac remodeling. These results reveal a novel role of Mst2 in stress-dependent cardiac hypertrophy and remodeling in the adult mouse and likely human heart.

The tumour suppressor Ras-association domain family protein 1A (RASSF1A) regulates TNF-α signalling in cardiomyocytes.

AIMS: Tumour necrosis factor-α (TNF-α) plays a key role in the regulation of cardiac contractility. Although cardiomyocytes are known to express the TNF-α receptors (TNFRs), the mechanism of TNF-α signal transmission is incompletely understood. The aim of this study was to investigate whether the tumour suppressor Ras-association domain family protein 1 isoform A (RASSF1A) modulates TNF-α signalling in cardiomyocytes. METHODS AND RESULTS: We used RASSF1A knockout (RASSF1A(-/-)) mice and wild-type (WT) littermates in this study. Acute stimulation with a low dose of TNF-α (10 µg/kg iv) increased cardiac contractility and intracellular calcium transients' amplitude in WT mice. In contrast, RASSF1A(-/-) mice showed a blunted contractile response. Mechanistically, RASSF1A was essential in the formation of the TNFR complex (TNFRC), where it functions as an adaptor molecule to facilitate the recruitment of TNFR type 1-associated death domain protein and TNFR-associated factor 2 to form the TNF-α receptor complex. In the absence of RASSF1A, signal transmission from the TNF-α receptor complex to the downstream effectors, such as cytoplasmic phospholipase A2 and protein kinase A, was attenuated leading to the reduction in the activation of calcium handling molecules, such as L-type Ca(2+) channel and ryanodine receptors. CONCLUSION: Our data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of the TNFRC and in signal transmission to the downstream targets.

Plasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain.

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac ß-adrenergic signaling.

Tumor suppressor Ras-association domain family 1 isoform A is a novel regulator of cardiac hypertrophy.

BACKGROUND: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy. METHODS AND RESULTS: A significant downregulation of RASSF1A expression was observed in hypertrophic mouse hearts, as well as in failing human hearts. To further investigate the role of RASSF1A in cardiac (patho)physiology, we used RASSF1A knock-out (RASSF1A(-)(/)(-)) mice and neonatal rat cardiomyocytes with adenoviral overexpression of RASSF1A. Ablation of RASSF1A in mice significantly enhanced the hypertrophic response to transverse aortic constriction (64.2% increase in heart weight/body weight ratio in RASSF1A(-)(/)(-) mice compared with 32.4% in wild type). Consistent with the in vivo data, overexpression of RASSF1A in cardiomyocytes markedly reduced the cellular hypertrophic response to phenylephrine stimulation. Analysis of molecular signaling events in isolated cardiomyocytes indicated that RASSF1A inhibited extracellular regulated kinase 1/2 activation, likely by blocking the binding of Raf1 to active Ras. CONCLUSIONS: Our data establish RASSF1A as a novel inhibitor of cardiac hypertrophy by modulating the extracellular regulated kinase 1/2 pathway.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium.

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Neuronal nitric oxide synthase signaling in the heart is regulated by the sarcolemmal calcium pump 4b.

BACKGROUND: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. METHODS AND RESULTS: We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the beta-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N omega-propyl-L-arginine) reduced the beta-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. CONCLUSIONS: These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.

Interleukin-6 is an afferent signal to the hypothalamo-pituitary-adrenal axis during local inflammation in mice.

The cytokines IL-1 and IL-6 are able to induce prostaglandin (PG)-dependent activation of the hypothalamo-pituitary-adrenal axis (HPAA) and are thought to play key roles in immune-neuroendocrine interactions during inflammation. The present study shows that inflammation induced by im injection of turpentine (TPS) in the hind limb of mice causes an increase in the plasma concentration of IL-6, but not that of IL-1 alpha or IL-1 beta, together with a prolonged (>18-h) activation of the HPAA. IL-6 plays a causal role in the TPS-induced elevation in HPAA activity, because the sustained (8-18 h) increases in 1) plasma corticosterone, 2) plasma ACTH, and 3) induction of c-Fos in the hypothalamic paraventricular nucleus are all markedly blunted in IL-6-deficient (IL-6(-/-)) mice. Peripheral administration of a neutralizing IL-6 antiserum inhibited the plasma corticosterone response of normal (C57BL/6) mice to hind limb inflammation to an extent similar to that seen in IL-6(-/-) mice, suggesting that the IL-6 responsible for the increased HPAA activity is produced, or acts, on the blood side of the blood-brain barrier. We also show that IL-6 in the circulation is induced almost exclusively at the local inflammatory site, where IL-1 beta is produced. Induction of IL-6 and activation of the HPAA are dependent upon prior activation of an IL-1 type I receptor, as both are inhibited in type I IL-1 receptor-deficient mice. Furthermore, hind limb inflammation induced cyclooxygenase-2 protein expression around the cerebrovasculature of normal (IL-6(+/+)), but not IL-6(-/-), mice. Based on these data, we propose that IL-6 is produced at the local inflammatory site under the control of IL-1 beta and is the circulating afferent signal that is in part responsible for elevated HPAA activity, possibly acting via eicosanoid production within the cerebrovasculature.