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Ischemia and reperfusion affect multiple elements of cardiomyocyte electrophysiology, especially within the mitochondria. We previously showed that in cardiac monolayers, upon reperfusion after coverslip-induced ischemia, mitochondrial inner membrane potential (ΔΨ) unstably oscillates between polarized and depolarized states, and ΔΨ instability corresponds with arrhythmias. Here, through confocal microscopy of compartment-specific molecular probes, we investigate the mechanisms underlying the post-ischemic ΔΨ oscillations, focusing on the role of Ca

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice—elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-l-arginine methyl ester (l-NAME)—recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α–XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF. iNOS-driven dysregulation of the IRE1α–XBP1 pathway leads to cardiomyocyte dysfunction in mice and recapitulates the systemic and cardiovascular features of human heart failure with preserved ejection fraction.

[Figure: see text].

Rationale: Heart failure with preserved ejection fraction (HFpEF) is a mortal clinical syndrome without effective therapies. We recently demonstrated in mice that a combination of metabolic and hypertensive stress recapitulates key features of human HFpEF. Objective: Using this novel preclinical HFpEF model, we set out to define and manipulate metabolic dysregulations occurring in HFpEF myocardium. Methods and Results: We observed impairment in mitochondrial fatty acid oxidation associated with hyperacetylation of key enzymes in the pathway. Downregulation of sirtuin 3 and deficiency of NAD + secondary to an impaired NAD + salvage pathway contribute to this mitochondrial protein hyperacetylation. Impaired expression of genes involved in NAD + biosynthesis was confirmed in cardiac tissue from patients with HFpEF. Supplementing HFpEF mice with nicotinamide riboside or a direct activator of NAD + biosynthesis led to improvement in mitochondrial function and amelioration of the HFpEF phenotype. Conclusions: Collectively, these studies demonstrate that HFpEF is associated with myocardial mitochondrial dysfunction and unveil NAD + repletion as a promising therapeutic approach in the syndrome.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP., Carboxyl terminus of Hsc70-interacting protein (CHIP) is proteostasis regulator. Here the authors show that CHIP-mediated protein turnover is enhanced by PKG-mediated phosphorylation, which results in attenuated cardiac ischemic proteotoxicity.

Background: Phosphodiesterase type-1 (PDE1) hydrolyzes cAMP and cGMP and is constitutively expressed in the heart, although cardiac effects from its acute inhibition in vivo are largely unknown. Existing data are limited to rodents expressing mostly the cGMP-favoring PDE1A isoform. Human heart predominantly expresses PDE1C with balanced selectivity for cAMP and cGMP. Here, we determined the acute effects of PDE1 inhibition in PDE1C-expressing mammals, dogs, and rabbits, in normal and failing hearts, and explored its regulatory pathways. Methods: Conscious dogs chronically instrumented for pressure-volume relations were studied before and after tachypacing-induced heart failure (HF). A selective PDE1 inhibitor (ITI-214) was administered orally or intravenously±dobutamine. Pressure-volume analysis in anesthetized rabbits tested the role of β-adrenergic and adenosine receptor signaling on ITI-214 effects. Sarcomere and calcium dynamics were studied in rabbit left ventricular myocytes. Results: In normal and HF dogs, ITI-214 increased load-independent contractility, improved relaxation, and reduced systemic arterial resistance, raising cardiac output without altering systolic blood pressure. Heart rate increased, but less so in HF dogs. ITI-214 effects were additive to β-adrenergic receptor agonism (dobutamine). Dobutamine but not ITI-214 increased plasma cAMP. ITI-214 induced similar cardiovascular effects in rabbits, whereas mice displayed only mild vasodilation and no contractility effects. In rabbits, β-adrenergic receptor blockade (esmolol) prevented ITI-214–mediated chronotropy, but inotropy and vasodilation remained unchanged. By contrast, adenosine A 2B -receptor blockade (MRS-1754) suppressed ITI-214 cardiovascular effects. Adding fixed-rate atrial pacing did not alter the findings. ITI-214 alone did not affect sarcomere or whole-cell calcium dynamics, whereas β-adrenergic receptor agonism (isoproterenol) or PDE3 inhibition (cilostamide) increased both. Unlike cilostamide, which further enhanced shortening and peak calcium when combined with isoproterenol, ITI-214 had no impact on these responses. Both PDE1 and PDE3 inhibitors increased shortening and accelerated calcium decay when combined with forskolin, yet only cilostamide increased calcium transients. Conclusions: PDE1 inhibition by ITI-214 in vivo confers acute inotropic, lusitropic, and arterial vasodilatory effects in PDE1C-expressing mammals with and without HF. The effects appear related to cAMP signaling that is different from that provided via β-adrenergic receptors or PDE3 modulation. ITI-214, which has completed phase I trials, may provide a novel therapy for HF.

Background: Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent clinical condition associated with significant morbidity, mortality and health care expenses. Yet, no effective treatment has been identified. We recently demonstrated that concomitant metabolic and hypertensive stress in mice elicited by a combination of high fat diet (HFD) and constitutive nitric oxide synthase inhibition by N [w] -nitro-l-arginine methyl ester (L-NAME) faithfully recapitulates the numerous and myriad features of human HFpEF (paper in press at Nature ). Methods: Wild type C57Bl6 mice were fed with HFD and L-NAME via drinking water for 5-8 weeks. Myocardial mitochondrial morphology was assessed by electron microscopy. Mitochondrial function was assessed by measuring oxygen consumption rates using an oxygen electrode. Protein expression and modification were assessed by Western blotting and immunoprecipitation. Results: Using this novel mouse model, we discovered significant impairment of mitochondrial fatty acid oxidation (FAO) associated with hyperacetylation of key FAO enzymes in HFpEF myocardium. Mechanistically, downregulation of Sirtuin3, the major mitochondrial deacetylase, and deficiency of its co-substrate nicotinamide adenine dinucleotide (NAD + ), culminated in mitochondrial protein hyperacetylation. Strikingly, supplementation with nicotinamide riboside (NR), a NAD + precursor, led to dramatic improvement of mitochondrial function, and importantly, amelioration of the HFpEF phenotype. Conclusion: In summary, we have unveiled that protein hyperacetylation-mediated mitochondrial dysfunction is a crucial mechanism of HFpEF pathogenesis. This is, to our knowledge, the first study identifying a specific signature of metabolic remodeling in HFpEF heart. We also demonstrated the therapeutic effect of NAD + repletion in a preclinical HFpEF model. In next steps, confirming this benefit in a clinical trial is warranted.

Background: The mouse is the most widely used mammal in experimental biology. Although many clinically relevant in vivo cardiac stressors are used, one that has eluded translation is long-term cardiac pacing. Here, we present the first method to chronically simulate and simultaneously record cardiac electrical activity in conscious mobile mice. We then apply it to study right ventricular pacing induced electromechanical dyssynchrony and its reversal (resynchronization). Methods and Results: The method includes a custom implantable bipolar stimulation and recording lead and flexible external conduit and electrical micro-commutator linked to a pulse generator/recorder. This achieved continuous pacing for at least 1 month in 77% of implants. Mice were then subjected to cardiac ischemia/reperfusion injury to depress heart function, followed by 4 weeks pacing at the right ventricle (dyssynchrony), right atrium (synchrony), or for 2 weeks right ventricle and then 2 weeks normal sinus (resynchronization). Right ventricular pacing–induced dyssynchrony substantially reduced heart and myocyte function compared with the other groups, increased gene expression heterogeneity (>10 fold) comparing septum to lateral walls, and enhanced growth and metabolic kinase activity in the late-contracting lateral wall. This was ameliorated by restoring contractile synchronization. Conclusions: The new method to chronically pace conscious mice yields stable atrial and ventricular capture and a means to dissect basic mechanisms of electromechanical physiology and therapy. The data on dyssynchrony and resynchronization in ischemia/reperfusion hearts is the most comprehensive to date in ischemic heart disease, and its similarities to nonischemic canine results support the translational utility of the mouse.

Treatment with nitroxyl (HNO) improves cardiac function in failing hearts by inducing release of SR Ca2+-ATPase from phospholamban (PLN), thereby enhancing Ca2+ reuptake. Keceli et al. use 15N-edited NMR spectroscopy to show that HNO achieves this by reversibly modifying PLN cysteines 41 and 46., Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca2+ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca2+ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca2+-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH2) that are less easily reversed by reductants, remain to be determined. Using an 15N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, 15N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

Background Patients with chronic kidney disease (CKD) and coincident heart failure with preserved ejection fraction (HFpEF) may constitute a distinct HFpEF phenotype. Osteopontin (OPN) is a biomarker of HFpEF and predictive of disease outcome. We recently reported that OPN blockade reversed hypertension, mitochondrial dysfunction, and kidney failure in Col4a3−/− mice, a model of human Alport syndrome. Objectives The purpose of this study was to identify potential OPN targets in biopsies of HF patients, healthy control subjects, and human induced pluripotent stem cell–derived cardiomyocytes (hiPS-CMs), and to characterize the cardiac phenotype of Col4a3−/− mice, relate this to HFpEF, and investigate possible causative roles for OPN in driving the cardiomyopathy. Methods OGDHL mRNA and protein were quantified in myocardial samples from patients with HFpEF, heart failure with reduced ejection fraction, and donor control subjects. OGDHL expression was quantified in hiPS-CMs treated with or without anti-OPN antibody. Cardiac parameters were evaluated in Col4a3−/− mice with and without global OPN knockout or AAV9-mediated delivery of 2-oxoglutarate dehydrogenase-like (Ogdhl) to the heart. Results OGDHL mRNA and protein displayed abnormal abundances in cardiac biopsies of HFpEF (n = 17) compared with donor control subjects (n = 12; p Conclusions Col4a3−/− mice present a model of HFpEF secondary to CKD wherein OPN and OGDHL are intermediates, and possibly therapeutic targets.

Dysregulation of Ca2+/calmodulin-dependent protein kinase (CaMK)II is closely linked with myocardial hypertrophy and heart failure. However, the mechanisms that regulate CaMKII activity are incompletely understood. Here we show that protein arginine methyltransferase 1 (PRMT1) is essential for preventing cardiac CaMKII hyperactivation. Mice null for cardiac PRMT1 exhibit a rapid progression to dilated cardiomyopathy and heart failure within 2 months, accompanied by cardiomyocyte hypertrophy and fibrosis. Consistently, PRMT1 is downregulated in heart failure patients. PRMT1 depletion in isolated cardiomyocytes evokes hypertrophic responses with elevated remodeling gene expression, while PRMT1 overexpression protects against pathological responses to neurohormones. The level of active CaMKII is significantly elevated in PRMT1-deficient hearts or cardiomyocytes. PRMT1 interacts with and methylates CaMKII at arginine residues 9 and 275, leading to its inhibition. Accordingly, pharmacological inhibition of CaMKII restores contractile function in PRMT1-deficient mice. Thus, our data suggest that PRMT1 is a critical regulator of CaMKII to maintain cardiac function., The mechanisms that regulate the activity of Ca2 +/calmodulin-dependent protein kinase II (CaMKII) in the context of heart failure are incompletely understood. Here the authors show that protein arginine methyltransferase 1 (PRMT1) prevents cardiac hyperactivation of CaMKII and heart failure development by methylating CaMKII at arginine residues 9 and 275.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using N

SummaryDecades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of the CM maturation that is subsequently required to generate adult myocytes remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent-stem-cell-derived CMs mature early in culture but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation.

Background— Pharmacological activation of cGMP-dependent protein kinase G I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG-activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGIα antiremodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood. Methods and Results— We performed a screen for myocardial proteins interacting with the PKGIα leucine zipper (LZ)–binding domain to identify myocardial-specific PKGI antiremodeling substrates. Our screen identified cardiac myosin–binding protein-C (cMyBP-C), a cardiac myocyte–specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGIα–cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGIα phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGIα, but not in cells transfected with LZ mutant PKGIα, containing mutations to disrupt LZ substrate binding. In mice subjected to left ventricular pressure overload, PKGI activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes. Conclusions— Taken together, these data support that PKGIα and cMyBP-C interact in the heart and that cMyBP-C is an anti remodeling PKGIα kinase substrate. This study provides the first identification of a myocardial-specific PKGIα LZ-dependent antiremodeling substrate and supports further exploration of PKGIα myocardial LZ substrates as potential therapeutic targets for heart failure.

On pathological stress, Wnt signaling is reactivated and induces genes associated with cardiac remodeling and fibrosis. We have previously shown that a cell surface receptor Cdon (cell-adhesion associated, oncogene regulated) suppresses Wnt signaling to promote neuronal differentiation however its role in heart is unknown. Here, we demonstrate a critical role of Cdon in cardiac function and remodeling. Cdon is expressed and predominantly localized at intercalated disk in both mouse and human hearts. Cdon-deficient mice develop cardiac dysfunction including reduced ejection fraction and ECG abnormalities. Cdon−/− hearts exhibit increased fibrosis and up-regulation of genes associated with cardiac remodeling and fibrosis. Electrical remodeling was demonstrated by up-regulation and mislocalization of the gap junction protein, Connexin 43 (Cx43) in Cdon−/− hearts. In agreement with altered Cx43 expression, functional analysis both using Cdon−/− cardiomyocytes and shRNA-mediated knockdown in rat cardiomyocytes shows aberrant gap junction activities. Analysis of the underlying mechanism reveals that Cdon−/− hearts exhibit hyperactive Wnt signaling as evident by β-catenin accumulation and Axin2 up-regulation. On the other hand, the treatment of rat cardiomyocytes with a Wnt activator TWS119 reduces Cdon levels and aberrant Cx43 activities, similarly to Cdon-deficient cardiomyocytes, suggesting a negative feedback between Cdon and Wnt signaling. Finally, inhibition of Wnt/β-catenin signaling by XAV939, IWP2 or dickkopf (DKK)1 prevented Cdon depletion-induced up-regulation of collagen 1a and Cx43. Taken together, these results demonstrate that Cdon deficiency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac fibrosis. Cdon exhibits great potential as a target for the treatment of cardiac fibrosis and cardiomyopathy.

Background Micro RNA (miRNA) is a type of noncoding RNA that can repress the expression of target genes through posttranscriptional regulation. In addition to numerous physiologic roles for mi RNA s, they play an important role in pathophysiologic processes affecting cardiovascular health. Previously, we reported that nuclear encoded microRNA (miR‐181c) is present in heart mitochondria, and importantly, its overexpression affects mitochondrial function by regulating mitochondrial gene expression. Methods and Results To investigate further how the miR‐181 family affects the heart, we suppressed miR‐181 using a miR‐181‐sponge containing 10 repeated complementary miR‐181 “seed” sequences and generated a set of H9c2 cells, a cell line derived from rat myoblast, by stably expressing either a scrambled or miR‐181‐sponge sequence. Sponge‐H9c2 cells showed a decrease in reactive oxygen species production and reduced basal mitochondrial respiration and protection against doxorubicin‐induced oxidative stress. We also found that miR‐181a/b targets phosphatase and tensin homolog ( PTEN ), and the sponge‐expressing stable cells had increased PTEN activity and decreased PI 3K signaling. In addition, we have used miR‐181a/b −/− and miR‐181c/d −/− knockout mice and subjected them to ischemia‐reperfusion injury. Our results suggest divergent effects of different miR‐181 family members: miR‐181a/b targets PTEN in the cytosol, resulting in an increase in infarct size in miR‐181a/b −/− mice due to increased PTEN signaling, whereas miR‐181c targets mt‐ COX 1 in the mitochondria, resulting in decreased infarct size in miR‐181c/d −/− mice. Conclusions The miR‐181 family alters the myocardial response to oxidative stress, notably with detrimental effects by targeting mt‐ COX 1 (miR‐181c) or with protection by targeting PTEN (miR‐181a/b).

Summary: Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases. : Pluripotent stem cell (PSC)-derived cells remain fetal like, and this has become a major impediment to modeling adult diseases. Cho et al. find that PSC-derived cardiomyocytes mature into adult cardiomyocytes when transplanted into neonatal rat hearts. This method can serve as a tool to understand maturation and pathogenesis in human cardiomyocytes. Keywords: cardiomyocyte, maturation, iPS, cardiac progenitor, neonatal, disease modeling, cardiomyopathy, ARVC, T-tubule, calcium transient, sarcomere shortening

Cyclic guanosine 3′,5′-monophosphate (cGMP) and its downstream target, protein kinase G (PKG or cGK), play central roles in cellular regulation and are important to cardiovascular homeostasis and disease pathophysiology. Cyclic GMP is synthesized via either nitric oxide (NO) or natriuretic peptide (NP) stimulation pathways, each coupled to corresponding cyclases, and catabolized by select members of the phosphodiesterase superfamily. Growing evidence now supports control of cGMP and PKG in distinct microdomains within the myocyte, which results in differential downstream targeting. This regional control stems from distinct localization of the relevant signaling components and their capacity to translocate in the cell under both physiological and pathophysiological conditions to further impact the net response. This chapter discusses current understanding of microdomain regulation of the cGMP/PKG pathway, as this information is important to optimally leverage their effects for the treatment of cardiovascular disease.

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone’s involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS–dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation.

Background Cyclic guanosine monophosphate‐protein kinase G‐phosphodiesterase 5 signaling may be disturbed in heart failure ( HF ) with preserved ejection fraction, contributing to cardiac remodeling and dysfunction. The purpose of this study was to manipulate cyclic guanosine monophosphate signaling using the dipeptidyl‐peptidase 4 inhibitor saxagliptin and phosphodiesterase 5 inhibitor tadalafil. We hypothesized that preservation of cyclic guanosine monophosphate cGMP signaling would attenuate pathological cardiac remodeling and improve left ventricular ( LV ) function. Methods and Results We assessed LV hypertrophy and function at the organ and cellular level in aortic‐banded pigs. Concentric hypertrophy was equal in all groups, but LV collagen deposition was increased in only HF animals. Prevention of fibrotic remodeling by saxagliptin and tadalafil was correlated with neuropeptide Y plasma levels. Saxagliptin better preserved integrated LV systolic and diastolic function by maintaining normal LV chamber volumes and contractility (end‐systolic pressure‐volume relationship, preload recruitable SW ) while preventing changes to early/late diastolic longitudinal strain rate. Function was similar to the HF group in tadalafil‐treated animals including increased LV contractility, reduced chamber volume, and decreased longitudinal, circumferential, and radial mechanics. Saxagliptin and tadalafil prevented a negative cardiomyocyte shortening‐frequency relationship observed in HF animals. Saxagliptin increased phosphodiesterase 5 activity while tadalafil increased cyclic guanosine monophosphate levels; however, neither drug increased downstream PKG activity. Early mitochondrial dysfunction, evident as decreased calcium‐retention capacity and Complex II ‐dependent respiratory control, was present in both HF and tadalafil‐treated animals. Conclusions Both saxagliptin and tadalafil prevented increased LV collagen deposition in a manner related to the attenuation of increased plasma neuropeptide Y levels. Saxagliptin appears superior for treating heart failure with preserved ejection fraction, considering its comprehensive effects on integrated LV systolic and diastolic function.

Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (beta-AR) stimulation in left ventricular myocytes and hearts. This modulation requires cyclic GMP synthesis via nitric oxide synthase (NOS)-NO stimulation, but upstream and downstream mechanisms remain un-defined. To determine this, adult cardiac myocytes from genetically engineered mice and controls were studied by video microscopy to assess sarcomere shortening (SS) and fura2-AM fluorescence to measure calcium transients (CaT). Enhanced SS from isoproterenol (ISO, 10 nM) was suppressed a parts per thousand yen50% by the PDE5A inhibitor sildenafil (SIL, 1 mu M), without altering CaT. This regulation was unaltered despite co-inhibition of either the cGMP-stimulated cAMP-esterase PDE2 (Bay 60-7550), or cGMP-inhibited cAMP-esterase PDE3 (cilostamide). Thus, the SIL response could not be ascribed to cGMP interaction with alternative PDEs. However, genetic deletion (or pharmacologic blockade) of beta 3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in beta 3-AR knockout (beta 3-AR(-/-)) myocytes as in controls. Downstream, cGMP stimulates protein kinase G (PKG), and we found contractile modulation by SIL required PKG activation and enhanced TnI phosphorylation at S23, S24. Myocytes expressing the slow skeletal TnI isoform which lacks these sites displayed no modulation of ISO responses by SIL. Non-equilibrium isoelectric focusing gel electrophoresis showed SIL increased TnI phosphorylation above that from concomitant ISO in control but not beta 3-AR(-/-) myocytes. These data support a cascade involving beta 3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial beta-adrenergic stimulation.

Cellular Ca(2+) signaling underlies diverse vital biological processes, including muscle contractility, memory encoding, fertilization, cell survival, and cell death. Despite extensive studies, the fundamental control mechanisms that regulate intracellular Ca(2+) movement remain enigmatic. We have found recently that activation of the (Na(+)+K(+))-ATPase markedly potentiates intracellular Ca(2+) transients and contractility of rat heart cells. Little is known about the pathway responsible for the activation of the (Na(+)+K(+))-ATPase-initiated Ca(2+) signaling. Here, we demonstrate a novel mechanism in which activation of the (Na(+)+K(+))-ATPase is coupled to increased L-type Ca(2+) channel function through a signaling cascade involving Src and ERK1/2 but not well established regulators of the channel, such as adrenergic receptor system or activation of PKA or CaMKII. We have also identified Ser(1928), a phosphorylation site for the alpha1 subunit of the L-type Ca(2+) channel that may participate in the activation of the (Na(+)+K(+))-ATPase-mediated Ca(2+) signaling. The findings reported here uncover a novel molecular cross-talk between activation of the (Na(+)+K(+))-ATPase and L-type Ca(2+) channel and provide new insights into Ca(2+) signaling mechanisms for deeper understanding of the nature of cellular Ca(2+) handling in heart.

We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca2+transport, and cytoplasmic Ca2+signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [14C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca2+-ATPase protein levels and Ca2+transport activity to ∼50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca2+signaling kinetics, measured with a Ca2+-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca2+signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca2+signaling is partially compensated by alternate Ca2+transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.

Introduction: The term “development” refers to the process of growing to maturity. While decades of progress in developmental cardiology contributed to our understanding of heart development, the efforts were mostly focused on early aspects of development such as cardiomyocyte (CM) differentiation, which is completed at mid-gestation. However, CM maturation, a later developmental process required to generate adult CMs, remains largely unknown. The lack of the knowledge may be attributed to the fact that the process typically takes place over a long period of time after terminal differentiation. Results: Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and found a large number of genes whose expression shifts gradually and continuously throughout the stages. Based on the datasets, we generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs). This analysis revealed discrete sets of key transcriptional regulators and pathways that are temporally activated or suppressed over the course of CM maturation. We developed a GRN-based program named MatStatCM that informs the status of CM maturation at the molecular level. The MatStatCM demonstrated that pluripotent stem cell-derived CMs undergo maturation early in culture, but are arrested at the late embryonic stage, accompanied by aberrant expression of key transcription factors. Conclusions: Our study provides a foundation for understanding CM maturation.

Background: Heart Failure (HF) is one of the main causes of morbidity and mortality in westernized countries but the molecular mechanisms underlying its development are still unclear. A paradigm-shifting view focuses on the accumulation of preamyloid oligomers (PAOs), similar to those observed in Alzheimer disease, as a potential mechanism of cardiac toxicity. We reported that differential desmin phosphorylation at serines (S) 27 and 31 could drive the formation of PAOs in the heart, in the absence of genetic mutations. We sought to establish the identity of the molecular seed triggering the nucleation of cardiac PAOs in an experimental model of HF and in cultured cardiac cells. Methods: Mice were subjected to transverse aortic constriction (TAC) for 4 weeks (FS% = 29.3±2.6, P=0.0001). Alternatively, neonatal rat ventricular myocytes were transduced with lentiviral vectors carrying alanine (A) or phospho-mimetic aspartate (D) desmin double mutants at S27 and S31, fused with GFP. Protein homogenates were subjected to western blot analysis with fluorescent co-staining using the A11 anti-PAOs and anti-desmin antibodies. Transduced cells were also subjected to live imaging to assess phenotype. Results: Co-western blot analysis of both TAC mice and phospho-mimetic mutant cells revealed the colocalization of PAOs with desmin modified (potentially cleaved) forms. Preamyloid oligomers and a desmin fragment were both increased in TAC mice vs. controls (2.8-fold, P=0.023 and 1.8-fold, P=0.038, respectively). The DD mutant, mimicking the doubly phosphorylated desmin that we hypothesized is the physiological form, showed a healthier phenotype in terms of number of spontaneously contracting cells (P=0.041) and incorporation of GFP-desmin at the Z-discs (P=0.0027), whereas the mono-phosphomimetic mutant (AD) resulted in the increase of desmin positive aggregates (P=0.0014). Conclusions: This preliminary evidence suggests that desmin modified forms represent the seed triggering the formation of cardiac PAOs, in the absence of genetic mutations. The accumulation of desmin PAOs could therefore represent an overarching mechanism underlying the deterioration of cardiac function in HF.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Purpose: Androgen ablation is the standard initial treatment for advanced prostate cancer; however, tumors eventually develop androgen independence and become incurable. Chemotherapy is commonly used after hormone treatment fails but has not shown significant survival benefit. Studies suggest that androgen ablation can select for a population of hormone-independent cells that are also relatively chemotherapy resistant. Thus, it may be therapeutically advantageous to target prostate cancer with chemotherapy before hormone ablation. This study was undertaken to determine the relative efficacy of such an approach in a preclinical model of prostate cancer. Experimental Design: Severe combined immunodeficient mice bearing human LNCaP prostate tumors were treated with docetaxel and/or surgical castration applied singly, concurrently, or in different sequences. Treatment efficacy was determined by tumor volume and growth delay measurements. The extent of apoptosis in tumors in response to treatments was assessed via terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assays. In addition, Western blots were done to study the relative expression of Bcl-2 and Bax in the tumors. Results: Docetaxel followed by castration showed the most potent antitumor effects. In contrast, with the exception of castration alone, castration followed by docetaxel produced the least antitumor activity. TUNEL assays confirmed that the density of apoptotic tumor cells was significantly greater for docetaxel followed by castration than for any other treatment. In tumors of mice treated with single modality therapies, Bax to Bcl-2 ratios decreased significantly after castration, whereas this ratio remained high after docetaxel treatment. Conclusion: A treatment sequence of docetaxel followed by hormone ablation may be more effective in treating prostate cancer than concurrent docetaxel/hormone therapy or hormone ablation followed by docetaxel.

The cGMP-dependent protein kinase-1α (PKG1α) transduces NO and natriuretic peptide signaling; therefore, PKG1α activation can benefit the failing heart. Disease modifiers such as oxidative stress may depress the efficacy of PKG1α pathway activation and underlie variable clinical results. PKG1α can also be directly oxidized, forming a disulfide bond between homodimer subunits at cysteine 42 to enhance oxidant-stimulated vasorelaxation; however, the impact of PKG1α oxidation on myocardial regulation is unknown. Here, we demonstrated that PKG1α is oxidized in both patients with heart disease and in rodent disease models. Moreover, this oxidation contributed to adverse heart remodeling following sustained pressure overload or Gq agonist stimulation. Compared with control hearts and myocytes, those expressing a redox-dead protein (PKG1α(C42S)) better adapted to cardiac stresses at functional, histological, and molecular levels. Redox-dependent changes in PKG1α altered intracellular translocation, with the activated, oxidized form solely located in the cytosol, whereas reduced PKG1α(C42S) translocated to and remained at the outer plasma membrane. This altered PKG1α localization enhanced suppression of transient receptor potential channel 6 (TRPC6), thereby potentiating antihypertrophic signaling. Together, these results demonstrate that myocardial PKG1α oxidation prevents a beneficial response to pathological stress, may explain variable responses to PKG1α pathway stimulation in heart disease, and indicate that maintaining PKG1α in its reduced form may optimize its intrinsic cardioprotective properties.

Aims: Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca2+ uptake and myofilament Ca2+ sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in a redox-dependent manner, improving Ca2+ handling in isolated myocytes/hearts. Results: Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln−/−) mice. Compared to WT, pln−/− myocytes displayed enhanced resting sarcomere shortening, peak Ca2+ transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca2+ transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln−/− cells/hearts. HNO enhanced SR Ca2+ uptake in WT but not pln−/− SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca2+-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. Innovation: HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. Conclusions: PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca2+ handling in failing hearts. Antioxid. Redox Signal. 19, 1185–1197.

Cardiac hypertrophy in response to pathologic stress is a major contributor to heart disease. Many studies have revealed that compartmentalized cyclic guanosine monophosphate (cGMP) regulation by phosphodiesterases (PDEs) can potently modify acute and chronic cardiac stress responses. PDE5a normally regulates cGMP generated from nitric oxide (NO)-stimulated soluble guanylate cyclase (sGC) but not natriuretic peptide (NP)-stimulated cGMP. Its inhibition blunts maladaptive hypertrophy and remodeling. Regulators of NP-cGMP remain uncertain. Here we reveal that PDE9a, a highly cGMP-specific PDE (×100 affinity vs PDE5a) that is expressed predominantly in the brain modulates myocyte cGMP as well, targeting NP-stimulated pools. PDE9a gene expression is observed in rat neonatal cardiac myocytes (RNCM) and adult mouse myocytes. Protein expression was identified in myocytes by immunohistochemistry, using gene silencing models as a negative control. PDE9a expression is upregulated by various hypertrophic stimuli (phenylephrine (PE) or endothelin-1 (ET-1), and observed in myocytes from pressure-overloaded whole myocardium. PDE9a inhibition with a selective antagonist (PF-9613) or gene silencing significantly abrogated PE- or ET-1-dependent upregulation of pathological-hypertrophy fetal genes and NFAT activity in RNCM, and PDE9a inhibition attenuated ET-1-induced hypertrophy in adult mouse myocytes. PF-9613 had no impact in cells lacking PDE9a (from knockout mice) or after gene silencing. Biochemical and cGMP-sensitive fluorescent probe studies shows PDE9a inhibition augments ANP- but not NO-dependent cGMP stimuli in RNCM and adult myocytes. PDE9a knockout (KO) mice were protected against hypertrophy/remodeling after pressure-overload, accompanied by a rise in myocardial cGMP. In PDE9a KO mice also exhibited less fibrosis and reduced expression of fibrotsis-related genes over wild-type mice. Together, these data identify PDE9a as a novel regulator of myocyte cGMP and hypertrophy that impacts a different compartmentalized cGMP pool to that by PDE5a. Our observations may provide a novel therapeutic approach in the treatment of heart failure.

The cyclic nucleotide cGMP and its corresponding activated kinase cGK-1 serve as a counterbalance to acute and chronic myocardial stress. cGMP hydrolysis by several members of the phosphodiesterase (PDE) superfamily, PDE1, PDE2, and PDE5, regulate this signaling in the heart. This review details new insights regarding how these PDEs modulate cGMP and cGK-1 to influence heart function and chronic stress responses, and how their inhibition may provide potential therapeutic benefits.

Our enjoyable collaboration with Dr. Jeffrey P. Froehlich focused on the role of phospholamban (PLN) in HNO-induced enhancement of SR Ca2+-ATPase (SERCA2a) activity. We hypothesized that given HNO thiophilic nature it modifies cysteine residues in PLN transmembrane domain, altering its interaction with SERCA2a, thus enhancing pump activity. When HNO, donated by Angeli's salt (AS), was administered to control isolated myocytes, it enhanced both sarcomere shortening and Ca2+ transient. However, when AS/HNO was applied to PLN-/- ventriculocytes, HNO inotropy was reduced by ≅ 50% (the remaining likely stemming from enhanced myofilament sensitivity to Ca2+). PLN centrality to HNO cardiotropic action was confirmed incubating SR vesicles from WT and PLN-/- mice with AS/HNO to measure ATP-dependent Ca2+ uptake by stopped-flow mixing. In WT, HNO increased Ca2+ uptake rate, but it failed to do so in PLN-/- vesicles. The role of cysteines in PLN emerged from studies using ER microsomes from Sf21 insect cells expressing SERCA2a±PLN (WT or Cys 36-41-46→Ala mutant) where we assessed SERCA2a dephosphorylation, a measure of E2P hydrolysis, i.e. a rate-limiting step of SERCA2a activity. AS/HNO augmented SERCA2a dephosphorylation in ER microsomes co-expressing SERCA2a and WT PLN, but this stimulation was absent in microsomes expressing SERCA2a and Cys 36-41-46→Ala mutant PLN. Thus, Jeff's elegant, creative and passionate approach helped us to show that PLN is essential for HNO-induced faster Ca2+ uptake by SERCA2a, suggesting that HNO action occurs, at least partly, via modifications of critical cysteines in PLN transmembrane domain. In Jeff's view, “forbidden” disulfide bonds in PLN are involved, and one of his legacies for us is unearthing the cysteine pairs that are involved in HNO-induced formation of an intramolecular bond that could distort the conformation of PLN, thus perturbing its interaction with SERCA2a and relieving the inhibition.

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca 2+ by the cardiac SR Ca 2+ pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphoryla- tion) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys f Ala mutant). The results show that activation of SERCA2a dephos- phorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca 2+ pump. Stimulation of the cardiac SR Ca 2+ pump (SERCA2a) 1 in isolated SR vesicles by cAMP/protein kinase A-dependent phosphorylation of phospholamban (PLN) was originally demonstrated by Tada et al. (1). Subsequent studies showed that phosphorylation of PLN at Ser 16 increases the apparent Ca 2+ affinity of SERCA2a (2), the rates of phosphorylation and dephosphorylation (2, 3), and the Vmax of Ca 2+ transport (3, 4). Phosphorylation of Ser 16 induces a change in the conformation of PLN (5, 6), which relieves its inhibition and activates the pump. Recent work with ex- pressed Ca 2+ pump proteins suggests that activation of the cardiac SR Ca 2+ pump following the relief of PLN inhibition involves changes in the kinetic behavior of the Ca 2+ -ATPase consistent with SERCA2a oligomerization (7). These changes include stabilization of the ADP-sensitive phosphoenzyme, E1P, and allosteric activation of dephosphorylation by ATP, resulting in an increased rate of turnover of the ADP- insensitive phosphoenzyme, E2P. We have recently shown that nitroxyl (HNO) donated by Angeli's salt (AS, Na2N2O3) activates the SR Ca 2+ pump in isolated murine cardiac myocytes (8). This effect is inde- pendent of � -adrenergic activation (9) and results from a direct effect of HNO on the SR Ca 2+ pump as evidenced by stimulation of Ca 2+ uptake by HNO in isolated murine heart SR vesicles (8). We hypothesize that HNO stimulates Ca 2+ uptake by covalently modifying critical thiol residues in the SERCA2a pump and/or its regulatory protein, PLN, altering the conformation of these proteins, and relieving the inhibi- tion of the pump. Chemical modification of protein thiols by HNO can proceed by two pathways leading to either (a) the formation of a sulfinamide (RS(O)NH2) when one thiol group is involved or (b) the formation of a disulfide (RSSR) plus hydroxylamine (H2NOH) when two thiols are in the proximity of each other (10, 11). The fact that HNO-induced activation of RyR2 receptors reconstituted in lipid planar bilayers can be reversed by DTT (8) suggests that disulfide bond formation may be important in activating Ca 2+ release. In this study, we investigated the mechanism of activation of the SR Ca 2+ pump by HNO using SERCA2a expressed in the absence or presence of PLN in High Five (HF) insect cell microsomes. To study Ca 2+ pump activation by HNO, we measured the kinetics of dephosphorylation of SERCA2a, which is accelerated by HNO. To test whether thiol residues in PLN are critical for activation, we carried out these experiments in microsomes expressing SERCA2a and null-Cys PLN (PLN(-C)) in which the three transmembrane (TM) domain cysteine residues were replaced with alanine. Immunoblotting with an anti-PLN antibody was used to test whether HNO-induced PLN oligomer formation contributes to the relief of inhibition of SERCA2a by PLN. The results show that PLN is necessary for activation of SERCA2a by HNO and that cysteine residues in the TM domain of PLN play a critical role in PLN-dependent HNO activation of the Ca 2+ pump.

Cells with increasing resistance to the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibitor thapsigargin (TG), ranging from 60-fold (PC3/TG 10 cells) to 1350-fold (PC3/TG 2000 cells), were derived from PC3 cells. SERCA2 is overexpressed in all PC3/TG cells but retains sensitivity to TG. siRNA-mediated downregulation of SERCA completely or partially reverses TG resistance in PC3/TG 10 or PC3/TG 2000 cells, respectively; thus SERCA overexpression mediates resistance in PC3/TG 10 cells but is not the only resistance mechanism in PC3/TG 2000 cells. By contrast, SERCA is not overexpressed in TG-resistant DU145/TG cells derived from DU145 cells. DU145/TG cells retain resistance while in PC3/TG cells resistance decreases upon removal of TG selection. The transport proteins PGP/BCRP/MRP1 and anti-apoptotic proteins Bcl2/Bcl XL are not involved in mediating resistance in either cell line. PARP and caspase 3 cleavage in response to other drugs demonstrate that the apoptotic pathways tested remain intact in these cells. Further, no cross-resistance occurs to other drugs. Thus, novel TG-specific resistance mechanisms are recruited by these cancer cells.

Impaired cGMP-PKG signaling may contribute to cardiomyocyte remodeling and cardiac dysfunction in heart failure with preserved ejection fraction (HFpEF) patients. The purpose of this study was to assess cardiomyocyte function and morphology in aortic-banded mini-swine displaying a HFpEF phenotype following manipulation of cGMP signaling via two mechanisms: 1) driving cGMP synthesis with the DPP4 inhibitor saxagliptin; and 2) preventing cGMP catabolism via the PDE5 inhibitor tadalafil. We hypothesized that preserving cGMP-PKG signaling would prevent cardiomyocyte dysfunction and remodeling. Contractile function was measured in enzymatically isolated cardiomyocytes electrically stimulated at three frequencies (0.25, 0.5, 1.0 Hz)from four groups: control (CON), aortic-banded (AB), AB saxagliptin-treated (AB-SAX), and AB tadalafil-treated (AB-TAD). Increased cGMP activity was only observed in AB-TAD animals, however, neither drug treatment increased PKG activity. A significant pacing-induced decrease in diastolic sarcomere length was observed in the AB and AB-TAD groups compared to CON. This finding, indicative of impaired diastolic relaxation, was prevented by saxagliptin treatment. Shortening amplitude decreased in AB cardiomyocytes with increasing pacing frequency, revealing a negative shortening-frequency relationship that was attenuated by both drug treatments. Length:width ratio was decreased in AB and AB-TAD animals with commensurate decreases in left ventricular (LV) end diastolic and end systolic volumes. Normal cardiomyocyte size and LV volumes were preserved in the AB-SAX group. Interestingly, all AB groups exhibited similar gross hypertrophic remodeling (heart weight:body weight ratio) despite differences in cardiomyocyte morphology. In conclusion, saxagliptin appears superior for preserving normal cardiomyocyte morphology and overall function versus tadalafil, independent of changes in cGMP-PKG activity.