Paving a pathway for large-scale utilization of genomics in precision medicine and population health.

Having worked with two large population sequencing initiatives, the separation between the potential for genomics in precision medicine and the current reality have become clear. To realize this potential requires workflows, policies, and technical architectures that are foreign to most healthcare systems. Many historical processes and regulatory barriers currently impede our progress. The future of precision medicine includes genomic data being widely available at the point of care with systems in place to manage its efficient utilization. To achieve such vision requires substantial changes in billing, reimbursement, and reporting as well as the development of new systemic and technical architectures within the healthcare system. Clinical geneticist roles will evolve into managing precision health frameworks and genetic counselors will serve crucial roles in both leading and supporting precision medicine through the implementation and maintenance of precision medicine architectures. Our current path has many obstacles that hold us back, leaving preventable deaths in the wake. Reengineering our healthcare systems to support genomics can have a major impact on patient outcomes and allow us to realize the long-sought promises of precision medicine.

HerediGene Population Study IT infrastructure: A model to support genomic research recruitment and precision public health.

The HerediGene Population Study is a large research study focused on identifying new genetic biomarkers for disease prevention, diagnosis, prognosis, and development of new therapeutics. A substantial IT infrastructure evolved to reach enrollment targets and return results to participants. More than 170,000 participants have been enrolled in the study to date, with 5.87% of those whole genome sequenced and 0.46% of those genotyped harboring pathogenic variants. Among other purposes, this infrastructure supports: (1) identifying candidates from clinical criteria, (2) monitoring for qualifying clinical events (e.g., blood draw), (3) contacting candidates, (4) obtaining consent electronically, (5) initiating lab orders, (6) integrating consent and lab orders into clinical workflow, (7) de-identifying samples and clinical data, (8) shipping/transmitting samples and clinical data, (9) genotyping/sequencing samples, (10) and re-identifying and returning results for participants where applicable. This study may serve as a model for similar genomic research and precision public health initiatives.

The Development of an Infrastructure to Facilitate the Use of Whole Genome Sequencing for Population Health.

The clinical use of genomic analysis has expanded rapidly resulting in an increased availability and utility of genomic information in clinical care. We have developed an infrastructure utilizing informatics tools and clinical processes to facilitate the use of whole genome sequencing data for population health management across the healthcare system. Our resulting framework scaled well to multiple clinical domains in both pediatric and adult care, although there were domain specific challenges that arose. Our infrastructure was complementary to existing clinical processes and well-received by care providers and patients. Informatics solutions were critical to the successful deployment and scaling of this program. Implementation of genomics at the scale of population health utilizes complicated technologies and processes that for many health systems are not supported by current information systems or in existing clinical workflows. To scale such a system requires a substantial clinical framework backed by informatics tools to facilitate the flow and management of data. Our work represents an early model that has been successful in scaling to 29 different genes with associated genetic conditions in four clinical domains. Work is ongoing to optimize informatics tools; and to identify best practices for translation to smaller healthcare systems.

Discovery of TITIN Gene Truncating Variant Mutations and 5-Year Outcomes in Patients With Nonischemic Dilated Cardiomyopathy.

Genetic factors play an important role in nonischemic dilated cardiomyopathy (NIDC). However, prime opportunities remain for genetic discovery and prognostic understanding. TITIN gene truncating variant mutations (TTNtv) are of interest because of their frequent appearance in NIDC series. We sought to discover known and novel TTNtv mutations in a NIDC cohort and assess 5-year outcomes. Patients with NIDC entered into the INSPIRE Registry with ≥3 years of follow-up were studied. Whole exome sequencing (WES) was performed using an Illumina Novaseq platform. Genetic analysis used Sentieon software and the GRCh38 human reference genome. Variant calls were annotated with ClinVar. Five-year outcomes were determined by functional assessment and ejection fraction (EF) as recovered (EF ≥50%), persistent (EF 21% to 49%), or progressive (left ventricular assist device, transplant, heart failure [HF] or arrhythmic death, or EF ≤20%). The study comprised 229 NIDC patients (age = 50 ± 15 years, 58% men). TTNtv's were discovered in 27 patients with 22 unique mutations; (7 known, 15 novel). TTNtv+ patients more frequently presented with severe NIDC (EF ≤20%) (p = 0.032). By 5-year, outcomes were worse in TTNtv+ patients (p = 0.027), and patients less often recovered (11% vs. 30%). Prognosis was similar with known and novel mutations. Nongenetic (e.g., environmental) cocausal risk factors for HF were frequently present, and these factors frequently appeared to act in concert with genetic variants to precipitate clinical HF. In conclusion, our study expands the library of likely pathogenic TTN mutations and increases our understanding of their clinical impact in association with other HF risk factors.

Cardiomyocyte substructure reverts to an immature phenotype during heart failure.

KEY POINTS: As reactivation of the fetal gene program has been implicated in pathological remodelling during heart failure (HF), we examined whether cardiomyocyte subcellular structure and function revert to an immature phenotype during this disease. Surface and internal membrane structures appeared gradually during development, and returned to a juvenile state during HF. Similarly, dyadic junctions between the cell membrane and sarcoplasmic reticulum were progressively 'packed' with L-type Ca2+ channels and ryanodine receptors during development, and 'unpacked' during HF. Despite similarities in subcellular structure, dyads were observed to be functional from early developmental stages, but exhibited an impaired ability to release Ca2+ in failing cardiomyocytes. Thus, while immature and failing cardiomyocytes share similarities in subcellular structure, these do not fully account for the marked impairment of Ca2+ homeostasis observed in HF. ABSTRACT: Reactivation of the fetal gene programme has been implicated as a driver of pathological cardiac remodelling. Here we examined whether pathological remodelling of cardiomyocyte substructure and function during heart failure (HF) reflects a reversion to an immature phenotype. Using scanning electron microscopy, we observed that Z-grooves and t-tubule openings at the cell surface appeared gradually during cardiac development, and disappeared during HF. Confocal and super-resolution imaging within the cell interior revealed similar structural parallels; disorganization of t-tubules in failing cells was strikingly reminiscent of the late stages of postnatal development, with fewer transverse elements and a high proportion of longitudinal tubules. Ryanodine receptors (RyRs) were observed to be laid down in advance of developing t-tubules and similarly 'orphaned' in HF, although RyR distribution along Z-lines was relatively sparse. Indeed, nanoscale imaging revealed coordinated packing of L-type Ca2+ channels and RyRs into dyadic junctions during development, and orderly unpacking during HF. These findings support a 'last in, first out' paradigm, as the latest stages of dyadic structural development are reversed during disease. Paired imaging of t-tubules and Ca2+ showed that the disorganized arrangement of dyads in immature and failing cells promoted desynchronized and slowed Ca2+ release in these two states. However, while developing cells exhibited efficient triggering of Ca2+ release at newly formed dyads, dyadic function was impaired in failing cells despite similar organization of Ca2+ handling proteins. Thus, pathologically deficient Ca2+ homeostasis during HF is only partly linked to the re-emergence of immature subcellular structure, and additionally reflects lost dyadic functionality.

Bone turnover markers are differentially affected by pre-analytical handling.

Given that bone turnover markers are often shipped to central laboratories, it is essential to be aware of factors that will affect stability. We have evaluated how sample type, time before separation of blood samples, and time between separation and analysis affect the stability of four bone turnover markers. INTRODUCTION: Bone turnover markers are often shipped to central laboratories for analysis, which require knowledge of the stability of the markers of interest in different sample materials. The aim of the current study was to evaluate how time before separation of blood samples and time between separation and analysis affect the stability of four bone turnover markers in serum and plasma samples. METHODS: Serum, EDTA, and Lithium heparin (LiHep) plasma samples from seven osteoporosis patients and three healthy controls were collected and stored at room temperature for up to 72 h before separation and analysis. After separation, samples were stored at room temperature for up to 72 h and re-analyzed. The bone turnover markers N-terminal pro-collagen type 1 extension pro-peptide (P1NP), bone-specific alkaline phosphatase (BAP), C-terminal teleopeptide cross links of collagen type 1 (CTX), and osteocalcin (OC) were analyzed using the automated iSYS IDS platform. RESULTS: P1NP and BAP were stable in both plasma and serum for 72 h before centrifugation. CTX levels were higher in EDTA plasma at all time points compared to LiHep plasma and serum. The use of EDTA plasma prolonged the stability of CTX as compared to LiHep plasma and serum. Osteocalcin showed high tendency to degrade in all sample types and concentrations were significantly lower after 24 h of storage. CONCLUSIONS: For the bone turnover markers P1NP and BAP, the use of both plasma and serum is recommended. Samples for CTX analysis should be taken as EDTA plasma. Samples for osteocalcin analysis can be taken in either type of plasma or serum, but should be analyzed within 3 h or preserved at - 18 °C.

Overexpression of integrin α11 induces cardiac fibrosis in mice.

AIM: To understand the role of the collagen-binding integrin α11 in vivo, we have used a classical approach of creating a mouse strain overexpressing integrin α11. A transgenic mouse strain overexpressing α11 in muscle tissues was analysed in the current study with special reference to the heart tissue. METHODS: We generated and phenotyped integrin α11 transgenic (TG) mice by echocardiography, magnetic resonance imaging and histology. Wild-type (WT) mice were subjected to aortic banding (AB) and the expression of integrin α11 was measured in flow cytometry-sorted cardiomyocytes and non-myocytes. RESULTS: TG mice developed left ventricular concentric hypertrophy by 6 months, with increased collagen deposition and reactivation of mRNA encoding foetal genes associated with cardiovascular pathological remodelling compared to WT mice. Masson's trichrome staining revealed interstitial fibrosis, confirmed additionally by magnetic resonance imaging and was found to be most prominent in the cardiac septum of TG but not WT mice. TG hearts expressed increased levels of transforming growth factor-ß2 and transforming growth factor-ß3 and upregulated smooth muscle actin. Macrophage infiltration coincided with increased NF-κB signalling in TG but not WT hearts. Integrin α11 expression was increased in both cardiomyocytes and non-myocyte cells from WT AB hearts compared to sham-operated animals. CONCLUSION: We report for the first time that overexpression of integrin α11 induces cardiac fibrosis and left ventricular hypertrophy. This is a result of changes in intracellular hypertrophic signalling and secretion of soluble factors that increase collagen production in the heart.

Concepts and relevance of genome-wide association studies.

Genome-wide association study (GWAS) technology has been a primary method for identifying the genes responsible for diseases and other traits for the past 10 years. Over 2,000 human GWAS reports now appear in the scientific journals. The technology is continuing to improve, and has recently become accessible to researchers studying a wide variety of animals, plants and model organisms. Here, we present an overview of GWAS concepts: the underlying biology, the origins of the method, and the primary components of a GWAS experiment.

Seasonal variation in accumulation of persistent organic pollutants in an Arctic marine benthic food web.

The aim of the present study was to investigate seasonal variation in persistent organic pollutant (POP) concentrations, as well as food-web biomagnification, in an Arctic, benthic marine community. Macrozoobenthos, demersal fish and common eiders were collected both inside and outside of Kongsfjorden, Svalbard, during May, July and October 2007. The samples were analysed for a selection of legacy chlorinated POPs. Overall, low levels of POPs were measured in all samples. Although POP levels and accumulation patterns showed some seasonal variation, the magnitude and direction of change was not consistent among species. Overall, seasonality in bioaccumulation in benthic biota was less pronounced than in the pelagic system in Kongsfjorden. In addition, the results indicate that δ(15)N is not a good predictor for POP-levels in benthic food chains. Other factors, such as feeding strategy (omnivory, necrophagy versus herbivory), degree of contact with the sediment, and a high dependence on particulate organic matter (POM), with low POP-levels and high δ(15)N-values (due to bacterial isotope enrichment), seem to govern the uptake of the different POPs and result in loads deviating from what would be expected consulting the trophic position alone.

Current status, between-year comparisons and maternal transfer of organohalogenated compounds (OHCs) in Arctic char (Salvelinus alpinus) from Bjørnøya, Svalbard (Norway).

High levels of organohalogenated compounds (OHCs) have been found in Arctic char from Lake Ellasjøen at Bjørnøya (Svalbard, Norway) compared to char from other arctic lakes. The first aim of the study was to investigate the OHC status, contaminant profile, and partitioning of OHCs between muscle and ovary tissue in spawning female char from the high-polluted Lake Ellasjøen and the low-polluted Lake Laksvatn. The second aim was to investigate if OHC levels in muscle tissue have changed over time. Between-lake comparisons show that the muscle levels (lipid weight) of hexachlorobenzene (HCB), chlordanes (∑CHLs), mirex, dichlorodiphenyltrichloroethanes (∑DDTs) and polychlorinated biphenyls (∑PCBs) were up to 36 times higher in char from Ellasjøen than in Laksvatn, and confirm that the char from Ellasjøen are still heavily exposed compared to char from neighboring lake. A higher proportion of persistent OHCs were found in Ellasjøen compared to Laksvatn, while the proportion of the less persistent OHCs was highest in Laksvatn. A between-year comparison of OHC levels (i.e., HCB, DDTs, PCBs) in female and male char shows higher levels of HCB in female char from Ellasjøen in 2009/2012 compared to in 1999/2001. No other between-year differences in OHC levels were found. Due to small study groups, findings associated with between-year differences in OHC levels should be interpreted with caution. OHCs accumulate in the lipid rich ovaries of spawning females, resulting in up to six times higher levels of OHCs in ovaries compared to in muscle (wet weight). The toxic equivalent (TEQ)-value for the dioxin-like PCBs (PCB-105 and -118) in ovaries of the Ellasjøen char exceeded levels associated with increased egg mortality in rainbow trout (Oncorhynchus mykiss). Hence, we suggest that future studies should focus on the reproductive health and performance abilities of the high-exposed population of char inhabiting Lake Ellasjøen.

Brain abnormalities in bipolar disorder detected by quantitative T1ρ mapping.

Abnormal metabolism has been reported in bipolar disorder, however, these studies have been limited to specific regions of the brain. To investigate whole-brain changes potentially associated with these processes, we applied a magnetic resonance imaging technique novel to psychiatric research, quantitative mapping of T1 relaxation in the rotating frame (T1ρ). This method is sensitive to proton chemical exchange, which is affected by pH, metabolite concentrations and cellular density with high spatial resolution relative to alternative techniques such as magnetic resonance spectroscopy and positron emission tomography. Study participants included 15 patients with bipolar I disorder in the euthymic state and 25 normal controls balanced for age and gender. T1ρ maps were generated and compared between the bipolar and control groups using voxel-wise and regional analyses. T1ρ values were found to be elevated in the cerebral white matter and cerebellum in the bipolar group. However, volumes of these areas were normal as measured by high-resolution T1- and T2-weighted magnetic resonance imaging. Interestingly, the cerebellar T1ρ abnormalities were normalized in participants receiving lithium treatment. These findings are consistent with metabolic or microstructural abnormalities in bipolar disorder and draw attention to roles of the cerebral white matter and cerebellum. This study highlights the potential utility of high-resolution T1ρ mapping in psychiatric research.

IL-18 neutralization during alveolar hypoxia improves left ventricular diastolic function in mice.

AIM: In patients, an association exists between pulmonary diseases and diastolic dysfunction of the left ventricle (LV). We have previously shown that alveolar hypoxia in mice induces LV diastolic dysfunction and that mice exposed to hypoxia have increased levels of circulating interleukin-18 (IL-18), suggesting involvement of IL-18 in development of diastolic dysfunction. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18. In this study, we hypothesized that neutralization of IL-18 during alveolar hypoxia would improve LV diastolic function. METHODS: Mice were exposed to 10% oxygen for 2 weeks while treated with IL-18BP or vehicle. Cardiac function and morphology were measured using echocardiography, intraventricular pressure measurements and magnetic resonance imaging (MRI). For characterization of molecular changes in the heart, both real-time PCR and Western blotting were performed. ELISA technique was used to measure levels of circulating cytokines. RESULTS: As expected, exposure to hypoxia-induced LV diastolic dysfunction, as shown by prolonged time constant of isovolumic relaxation (τ). Improved relaxation with IL-18BP treatment was demonstrated by a significant reduction towards control τ values. Decreased levels of phosphorylated phospholamban (P-PLB) in hypoxia, but normalization by IL-18BP treatment suggest a role for IL-18 in regulation of calcium-handling proteins in hypoxia-induced diastolic dysfunction. In addition, MRI showed less increase in right ventricular (RV) wall thickness in IL-18BP-treated animals exposed to hypoxia, indicating an effect on RV hypertrophy. CONCLUSION: Neutralization of IL-18 during alveolar hypoxia improves LV diastolic function and partly prevents RV hypertrophy.

Rex (encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough is a repressor of sulfate adenylyl transferase and is regulated by NADH.

Although the enzymes for dissimilatory sulfate reduction by microbes have been studied, the mechanisms for transcriptional regulation of the encoding genes remain unknown. In a number of bacteria the transcriptional regulator Rex has been shown to play a key role as a repressor of genes producing proteins involved in energy conversion. In the model sulfate-reducing microbe Desulfovibrio vulgaris Hildenborough, the gene DVU_0916 was observed to resemble other known Rex proteins. Therefore, the DVU_0916 protein has been predicted to be a transcriptional repressor of genes encoding proteins that function in the process of sulfate reduction in D. vulgaris Hildenborough. Examination of the deduced DVU_0916 protein identified two domains, one a winged helix DNA-binding domain common for transcription factors, and the other a Rossman fold that could potentially interact with pyridine nucleotides. A deletion of the putative rex gene was made in D. vulgaris Hildenborough, and transcript expression studies of sat, encoding sulfate adenylyl transferase, showed increased levels in the D. vulgaris Hildenborough Rex (RexDvH) mutant relative to the parental strain. The RexDvH-binding site upstream of sat was identified, confirming RexDvH to be a repressor of sat. We established in vitro that the presence of elevated NADH disrupted the interaction between RexDvH and DNA. Examination of the 5' transcriptional start site for the sat mRNA revealed two unique start sites, one for respiring cells that correlated with the RexDvH-binding site and a second for fermenting cells. Collectively, these data support the role of RexDvH as a transcription repressor for sat that senses the redox status of the cell.

Reduced visfatin levels in aortic stenosis increase after aortic valve replacement and may contribute to reverse left ventricular remodelling.

AIM: Visfatin may play a part in reverse left ventricular remodelling. Using a mouse model of reversible left ventricle pressure overload, we examined if visfatin was altered in the myocardium. Furthermore, we addressed this issue in patients with aortic stenosis (AS) and examined whether visfatin levels are related to reverse remodelling following aortic valve replacement (AVR). METHODS: Myocardial visfatin was analysed after aortic banding (AB) and debanding (DB) in mice and compared to sham operated animals. Myocardial visfatin was measured in biopsies from patients undergoing AVR and compared to controls. Serum visfatin was measured before and after AVR in patients with AS and correlated with echocardiographic measurments of cardiac morphology and function. RESULTS: Four weeks after AB, myocardial visfatin protein was reduced by 50% compared to sham. Three days after DB, myocardial protein levels increased significantly. Myocardial visfatin and serum visfatin levels were reduced by 23% and 64%, respectively, in patients with AS compared to controls. Twelve months after AVR, serum visfatin levels increased compared to preoperative values and correlated negatively with degree of left ventricular hypertrophy. CONCLUSION: Myocardial visfatin and serum visfatin levels are reduced by cardiac pressure overload. Visfatin levels increase after correction of pressure overload and may play a part in postoperative reverse remodelling.

Impaired left ventricular mechanical and energetic function in mice after cardiomyocyte-specific excision of Serca2.

Sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA)2 transports Ca2+ from the cytosol into the sarcoplasmic reticulum of cardiomyocytes and is essential for maintaining myocardial Ca2+ handling and thus the mechanical function of the heart. SERCA2 is a major ATP consumer in excitation-contraction coupling but is regarded to contribute to energetically efficient Ca2+ handling in the cardiomyocyte. Previous studies using cardiomyocyte-specific SERCA2 knockout (KO) mice have demonstrated that decreased SERCA2 activity reduces the Ca2+ transient amplitude and induces compensatory Ca2+ transport mechanisms that may lead to more inefficient Ca2+ transport. In this study, we examined the relationship between left ventricular (LV) function and myocardial O2 consumption (MVo2) in ex vivo hearts from SERCA2 KO mice to directly measure how SERCA2 elimination influences mechanical and energetic features of the heart. Ex vivo hearts from SERCA2 KO hearts developed mechanical dysfunction at 4 wk and demonstrated virtually no working capacity at 7 wk. In accordance with the reported reduction in Ca2+ transient amplitude in cardiomyocytes from SERCA2 KO mice, work-independent MVo2 was decreased due to a reduced energy cost of excitation-contraction coupling. As these hearts also showed a marked impairment in the efficiency of chemomechanical energy transduction (contractile efficiency, i.e, work-dependent MVo2), hearts from SERCA2 KO mice were found to be mechanically inefficient. This ex vivo evaluation of mechanical and energetic function in hearts from SERCA2 KO mice brings together findings from previous experimental and mathematical modeling-based studies and demonstrates that reduced SERCA2 activity not only leads to mechanical dysfunction but also to energetic dysfunction.

The cardiokine secreted Frizzled-related protein 3, a modulator of Wnt signalling, in clinical and experimental heart failure.

OBJECTIVES: Experimental studies have shown involvement of Wnt signalling in heart failure (HF). We hypothesized that secreted frizzled-related protein 3 (sFRP3), a modulator of Wnt signalling, is related to the progression of HF. DESIGN: Circulating sFRP3 was measured in 153 HF patients and compared with 25 healthy controls. The association of sFRP3 with mortality was evaluated in 1202 patients (GISSI-HF trial). sFRP3 mRNA expression was assessed in failing human and murine left ventricles (LV), and cellular localization was determined after fractioning of myocardial tissue. In vitro studies were carried out in cardiac fibroblasts subjected to cyclic mechanical stretch. RESULTS: (i) Heart failure patients had significantly raised serum sFRP3 levels compared with controls, (ii) during a median follow-up of 47 months, 315 patients died in the GISSI-HF substudy. In univariable Cox regression, tertiles of baseline sFRP3 concentration were significantly associated with all-cause and cardiovascular mortality. After adjustment for demographic and clinical variables, but not for CRP and NT-proBNP, the associations with mortality remained significant for the third tertile (all-cause, HR 1.45, P = 0.011; cardiovascular, HR 1.66, P = 0.003), (iii) sFRP3 mRNA expression was increased in failing human LV, with a decline following LV assist device therapy. LV from post-MI mice showed an increased sFRP3 mRNA level, particularly in cardiac fibroblasts, and (iv) mechanical stretch enhanced sFRP3 expression and release in myocardial fibroblasts. CONCLUSION: There is an association between increased sFRP3 expression and adverse outcome in HF, suggesting that the failing myocardium itself contributes to an increase in circulating sFRP3.

Bacterial skin commensals and their role as host guardians.

Recent years' investigations of the co-evolution and functional integration of the human body and its commensal microbiota have disclosed that the microbiome has a major impact on physiological functions including protection against infections, reaction patterns in the immune system, and disposition for inflammation-mediated diseases. Two ubiquitous members of the skin microbiota, the Gram-positive bacteria Staphylococcus epidermidis and Propionibacterium acnes, are predominant on human epithelia and in sebaceous follicles, respectively. Their successful colonisation is a result of a commensal or even mutualistic lifestyle, favouring traits conferring persistency over aggressive host-damaging properties. Some bacterial properties suggest an alliance with the host to keep transient, potential pathogens at bay, such as the ability of S. epidermidis to produce antimicrobials, or the production of short-chain fatty acids by P. acnes. These features can function together with host-derived components of the innate host defence to establish and maintain the composition of a health-associated skin microbiota. However, depending largely on the host status, the relationship between the human host and S. epidermidis/P. acnes can also have parasitic features. Both microorganisms are frequently isolated from opportunistic infections. S. epidermidis is a causative agent of hospital-acquired infections, mostly associated with the use of medical devices. P. acnes is suspected to be of major importance in the pathogenesis of acne and also in a number of other opportunistic infections. In this review we will present bacterial factors and traits of these two key members of our skin microbiota and discuss how they contribute to mutualistic and parasitic properties. The elucidation of their roles in health-promoting or disease-causing processes could lead to new prophylactic and therapeutic strategies against skin disorders and other S. epidermidis/P. acnes-associated diseases, and increase our understanding of the delicate interplay of the skin microbiota with the human host.

SERCA2 activity is involved in the CNP-mediated functional responses in failing rat myocardium.

BACKGROUND AND PURPOSES: Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca(2+) ATPase2 (SERCA2) activity is essential for these responses. EXPERIMENTAL APPROACH: Contractility, cGMP levels, Ca(2+) transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction. KEY RESULTS: CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser(16) and troponin I (TnI) Ser(23/24) phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca(2+) transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca(2+) channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene. CONCLUSIONS AND IMPLICATIONS: We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.