History of the development of isolated heart perfusion experimental model and its pioneering role in understanding heart physiology.

The isolated heart perfusion model, a fundamental tool in cardiovascular research, has evolved significantly since its inception in the late 19th century. This review traces the development of the isolated heart model, from its early adaptations by pioneers such as Langendorff and Starling to modern advancements by researchers like Morgan and Neely. We discuss the various applications of the model in pharmacological testing, disease modeling, and educational settings, emphasizing its crucial role in understanding cardiac function and disease mechanisms. Recent technological enhancements, including high-resolution imaging, integration with bioengineering, and advanced genomic and proteomic analyses, have significantly broadened the capabilities of these models. Looking forward, we explore potential future developments such as the integration of precision medicine, stem cell research, and artificial intelligence, which promise to revolutionize the use of isolated heart perfusion models. This review highlights the model's crucial role in bridging experimental research and clinical applications.

Guidelines for assessing maternal cardiovascular physiology during pregnancy and postpartum.

Maternal mortality rates are at an all-time high across the world and are set to increase in subsequent years. Cardiovascular disease is the leading cause of death during pregnancy and postpartum, especially in the United States. Therefore, understanding the physiological changes in the cardiovascular system during normal pregnancy is necessary to understand disease-related pathology. Significant systemic and cardiovascular physiological changes occur during pregnancy that are essential for supporting the maternal-fetal dyad. The physiological impact of pregnancy on the cardiovascular system has been examined in both experimental animal models and in humans. However, there is a continued need in this field of study to provide increased rigor and reproducibility. Therefore, these guidelines aim to provide information regarding best practices and recommendations to accurately and rigorously measure cardiovascular physiology during normal and cardiovascular disease-complicated pregnancies in human and animal models.

Molecular and cellular neurocardiology in heart disease.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.

Comparative specialization of intrinsic cardiac neurons in humans, mice, and pigs.

Intrinsic cardiac neurons (ICNs) play a crucial role in the proper functioning of the heart; yet a paucity of data pertaining to human ICNs exists. We took a multidisciplinary approach to complete a detailed cellular comparison of the structure and function of ICNs from mice, pigs, and humans. Immunohistochemistry of whole and sectioned ganglia, transmission electron microscopy, intracellular microelectrode recording and dye filling for quantitative morphometry were used to define the neurophysiology, histochemistry, and ultrastructure of these cells across species. The densely packed, smaller ICNs of mouse lacked dendrites, formed axosomatic connections, and had high synaptic efficacy constituting an obligatory synapse. At Pig ICNs, a convergence of subthreshold cholinergic inputs onto extensive dendritic arbors supported greater summation and integration of synaptic input. Human ICNs were tonically firing, with synaptic stimulation evoking large suprathreshold excitatory postsynaptic potentials like mouse, and subthreshold potentials like pig. Ultrastructural examination of synaptic terminals revealed conserved architecture, yet small clear vesicles (SCVs) were larger in pigs and humans. The presence and localization of ganglionic neuropeptides was distinct, with abundant VIP observed in human but not pig or mouse ganglia, and little SP or CGRP in pig ganglia. Action potential waveforms were similar, but human ICNs had larger after-hyperpolarizations. Intrinsic excitability differed; 93% of human cells were tonic, all pig neurons were phasic, and both phasic and tonic phenotypes were observed in mouse. In combination, this publicly accessible, multimodal atlas of ICNs from mice, pigs, and humans identifies similarities and differences in the evolution of ICNs.

Characterizing the Causal Pathway From Childhood Adiposity to Right Heart Physiology and Pulmonary Circulation Using Lifecourse Mendelian Randomization.

BACKGROUND: Observational epidemiological studies have reported an association between childhood adiposity and altered cardiac morphology and function in later life. However, whether this is due to a direct consequence of being overweight during childhood has been difficult to establish, particularly as accounting for other measures of body composition throughout the lifecourse can be exceptionally challenging. METHODS AND RESULTS: In this study, we used human genetics to investigate this using a causal inference technique known as lifecourse Mendelian randomization. This approach allowed us to evaluate the effect of childhood body size on 11 measures of right heart and pulmonary circulation independent of other anthropometric traits at various stages in the lifecourse. We found strong evidence that childhood body size has a direct effect on an enlarged right heart structure in later life (eg, right ventricular end-diastolic volume: ß=0.24 [95% CI, 0.15-0.33]; P=3×10-7) independent of adulthood body size. In contrast, childhood body size effects on maximum ascending aorta diameter attenuated upon accounting for body size in adulthood, suggesting that this effect is likely attributed to individuals remaining overweight into later life. Effects of childhood body size on pulmonary artery traits and measures of right atrial function became weaker upon accounting for adulthood fat-free mass and childhood height, respectively. CONCLUSIONS: Our findings suggest that, although childhood body size has a long-term influence on an enlarged heart structure in adulthood, associations with the other structural components of the cardiovascular system and their function may be largely attributed to body composition at other stages in the lifecourse.

Sex-specific structural and functional cardiac remodeling during healthy aging assessed by cardiovascular magnetic resonance.

BACKGROUND: Aging as a major non-modifiable cardiac risk factor challenges future cardiovascular medicine and economic demands, which requires further assessments addressing physiological age-associated cardiac changes. OBJECTIVES: Using cardiovascular magnetic resonance (CMR), this study aims to characterize sex-specific ventricular adaptations during healthy aging. METHODS: The population included healthy volunteers who underwent CMR at 1.5 or 3 Tesla scanners applying cine-imaging with a short-axis coverage of the left (LV) and right (RV) ventricle. The cohort was divided by sex (female and male) and age (subgroups in years): 1 (19-29), 2 (30-39), 3 (40-49), and 4 (≥50). Cardiac adaptations were quantitatively assessed by CMR indices. RESULTS: After the exclusion of missing or poor-quality CMR datasets or diagnosed disease, 140 of 203 volunteers were part of the final analysis. Women generally had smaller ventricular dimensions and LV mass, but higher biventricular systolic function. There was a significant age-associated decrease in ventricular dimensions as well as a significant increase in LV mass-to-volume ratio (LV-MVR, concentricity) in both sexes (LV-MVR in g/ml: age group 1 vs. 4: females 0.50 vs. 0.57, p=0.016, males 0.56 vs. 0.67, p=0.024). LV stroke volume index decreased significantly with age in both sexes, but stronger for men than for women (in ml/m2: age group 1 vs. 4: females 51.76 vs. 41.94, p<0.001, males 55.31 vs. 40.78, p<0.001). Ventricular proportions (RV-to-LV-volume ratio) were constant between the age groups in both sexes. CONCLUSIONS: In both sexes, healthy aging was associated with an increase in concentricity and a decline in ventricular dimensions. Furthermore, relevant age-related sex differences in systolic LV performance were observed.

Reduction of left ventricular diastolic pressure as a key regulator of infarct coronary flow under mechanical left ventricular support.

Restoring ischaemic myocardial tissue perfusion is crucial for minimizing infarct size. Acute mechanical left ventricular (LV) support has been suggested to improve infarct tissue perfusion. However, its regulatory mechanism remains unclear. We investigated the physiological mechanisms in six Yorkshire pigs, which were subjected to 90-min balloon occlusion of the left anterior descending artery. During the acute reperfusion phase, LV support using an Impella heart pump was initiated. LV pressure, coronary flow and pressure of the infarct artery were simultaneously recorded to evaluate the impact of LV support on coronary physiology. Coronary wave intensity was calculated to understand the forces regulating coronary flow. Significant increases in coronary flow velocity and its area under the curve were found after mechanical LV support. Among the coronary flow-regulating factors, coronary pressure was increased mainly during the late diastolic phase with less pulsatility. Meanwhile, LV pressure was reduced throughout diastole resulting in significant and consistent elevation of coronary driving pressure. Interestingly, the duration of diastole was prolonged with LV support. In the wave intensity analysis, the duration between backward suction and pushing waves was extended, indicating that earlier myocardial relaxation and delayed contraction contributed to the extension of diastole. In conclusion, mechanical LV support increases infarct coronary flow by extending diastole and augmenting coronary driving pressure. These changes were mainly driven by reduced LV diastolic pressure, indicating that the key regulator of coronary flow under mechanical LV support is downstream of the coronary artery, rather than upstream. Our study highlights the importance of LV diastolic pressure in infarct coronary flow regulation. KEY POINTS: Restoring ischaemic myocardial tissue perfusion is crucial for minimizing infarct size. Although mechanical left ventricular (LV) support has been suggested to improve infarct coronary flow, its specific mechanism remains to be clarified. LV support reduced LV pressure, and elevated coronary pressure during the late diastolic phase, resulting in high coronary driving pressure. This study demonstrated for the first time that mechanical LV support extends diastolic phase, leading to increased infarct coronary flow. Future studies should evaluate the correlation between improved infarct coronary flow and resulting infarct size.

Effects of pericardiocentesis on renal function and cardiac hemodynamics.

BACKGROUND: Previous case studies have reported reversal of acute renal failure after pericardiocentesis in pericardial effusion. This study examines the effects of pericardiocentesis on preprocedural low cardiac output and acute renal dysfunction in patients with pericardial effusion. METHODS: This is a retrospective study of 95 patients undergoing pericardiocentesis between 2015 and 2020. Pre- and post-procedure transthoracic echocardiograms (TTE) were reviewed for evidence of cardiac tamponade, resolution of pericardial effusion, and for estimation of right atrial (RA) pressure and cardiac output. Laboratory values were compared at presentation and post-procedure. Patients on active renal replacement therapy were excluded. RESULTS: Ninety-five patients were included for analysis (mean age 62.2 ± 17.8 years, 58% male). There was a significant increase in glomerular filtration rate pre- and post-procedure. Fifty-six patients (58.9%) had an improvement in glomerular filtration rate after pericardiocentesis (termed "responders"), and these patients had a lower pre-procedure glomerular filtration rate than "non-responders." There was a significant improvement in estimated cardiac output and right atrial pressure for patients in both groups. Patients who had an improvement in renal function had significantly lower pre-procedural diastolic blood pressure and mean arterial pressure. CONCLUSIONS: Pericardial drainage may improve effusion-mediated acute renal dysfunction by reducing right atrial pressure and thus systemic venous congestion, and by increasing forward stroke volume and perfusion pressure.

Cannon A wave validation as a diagnostic tool in paroxysmal supraventricular tachycardias.

OBJECTIVE: The presence of cannon A waves, the so called "frog sign", has traditionally been considered diagnostic of atrioventricular nodal re-entrant tachycardia (AVNRT). Nevertheless, it has never been systematically evaluated. The aim of this study is to assess the independent diagnostic utility of cannon A waves in the differential diagnosis of supraventricular tachycardias (SVTs). METHODS: We prospectively included 100 patients who underwent an electrophysiology (EP) study for SVT. The right jugular venous pulse was recorded during the study. In 61 patients, invasive central venous pressure (CVP) was registered as well. CVP increase is thought to be related with the timing between atria and ventricle depolarization; two groups were prespecified, the short VA interval tachycardias (including typical AVNRT and atrioventricular reciprocating tachycardia (AVRT) mediated by a septal accessory pathway) and the long VA interval tachycardias (including atypical AVNRT and AVRT mediated by a left free wall accessory pathway). RESULTS: The relationship between cannon A waves and AVNRT did not reach the statistical significance (OR: 3.01; p = .058); On the other hand, it was clearly associated with the final diagnosis of a short VA interval tachycardia (OR: 10.21; p < .001). CVP increase showed an inversely proportional relationship with the VA interval during tachycardia (b = -.020; p < .001). CVP increase was larger in cases of AVNRT (4.0 mmHg vs. 1.2 mmHg; p < .001) and short VA interval tachycardias (3.9 mmHg vs. 1.2 mmHg; p < .001). CONCLUSION: The presence of cannon A waves is associated with the final diagnosis of short VA interval tachycardias.

Expanding the Frontiers of Guardian Antioxidant Selenoproteins in Cardiovascular Pathophysiology.

Significance: Physiological levels of reactive oxygen and nitrogen species (ROS/RNS) function as fundamental messengers for many cellular and developmental processes in the cardiovascular system. ROS/RNS involved in cardiac redox-signaling originate from diverse sources, and their levels are tightly controlled by key endogenous antioxidant systems that counteract their accumulation. However, dysregulated redox-stress resulting from inefficient removal of ROS/RNS leads to inflammation, mitochondrial dysfunction, and cell death, contributing to the development and progression of cardiovascular disease (CVD). Recent Advances: Basic and clinical studies demonstrate the critical role of selenium (Se) and selenoproteins (unique proteins that incorporate Se into their active site in the form of the 21st proteinogenic amino acid selenocysteine [Sec]), including glutathione peroxidase and thioredoxin reductase, in cardiovascular redox homeostasis, representing a first-line enzymatic antioxidant defense of the heart. Increasing attention has been paid to emerging selenoproteins in the endoplasmic reticulum (ER) (i.e., a multifunctional intracellular organelle whose disruption triggers cardiac inflammation and oxidative stress, leading to multiple CVD), which are crucially involved in redox balance, antioxidant activity, and calcium and ER homeostasis. Critical Issues: This review focuses on endogenous antioxidant strategies with therapeutic potential, particularly selenoproteins, which are very promising but deserve more detailed and clinical studies. Future Directions: The importance of selective selenoproteins in embryonic development and the consequences of their mutations and inborn errors highlight the need to improve knowledge of their biological function in myocardial redox signaling. This could facilitate the development of personalized approaches for the diagnosis, prevention, and treatment of CVD. Antioxid. Redox Signal. 40, 369-432.

Is the Ventilatory Efficiency in Endurance Athletes Different?-Findings from the NOODLE Study.

Background: Ventilatory efficiency (VE/VCO2) is a strong predictor of cardiovascular diseases and defines individuals' responses to exercise. Its characteristics among endurance athletes (EA) remain understudied. In a cohort of EA, we aimed to (1) investigate the relationship between different methods of calculation of VE/VCO2 and (2) externally validate prediction equations for VE/VCO2. Methods: In total, 140 EA (55% males; age = 22.7 ± 4.6 yrs; BMI = 22.6 ± 1.7 kg·m-2; peak oxygen uptake = 3.86 ± 0.82 L·min-1) underwent an effort-limited cycling cardiopulmonary exercise test. VE/VCO2 was first calculated to ventilatory threshold (VE/VCO2-slope), as the lowest 30-s average (VE/VCO2-Nadir) and from whole exercises (VE/VCO2-Total). Twelve prediction equations for VE/VCO2-slope were externally validated. Results: VE/VCO2-slope was higher in females than males (27.7 ± 2.6 vs. 26.1 ± 2.0, p < 0.001). Measuring methods for VE/VCO2 differed significantly in males and females. VE/VCO2 increased in EA with age independently from its type or sex (ß = 0.066-0.127). Eleven equations underestimated VE/VCO2-slope (from -0.5 to -3.6). One equation overestimated VE/VCO2-slope (+0.2). Predicted and observed measurements differed significantly in nine models. Models explained a low amount of variance in the VE/VCO2-slope (R2 = 0.003-0.031). Conclusions: VE/VCO2-slope, VE/VCO2-Nadir, and VE/VCO2-Total were significantly different in EA. Prediction equations for the VE/VCO2-slope were inaccurate in EA. Physicians should be acknowledged to properly assess cardiorespiratory fitness in EA.

The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila.

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

Looking Back, Going Forward: Understanding Cardiac Pathophysiology from Pressure-Volume Loops.

Knowing cardiac physiology is essential for health care professionals working in the cardiovascular field. Pressure-volume loops (PVLs) offer a unique understanding of the myocardial working and have become pivotal in complex pathophysiological scenarios, such as profound cardiogenic shock or when mechanical circulatory supports are implemented. This review provides a comprehensive summary of the left and right ventricle physiology, based on the PVL interpretation.

Long-term outcomes after heart transplantation in adult patients with univentricular versus biventricular congenital heart disease.

OBJECTIVES: Heart transplantation (HT) is the only life-extending therapy in adults with congenital heart disease (CHD) and end-stage heart failure. HT is considered at high risk in complex CHD given the anatomical complexity and past medical history. Little is known about long-term outcomes after HT in these patients. We aimed to evaluate early and long-term outcomes after HT in adult patients with univentricular versus biventricular CHD. METHODS: This multicentre retrospective cohort study included all adult CHD patients who underwent HT between 1988 and 2021 in 3 tertiary centres. Factors associated with early (<30 days) and conditional long-term survival were assessed in the entire cohort. RESULTS: Over a mean follow-up of 10.1 ± 7.8 years, 149 patients were included, of whom 55 (36.9%) had univentricular CHD. Sixty-four patients died during follow-up including 47 deaths before discharge from hospital. In multivariable analysis, univentricular physiology and female recipient gender were independently associated with a higher risk of early mortality (odds ratio 2.99; 95% confidence interval [1.33-6.74] and odds ratio 2.76; 95% confidence interval [1.23-6.20], respectively). For patients who survived the early period, conditional long-term survival was excellent for both groups and was not different between 2 groups (P = 0.764). CONCLUSIONS: Adult CHD patients have a high incidence of overall mortality due to a high rate of early mortality. Univentricular physiology was associated with a significant increased risk of early death compared to biventricular physiology. However, late mortality was excellent and no longer different between the 2 physiologies.

α7 nicotinic receptors play a role in regulation of cardiac hemodynamics.

The α7 nicotinic receptors (NR) have been confirmed in the heart but their role in cardiac functions has been contradictory. To address these contradictory findings, we analyzed cardiac functions in α7 NR knockout mice (α7-/-) in vivo and ex vivo in isolated hearts. A standard limb leads electrocardiogram was used, and the pressure curves were recorded in vivo, in Arteria carotis and in the left ventricle, or ex vivo, in the left ventricle of the spontaneously beating isolated hearts perfused following Langedorff's method. Experiments were performed under basic conditions, hypercholinergic conditions, and adrenergic stress. The relative expression levels of α and ß NR subunits, muscarinic receptors, ß1 adrenergic receptors, and acetylcholine life cycle markers were determined using RT-qPCR. Our results revealed a prolonged QT interval in α7-/- mice. All in vivo hemodynamic parameters were preserved under all studied conditions. The only difference in ex vivo heart rate between genotypes was the loss of bradycardia in prolonged incubation of isoproterenol-pretreated hearts with high doses of acetylcholine. In contrast, left ventricular systolic pressure was lower under basal conditions and showed a significantly higher increase during adrenergic stimulation. No changes in mRNA expression were observed. In conclusion, α7 NR has no major effect on heart rate, except when stressed hearts are exposed to a prolonged hypercholinergic state, suggesting a role in acetylcholine spillover control. In the absence of extracardiac regulatory mechanisms, left ventricular systolic impairment is revealed.

Modulating cardiac physiology in engineered heart tissue with the bidirectional optogenetic tool BiPOLES.

Optogenetic actuators are rapidly advancing tools used to control physiology in excitable cells, such as neurons and cardiomyocytes. In neuroscience, these tools have been used to either excite or inhibit neuronal activity. Cell type-targeted actuators have allowed to study the function of distinct cell populations. Whereas the first described cation channelrhodopsins allowed to excite specific neuronal cell populations, anion channelrhodopsins were used to inhibit neuronal activity. To allow for simultaneous excitation and inhibition, opsin combinations with low spectral overlap were introduced. BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing) is a bidirectional optogenetic tool consisting of the anion channel Guillardia theta anion-conducting channelrhodopsin 2 (GtACR2 with a blue excitation spectrum and the red-shifted cation channel Chrimson. Here, we studied the effects of BiPOLES activation in cardiomyocytes. For this, we knocked in BiPOLES into the adeno-associated virus integration site 1 (AAVS1) locus of human-induced pluripotent stem cells (hiPSC), subjected these to cardiac differentiation, and generated BiPOLES expressing engineered heart tissue (EHT) for physiological characterization. Continuous light application activating either GtACR2 or Chrimson resulted in cardiomyocyte depolarization and thus stopped EHT contractility. In contrast, short light pulses, with red as well as with blue light, triggered action potentials (AP) up to a rate of 240 bpm. In summary, we demonstrate that cation, as well as anion channelrhodopsins, can be used to activate stem cell-derived cardiomyocytes with pulsed photostimulation but also to silence cardiac contractility with prolonged photostimulation.

Using DeepLabCut for markerless cardiac physiology and toxicity estimation in water fleas (Daphnia magna).

Daphnia magna is one species of water flea that has been used for a long time for ecotoxicity studies. In addition, Daphnia has a myogenic heart that is very useful for cardiotoxicity studies. Previous attempts to calculate the cardiac parameter endpoints in Daphnia suffer from the drawback of tedious operation and high variation due to manual counting errors. Even the previous method that utilized deep learning to help the process suffer from either overestimation of parameters or the need for specialized equipment to perform the analysis. In this study, we utilized DeepLabCut software previously used for animal pose tracking and demonstrated that ResNet_152 was the best fit for training the network. The trained network also showed comparable results with ImageJ and Kymograph, which was mostly done manually. In addition to that, several macro scripts in either Excel or Python format were developed to help summarize the data for faster analysis. The trained network was then challenged to analyze the potential cardiotoxicity of imidacloprid and pendimethalin in D. magna, and it showed that both pesticides cause alteration in their cardiac performance. Overall, this method provides a simple and automatic method to analyze the cardiac performance of Daphnia by utilizing DeepLabCut. The method proposed in this paper can contribute greatly to scientists conducting fast and accurate cardiotoxicity measurements when using Daphnia as a model.

Expression Levels of the Tnni3k Gene in the Heart Are Highly Associated with Cardiac and Glucose Metabolism-Related Phenotypes and Functional Pathways.

BACKGROUND: Troponin-I interacting kinase encoded by the TNNI3K gene is expressed in nuclei and Z-discs of cardiomyocytes. Mutations in TNNI3K were identified in patients with cardiac conduction diseases, arrhythmias, and cardiomyopathy. METHODS: We performed cardiac gene expression, whole genome sequencing (WGS), and cardiac function analysis in 40 strains of BXD recombinant inbred mice derived from C57BL/6J (B6) and DBA/2J (D2) strains. Expression quantitative trait loci (eQTLs) mapping and gene enrichment analysis was performed, followed by validation of candidate Tnni3k-regulatory genes. RESULTS: WGS identified compound splicing and missense T659I Tnni3k variants in the D2 parent and some BXD strains (D allele) and these strains had significantly lower Tnni3k expression than those carrying wild-type Tnni3k (B allele). Expression levels of Tnni3k significantly correlated with multiple cardiac (heart rate, wall thickness, PR duration, and T amplitude) and metabolic (glucose levels and insulin resistance) phenotypes in BXDs. A significant cis-eQTL on chromosome 3 was identified for the regulation of Tnni3k expression. Furthermore, Tnni3k-correlated genes were primarily involved in cardiac and glucose metabolism-related functions and pathways. Genes Nodal, Gnas, Nfkb1, Bmpr2, Bmp7, Smad7, Acvr1b, Acvr2b, Chrd, Tgfb3, Irs1, and Ppp1cb were differentially expressed between the B and D alleles. CONCLUSIONS: Compound splicing and T659I Tnni3k variants reduce cardiac Tnni3k expression and Tnni3k levels are associated with cardiac and glucose metabolism-related phenotypes.