Academic education in environmental chemistry in Europe-addressing future challenges in research and regulation.

Following the tradition of the 16th International Conference on Chemistry and the Environment (ICCE) in Oslo, Norway (2017), and the subsequent 17th ICCE in Thessaloniki, Greece (2019), a follow-up session on higher education in environmental science was organized at the 18th ICCE in Venice, Italy (June 2023). The aim of the session was to stimulate the exchange of experiences and knowledge on graduate and post-graduate level educational programmes, including their development, prioritization, and implementation. The session discussed the integration of practical training activities, which included the integration of environmental chemistry in various bachelor's and master's programmes. The aim was to demonstrate the versatility of environmental chemistry as an interdisciplinary scientific discipline that allows the development of essential green skills for developing sustainable strategies for the environmental experts of tomorrow. Furthermore, during the session, a survey was conducted among the conference participants to collect attitudes and reflections from the audience on the education of environmental chemistry (and related fields).

The W792R HCM missense mutation in the C6 domain of cardiac myosin binding protein-C increases contractility in neonatal mouse myocardium.

Missense mutations in cardiac myosin binding protein C (cMyBP-C) are known to cause hypertrophic cardiomyopathy (HCM). The W792R mutation in the C6 domain of cMyBP-C causes severe, early onset HCM in humans, yet its impact on the function of cMyBP-C and the mechanism through which it causes disease remain unknown. To fully characterize the effect of the W792R mutation on cardiac morphology and function in vivo, we generated a murine knock-in model. We crossed heterozygous W792RWR mice to produce homozygous mutant W792RRR, heterozygous W792RWR, and control W792RWW mice. W792RRR mice present with cardiac hypertrophy, myofibrillar disarray and fibrosis by postnatal day 10 (PND10), and do not survive past PND21. Full-length cMyBP-C is present at similar levels in W792RWW, W792RWR and W792RRR mice and is properly incorporated into the sarcomere. Heterozygous W792RWR mice displayed normal heart morphology and contractility. Permeabilized myocardium from PND10 W792RRR mice showed increased Ca2+ sensitivity, accelerated cross-bridge cycling kinetics, decreased cooperativity in the activation of force, and increased expression of hypertrophy-related genes. In silico modeling suggests that the W792R mutation destabilizes the fold of the C6 domain and increases torsion in the C5-C7 region, possibly impacting regulatory interactions of cMyBP-C with myosin and actin. Based on the data presented here, we propose a model in which mutant W792R cMyBP-C preferentially forms Ca2+ sensitizing interactions with actin, rather than inhibitory interactions with myosin. The W792R-cMyBP-C mouse model provides mechanistic insights into the pathology of this mutation and may provide a mechanism by which other central domain missense mutations in cMyBP-C may alter contractility, leading to HCM.

Lactate- and immunomagnetic-purified hiPSC-derived cardiomyocytes generate comparable engineered cardiac tissue constructs.

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared with magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs affects the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed that lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for 4 weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transient measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates that lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and it suggests that lactate purification does not result in an irreversible change in a hiPSC-CM phenotype.

Neutral sphingomyelinase regulates mechanotransduction in human engineered cardiac tissues and mouse hearts.

Cardiovascular disease is the leading cause of death in the USA and is known to be exacerbated by elevated mechanical stress from hypertension. Caveolae are plasma membrane structures that buffer mechanical stress but have been found to be reduced in pathological conditions associated with chronically stretched myocardium. To explore the physiological implications of the loss of caveolae, we used human engineered cardiac tissue (ECT) constructs, composed of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived cardiac fibroblasts, to develop a long-term cyclic stretch protocol that recapitulates the effects of hypertension on caveolae expression, membrane tension, and the ß-adrenergic response. Leveraging this new stretch protocol, we identified neutral sphingomyelinases (nSMase) as mechanoregulated mediators of caveolae loss, ceramide production and the blunted ß-adrenergic response in this human cardiac model. Specifically, in our ECT model, nSMase inhibition via GW4869 prevented stretch-induced loss of caveolae-like structures, mitigated nSMase-dependent ceramide production, and maintained the ECT contractile kinetic response to isoprenaline. These findings are correlated with a blood lipidomic analysis in middle-aged and older adults, which revealed an increase of the circulating levels of ceramides in adults with hypertension. Furthermore, we found that conduction slowing from increased pressure loading in mouse left ventricle was abolished in the context of nSMase inhibition. Collectively, these findings identify nSMase as a potent drug target for mitigating stretch-induced effects on cardiac function. KEY POINTS: We have developed a new stretch protocol for human engineered cardiac tissue that recapitulates changes in plasma membrane morphology observed in animal models of pressure/volume overload. Stretch of engineered cardiac tissue induces activation of neutral sphingomyelinase (nSMase), generation of ceramide, and disassembly of caveolae. Activation of nSMase blunts cardiac ß-adrenergic contractile kinetics and mediates stretch-induced slowing of conduction and upstroke velocity. Circulating ceramides are increased in adults with hypertension, highlighting the clinical relevance of stretch-induced nSMase activity.

Lactate and Immunomagnetic-purified iPSC-derived Cardiomyocytes Generate Comparable Engineered Cardiac Tissue Constructs.

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACs-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. After purification, hiPSC-CMs were combined with hiPSC-cardiac fibroblasts to create 3D hiPSC-ECT constructs maintained in culture for four weeks. There were no structural differences observed, and there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force, Ca 2+ transients, and ß-adrenergic response revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in any protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable molecular and functional properties, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype.

cMyBP-C ablation in human engineered cardiac tissue causes progressive Ca2+-handling abnormalities.

Truncation mutations in cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM). Heterozygous carriers present with classical HCM, while homozygous carriers present with early onset HCM that rapidly progress to heart failure. We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes derived from these isogenic lines were used to generate cardiac micropatterns and engineered cardiac tissue constructs (ECTs) that were characterized for contractile function, Ca2+-handling, and Ca2+-sensitivity. While heterozygous frame shifts did not alter cMyBP-C protein levels in 2-D cardiomyocytes, cMyBP-C+/- ECTs were haploinsufficient. cMyBP-C-/- cardiac micropatterns produced increased strain with normal Ca2+-handling. After 2 wk of culture in ECT, contractile function was similar between the three genotypes; however, Ca2+-release was slower in the setting of reduced or absent cMyBP-C. At 6 wk in ECT culture, the Ca2+-handling abnormalities became more pronounced in both cMyBP-C+/- and cMyBP-C-/- ECTs, and force production became severely depressed in cMyBP-C-/- ECTs. RNA-seq analysis revealed enrichment of differentially expressed hypertrophic, sarcomeric, Ca2+-handling, and metabolic genes in cMyBP-C+/- and cMyBP-C-/- ECTs. Our data suggest a progressive phenotype caused by cMyBP-C haploinsufficiency and ablation that initially is hypercontractile, but progresses to hypocontractility with impaired relaxation. The severity of the phenotype correlates with the amount of cMyBP-C present, with more severe earlier phenotypes observed in cMyBP-C-/- than cMyBP-C+/- ECTs. We propose that while the primary effect of cMyBP-C haploinsufficiency or ablation may relate to myosin crossbridge orientation, the observed contractile phenotype is Ca2+-mediated.

Quantification of Advective Transport Phenomena to Better Understand Dispersion in the Field.

Observation of dispersion in field situations has left three issues that may be better understood by applying advective transport phenomena. (1) In some experiments, the longitudinal dispersivity becomes constant with increasing pathlength and in other cases it remains growing. (2) Dispersivities reported from multiple comprehensive observations at a single site differ at similar pathlength in some cases more than a factor two. (3) The observed difference between the plume fronts and plume tails is not represented in the reported parameters. The analytic equations for advective transport phenomena at macroscale of De Lange (2020) describe the thickness of the affected flow-tube and the spread of the plume front and tail. The scale factor defines the size of the averaging domain and so of the initial phase. The new macroscale correlation coefficient relates the growth of the longitudinal dispersivity beyond the initial phase to the aquifer heterogeneity. Using stochastic parameters for the aquifer heterogeneity, the parameters are quantified at 14 field experiments in the United States, Canada and Europe enabling the comparison of calculated and reported final dispersivities. Using the quantified parameters, 146 reported and calculated dispersivities along the traveled paths show a good match. A dispersivity derived from the local plume growth may differ a factor of two from the aquifer-representative value. The growths of plume fronts and tails between two plume stages are assessed in 14 cases and compared to calculated values. Distinctive parameters for the plume front and tail support better understanding of field situations. A user-ready spreadsheet is provided.

Human iPSC-engineered cardiac tissue platform faithfully models important cardiac physiology.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and the intact human myocardium. Fulfilling this potential is hampered by their relative immaturity, leading to poor physiological responsiveness. hiPSC-CMs grown in traditional two-dimensional (2D) culture lack a t-tubular system, have only rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as an energy substrate. Culturing hiPSC-CM in a variety of three-dimensional (3D) environments and the addition of nutritional, pharmacological, and electromechanical stimuli have proven, to various degrees, to be beneficial for maturation. We present a detailed assessment of a novel model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are cocultured in a 3D fibrin matrix to form engineered cardiac tissue constructs (hiPSC-ECTs). The hiPSC-ECTs are responsive to physiological stimuli, including stretch, frequency, and ß-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Furthermore, transcript levels of various genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a relatively short period of time in culture (6 wk vs. 2 wk in hiPSC-ECTs). A comparison of the hiPSC-ECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to aid selection of the most appropriate platform for the research question at hand. Important and noteworthy aspects of this human cardiac model system are its reliance on "off-the-shelf" equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological, and electromechanical stimuli that may elicit unintended effects on function.NEW & NOTEWORTHY This study seeks to provide an in-depth assessment of contractile performance of human iPSC-derived cardiomyocytes cultured together with fibroblasts in a 3-dimensional-engineered tissue and compares performance both over time as cells mature, and with corresponding measures found in the literature using alternative 3D culture configurations. The suitability of 3D-engineered human cardiac tissues to model cardiac function is emphasized, and data provided to assist in the selection of the most appropriate configuration based on the target application.

Functionally Integrated Top-Down Proteomics for Standardized Assessment of Human Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues.

Three-dimensional (3D) human induced pluripotent stem cell-derived engineered cardiac tissues (hiPSC-ECTs) have emerged as a promising alternative to two-dimensional hiPSC-cardiomyocyte monolayer systems because hiPSC-ECTs are a closer representation of endogenous cardiac tissues and more faithfully reflect the relevant cardiac pathophysiology. The ability to perform functional and molecular assessments using the same hiPSC-ECT construct would allow for more reliable correlation between observed functional performance and underlying molecular events, and thus is critically needed. Herein, for the first time, we have established an integrated method that permits sequential assessment of functional properties and top-down proteomics from the same single hiPSC-ECT construct. We quantitatively determined the differences in isometric twitch force and the sarcomeric proteoforms between two groups of hiPSC-ECTs that differed in the duration of time of 3D-ECT culture. Importantly, by using this integrated method we discovered a new and strong correlation between the measured contractile parameters and the phosphorylation levels of alpha-tropomyosin between the two groups of hiPSC-ECTs. The integration of functional assessments together with molecular characterization by top-down proteomics in the same hiPSC-ECT construct enables a holistic analysis of hiPSC-ECTs to accelerate their applications in disease modeling, cardiotoxicity, and drug discovery. Data are available via ProteomeXchange with identifier PXD022814.

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics.

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.

Advective Transport Phenomena to Better Understand Dispersion in Field and Modeling Practice.

The absence of recent research on dispersion in engineering applications indicates the need for a description that is more focused on field and modeling practice. Engineers may benefit from simple calculation tools allowing them to understand the processes encountered in the field. Based on a conceptual model for advective transport through an elongated conductivity zone, for example, in fluvial sediments, explicit expressions are presented for macro-scale phenomena: (1) the different travel distances of water particles traveling in laminar flow through and adjacent to a single zone with conductivity higher or lower than that of the aquifer; (2) the affected thickness of the bundle of flowlines; (3) the distinction of inflow, outflow, and through-flow sections; (4) the development of a plume front vs. that of a tail; (5) conservation of mass causing water particles to travel both slower and faster than the aquifer average velocity while passing a single zone. The spread derived from a spatial distribution in a field experiment relates to the geometric mean of the spreads of the plume front and tail. The results obtained for a single conductivity zone are expanded for a general aquifer that is characterized by stochastic parameters. A fundamental new expression describes the dispersive mass flux as the product of the advective volume shift and the related local concentration difference. Contrary to Fickian theory, the dispersive mass flux in both the front and tail of a plume in highly heterogeneous aquifers is limited. In modeling, the advective volume shift is proportional to the cell size.

An Unbiased Proteomics Method to Assess the Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: Human pluripotent stem cell (hPSC)-derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes. OBJECTIVE: We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation. METHODS AND RESULTS: Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. CONCLUSIONS: We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.

Transcriptome Analysis of Cardiac Hypertrophic Growth in MYBPC3-Null Mice Suggests Early Responders in Hypertrophic Remodeling.

Rationale: With a prevalence of 1 in 200 individuals, hypertrophic cardiomyopathy (HCM) is thought to be the most common genetic cardiac disease, with potential outcomes that include severe hypertrophy, heart failure, and sudden cardiac death (SCD). Though much research has furthered our understanding of how HCM-causing mutations in genes such as cardiac myosin-binding protein C (MYBPC3) impair contractile function, it remains unclear how such dysfunction leads to hypertrophy and/or arrhythmias, which comprise the HCM phenotype. Identification of early response mediators could provide rational therapeutic targets to reduce disease severity. Our goal was to differentiate physiologic and pathophysiologic hypertrophic growth responses and identify early genetic mediators in the development of cardiomegaly in the cardiac myosin-binding protein C-null (cMyBP-C-/-) mouse model of HCM. Methods and Results: We performed microarray analysis on left ventricles of wild-type (WT) and cMyBPC-/- mice (n = 7 each) at postnatal day (PND) 1 and PND 9, before and after the appearance of an overt HCM phenotype. Applying the criteria of ≥2-fold change, we identified genes whose change was exclusive to pathophysiologic growth (n = 61), physiologic growth (n = 30), and genes whose expression changed ≥2-fold in both WT and cMyBP-C-/- hearts (n = 130). Furthermore, we identified genes that were dysregulated in PND1 cMyBP-C-/- hearts prior to hypertrophy, including genes in mechanosensing pathways and potassium channels linked to arrhythmias. One gene of interest, Xirp2, and its protein product, are regulated during growth but also show early, robust prehypertrophic upregulation in cMyBP-C-/- hearts. Additionally, the transcription factor Zbtb16 also shows prehypertrophic upregulation at both gene and protein levels. Conclusion: Our transcriptome analysis generated a comprehensive data set comparing physiologic vs. hypertrophic growth in mice lacking cMyBP-C. It highlights the importance of extracellular matrix pathways in hypertrophic growth and early dysregulation of potassium channels. Prehypertrophic upregulation of Xirp2 in cMyBP-C-/- hearts supports a growing body of evidence suggesting Xirp2 has the capacity to elicit both hypertrophy and arrhythmias in HCM. Dysregulation of Xirp2, as well as Zbtb16, along with other genes associated with mechanosensing regions of the cardiomyocyte implicate stress-sensing in these regions as a potentially important early response in HCM.

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

Novel Adult-Onset Systolic Cardiomyopathy Due to MYH7 E848G Mutation in Patient-Derived Induced Pluripotent Stem Cells.

A novel myosin heavy chain 7 mutation (E848G) identified in a familial cardiomyopathy was studied in patient-specific induced pluripotent stem cell-derived cardiomyocytes. The cardiomyopathic human induced pluripotent stem cell-derived cardiomyocytes exhibited reduced contractile function as single cells and engineered heart tissues, and genome-edited isogenic cells confirmed the pathogenic nature of the E848G mutation. Reduced contractility may result from impaired interaction between myosin heavy chain 7 and cardiac myosin binding protein C.

Linking metabolic and contractile dysfunction in aged cardiac myocytes.

Aging is associated with declining cardiac contractile function as well as changes in metabolism and mitochondrial function. The relationship between age-related changes in cardiac metabolism and declining cardiac contractile function has not been determined. In order to define the role energetics play in changes in contractile function, we measured mitochondrial NADH, [NADH]m, during continuous contractions of isolated left ventricular myocytes from young (Y) and old (O) FBN rats. Second, we explored the role of metabolic disruption with rotenone and increased workload with isoproterenol (ISO) had on age-related changes in myocytes shortening. Single, intact myocytes were stimulated for 10 min of continuous contraction at either 2 Hz or 4 Hz while being perfused with Ringer's solution. Properties of shortening (peak shortening and rate of shortening) were measured at the onset (T0) and after 10 min (T10) of continuous contraction, and the decline in shortening over time (T10/T0) was determined. Although young and old myocytes had similar contractile function under resting conditions, old myocytes demonstrated decrements in [NADH]m during continuous stimulation, while young myocytes maintained constant [NADH]m over this time. In addition, old myocytes exhibited impaired contractile function to a workload (ISO) and metabolic (rotenone) stress compared to young myocytes. Taken together, these results demonstrated that old myocytes are susceptible to stress-induced contractile dysfunction which may be related to altered cellular energetics.

Increased Postnatal Cardiac Hyperplasia Precedes Cardiomyocyte Hypertrophy in a Model of Hypertrophic Cardiomyopathy.

Rationale: Hypertrophic cardiomyopathy (HCM) occurs in ~0.5% of the population and is a leading cause of sudden cardiac death (SCD) in young adults. Cardiomyocyte hypertrophy has been the accepted mechanism for cardiac enlargement in HCM, but the early signaling responsible for initiating hypertrophy is poorly understood. Mutations in cardiac myosin binding protein C (MYBPC3) are among the most common HCM-causing mutations. Ablation of Mybpc3 in an HCM mouse model (cMyBP-C-/-) rapidly leads to cardiomegaly by postnatal day (PND) 9, though hearts are indistinguishable from wild-type (WT) at birth. This model provides a unique opportunity to explore early processes involved in the dramatic postnatal transition to hypertrophy. Methods and Results: We performed microarray analysis, echocardiography, qPCR, immunohistochemistry (IHC), and isolated cardiomyocyte measurements to characterize the perinatal cMyBP-C-/- phenotype before and after overt hypertrophy. cMyBP-C-/- hearts showed elevated cell cycling at PND1 that transitioned to hypertrophy by PND9. An expanded time course revealed that increased cardiomyocyte cycling was associated with elevated heart weight to body weight ratios prior to cellular hypertrophy, suggesting that cell cycling resulted in cardiomyocyte proliferation. Animals heterozygous for the cMyBP-C deletion trended in the direction of the homozygous null, yet did not show increased heart size by PND9. Conclusions: Results indicate that altered regulation of the cell cycling pathway and elevated proliferation precedes hypertrophy in the cMyBP-C-/- HCM model, and suggests that increased cardiomyocyte number contributes to increased heart size in cMyBP-C-/- mice. This pre-hypertrophic period may reflect a unique time during which the commitment to HCM is determined and disease severity is influenced.

Molecular Approaches in HFpEF: MicroRNAs and iPSC-Derived Cardiomyocytes.

Heart failure with preserved left ventricular ejection fraction (HFpEF) has emerged as one of the largest unmet needs in cardiovascular medicine. HFpEF is increasing in prevalence and causes significant morbidity, mortality, and health care resource utilization. Patients have multiple co-morbidities which contribute to the disease complexity. To date, no effective treatment for HFpEF has been identified. The paucity of cardiac biopsies from this patient population and the absence of well-accepted animal models limit our understanding of the underlying molecular mechanisms of HFpEF. In this review, we discuss combining state-of-the-art technologies of microRNA profiling and human induced pluripotent cell-derived cardiomyocytes (iPSC-CMs) in order to uncover novel molecular pathways that may contribute to the development of HFpEF. Here, we focus the advantages and limitations of microRNA profiling and iPSC-CMs as a disease model system to discover molecular mechanisms in HFpEF.

Cotranslational association of mRNA encoding subunits of heteromeric ion channels.

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes.

Costs of food waste in South Africa: Incorporating inedible food waste.

The economic, social and environmental costs of food waste are being increasingly recognised. Food waste consists of both edible and inedible components. Whilst wastage of edible food is problematic for obvious reasons, there are also costs associated with the disposal of the inedible fraction to landfill. This is the third in a series of papers examining the costs of food waste throughout the value chain in South Africa. The previous papers focused on the edible portion of food waste. In this paper, costs associated with inedible food waste in South Africa are estimated, in terms of the value foregone by not recovering this waste for use in downstream applications, such as energy generation or composting; as well as costs associated with disposal to landfill. Opportunity costs are estimated at R6.4 (US$0.64) billion per annum, or R2668 (US$266) per tonne. Adding this to the previous estimate for edible food waste of R61.5 billion per annum (in 2012 prices; equivalent to R65 billion in 2013 prices) results in a total opportunity cost of food waste in South Africa (in terms of loss of a potentially valuable food source or resource) of R71.4 (US$7.14) billion per annum, or R5667 (US$567) per tonne. Thereafter, estimates of the costs associated with disposal of this food waste to landfill, including both financial costs and externalities (social and environmental costs), are taken into account. These costs amount to R255 (US$25) per tonne, giving rise to a total cost of food waste in South Africa of R75 billion (US$7.5 billion) per annum, or R5922 (US$592) per tonne. This is equivalent to 2.2% of South Africa's 2013 GDP.