The cardiomyocyte origins of diastolic dysfunction: cellular components of myocardial "stiffness".

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca2+ flux and Ca2+ levels in diastole, the troponin-tropomyosin complex role as a Ca2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

Altered cardiac structure and function is related to seizure frequency in a rat model of chronic acquired temporal lobe epilepsy.

OBJECTIVE: This study aimed to prospectively examine cardiac structure and function in the kainic acid-induced post-status epilepticus (post-KA SE) model of chronic acquired temporal lobe epilepsy (TLE), specifically to examine for changes between the pre-epileptic, early epileptogenesis and the chronic epilepsy stages. We also aimed to examine whether any changes related to the seizure frequency in individual animals. METHODS: Four hours of SE was induced in 9 male Wistar rats at 10 weeks of age, with 8 saline treated matched control rats. Echocardiography was performed prior to the induction of SE, two- and 10-weeks post-SE. Two weeks of continuous video-EEG and simultaneous ECG recordings were acquired for two weeks from 11 weeks post-KA SE. The video-EEG recordings were analyzed blindly to quantify the number and severity of spontaneous seizures, and the ECG recordings analyzed for measures of heart rate variability (HRV). PicroSirius red histology was performed to assess cardiac fibrosis, and intracellular Ca2+ levels and cell contractility were measured by microfluorimetry. RESULTS: All 9 post-KA SE rats were demonstrated to have spontaneous recurrent seizures on the two-week video-EEG recording acquired from 11 weeks SE (seizure frequency ranging from 0.3 to 10.6 seizures/day with the seizure durations from 11 to 62 s), and none of the 8 control rats. Left ventricular wall thickness was thinner, left ventricular internal dimension was shorter, and ejection fraction was significantly decreased in chronically epileptic rats, and was negatively correlated to seizure frequency in individual rats. Diastolic dysfunction was evident in chronically epileptic rats by a decrease in mitral valve deceleration time and an increase in E/E` ratio. Measures of HRV were reduced in the chronically epileptic rats, indicating abnormalities of cardiac autonomic function. Cardiac fibrosis was significantly increased in epileptic rats, positively correlated to seizure frequency, and negatively correlated to ejection fraction. The cardiac fibrosis was not a consequence of direct effect of KA toxicity, as it was not seen in the 6/10 rats from separate cohort that received similar doses of KA but did not go into SE. Cardiomyocyte length, width, volume, and rate of cell lengthening and shortening were significantly reduced in epileptic rats. SIGNIFICANCE: The results from this study demonstrate that chronic epilepsy in the post-KA SE rat model of TLE is associated with a progressive deterioration in cardiac structure and function, with a restrictive cardiomyopathy associated with myocardial fibrosis. Positive correlations between seizure frequency and the severity of the cardiac changes were identified. These results provide new insights into the pathophysiology of cardiac disease in chronic epilepsy, and may have relevance for the heterogeneous mechanisms that place these people at risk of sudden unexplained death.

Pericardial adipose and aromatase: A new translational target for aging, obesity and arrhythmogenesis?

A correlation exists between the extent of pericardial adipose and atrial fibrillation (AF) risk, though the underlying mechanisms remain unclear. Selected adipose depots express high levels of aromatase, capable of converting androgens to estrogens - no studies have investigated aromatase occurrence/expression regulation in pericardial adipose. The Women's Health Initiative reported that estrogen-only therapy in women elevated AF incidence, indicating augmented estrogenic influence may exacerbate cardiac vulnerability. The aim of this study was to identify the occurrence of pericardial adipose aromatase, evaluate the age- and sex-dependency of local cardiac steroid synthesis capacity and seek preliminary experimental evidence of a link between pericardial adipose aromatase capacity and arrhythmogenic vulnerability. Both human atrial appendage and epicardial adipose exhibited immunoblot aromatase expression. In rodents, myocardium and pericardial adipose aromatase expression increased >20-fold relative to young controls. Comparing young, aged and aged-high fat diet animals, a significant positive correlation was determined between the total aromatase content of pericardial adipose and the occurrence/duration of triggered atrial arrhythmias. Incidence and duration of arrhythmias were increased in hearts perfused with 17ß-estradiol. This study provides novel report of pericardial adipose aromatase expression. We show that aromatase expression is remarkably upregulated with aging, and aromatase estrogen conversion capacity significantly elevated with obesity-related cardiac adiposity. Our studies suggest an association between adiposity, aromatase estrogenic capacity and atrial arrhythmogenicity - additional investigation is required to establish causality. The potential impact of these findings may be considerable, and suggests that focus on local cardiac steroid conversion (rather than systemic levels) may yield translational outcomes.

BGP-15 Improves Aspects of the Dystrophic Pathology in mdx and dko Mice with Differing Efficacies in Heart and Skeletal Muscle.

Duchenne muscular dystrophy is a severe and progressive striated muscle wasting disorder that leads to premature death from respiratory and/or cardiac failure. We have previously shown that treatment of young dystrophic mdx and dystrophin/utrophin null (dko) mice with BGP-15, a coinducer of heat shock protein 72, ameliorated the dystrophic pathology. We therefore tested the hypothesis that later-stage BGP-15 treatment would similarly benefit older mdx and dko mice when the dystrophic pathology was already well established. Later stage treatment of mdx or dko mice with BGP-15 did not improve maximal force of tibialis anterior (TA) muscles (in situ) or diaphragm muscle strips (in vitro). However, collagen deposition (fibrosis) was reduced in TA muscles of BGP-15-treated dko mice but unchanged in TA muscles of treated mdx mice and diaphragm of treated mdx and dko mice. We also examined whether BGP-15 treatment could ameliorate aspects of the cardiac pathology, and in young dko mice it reduced collagen deposition and improved both membrane integrity and systolic function. These results confirm BGP-15's ability to improve aspects of the dystrophic pathology but with differing efficacies in heart and skeletal muscles at different stages of the disease progression. These findings support a role for BGP-15 among a suite of pharmacological therapies for Duchenne muscular dystrophy and related disorders.

Telomere dynamics during aging in polygenic left ventricular hypertrophy.

Short telomeres are associated with increased risk of cardiovascular disease. Here we studied cardiomyocyte telomere length at key ages during the ontogeny of cardiac hypertrophy and failure in the hypertrophic heart rat (HHR) and compared these with the normal heart rat (NHR) control strain. Key ages corresponded with the pathophysiological sequence beginning with fewer cardiomyocytes (2 days), leading to left ventricular hypertrophy (LVH) (13 wk) and subsequently progression to heart failure (38 wk). We measured telomere length, tissue activity of telomerase, mRNA levels of telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc), and expression of the telomeric regulator microRNA miR-34a. Cardiac telomere length was longer in the HHR compared with the control strain at 2 days and 38 wk, but shorter at 13 wk. Neonatal HHR had higher cardiac telomerase activity and expression of Tert and miR-34a. Telomerase activity was not different at 13 or 38 wk. Tert mRNA and Terc RNA were overexpressed at 38 wk, while miR-34a was overexpressed at 13 wk but downregulated at 38 wk. Circulating leukocytes were strongly correlated with cardiac telomere length in the HHR only. The longer neonatal telomeres in HHR are likely to reflect fewer fetal and early postnatal cardiomyocyte cell divisions and explain the reduced total cardiomyocyte complement that predisposes to later hypertrophy and failure. Although shorter telomeres were a feature of cardiac hypertrophy at 13 wk, they were not present at the progression to heart failure at 38 wk.

Dietary omega-6 fatty acid replacement selectively impairs cardiac functional recovery after ischemia in female (but not male) rats.

A definitive understanding of the role of dietary lipids in determining cardioprotection (or cardiodetriment) has been elusive. Randomized trial findings have been variable and sex specificity of dietary interventions has not been determined. In this investigation the sex-selective cardiac functional effects of three diets enriched by omega-3 or omega-6 polyunsaturated fatty acids (PUFA) or enriched to an equivalent extent in saturated fatty acid components were examined in rats after an 8-wk treatment period. In females the myocardial membrane omega-6:omega-3 PUFA ratio was twofold higher than males in the omega-6 diet replacement group. In diets specified to be high in omega-3 PUFA or in saturated fat, this sex difference was not apparent. Isolated cardiomyocyte and heart Langendorff perfusion experiments were performed, and molecular measures of cell viability were assessed. Under basal conditions the contractile performance of omega-6 fed female cardiomyocytes and hearts was reduced compared with males. Omega-6 fed females exhibited impaired systolic resilience after ischemic insult. This response was associated with increased postischemia necrotic cell damage evaluated by coronary lactate dehydrogenase during reperfusion in omega-6 fed females. Cardiac and myocyte functional parameters were not different between omega-3 and saturated fat dietary groups and within these groups there were no discernible sex differences. Our data provide evidence at both the cardiac and cardiomyocyte levels that dietary saturated fatty acid intake replacement with an omega-6 (but not omega-3) enriched diet has selective adverse cardiac effect in females. This finding has potential relevance in relation to women, cardiac risk, and dietary management.

Mechanical loading reveals an intrinsic cardiomyocyte stiffness contribution to diastolic dysfunction in murine cardiometabolic disease.

Cardiometabolic syndromes including diabetes and obesity are associated with occurrence of heart failure with diastolic dysfunction. There are no specific treatments for diastolic dysfunction and therapies to manage symptoms have limited efficacy. Understanding of the cardiomyocyte origins of diastolic dysfunction is an important priority to identify new therapeutics. The investigative goal was to experimentally define in vitro stiffness (stress/strain) properties of isolated cardiomyocytes derived from rodent hearts exhibiting diastolic dysfunction in vivo in response to dietary induction of cardiometabolic disease. Mice fed a High Fat/Sugar Diet (HFSD vs control) for at least 25 weeks exhibited glucose intolerance, obesity and diastolic dysfunction (echo E/e'). Intact paced cardiomyocytes were functionally investigated in three conditions: non-loaded, loaded and stretched. Mean stiffness of HFSD cardiomyocytes was 70% higher than control. The E/e' doppler ratio for the origin hearts was elevated by 35%. A significant relationship was identified between in vitro cardiomyocyte stiffness and in vivo dysfunction severity. With conversion from non-loaded to loaded condition, the decrement in maximal sarcomere lengthening rate was more accentuated in HFSD cardiomyocytes (vs control). With stretch, the Ca 2+ transient decay time course was prolonged. With transition from 2-4Hz pacing, HFSD cardiomyocyte stiffness was further increased, yet diastolic Ca 2+ rise was 50% less than control. Collectively, these findings demonstrate that a component of cardiac diastolic dysfunction in cardiometabolic disease is derived from intrinsic cardiomyocyte mechanical abnormality. Differential responses to load, stretch and pacing suggest that a previously undescribed alteration in myofilament-Ca 2+ interaction contributes to cardiomyocyte stiffness in cardiometabolic disease. KEY POINTS: Understanding cardiomyocyte stiffness components is an important priority for identifying new therapeutics for diastolic dysfunction, a key feature of cardiometabolic disease. In this study cardiac function was measured in vivo (echocardiography) for mice fed a high-fat/sugar diet (HFSD, ≥25weeks) and performance of intact isolated cardiomyocytes derived from the same hearts was measured during pacing under non-loaded, loaded and stretched conditions in vitro . Using a calibrated cardiomyocyte stretch protocol, stiffness (stress/strain) was elevated in HFSD cardiomyocytes in vitro and correlated with diastolic dysfunction (E/e') in vivo . The HFSD cardiomyocyte Ca 2+ transient decay was prolonged in response to stretch, and stiffness was accentuated in response to pacing increase while the rise in diastolic Ca 2+ was attenuated. These findings suggest that stretch-dependent augmentation of the myofilament-Ca 2+ response during diastole partially underlies elevated cardiomyocyte stiffness and diastolic dysfunction of hearts of animals with cardiometabolic disease.

Localized cardiomyocyte lipid accumulation is associated with slowed epicardial conduction in rats.

Transmural action potential duration differences and transmural conduction gradients aid the synchronization of left ventricular repolarization, reducing vulnerability to transmural reentry and arrhythmias. A high-fat diet and the associated accumulation of pericardial adipose tissue are linked with conduction slowing and greater arrhythmia vulnerability. It is predicted that cardiac adiposity may more readily influence epicardial conduction (versus endocardial) and disrupt normal transmural activation/repolarization gradients. The aim of this investigation was to determine whether transmural conduction gradients are modified in a rat model of pericardial adiposity. Adult Sprague-Dawley rats were fed control/high-fat diets for 15 wk. Left ventricular 300 µm tangential slices were generated from the endocardium to the epicardium, and conduction was mapped using microelectrode arrays. Slices were then histologically processed to assess fibrosis and cardiomyocyte lipid status. Conduction velocity was significantly greater in epicardial versus endocardial slices in control rats, supporting the concept of a transmural conduction gradient. High-fat diet feeding increased pericardial adiposity and abolished the transmural conduction gradient. Slowed epicardial conduction in epicardial slices strongly correlated with an increase in cardiomyocyte lipid content, but not fibrosis. The positive transmural conduction gradient reported here represents a physiological property of the ventricular activation sequence that likely protects against reentry. The absence of this gradient, secondary to conduction slowing and cardiomyocyte lipid accumulation, specifically in the epicardium, indicates a novel mechanism by which pericardial adiposity may exacerbate ventricular arrhythmias.

Cardiac mechanical efficiency is preserved in primary cardiac hypertrophy despite impaired mechanical function.

Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca2+ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca2+ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca2+ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy.

Systematic quantification of histologic ventricular fibrosis in isolated mitral valve prolapse and sudden cardiac death.

BACKGROUND: Cardiac fibrosis in mitral valve prolapse (MVP) is implicated in the development of sudden cardiac death (SCD); however, the pattern remains poorly characterized. OBJECTIVE: The purpose of this study was to systematically quantify left and right ventricular fibrosis in individuals with isolated MVP and SCD (iMVP-SCD), whereby other potential causes of death are excluded, compared to a control cohort. METHODS: Individuals with iMVP-SCD were identified from the Victorian Institute of Forensic Medicine, Australia, and matched for age, sex, and body mass index to control cases with noncardiac death. Cardiac tissue sections were analyzed to determine collagen deposition in the left ventricular free wall (anterior, lateral, and posterior portions), interventricular septum, and right ventricle. Within the iMVP-SCD cases, the endocardial-to-epicardial distribution of fibrosis within the left ventricle was specifically characterized. RESULTS: Seventeen cases with iMVP-SCD were matched 1:1 with 17 controls, yielding 149 samples and 1788 histologic regions. The iMVP-SCD group had increased left ventricular (anterior, lateral, and posterior; all P <.001) and interventricular septum fibrosis (P <.001), but similar amounts of right ventricular fibrosis (P = .62) compared to controls. In iMVP-SCD, left ventricular fibrosis was significantly higher in the lateral and posterior walls compared to the anterior wall and interventricular septum (all P <.001). Within the lateral and posterior walls, iMVP-SCD cases had a significant endocardial-to-epicardial gradient of cardiac fibrosis (P <.01) similar to other known conditions that cause cardiac remodeling. CONCLUSION: Our study indicates that nonuniform left ventricular remodeling with both localized and generalized left ventricular fibrosis is important in the pathogenesis of SCD in individuals with MVP.

Targeted GLUT-4 deficiency in the heart induces cardiomyocyte hypertrophy and impaired contractility linked with Ca(2+) and proton flux dysregulation.

There is clinical evidence to suggest that impaired myocardial glucose uptake contributes to the pathogenesis of hypertrophic, insulin-resistant cardiomyopathy. The goal of this study was to determine whether cardiac deficiency of the insulin-sensitive glucose transporter, GLUT4, has deleterious effect on cardiomyocyte excitation-contraction coupling. Cre-Lox mouse models of cardiac GLUT4 knockdown (KD, 85% reduction) and knockout (KO, >95% reduction), which exhibit similar systemic hyperinsulinemic and hyperglycemic states, were investigated. The Ca(2+) current (I(Ca)) and Na(+)-Ca(2+) exchanger (NCX) fluxes, Na(+)-H(+) exchanger (NHE) activity, and contractile performance of GLUT4-deficient myocytes was examined using whole-cell patch-clamp, epifluorescence, and imaging techniques. GLUT4-KO exhibited significant cardiac enlargement characterized by cardiomyocyte hypertrophy (40% increase in cell area) and fibrosis. GLUT4-KO myocyte contractility was significantly diminished, with reduced mean maximum shortening (5.0+/-0.4% vs. 6.2+/-0.6%, 5 Hz). Maximal rates of shortening and relaxation were also reduced (20-25%), and latency was delayed. In GLUT4-KO myocytes, the I(Ca) density was decreased (-2.80+/-0.29 vs. -5.30+/-0.70 pA/pF), and mean I(NCX) was significantly increased in both outward (by 60%) and inward (by 100%) directions. GLUT4-KO expression levels of SERCA2 and RyR2 were reduced by approximately 50%. NHE-mediated H(+) flux in response to NH(4)Cl acid loading was markedly elevated GLUT4-KO myocytes, associated with doubled expression of NHE1. These findings demonstrate that, independent of systemic endocrinological disturbance, cardiac GLUT4 deficiency per se provides a lesion sufficient to induce profound alterations in cardiomyocyte Ca(2+) and pH homeostasis. Our investigation identifies the cardiac GLUT4 as a potential primary molecular therapeutic target in ameliorating the functional deficits associated with insulin-resistant cardiomyopathy.

Cell volume control in phospholemman (PLM) knockout mice: do cardiac myocytes demonstrate a regulatory volume decrease and is this influenced by deletion of PLM?

In addition to modulatory actions on Na+-K+-ATPase, phospholemman (PLM) has been proposed to play a role in cell volume regulation. Overexpression of PLM induces ionic conductances, with 'PLM channels' exhibiting selectivity for taurine. Osmotic challenge of host cells overexpressing PLM increases taurine efflux and augments the cellular regulatory volume decrease (RVD) response, though a link between PLM and cell volume regulation has not been studied in the heart. We recently reported a depressed cardiac contractile function in PLM knockout mice in vivo, which was exacerbated in crystalloid-perfused isolated hearts, indicating that these hearts were osmotically challenged. To address this, the present study investigated the role of PLM in osmoregulation in the heart. Isolated PLM wild-type and knockout hearts were perfused with a crystalloid buffer supplemented with mannitol in a bid to prevent perfusate-induced cell swelling and maintain function. Accordingly, and in contrast to wild-type control hearts, contractile function was improved in PLM knockout hearts with 30 mM mannitol. To investigate further, isolated PLM wild-type and knockout cardiomyocytes were subjected to increasing hyposmotic challenges. Initial validation studies showed the IonOptix video edge-detection system to be a simple and accurate 'real-time' method for tracking cell width as a marker of cell size. Myocytes swelled equally in both genotypes, indicating that PLM, when expressed at physiological levels in cardiomyocytes, is not essential to limit water accumulation in response to a hyposmotic challenge. Interestingly, freshly isolated adult cardiomyocytes consistently failed to mount RVDs in response to cell swelling, adding to conflicting reports in the literature. A proposed perturbation of the RVD response as a result of the cell isolation process was not restored, however, with short-term culture in either adult or neonatal cardiomyocytes.

Sex differences in cardiac muscle responsiveness to Ca2+ and L-type Ca2+ channel modulation.

This study investigated sex-related differences in rat papillary muscle force generation in response to altered extracellular [Ca2+] ([Ca2+](o), 0.2 to 5.0 mM) and to L-type Ca2+ channel modulators (nifedipine and Bay K8644). At all [Ca2+]o examined, contractile force was significantly greater in male than female papillary muscles. The [Ca2+]o required for 50% maximum force was significantly lower in male [0.34+/-0.06 mM] than female [0.61+/-0.10 mM] papillary muscles. Nifedipine decreased contractile force in papillary muscles of both sexes in a concentration-dependent manner, but the extent of the contractile depression was more marked in male papillary muscles at all nifedipine concentrations examined. BayK 8644 produced a concentration-dependent increase in contractile force in male papillary muscles but notably, not in female papillary muscles. These findings show that sex differences in myocardial mechanical function are associated with sex-specific modulation of L-type Ca2+ channel responsiveness. Thus, the L-type Ca2+ channel could represent an important cellular locus from which sex-based differences in myocardial excitation-contraction coupling arise.

Diastolic dysfunction is more apparent in STZ-induced diabetic female mice, despite less pronounced hyperglycemia.

Diabetic cardiomyopathy is a distinct pathology characterized by early emergence of diastolic dysfunction. Increased cardiovascular risk associated with diabetes is more marked for women, but an understanding of the role of diastolic dysfunction in female susceptibility to diabetic cardiomyopathy is lacking. To investigate the sex-specific relationship between systemic diabetic status and in vivo occurrence of diastolic dysfunction, diabetes was induced in male and female mice by streptozotocin (5x daily i.p. 55 mg/kg). Echocardiography was performed at 7 weeks post-diabetes induction, cardiac collagen content assessed by picrosirius red staining, and gene expression measured using qPCR. The extent of diabetes-associated hyperglycemia was more marked in males than females (males: 25.8 ± 1.2 vs 9.1 ± 0.4 mM; females: 13.5 ± 1.5 vs 8.4 ± 0.4 mM, p < 0.05) yet in vivo diastolic dysfunction was evident in female (E/E' 54% increase, p < 0.05) but not male diabetic mice. Cardiac structural abnormalities (left ventricular wall thinning, collagen deposition) were similar in male and female diabetic mice. Female-specific gene expression changes in glucose metabolic and autophagy-related genes were evident. This study demonstrates that STZ-induced diabetic female mice exhibit a heightened susceptibility to diastolic dysfunction, despite exhibiting a lower extent of hyperglycemia than male mice. These findings highlight the importance of early echocardiographic screening of asymptomatic prediabetic at-risk patients.

Cardiomyocyte Functional Etiology in Heart Failure With Preserved Ejection Fraction Is Distinctive-A New Preclinical Model.

BACKGROUND: Among the growing numbers of patients with heart failure, up to one half have heart failure with preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF is a substantial and escalating unmet clinical need-and the lack of HFpEF-specific animal models represents a major preclinical barrier in advancing understanding of HFpEF. As established treatments for heart failure with reduced ejection fraction (HFrEF) have proven ineffective for HFpEF, the contention that the intrinsic cardiomyocyte phenotype is distinct in these 2 conditions requires consideration. Our goal was to validate and characterize a new rodent model of HFpEF, undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology. METHODS AND RESULTS: The selectively inbred Hypertrophic Heart Rat (HHR) strain exhibits adult cardiac enlargement (without hypertension) and premature death (40% mortality at 50 weeks) compared to its control strain, the normal heart rat. Hypertrophy was characterized in vivo by maintained systolic parameters (ejection fraction at 85%-90% control) with marked diastolic dysfunction (increased E/E'). Surprisingly, HHR cardiomyocytes were hypercontractile, exhibiting high Ca2+ operational levels and markedly increased L-type Ca2+ channel current. In HHR, prominent regions of reparative fibrosis in the left ventricle free wall adjacent to the interventricular septum were observed. CONCLUSIONS: Thus, the cardiomyocyte remodeling process in the etiology of this HFpEF model contrasts dramatically with the suppressed Ca2+ cycling state that typifies heart failure with reduced ejection fraction. These findings may explain clinical observations, that treatments considered appropriate for heart failure with reduced ejection fraction are of little benefit for HFpEF-and suggest a basis for new therapeutic strategies.

Quantitative polarized phase microscopy for birefringence imaging.

A novel application of quantitative phase imaging under linearly polarized light is introduced for studying unstained anisotropic live cells. The method is first validated as a technique for mapping the two-dimensional retardation distribution of a well-characterized optical fiber and is then applied to the characterization of unstained isolated cardiac cells. The experimental retardation measurements are in very good agreement with the established Brace-Köhler method, and additionally provide spatially resolved cell birefringence and phase data.

Friedreich's ataxia induced pluripotent stem cell-derived cardiomyocytes display electrophysiological abnormalities and calcium handling deficiency.

We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.

Experimental and Human Evidence for Lipocalin-2 (Neutrophil Gelatinase-Associated Lipocalin [NGAL]) in the Development of Cardiac Hypertrophy and heart failure.

BACKGROUND: Cardiac hypertrophy increases the risk of developing heart failure and cardiovascular death. The neutrophil inflammatory protein, lipocalin-2 (LCN2/NGAL), is elevated in certain forms of cardiac hypertrophy and acute heart failure. However, a specific role for LCN2 in predisposition and etiology of hypertrophy and the relevant genetic determinants are unclear. Here, we defined the role of LCN2 in concentric cardiac hypertrophy in terms of pathophysiology, inflammatory expression networks, and genomic determinants. METHODS AND RESULTS: We used 3 experimental models: a polygenic model of cardiac hypertrophy and heart failure, a model of intrauterine growth restriction and Lcn2-knockout mouse; cultured cardiomyocytes; and 2 human cohorts: 114 type 2 diabetes mellitus patients and 2064 healthy subjects of the YFS (Young Finns Study). In hypertrophic heart rats, cardiac and circulating Lcn2 was significantly overexpressed before, during, and after development of cardiac hypertrophy and heart failure. Lcn2 expression was increased in hypertrophic hearts in a model of intrauterine growth restriction, whereas Lcn2-knockout mice had smaller hearts. In cultured cardiomyocytes, Lcn2 activated molecular hypertrophic pathways and increased cell size, but reduced proliferation and cell numbers. Increased LCN2 was associated with cardiac hypertrophy and diastolic dysfunction in diabetes mellitus. In the YFS, LCN2 expression was associated with body mass index and cardiac mass and with levels of inflammatory markers. The single-nucleotide polymorphism, rs13297295, located near LCN2 defined a significant cis-eQTL for LCN2 expression. CONCLUSIONS: Direct effects of LCN2 on cardiomyocyte size and number and the consistent associations in experimental and human analyses reveal a central role for LCN2 in the ontogeny of cardiac hypertrophy and heart failure.

Male and female hypertrophic rat cardiac myocyte functional responses to ischemic stress and ß-adrenergic challenge are different.

BACKGROUND: Cardiac hypertrophy is the most potent cardiovascular risk factor after age, and relative mortality risk linked with cardiac hypertrophy is greater in women. Ischemic heart disease is the most common form of cardiovascular pathology for both men and women, yet significant differences in incidence and outcomes exist between the sexes. Cardiac hypertrophy and ischemia are frequently occurring dual pathologies. Whether the cellular (cardiomyocyte) mechanisms underlying myocardial damage differ in women and men remains to be determined. In this study, utilizing an in vitro experimental approach, our goal was to examine the proposition that responses of male/female cardiomyocytes to ischemic (and adrenergic) stress may be differentially modulated by the presence of pre-existing cardiac hypertrophy. METHODS: We used a novel normotensive custom-derived hypertrophic heart rat (HHR; vs control strain normal heart rat (NHR)). Cardiomyocyte morphologic and electromechanical functional studies were performed using microfluorimetric techniques involving simulated ischemia/reperfusion protocols. RESULTS: HHR females exhibited pronounced cardiac/cardiomyocyte enlargement, equivalent to males. Under basal conditions, a lower twitch amplitude in female myocytes was prominent in normal but not in hypertrophic myocytes. The cardiomyocyte Ca(2+) responses to ß-adrenergic challenge differed in hypertrophic male and female cardiomyocytes, with the accentuated response in males abrogated in females-even while contractile responses were similar. In simulated ischemia, a marked and selective elevation of end-ischemia Ca(2+) in normal female myocytes was completely suppressed in hypertrophic female myocytes-even though all groups demonstrated similar shifts in myocyte contractile performance. After 30 min of simulated reperfusion, the Ca(2+) desensitization characterizing the male response was distinctively absent in female cardiomyocytes. CONCLUSIONS: Our data demonstrate that cardiac hypertrophy produces dramatically different basal and stress-induced pathophenotypes in female- and male-origin cardiomyocytes. The lower Ca(2+) operational status characteristic of female (vs male) cardiomyocytes comprising normal hearts is not exhibited by myocytes of hypertrophic hearts. After ischemia/reperfusion, availability of activator Ca(2+) is suppressed in female hypertrophic myocytes, whereas sensitivity to Ca(2+) is blunted in male hypertrophic myocytes. These findings demonstrate that selective intervention strategies should be pursued to optimize post-ischemic electromechanical support for male and female hypertrophic hearts.